Glycoconj J (2013) 30:281–461
DOI 10.1007/s10719-013-9474-x
GLYCO 22
XXII International Symposium on Glycoconjugates
Abstracts
June 23–28, 2013
Dalian, China
中国 大连
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Organising Committee
Co-Chairs
Jianing Zhang (Dalian, China)
Xinmiao Liang (Dalian, China)
Scientific Program Committee
Co-Chairs
Yukui Zhang (Dalian, China)
Jianxin Gu (Shanghai, China)
Committee
Jinku Bao (Chengdu, China)
Kan Ding (Shanghai, China)
Yuguang Du (Dalian, China)
Yuguo Du (Beijing, China)
Xiaodong Gao (Wuxi, China)
Zhongwu Guo (Jinan, China)
Cheng Jin (Beijing, China)
Haojie Lu (Shanghai, China)
Xiaohong Qian (Beijing, China)
Aiguo Shen (Nantong, China)
Lei Wang (Tianjin, China)
Zhongfu Wang (Xi’an, China)
Min Xiao (Jinan, China)
Pengyuan Yang (Shanghai, China)
Qiu Yan (Dalian, China)
Xinshan Ye (Beijing, China)
Biao Yu (Shanghai, China)
Chuyi Yu (Beijing, China)
Guangli Yu (Qingdao, China)
Xiaolian Zhang (Wuhan, China)
Yan Zhang (Shanghai, China)
Yifa Zhou (Changchun, China)
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The International Glycoconjugate Organisation
Iain B. H. Wilson, President
Jianxin Gu, President-elect
Gerald W. Hart, Immediate Past-president
Sandro Sonnino, Secretary
Thierry Hennet, Treasurer
National Representatives
Pedro Bonay (Spain)
Nicolai Bovin, Бовин Николай (Russia)
Inka Brockhausen (Canada)
Jin Won Cho (Korea)
Henrik Clausen (Denmark)
José Luis Daniotti (Argentina)
Anne Dell (UK)
Jukka Finne (Finland)
Paul Gleeson (Australia)
Jianxin Gu 顾建新 (China)
Gerald Hart (USA)
Thierry Hennet (Switzerland)
Jaroslav Katrlík (Slovakia)
Gordan Lauc (Croatia)
Hakon Leffler (Sweden)
Jean-Claude Michalski (France)
Paulo Mourão (Brasil)
Andrei Petrescu (Romania)
Celso Albuquerque Reis (Portugal)
Werner Reutter (Germany)
Sandro Sonnino (Italy)
Avadhesha Surolia (India)
Ken Kitajima 北島健 (Japan)
Maciej Ugorski (Poland)
Johannes F.G. Vliegenthart (The Netherlands)
Iain Wilson (Austria)
Michaela Wimmerová (Czech Republic)
Albert M. Wu 吳明道 (Taiwan)
Lode Wyns (Belgium)
Yehiel Zick (Israel)
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Past Presidents
Eugene. A. Davidson (USA)
Alan B. Foster (UK)
Paul Gleeson (Australia)
Mary Catherine Glick (USA)
Gerald W. Hart (USA)
Colin Hughes (UK)
Roger W. Jeanloz (USA)
Akira Kobata 木幡陽 (Japan)
Jerzy Koscielak (Poland)
Bengt Lindberg (Sweden)
Jean Montreuil (France)
Jürgen Roth (Switzerland)
Harry Schachter (Canada)
Roland Schauer (Germany)
Nathan Sharon (Israel)
Avadhesha Surolia (India)
Guido Tettamanti (Italy)
Johannes F.G. Vliegenthart (The Netherlands)
Tamio Yamakawa 山川民夫 (Japan)
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International Glycoconjugate Organisation Award 2013
Professor Kurt DRICKAMER
Kurt Drickamer is a household name in the glycobiology field for his seminal discovery of and subsequent work on calciumdependent or “C-type” lectin domains. The succession of field-leading discoveries that Drickamer has made during the last
30 years, and the insights they afford, has had incomparable influence on the forefront of research in several disciplines,
including biochemistry, structural biology, immunology and microbiology.
Beginning with his 1981 JBC publication on the hepatic glycoprotein receptor, which controls serum-glycoprotein turnover by
recognising changes with age in their carbohydrate moieties, Drickamer initiated molecular analysis of what turned out to be a huge
family of glycan-binding lectins. After this pioneering (single-authored!) study, he went on to demonstrate that the soluble mannosebinding protein (MBP) (alternatively called the mannose-binding lectin or MBL) of blood and lymph also contains CRDs. In this
protein he showed the CRDs are linked to collagen-like sequences, as is also the case for pulmonary surfactants. Thereby he also
saw the functional parallel between mannose-binding protein and C1q: the former detecting pathogens to initiate innate immunity,
the latter initiating adaptive immunity through detection of pathogen-specific antibody bound to the pathogen. This was a major
contribution to our understanding of innate immunity in a time (i.e. pre-Toll receptors) when innate immunity was not all the rage.
From these seminal observations of CRDs in soluble and cell-surface receptors with different functions in blood homeostasis and
immunity, Drickamer predicted that CRDs were likely to be a feature shared by many other carbohydrate receptors. To test this
hypothesis he initiated systematic investigation and classification of glycan-binding receptors based on the presence of different
types of CRDs. From comparison of the CRDs in those C-type lectins that were discovered using traditional assays of biological
function, Drickamer had defined the sequence motifs that typify this class of CRDs, which enabled him to identify several CRDcontaining proteins that were not known to bind carbohydrates. He pioneered the scanning of whole-genome sequences to
identify novel, uncharacterised proteins that contain CRDs and are thus candidates for having glycan-binding function.
Complementing this approach for the identification of candidate lectins, Drickamer has been a leader in using glycan array
technology to test such candidates for their capacity to bind glycans and determine their sugar specificities.
He is also well known for co-writing, with his colleague and collaborator Maureen Taylor, the text book “Introduction to
Glycobiology”, now in its third edition. Their book enunciates the established and emerging principles of glycobiology in a
manner accessible to students and the broader scientific community and has been translated into Chinese, Japanese, and Korean.
To summarise, Drickamer is a scientist who has both made seminal ‘text book’ discoveries and followed up on them to become the
leading authority on CRDs and their role in cell-cell and cell-microbe recognition. Moreover he unselfishly gives an enormous
amount of his time supporting international glycobiology—for example he served as the deputy chair of the Consortium for
Functional Glycomics Steering Committee for a decade and personally made substantial contributions to the CFG Paradigm Pages.
Since 2005 he is Professor of Biochemistry at Imperial College London and in 2012 he received the Karl Meyer Award from the
Society for Glycobiology. In 2013 he has been selected to be recipient of the International Glycoconjugate Organisation Award.
Michael A.J. Ferguson
University of Dundee, Scotland
IGO Award Recipient 1999
Iain B. H. Wilson
Universität für Bodenkultur Wien, Austria
IGO President 2011–2013
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The IGO Young Glycoscientist Awards 2013 supported by Genos
Dr. Amanda LEWIS
Dr. Amanda Lewis completed her Ph.D. and postdoctoral studies at the University of California San Diego, under the
mentorship of Ajit Varki and Victor Nizet respectively. Training at the intersection of glycobiology and host-microbe
interactions led her to an Assistant Professor position at Washington University School of Medicine in St. Louis, USA
where she has built a successful research program supported by multiple foundation and government grants. The Lewis lab
investigates mechanisms of host sialic acid mimicry and sialoglycan degradation in host-bacterial interactions within the
female urogenital tract. Dr. Lewis’ work on Group B Streptococcus (GBS), the leading cause of sepsis and meningitis in
newborns, has contributed to our understanding of mechanisms by which the bacterium uses its sialic acid-containing capsule
to survive in the maternal urogenital tract and the infant bloodstream. Dr. Lewis also led studies showing that the presence of
GBS and its ability to precisely mimic host sialic acids influences the severity of urinary tract infection (UTI) caused by E.
coli, the most common cause of human UTI. Genomic and biochemical investigations by Dr. Lewis and colleagues suggest
that bacterial mimicry of host sialic acids may be a more common theme in bacterial immune evasion than previously
recognized.
The Lewis lab has also made recent strides in understanding an enigmatic polymicrobial imbalance of the vaginal microbiota
known as bacterial vaginosis (BV)—a condition linked with preterm birth and other pregnancy complications. Lewis’ group
has demonstrated new biochemical and cellular phenotypes of BV and is building new experimental models of the condition
by combining approaches in genomics, microbiology, biochemistry, and infection studies. Recent findings demonstrate that a
controversial contributor in BV, Gardnerella vaginalis, is sufficient to cause BV-like phenotypes in an animal model of
infection and to engage in degradation, foraging, and depletion of mucus barrier sialoglycans. Ongoing studies in the Lewis
lab explore novel contexts and mechanisms of bacterial glycan mimicry, roles of bacterial glycosidases in colonization and
infection, and concepts in glyco-ecology within the female reproductive tract.
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Dr. Wen YI
Dr. Wen Yi received his bachelor degree from Fudan University in China in 2002. He went to the U.S for graduate study, and
in 2008 obtained his Ph.D. degree in Biochemistry from the Ohio State University under the guidance of Prof. Peng G. Wang.
His doctoral research mainly focused on chemo-enzymatic synthesis of complex carbohydrates, and in vitro characterization
of bacterial polysaccharide biosynthesis. From 2008 to 2012, he conducted his postdoctoral training with Prof. Linda C.
Hsieh-Wilson in California Institute of Technology, where he carried out studies to understand the role of O-GlcNAcylation
in regulating cancer metabolism and cell growth. After finishing his postdoctoral training, he moved back to China and
accepted a faculty position at the College of Life Sciences in Zhejiang University. He was awarded multiple prestigious
research grants, including the Concept Award from the Department of Defense Breast Cancer Research Program, and the
Postdoctoral Fellowship from the Tobacco-Related Disease Research Program. He was recently elected to the Thousand
Young Talents Program, a prestigious award given by the Chinese government for recruiting outstanding young talents from
overseas.
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Plenary Lectures
001: A structural and genomic view of receptors for cell
surface glycans
Kurt Drickamer; Department of Life Sciences, Imperial
College, London SW7 2AZ, United Kingdom
k.drickamer@imperial.ac.uk
Mammalian receptors that recognise specific oligosaccharides
represent an important way in which information in glycan
structures can be decoded. We are interested in defining the
complement of human glycan-binding receptors and understanding how they function. Our focus is on receptors that
contain calcium-dependent carbohydrate-recognition domains.
Biochemical, structural and glycan array studies have revealed
multiple different mechanisms by which these receptors bind
selectively to different types of glycans. In receptors found on
macrophages, dendritic cells and endothelial cells in the innate
immune system, the binding mechanisms often favour interaction with broad classes of sugar structures, facilitating selective
interaction with viral, bacterial and fungal pathogens. In receptors that mediate either glycoprotein trafficking in cells and
organisms or cell-cell adhesion, the binding sites are more
restrictive, leading to interactions with a much more limited
set of oligosaccharides. Knowledge of the structures of
carbohydrate-recognition domains has led to identification of
novel glycan-binding receptors. Investigation of these receptors and their target ligands has allowed us to proposal novel
roles for carbohydrate-mediated communication between cells.
002: Glycomics and glycoproteomics: Windows to
glycan function
Anne Dell; Department of Life Sciences, Imperial College
London, SW7 2AZ, UK
a.dell@imperial.ac.uk
Ultra-high sensitivity mass spectrometric strategies incorporating MALDI-MS/MS and nano-electrospray(ES)-MS/MS
enable very complex mixtures of glycans and glycopeptides
from biological extracts of cells and tissues to be studied
thereby revealing the types of glycans present and, importantly, providing clues to structures that are likely to be
functionally important. Glycomic methodologies seek to
define the total N-glycan and/or O-glycan repertoire in a
biological sample, whilst glycoproteomic strategies are
concerned with the analysis of glycopeptides in order to
define heterogeneity at individual glycosylation sites.
Data emerging from our glycomic and glycoproteomic
programmes of collaborative research, which are helping
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to provide new insights into the functions of glycans in
health and disease, will be described. Exemplar projects in
the fields of human reproduction, pathogen-host interactions, glycoimmunology and congenital disorders of glycosylation will be discussed.
In addition our progress on the development of informatic
tools to manage the large volumes of data being acquired
will be outlined.
Acknowledgements: My research is primarily supported by
the Biotechnology and Biological Sciences Research Council. The work described in this talk is a group effort and I am
grateful to my Imperial College colleagues for their contributions. I also thank our numerous collaborators worldwide
for giving us the opportunity to exploit our technology in
many biological fields.
003: The complex interplay of “yin and yang” in branched
N-glycans: from bisecting GlcNAc to core fucose
Naoyuki Taniguchi; Global Research Cluster, Systems
Glycobiology Research Group and RIKEN-Max Planck
Joint Research Center, Wako, 351-0198, Japan
tani52@wd5.so-net.ne.jp
Glycosyltransferases, which catalyze the biosynthesis of Nglycan branching, have different kinds of functions as our
group and other groups have reported. Among them GnT
(UDP-N-acetylglucosaminyltransferases)-III, IV, V, IX
(Vb), and Fut8 (alpha1,6-fucosyltransferase) play key roles
in various physiological and pathological situations. These
enzymatic products such as bisecting GlcNAc, beta1,6GlcNAc branching and core fucose are attached to specific
target proteins, including E-cadherin, integrin RPTP (receptor protein tyrosine phosphatase)-beta, TGF (transforming
growth factor)-beta receptors, EGF (epidermal growth factor) receptors, glucose transporters, and play key roles in
various diseases such as the development of cancer, type II
diabetes, COPD (chronic obstructive pulmonary disease)
and neurodegenerative diseases such as Alzheimer’s disease
and schizophrenia etc. Serum proteins such as AFP (alpha
fetoprotein) are also being used or planned for use as cancer
biomarkers. Moreover glycan modifications, such as core
fucose, result in an enhancement in the activity of ADCC
(antibody dependent cellular cytotoxicity) for antibody therapy against leukemia and several other diseases. On the other
hand, the deletion of core fucose in mice is semi-lethal and the
survivors develop emphysema. In humans and mice, decreased levels of core fucose result in the development of
sensitivity to emphysema and COPD, respectively. Bisecting
GlcNAc was found to have a suppressive action against
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cancer metastasis and to be involved in the development of a
protective effect against Alzheimer’s disease. In some cases,
the addition of bisecting GlcNAc in the brain results in the
opposite behavior. In the case of GnT-V, its activity is high in
most cancers with metastatic properties, whereas it is decreased in some.
In this lecture I will present an overview of the complex and
yin and yang behavior of branched N-glycans when added
to target proteins.
004: Cytoplasmic Glycosylation Targets a Polyubiquitin
Ligase to Control Oxygen Sensing in Protists
Christopher M. West1, M. Osman Sheikh1, Christopher
M. Schafer1, Hanke Van Der Wel1, and Ira Blader2;
1
Departments of Biochemistry & Molecular Biology,
and 2Microbiology & Immunology, Oklahoma Center
for Medical Glycobiology, University of Oklahoma
Health Sciences Center, Oklahoma City, Oklahoma 73104
USA
Cwest2@ouhsc.edu
Studies on a novel form of cytoplasmic glycosylation in the
social amoeba Dictyostelium have led to the discovery of an
O2-sensing mechanism related to that of humans, with a
novel twist that instead of generating a degron for the
polyubiquitination of the transcriptional factor hypoxia inducible factor alpha (HIFα), the ubiquitin ligase itself appears to be regulated. The target of this modification is a
single proline on Skp1, an adaptor subunit of the large SCF
(Skp1/cullin-1/F-box protein) family of E3 ubiquitin ligases
that control physiological and developmental timing via
targeted protein degradation. Genetic studies show that
prolyl hydroxylation and five sequential glycosylation
reactions modulate O2-sensing by a mechanism that does
not affect Skp1 stability. Cellular and biochemical studies
reveal that modifications affect Skp1 conformation and
promote binding to F-box proteins, the first step in SCF
assembly. The terminal glycosyltransferase is particularly influential, contributing both positive enzymatic and
negative non-enzymatic functions to pathway activity
and Skp1 function. Key elements of the pathway have
conserved O2-sensing functions in other protists including the agent for human toxoplasmosis, Toxoplasma
gondii. The findings suggest a model in which developmental progression is tuned by environmental cues
via a multi-step hydroxylation/glycosylation pathway
that controls the rate of assembly of E3SCFUb-ligases
which, in turn, influence lifetimes of specific regulatory
targets according to the genetic program of F-box protein expression.
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005: Complicated N-linked glycans in simple organisms
Iain B. H. Wilson, Shi Yan (闫 石), Alba Hykollari, Birgit
Schiller, Dubravko Rendic, Simone Kurz and Katharina
Paschinger; Department für Chemie, Universität für
Bodenkultur Wien, 1190 Wien, Austria
iain.wilson@boku.ac.at
Although countless genomes have now been sequenced, the
glycomes of the vast majority of eukaryotes still present a
series of unmapped frontiers. However, strides are being
made in a few groups of invertebrate and unicellular organisms as regards their N-glycans and N-glycosylation pathways. Thereby, the traditional classification of glycan
structures inevitably approaches its boundaries. Indeed, the
glycomes of these organisms are rich in surprises including
a multitude of modifications of the core regions of Nglycans and unusual antennae. The trifucosylation of nematode N-glycan cores can now, from our data, be explained
enzymologically; the pentosylation of trichomonad and
Acanthamoeba glycans is based on different Man5GlcNAc2
or Man8-9GlcNAc2 based scaffolds; zwitterionic glycans
with phosphorylcholine or phosphorylethanolamine are
found in nematodes, cestodes and some Trichomonas
strains; phosphate and sulphate are modifications of glycans
from one trichomonad strain, from Dicytostelium, from oysters and possibly even insects. From the actually rather
limited glycomic information we have, it is nevertheless
obvious that the biotechnological, developmental and immunological relevance of these modifications, especially in
insect cell lines, model organisms or parasites means that
deciphering unusual glycomes is of more than just academic
interest.
006: Targeting siglecs for modulation of immune
responses
Mathew S. Macauley1, Fabian Pfrengle1, Christoph
Rademacher1, Corwin M. Nycholat1, Cory D. Rillahan,
Andrew J. Gale 2 , Annette von Drygalski 2,3 , and
James C. Paulson1; 1Departments of Cell and Molecular
Biology, and Chemical Physiology, and 2Department of
Molecular Experimental Medicine, The Scripps Research Institute, La Jolla, CA, 92037 USA; 3Division of Hematology/
Oncology, School of Medicine, University of California,
San Diego, La Jolla, CA, 92037, USA
jpaulson@scripps.edu
The sialic acid binding immunoglobulin lectin (siglec) family of cell adhesion molecules are differentially expressed on
white blood cells that confer innate and adaptive immune
responses. Using rational design and high-throughput
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synthesis and screening of sialoside analogs we have developed siglec ligands that selectively recognize a single siglec,
suitable for in vivo targeting of liposomal nanoparticles loaded
with a therapeutic agent or immune modifier to siglec bearing
cells. Using this platform, we have investigated the roles of B
cell siglecs, CD22 and Siglec-G, in modulation of B cell
receptor signaling. CD22 and Siglec-G are well documented
as inhibitory co-receptors of the B cell receptor. We hypothesized that the inhibitory activities of CD22 and Siglec-G
cooperate to tolerize B cells that recognize a cell surface
autoantigen, as a result of their recruitment to the site of the
immunological synapse via sialic acid containing ligands on
the antigen bearing cell. To test this, we generated liposomes
displaying an antigen and siglec ligands to mimic a cell
expressing an autoantigen. Remarkably, injection of the liposomes into mice results in induction of apoptosis in reactive B
cells, and the mice are then incapable of mounting an antibody
response to that antigen in a subsequent challenge. Since
development of inhibitory antibodies to FVIII is a serious
problem in treatment of hemophilia A patients, we investigated the potential of this approach for inducing tolerance to
FVIII in a hemophilia mouse model. Our tolerogenic liposomes prevented formation of inhibitory FVIII antibodies,
allowing for effective administration of FVIII to hemophilia
mice to prevent bleeding. Further studies have documented
that the same tolerogenic mechanism is induced by natural
siglec ligands present on cells that contain a membrane bound
antigen. Thus, through a combination of genetic and biochemical approaches, we have obtained strong evidence for a major
function of the B cell siglecs as receptors that recognize sialic
acid as ‘self’ to induce apoptosis in autoreactive B cells for
maintenance of peripheral tolerance. Exploiting this mechanism has therapeutic potential in the areas of autoimmunity,
allergies, and biotherapeutics (NIH grants AI050143,
AI099141, CA013889 and HFSP Fellowship
LT001099/2010-L).
007: Large-scale identification of in vivo target proteins
of glycosyltransferases by Lectin-IGOT-LC/MS, an
LC/MS-based glycoproteomic approach using knock-out
mouse
Hisashi Narimatsu; Research Center for Medical
Glycoscience, National Institute of Advanced Industrial
Science and Technology (AIST), 1-1-1 Umezono,
Tsukuba, Ibaraki 305-8568, Japan
Model organisms containing deletion or mutation in a
glycosyltransferase-gene exhibit various physiological abnormalities, suggesting that specific glycan motifs on certain
proteins play important roles in vivo. Identification of the in
Glycoconj J (2013) 30:281–461
vivo target proteins of each glycosyltransferase is the key to
understand the roles of glycans. Here, we demonstrated the
large scale identification of the target proteins specific
for several glycosyltransferases by Lectin-IGOT-LC/MS
glycoproteomic approach.
A l t h o u g h β 4 G a l T- I i s t h e m o s t c h a r a c t e r i z e d
glycosyltransferase, its distinctive contribution to β1,4galactosylation has been hardly described so far. We identified a large number of the target proteins specific for 4GalTI by comparative analysis of 4GalT-I-deleted and wild-type
mice using the LC/MS-based technique with the isotopecoded glycosylation site-specific tagging (IGOT) of lectincaptured N-glycopeptides.
In addition, we are now determining the large number of
target proteins of some glycosyltarnsferases, such as b3GnT,
b3Gnt5 (polyLacNAc synthases) and two LacdiNAc(LDN)
synthases. Our approach to identify the target proteins offers
unique information regarding the molecular characteristics
common to the target proteins, which facilitate understanding of the mechanism that controls protein glycosylation
in vivo.
008: Roles of O-glycans in Cellular Interactions and
Cancer
Richard D. Cummings, Yingchun Wang, Xiaokun Ding,
Sean Stowell, and Tongzhong Ju; Department of
Biochemistry, The Emory Glycomics Center, Emory
University School of Medicine, Atlanta, GA
Altered expression of glycan structures is seen in many
human diseases and disorders, including many types of
cancers. A common modification of membrane and secreted
glycoproteins is O-GalNAc type O-glycans linked to
Ser/Thr residues. These O-GalNAc O-glycans are normally
extended, and occur as branched, sialylated, sulfated, and/or
fucosylated forms. Our studies show that such O-glycans are
essential for normal development and are involved in numerous cellular interactions. In addition, normal O-GalNAc
O-glycans are essential to for normal hemostasis, and truncations of O-glycans result in altered blood cell and endothelial cell functions, including abnormal hemostasis from
loss of functions in platelets and Von Willebrand factor. In
some human diseases in human diseases such O-GalNAc
glycans are be truncated. Among the most common tumorassociated carbohydrate antigens are the Tn and Sialyl Tn
antigen. Extension of the Tn antigen (GalNAcα1-Ser/Thr),
which is a normal precursor for extended O-glycans, is under
the control of two key genes, the X-linked gene encoding
Cosmc (Xq24) and the autosomal gene encoding the T-
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synthase (7p14-p13). Expression of functional T-synthase, a
β3-galactosyltransferase that is required for formation of normal extended core 1-derived O-glycans of the structure
Galβ1-3GalNAcα1-Ser/Thr, depends on the expression of
the unique molecular chaperone Cosmc. Our studies show
that Cosmc is a resident protein in the endoplasmic reticulum
that prevents oligomerization of the T-synthase into inactive
forms that are targeted for degradation, and promotes formation of active T-synthase dimers that leave the ER to function
in the Golgi apparatus. Immunohistochemical screening of a
wide variety of human tumor specimens in tissue microarrays
with specific monoclonal antibody to the Tn antigen show that
it is expressed in a majority of human carcinomas. There are
multiple mechanisms that cause Tn expression, including
acquired loss-of-function of Cosmc. Thus, the expression of
normal O-glycans are required in a surprising number of
biological systems and leads to normal glycoprotein function
and expression. Moreover, expression of the Tn antigen is
uniquely associated with neoplastic transformation and
Cosmc is implicated as a major contributor to the expression
of altered O-glycans in neoplasia.
009: General Tactics and New Glycosylation Methods
for the Assembly of Naturally Occurring Glycosides
Biao Yu; State Key Laboratory of Bio-organic and Natural
Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, China
byu@mail.sioc.ac.cn,
The secondary metabolites (or natural products), such as flavonoids, steroids, triterpenes, lipids, and antibiotics, are frequently post-modified with saccharides. These ubiquitous glycosides
are extremely diverse in structures and functions. Purification
of a homogeneous glycoside from the nature sources, especially in an appreciable amount, is always difficult. Chemical
synthesis provides a feasible access to the homogenous glycosides and their congeners. This lecture discusses the general
tactics for the synthesis of the diverse glycosides and the newly
developed gold(I)-catalyzed glycosylation method for installation of saccharides onto aglycones which are poorly nucleophilic or extremely labile to acid or electrophile.
Reference:
Assembly of naturally occurring glycosides, evolved tactics
and glycosylation methods. B. Yu, J. Sun, X. Yang, Acc.
Chem. Res. 2012, 45, 1227-1236.
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010: Linking Nutrients to Signaling: Many Kinases are
Regulated by O-GlcNAcylation
Gerald W. Hart1; 1Dept. of Biol. Chem., Johns Hopkins
Univ., Sch. of Medicine, 725 N. Wolfe St., Baltimore,
MD 21205
gwhart@jhmi.edu
Dynamic O-GlcNAcylation serves as a major sensor of
cellular nutrient status, and has extensive crosstalk with
phosphorylation. O-GlcNAc also crosstalks with other
abundant post-translational modifications, such as
ubiquitination, methylation, and acetylation. This extensive
crosstalk regulates transcription, signaling and cellular metabolism in response to nutrient status. O-GlcNAcylation
plays a fundamental role in gene transcription at many
levels, including regulation of the basal machinery, as well
as modifying the interactions and localizations of transcription factors. Prolonged excess O-GlcNAcylation, as occurs
in diabetes, underlies fundamental mechanisms of glucose
toxicity. Abnormal O-GlcNAcylation is associated with neurodegenerative disease and is elevated many types of cancer.
Recent glycomic analyses have found that many kinases are
both modified and regulated by O-GlcNAcylation. For
example, O-GlcNAcylation either inhibits or activates,
depending upon the kinase, and can alter substrate specificity, as exemplified by casein kinase II. Hyper-OGlcNAcylation of mitochondrial proteins in diabetic tissues
contributes directly to production of reactive oxygen species (ROS). Mitochondrial O-GlcNAc transferase (OGT) is
strikingly elevated and mis-localized in cardiac mitochondria from diabetic rats. O-GlcNAcylation is very abundant
at nerve terminals, is abundant on myriad synaptic vesicle
proteins, and appears to play a direct role in learning and
memory. OGT is highly enriched in the post-synaptic
density. Supported by NIH R01CA42486, R01DK61671;
N01-HV-00240; P01HL107153 and the Patrick C. Walsh
Prostate Cancer Research Fund. Dr. Hart receives a share
of royalty received by the university on sales of the CTD
110.6 antibody, which are managed by JHU
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IGO Young Glycoscientist Awards
011: Degradation, Foraging and Depletion of Mucus
Sialoglycans by the Vagina-Adapted Actinobacterium
Gardnerella vaginalis
Warren G. Lewis, Lloyd S. Robinson, Nicole M Gilbert,
and Amanda L. Lewis; Washington University in St. Louis,
United States
mandylew@gmail.com
Bacterial vaginosis (BV) is a polymicrobial imbalance of the
vaginal microbiota associated with reproductive infections,
preterm birth, and other adverse health outcomes. Sialidase
activity in vaginal fluids is diagnostic of BV and sialic acidrich components of mucus have protective and immunological roles. However, while mucus degradation is believed to
be important in the etiology and complications associated
with BV, the role(s) of sialidases and the participation of
individual bacterial species in the degradation of mucus
barriers in BV have not been investigated. Here we demonstrate that the BV-associated bacterium Gardnerella vaginalis
uses sialidase to break down and deplete sialic-acid-containing
mucus components in the vagina. Biochemical evidence using
purified sialoglycan substrates supports a model in which 1) G.
vaginalis extracellular sialidase hydrolyzes mucosal
sialoglycans, 2) liberated sialic acid (N-acetylneuraminic acid)
is transported into the bacterium, a process inhibited by excess
N-glycolylneuraminic acid, and 3) sialic acid catabolism is
initiated by an intracellular aldolase/lyase mechanism. G.
vaginalis engaged in sialoglycan foraging in vitro, in the presence of human vaginal mucus, and in vivo, in a murine vaginal
model, in each case leading to depletion of sialic acids. Comparison of sialic acid levels in human vaginal specimens also
demonstrated significant depletion of mucus sialic acids in
women with BV compared to women with a “normal”
lactobacilli-dominated microbiota. Taken together, these studies show that G. vaginalis utilizes sialidase to support the
degradation, foraging, and depletion of protective host mucus
barriers, and that this process of mucus barrier degradation and
depletion also occurs in the clinical setting of BV.
Glycoconj J (2013) 30:281–461
012: Protein O-GlcNAcylation Regulates Cancer
Metabolism and Cell Growth
Wen Yi1,2, Peter Clark2, Daniel Mason3, Eric Peters3,
Edward Driggers4, and Linda C. Hsieh-Wilson2; 1College
of Life Sciences of Zhejiang University, Hangzhou,
Zhejiang 310058, China; 2Division of Chemistry and
Chemical Engineering, California Instititute of Technology,
Pasadena, CA 91125, USA; 3Genomics Institute of the
Novartis Research Foundation, San Diego, CA 92121, USA;
4
Agios Pharmaceuticals, 38 Sidney Street, Cambridge, MA
02139, USA
wyi@zju.edu.cn
The dynamic post-translational modification of intracellular
proteins with O-linked β-N-acetylglucosamine (O-GlcNAc)
has recently emerged as an important regulator of biological
processes, including transcription, translation, stress response,
and insulin signaling. More recently, O-GlcNAcylation is also
implicated in tumor pathogenesis. For instance, O-GlcNAc
levels are increased in multiple tumor types, and O-GlcNAc
ablation has caused inhibition of tumor growth and cancer
metastasis. However, the functional role of O-GlcNAc in
cancer development and progression is largely unknown. Here
we demonstrate that O-GlcNAcylation of a key glycolytic
enzyme phosphofructokinase 1 (PFK1) plays an important
role in regulating cancer cell growth and metabolism. Glycosylation inhibited PFK1 activity and redirected glucose flux
through the pentose phosphate pathway, thereby conferring a
selective growth advantage to cancer cells. Blocking glycosylation of PFK1 reduced cancer cell proliferation in vitro and
impaired tumor formation in vivo. These studies reveal a
previously uncharacterized mechanism for the regulation of
metabolic pathways in cancer and a possible target for therapeutic intervention.
Mon-Glycoproteomics
013: Keynote Lecture: Oligosaccharide Microarrays:
Past, Present and Future Tools in Glycoscience
Ten Feizi; The Glycosciences Laboratory, Imerial College
London, W12 0NN, United Kingdom
t.feizi@Imperial.ac.uk
Oligosaccharide microarrays have revolutionized the molecular dissection of carbohydrate-protein interactions.
With the increased awareness that oligosaccharides are
involved in diverse molecular interactions in health and,
directly or indirectly, in the majority of disease processes,
oligosaccharide microarrays have become essential tools in
the biomedical sciences.
Glycoconj J (2013) 30:281–461
Dating from work with human Mycoplasma pneumoniae
infection and the monoclonal antibodies that it elicits to its
carbohydrate receptors, our speciality has been the discovery of oligosaccharide ligands for proteins involved in innate and acquired immunity, pathogen-host interactions and
in endogenous recognition systems.
The oligosaccharide microarray system we established in
2002 for sequence-defined oligosaccharides is based on
the neoglycolipid (NGL) technology, which we introduced
in 1980s whereby oligosaccharides chemically linked to a
long-chain lipid are immobilized and probed for proteinrecognition in conjunction with sequence determination by
mass spectrometry. I will present highlights among them
discoveries of hitherto unsuspected ligands arising from
screening analyses; these include: host-cell binding proteins
for infective agents such as Simian Virus 40 (SV40), human
JC virus, the pandemic H1N1 2009 influenza virus, parasites of the Apicomplexan family that include Toxoplasma
gondii, and a monoclonal antibody AE3 directed to a
cancer-associated epithelial antigen. I will also discuss designer arrays such as those that enabled the discovery of
sulphated ligands for the selectins, assignments of those for
Dectin-1 and other glucan-recognition systems of biological
and medical importance, including malectin, a newly discovered resident protein in the endoplasmic reticulum of all
animals, and potentially therapeutic antibodies elicited following experimental vaccination with fungal polysaccharides. I
will proceed to discuss our aspirations for metaglycomic
arrays in the future.
The Carbohydrate Microarray Facility in the Glycosciences
Laboratory is a Wellcome Trust-supported Biomedical
Resource to serve the biomedical community.
014: Age- and sex-associated differences in the
glycopatterns of human salivary glycoproteins and their
roles against influenza A virus
Yannan Qin1, Yaogang Zhong1, Minzhi Zhu1, Liuyi
Dang1, Zhuo Chen1, Hanjie Yu1, Xiurong Wang2, Hua
Zhang3, Zheng Li1; 1Laboratory for Functional Glycomics,
College of Life Sciences, Northwest University, Xi’an,
710069, China, 2National Key Laboratory of Veterinary
Biotechnology, Harbin Veterinary Research Institute,
Chinese Academy of Agricultural Science, Harbin, 150001,
China, 3Department of Oncology, Shaanxi Provincial
People’s Hospital, Xi’an, 710068, China
zhengli@nwu.edu.cn
Recent studies have elucidated that expression of certain
glycoproteins in human saliva are increased or decreased
293
according to age, meanwhile, human saliva may inhibit viral
infection and prevent viral transmission. However, little is
known about the age- and sex-associated differences in the
glycopatterns of human salivary glycoproteins and their
significant roles against influenza A virus (IVA). Here we
investigate the glycopatterns of human salivary glycoproteins with 180 healthy saliva samples divided into six
age/sex groups using lectin microarrays and fabricate saliva
microarrays to validate the terminal carbohydrate moieties
of glycoproteins in individual saliva samples. Furthermore,
we assess the binding activity of saliva against two strains of
influenza A (H9N2) virus. We find that seven lectins (e.g.,
MAL-II and SNA) show significant age differences in both
females and males, and seven lectins (e.g., WFA and STL)
show significant sex differences in children, adults and
elderly people. Interestingly, we observe that elderly individuals have the strongest resistance to IVA mainly by
presenting more terminal α2-3/6-linked sialic acid residues
in their saliva, which bind with the influenza viral hemagglutinations. We conclude that age- and sex- associated
differences in the glycopatterns of human salivary glycoproteins may provide pivotal information to help understand
some age related diseases and physiological phenomenon.
015: Analysis of glycan and lectin arrays printed on
functionalized ITO slides by Mass Spectrometry and
Fluorescence
Ana Beloqui1, Javier Calvo2, Manuel Martin-Lomas1,3,
Niels-Christian Reichardt1,3; 1Biofunctional
Nanomaterials Unit, CICbiomaGUNE, Donostia-San
Sebastian, 20009, Spain, 2Mass Spectrometry Platform,
CICbiomaGUNE, Donostia-San Sebastian, 20009, Spain,
3
CIBER-BBN, Donostia-San Sebastian, 20009, Spain
nreichardt@cicbiomagune.es
Microarrays are now the preferred format for most highthroughput screening applications in genomics, proteomics
and glycomics and the ability to analyze them by more than
a single readout method is desirable to broaden their applications.[1] We have prepared micrometer sized glycan and
lectin arrays on functionalized indium tin oxide (ITO) slides
that can be analyzed by fluorescence spectroscopy, MALDITof-MS and optical microscopy without a change of the
format. No prior tagging of analytes is required[2] and ligand
libraries can be printed directly onto a non-covalent sandwich composed of activated bi-dentate lipids embedded in a
hydrophobic layer conjugated to ITO. These array surfaces
are transparent, conductive and very resistant to repeated
aqueous washing steps. Moreover, this effective ligand immobilization by hydrophobic interactions provided the signal intensity required for the mass spectrometric readout of
294
micrometer-sized spots including the structural assignment
of enzymatic modifications by MS/MS fragmentation and
peptide sequencing by MALDI-Tof MS.
To demonstrate its scope this platform was applied to study
the action of seven recombinant glucosyltransferases on
multi-antennary N-glycan acceptors, assign the specificity of
a fucosyltransferase by on-chip product fragmentation, profile
the proteins bound to a lectin array by mass spectrometry and
to identfy a lectin bound to a glycan array by on-chip tryptic
digestion and in situ sequencing of peptide fragments.
[1] a)P. Jonkheijm, D. Weinrich, H. Schröder, C. M.
Niemeyer, H. Waldmann, Angew. Chem. Int. Ed. 2008,
47, 9618–9647; b)A. J. Vegas, J. H. Fuller, A. N.
Koehler, Chem. Soc. Rev. 2008, 37, 1385–1394.
[2] a)F. A. Jaipuri, B. Y. Collet, N. L. Pohl, Angew. Chem.
Int. Ed. 2008, 47, 1707–1710; b)S.-H. Chang, J.-L. Han, S.
Y. Tseng, H.-Y. Lee, C.-W. Lin, Y.-C. Lin, W.-Y. Jeng, A. H.
J. Wang, C.-Y. Wu, C.-H. Wong, J. Am. Chem. Soc. 2010,
132, 13371–13380; c)T. R. Northen, J.-C. Lee, L. Hoang, J.
Raymond, D.-R. Hwang, S. M. Yannone, C.-H. Wong, G.
Siuzdak, Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 3678–3683.
016: Identification of metastasis related glycans on
breast cancer cells
Shu-Min Zhou 1,2 , Li Cheng 1,2 , Shu-Juan Guo 1,2 ,
Sheng-Ce Tao1,2†; 1Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine
(Ministry of Education), Shanghai Jiao Tong University,
Shanghai 200240, China, 2State Key Laboratory of Oncogenes and Related Genes, Shanghai, 200240, China
taosc@sjtu.edu.cn (S.-C. T.)
Membrane-localized glycans which usually conjugated to
proteins and lipids are modifiers of cellular motility processes and thus are of crucial relevance for the onset of cancer
metastasis. The aim of this study was to identify altered
glycans which take the responsibilities in the metastasis of
breast carcinoma cells. To this end, human breast carcinoma
cell lines, MDA-MB-231 and its single cell progenies
(SCPs) of different metastatic abilities were examined by
lectin microarray with 91 lectins. As a result, we found
lectins, PTL II, RCA-I and black bean crude express an
increasing binding with the rising of metastatic ability of
breast cancer cells. In contrary, lectins, VFA, PSA and
WGA express a decreasing binding with the rising of
metastatic ability of the same cells. And most interestingly, WFA, a lectin binds to glycans terminating in N acetylgalactosamine linked alpha or beta to the 3 or 6
position of galactose, showed a different binding to the
Glycoconj J (2013) 30:281–461
breast cancer cells of close metastatic ability, but different
organ-specificities, which indicate that the glycans recognized
by WFA plays a role in the tropism of metastatic breast tumor
cells. To our knowledge, we are the first one to show the
global picture of membrane- localized glycans related to tumor metastasis in breast cancer. We believe the results from
this study will help us to pave the way for studying the
correlation of aberrant glycans and breast cancer metastasis,
and the underlying mechanism.
References:
1. Tao SC, Li Y, Zhou J, Qian J, Schnaar RL, Zhang Y,
Goldstein IJ, Zhu H, Schneck JP. Lectin microarrays identify cell-specific and functionally significant cell surface glycan markers. Glycobiology. (2008); 18(10):761–9.
2. Zhou SM, Cheng L, Guo SJ, Zhu H, Tao SC. Lectin
microarrays: a powerful tool for glycan-based biomarker
discovery. Comb Chem High Throughput Screen.(2011);
14(8):711–9.
3. Yang L, Guo S, Li Y, Zhou S, Tao S. Protein microarrays
for systems biology. Acta Biochim Biophys Sin (Shanghai).
(2011); 43(3):161–71.
4. Sethi N, Dai X, Winter CG, Kang Y. Tumor-derived
JAGGED1 promotes osteolytic bone metastasis of breast
cancer by engaging notch signaling in bone cells. Cancer
Cell (2011); 19(2):192–205.
017: A Computational Framework for Identification and
Quantification of Intact Glycopeptides in Complex
Proteome Samples
Haixu Tang1*, Anoop Mayampurath1, Chuan-yih Yu1,
Ehwang Song2 and Yehia Mechref2,*; 1School of Informatics & Computing, Indiana University, Bloomington IN
USA 47408, 2Department of Chemistry, Texas Tech University, Lubbock TX USA 79409
hatang@indiana.edu (Haixu Tang) or Yehia Mechref:
yehia.mechref@ttu.edu
Glycosylation is an important protein modification that involves the attachment of sugars to amino acid residues. The
functional roles of glycoproteins include cell signaling and
immune response. Thus, understanding the structure of
sugars and effects of glycosylation are vital for developing
indicators of disease. Although computational methods
based on mass spectrometry data have proven to be effective
in monitoring changes in the glycome, developing such
methods for the glycoproteome can be challenging, largely
due to the inherent complexity in studying glycan structures
in tandem with corresponding glycosylation sites. Here, we
present a computational framework for identifying intact
glycopeptides, i.e. glycopeptides with glycans attached to
Glycoconj J (2013) 30:281–461
their glycosylation sites, in complex proteome samples.
Scoring algorithms are presented for tandem mass spectra
of glycopeptides resulting from Collision-Induced Dissociation (CID), High-energy C-trap Dissociation (HCD) and Electron Transfer Dissociation (ETD) fragmentation methods. An
empirical false-discovery rate (FDR) estimation method is
derived based on a target-decoy search approach to assign
confidence to the identified glycopeptides. The power of our
method is further enhanced when multiple datasets are pooled
together from technical and biological replicates. Using this
framework, a considerable number (~100) of highly confident
glycopeptides are identified from complex human serum proteome samples using conventional liquid chromatography
mass spectrometry platforms without any further glycopeptide
enrichment. Finally, we developed a novel linear statistical
model for quantifying and comparing site-specific glycosylation events across multiple samples. We applied the model to
an esophagus cancer study based on blood serum samples to
discover potential glyco-biomarkers that show significant
abundance alteration at glycopeptide level, but not at the
corresponding whole glycoprotein levels. A total of five glycoproteins were detected in this study showing significantly
different abundances at site-specific glycosylation levels within cancer/control samples, indicating that our method is ready
to be used for the discovery of biomarkers on site-specific
protein glycosylations for human disease. Our framework was
implemented in a new software tool called GlycoFragWork,
which analyzes multiple pre-aligned LC-MS/MS datasets, and
reports a list (termed as a glycomap) of identified intact
glycopeptides with their mass, elution time and abundances.
The software will be freely available at the conference.
018: Keynote Lecture: Enabling glycoanalysis with informatics: discovery of glycans involved in infection
Arun Everest-Dass, Wai Yuen Cheah, Terry
Nguyen-Khuong, Liisa Kautto, Robyn Petersen,
Frederique Lisacek, Daniel Kolarich, Matthew P.
Campbell, Nicolle H Packer; Macquarie University,
Australia
arun.dass@mq.edu.au
As has been the case in proteomics and genomics, an essential
requirement for glycomics and glycoproteomics to progress
out of our relatively small community into the greater scientific sphere is the development of informatics tools to interpret
and store diverse experimental glycan data and enable public
accessibility. Ultimately the analytical data must relate to the
function of these glycans and their glycoconjugates.
There currently are three main analytical approaches to the
analysis of protein glycosylation which are at various levels
295
of technological development: Glycomics (the global structural analysis of N- and O-glycans attached to proteins);
Glycoprotein (glycan structure and site analysis of a single
protein); and Glycoproteomics (glycan structure and site
analysis of complex mixtures of glycoproteins).
Glycomics analysis still gives the most detailed structural
information in terms of the branching, linkage and overall
topology of the surface of the cell as it presents to the
surrounding environment. In fact all the proteins produced
in one cell type carry the same glycomic profile, since all are
products of the same intracellular glycosylation machinery.
In particular, the human epithelial cells and secreted fluids
display a dense, heterogeneous array of cell specific glycan
structures to invading microorganisms. I will present the
analytical strategy and informatics tools we are developing
for glycomics analysis, and give some examples of how
specific structures affect the adhesion of different microbes.
019: The use of Glycoproteomics for the discovery of
biomarkers of HCC-From GP73 to the development of
biomarkers of AFP negative cancer
Anand Mehta1, Mary Ann Comunale1, Mengjun Wang1,
Lucy Betesh 1 , Timothy Block 1 ; 1 Drexel University
College of Medicine, Doylestown, PA 18902, United States
of America.
anand.mehta@drexelmed.edu
Hepatocellular carcinoma (HCC), caused by infection with
hepatitis B virus (HBV) (and/or hepatitis C virus), is one of
the most common solid malignancies worldwide. Despite
advances in medical technology, the 5-year survival rate is
8–13 %, likely due to the fact that the majority of patients
with HCC are diagnosed at advanced stage. Currently,
alpha-feto protein (AFP) is used in combination with several
imaging methodologies to help identify HCC. Unfortunately, AFP is elevated only in 40–60 % of patient with HCC,
limiting its usefulness. In this setting of a significant increase in the number of patients with HCC, early detection
and treatment are vital to improve outcome. The association
of carbohydrates with cancer has long been known and we
were one of the first groups to combine glycomic strategies
with proteomic methods to identify modified glycoproteins
in cancer. One such protein was Golgi protein 73 (GP73),
which has shown great promise as a biomarker of HCC and
is now commercially available in China.
However, GP73 has limitations and is better in the detection
of AFP positive cancers than in the detection of AFP negative cancers. Thus in this study, we have performed biomarker discovery in AFP and GP73 negative, HCC positive
296
individuals. Our discovery effort involved the examination
of the N-linked glycans on serum proteins following resolution via two dimensional electrophoresis (2DE). This approach has led to the discovery of specific change in
glycosylation that were associated with AFP negative
HCC. The changes that were observed included increased
levels of alpha 2,6 linked sialic acid and increased level of
branching on several proteins. These specific glycoforms
were subsequently analyzed in the serum of a large patient
cohort. Together, with AFP, these new biomarkers could be
used to identify close to 100 % of those with HCC.
020: LC-MS/MS Quantification of Neu5Ac, Neu5Gc and
KDN Levels in Urine and Potential Health & Disease in
3~5 Years Old Children
Yue Chen1, Lili Pan1, Ni Liu1, Frederic A. Troy II1,2,
Bing Wang1,3; 1School of Medicine, Xiamen University,
Xiamen City 361102, China; 2Department of Biochemistry
and Molecular Medicine, University of California School of
Medicine, Davis, CA 95616, USA; 3School of Molecular
Biosciences, The University of Sydney, NSW 2006, Australia
bing.wang@sydney.edu.au/bwang@xmu.edu.cn
Sialic acids (Sias) are a diverse family of sugars based on
the parent compound, neuraminic acid, a nine-carbon sugar
acid. N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acids (Neu5Gc), are the two
major forms of Sia in mammal. A less common Sia is
ketodeoxynonulosonic acid (KDN) that occurs frequently
in lower vertebrates. Previous studies show that Sia is an
essential nutrient for neural structure and function. Red meat
and dairy products contain relatively higher Sias, including
non-human Sias Neu5Gc, which correlate with human inflammatory diseases including atherosclerosis and cancer,
but the metabolic fate and health impact in children remains
unknown. Our aim is to quantify the levels of urinary Sias,
Neu5Ac, Neu5Gc, KDN, and to discuss health & disease
risk of preschool children in China.
Spot urine was collected from 386 healthy children at the
ages of 3 (n=108), 4 (n=144) and 5 (n=134) at 6:30~7:00,
11:30~12:00, and 16:30~17:00. Children were provided
breakfast, lunch and afternoon dessert from 7:30 to 17:30
and food intake was recorded on the day of urine collection.
Sia levels were quantified using LC-MS/MS. This study was
approved by the Human Ethics Committee of Xiamen University, with written informed consent from all subjects.
Our results showed that total urinary Sia levels in healthy
preschool children ranged from 40~79 mmol Sia/mol creatinine. The predominated form of urinary Sia levels were
Glycoconj J (2013) 30:281–461
conjugated Neu5Ac (~70.8 %), followed by free Neu5Ac
(~21.3 %), conjugated KDN (~4.2 %) and free KDN
(~3.7 %). Neu5Gc was detected in the urine of only one
4 year-old girl. Total urinary Sia levels were highest in the
morning and declined over time in the 4 and 5 year-old
children (P<0.05), but not 3 year-old children. We also
found 3 year-old children had a relatively higher Sia intake
at breakfast, lunch and dessert than other age-groups, their
urinary Sia concentration was lower than that of 5 year-old
children. The age difference of urinary Sia suggests that metabolism and utilization rates of dietary Sia are age dependent.
This study was supported by a research grant from the
School of Medicine of Xiamen University and Nestle
Research Center, Beijing.
021: Quantitative Glycomics Reveals specific N-glycans
change of cell-secreted proteomes in Metastasis of
Epithelial Ovarian Cancer
Xingwang Zhang1, Yisheng Wang2, Yifan Qian1, Shifang
Ren1, Congjian Xu2, Jianxin Gu1; 1Key Laboratory of
Glycoconjugate Research Ministry of Public Health;
Department of Biochemistry and Molecular Biology, Shanghai
Medical College, Fudan University, Shanghai, P.R. China,
200032, 2Obstetrics and Gynecology Hospital, Department of
Obstetrics and Gynecology of Shanghai Medical School,
Fudan University, Shanghai, P.R. China, 200032
renshifang@fudan.edu.cn
It has been known that glycans which conjugated to proteins
of secretome play a prominent role in cell signaling, communication and migration. Quantitative and/or qualitative
variations in glycans may result in aberrant glycosylation
patterns, which are linked to cancer metastasis. In this study,
we attempted to identify alterations of N-glycans involved
in tumor metastasis in SKOV3(human serous ovarian cancer
cell line) and its high metastatic derivative cell line. Using
quantitative glycomics technology, we determined the detailed N-glycan profile of secreted proteins from this two
ovarian cancer cell lines. Interestingly, the N-linked
glycomic profiles revealed that the levels of some specific
glycoforms like tri-antennary glycans that were identified
altered in SKOV3ip compared to high metastastic derivative
cell line. Real time PCR, western blotting analysis, transwell
experiments and immunohistochemical study were further
employed to validated these specific changes. All the results
were in accordance with the change of the N-glycans revealed by quantitative glycomics methods. With the
methods, we revealed specific N-glycans changes of cellsecreted proteomes associated with metastasis of epithelial
ovarian cancer. Our data suggest alterations of these
Glycoconj J (2013) 30:281–461
glycoforms in human ovarian cancer cells correlate with cancer metastasis, and their glycan subclass could poteintially be
used as a biomarker for metastatic prognosis. Also, this study
are helpful to understand the mechanism of metastasis of
epithelial ovarian cancer.
022: Monoclonal IgM cryoglobulins show patient specific
glycosylation patterns that differ from average polyclonal
human IgM
1,2
Daniel Kolarich, 2,3Pia H. Jensen, 4Mariel Bartley,
Vicky Vallas, 4William Farrugia, 2Nicolle H. Packer,
4
Paul A. Ramsland; 1Max Planck Institute of Colloids
and Interfaces, Department of Biomolecular Systems,
14424 Potsdam, Germany, 2Department of Chemistry and
Biomolecular Sciences, Macquarie University, Sydney,
NSW 2109, Australia, 3Institute of Molecular Medicine,
University of Southern Denmark, 5230 Odense, Denmark,
4
Centre for Immunology, Burnet Institute, Melbourne, VIC
3004, Australia
daniel.kolarich@mpikg.mpg.de
4
Cryoglobulinaemia occurs in many clinical settings and is
characterised by the presence of serum cryoglobulins (IgM
and/or IgG) which form insoluble aggregates in the cold [1].
The clinical severity varies widely and correlates with the type
of cryoglobulin (monoclonal or mixed Igs) and the physical
nature of the aggregates (precipitate, gel or crystal). We present a comprehensive glycoproteomic study of two monoclonal
IgM cryoglobulins (Pot [precipitating] and Yvo [gelling])
isolated from plasma of patients with Waldenström’s macroglobulinemia [2], and compare this data with commercially
available polyclonal IgM isolated from pooled normal human
plasma.
Porous Graphitized Carbon (PGC) LC-ESI-IT-MS/MSn
glyco-profiling of the glycan structures [3] revealed a significant difference between the two cryoglobulin IgMs and
normal IgM. The majority of N-glycans in all three samples
contained bisecting GlcNAc and core α1,6 fucosylation.
Both the Pot and Yvo IgM cryoglobulins showed a higher
amount of neutral N-linked oligosaccharides, with considerable amounts of oligomannosidic and hybrid type structures, which were found to just constitute only a minor
fraction of the normal IgM N-glycans. There was no indication of cryoglobulin O-glycosylation.
Analysis of IgM heavy chain glycopeptides was performed
after HILIC enrichment using LC-ESI-Q-TOF-MS and LCESI-IT-MS/MSn as described previously [4]. Combining
data derived from Collision Induced Dissociation (CID)
and Electron Transfer Dissociation (ETD) experiments
297
enabled simultaneous identification and characterisation
of peptide sequences and the glycan(s) structures at
specific attachment sites. In addition, the N-glycan distribution at specific sites on the peptide was determined
from the MS-scan spectra using the software tool
GlycoSpectrumScan (www.glycospectrumscan.org) [5]. The
three N-glycosylation sites closer to the N-terminus of the
protein were found to carry mostly complex type N-glycans,
whereas the more C-terminally positioned sites preferentially carried high mannose type structures. Despite
overall structural N-glycan differences this glycan type
distribution pattern was similar in all three IgM preparations analysed. This study presents the first comprehensive glycoproteomic characterisation of IgM heavy
chains clearly showing that glycosylation of this important immunoglobulin is affected in disease.
References:
[1] Ramsland PA, Terzyan SS, Cloud G, Bourne CR,
Farrugia W, Tribbick G, Geysen HM, Moomaw CR,
Slaughter CA, Edmundson AB., Biochem J. 2006;
395(3):473–81
[2] Vallas V, Farrugia W, Raison RL, Edmundson AB,
Ramsland PA. J Mol Recognit. 2007; 20(2):90–6.
[3] Jensen PH, Karlsson NG, Kolarich D, Packer NH., Nat
Protoc. 2012; 7(7):1299–310.
[4] Kolarich D, Jensen PH, Altmann F, Packer NH.; Nat
Protoc. 2012; 7(7):1285–98
[5] Deshpande N, Jensen PH, Packer NH, Kolarich D.; J
Proteome Res. 2010; 9(2):1063–75.
023: Keynote Lecture: Mass spectrometry-based strategies for qualitative and quantitative glycoproteomics
Haojie Lu and Pengyuan Yang; Department of Chemistry
and Institutes of Biomedical Sciences, Shanghai, 200032,
China
luhaojie@fudan.edu.cn
As one of the most ubiquitous post-translation modifications, glycosylation is involved in a variety of physiological
and pathological processes. Qualitative and quantitative of
the glycoproteome, which involves the identification and
quantitation of protein components in various biological
systems, therefore becomes an important area for biomarker
research.
However, the huge dynamic range of proteins expressed in
the complex biological always masks the detection of the
inherent low abundance glycoproteins, and the glycan
microheterogeneity further reduces the relative amount of
glycopeptides and decreases the detection sensitivity. To
298
gain thorough insights into glycosylation and elucidate the
functional relationship among proteins, efficient separation
of glycoproteins for their analysis is absolutely necessary.
Therefore, one of our research interests is the development of
novel methods for highly specific and efficient capture of
glycoproteins. Different kinds of boronic acid-functionalized
nanomaterials have been developed and successfully applied
to glyco-specific enrichment in large-scale glycosylation identification. Sensitivity for glycoproteins was improved by 1~2
orders using these newly synthesized nanocomposites. Especially, 165 new N-glycosylation sites were identified by our
new methods. The other research interests of our group focuses on developing mass-spectrometry based strategies for the
relative quantification of glycoproteins as well as the changes
of occupancy of N-glycosylation at specific sites between
healthy and diseased individuals. 18O atom incorporated into
the N-glycosylation site of asparagines-linked sugar chains
specifically via a N-glycosidase F (PNGase F)-mediated hydrolysis served as one isotope-code. Other labeling methods,
for example 18O or iTRAQ, introduced an additional isotopecode. Thus, the relative quantities of N-glycosylated and its
parent protein levels were obtained simultaneous by measuring the intensity ratios of 18O/16O for glycosylated peptides
and 18O/16O or 114/115/116/117 for nonglycosylated peptides, respectively. A comparison of these two ratios was
utilized to evaluate the changes of occupancy of Nglycosylation at specific sites. For example, N-glycosylation
site ratios on serum haptoglobin (Hp) beta-chain in healthy
individuals as well as patients with hepatitis B virus (HBV),
liver cirrhosis (LC) and hepatocellular carcinoma (HCC) were
quantified and glycosite ratios of VVLHPN#YSQVDIGLIK
were observed to change significantly in HCC patients compared with LC and HBV patients.
024: Characterization of the Yeast Glycoprotoeme by
Using Hydrophilic Affinity Enrichment Coupled to Mass
Spectrometry
Long Yu1, Xinmiao Liang1; 1Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, 116023,
China
souron@126.com
Glycosylation, which is a common and important posttranslational modification of proteins in eukaryotic origanisms,
involves in many cellular activities. It is essential to acquire the
structure information of glycosylation, i.e., the glycosylation
site and the attached glycan structure, prior to understand
the biological functions of glycosylation. We developed a
bottom-up glycoproteomic strategy based on the hydrophilic
affinity enrichment, where a customized hydrophilic material named Click TE-Cys was used for the selective
Glycoconj J (2013) 30:281–461
enrichment of glycopeptides. We first evaluated the selectivity and the glycosylation site coverage of Click TE-Cys by
using the immunoglobulin G (IgG) and horseradish peroxidase (HRP) as model glycoproteins, respectively. We have
identified 21 IgG glycopeptides in the glycopeptide fraction
enriched by Click TE-Cys from the IgG digest mixed with
human serum albumin digest (w/w, 1:10). We also identified
11 glycopeptides with different peptide backbone from the
enriched HRP glycopeptide fraction. Thereafter, we applied
the enrichment method to the lysate of the baker’s yeast
Saccharomyces cerevisiae, where 174 glycosylation sites
from 102 glycoproteins were identified. By comparison, only
48 glycosylation sites from 31 glycoproteins were identified
from the yeast glycopeptide fraction enriched by concanavalin
A-immobilized lectin affinity chromatography. These results
indicated the superior selectivity and glycosylation site coverage of the Click TE-Cys material. We hope the Click TECys material and the corresponding enrichment method could
be a powerful tool for the glycoproteomics analysis.
025: The GlycoFilter: A Simple and Comprehensive
Sample Preparation Platform for Proteomics, NGlycomics and Glycosylation Site Assignment
Hui Zhou1, John W. Froehlich, Andrew C. Briscoe,
Richard S. Lee; 1Department of Urology and The Proteomics Center, Boston Children’s Hospital and Harvard Medical
School, Boston, MA 02115 USA
Hui.Zhou@childrens.harvard.edu
N-glycosylation, one of the most abundant protein posttranslational modifications, is highly involved in many biological and pathological processes. The amount of glycoproteins, glycosylation site occupancy, and the glycoforms are
well known to alter between normal and disease. Unfortunately, current strategies to study N-glycoproteins in complex
samples are often distinct, focusing on either N-glycans, or
N-glycosites enriched by sugar-based techniques such as the
hydrazide beads or lectins. In this study we report a simple and
rapid sample preparation platform, the GlycoFilter, which
allows a comprehensive characterization of N-glycans, Nglycosites, and proteins in a single workflow. Both PNGase
F catalyzed de-N-glycosylation and trypsin digestions are
accelerated by microwave irradiation and performed sequentially in a single spin filter. Both N-glycans and peptides
(including de-N-glycosylated peptides) are separately collected by filtration. The condition to effectively collect complex
and heterogeneous N-glycans, including those with multiple
sialic acid residues, was established on model glycoproteins,
bovine ribonuclease B, bovine fetuin, and human serum IgG.
With this platform, the N-glycome, and proteome of human
urine and plasma were characterized. Furthermore, a total of
Glycoconj J (2013) 30:281–461
912 and 318 N-glycosites were identified from three pairs of
urine and plasma samples, respectively. Many sites were
defined unambiguously as partially occupied by the detection
of both their sugar-modified and non-sugar-modified peptides
(149 from urine and 75 from plasma), demonstrating that
partial occupancy of N-glycosylation occurs frequently, even
in healthy adults. Given the likely high prevalence and variability of partial occupancy, an unbiased approach that captures both the sugar-occupied and non-sugar occupied peptide
will lead to improved glycoprotein quantifications.
026: Selective Enrichment of N-linked Glycopeptides
Using Hydrophilic Interaction Chromatography-based
Materials
Xiuling Li*, Bingcheng Yang, Yanyan Zhao, Xinmiao
Liang; Key Lab of Separation Science for Analytical
Chemistry, Dalian Institute of Chemical Physics, Chinese
Academy of Sciences, Chinese Academy of Science,457
Zhongshan Road, Dalian 116023, China
lixiuling@dicp.ac.cn
N-linked protein glycosylation is the most common posttranslational modification (PTM). However, characterizing
this PTM is difficult for mass spectrometry (MS) because of
low concentration of glycopeptides and suppression effect of
non-glycosylated peptides. Therefore it is imperative to enrich
glycopeptides prior to MS analysis. Hydrophilic interaction
chromatography (HILIC) in solid phase extraction (SPE)
mode has been increasingly employed to enrich glycopeptides
in the last few years. Various types of N-linked glycopeptides
can be selectively separated from non-glycosylated peptides
and the glycosylation heterogeneity coverage is improved.
However, the enrichment selectivity for glycopeptides is moderate because of co-elution of non-glycosylated peptides
containing multiple serine/threonine residues or larger molecular weight. In order to improve the enrichment selectivity of
glycopeptides, two types of novel HILIC-based materials
including Click aspartic acid (Click Asp) and Click OEGCD were synthesized in our group and applied to enrich Nlinked glycopeptides. For the case of Click OEG-CD, Nlinked glycopeptides could be selectively enriched through
reversed phase depletion coupled with hydrophilic affinity
enrichment. Compared to single HILIC mode, the method
exhibited remarkably higher selectivity for glycopeptides.
Concerning Click Asp, It demonstrated good hydrophilic
property for separation of highly polar compounds. ClickAsp based HILIC SPE was observed to efficiently bind both
neutral and sialylated glycopeptides with short to long peptide
chain. The application of Click-Asp to simulated proteomic
samples proved its specialty toward glycopeptides. The high
hydrophilicity of these Click-based HILIC materials result
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from their unique structure constructed via simple click chemistry. We believe these HILIC materials will become promising
tools in glycoproteomic studies.
027: Glycoproteomics in the diagnosis and understanding
the pathogenesis of Rheumatoid Arthritis
Ashish Saroha1,3, Sagarika Biswas1, Sarvanan Kumar1,
Hasi R Das1, Bishnu P Chatterjee2*; 1Glycomics and
Molecular Medicine Division, CSIR-Institute of Genomics
and Integrative Biology, Delhi-110007, India, 2Department
of Natural Science, West Bengal University of Technology,
Salt Lake, Kolkata-700064, India, 3Department of Biological
Chemistry, Weizmann Institute of Science, Rehovot-76100,
Israel.
cbishnup@gmail.com
Glycosylation studies of plasma proteins can provide information about the onset and progression of disease. Rheumatoid Arthritis (RA) is an autoimmune, chronic systemic
inflammatory disease characterized by swelling of synovial
joint leading to there deformation and destruction. The most
notable changes in the development of RA are alteration of
glycosylation in plasma proteins and there level of expression. The increased level of agalctosylated IgG (IgG0) correlates with the disease severity of RA and thus showing its
role in disease pathogenesis. Our study demonstrates plasma
glycoproteins enrichment by wheat germ agglutinin (WGA)
followed by 2DE. The protein spots were identified by
MALDI-TOF-MS. Database search of MS-MS spectra identified alpha 1-acid glycoprotein (AGP), haptoglobin alpha 2
(Hp-α2) and haptoglobin beta 2 (Hp-β) chain respectively.
High performance anion-exchange chromatography with
pulse amperometric detection (HPAEC-PAD) revealed the
presence of significantly higher levels of GlcN (p=0.02),
Gal (p=0.01) and Man (p=0.0) in plasma of AGP in RA
patients. Similarly by HPAEC-PAD analysis showed the
decreased amount of Man (p<0.5) in Hp-β chain of RA
patients. These results were further validated by ELISA using
ConA lectin which showed higher binding of ConA with AGP
in RA plasma indicating possible increased expression of
biantenary glycan. In another study O-linked glycoproteins
in plasma of RA patients enriched by jacalin affinity chromatography followed by 2DE revealed a number of differentially
expressed protein spots as compared to healthy control. Eighteen protein spots were found to have statistically significant
(p<0.5) difference in their expression level from four sets of
gel and were identified by MALD-TOF-MS. Most of the
proteins were identified to be O glycosylated by Net-O-Gly
3.1 algorithm. Among these the alpha 2-HS glycoprotein
(A2HSG) were found to be down regulated whereas inter
alpha trypsin inhibitor 4 (ITIH4) was up regulated and this
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was validated by western blotting. Thus glycoproteomics is a
promising approach to discover novel glyco-markers in diagnosis of RA and understanding the disease pathogenesis.
Biosynthesis & Metabolism of Glycoconjugates
028: Keynote Lecture: Development of Glyco-enzyme
Inhibitors
Chun-Hung Lin1; 1Institute of Biological Chemistry,
Academia Sinica, Taipei 115 Taiwan
chunhung@gate.sinica.edu.tw
Fucosyltransferases (FucTs) usually catalyze the final step of
glycosylation and are critical to many biological processes.
High levels of specific FucT activities are often associated
with various cancers. Here I will discuss the development of a
chemoenzymatic method for synthesizing a library of guanosine diphosphate β-L-fucose (GDP-Fuc) derivatives, followed
by in situ screening for inhibitory activity against bacterial and
human α-1,3-FucTs. Several compounds incorporating appropriate hydrophobic moieties were identified from the initial
screening in a recent publication of Advanced Synthesis &
Catalysis in 2012 (volume 354, pages 1750–1758). These
compounds were then individually synthesized, purified and
characterized in detail for their inhibition kinetics. Out of these
products, one had a Ki of 29 nM for human FucT-VI, and is
269 and 11 times more selective than for Helicobacter pylori
FucT (Ki =7.8 μM) and for human FucT-V (Ki =0.31 μM).
029: Sequencing pond life in the search for CAZymes
Ellis O’Neill, a Martin Trick, a Bernard Henrissat, b
Robert A. Field a*; a Depts of Biological Chemistry and
Computational and Systems Biology, John Innes Centre,
Norwich, NR4 7UH, UK, b Lab Architecture et Fonction
des Macromolécules Biologiques, Aix-Marseille Université,
CNRS, UMR 7257, 13288 Marseille cedex 9, France
rob.field@jic.ac.uk
Euglena gracilis is a metabolically flexible protozoan alga with
a complicated evolutionary history. Genetically, it is more closely related to the African trypanosome, Trypanosoma brucei, than
it is to many other algae. Euglena is capable of growing to very
high cell density and is capable of both photosynthesis and
absorbing exogenous nutrients. It contains high levels of vitamins and essential amino acids and it produces polyunsaturated
fatty acids. In contrast to plants, the storage polysaccharide in
Euglena is paramylon, an insoluble β-1,3-glucan with interesting biomedical properties. Unexpectedly, the cell surface of
Euglena is proposed to contain few carbohydrates. In order to
Glycoconj J (2013) 30:281–461
gain greater understanding about the metabolic potential of
Euglena, we have obtained de novo transcriptome sequencing
data as a surrogate for full genome sequencing. The
transcriptome sequences contain a wide variety of carbohydrate
active enzymes, including many involved in β-glucan metabolism. An equally wide range of metabolic pathways are also
encoded, including several natural product biosynthesis pathways that are unprecedented in these organisms.
030: 1,3/6-Linked Galactotrioses for the Identification of
Arabinogalactan Biosynthetic Enzymes
Matilde Aguilar-Moncayo, Stephan Goetz, Robert A.
Field.*; Department of Biological Chemistry, John Innes
Centre, Norwich, NR4 7UH, United Kingdom
matilde.aguilar-moncayo@jic.ac.uk
Arabinogalactan-proteins (AGPs) are an abundant class of
plant cell surface proteoglycans, which have been implicated
in many processes involved in plant growth and development.
The structure of AGPs contains highly complex and diverse
polysaccharide units (arabinogalactan) with a β-(1→3)-Dgalactan backbone and β-(1→6)-D-galactan side chains.
In order to identify and characterize glycosyltransferases
(GTs) involved in the biosynthesis of arabinogalactans,
a methodology recently developed in our research group
was applied. In this methodology, APTS-labelled oligosaccharides were used as suitable glycosyl acceptors in
biochemical assays catalysed by GTs of plant cell membrane extracts, allowing the high sensitivity analysis of the
resulting products by capillary electrophoresis with laserinduced fluorescence detection (CE-LIF).
In combination with the chemical synthesis of different
galactotrioses as standards, previously unreported β-(1→3)galactosyltransferases in the model plant Arabidopsis thaliana
could be identified with the use of this methodology. Here we
describe the preparation of three β-(1→3)/(1→6)-linked
galactotrioses, their APTS-labelling and analysis by CE. The
comparison of the elution times obtained for the synthetic
standards and the products formed in biochemical reactions
catalysed by over-expressed candidate galactosyltransferases
confirmed the identity of the new glycosidic linkages.
031: Synthesis of sialyllactose derivatives by bio-conversion
Jae Kyung Sohng; Institute of Biomolecule Reconstruction
(iBR), Department of Pharmaceutical Engineering, Sun
Moon University, #100, Kalsan-ri, Tangjeong-myeon,
Asansi, Chungnam 336-708, Korea
sohng@sunmoon.ac.kr
Glycoconj J (2013) 30:281–461
Sialylated sugar chains are present at the cell surface of
various animal species. Due to their position, they are
thought to serve important roles in a large variety of biological functions such as cell–cell and cell–substrate interactions,
bacterial and virus adhesion, and protein targeting. We present
a bio-conversion process for the conversion of N-acetylglucosamine and CMP into CMP-neuraminic acid with five
enzymes. Key enzyme is N-acyl-D-glucosamine 2-epimerase
from Baceroides fragilis. 2,3-Sialyllactose and 2,6-sialyllactose
are synthesized by one-pot reaction from lactose, N-acetylglucosamine and CMP with CMP recycling. Vancomycin
sialoside, biotin-sialoside, flavonoid sialoside and multiligand sialosides were synthesized by glycosylation,
galactosylation and sialydation with CMP-neuraminic acid.
Acknowledgement: This research was supported by the
Converging Research Center Program through the National
Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (20090082333).
032: Keynote Lecture: C. elegans DPY-19 is a Cmannosyltransferase
Hans Bakker; Cellular Chemistry, Hannover Medical
School, Hannover, 30625, Germany
Bakker.Hans@mh-hannover.de
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a WXXWXXW sequence. Lack of mannosylation of MIG21 in the dpy-19 mutant is most likely responsible for the
neuroblast migration phenotype. Dpy-19 and mig-21 mutants have the same neuroblast migration defect.
033: In vitro catalytic properties of ER α-mannosidase I
Jun-ichi Aikawa1, Yoichi Takeda 2, Ichiro Matsuo3 ,
Yukishige Ito1, 2; 1Synthetic Cellular Chemistry Laboratory,
RIKEN, 2-1 Hirosawa, Wako, Saitama, 351-0198 JAPAN,
2
ITO Glycotrilogy Project, ERATO, JST, 2-1 Hirosawa,
Wako, Saitama, 351-0198 JAPAN, 3Graduate School of
Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu,
Gunma, 376-8515 JAPAN
aikawa@riken.jp
Backgrounds: Processing of asparagine-linked sugar chains
of newly synthesized glycoproteins is essential for those glycoproteins to be delivered to the final destination. In this
process, trimming reaction of α-1,2 linked mannose moieties
is catalyzed by members of glycosylhydrolase family 47,
which include ER α-1,2 mannosidase I (ERManI), Golgi
α-1,2 mannosidase IA-C, and EDEM1-3.
C-mannosylation of tryptophan residues of the consensus
sequence WXXW forms a unique type of glycosylation
next to the more known N- and O-glycosylation. It is an
abundant type of glycosylation typically, but not exclusively, occurring on proteins with thrombospondin type I
repeats and on cytokine type I receptors. Mutagenesis of
the WSXWS motif in cytokine receptors already
established an important role of this motif for receptor
function. The genetic base of the enzyme catalyzing Cmannosylation was so far elusive.
We had synthesized various high-mannose type oligosaccharides by our convergent methods and utilized them to
analyze biochemical characteristics of proteins involved in
ER quality control1. As a part of the studies, we had revealed that a recombinant form of human ERManI had an
activity to generate smoothly Man 6-5 GlcNAc 2 from
Man9GlcNAc22. In addition, hERManI removed more mannose moieties from some glycoproteins, when they had been
exposed to denaturing conditions2, 3. In this study, we have
examined to reveal influencial factors and reaction conditions which could accelerate trimming of mannoses from the
oligosaccharides by ERManI in living organisms.
We identified C. elegans DPY-19 as the C-mannosyltransferase.
DPY-19 is homologous to the catalytic subunit of
oligosaccharyltransferase responsible for N-glycosylation.
The overall membrane topology is identical and amino acids
involved in the binding of the common dolichol-phosphate
moiety of the donor are conserved. Overall identity is,
however, very limited.
Methods: Pyridylaminated GlcMan9GlcNAc2 was prepared from oligosaccharides in chicken immunoglobulin
Y3. Mannotrioses of Man(α-1,2)Man(α-1,2)Man-dansyl2,
Man(α-1,2)Man(α-1,3)Man-dansyl, and Man(α-1,2)Man(α-1,6)Man-dansyl were synthesized. A solution of
polyethyleneglycol (PEG) was included in each demannosylation reaction2.
DPY-19 was already known from a C. elegans mutant
defective in neuroblast migration. In vitro assays showed
that the dpy-19 mutant is devoid of C-mannosyltransferase
activity. Moreover, by recombinant expression of DPY-19
and mass spectrometric analysis, we could show Cmannosylation of C. elegans proteins UNC-5 and MIG-21,
which both contain two thrombospondin type I repeats with
Results: First, chemicals to induce macromolecular
crowding effects were included in the reaction. As a result,
removal of mannoses from GlcMan9GlcNAc2-PA was markedly accelerated in the presence of 50 % PEG 400. The
result may suggest that de-mannosylation by hERManI
could be enhanced under macromolecular crowding
circumstances.
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Second, we have revealed that all three mannotrioses were
susceptible to hERManI. The reactivity of hERManI to
Man(α-1,2)Man(α-1,6)Man was, however, much lower
than the two other compounds, suggesting that the efficiency of de-mannosylation by hERManI was highly dependent
on the structure at the non-reducing end. When reactions
were performed at pH 5–7, maximum processing of those
compounds was observed around pH 5.0. In addition,
hERManI exhibited highest efficiency at pH 5.5 of demannosylation from heat-treated immunoglobulin Y. These
results indicated that de-mannosylation by hERManI was
accelerated under acidic conditions.
1
Takeda, Y. et al. Curr. Opin. Chem. Biol. 13, 582–591
(2009)
2
Aikawa, J. et al. Glycoconj. J, 29, 35–45 (2012)
3
Aikawa, J. et al. 26th International Carbohydrate Symposium
(2012)
034: Expression regulation of glycosyltransferase gene
β3Gn-T8 by transcription factor c-jun in gastric cancer
cell line SGC-7901
Zhi Jiang1, Lan Xu1, Ying-hui Zhou1, Shi-liang Wu*1;
Department of Biochemistry and Molecular Biology, School
of Medicine,Soochow University, Suzhou, China 215123
wushiliang@suda.edu.cn
Objective: To clarify the expression regulation of
glycosyltransferase gene β3Gn-T8 by transcription factor cjun in gastric cancer cell line SGC-7901. Methods: Online
prediction of the transcription factor c-jun binding sites in the
β3Gn-T8 promoter region was performed with a transcription
factor database. Eight β3Gn-T8 promoter segments and one
mutated segment were cloned by PCR from the gastric cancer
cell line SGC-7901 genomic DNA. Then, they were used to
construct recombinant plasmids with luciferase reporter gene
respectively. The above β3Gn-T8 promoter segment reporter
gene expression vectors, as well as an internal control plasmid
pRL-SV40 and transcription factor expression vector pCIneo-jun were co-transfected into the SGC-7901 cells. A
Dual-Luciferase Reporter Assay System was used to detect
the activation of the β3Gn-T8 promoter by the transcription
factor c-jun. Whether the activation relied on the c-jun
binding site (-561/+8) of β3Gn-T8, and whether it was
dose-dependent on c-jun was also observed. Furthermore,
the binding of c-jun protein to β3Gn-T8 DNA in gastric
cancer cells was detected using chromatin immunoprecipitation (ChIP). Results: The transcription factor c-jun binding sites resided within the β3Gn-T8 promoter region and
could activate the transcriptional of β3Gn-T8. pGL3-luc
(-561/+8) showed the highest luciferase activity, which
Glycoconj J (2013) 30:281–461
was dose-dependent on c-jun. The ChIP results verified the
binding of c-jun to β3Gn-T8 DNA fragments. Conclusion:
The transcription factor c-jun is involved in the expression
regulation of β3Gn-T8 in the gastric cancer cell line
SGC-7901.
035: Epigenetic regulation of glycosyltransferase gene
expression
Yasuhiko Kizuka1,2, Shinobu Kitazume1,2, Naoyuki
Taniguchi1, 2, 3; 1Disease Glycomics Team, 2RIKEN-Max
Planck Joint Research Center, RIKEN, Wako, 351-0198,
Japan; 3Alliance Laboratory of ISIR, Osaka Univ. and
RIKEN, Ibaraki, 567-0047, Japan.
y.kizuka@riken.jp
Glycan expression is regulated by several factors such as
nucleotide-sugars, acceptor substrate molecules, glycosidase
etc. Expression level of glycosyltransferase is a key factor to
control glycan expression, and the tissue-specific regulation
of glycosyltransferase expression is indispensable for distinct glycan biosynthesis in tissues. However, it is poorly
understood how each glycosyltransferase is expressed in a
tissue specific manner, suggesting that unidentified mechanisms exist to regulate glycosyltransferase gene expression.
Recently, we have reported that the expression of one of
brain-specific glycosyltransferases, GnT-IX (also designated
as GnT-Vb), is epigenetically regulated at chromatin level
(Kizuka et al. J. Biol. Chem. 2011). Particularly, active and
repressive histone modification marks are well-correlated
with the GnT-IX mRNA expression level in a tissue or
cell-type specific manner, indicating that brain specific
GnT-IX expression is regulated by an epigenetic mechanism. So far, however, little is known about detailed mechanisms of how glyco-genes including GnT-IX are regulated
epigenetically.
In this study, we investigated epigenetic mechanisms of how
chromatin of GnT-IX gene is activated. In addition, epigenetic regulation mechanisms of other glycosyltransferase
genes are also analyzed including GnT-III, -V and Fut8. As
target epigenetic factors, we focused on 1) HDAC (histone
deacetylase) that downregulates histone acetylation, 2)
histone methylation particularly at histone H3 lysine 27
that is involved in gene repression, 3) TET (ten-eleven
translocation) that is required for hydroxylation of methylated DNA. As a result, we found that histone acetylation
selectively activates GnT-IX gene among the glyco-genes
tested while histone methylation down-regulates several
glycosyltransferase genes in a cell type specific manner. In
addition, it was revealed that TET enzyme is involved in
Glycoconj J (2013) 30:281–461
activation of the specific GnT genes. These results indicate
that glycosyltransferase genes are systematically regulated
by multiple epigenetic mechanisms and that each gene is
distinctly regulated by different epigenetic factors.
036: Keynote Lecture: Plasma membrane glycohydrolases
and new strategies for medical dignosis and therapeutic
treatments
Sandro Sonnino, Nicoletta Loberto, Rosaria Bassi,
Vanna Chigorno, Alessandro Prinetti and Massimo
Aureli; Department of Medical Biotechnology and Translational Medicine, University of Milan, Italy
The following enzymes of the sphingolipid metabolism have
been found associated to the external leaflet of the plasma
membranes: sialidase, sialyl transferase, -hexosaminidase,
-galactosaminyl transferase, β-galactosidase, β-glucosidase,
sphingomyelinase, sphingomyelin synthase.
The association of glycosphingolipid metabolic enzymes
with the plasma membranes, the information on their
activity on natural substrates in living cells, the availability of couples of enzymes capable to catalyze opposite
reactions, suggest that changes of glycosphingolipid
structures at the plasma membrane could be the way to
change rapidly the GSL plasma membrane concentration,
to modify the glycosphingolipid pattern and to modulate
cell functions.
All the enzymatic activities found associated to the plasma
membranes result strictly related to the extracellular pH,
being in general optimal under mild acidic conditions. Mild
acidic conditions are found in specific membrane domains
known as lipid rafts, where glycosphingolipids are highly
enriched together with cholesterol, receptor proteins, proteins involved in cell signaling, and antiports or general ion
exchange proteins. Several proton pumps are present on the
cell surface and inserted into lipid rafts together with the
glycohydrolases. Thus their activity and the following catabolic process at the cell surface, that under particular conditions can lead to apoptosis, can be increased by activating
the proton pump and decreasing the local pH of their extracellular environment. In addition to this, it has been demonstrated that a cross talk among the glyoshingolipid
hydrolases exists. This introduces the possibility to pharmacologically activate a single glycohydrolase to activate a
series of enzymes working in sequence.
Changes of the cell surface glycosphingolipid hydrolase
activities in sphingolipidosis and during radiotherapy will
be presented and discussed.
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037: Exploring the “life” of N-glycans on glycoproteins
Tadashi Suzuki; Glycometabolome Team, Systems
Glycobiology Research Group, RIKEN Advanced Science
Institute (ASI), Wako, Saitama 351-0198, JAPAN
tsuzuki_gm@riken.jp
Cell surface glycans can be regarded as a “face” of cells, and
their structures are known to change depending on developmental stages or environment. Therefore cell surface glycans
are utilized for identification of stem cells such as iPS/ES
cells, or as valuable biomarkers in diagnosis/detection of
cancer. To understand the molecular mechanism of “change
of face”, biosynthetic pathway for glycan chains on
glycoproteins/glycolipids has been, in most part, revealed,
while there are still many unresolved issues on their
transport/degradation aspect.
We have been studying enzymes involved in the formation
and degradation of free oligosaccharides derived from the
N-glycans or their precursor (dolichol-linked oligosaccharides) in the cytosol, i.e. peptide:N-glycanase (PNGase) (1),
endo-β-N-acetylglucosaminidase (ENGase)(2), and αmannosidase (Man2C1) (3). These enzymes constitute a
major pathway for the non-lysosomal catabolic processing
of free oligosaccharides in mammalian cells. We have
shown that free oligosaccharides in yeast can serve as a
read-out of N-glycan processing for misfolded glycoproteins
during a process called ER-associated degradation (ERAD)
(4, 5).
In this symposium, current knowledge for catabolic pathway
of glycans in S. cerevisiae will be presented. We will also
present our new approach to visualize “glycoforms” of proteins of interest using transmembrane FRET (fluorescence
resonance energy transfer) technique. This technique will
help us understand how glycan structures can affect the
stability and/or intra/intercellular trafficking of carrier proteins. We will also present our new approach to visualize
“glycoforms” of proteins of interest using transmembrane
FRET (fluorescence resonance energy transfer) technique
(6). This technique will help us understand how glycan
structures can affect the stability and/or intra/intercellular
trafficking of carrier proteins (6, 7).
References
(1) Suzuki, T., et al. J. Cell Biol. 149, 1039 (2000); (2)
Suzuki, T., et al. Proc. Natl. Acad. Sci. USA 99, 9691
(2002); (3) Suzuki, T., et al. Biochem. J. 400, 33 (2006);
(4) Hirayama, H., et al. J. Biol. Chem. 285, 12390 (2010);
(5) Hosomi, A., et al. J. Biol. Chem. 285, 24324 (2010); (6)
Haga, Y., et al. Nature Commun. 3, 907 (2012); (7) Haga,
Y., et al. J. Biol. Chem. 286, 31320 (2011).
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038: Inhibition of nucleotide sugar transport in
Trypanosoma brucei alters surface glycosylation
039: POFUT1/2 Promotes Embryonic Cell Adhesion and
Migration at Fetal-Maternal Interface
Li Liu,1 Yu-Xin Xu,1** 1 Kacey L. Caradonna,2 Emilia
K. Kruzel 3, Barbara A. Burleigh2, James D. Bangs 3,
and Carlos B. Hirschberg1; 1Dept. of Molecular and Cell
Biology, Boston University Goldman School of Dental Medicine, Boston, MA 02118, USA, 2Dept.of Immunology and
Infectious Diseases, Harvard School of Public Health, Boston,
MA 02115, USA, 3Dept. of Microbiology and Immunology,
School of Medicine and Biomedical Sciences, University at
Buffalo, SUNY, Buffalo, NY 14214, USA, 1**Present address: Center for Human Genetic Research and Cardiovascular Research Center, Massachusetts General Hospital, Boston,
MA 02114, USA
liul@bu.edu
Shuai Liu1, Xuesong Yang1, Jiao Wang1, Xiaoqi Wang2,
Qiu Yan1; 1Department of Biochemistry and Molecular
Biology, Dalian Medical University, Liaoning Provincial
Core Lab of Glycobiology and Glycoengineering, Dalian
116044, China; 2Department of Dermatology, Northwestern
University Feinberg School of Medicine, Chicago, IL
60611, USA
yanqiu63@126.com
Nucleotide sugar transporters (NSTs) are indispensible for the
biosynthesis of glycoproteins by providing the nucleotide
sugars needed for glycosylation in the lumen of the Golgi
apparatus. Mutations in NST genes cause human and cattle
diseases and impaired cell walls of yeast and fungi. Information
regarding their function in the protozoan parasite, Trypanosoma
brucei, a causative agent of African trypanosomiasis, is unknown. Here, we characterized the substrate specificities of
four NSTs, TbNST1-4, which are expressed in both the insect
procyclic form (PCF) and the mammalian bloodstream form
(BSF) stages. TbNST1/2 transport UDP-Gal/UDP-GlcNAc,
TbNST3 transports GDP-Man, and TbNST4 transports UDPGlcNAc, UDP-GalNAc and GDP-Man. TbNST4 is the first
NST shown to transport both pyrimidine and purine nucleotide
sugars and is demonstrated here to be localized at the Golgi
apparatus. RNAi-mediated silencing of TbNST4 in PCF caused
underglycosylated surface glycoprotein EP-procyclin. Similarly, defective glycosylation of the variant surface glycoprotein
(VSG221) as well as the lysosomal membrane protein, p67 was
observed in ΔTbNST4 BSF T. brucei. Relative infectivity
analysis showed that defects in glycosylation of the surface
coat resulting from TbNST4 deletion were insufficient to impact the ability of this parasite to infect mice. Notably, the fact
that inactivation of a single NST gene results in measurable
defects in surface glycoproteins in different life cycle stages of
the parasite, highlights the essential role of NST(s) in glycosylation of T. brucei. Thus, results presented in this study provide a
framework for conducting functional analyses of other NSTs
identified in T. brucei.
Implantation is a complex developmental event that is
initiated by the recognition and adhesion of the embryo
to the endometrial epithelium. The fucosylation of glycoproteins both on the embryonic and uterine epithelial
cell surface plays critical roles in the molecular interactions at fetal-maternal interface. Our previous study
shows that the occurrence of N-linked fucosylated sugar
chain, such as sialyl LeX (sLeX), is closely related to the
adhesive potential of embryo to the endometrium. However the function of O-fucosylation in this process is not
still clearly stated till now. In this study, the expression
of protein O-fucosyltransferases 1 and 2 (POFUT1/2),
which are the two subtypes of synthetic enzymes transferring GDP-Fuc to the Ser/Thr residues of the specific
substrate protein molecules, was detected in human embryonic cells (Jar) and human uterine epithelial cells
(RL95-2 and HEC), respectively. The results showed that
both embryonic cells and uterine epithelial cells
expressed POFUT1/2 by RT-PCR, Western blot and immunofluorescent assays. The POFUT1/2 expression level
was dose-dependently regulated by leukemia inhibitory
factor (LIF) in embryonic cells, and was correlated to the
adhesive potential detected by adhesive percentage in
vitro implantation model constituting by Jar and RL952. Furthermore, the elevated POFUT1/2 increased the
invasive capability of embryonic cells by migration and
matrix-degradation assays. The primarily study in mouse
also showed that the level of POFUT1/2 in murine embryos
changed dynamically with the different stages of embryo
development. Conclusion: POFUT1/2 is expressed at fetalmaternal interface, and the elevated POFUT1/2 is related to
the adhesion, invasion as well as developmental processes of
embryo. They may be the important markers of reproductive
functions and further study is needed.
Glycoconj J (2013) 30:281–461
Tue-Development & Differentiation
040: Keynote Lecture: Transcriptome and biochemical
analysis reveals that suppression of GPI-anchor synthesis
leads to autophagy and necroptosis in Aspergillus fumigatus
Jianghong Yan, Ting Du, Wan Zhao, Cheng Jin*; State
Key Laboratory of Mycology, Institute of Microbiology,
Chinese Academy of Sciences, Beijing 100101, China
jinc@im.ac.cn
Previously, it has been shown that GPI proteins are required
for cell wall synthesis and organization in Aspergillus
fumigatus, a human opportunistic pathogen causing lifethreatening invasive aspergillosis (IA) in immunocompromised population. Blocking GPI anchor synthesis leads to
severe phenotypes such as cell wall defects, increased cell
death, and attenuated virulence. However, the mechanism by
which the phenotypes are induced is unclear. To gain insight
into global effects of GPI anchoring in A. fumigatus, in this
study a conditional expression mutant was constructed and a
genome wide transcriptome analysis was carried out. Our
results suggested that suppression of GPI anchor synthesis
mainly led to activation of the phophatidylinositol (PtdIns)
signaling and ER stress. Biochemical and morphological evidences showed that autophagy and increased necroptosis
were induced in response to suppression of the GPI anchor
synthesis. Based on our results, we proposed that activation of
PtdIns3K and increased cytosolic Ca2+, which was induced
by both ER stress and PtdIns signaling, acted as the main
effectors to induce autophagy and necroptosis.
This work was funded by the National Natural Science
Foundation of China (31030025) to C. Jin.
041: Differential Targeting of the Dolichol Pathway
Develops Uneven Cellular Responses
Zhenbo Zhang1, Aditi Banerjee1, Elena A. Carasquillo1,
Krishna Baksi2 and Dipak K. Banerjee1; 1Department of
Biochemistry, School of Medicine, University of Puerto
Rico, Medical Sciences Campus, San Juan, PR 009365067, USA; and 2Department of Anatomy and Cell Biology,
School of Medicine, Universidad Central del Caribe,
Bayamon, PR 00960-3001, USA
dipak.banerjee@upr.edu
Assembly of Glc3Man9GlcNAc2 on dolichol is the hallmark
of asparagine-linked (N-linked) protein glycosylation as the
angiogenesis for tumor progression and metastasis. The
process is evolutionary conserved and contributes to the
structural and functional integrity of the N-linked
305
glycoproteins. Dynamics of the N-glycosylation process indicates the presence of a cross-talk between
mannosyphospho dolichol synthase (DPMS) and N-acetylglucosaminyl 1-phosphate transferase (GPT). DPMS has a
conserved phosphorylation motif and has been found to in
all Dpm1 gene sequenced from 35 different species, viz.,
protozoan parasite to human. Phosphorylation activation of
DPMS accelerates the Glc3Man9GlcNAc2-PP-Dol (LLO)
biosynthesis and turnover, and consequently the protein Nglycosylation and capillary endothelial cell proliferation,
i.e., angiogenesis. GPT activity is also increased. Cells
transfected with overexpressing DPMS plasmid enhances
the expression of GlcNAc-β-(1,3)-GlcNAc)1-4-β-GlcNAcNeuAc containing glycans and increased angiogenesis. Removal of the phosphorylation motif by site-directed mutagenesis prevents DPMS activation and reduction of S. cerevisiae
proliferation. Similarly, silencing of DPMS with shRNA
down regulates glycan expression, angiogenesis as well as
Matrigel™ invasion. Reduced PCNA, Bcl-2 and caspase-3
expression supports that DPMS silencing affects the cell cycle
progression without inducing apoptosis. Dol-P-Man is an
activator of GPT, and tunicamycin is a competitive inhibitor
of GPT. When treated, tunicamycin inhibits DPMS activity in
capillary endothelial cells without affecting the protein or the
mRNA. The results are G1 arrest and the induction of apoptosis. Silencing of the transcription factor E2F1 has no
additive or synergistic effect. Mechanistic details support
development of “ER stress” and the cell deaths is due to
unfolded protein response (upr). Raman Spectroscopy of
the proteome at C-O stretching supports protein denaturation. cDNA microarray analysis indicates that among
the affected genes, nearly 20 % are transcription factors.
The developmentally regulated genes such as c-fos, c-myc
and c-jun are all upregulated but not their down-stream
regulators. In vivo, tunicamycin inhibits angiogenesis in
Matrigel™ implants in athymic Balb/c (nu/nu) mice and
prevents breast tumor progression. There is ER stress in
tumor blood vessels, and reduced N-glycosylation correlates narrowing of the vessel diameter. Supported by
grants from the NIH U54-CA096297 (DKB), Susan G.
Komen for the Cure BCTR0600582) (DKB) and
NIH/NIMHD 8G12MD007583 (KB).
306
042: GPI-anchor synthesis is indispensable for the
germline development of the nematode Caenorhabditis
elegans
Daisuke Murata1,2,3 , Kazuko H. Nomura 1,2 , Ayako
Matsuda1, Sayaka Akiyoshi1, Katsufumi Dejima1,2,4,Lance
Wells 5 , Karen L. Abbott 6 , Nana Kawasaki2,7 , Keiko
Gengyo-Ando 2 , 8 , 9 ,Eriko Kage-Nakadai 8 , Shohei
Mitani2,8, Kazuya Nomura1,2; 1 Department of Biological
Sciences, Kyushu University Graduate School, Fukuoka
812-8582, Japan; 2 Core Research for Evolutional Science
and Technology, Kawaguchi 332-0012, Japan; 3 Department
of Pharmacology, Graduate School of Medicine, Osaka
University, Osaka 565-0871, Japan; 4 JSPS Postdoctoral
Fellow for Research Abroad, Division of Biological Sciences,
University of California, San Diego, La Jolla California
92093-0368, USA; 5 Department of Chemistry, University
of Georgia, Athens, Georgia 30605, USA; 6 Complex Carbohydrate Research Center, University of Georgia, Athens,
Georgia 30605; 7 Division of Biological Chemistry and
Biologicals, National Institute of Health Sciences, Tokyo
158-8501, Japan; 8 Department of Physiology, Tokyo
Women’s Medical University School of Medicine, Tokyo
162-8666, Japan; 9 Saitama University Brain Science
Institute, Saitama 338-8570, Japan
knomuscb@kyushu-u.org
GPI (glycosylphosphatidylinositol)-anchor attachment is
one of the most common post-translational protein modifications, widely used in organisms from archaebacteria to
humans. Pathogens such as Trypanosoma cruzi use GPIanchor variants to escape from host immune surveillance,
and the enzymes involved in their synthesis are potential
drug targets for the treatment and prevention of disease.
GPI-anchored proteins are concentrated in lipid rafts on
the cell surface or membrane nanodomains and play essential roles in signal transduction in multicellular organisms.
Using C. elegans, we determined that GPI-anchored proteins are present in germline cells as well as distal tip cells
(DTCs), which are essential for maintenance of the germline
stem cell niche. We identified 24 C. elegans orthologs of
human genes involved in GPI-anchor synthesis by PSIBLAST search and used RNAi and gene knockout strategies
to inhibit their functions. Inhibition of genes involved in
various steps of GPI-anchor synthesis resulted in abnormal
development of oocytes and early embryos, and both lethal
and sterile phenotypes were observed. The PIG-A gene
codes for the major catalytic subunit (subunit A) of the
phosphatidylinositol N-acetylglucosaminyltransferase complex (EC 2.4.1.198), which catalyses the first step of GPIanchor synthesis. Mutations in this gene would thus inhibit
all GPI-anchor synthesis. By isolating a deletion mutant
allele of piga-1 gene (C. elegans PIG-A ortholog), we
Glycoconj J (2013) 30:281–461
showed that GPI-anchor synthesis is indispensable for the
normal development of germline cells. Lack of GPI-anchor
synthesis resulted in 100 % lethality with decreased mitotic
germline cell number, abnormal oocyte formation and death
of cleaving eggs. Using cell specific rescue of the null allele,
we showed that expression of PIGA-1 in DTCs or in gonadal sheath cells is sufficient for germline cells to develop
normally. These results clearly demonstrate that GPI anchor
synthesis is indispensable for the normal development of
oocytes and embryos in C. elegans. To elucidate the molecular mechanism of GPI-anchored proteins in germline development, we concentrated GPI-anchored proteins from
wild type and DTC-rescued worms with alpha toxin column
and PI-PLC treatment followed by nanoLC/MS/MS. Results
of RNAi of these identified protein genes will be discussed
in the paper.
043: Novel Marker Antibodies for Human iPS/ES Cells
and their Application
Kawasaki, T.1 Nakao, H.1 Matsumoto, S.1 Toyoda, H.2
Kusuda Furue, M.3 and Kawasaki, N.1; 1Research Center
for Glycobiotechnology, Ritsumeikan University, Shiga
525-8577, Japan; 2Laboratory of Bio-analytical Chemistry,
Faculty of Pharmaceutical Sciences, Ritsumeikan University, Shiga 525-8577, Japan; 3Laboratory of Cell Cultures,
Department of Disease Bioresources, National Institute of
Biomedical Innovation, Ibaraki, Osaka 567-0085, Japan
tkawasak@fc.ritsumei.ac.jp
Carbohydrate recognizing antibodies are very useful tools
for monitoring the specific glycans on a specific cell with
high sensitivity and sharp specificity. Among the conventional human iPS (hiPS)/ES marker antibodies, SSEA-3 and
SSEA-4 recognize specifically globosides, and TRA-1-60
and TRA-1-81 recognize keratan sulfate. However, most of
these antibodies were generated against human embryonal
carcinoma (hEC) cells. Consequently, these antibodies recognize those glycans, which are common to hiPS/ES and
hEC cells. In order to obtain antibodies specific to hiPS
cells, we immunized mice with a hiPS cell line, Tic, and
selected hiPS-cell positive hybridomas. These hybridomas
were then subjected to screening with hEC cells and hEC
positive hybridomas were excluded. Upon Western blotting
with antibody R-10G, one of these hiPS/ES specific antibodies, following SDS-PAGE, the Tic cell lysate indicated
one major but broad immunoreactive band at the position
corresponding to over 250 kDa. The antigen protein was
isolated from the Tic cell lysate by using an affinity column
of R-10G. The epitope was identified as keratan sulfate with
little over-sulfation, since digestion of the R-10G antigen
with keratanase, keratanase II and endo-beta-galactosidase
Glycoconj J (2013) 30:281–461
307
reduced completely the immunoractivity of the antigen protein. The carrier protein of the R-10G epitope on hiPS cells
was identified as podocalyxin by liquid chromatography/mass
spectrometry (LC/MS/MS) analysis of the R-10G positiveprotein band material obtained on SDS-PAGE. The R-10G
epitopes differ from those recognized by other human
pluripotent-cell marker antibodies such as TRA-1-60 and
TRA-1-81 and from high-sulfated keratan sulfaterecognizing antibodies such as 5D4 and BCD4, indicating
that the R-10G should be a new useful tool for studying the
roles of glycans on the cell surfaces. The R-10G epitope is
distributed heterogeneously on hiPS cells, suggesting that a
single colony of undifferentiated hiPS cells consists of different cell subtypes. Survey of a human tissue array indicated
that the R-10G epitope is not confined to hiPS/ES cells but is
expressed in the adult brain and cerebellum.
biosynthesis pathways of Psl, Pel, alginate, and LPS compete
for common sugar precursors. As AlgC is the only enzyme that
provides precursors for each of these exopolysaccharides, we
propose that AlgC is a key checkpoint enzyme that coordinates
the total amount of exopolysaccharide biosynthesis by controlling sugar precursor pool. Our data also provide a plausible
strategy that P. aeruginosa utilizes to modulate its biofilm
matrix exopolysaccharides.
044: The role of multiple exopolysaccharides in the
communities of Pseudomonas aeruginosa and their
coordinate regulation
Differentiation system of intestinal epithelia cells develops
from normal stem cells residing crypt of the intestinal cells.
From these, enterocytes, mucus secreting cells, and endocrine
cells will be developed during the journey to the top of the villi.
It has been shown that stem cells to normal differentiated cells
develop into these cell types while stem cells maintain selfrenewal. To determine the roles of heparan sulfate in intestinal
cell development, we utilized Lgr5-driven inducible gene
knockout, which was generated by Hans Clevers (Nature;
459: 262–265, 2009). After treatment on mouse with tamoxifen, heparan sulfate was significantly abrogated. As a result,
the height of villus was shorter than EXT1wt/wt control mice,
and resulted in fewer enterocyte and endocrine cells. Surprisingly, Ki-67 staining level was diminished in crypt cells of
EXT1 knockout mice, suggesting that intestinal stem cells
require heparan sulfate in stem cell proliferation, but heparin
sulfate requirement was not obvious in enterocytes and endocrine cells in the villus domain.
Luyan Ma; State Key Laboratory of Microbial Resources,
Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100101, China
luyanma27@im.ac.cn
Biofilms are surface-associated communities of microorganisms encased in an extracellular substances matrix.
Exopolysaccharide is a critical biofilm matrix component.
Pseudomonas aeruginosa can produce several biofilm matrix
exopolysaccharides that include alginate, Psl, and Pel, which
each has contributions in biofilm. The overproduction of
exopolysaccharide alginate leads to the mucoid phenotype of
P. aeruginosa, which is believed to play a role in the bacteria’s
resistance to antibiotics and to evade host defense mechanisms.
Our data shows that alginate occupy a lot of space in biofilm,
resulting in the muciod biofilm with high biofilm thickness, but
little biomass. Psl is an essential matrix component for
nonmucoid and mucoid P. aeruginosa to initiate and maintain
biofilms. Pel is a glucose-rich exopolysaccharide, which is
required to form air-liquid interface biofilms (pellicles). We
demonstrated that AlgC, a key enzyme that provides sugar
precursors for the synthesis of alginate and lipopolysaccharides
(LPS) is also required for both Psl and Pel production. We
showed that forced-synthesis of Psl in alginate-producing mucoid bacteria reduced alginate production but this was not due
to transcription of the alginate biosynthesis-operon. Likewise,
when either alginate or Psl were overproduced, levels of Bband LPS decreased. Induction of Pel resulted in a reduction of
Psl levels. Since the effects of reduced exopolysaccharide
synthesis when another is overproduced didn’t appear to be
regulated at the transcriptional level, this suggests that the
045: Keynote Lecture: Roles of Heparan Sulfate in Normal and Cancer Stem Cells in intestine
Minoru Fukuda; Glycobiology Unit, Tumor Microenvironment Program, Cancer Center, Sanford-Burnham Medical Research Institute, La Jolla California 92037, USA
minoru@sanfordburnham.org
In EXT1 knockout mouse, azoxymethane (AOM) and dextran
sodium sulfate induced only adenoma while adenocarcinomas
were apparently produced in wild-type mouse counterparts.
The results strongly suggest that cancer stem cells also require
heparan sulfate to produce progenies. Further work is necessary to determine the role of heparan sulfate in colon cancer
formation, possibly by utilizing organoid culture. The work is
support by NCI grant, P01CA71932
308
Glycoconj J (2013) 30:281–461
046: 3-O-sulfated heparan sulfate structure contributes
to the differentiation of mouse embryonic stem cells by
activating Fas signaling
and indicates a novel signaling pathway for differentiation
in mouse ES cells.
Shoko Nishihara1, Kazumi Hirano1, Norihiko Sasaki1,
Toin H. Van Kuppevelt 2 , Taichi Miura 1 , Tomomi
Ichimiya1; 1Laboratory of Cell Biology, Department of Bioinformatics, Faculty of Engineering, Soka University, 1-236
Tangi-cho, Hachioji, Tokyo 192-8577, Japan; 2Department of
Biochemistry, Nijmegen Center for Molecular Life Sciences,
Radboud University Nijmegen Medical Centre, Nijmegen,
Netherlands
shoko@soka.ac.jp
047: The expression of chondroitin sulphate glycosaminoglycans in the development of human foetal articular
cartilage
Embryonic stem cells (ES cells) are derived from the inner cell
mass of pre-implantation blastocysts, have the characteristic
features of self-renewal and pluripotency, and are able to
differentiate into all cell types deriving from the different germ
layers. Maintenance of self-renewal and pluripotency in mouse
ES cells is regulated by the balance between several extrinsic
signaling pathways. Recently, we demonstrated that heparan
sulfate (HS) chains play important roles in the maintenance
and differentiation of mouse ES cells by regulating extrinsic
signaling. Sulfated HS structures are modified by various
sulfotransferases during differentiation of mouse ES cells.
However, the significance of specific HS structures during
their differentiation remains unclear. Therefore, we performed
a screen by overexpression of various sulfotransferases in
mouse ES cells and found that HS 3-O-sulfotransferases
(3OSTs) contribute to the differentiation of mouse ES cells.
The 3-O-sulfated HS structure synthesized by 3OSTs and
recognized by the antibody HS4C3 increased during differentiation of mouse ES cells. Expression of Fas on the cell surface
of the differentiated cells also increased. Overexpression of the
3-O-sulfated HS structure in mouse ES cells induced apoptosis
and spontaneous differentiation even in the presence of LIF
and serum. Furthermore, knockdown of 3OST and inhibition
of Fas signaling reduced the potential for differentiation into
the three germ layers during embryoid body formation. These
data showed that the 3-O-sulfated HS structure was required
for differentiation of mouse ES cells. Up-regulation of the 3-Osulfated HS structure resulted in the recruitment of Fas from
the cytoplasm to lipid rafts on the cell surface followed by
activation of Fas signaling. The 3-O-sulfated HS structure
interacted with a region that included the heparin-binding
domain of Fas. Reduced self-renewal capability in cells
overexpressing 3OST resulted from the degradation of Nanog
by activated caspase-3, which is downstream of Fas signaling,
and was rescued by the inhibition of Fas signaling.
This is the first demonstration that activation of Fas signaling is mediated by an increase in 3-O-sulfated HS structure
Siyuan Li1, Rongliang Xue1, Antony J Hayes2, Caterson
Bruce2, Clare E. Hughes2 & Junling Cao3; 1The Department of Anaesthesia, The Second Hospital of Xi’an Jiaotong
University, Xi’an, 710004, China; 2The Department of Pathophysiology and Repair, School of Biosciences, Cardiff
University, CF10 3AX, UK; 3The Institute of Endemic
Diseases, Medical School of Xi’an Jiaotong University,
Xi’an, Shaanxi, 710061,China
foxlsy@163.com
Introduction: Our previous studies have illustrated that
novel chondroitin sulphate sulphation motifs in glycosaminoglycans (GAGs) on proteoglycans can be used to identify
early stages of stem/progenitor cell differentiation in the
articular cartilage. However, the spatial-temporal expression
profiles of these novel chondroitin sulphate sulphation motifs in GAGs during the development of human articular
cartilage are largely unknown.
Materials & Methods: Articular cartilage from figure joints
was obtained from aborted human fetuses aged 25 and
32 weeks, respectively. Articular cartilage from normal human figure joints aged 2-year-old was used as a postnatal
control. The expression of novel chondroitin sulphated
sulphation motifs including 3B3, 4C3, 6C3 and 7D4 were
investigated immunohistochemically.
Results: 7D4 and 6C3 positive staining was observed in the
articular cartilage from both foetal (25-week-old & 32week-old) and juvenile figure joints (2-year-old). In 25week-old foetal figure joints, 7D4 positive staining was
found in the inter-territorial matrix in the surface layer of
articular cartilage, and the cartilage near synovium. Differently, intensive 7D4 positive staining was observed in the
superficial, upper middle zone, and deep zone of articular
cartilage near subchondrol bone in the figure joints from 32week-old foetus. Interestingly, 7D4 positive staining was
only localised in the territorial but not inter-territorial matrix
around some cell clusters in the deep zone of articular
cartilage from the juvenile figure joints. 6C3 positive
staining mainly localised in the territorial matrix of surface
& middle zone of articular cartilage from 25-week-old foetal
figure joints. However, its positive staining expanded to the
inter-territorial matrix in the surface and deep zone of articular cartilage from the 32-week-old figure joints. In the
Glycoconj J (2013) 30:281–461
juvenile figure joints, 6C3 positive staining can be observed
in both territorial and inter-territorial matrix across the
whole depth of articular cartilage. There was no or very
weak 3B3 or 4C3 positive staining in the articular cartilage
from these figure joints.
Conclusion: The different expression profiles of these novel
chondroitin sulphated sulphation motifs indicated their dynamics of distributions during articular cartilage development, which may be associated with distinct chondroitin
sulphated proteoglycans in the extracellular matrix in the
articular cartilage, and tissue mature as well as homeostasis.
048: Direct interaction of FGF2 with polysialic acid
regulates the cell growth, but not cell survival
Sayaka Ono1, Masaya Hane1,2, Ken Kitajima1,2, and
Chihiro Sato1,2; 1Bioscience and Biotechnology Center,
Nagoya University; 2Program for Leading Graduate Schools
IGER, Nagoya University, Nagoya 464-8601, Japan
chi@agr.nagoya-u.ac.jp
Polysialic acid (polySia) is a unique polymer that spaciotemporally modifies the neural cell adhesion molecule
(NCAM) in vertebrates. Recently, we demonstrated that
polySia functions not only as an anti-cell adhesion molecule, but also as a reservoir for neurologically important
molecules such as neurotrophic factors and neurotransmitters. In this study, to gain further insight into the reservoir
function of polySia, we focused on FGF2 as a critical
molecule in neurogenesis and neural functions. We first
demonstrated the direct interaction between polySia and
FGF2 by native-PAGEs, gel-filtration, and surface plasmon
resonance (SPR) methods. Actually, polySia formed a large
complex with FGF2, and the minimum chain length of
polySia for the binding was 17 sialic acid residues. Heparan
sulfate (HS), which is a well-known FGF2-binding glycosaminoglycan, also formed a large complex with FGF2; however, the size and structural feature of the FGF2 complexes
were completely different between polySia and HS. To understand the effects of the expression of cell surface polySia
on FGF2-dependent physiological effects, the cells expressing
non-polysialylated NCAM were transfected with plasmids
encoding either polysialyltransferase ST8SiaII/STX or
ST8SiaIV/PST genes and analyzed for cell survival and cell
growth under FGF2 treatment. Interestingly, polySia-NCAMexpressing cells inhibited the FGF2-dependent cell growth
without affecting the cell survival. These results suggest that
the FGF2-polySia complex stimulates a new signal pathway
via FGF receptor (FGFR) different than the FGF2-HS complex that triggers conventional FGFR signaling to enhance the
cell proliferation.
309
049: Novel regulation of the intracellular Ca 2+ by
microdomain-localized GPI-anchored glycans in animal
sperm
Ken Kitajima 1,2,3 , Takeru Kanazawa 1,2,3 , Estelle
Garénaux1, Waraporn Kasekarn1, Kazuki Hori2, Andrés
D. Maturana2, Hiroshi Yasue4, Chihiro Sato1,2,3; 1Bioscience & Biotechnology Center, Nagoya University; 2Graduate
School of Bioagricultural Sciences, Nagoya University; 3Program for Leading Graduate Schools IGER, Nagoya University, Nagoya 464-8601, Japan; 4Transgenic Animal Research
Center, NIAS, Tsukuba 305-8602, Japan
kitajima@agr.nagoya-u.ac.jp.
Although membrane microdomains (or lipid rafts) are well
recognized as glycosphingolipid-enriched membrane areas involved in interactions and the subsequent signal transductions,
very few studies have focused on biological roles of glycan
chains of proteins on the microdomains. In 1999, we reported
the first biochemical characterization of sperm microdomains
from sea urchin [Ohta et al. 1999; 2000]. Many reports have
since been made on gamete microdomains in various species.
Of glycoproteins of sea urchin sperm microdomains, a heavily
glycosylated protein localized in sperm flagella, named
flagellasialin, is involved in sperm motility by regulating an
intracellular Ca2+, [Ca2+]i. Flagellasialin is a GPI-anchored
protein that contains a high content of glycans (80–90 % by
weight) on a short polypeptide (7.2 kDa). We have recently
reported that flagellasialin is involved in Ca2+-dependent
mechanosensory behavior of sea urchin sperm [Kambara et
al. 2011]. Regulation of the [Ca2+]i is an important physiological event in animal sperm. However, no report has been made
on how heavily glycosylated proteins like flagellasialin are
involved in Ca2+ regulation through the microdomain. Therefore, we asked if this type of glycoprotein is also present in
sperm of other animals. Here we report the presence of such a
glycoprotein in pig sperm. The glycoprotein is named WGA-gp
due to its high binding property with wheat germ agglutinin
(WGA) [Kasekarn et al. 2012]. WGA-gp is a highly
glycosylated GPI-anchored protein and shares the unique features in localization, structure, and potential function with
flagellasialin. Several lines of evidence demonstrated that
WGA-gp is identified as CD52, which is known to be a
mammals-specific protein. Interestingly, the structure of mature
protein and glycans is extensively different among CD52 molecules. Furthermore, the [Ca2+]i started oscillating at sperm
population level, when sperm were treated with a monoclonal
anti-WGA-gp antibody (4D1). The transient increase of [Ca2+]i
was observed at a single cell level. This is a mAb.4D1-specific
event, because no other antibody recognizing sperm glycan
epitopes induced the [Ca2+]i change. Therefore, the presence
of carbohydrate-enriched flagellar proteins involved in [Ca2+]i
regulation may be a common feature among animal sperm.
310
Infection & Immunity
050: Keynote Lecture: Genes affecting glycosylation
of human IgG show pleiotropy with autoimmune
diseases and haematological cancers
Gordan Lauc; University of Zagreb, Zagreb 10000, Croatia
glauc@pharma.hr
Glycosylation of immunoglobulin G (IgG) influences IgG
effector function by modulating binding to different Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans in plasma of
2,247 individuals from four European populations. After
isolating IgG from human plasma, N-glycans were released
and analyzed by ultra performance liquid chromatography
(UPLC). Genome-wide association study (GWAS) identified 9 genome-wide significant loci (P<2.27×10−9). Four
loci contained genes encoding glycosyltransferases
(ST6GAL1, B4GALT1, FUT8 and MGAT3), while the
remaining 5 contained genes that have not been previously
implicated in protein glycosylation (IKZF1, IL6STANKRD55, ABCF2-SMARCD3, SUV420H1, and
SMARCB1-DERL3). However, most of them have been
strongly associated with autoimmune and inflammatory
conditions (e.g., systemic lupus erythematosus, rheumatoid
arthritis, ulcerative colitis, Crohn’s disease, diabetes type 1,
multiple sclerosis, Graves’ disease, celiac disease, nodular
sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma).
Follow-up functional experiments in haplodeficient Ikzf1
knock-out mice showed the same general pattern of
changes in IgG glycosylation as identified in the metaanalysis. As IKZF1 was associated with multiple IgG Nglycan traits, we explored biomarker potential of affected
N-glycans in 101 cases with SLE and 183 matched
controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve =
0.842). Our study indicates that IgG glycosylation is
regulated by a complex network of genes through still
unknown mechanisms. The observed pleiotropy with
autoimmune diseases and haematological cancer indicates
that individual variations in IgG glycosylation affect IgG
function and contribute to disease development and
progression.
Glycoconj J (2013) 30:281–461
051: Glycobiology aspects of the periodontal pathogen
Tannerella forsythia
Gerald Posch1, Andrea Koerdt1, Laura Neumann2,
Friedrich Altmann 2 , Paul Messner 1 , Christina
Schäffer 1 ; 1 Department of NanoBiotechnology,
NanoGlycobiology Unit, Universität für Bodenkultur Wien,
Muthgasse 11, A-1190 Vienna, Austria; 2Department of
Chemistry, Universität für Bodenkultur Wien, Muthgasse 18,
A-1190 Vienna, Austria
christina.schaeffer@boku.ac.at
Glycosylation of proteins in bacteria is becoming increasingly documented, including both O- and N-linked glycosylation, with bacterial glycosylation profiles exceeding by
far those known from eukaryotes.
For the recently investigated periodontal pathogen
Tannerella forsythia glycobiology plays a pivotal role, affecting the cellular integrity of the bacterium, its life-style,
and its virulence potential. The bacterium possesses a
unique Gram-negative cell envelope with a glycosylated
surface (S-) layer as outermost decoration. This S-layer is
formed by self-assembly oft two S-layer glycoproteins into a
square 2D lattice with a spacing of ~10 nm which is proposed to be anchored to the cell envelope via a rough
lipopolysaccharide.
The T. forsythia S-layer glycan was elucidated by a
combination of electrospray ionization-tandem mass
spectrometry and nuclear magnetic resonance spectroscopy as an oligosaccharide with the structure 4-MeO-βManpNAcCONH 2 -(1→3)-[Pse5Am7Gc-(2→4)-]-βManpNAcA-(1→4)-[4-MeO-α-Galp-(1→2)-]-αFucp-(1→4)-[α-Xylp-(1→3)-]-β-GlcpA-(1→3)-[βDigp-(1→2)-]-α-Galp and is linked to distinct serine
and threonine residues within the D(S/T)(A/I/L/M/T/V)
amino acid motif. This S-layer glycan impacts the life
style of T. forsythia because increased biofilm formation
of an UDP-N-acetylmannosaminuronic acid dehydrogenase mutant can be correlated with the presence of
truncated S-layer glycans devoid of the charged glycan
branch. In addition, several other proteins of T. forsythia
are modified with that specific oligosaccharide. Proteomics identified two of them as being among previously
classified antigenic outer membrane proteins that are upregulated under biofilm conditions, in addition to two
predicted antigenic lipoproteins. Theoretical analysis of
the S-layer O-glycosylation of T. forsythia indicated the
involvement of a 6.8-kb gene locus that is conserved
among different bacteria from the Bacteroidetes phylum.
Together, these findings reveal the presence of a protein
O-glycosylation system in T. forsythia that is essential
Glycoconj J (2013) 30:281–461
for creating a rich glycoproteome pinpointing a possible
relevance for the virulence of this bacterium.
While the S-layer has also been shown to be a virulence
factor and to delay the bacterium’s recognition by the innate
immune system of the host, the contribution of glycosylation to modulating host immunity is currently unraveling.
Financial support came form by the Austrian Science Fund
FWF projects P20605-B12 and P21954-B20 (to C.S.) and
the PhD programme “BioToP - Biomolecular Technology of
Proteins” (Austrian Science Fund, FWF project W1224).
052: The Impact of Carbohydrate Microarrays in Studies
of the Molecular Basis of Virus-host Interactions
Yan Liu1, Angelina S. Palma1,2, Wengang Chai1, Kerstin
Reiss3, Ursula Neu3, Luisa Stroeh3, Thilo Stehle3, Feizi
Ten1; 1 Glycosciences Laboratory, Department of Medicine,
Imperial College London, London, W12 0NN, UK,
2
REQUIMTE, Departamento de Química, Universidade
Nova de Lisboa, 2829-516 Caparica, Portugal, 3Interfaculty
Institute of Biochemistry, University of Tuebingen,
Hoppe-Seyler-Str. 4, D-72076 Tuebingen, Germany
yan.liu2@imperial.ac.uk
Carbohydrate microarrays are important tools in elucidation of
carbohydrate receptors involved in virus–host interactions.
The neoglycolipid (NGL)-based oligosaccharide microarray
system is among the relatively few in the world that are
beyond proof-of-concept. The ability to readily broaden glycan libraries by incorporating both natural and chemically
synthesized oligosaccharides, and the clustered and flexible
presentation of non-covalently immobilized lipid-linked
probes with an element of mobility, are key advantages that
render the NGL system powerful in providing information on
the molecular basis of the virus-host interactions. The NGL
system has the versatility not only in that it lends itself well to
analyses of the carbohydrate-mediated interactions of whole
viruses, virus-like particles and recombinantly-expressed viral
attachment proteins, but also in that the NGLs can be incorporated into live cells for infectivity assays to corroborate the
biological significance of in vitro binding data. There have
been contributions to a number of receptor-binding studies of
human pathogenic viruses, such as the pandemic 2009 H1N1
and H5N1 ‘avian’ influenza viruses isolated from infected
humans, and the human polyomavirus JCV which, in immunocompromised individuals, causes a fatal demyelinating disease, progressive multifocal leukoencephalopathy.
This communication will be focused on recent applications
of the NGL-based microarray system in pinpointing the
311
oligosaccharide receptors for the capsid proteins of viruses
that belong to the reoviridae and the polyoma families. In
contrast to the haemagglutinin of influenza virus that can
bind to a broad spectrum of sialyl-glycans, these viral proteins exhibit narrow specificities for sialylated oligosaccharide sequences, among around 120 in the microarrays. I
will highlight the discovery of the ganglioside GM2
glycan as a receptor for the attachment protein Sigma
1 of reovirus strain type 1 Lang (T1L). New findings
with B-lymphotropic polyomavirus (LPV) and several
recently discovered polyomaviruses will also be described. These findings using microarrays, together with
protein structural data, are providing information crucial
to our understanding the viral tropisms, and also opportunities for the design of anti-viral agents.
053: Interaction of lectins from pathogens with bacterial
surfaces visualised by fluorescence microscopy
Lenka Malinovská 1,2 , Lenka Adamová 2 , Michaela
Wimmerová1,2,3; 1CEITEC – Central-European Institute
of Technology, Masaryk University, Kamenice 5, 625 00,
Brno, Czech Republic, 2National Centre for Biomolecular
Research, Fac of Science, Masaryk University, Kamenice 5,
625 00, Brno, Czech Republic, 3Department of Biochemistry,
Fac of Science, Masaryk University, Kamenice 5, 625 00,
Brno, Czech Republic
michaw@chemi.muni.cz
Lectins are of non-immune origin occurring in all organisms
and executing various functions. Microbial carbohydratebinding proteins, lectins, can recognize saccharides on host
cells (glycoproteins or glycolipides) and mediate adhesion
to the host cells or mucosal surfaces.
Burkholderia cenocepacia and Pseudomonas aeruginosa are
opportunistic human pathogens that infect immunocompromised people and are dangerous for example for cystic fibrosis
(CF) patients. Both B. cenocepacia and P. aeruginosa produce
several soluble lectins located in cytoplasm but significant
part of lectins is exported and attached to bacterial surface
by an unknown mechanisms. Lectins PA-IL and PA-IIL from
P. aeruginosa are proved virulence factors. Soluble lectins
from B. cenocepacia are homologues of PA-IIL and are also
considered as virulence factors. Lectins differ in fine carbohydrate specificity but all of them could bind heptose and
heptose derivatives. Thus, lipopolysaccharides (LPS) from B.
cenocepacia could be suitable targets for lectins and anchor
them directly on the bacterial surface.
We labelled lectins with fluorescent labels to follow
their ability to bind to surfaces of B. cenocepacia
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(single cells and biofilm) using fluorescence microscopy. After incubation with lectins-FITC conjugates, both
cells and biofilm provided visible fluorescence signal
however differently for individual lectins. Binding to
LPS could therefore serve as direct attachment of
lectins on microbial surfaces which is necessary for
adhesion functions.
This work was supported by the European Community’s
Seventh Framework Programme under the European Regional Development Fund (CZ.1.05/1.1.00/02.0068) and
Capacities specific programme (286154).
054: Ficolin-2 inhibits HCV infection, while ApoE3 mediates viral immune escape
Yinglan Zhao1, Yushan Ren1, Xuping Zhang1, Ping
Zhao2, Xiao-Lian Zhang1*; 1 State Key Laboratory of
Virology, Department of Immunology, Hubei Province
Key Laboratory of Allergy and Immunology, Wuhan University School of Medicine, Wuhan 430071, China, 2 Department of Microbiology, Second Military Medical
University, Shanghai 200433 China
zhangxiaolian@whu.edu.cn
Ficolin-2 is a human serum complement lectin with a
similar structure to Mannan-binding lectin (MBL) and
has been implicated in anti-bacterial innate immunity.
However, the role of ficolin-2 in viral infection remains
elusive. In this study, we found that ficolin-2 inhibited
and neutralized HCV cell culture (HCVcc) infection to
Huh7.5.1 cells, blocking HCVcc attachment to the receptors of low-density lipoprotein receptor (LDLR) and
the scavenger receptor (SR-B1), but weakly interfered
with attachment to CD81, and did not interfere with
attachment to the claudin-1 and occludin receptors. Cterminal fibrinogen domain (113-313aa) of ficolin-2 was
the critical binding region of HCV E1E2 N-glycans and
played a critical role in anti-HCV activity. In addition,
we found ApoE3 could block the interaction between
ficolin-2 and HCV through competitively binding to
HCV-E1E2 heterodimers but not by directly interacting
with ficolin-2. Extrogenous ApoE3 blocked ficolin-2’s
effect, while knockdown of endogenous ApoE3 decreased HCV infection and increased L-ficolinmediated anti-viral activity. This is the first report demonstrating the beneficial therapeutic effects of ficolin-2
against HCV infection. Our findings provide new insights into the important role of ApoE3 in blocking
the anti-viral activities of the lectin ficolin-2 and the
immune escape mechanism mediated by ApoE3 during
HCV chronic infection.
Glycoconj J (2013) 30:281–461
055: Keynote Lecture: Role of Glycolipids in Innate
Immune Response
Kazuhisa Iwabuchi1,2; 1Infection Control Nursing, Graduate School of Health Care and Nursing, Juntendo University,
Urayasu 279-0023, Japan, 2 Institute for Environmental and
Gender-Specific Medicine, Graduate School of Medicine,
Juntendo University, Urayasu 279-0021, Japan.
iwabuchi@juntendo.ac.jp
The innate immune system is the first line of defense against
pathogenic microorganisms. Professional phagocytes, such as
neutrophils and macrophages, are essential for this system. It
used to be thought that the innate immune system comprised
mechanisms to defend the host from infection by microorganisms in a non-specific manner. However, recent studies indicate this to be untrue. The mechanism of the innate immune
system involves interactions of specific molecules. Host cells
express several kinds of glycolipids on their surfaces. Because
of their physicochemical characteristics, glycolipids tend to
form clusters with cholesterol as membrane microdomains on
plasma membranes. Membrane microdomains have been
shown to be involved in several biological functions. Despite
the role of the microdomains in activation of these biological
functions, host membrane microdomains, especially glycolipids, are targeted by intracellular pathogens at different
points in the infective process as gateways for entry into the
cell in order to create environments in which to replicate
avoiding host immune responses. In this light, glycolipids
are the main players among the microdomain components in
host-microorganism interactions.
Here I would like to introduce the role of glycolipids in innate
immuno responses of neutrophils. Microorganisms are
engulfed by neutrophils via PRRs under non-opsonized conditions. Engulfed microorganisms are internalized within intracellular vacuolar compartments called phagosomes;
thereafter, these phagosomes sequentially fuse with lysosomes, which contain proteolytic enzymes. Pathogenic
mycobacteria, such as Mycobacterium tuberculosis, have
been shown to avoid fusion with lysosomes, resulting in their
escape from killing by phagocytes. A neutral glycolipid,
lactosylceramide (LacCer) has been shown to bind specifically to several types of pathogenic microorganisms, including
Escherichia coli, Bacillus dysenteriae, and Candida albicans.
LacCer is highly expressed on human phagocytes, and forms
membrane microdomains coupled with Src family kinase
Lyn on plasma membranes of those cells as pattern recognition receptors (PRRs). LacCer has been demonstrated to
mediate neutropihl chemotaxis, phagocytosis and superoxide generation. Recently, we found that neutrophils phagocytosed mycobacteria via the carbohydrate-carbohydrate
interactions between LacCer-enriched microdomains and
Glycoconj J (2013) 30:281–461
lipoarabinomannan (LAM) expressing on mycobacteria
under non-opsonized conditions. Importantly, pathogenic
mycobacteria utilized LacCer-enriched microdomains to
arrest phagolysosome formation.
056: Reveal the molecular basis of N-glycan core
chitobiose modifications
Shi Yan1, Sonia Serna2, Niels-Christian Reichardt2,
Katharina Paschinger1 and Iain B. H. Wilson1; 1Department für Chemie, Universität für Bodenkultur, A-1190
Wien, Austria; 2 Biofunctional Nanomaterials Unit,
CICbiomaGUNE, 20009 San Sebastian, Spain
shi.yan@boku.ac.at
Fucosylation is one of the well-recognized N-glycan
modifications in eukaryotes. Whereas in vertebrates,
α1,6-linked fucose is the sole core modification of Nglycan, studies have shown that the “lower organisms”
tend to produce even complicated core structures with
multiple fucose and galactosylated fucose residues. The
core chitobiose of sheep parasite Haemonchus contortus
displays up to three fucoses, including an usual α1,3
fucose moiety linked to the distal GlcNAc; the same
modification is present in the free-living model organism
Caenorhabditis elegans as well as the pig parasites Ascaris suum and Oesophagostomum dentatum. To date, C.
elegans core α1,3 fucosyltransferase-1 (FUT-1) and core
α1,6 fucosyltransferase-8 (FUT-8) are proven to strictly
modify the proximal GlcNAc, and no FUT has been
found responsible for the distal GlcNAc fucosylation.
Since the core α1,3 fucose is immunogenic to mammals,
α1,3 fucosylation on the distal GlcNAc might enhance
the immunogenicity of any parasite glycoproteins. Therefore, it is of interest to identify the “third” core FUT. We
employed MALDI-TOF MS and a glycoarray platform
with 22 immobilised glycan structures to investigate the
enzymatic activity of recombinant FUTs from C. elegans.
A homologue of core α1,3 fucosyltransferase was identified, which is capable to fucosylate the distal GlcNAc of
the trimannosyl N-glycan core. In combination with other
FUTs and hexosaminidase, sequential modifications on a
chemically synthesised N-glycan yielded a tri-fucosylated
structure; NMR data showed this structure is modified
with α1,3 fucose and α1,6 fucose on proximal GlcNAc,
and α1,3 fucose on the distal GlcNAc. We also proved
that certain biosynthesis pathways have to be followed to
achieve a tri-fucosylated N-glycan core.
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057: A Trick of Galectins Interaction with Influenza Virus
Evgeny S. Chernyy1, Hans-J. Gabius2, Nicolai V. Bovin1;
Shemyakin Instutute of Bioorganic Chemistry RAS, Moscow,
117997, Russia; 2Faculty of Veterinary Medicine, LudwigMaximilians-University, Munich, D-80539, Germany
bovin@carb.ibch.ru
1
Influenza virus is known to dock onto sialoglycans at the
surface of host cells; recent data suggest the involvement of
additional targets in starting viral infection. Of note, the viral
neuraminidase generates an enormously high density of terminal galactose residues on the surface of virions. Thus, an
interaction of the influenza virus with mammalian gelatins
(gals) is a possibility. Six virus strains, three cell lines (as host
cells), and panels of human and chicken gals were studied. Gals
indeed promote virus binding in a dose-dependent manner, but
they do not influence the internalization stage. Gals are able to
restore the ability of influenza virus to infect desalinated cells
up to the level of completely sialylated surfaces. The extent of
adhesion increase depends both on glycan structure and gal
nature. The most intriguing yet is the strong inhibition by gals
on natural anti-influenza virus antibodies: functional activity of
viral haemagglutinin and neuraminidase was not blocked by
loaded gals, whereas antigenic determinants were masked.
058: The galactic CvGal1 from the eastern oyster
(Crassostrea virginica) binds to blood group A oligosaccharides on the hemocyte surface
Chiguang Feng‡, Anita Ghosh§, Mario A. Bianchet§,
Barbara Giomarelli ‡ , Surekha Sridhar ‡ , Aditi
Banerjee‡, Mohammed Amin¶, Lai-Xi Wang¶, Iain B.
H. Wilson†, and Gerardo R. Vasta‡; ‡Department of Microbiology and Immunology, University of Maryland
School of Medicine, and Institute of Marine and Environmental Technology, Columbus Center, 701 East Pratt Street,
Baltimore, MD 21202, USA, § Department of Neurology,
The Johns Hopkins University School of Medicine, 725
North Wolfe Street, Baltimore, MD 21205, USA, ¶ Institute
of Human Virology and Department of Biochemistry &
Molecular Biology, University of Maryland School of
Medicine, 725 West Lombard Street, Baltimore, MD
21201, USA, †Department für Chemie, Universität für
Bodenkultur, A-1190 Wien, Austria
GVasta@som.umaryland.edu
The protozoan parasite Perkinsus marinus causes “Dermo”
disease in the eastern oyster (Crassostrea virginica), and is
responsible for catastrophic damage to shellfisheries and the
estuarine environment in North America. The parasite’s
mechanism(s) for entry into the host’s blood cells
314
(hemocytes) have been largely unknown. We identified and
characterized in oyster hemocytes a galactic (CvGal1) of
unique carbohydrate-recognition domain organization that,
unlike most mammalian gelatins, recognizes both endogenous
and exogenous carbohydrate ligands. CvGal1 displays four
CRDs in which conserve seven of the nine amino acid residues
that bind ligand in bovine galectin-1. The carbohydrate specificity of CvGal1, previously analyzed by hemagglutinationinhibition studies, was further characterized by a glycan array
(Consortium for Functional Glycomics) and confirmed that
CvGal1 binds preferentially to glycans containing nonreducing terminal N-acetylgalactosamine, fucosylated (α1-2)
galactose, and particularly blood group A oligosaccharides.
Antibodies specific for CvGal1 showed that attached and
spread hemocytes released soluble CvGal1, some of which
specifically binds to the hemocyte surface. Glycomic analysis
of the hemocyte surface revealed the presence of blood group
A oligosaccharides. CvGal1 binds to a variety of microorganisms and phytoplankton components, but preferentially to
Perkinsus spp trophozoites. Pre-treatment of oyster hemocytes
with anti-CvGal antibodies reduced trophozoite uptake, revealing that CvGal1 is a phagocytic receptor for P. marinus. We
propose that P. marinus has evolved to adapt its glycocalyx to
be selectively recognized by CvGal1 over algal food or bacterial pathogens, thereby subverting the oyster’s innate
immune/feeding recognition mechanism to gain entry into
the host cells [Supported by grants IOS 0822257 and IOS
1063729 from the National Science Foundation to G.R.V.].
059: Natural anti-glycan antibodies of mammals and
birds
Nadezhda V. Shilova1, Nailya R. Khasbiullina1 Nicolai V.
Bovin1; 1M.M. Shemyakin and Y.A. Ovchinnikov Institute
of Bioorganic Chemistry, Russian Academy of Sciences,
Moscow, Russia.
shilova_nv@carb.ibch.ru
Natural anti-glycan antibodies (nAbs) are important part of
innate immune system, but at the same time they are least of
all studied. Their role and origin are still poor understood. Bloodgroup related antigens, tumor-associated and xeno-antigens are
well-known counter-part of human nAbs. Anti-glycan nAbs
pattern of healthy donors have been investigated with help of
PGA. It was shown that about half of typical mammalian glycans
bound human serum IgG and IgM, most of the specificities are
not described yet. Discrete PGA data about anti-glycan nAbs of
other mammals as well as birds were summarized (most of them
are available from http://www.functionalglycomics.org), and
detailed analysis was performed. Baseline and control
data from mice, rats, primates and hens were taken into
account and compared with humans.
Glycoconj J (2013) 30:281–461
Primates’ (baboons) pattern of nAbs is mostly cognate to
human: number of detected specificities and scope of repertoire were close to humans. Anti-glycan nAbs repertoire of
mice, rats and hens was dramatically shrunk, top-binding
glycans are different. Common epitopes recognized by nAbs
such as chitooligosaccharides, L-rhamnose, L-fucose, and
nAbs to core structures can be mentioned. It’s argued for
allied origin of nAbs. Note that all examined species have
own features in nAbs repertoires which reflect their individual immunological “history” as humans have. The information about the natural anti-glycan antibodies repertoire is
necessary because laboratory mammals and birds are widely
used as immunological models.
Wed-Glyco(bio)technology
060: Keynote Lecture: Synthetic and biological studies of
GPIs and GPI-anchored proteins
Zhongwu Guo1, 2; 1National Glycoengineering Research
Center, Shandong University, Ji’nan 250100, China;
2
Department of Chemistry, Wayne State University, Detroit,
Michigan 48202, USA
zwguo@chem.wayne.edu, zwguo@sdu.edu.cn
Many surface proteins and glycoproteins are anchored to the
cell membrane through glycosylphospha-tidylinositols
(GPIs), a group of complex glycolipids sharing a conserved
core structure: H2N-CH2CH2-OPO3-6-α-Man-(1→2)-αMan-(1→6)-α-Man-(1→4)-α-GlcNH2(1→6)-inositol-1OPO3-glycerolipid. All GPI-anchored proteins and glycoproteins have their polypeptide C-termini linked to the conserved phosphoethanolamine moiety. GPI-anchored proteins
and glycoproteins play a critical role in various biological and
pathological processes. To study these events, it is essential to
have access to GPIs and GPI-anchored proteins and glycoproteins in homogeneous and structurally well-defined forms,
which is a significant challenge. Our research aims at developing methodologies for the synthesis of natural and functionalized GPI anchors and GPI-peptide, glycopeptide, protein
and glycoprotein conjugates, as well as studying GPI biology
using synthetic GPIs and GPI conjugates.
We have developed a highly convergent strategy for GPI anchor synthesis utilizing a phospholipidated pseudodisaccharide
as the common key intermediate, and applied it to the synthesis
of many GPIs and GPI analogs. With p-methoxybenzyl group
as a permanent protection of hydroxyl groups, the strategy was
also applied to preparing GPIs with unsaturated lipids and other
functionalities that enabled their further modifications and visualization. We have also developed chemical and enzymatic
methods for the synthesis of structurally defined GPI-linked
Glycoconj J (2013) 30:281–461
peptides, glycopeptides, and proteins. For the chemical synthesis, extensively protected GPIs with free conserved
phosphoethanolamine moiety and peptides or glycopeptides
having free C-termini were regiospecifically coupled by a
conventional condensation reaction. For the enzymatic synthesis, free GPI anchors and peptides, glycopeptides and proteins were effectively ligated by sortase A, a transpeptidase
used by bacteria to anchor surface proteins to the cell wall. We
have demonstrated that sortase A could accept GPIs having
one or multiple glycine residues attached to the conserved
phosphoethanolamine moiety as substrates and couple them
with peptides, glycopeptides or proteins. Sortase A could also
cyclize peptides and glycopeptides to form macrocyclic peptides and glycopeptides or attach proteins to liposomes. The
synthetic GPIs, functionalized GPIs, and various GPI conjugates have been used to study how bacterial toxins interact
with GPIs and how GPIs distribute in membranes, and to
study the structure-activity relationships of GPIs and cell
surface GPIomics as well.
061: Engineering of Cyclodextrin Glucanotransferase as
an O-Glycoligase
Chao Li1, Hee-Jeong Ahn1, Jin-Hyo Kim2, Young-Wan
Kim1; 1Department of Food and Biotechnology, Korea University, Sejong, 339-700, Korea; 2Chemical Safety Division,
National Academy of Agricultural Sciences, Rural Development Administration, Suwon, 441-701, Korea
ywankim@korea.ac.kr
O-Glycoligase approach has been reported as a new category
for the enzymatic synthesis of O-glycosidic linkages using a
retaining glycoside hydrolase mutant whose hydrolysis activity
is markedly reduced by the mutation of its general acid/base
catalytic residue but its transglycosylation activity still
maintained. In this study, a cyclodextrin glucanotransferase
mutant with an alanine residue at its general acid/base catalyst
position (CGT-E284A) was constructed, and thus no obvious
hydrolytic function but significant transfer activity was
detected using α-maltosyl fluoride (αG2F) and 4-nitrophenyl
β-D-maltoside (4-NPβG2) as the substrates. In order to improve the transglycosylation activity, an E284X library was
created by site-directed saturation mutagenesis at the position
of its acid/base catalyst. Through thin-layer chromatography
analysis of the reaction mixtures, two mutants were screened;
One is a mutant with a glycine residue at Glu284 (CGT-E284G)
showing better activity than the original alanine mutant, and the
other is a mutant with a serine residue (CGT-E284S) with
severe hydrolytic activity. Kinetic analysis revealed that the
transglycosylation activities of CGT-E284A and CGT-E284G
were inhibited at high substrate concentrations of acceptor
sugars above 0.8 mM. By contrast, the typical saturation
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kinetics mode was observed upon varying the donor (αG2F)
concentration at a fixed acceptor concentration (0.8 mM). The
catalytic efficiencies (kcat/KM) of CGT-E284G were improved
by 3 and 5 folds compared to those of CGT-E284A using 4NPβG2 and 4-nitrophenyl β-D-glucopyranoside as the sugar
acceptors, respectively. Transglycosylation yield of the reaction using αG2F and 4-NPβG2 at 1 mM each was about
80 %, and NMR analysis confirmed that the transfer
product was 4-nitrophenyl β-D-maltotetraoside.
062: Biotransformation of Ginseng Pectin and Its
Application in Structure-activity Relationship Study
Yifa Zhou, Di Wu, Lin Sun; School of Life Sciences,
Northeast Normal University, Changchun 130024, PR
China
zhouyf383@nenu.edu.cn
Pectins are the active components of Ginseng, including HG,
RG-I, RG-II and AG types. Pectins have very complex structures and diverse activities. It is difficult to study the structures
and the activities of their structure domains. In the present
work, we tried to transform pectin using micros and enzymes
to produce some different domains of pectin molecules, in order
to further study their structures and activities relationship.
Ginseng pectin was purified and hydrolyzed by endopolygalacturonase to produce several HG, RG-I and AG
fragments which have been purified and analyzed. The results showed that HG fragments were highly esterified and
RG-I domains were good inhibitors of galactin-3. Ginseng
pectin was first de-esterified by alkali, and then hydrolyzed
by endo-polygalacturonase. The hydrolysates were fractionated by a combination of ion-exchange and size exclusion
chromatography, and four de-esterified RG-I domains were
obtained. They were all composed of GalA, Rha, Ara and
Gal as the main components, and the ratios of Rha/GalA
were between 0.28 and 0.94. The molecular weights of these
fractions were between 7.7×103−1.2×105 Da. IR and NMR
spectra indicated that they were nearly no methyl-ester and
acetyl groups in these fractions. NMR results indicated that
the four fractions were RG-I type pectins, and two of them
contained arabinogalactan II side chains while the others
contained arabinogalactan I side chains.
The numbers of commercial available pectinases are limited.
Therefore, producing pectinase fungi were used to transform
pectin. Ginseng pectin was transformed by Aspergillus Niger
and Penicillium oxalicum which could ferment pectinases, to
produce a series of pectic fragments with molecular weight
from 1 kDa to 10 kDa, containing Rha, GalA, Gal, Ara and
Glc. Comparatively, sunflower pectin was also biotransformed
316
to produce different fragments of pectin molecules. The combining results indicated that ginseng pectins were degraded by
different fungi to produce different pectin molecule domains.
And different pectins were digested by the same fungus to
produce different fragments of pectin. Thus, using biotransformation may constitute a new way for studying structureactivity relationship of pectin.
This work was supported by the Chinese New Drug Creation
and Manufacturing Program(2012ZX09502001-001), the National Natural Science Foundation of China(No: 31170770).
063: Characterization of The Anti-HIV-1 Mechanism of
a Pseudoproteoglycan Produced by Conjugating
Unsulfated Dextran with Poly-L-lysine
Haruko Ogawa1,Fumie Kano1, Takahiro Otsuki2, Hiroo
Hoshino2, Kosuke Nakamura1, Haruyo Mori3, Hiromi
Sakagami1; 1Ochanomizu University, Tokyo, 112-8610,
Japan, 2Gunma University, Gunma 371-8511, Japan, 3Osaka
Prefectural Institute of Public Health, Osaka 537-0025, Japan.
ogawa.haruko@ocha.ac.jp
We reported that pseudoproteoglycan (pseudoPG), DexPLL, which had been synthesized by conjugating unsulfated
dextran (Dex) with poly-L-lysine (PLL). Dex-PLL was
found to have remarkable anti-HIV-1 activity against both
the macrophage-tropic R5 virus Ba-L and T-cell line tropic
X4 virus IIIB strains, although neither PLL nor dextran has
such activity1). Conjugation of PLL with dextran was found
to produce an antiviral effect showing the chain-lengths
dependency in three kinds of assay systems including a
human CD4+ T-cell line. The pseudoPG synthesized using
10 kDa PLL and 10 kDa dextran showed EC50 4–40 times
lower than that of sulfated dextran or heparin against Ba-L
and EC50 equal to that against IIIB virus, indicating that
Dex-PLL was more effective against R5 virus than sulfated
polysaccharides. Dex-PLL suppressed a clinically isolated
R5 virus from primary peripheral blood mononuclear cells.
Dex-PLL significantly inhibited virus adsorption to cells
and subsequent virus entry into the cells. On the smear
preparations of uninfected or IIIB-infected MOLT-4 Tlymphocytes, Dex-PLL inhibited the reactivities of the IgGs
in AIDS patient’s serum to the IIIB viruses budding on
infected cells but not to uninfected cells, indicating that
Dex-PLL bound to the IIIB viruses. Flow cytometry using
budding MOLT-4-IIIB cells supported that Dex-PLL bound
to the virus at a site different from that of heparin.
Confocal laser-scanning microscopy demonstrated that
Alexa488-labelled Dex-PLL bound to the surface of human
Glycoconj J (2013) 30:281–461
glioma NP-2 cells at 4 °C and 37 °C, and entered into a
cytoplasmic matrix to localize near nuclear region at 37 °C, while
N-acetylation of Dex-PLL abolished the endocytosis and the
HIV-1-suppressing activity. On membrane analysis using separated HIV-1 proteins, Dex-PLL was found to bind to viral core
proteins but not to envelope glycoproteins including GP160.
These results suggest that Dex-PLL may have unique multiple
preventive mechanisms against HIV-1 at not only adsorption and
entry to the cells, but at the intracellular steps after denucleation
of the virus. Elucidating the action mechanisms of Dex-PLL will
provide new insights into the molecular processes of the viral
infection to develop a novel anti-HIV-1 treatment.
1)
Nakamura K., et al., Antiviral Res.,94 (2012)89–97.
064: A novel approach to studying the protein–carbohydrate interactions employing cancer cells grown on a
QCM biosensor surface
Xueming Li1, Yuxin Pei1, Teodor Aastrup2, Zhichao
Pei1,2,*; 1First author’s affiliation, City, Zip code, Country;
2
Second author’s affiliation, City, Zip code, Country;
1
College of Science, Northwest A&F University, Yangling,
Shaanxi, 712100, China, 2Attana, SE-11419, Stockholm,
Sweden
peizc@nwsuaf.edu.cn
A novel approach to studying the protein–carbohydrate interactions on the surface of cancer cells using a QCM (quartz crystal
microbalance) biosensor was developed, where a PANC-1 cell
line and a SKOV-3 cell line were grown onto polystyrenecoated quartz crystals. To evaluate lectin–cancer cell interactions, the kinetics of binding of a diverse range of lectins were
evaluated. The binding and dissociation between the lectins and
the cells as well as the inhibition of the binding by monosaccharides were monitored in real time; the kinetic parameters of
the interaction of ConA with SKOV-3 cells were studied. The
real-time lectin screening of a range of lectins enabled the
accurate study of the glycosylation changes between cells, such
as the changes associated with cancer progression and development. This methodology provides a novel tool to study cell
surface glycosylation which binding events take place at the cell
surface, more closely mimicking an in vivo system.
References
1. Sharon N., Lis H. 2004, Glycobiology. 14 (11), 53R-62R.
2. Elovsson K, Pei Z, AastrupT. Am. Lab. 2011, 6: 13.
3. Dahlbäck M, Jørgensen LM, Nielsen MA, Clausen TM,
Ditlev SB, Resende M, Pinto VV, Arnot DE, Theander TG,
Salanti A. J. Biol. Chem. 2011, 286:18, 15908–15917
4. Pei Z, Saint-Guirons J, Käck C, Ingemarsson B, Aastrup
T. Biosens. Bioelectron. 2012, 35, 200–205
Glycoconj J (2013) 30:281–461
317
065: Structure-Function Studies on Glycosyltransferases:
Design of Novel Glycosyltransferases for the Site-Specific
Conjugation of Biomolecules
066: High-throughput screening for discovering glycosidase from metagenomic libraries using droplet-based
microfluidics
Pradman Qasba, Boopathy Ramakrishnan and Natalia
Mercer; National Cancer Institute, National Institutes of
Health, United States
qasbapr@helix.nih.gov
Xiaohui Wang1, 2, Runtao Zhong1, 3, Bingcheng Lin1, 3,
Yuguang Du*1, 3; 1Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian, 116023, China;
2
University of Chinese Academy of Sciences, Beijing,
100049, China; 3Research Center of Lab on a Chip, Dalian,
China
duyg@dicp.ac.cn
Outer surface of a cell is decorated with information-rich
complex oligosaccharides (glycans) of glycoproteins, glycolipids, and proteoglycans which participate in several
biological processes. Cell’s glycophenotype changes with
the cellular growth, development and differentiation as well
as in the pathological state such as inflammation and cancer.
In the growing glycomics field, there is a need for the development of new strategies for detecting and identifying the
glycan moieties associated with the cellular status. The glycan
chains are synthesized by a large family of enzymes called
Glycosyltransferases (GT) that transfer a sugar moiety from a
sugar nucleotide donor to an acceptor molecule. The structural
information on GTs from our laboratory has shown that there
are flexible loops that undergo marked conformational changes
upon binding of metal ion and sugar-nucleotides and simultaneously create at the C-terminal region of the flexible loop an
oligosaccharide acceptor binding site that did not exist before.
The loop acts as a lid covering the bound donor substrate.
Furthermore, there are few residues in the catalytic pocket of
GTs that determine their sugar donor specificity. Mutation of
these residues broadens their donor sugar specificities, including towards the sugars carrying a chemical handle such as keto
or azido group at C-2. Furthermore, the N-acetyl group of the
donor sugars, e. g GalNAc and GlcNAc, binds in a cavity of
their respective glycosyltransferases that can also accommodate a sugar analogue with a chemical handle. The transfer of a
modified sugar residue that has a chemical handle by the
mutant or wild type GTs to a specific sugar residue on a
glycoconjugate has made it possible to develop a highly
sensitive chemoenzymatic method for detecting and isolating glycan biomarkers. The mutant and wild type
glycosyltransferases, currently being produced in our laboratory, are turning out as powerful tools for investigating
the cell’s glycophenotype with high specificity.
Glycoside hydrolases (also called glycosidases) catalyze the
hydrolysis of the glycosidic linkage to release smaller
sugars. They are extremely common enzymes with roles in
nature including degradation of biomass, in anti-bacterial
defense strategies, in pathogenesis mechanisms and in normal cellular function. Together with glycosyltransferases,
glycosidases form the major catalytic machinery for the
synthesis and breakage of glycosidic bonds. Metagenomics
has emerged as an alternative approach to conventional
microbial screening that allows exhaustive screening of
microbial genomes in their natural environments. It has been
proven to be a powerful tool to isolate new enzymes and
drugs of industrial applications. However, searching for
desired traits from metagenomes is often problematic because of the lack of ultrahigh-throughput and cost-effective
screening approaches.
This work aims to establish a general ultrahighthroughput screening platform using droplet-based
microfluidics that overcomes the limitations mentioned
above. To demonstrate its performance, we have been
identifying new types of chitinases exhibiting more
efficient and specific enzymatic activity of breaking
down glycosidic bonds in chitin. Transformed E.coli
cells in a metagenomic library obtained from deep sea
were encapsulated individually into aqueous picolitervolume droplets, dispersed in inert carrier oil and then
cultured off chip. After expression of the encoded
enzymes, fluorogenic substrates were added into each
droplet and incubated with the contents in the droplet.
Finally, the droplets were sorted based on their own
fluorescent intensities, which represent the activities of
the expressed enzymes, and the sequences of the functional genes can be recovered from the E.coli cells
contained in collected droplets. The droplet-screening
frequency is in the range of hundreds to thousands of
droplets per second. Compared to screening using microtiter plate-based systems, the volume and cost of the
reagents are reduced by almost 104~105-fold, allowing
the screening of 106~107 genes using only microliterscale reagents.
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067: Keynote Lecture: N-GlcNAc and O-GlcNAc Modification of Plant Proteins: Origin and Function
Neil E. Olszewski1; 1University of Minnesota, St. Paul,
MN, 55108, USA
neil@umn.edu
Plant proteins can be post-translationally modified with
monosaccharides consisting of N- or O-linked β-D-Nacetylglucosamine (GlcNAc). In Arabidopsis thaliana, two
enzymes, SPINDLY (SPY) and SECRET AGENT (SEC)
modify serine and threonine residues of nuclear and cytosolic proteins with O-linked GlcNAc. spy mutants have
defects in hormone signaling, light signaling, response to
stress and the circadian clock, indicating a role for OGlcNAc modifications in many plant processes. While sec
mutants have only subtle phenotypes, sec spy double mutant
embryos die before completing seed development suggesting
that SEC and SPY have overlapping functions and indicating
that O-GlcNAc modification is required for seed development. SEC modifies the capsid protein of Plum pox virus
(PPV) and sec mutants are weakly resistant to PPV suggesting
that PPV co-opts the host O-GlcNAc modification machinery
to enhance its infectivity. Recently, a number of arabidopsis
proteins have been found to be N-GlcNAc modified at the Asn
of the Asn-X-Ser/Thr sequon. These proteins transit through
the secretory system suggesting the modification may be
produced when N-linked glycans are removed. Consistent
with this hypothesis but also suggesting that N-GlcNAc modifications arise by multiple mechanisms, N-GlcNAc modification of some, but not all, proteins is blocked in endo-β-Nacetylglucosaminidase mutants. Recent results on regulation
of plant processes by N- and O-GlcNAc modification will be
presented.
068: Marine oligosaccharides production and application
in plant protection
Heng Yin, Xiaoming Zhao, Yuguang Du; Dalian Institute
of Chemical Physics, Chinese Academy of Sciences, Dalian,
116023, China
duyg@dicp.ac.cn
There are lots of polysaccharides resources in the sea. Many
papers reported that these polysaccharides have several functions. But some of these polysaccharides are unsoluble in water
and inconvenient for application. So we identified some glycosidases to hydrolysis the polysaccharides (chitin, chitosan,
alginate, carrageenan and etc.,) to soluble oligosaccharides.
Furthermore, we developed a rapid and novel method to
separate and purify the different DP (degree of polymerzation)
Glycoconj J (2013) 30:281–461
oligosaccharides by using hydrophilic interaction liquid chromatography. Now, more than 1,000 oligosaccharides mixtures
and more than 200 specific oligosaccharides with clear structure were identified in our lab.
As functional materials with excellent absorption and high
bio-activity, marine oligosaccharides are widely used in
pharmaceutical, food materials, cosmetic and agriculture.
As plant elicitors or called plant disease vaccines, marine
oligosaccharides have the ability to induce plant immunity
system to resist plant diseases and stresses. Based on our
experiments and other literatures, we conclude that there are
five steps in the process of marine oligosaccharides activated plant defense: signal reception by receptor, secondary
signal messengers activation, plant hormones accumulation,
responded genes regulation and proteins expression,
defense-related secondary metabolites production.
Some oligosaccharides based bio-pesticides and plant
growth regulators were developed by our lab. These bioreagents are widely used in some provinces of China. They
have good effect on food crops, economic crops, vegetables,
fruits and horticultural plants.
069: The mitogenic and antioxidant properties of
Andrias davidianus glycopeptides
Li Wei1, Min Qu2, Changqing Tong3, Qiao Jin1, Wenli
Wang1, Xinying Yu1, and Lukyanov Pavel4; 1College of
Food Engineering, Dalian Ocean University, Dalian,
Liaoning, 116023 China, 2College of Food Science, Shenyang
Agriculture University, Dalian, Liaoning, 116023 China, 3Alkali Soil Natural Environmental Science Center, Northeast
Forestry University, Harbin, Heilongjiang, 150040 China,
4
G.B.Elyakov Pacific Institute of Bioorganic Chemistry, Far
East Branch of the Russian Academy of Science, Vladivostok,
690022 Russia
paluk@mail.ru
The Andrias davidianus glycopeptides (ADGPs) was prepared from skin secretion of Chinese giant salamander.
MALDI-TOF mass spectrometry showed that molecular
weights of hydrolysates were below 3.5 kDa. ADGPs are
stabile at 0.1 M HCl, trypsin and pepsin hydrolysis. The
glycopeptides–FITC binding with the surface of human
epithelial cells was investigated by fluorescence microscope. Mitogenic activity of ADGPs on human epithelial
cells was studied by the MTT. ADGPs show potent mitogenic effect on human epithelial cells, with the highest cell
survival increase of 29 % and 48 % for a ADGPs concentration of 6 μg/mL and 98 μg/mL, respectively. Glycopeptides have strong antioxidant properties, and normalize NO-
Glycoconj J (2013) 30:281–461
319
synthase activity in human peripheral white blood cells
stimulated with lipopolysaccharide, inhibit hyperproduction
of reactive oxygen species (ROS) in them.
microbes. These include innate and acquired immunity to
pathogens, modulation of immune responses, pathogen virulence, and plant cell wall biodegradation.
The work was supported by FEB RAS (09-I-P24-05, Russia)
and NSFC (31071612 and 21075012, China).
The molecular features of the glucan-protein interactions
include specific glucose linkages, sequence and chain
length. In this communication we demonstrate a strategy
of negative-ion electrospray tandem mass spectrometry with
collision-induced dissociation for high-sensitivity determination of linkage (1,2-, 1,3-, 1,4- and 1,6-) and sequence
(linear and branched) in major types of glucooligosaccharide chains, and the combined use of a designer gluco-oligosaccharide microarray for binding analyses
of the specificities of glucan recognition.
070: Chitosan Oligosaccharides: A New Feed Additive
Qingsong Xu 1, Yuguang Du 1* ; 1 Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian
116023, PR China.
articles1805@gmail.com
There is increasing urgency in the livestock production business worldwide to develop good alternatives to the in-feed
antibiotics that have been a significant component of intensive
livestock production for many years. Although the antibiotics
are powerful agents to control enteric animal disease, the same
antibiotics are also the bottleneck restricting the development
of Chinese livestock. Chitosan oligosaccharide (COS) prepared from chitosan is a oligosaccharide with free amino
group derived through deacetylation from chitin. It has been
used as immunity regulator, antimicrobial agent, health food,
feed additive, etc. This paper summarized recent progress
made on chitosan oligosaccharide feed additive, including:
preparation of chitosan oligosaccharide; application in the
livestock; application in the poultry; application in the fisheries; mechanism of action of chitosan oligosaccharide. Based
on our former results, we deduce that the mode of chitosan
oligosaccharide act on animal health, So we conclude that
chitosan oligosaccharide is a potential feed additive to substitute the in-feed antibiotics.
071: Probing Glucan Oligosaccharide Sequences by
Negative-Ion Electrospray Tandem Mass Spectrometry
and their Recognition by Glucan-Binding Proteins using
a Designer Microarray
Angelina S. Palma1,2, Yan Liu1, Hongtao Zhang1, Yibing
Zhang1, Barbara Mulloy1, Ten Feizi1, Wengang Chai1;
1
Glycosciences Laboratory, Department of Medicine, Imperial College London, London W12 0NN, UK, 2REQUIMTE,
Departamento de Química, Universidade Nova de Lisboa,
2829-516 Caparica, Portugal
w.chai@imperial.ac.uk
Glucans are homopolymers of D-glucose and are constituents of fungal, bacterial, and plant cell walls. Their specific
recognition by glucan-binding proteins is important in mediating diverse biological processes in animals, plants and
We have established a comprehensive gluco-oligosaccharide
library, prepared from glucan polysaccharides of various
fungal, plant and bacterial origins and from chemical synthesis, and constructed a gluco-oligosaccharide microarray,
based on the neoglycolipid-technologyfor generating lipidlinked probes. The microarray comprises ~150 probes: linear sequences with 1,2-, 1,3-, 1,4- and 1,6-linkages of αand β-configurations ranging from 2 up to 16mers, with
mixed β1,3/β1,4- and α1,4/α1,6-linkages, and also
branched β1,3/β1,6-sequences.
The prototype gluco-oligosaccharide microarray was instrumental in assigning oligosaccharides recognized by the major receptor of the innate immune system for fungal
pathogens, Dectin-1, and by murine monoclonal antibodies
elicited using glucan-based vaccines. We now extend our
study by using the more diverse microarray to investigate a
panel of 16 glucan-binding proteins, including mammalian
lectins, monoclonal anti-glucan antibodies and microbial
carbohydrate-binding modules of glycoside hydrolases.
The microarray analyses reveal different patterns of glucan
recognition among these proteins.
For proteins and gluco-poly- and oligosaccharides, our
grateful acknowledgements are due to the following
collaborators: Alisdair Boraston (Victoria); Gordon
Brown (Aberdeen); Antonio Cassone & Antonella
Torosantucci (Rome); Andres Ciocchini (Buenos Aires);
Harry Ensley (Tulane); Harry Gilbert (New Castle);
Jianxin Gu (Shanghai); Yvette van Kooyk (Amsterdam);
Eckhard Loos (Regensburg); Ana Luís, Ana-Luísa Carvalho
& Carlos Fontes (Lisbon); Barry McCleary (Wicklow);
Takashi Tonozuka (Tokyo); Denong Wang (Stanford); David
Williams (Johnson City); Guangli Yu (Qingdao); and Lina
Zhang (Wuhan).
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072: Exploring sugar code via macromolecular
self-assembly
Guosong Chen; State Key Laboratory of Molecular
Engineering of Polymers and Department of Macromolecular
Science, Fudan University, 220 Handan Rd., Shanghai, China.
guosong@fudan.edu.cn
Carbohydrates are the most abundant organic species in the
world and also the third most important biological macromolecules after nucleic acids and proteins. The self-assembly of
DNA and proteins make a significant contribution to our lives
and they have been employed to make functional selfassembled materials recently. Compared to the development
of DNA and proteins, our knowledge and manipulation to the
self-assembly of carbohydrates as well as their functionality is
quite limited. The major obstacle for our understanding of
carbohydrates is their complicated chemical structure of oligosaccharides and perplexing glycoforms and microhetrogeneity
on proteins, which currently is a problematic and long-term
task. Under this circumstance, macromolecular self-assembly
might provide an alternate insight to this problem. Many of the
biological functions of carbohydrates are related to their
binding ability to proteins, especially for the sugars on the cell
surface, which are called glycocalyx. By using our macromolecular self-assembly method, vesicles with a heavy layer of
glycopolymers on their outer surface and inner surface
were obtained, which could mimic the structure and
function of glycocalyx. Besides, the molecular recognition of lectin and sugars were employed to construct
self-assembled nanoparticles as well.
073: Two-step Hydrolysis for Preparing Low- and
High-molecular Weight β-1,3-glucan Oligosaccharides
from Curdlan
Jing Li 1, Li Zhu 2, Zhi-Yong Zheng 1, Xiao-Bei Zhan 1, 2, *,
Chi-Chung Lin 1, Hong-Tao Zhang1; 1 Key Laboratory of
Carbohydrate Chemistry and Biotechnology of Ministry of
Education, School of Biotechnology, Jiangnan University,
Wuxi, Jiangsu 214122, China; 2 Jiangsu Rayguang Biotech
Co. Ltd., Wuxi, Jiangsu 214125, China
xbzhan@yahoo.com (X.-B. Zhan)
Bioactive β-1,3-glucan oligosaccharides (GOSs) are increasingly important for many biomedical applications.
The simple and reproducible processes for the preparation
of high and low molecular weight GOSs with degree of
polymerization (DP) from 2 to 10 (identified by ESI-MS
and TLC) by 1 mol/L sulfuric acid hydrolysis of curdlan
aqueous suspension at various temperature and time were
Glycoconj J (2013) 30:281–461
developed. A putative structure of the curdlan insoluble
particles in acid suspension is proposed. It is hypothesized
that the curdlan particle consists of exposed and relaxing
exterior surface susceptible to acid hydrolysis and compact
and water-insoluble core hindering hydrolysis. Consequently, two-step acid hydrolysis processes were developed for
the GOSs. Combination of hydrolysis processes at 70 °C,
6 h then 80 °C, 3 h is effective for obtaining low molecular
weight GOSs (DP 2 to 6). For high molecular weight GOSs
(DP 7 to 10), the optimal hydrolyses condition is 70 °C, 4 h
and then 80 °C, 1 h.
Thu-Carbohydrate & Disease
074: Keynote Lecture: Alterations of glycosylation in
gastric carcinogenesis
Celso A. Reis1,2,3; 1Institute of Molecular Pathology and
Immunology of the University of Porto (IPATIMUP),
Portugal, 2Instituto de Ciências Biomédicas Abel Salazar
(ICBAS-UP), Portugal; and 3Medical Faculty, University
of Porto, Portugal.
celsor@ipatimup.pt
Altered glycosylation of cell surface membrane and secreted
glycoconjugates is a common modification in cancer.
In the gastric mucosa major alterations are observed during
the process of gastric carcinogenesis. Helicobacter pylori
attachment to human gastric mucosa is mediated by bacterial
adhesins, such as BabA, that recognizes neutral histo-blood
group antigens expressed by gastric epithelial cells. Persistent
H. pylori colonization promotes gastric mucosa expression of
sialylated glycan structures. These include receptors for the
bacterial sialic acid binding adhesin (SabA). We have demonstrated in human gastric tissues and in cell models that H.
pylori infection induces the expression of glycosyltranferases,
namely B3GNT5, involved in the biosynthesis of sialylated
Lewis antigens leading to an increased H. pylori adhesion.
Furthermore, major glycosylation alterations are observed in
pre-cancerous and cancerous lesions of the gastric mucosa,
such as the aberrant expression of simple mucin-type carbohydrate antigen Sialyl-Tn due to the overexpression of
ST6GalNAc-I.
The increased expression of sialylated glycan structures in
gastric cancer is associated with more aggressive phenotypes.
Gastric cancer cells showing increased expression of ST3GalIV and sialyl-Lewis X antigen display an increased activation
of signaling pathways leading to more aggressive cancer cell
behavior. Furthermore, alterations on N-glycosylation of E-
Glycoconj J (2013) 30:281–461
cadherin have been shown to be crucial in gastric cancer
progression. Altogether these alterations constitute a
source of cancer biomarkers with clinical application.
These findings show that activation and modulation of the
glycosylation pathways in gastric cancer cells play key roles
in the process of carcinogenesis and in the biology of the
cancer cell.
075: The modification of aggrecan sulphation in articular
cartilage from KBD children
Mingxiu Luo1, Jinghong Chen1, Siyuan Li1, Clare E
Hughes2, Bruce Caterson2 & Junling Cao1*; 1Institute
of Endemic Diseases, Xi’an Jiaotong University College of
Medicine; Key Laboratory of Environment and Genes related
to Diseases (Xi’an Jiaotong University), Ministry of Education, Xi’an, 710061, Shaanxi, P.R. China; 2Connective Tissue
Biology Laboratories, Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff,
Wales, UK CF10 3AX
caojl@mail.xjtu.edu.cn
Objectives: The sulfate of the proteoglycan is a necessary
process in cartilage matrix formation. In this study, we tried
to investigate the potential role of the sulfate modification of
cartilage proteoglycan on the pathogenesis of Kaschin–Beck
disease (KBD) children.
Materials and Methods: Samples of articular cartilage
were divided into 2 groups: control children (5 samples
from 5 cases), and children with KBD (5 samples from
5 cases). The morphology and pathology of hand joint
cartilage of KBD children were examined by light microscopy and the expression of proteoglycans was determined by histochemical staining. Aggrecan, PAPSS2
(3′-phosphoadenosine-5′-phosphosulfate synthetase 2),
PAPST1 (3′-phosphoadenosine-5′-phosphosulfate transporter
1), CHST15 (Carbohydrate (N-acetylgalactosamine 4-sulfate
6-O) sulfotransferases 15), ARSB (Arylsulfatase B) and
GALNS (N-acetylgalactosamine-6-sulfate sulfatase) localization were performed by inmmunohistochemitry. Analysis of
proteoglycan degradation products was performed by immunohistochemistry using antibodies 1B5, 2B6 and 3B3(+),
which are specific for the sulphation patterns of CS (chondroitin sulfate) substituted on the aggrecan core protein. Articular
cartilage was classified in 3 zones, and positive findings were
counted by light microscopy for cytoplasmic staining by antibodies of PAPSS2, PAPST1, CHST15, ARSB and GALNS.
Results: There were lesser amounts of aggrecan in the ECM
(extracellular matrix) of the articular cartilage of KBD
321
children and the sulphation patterns of CS substituted on
the aggrecan core protein in KBD articular cartilage proteoglycans were altered. The percentages of chondrocytes
staining for PAPSS2, PAPST1 and CHST15 in the upper(18.38 % ± 3.22 %, 16.79 %±3.42 % and 13.14 %±
1.17 % respectively) and middle (12.47 %±1.47 %, 7.35 %±
1.42 % and 6.75 %±0.61 % respectively) (p<0.05) zone of
KBD children were significantly lower than in controls, while
the percentage of chondrocytes staining for the ARSB and
GALNS was significantly higher than in controls in the upper(43.79 %±11.05 %, 35.53 %±4.24 % respectively) and
middle zone (27.12 %±3.45 %, 14.45 %±1.53 % respectively)
of articular cartilage.
Conclusion: The decreased sulfation of aggrecan maybe
related to several enzymes about the sulfated modification
in KBD patients. Our results suggest that the altered sulfate
modification of cartilage proteoglycan has an important
effect on the pathogenesis of KBD. Our study may provide
strong evidence for further studying the etiology and pathogenesis of KBD.
This research was supported by the National Natural Science
Foundation of China (Nos.31070725,30872187 and
30471499).
076: Developmental process and tumor progression are
mediated by carbohydrate to carbohydrate interaction
Sen-itiroh Hakomori1,2 and Kazuko Handa2; 1Depts of
Pathobiology and Global Heath, Univ. of Washington,
Seattle, WA, USA; 2Pacific Northwest Research Institute,
Seattle, WA 98122, USA,
hakomori@u.washington.edu
Various types of cell-cell interactions and signal transductions were found to be mediated by protein to protein
interaction (PPI), or protein to carbohydrate interaction
(CPI), and these processes were well established by many
previous studies. In addition to the interactions, carbohydrate to carbohydrate interaction (CCI) has been found as a
novel molecular interaction, which is involved in cell-cell
interactions, signal transductions and cell phenotype
changes: (i) Compaction process at the “morula stage” of
embryos is mediated by Lex to Lex interaction leading to
development into “blastocyst”. In this process, Lex binding
proteins were not involved. (ii) Interaction between GM3
and Gg3 or GM3 and LacCer has been suggested to be
involved in adhesion of certain tumor cells to endothelial
cells, which is considered a crucial step in metastasis of the
tumor cells; and the metastasis was inhibited by mimetics of
Gg3 or LacCer. (iii) In contrast to CCI mediating cell-cell
322
interaction, CCI between molecules expressed within the
same cell surface membrane microdomain was termed cisCCI. The interaction of ganglioside GM3 to multi (>3)
GlcNAc termini of N-linked glycans of epidermal growth
factor receptor (EGFR) has been indicated as the molecular
mechanism for the inhibitory effect of GM3 on EGFR
activation. Also, the complex of GM3 and GM2 has been
shown to inhibit the activation of hepatocyte growth factor
receptor, cMet, through its association with tetraspanin
CD82. Studies on CCI, especially using quantitative biophysical methodologies, have been accumulating.
077: Gene Regulations during Apoptotic Induction in
Metastatic Breast Cancer and in Embryonic Brain
Cells
Subhash Basu, Manju Basu and Rui Ma; Department of
Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, Indiana 46556
sbasu@nd.edu
New apoptotic agents identified during our recent studies
can be employed as a new generation of anti-cancer drugs
after being properly delivered to the patients. In order to
study glyco-gene regulation, we used four clonal metastatic
cancer cells of colon and breast cancer tissue origin (Colo205, SKBR-3, MDA-468, and MCF-3). The glyco-genes for
synthesis of SA-Le x and SA-Le a (which contain Nacetylglucosamine, sialic acid, and fucose) in these cells
were modulated differently at different phases induced by
D-PDMP (inhibitor of glucosylceramide biosynthesis),
Betulinic Acid (a triterpinoid isolated from the bark of
certain trees and used for cancer treatment in China), Tamoxifen (a drug in use in the west for treatment of early
stages of the disease in breast cancer patients in China), and
cis-platin (an inhibitor of DNA biosynthesis used for testicular cancer patients) when used for induction of apoptosis in
the above mentioned cell lines. Within 2 to 6 h, transcriptional
modulation of a number of glyco-genes was observed by DNA
micro-array (containing over 300 glyco genes attached to the
glass cover slips) studies. Under a long incubation time (24 to
48 h) almost all of the glyco-genes were downregulated. The
cause of these glyco-gene regulations during apoptotic induction in metastatic carcinoma cells is unknown and needs future
investigations for further explanations. At least six GSLglycosyltransferase activities (GLTs) of Basu-Roseman pathway catalyzing the biosynthesis of GD1a gangliosides have
been characterized in developing chicken brains. Most of these
glyco-genes are expressed in the early stages (7 to 17 days) of
brain development and lowered in the adult stage, but the cause
of reduction of enzymatic activities of these GLTs in the adult
stages is also not known. Our recent attempt of in vitro
Glycoconj J (2013) 30:281–461
Glyco-gene regulation in metastatic cells may lead us to explain
the in vivo Glyco- gene regulation in normal or diseased animal
organs.
078: Galectin-binding fine specificities, from small
saccharides to glycoproteins—mechanisms, evolution,
functions and medical use
Michael Carlsson 1 , Ulf, Ulf Ryde 2 , Derek Logan 3 ,
Mikael Akke4, Ulf J Nilsson5, Hakon Leffler1; 1Laboratory Medicine; 2Theoretical Chemistry; 3Biochemistry and
Structural Biology; 4 Biophysical Chemistry; 5 Organic
Chemistry5, Lund University, SE 22362 Lund, Sweden
hakon.leffler@med.lu.se
Methods. Galectin-ligand interactions have been analyzed
by fluorescence anisotropy, affinity chromatography and
other binding-assays, and interactions have been mapped
using galactic mutants, galactic orthologues from different
species, and an array of different natural and synthetic
ligands. For galectin-3, the structure and dynamics of complexes with small ligands have been analyzed by NMRspectroscopy, ultra high resolution (~0.85 Å) X-raycrystallography and neutron crystallography.
Mechanism. The conserved galactic-defining amino acid motif provides a preorganized binding site for interaction with
galactosides, but an increase in conformational entropy of the
protein upon ligand binding is also a newly discovered contributor to the affinity, as large as the enthalpy contributed by
the previously well-known hydrogen bonds and van der Waals
interaction. Interactions in neighboring sites may enhance or
decrease the affinity in different ways for different gelatins,
and it is possible to alter fine specificity by mutagenesis.
Selectivity and affinity of gelatins for natural glycoproteins,
such as found in human serum, is much higher than expected
from the structure of their glycans. This suggests that the
interaction also involves the specific context of the glycan in
the protein and possible interaction with protein parts.
Functions and relationship to physiological regulation and
disease. Different aspects of galactic fine specificity appear
to be important in different contexts, for example, the tolerance of galectin-3 for 2-3 sialylated galactosides is not
required for its activation of neutrophils but it is for its
binding to serum glycoproteins. Galectin fine specificity
determines intracellular traffic of the galactic itself and of
bound glycoproteins, as shown for transferrin and
haptoglobin-hemoglobin complexes, after their uptake into
their respective target tissue cells. Increase or decrease of
specific galactic-binding glycoforms in serum correlates in
different ways with diseases.
Glycoconj J (2013) 30:281–461
Galectin inhibitors for medical use. The understanding of
galactic fine specificity has made it possible to design highly
potent (Kd in low nM range) and selective small molecule
galactic inhibitors. Modified citrus pectin and other plant
polysaccharides were found to have low or no affinity for
the canonical galactic carbohydrate-binding site of a range
of gelatins, which puts into question their proposed mechanism of action as specific galactic inhibitors.
079: Caveolin-1 up-regulates ST6Gal-I expression and
integrin α2,6-sialylation to promote integrin α5β1dependent hepatocarcinoma cell adhesion and FAK
signaling
Shujing Wang 1*,Shengjin Yu 1, Jianhui Fan 1, Linhua
Liu 1, Lijun Zhang 1, Nanyang Li 1, Jianing Zhang 1*;
1
Department of Biochemistry, Institute of Glycobiology,
Dalian Medical University, Dalian 116044, Liaoning
Province, China
*Correspondence to: Jianing Zhang; Shujing Wang.
jnzhang@dlmedu.edu.cn; wangshujing82101@sina.com.
The alternations of cell surface sialylation play an important
role in tumorigenesis and development. Although an increase in α2,6-sialylation is observed in hepatocellular carcinogenesis (HCC), the regulation mechanisms and roles
involved remain unclear. Caveolin-1 is a major structural
protein of caveolae and involved in many biological processes including protein glycosylation. Hca-F and Hepa1-6
are mouse hepatocarcinoma cell lines with high and low
malignant potential, respectively. This study showed that
caveolin-1 overexpression in Hepa1-6 cells up-regulated
sialyltransferase ST6Gal-I expression and activated FAKmediated adhesion signaling, and down-regulation of
ST6Gal-I attenuated caveolin-1-induced increase in the cell
adhesive ability to fibronectin. Conversely, caveolin-1
knockdown in Hca-F cells inhibited ST6Gal-I expression
and FAK signaling-mediated cell adhesion to fibronectin.
Re-expression of wild-type caveolin-1 or ST6Gal-I rescued
the decreased ST6Gal-I expression and adhesion of Hca-F
cells caused by caveolin-1 silencing. Further studies demonstrated caveolin-1 up-regulated cell surface α2,6-linked
sialic acid via stimulating ST6Gal-I transcription. Cell surface α2,6-sialylation was required for integrin α5β1dependent cell adhesion to fibronectin, and an increase in
α2,6-linked sialic acid on α5-subunit facilitated fibronectinmediated FAK phosphorylations. Taken together, these results indicate for the first time that caveolin-1 can upregulate ST6Gal-I expression and integrin α2,6-sialylation,
and further contribute to promoting mouse hepatocarcinoma
cell adhesion to fibronectin. This study provides new insights into the biological functions of caveolin-1 and the
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significance of sialylation modification for integrin α5β1 in
HCC metastasis. This work was supported by grants from
the Major State Basic Research Development program of
China (2012CB822103), and National Natural Science
Foundation of China (31000372, 31170774 and 31000618).
080: Roles of sialidase in cancer initiation and progression
Taeko Miyagi 1, Kohta Takahashi1, Masahiro Hosono 2,
Koji Yamamoto1, Kazuo Nitta2; Divisions of 1Cancer
Glycosylation Research and 2 Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology,
Tohoku Pharmaceutical University, Japan
tmiyagi@tohoku-pharm.ac.j
Plasma membrane-associated sialidase (NEU3), a key enzyme for ganglioside degradation, is remarkably upregulated in several human cancers, and its up-regulation
leads to increased cell invasion, motility and survival of
cancer cells (1). Up-regulation of NEU3 is also of importance for the promotion stage of colorectal carcinogenesis in
vivo, from experiments using NEU3 transgenic (2) and
Neu3-deficient mice (3), possibly via activation of EGF
signaling. These results suggest that alteration in the
sialidase expression thus may be a crucial defining factor
in malignancy. We then analyzed whether and how NEU3
contributes to cancer promotion as well as to progression.
When NEU3 was transfected into NIH3T3-cells, NEU3
exhibited significant increase in colonogenic growth,
clonogenicity on soft agar and in vivo tumor growth in the
presence of EGF, as compared with vector control. Although endogenous level of EGFR in the cells is extremely
low, NEU3 enhances markedly its phosphorylation, and
probably mostly determines EGFR activation. In the cells,
NEU3 also stimulated ERK and Akt, whereas the NEU3mediated activation was largely abrogated by PP2 (a src
inhibitor) or AG1478 (an EGFR inhibitor), suggesting that
NEU3 is indeed involved in tumorigenesis through EGFR/src
signaling pathway. Furthermore, NEU3-silencing in colon
cancer cells resulted in significant decrease in clonogenic
growth, chemosensitivity to oxaliplatin, clonogenicity on soft
agar and in vivo tumor growth, along with down-regulation of
stemness genes and Wnt–related genes. Further analyses revealed that NEU3 enhanced phosphorylation of the Wnt receptor LRP6 and consequently β-catenin activation by
accelerating complex formation with LRP6 and recruitment
of GSK3β and Axin, whereas its silencing exerted the opposite effects. These results suggest that NEU3 contributes to
tumorigenic potential of cancer cells by regulating Wnt/βcatenin signaling and/or EGFR signaling. The detailed relationship between these signaling pathways remains to be
solved.
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(1) Kawamura, S. et al. Cell Death Differ. 19, 170, 2012 (2)
Shiozaki, K. et al. Cancer Sci. 100, 588–594, 2009 (3)
Yamaguchi, K., et al. PLoS ONE 7, e41132, 2012
081: Keynote Lecture: Protein O-mannosylation and its
pathological role
Tamao Endo; Glycobiology Research Group, Tokyo Metropolitan Institute of Gerontology, Tokyo 173-0015, JAPAN
endo@tmig.or.jp
Glycosylation is the most common post-translational modification of proteins. The past decade of research on glycan
function has revealed the etiology of a growing number of
human genetic diseases with aberrant glycan formation.
Protein O-mannosylation is important in muscle and brain
development. α-Dystroglycan (α-DG) is a highly
glycosylated surface membrane protein. The main glycan of
α-DG was found to be O-mannosylglycan. We identified and
characterized glycosyltransferases, protein O-mannose β1,2N-acetylglucosaminyltransferase (POMGnT1) and protein Omannosyltransferase 1 (POMT1) and its homolog, POMT2
are involved in O-mannosyl glycan synthesis. Then
POMGnT1 is found to be responsible for muscle-eye-brain
disease (MEB) and POMT1/2 are for Walker-Warburg syndrome (WWS). MEB and WWS are congenital muscular
dystrophies with brain malformation and structural eye abnormalities. In addition, defects in glycosylation of α-DG are also
the cause of other four muscular dystrophies, e.g., Fukuyamatype congenital muscular dystrophy (FCMD caused by
fukutin), congenital muscular dystrophy type 1C (MDC1C
caused by FKRP), limb-girdle muscular dystrophy 2I
(LGMD2I caused by FKRP), and congenital muscular dystrophy type 1D (MDC1D caused by LARGE) since highly
glycosylated α-DG was also found to be selectively deficient
in the skeletal muscle of these patients. These all are named
dystroglycanopathies. Elucidation of the intrinsic characters
of these gene products improves out understanding of
the pathomechanisms of these diseases. Recently, a
phosphodiester-linked modification on an O-mannose was
critical for laminin-binding and LARGE was found to have
xylosyl- and glucuronyltransferase activity. However, the
functions of fukutin and FKRP are largely unknown. Additionally, new genes for dystroglycanopathies, ISPD and
DPM3, were reported. Interestingly, both affected Omannosylation.
Co llectively, details of m olecular pathology of
dystroglycanopathies are still unclear and structures and processing of O-mannosylglycans are highly complicate. I will
focus on the relation between aberrant glycosylation of α-DG
Glycoconj J (2013) 30:281–461
and congenital muscular dystrophies. Possible regulatory mechanism of protein O-mannosylation will also be discussed.
082: A Rationally Designed Glycomimetic Antagonist of
E, P, and L-selectins Inhibits Functional Biomarkers in
Sickle Cell Disease Patients in a Phase 1 Clinical Trial
John T. Patton1, Daniel D. Myers2, Scott I. Simon3, Ted
Wun 4 , Helen Thackray 1 and John L. Magnani 1 ;
1
GlycoMimetics Inc., Gaithersburg MD 20879 U.S.A.,
2
Univ. Michigan, Ann Arbor MI, USA, 3Univ California
Davis, Davis, CA USA, 4Univ California Davis School of
Medicine, Sacramento, CA, USA.
jmagnani@glycomimetics.com
The selectins are a family of carbohydrate binding proteins
that have traditionally been studied as adhesion molecules
involved inflammatory diseases and cancer. More recently
they have been shown to directly activate cells resulting in
downstream effects functioning in a variety of diseases
thereby making them novel targets for drug development.
We rationally designed a small molecule glycomimetic antagonist based on the bioactive conformation of the native
ligand (sialyl Lex) in the E-selectin carbohydrate recognition
domain combined with a sulfated domain required for optimal binding to L- and P-selectins. The resulting pan selectin
antagonist (GMI-1070) showed efficacy in many animal
models including vaso-occlusive crisis in sickle cell disease
where the selectins are known to play a critical role. GMI1070 strongly blocks E-selectin-mediated activation of neutrophils as determined by inhibition of expression of high
affinity CD18 with an IC50 of 0.5 μM, a value about 10 fold
lower than the IC50 (5.5 μM) required to inhibit cell adhesion in a flow chamber. E-selectin also functions in thrombus formation and GMI-1070 dosed at 20 mg/kg BID
potently inhibits the weight of thrombus formed at day 2
(90.6 %, p<0.01) and day 6 (83.6 %, p<0.01) post injury in
a venous thrombosis mouse model.
Patients with sickle cell disease (SCD) exhibit elevated biomarkers of adhesion, neutrophil activation (Mac-1, LFA-1),
and thrombosis (TF, TAT). These E-selectin-mediated
mechanisms play roles in the life-threatening vasoocclussive crises (VOC) periodically experienced by SCD
patients. GMI-1070 was administered in a Phase 1 study to
SCD patients (n=15) not in VOC with an IV loading dose of
20 mg/kg followed by a second dose 10 h later at 10 mg/kg.
Blood sample were taken from patients prior to dosing and
at 4,8,24 and 48 h after the loading dose. GMI-1070
significantly reduced cell adhesion markers at 8 h (sEselectin, p=0.004; sP-selectn, p=0.028; sICAM-1, p=
0.004), activation markers on monocytes at 4,8,24 and
Glycoconj J (2013) 30:281–461
48 h (Mac-1 p=0.002, 0.001, 0.008, 0.004; LFA-1 p=
0.004, 0.004, 0.012, 0.008) and markers of thrombosis
at 4 and 8 h (TF p=0.009, 0.05) and at 4,8,24 and 48 h
(TAT p=<0.001, <0.001, <0.001, 0.002). These results
suggest that GMI-1070 is affecting its target in patients
and supports further clinical evaluations.
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membrane retention in hepatocellular carcinoma, which identifies MGAT5/gp130/STAT3 signal activation as potential
therapeutic targets for personal intervention with HBVassociated HCC patients.
084: Serum glycome profiling - a biomarker for diagnosis
of ovarian cancer
083: Hepatitis B virus sensitizes IL-6 stimulation and
confers sorafenib resistance via N-glycosylation-mediated
gp130 membrane retention in hepatocellular carcinoma
Jiejie Xu, Haiou Liu, Jianxin Gu; Department of Biochemistry and Molecular Biology, School of Basic Medical
Sciences, Fudan University, Shanghai, China.
jiejiexu@fudan.edu.cn
Chronic active hepatitis (CAH) induced by persistent hepatitis B virus (HBV) infection remains the major etiologic
risk factor for hepatocellular carcinoma (HCC) in China.
Cytokine bath with interleukin 6 (IL-6) secreted mainly by
activated Kuffer cells (KCs) in local liver microenvironment
dominates inflammatory response of hepatocytes during
chronic hepatitis. Previous studies have indicated the crucial
role of IL-6 upregulation in hepatocarcinogenesis due to
HBV infection. However, alterations in stimulatory response
to IL-6 of hepatocytes undergoing chronic HBV infection
remain obscure. In this study, we found that HBV infection
sensitized hepatoma cells to IL-6 stimulation, which was
dependent on membrane retention of glycoprotein 130
(gp130) on hepatoma cells due to HBV X protein (HBx)
expression. Moreover, HBV infection and HBx expression
were found to increase gp130 membrane stability due to
MGAT5-mediated N-glycosylation. HBV infection and
HBx expression could also upregulate MGAT5 transcription
through ERK/Ets-1 signal activation. More importantly, HBV
infection could confer hepatoma cells with tumor stemness
and sorafenib resistance via N-glycosylation-mediated gp130
activation. Genetic ablation and pharmacological inhibition of
MGAT5/gp130/STAT3 pathway decreased tumorigenicity
and sorafenib resistance of hepatoma cells with HBV infection
and HBx expression. Immunohistochemial staining in clinical
hepatoma samples showed that MGAT5/gp130/STAT3 activation interlinks HBV infection with hepatoma stemness and
poor overall survival in HCC patients. Our results reveal that
HBV infection sensitizes IL-6 stimulation and confers
sorafenib resistance via N-glycosylation-mediated gp130
Karina Biskup1, Elena I. Braicu2, Jalid Sehouli2, Christina
Fotopoulou 2, Rudolf Tauber1, Markus Berger1 and
Véronique Blanchard1;1Institute of Laboratory Medicine,
Clinical Chemistry and Pathobiochemistry, Charité Medical
University, Charité Platz 1, 10117 Berlin, Germany;
2
Department of Gynecology, Charité Medical University,
Augustenburger Platz 1, 13353 Berlin, Germany
It has been known that protein glycosylation plays an
important role in many biological and biochemical processes. Glycosylation has been shown to change during
cancer initiation and progression. Ovarian cancer is
mostly diagnosed after the development of metastases.
As a consequence, prognoses and overall survival are
very poor. In the case of Epithelial Ovarian Cancer
(EOC), the common clinically used tumor markers such
as CA-125 are known to have poor sensitivity as they
often fail to detect the disease at its early stages. Therefore there is a high need for more suited biomarkers. The
aim of the work was to analyse the serum N-glycome of
EOC patients in order to identify a new potential serum
biomarker.
N-glycans were released from serum glycoproteins by
PNGase F digestion, purified in a solid phase extraction,
permethylated und subsequently analyzed by MALDI-TOF
mass spectrometry. 87 samples from preoperative EOC patients and 33 samples from age-matched healthy women
were enrolled in this study. Statistical analyses were carried
out using the SPSS 18.0 software. A GLYCOV value was
calculated from the structures that were over- and
underexpressed.
We were able to identify statistical N-glycome differences
between primary ovarian cancer and control sera, giving
better results than those of the established tumor marker
CA-125, and for this reason could potentially be used as a
biomarker.
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085: Solubule smyloid precrsor protein 770 is secreted in
a O-glycosylated form from inflamed endothelial cells
Shinobu Kitazume 1 , Akiomi Yoshihisa 2 , Takayoshi
Yamaki2, Masayoshi Oikawa2, Yuriko Tachida1, Kazuko
Ogawa1, Rie Imamaki1, Yoshiaki Hagiwara3, Noriaki
Kinoshita 3 , Yasuchika Takeishi 2 , Nobuhisa Iwata 5 ,
Takaomi Saido5, Naomasa Yamamoto6 and Naoyuki
Taniguchi1; 1Disease Glycomics Team, RIKEN Advanced
Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan; 2Department of Advanced Cardiac Therapeutics,
Cardiology and Hematology, Fukushima Medical University,
1 Hikarigaoka, Fukushima 960-1295, Japan; 3Department of
Biological Sciences, Immuno-Biological Laboratories Co.
Ltd., 1091-1 Naka, Fujioka, Gunma 375-0005, Japan;
4
Laboratory for Proteolytic Neuroscience, RIKEN Brain
Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198,
Japan; and 5Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, 31-1 Sankakudo, Tomita,
Koriyama, Fukushima 963-8611, Japan.
shinobuk@riken.jp
Deposition of amyloid β (Aβ) in the brain is closely associated with Alzheimer’s disease (AD). Aβ is generated from
amyloid precursor protein (APP) by the actions of β- and γsecretases. In addition to Aβ deposition in the brain parenchyma, deposition of Aβ in cerebral vessel walls, termed
cerebral amyloid angiopathy (CAA), is observed in more
than 80 % of AD individuals. The mechanism for how Aβ
accumulates in blood vessels remains largely unknown. In
the present study, we show that brain endothelial cells
expressed APP770, a differently spliced APP mRNA isoform from neuronal APP695, and produced Aβ40 and
Aβ42. Furthermore, we found that the endothelial APP770
had sialylated core 1 type O-glycans. Interestingly, Οglycosylated APP770 was preferentially processed by both
α- and β-cleavage and secreted into the media, suggesting
that the O-glycosylation and APP processing involved related pathways. By immunostaining of human brain sections
with an anti-APP770 antibody, we found that APP770 was
expressed in vascular endothelial cells. We then established
sandwich ELISA system to detect sAPP770 in human serum
samples. We observed that inflammatory cytokines significantly enhanced sAPP770 secretion by endothelial cells.
Furthermore, we unexpectedly found that sAPP770 was
rapidly released from activated platelets. Our finding showing that plasma sAPP770 is significantly higher in acute
coronary syndrome (ACS) patiens raise the possibility that
sAPP770 can be a useful biomarker for ACS.
References
1. S. Kitazume, A. Yoshihisa, T. Yamaki, M. Oikawa, Y.
Tachida, K. Ogawa, R. Imamaki, Y. Hagiwara, N. Kinoshita,
Glycoconj J (2013) 30:281–461
Y. Takeishi, K. Furukawa, N. Tomita, H. Arai, N. Iwata, T.
C. Saido, N. Yamamoto, and N. Taniguchi (2012) Soluble
amyloid precursor protein 770 is released from inflamed
endothelial cells and activated platelets: a novel biomarker
for acute coronary syndrome. J. Biol. Chem. (2012) 287,
40817–40825.
2. S. Kitazume, Y. Tachida, M. Kato, Y. Yamaguchi, T.
Honda, Y. Hashimoto, Y. Wada, T. Saito, N. Iwata, T. Saido
and N, Taniguchi (2010) Brain endothelial cells produce
amyloid β from amyloid precursor protein 770 and preferentially secrete the O-glycosylated form J. Biol. Chem. 285,
40097–40103.
086: Sialin as a Therapeutic Target for Solid Tumors
Roger A Laine1, Ioan Horia Negulescu2; 1Department of
Biological Sciences, Louisiana State University and A&M
College, and 1,2TumorEnd, LLC Baton Rouge, Louisiana,
USA 70803
rogerlaine@gmail.com
The HP59 lectin gene contains, entirely within its coding
region, the Sialin Gene SLC17A5, chromosome 6, solute
carrier family 17 (anion/sugar transporter), member 5,
which codes for Sialin. Sialin is a lysosomal membrane
sialic acid/acidic amino acid transporter, also important in
CNS myelination. HP59 has a transcription initiation site
300 bp upstream of the Sialin Gene SLC17A5, and only in
humans encodes 41 additional amino acids at the Aminoterminus of Sialin. In humans, there is a functional upstream
transcription initiation site and an alternate start codon for
HP59, incorporating the entire Sialin gene product. An
uniquely human capillary endothelial lumen-expressed form
of Sialin was termed HP59/SP55 by Hellerqvist, et al.,
discovered independently as the receptor for Group B streptococcal 270 kDa polysaccharide toxin, (GBS Toxin,
CM101). CM101 binds HP59/SP55 on the luminal face of
anoxic driven capillary membranes as a lectin receptor of
unknown specificity and function. HP59, having 7 or 12
transmembrane domains, is expressed in the neonate lung in
the first 5 days after birth. GBS Toxin (CM101), secreted
during sepsis, is the etiologic agent for “Early Onset Disease, to which the neonate is sensitive 5 days post partum
during rapid anoxic driven lung vascularization. The
CM101-HP59 lectin complex activates complement C3b,
initiating an inflammatory cytokine cascade which recruits
CD69+, activated granulocytes to destroy HP59 capillaries
and surrounding lung tissue causing a 50 % mortality. Post
partum in humans, rodents, canines, equines and primates
later than 5 days, HP59/SP55 (Sialin) is expressed in wound
healing and tumor angiogenesis. Sialin is also expressed on
Salivary Acinar Cells plasma membranes, transporting
Glycoconj J (2013) 30:281–461
nitrate, and in Aspartergenic Synaptic Vesicles transporting
aspartate. The homologous Sialin form SP55 is in the genomes of all mammals. Ungulates and felines express
HP55-sialin in lung capillaries, susceptible to GBS toxin
as adult animals. CM101 has been shown in a published
Phase I, FDA-IND-approved clinical trial to have clinical
safety and 33 % effectivity on select stage IV cancer patients, specifically targeting tumor vasculature. Endothelial
involvement of CM101 binding to HP59 is indicated by
levels of Soluble E Selectin. References can be found under
HP59 in Wikipedia or at www.tumorend.com.
087: O-glycome of Ascaris suum and Acanthocheilonema
viteae
Chunsheng Jin, Iain H Wilson, William Harnett and
Niclas Karlsson; Department of Medical Biochemistry, Institute of Biomedicine, Göteborg University
chunsheng.jin@medkem.gu.se
O-glycan structures from free-living nematode
Caenorhabditis elegans, porcine parasitic nematode
(roundworms) Ascaris suum, and rodent filarial nematode
Acanthocheilonema viteae, were analyzed by LC-MS. As
for C. elegans, several new fucosylated O-glycans were
found together with glycans modified with hexuronic
acid (HexA). However, phosphocholine (PC)-linked Oglycan was detected in low amount. In contrast to C.
elegans, the predominant O-glycans isolated from A.
suum and A. viteae were modified by PC.
327
chromatography on amylose-Sepharose. RecHPA exhibited
almost the same carbohydrate-binding specificity as PPA,
indicating that mammalian pancreatic α-amylases share a
common carbohydrate-binding activity. Immunostaining revealed that both α-amylases are located at the BBM in the
porcine duodenum. The binding was inhibited by mannan but
not galactan, indicating that α-amylases bind carbohydratespecifically to BBM. The ligands for α-amylase were identified by combining ligands purified by a PPA-Sepharose column, PAGE, and LC/MS/MS. The ligands identified for PPA
in BBM included sucrase-isomaltase (SI) and Na+/glucosecotransporter 1 (SGLT1), both of which take part in assimilating glucose. Bindings of SI and SGLT1 in BBM to PPA
were dose-dependent and inhibited by mannan. Assays using
freshly prepared BBM vesicles revealed functional changes in
PPA and its ligands: the starch-degrading activity of PPA and
maltose-degrading activity of SI were enhanced 240% and
175%, respectively, while Glc uptake by SGLT1 was markedly reduced by PPA at high, but physiologically possible,
concentrations after eating food. The effect of PPA on [14C]Glc uptake by BBM vesicles was attenuated by the addition of
mannose-specific lectins, especially Galanthus nivalis lectin,
supporting the involvement of a mannose-specific interaction.
The interaction of α-amylase with N-glycans in the BBM
activated starch degradation to produce much more Glc on
one hand, while suppressing a sharp increase in Glc absorption on the other1). Therefore, α-amylase plays a key role in
regulating Glc assimilation to maintain blood homeostasis
through carbohydrate-specific interaction in the intestine.
1)
088: Another method of controlling Glc assimilation at
high α-amylase concentrations in intestine through
N-glycan-specific interactions
K. Date1, N. Kawasaki2, N. Hashii2, H Sakagami1, H.
Ogawa1; 1Ochanomizu University, Tokyo, 112-8610, Japan;
2
National Institute of Health Sciences, Tokyo, 158-8501, Japan
date.kimie@ocha.ac.jp
Pancreatic α-amylase plays dual modulatory roles in digestion and absorption of D-glucose (Glc) to achieve blood
sugar homeostasis1). We previously reported that porcine
pancreatic α-amylase (PPA) binds to the N-glycans of glycoproteins2). In this study, we discovered a new system that
controls Glc assimilation by interaction between pancreatic
α-amylase and the N-glycans of glycoproteins in the luminal brush-border membrane (BBM) of enterocytes.
Recombinant human pancreatic α-amylase (recHPA) was
expressed in yeast and purified by single-step affinity
Asanuma-Date K. et al., J. Biol. Chem., 287, 23104–
23118 (2012)
2)
Matsushita, H. et al., J. Biol. Chem., 277, 4680–4686 (2002)
Structural & Chemical Glycobiology
and Glycomics
089: Keynote Lecture: Glycomics technologies identify
stem cell and cancer biomarkers
Michael Pierce, Samuel Dolezal, Hua-bei Guo, Jin-Kyu
Lee, Michael Tiemeyer, Lance Wells, and Karen Abbott;
University of Georgia Cancer Center and Complex Carbohydrate Research Center, Athens, GA 30605, USA
hawkeye@uga.edu
Cell surface glycan expression is highly regulated during
embryonic stem cell and iPS cell differentiation, as well as
during oncogenesis. Our team has developed novel
Glycomics technologies that facilitate the discovery of
328
glycoproteins and glycans that can serve as stage-specific or
disease-specific biomarkers, which can then be further investigated to determine possible functional significance. We
have identified a glycan that is expressed on cells of various
lineages after human or mouse embryonic stem cells or iPS
cells are induced to differentiate. Blocking expression of this
glycan inhibits or blocks differentiation. In addition, we have
developed methodologies for targeted glycoproteomics that
have identified markers for several types of cancers, including
invasive breast, pancreatic, and ovarian carcinomas. In the
case of pancreatic cancer, we have identified a unique glycan
epitope with high sensitivity and specificity for pancreatic
adenocarcinoma. In preliminary experiments we can detect
this epitope in blood from patients with this cancer. For
invasive ductal breast carcinoma, we have identified a novel
glycoform of periostin that is expressed during the EMT.
During breast ductal carcinoma oncogenesis, periostin is secreted and functions in the matrix as a niche substituent for
cancer stem cells. Moreover, stromal cells surrounding the
breast tumor cells are induced to produce periostin, providing
an amplification step. Initial experiments suggest that serum
levels of this specific glycoform of periostin may serve as an
accurate biomarker of invasive breast carcinoma and can
distinguish serum from cancer patients and those that are
non-diseased or have fibrocystic breast disease.
090: Mapping the glycome of Erythropoietin
Sureyya Ozcan1, Myung Jin Oh1, Rudolf Grimm1,2, and
Hyun Joo An1; 1Graduate School of Analytical Science and
Technology (GRAST), Chungnam National University,
Daejeon, Republic of Korea 2Agilent Technologies, Inc.,
Santa Clara, California 95052, USA,
sureyya.ozcan@gmail.com Tel: 82-42-821-8547, Fax:
82-42-821-8551
Erythropoietin is heavily glycosylated therapeutic glycoprotein that stimulates red blood cell production. Increasing role
of complex therapeutic proteins in immune based therapies
requires further characterization in many aspects. The quality,
safety, and potency of recombinant erythropoietins are determined largely by their glycosylation. Small variations in cell
culture conditions can significantly affect the glycosylation,
and therefore the efficacy, of recombinant erythropoietins. We
have determined the glycosylation sites and glycan heterogeneity with isomer-specific and quantitative glycomic analysis
of recombinant erythropoietins isolated from Chinese hamster
ovary (CHO) cells and mammalian cells. The platform was
used to profile native N and O-glycans from three production
batches of darbepoetin alfa (aka NESP), a common form of
Glycoconj J (2013) 30:281–461
recombinant erythropoietin and three production batches of
epoetin alfa. Glycosylation sites of corresponding glycoproteins are also profiled to show extensive glycan heterogeneity.
Results were verified by independent orthogonal analysis with
both MALDI-TOF and nano-LC/Q-TOF MS. We found
multi-antennary N-glycans with high levels of sialylation, Oacetylation, and dehydration in CHO cell. The detailed structures of these oligosaccharides were successfully characterized by tandem MS. This platform may be applied to quality
control and batch analysis of not only recombinant erythropoietin but also other complex, glycosylated biotherapeutics
and biosimilars.
091: Glycomics analysis using solid-phase glycan extraction
on chip and chipLC-MS
Shuang Yang1, Yan Li2, Hanching Chiu1, Shadi Toghi
Eshghi1, Don L. DeVoe3, and Hui Zhang1C; 1Department
of Pathology, Johns Hopkins University, Baltimore, MD
21231; 2Institute of Biophyscis, Chinese Academy of
Sciences, Beijing, China 100101; 3Department of Mechanical
Engineering, University of Maryland, College Park, MD 20742
hzhang32@jhmi.edu
Glycosylation is one of the most common protein modifications and plays significant roles in many biological processes,
including regulation of transcription and translation, signal
transduction, cell proliferation, cell interactions and protein
degradation. Aberrant glycosylation is reported to associate
with many diseases such as cancers and immune disorders.
Unlike the protein synthesis, the glycans attached to glycoproteins are enormously complex due to the non-template
dependent synthesis of glycans. As a result, the comprehensive analysis of glycans from biological or clinical samples is
an unmet technical challenge. Development of the highthroughput method will facilitate the glycomic analysis.
In this study, we developed a novel method for the highthroughput analysis of glycans from glycoproteins using
Solid-Phase Glycan Extraction (SPGE) on chip, coupling with
chipLC-mass spectrometry (MS) in an integrated microfluidic
system. Proteins were immobilized on a solid support and
other non-conjugated molecules were removed. Sialylated
glycans were acetylated on solid support to enhance their
stabilization and hydrophobicity. N-Glycans were then enzymatically released by PNGase F and then separated in a
microfluidics device that was packed with porous graphitized
carbon particles; O-glycans were then chemically cleaved by
β-elimination and separated by chipLC. The glycans were
analyzed by mass spectrometry (MS) and verified by tandem
Glycoconj J (2013) 30:281–461
mass spectrometry (MS/MS). We applied this technology on
human serum and showed high glycan coverage, specificity,
and sensitivity.
This method was used to analyze glycans from mouse heart
and serum. Comparative analysis of extracted glycans from
heart tissues, proximal serum, and distal serum showed that
unique glycan structures were identified in heart but not in
the proximal serum or blood serum.
092: Whole-Body Imaging of Glycan-Dependence on
Tumor Metastasis
Katsunori Tanaka1,2, Kenta Moriwaki3, Eiji Miyoshi3,
and Koichi Fukase1; 1Department of Chemistry, Graduate
School of Science, Osaka University, Toyonaka-shi, Osaka,
560-0043, Japan, 2RIKEN, Wako-shi, Saitama, 351-0198,
Japan, 3Department of Molecular Biochemistry and Clinical
Investigation, Osaka University, Suita-shi, Osaka, 5650871, Japan
kotzenori@riken.jp
We h a v e r e c e n t l y d e v e l o p e d t h e r a p i d 6 π azaelectrocyclization to efficiently and conveniently introduce the 68Ga-DOTA or the fluorescent groups to the
amino groups of the proteins and on the live cell
surface via a reaction with unsaturated aldehyde probes
under the quite mild conditions (Angew. Chem. Int. Ed.
329
2008, 47, 102–105). The efficient labeling procedure led
to the successful imaging of the in vivo dynamics of
glycoproteins (Angew. Chem. Int. Ed. 2008, 47, 102–
105), glycoclusters (Angew. Chem. Int. Ed. 2010, 49, 8195–
8200), and lymphocytes (J. Carbohydr. Chem. 2010, 29, 118–
132), which could precisely be controlled by the surface Nglycan structures, e.g, non-reducing sialic acids.
In further applying our azaelectrocyclization chemistry to
visualize the glycan dependence on cell trafficking of biological importance, we investigated a whole-body fluorescence imaging of tumor metastasis (Bioorg. Med. Chem.
2013, 21, 1074–1077). Two kinds of cancer cell lines, i.e.,
human gastric cancer MKN45 and human colon cancer
HCT116, and their transfected versions with the surface
glycan-related genes, i.e., MKN45 transfected with GnT-V
(β(1-6)N-acetylglucosaminyltransferase) and HCT116
transfected with GMDS (GDP-mannose-4,6-dehydrase),
were fluorescently labeled by the azaelectrocyclization
without affecting the cell viability, and their trafficking and
metastasis in BALB/c nude mice were imaged. The metastasis properties depending on glycan structures on their cell
surfaces, which were investigated previously in details
through in vitro and invasive vivo experiments (J. Biol.
Chem. 2002, 277, 16960; J. Biol. Chem. 2011, 286,
43123), was clearly visualized over a month; namely, the
increased polylactosamine structure by overexpression of
GnT-V in MKN45 cells and the deficiency of fucosylation in
the mock HCT116 cells, enhances the metastatic potential.
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Glycoconj J (2013) 30:281–461
093: Human serum N-glycome: milestone to the Human
Glycome Atlas
094: Endowing Metabolic Glycan Labeling with
Specificity
Chanyoung Han 1, Hyun-Joo An1, Carlito Lebrilla2,
Jeahan Kim3*; 1 Graduate School of Analytical Science
and Technology, Chungnam National University, Daejeon,
Korea, 2 Department of Chemistry, University of California,
Davis, USA, 3 Department of Food and Nutrition,
Chungnam National University, Daejeon, Korea
jaykim@cnu.ac.kr
Wei Lin, Ran Xie, Senlian Hong, Jie Rong, and Xing
Chen; Department of Chemical Biology, College of Chemistry and Molecular Engineering, Peking University, Beijing,
100871, China
xingchen@pku.edu.cn
Glycan moieties on biological molecules play crucial roles
engaging in their stability, functionality and regulation. The
changes of glycosylation on proteins or lipids, thus, caused
or occurred by the abnormal conditions of biological system
such as disease. Genomics, transcriptomics and proteomics
enable to draw a big picture of human biological network,
yet a huge part of their dynamic interactions or regulations
are not completely understood. Despite the necessities to
elucidate the structural and positional information of glycosylation, the progress on high throughput analysis of
glycans has been hindered by the heterogeneity and
complexity of glycosylation site or isomeric structure.
Nonetheless, recent advancement in mass spectrometry
can routinely provide various information of glycan such
that a compositional glycan profile in a single analysis or
a quantitative isomeric separations in combination with
porous graphite carbon column. Aiming to link the mass
spectrometric data of glycosylation in human specimen to
their systemic figures within the biological context, we
start build up the database called Human Glycom Atlas
(HUG-A). N-glycan in human serum was chosen for the
first target since the quantitative and qualitative changes
of glycosylation has been reported to reflect the host
medical conditions.
Total N-glycan was released from the serum of 100 controls
(non-cancer) and 100 lung cancer patient followed by the
fractionation using PGC cartridge. Three fractions (release
by 10 %, 20 %, and 40 % acetonitrile each) were then
analyzed by MALDI-TOF/TOF for overall profile and
nanoLC/Chip Q-TOF for isomeric separation. Overall, 108
glycans were identified based on their accurate mass and
isomeric separations and the glycan synthetic correlation enabled drawing a serum N-glycan map that similar to the
metabolic network. Among them, 17, 9 and 11 glycans were
major glycan that comprise 90 % of total peak intensity of
10 % 20 % and 40 %, respectively. Several major glycans
were showed more than one fraction, however, the nanoLC
Chip Q-TOF confirmed that they have same chemical structures. Interestingly, 33 glycans, mostly major glycans, were
present regardless of medical condition of samples such as
cancer, age or sex albeit the peak intensities varied.
Glycosylation plays a key role in mediating molecular recognition, development, and cell signaling. The metabolic
glycan labeling technique has recently emerged as an appealing approach for detecting and imaging glycans on live
cells and within living animals. However, this technique
lacks cell-type selectivity and protein specificity. Here, we
present the development of a cell-specific metabolic glycan
labeling strategy using azidosugars encapsulated in ligandtargeted liposomes. The ligands are designed to bind specific cell-surface receptors that are only expressed or upregulated in target cells, which mediates the intracellular
delivery of azidosugars. The delivered azidosugars are metabolically incorporated into cell-surface glycans, which are
then imaged via a bioorthogonal reaction. We will also
present a FRET-based strategy for imaging glycans attached
to a specific protein on live cell surface.
Reference:
01. Xie, R.; Hong, S.; Feng, L.; Rong, J.; Chen, X. “CellSelective Metabolic Glycan Labeling Based on LigandTargeted Liposomes” J. Am. Chem. Soc. 2012, 134, 9914–
9917.
02. Rouhanifard, S. H.; Xie, R.; Zhang, G.; Sun, X.; Chen,
X.; Wu, P. “Detection and Isolation of Dendritic Cells Using
Lewis X-Functionalized Magnetic Nanoparticles”
Biomacromolecules 2012, 13, 3039–3045.
03. Hao, Z.; Hong, S.; Chen, X.; Chen, R. P. “Introducing
Bioorthogonal Functionalities into Proteins in Living Cells”
Acc. Chem. Res. 2011, 44, 742–751.
095: A Novel Membrane Electrophoresis Compatible
with Glycoconjugate Analysis
Weijie Dong, Yu-ki Matsuno, Akihiko Kameyama;
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba,
305–8568, Japan
aki-kameyama@aist.go.jp
A number of electrophoresis techniques are available to
researchers in the life sciences. These include gel electrophoresis, capillary electrophoresis (CE), or its miniaturized
technology, chip electrophoresis. Membrane electrophoresis
Glycoconj J (2013) 30:281–461
such as paper electrophoresis is now largely obsolete. However, cellulose acetate membrane electrophoresis is widely
used for a clinical laboratory test even now as a simple and
robust method. Unlike modern electrophoresis such as 2Delectrophoresis and CE, the membrane electrophoresis does
not require skilled techniques and any expensive instruments. That might be one of the reasons why this method
has been widely used in clinical labs even though the resolving power is low.
Recently, we have developed a novel membrane electrophoresis, termed supported molecular matrix electrophoresis
(SMME), in which hydrophilic polymer such as polyvinyl
alcohol (PVA) soaking into porous poly(vinylidene
difluoride) (PVDF) membrane is used as separation
medium. Although SMME is as easy and cheap as
cellulose acetate membrane electrophoresis, it has several advantages arising from the use of the PVDF membrane as follows: compatible with glycan analysis
following β-elimination, direct immunostaining the
membrane just after electrophoresis, resistant to various
chemical treatments, low electro-osmotic flow, and
lectins are attachable through hydrophobic interaction.
As application of SMME, we here report human serum
fractionation and compare the results with that obtained
by cellulose acetate membrane electrophoresis. Moreover, analysis of glycans of each fraction is demonstrated. Additionally, we have reported that SMME could
also be used for analysis of mucins and mucopolysaccharides. Alcian blue can sensitively stain these acidic
molecules but not mucins with low acidic glycan content, such as porcine gastric mucin (PGM). To overcome
this problem, we developed a novel staining method,
succinylation-Alcian blue staining. In this method, the
glycans of the mucin molecules are modified with
succinic anhydride, thereby enabling them to be stained
with Alcian blue. Furthermore, we demonstrate the compatibility of this novel staining procedure with glycan
analysis using PGM as a model mucin.
096: Expression and purification of recombinant singlesubunit oligosaccharyltransferase from Leishmania
braziliensis
Toshihiko Kitajima, Markus Aebi; ETH Zürich, Zürich,
8093, Switzerland
kitajima@micro.biol.ethz.ch
Oligosaccharyltransferases (OSTs) mediate the en bloc
transfer of N-glycan intermediates onto asparagine residue
of nascent protein in glycosylation sequons (Asn-XaaThr/Ser, Xaa≠Pro). The enzymes are typically heteromeric
331
complexes composed of several membrane-associated subunits, in which Stt3 is highly conserved as catalytic core.
The difficulties in preparation of the enzyme have hampered
in vitro characterization and its ternary structure determination. Recent progress of genome-sequencing analysis revealed that the species within the Kinetoplastida, such as
Leishmania and Trypanosoma, lack the genes encoding
non-catalytic subunits of OST, but they code for several
different Stt3 proteins [1]. Recent studies demonstrated that
protozoan OSTs can complement yeast stt3 mutant without
formation of heteromeric complex with yeast OST [2]. In
this study, we show the expression and purification of
single-subunit OST from Leishmania braziliensis
(LbStt3_1). Recombinant genes encoding full length
LbStt3_1 proteins were expressed in Leishmania tarentolae
under the control of a constitutive promoter. Solubilization
and purification conditions were optimized. The purified
protein is active with fluorescent-labeled peptide as acceptor
and lipid-linked chitobiose as donor. We will report our
recent progress on the characterization of recombinant
LbStt3_1 protein.
References: [1] Kelleher, D. J., et al., (2007) J. Cell Biol.
177, 29–37. [2] Nasab, F. P., et al., (2008) Mol. Biol. Cell
19, 3758–3768.
097: Donor substrate specificity provides insights into
sugar donor-OGT recognition and discriminatory
proteomic substrate analysis of sOGT and ncOGT
Hui Yan, Xiaofeng Ma, Xiaoyan Liu, Feng Zhou, Yuqiu
Wang, Lianwen Zhang*; College of pharmacy, Nankai
University, 300071, Tianjin, China
lianwen@nankai.edu.cn
O-linked β-N-acetylglucosamine transferase (OGT) plays
an important role in the glycosylation of proteins involving
in various cellular events. Three isoforms of human OGT
(sOGT, mOGT and ncOGT) are distinct in their N-terminal
TPR domain, but share the same C-termianl catalytic domain. For all these isoforms, UDP-GlcNAc is a popular
sugar donor though other sugar donors such as UDPGlcNAz can also be used ncOGT.
We have synthesized a set of donor substrate derivatives
and tested them with two isoforms of OGT: sOGT and
ncOGT. When CKII3K, a peptide from casein kinase,
was used as acceptor substrate, five donor substrate derivatives (UDP-GlcNAz, UDP-GlcNPr, UDP-4DeoGlcNAc, UDP-6Deo-GlcNAc, UDP-6Deo-GalNAc) could
be accepted in sOGT-mediated glycosylation reaction.
While using an FITC labeled octapeptide, including the
332
above five derivatives, other three derivatives (UDPGlcNPh, UDP-GlcNBu, UDP-GlcNGc) could also be accepted by sOGT, suggesting that (1) sOGT is ambiguous
in recognizing sugar donor derivatives, and (2) the sugar
donor-sOGT recognition could be affected by different
acceptor substrates. Moreover, ncOGT and sOGT could
recognize different panel of sugar donor derivatives.
UDP-GlcNPh and UDP-GlcNTFA could be accepted by
sOGT and ncOGT, respectively. Some derivatives (UDPGlcNGc, UDP-GlcNAz, UDP-GlcNBu, UDP-GlcNPr)
could be accepted by both the isoforms, but their binding
capacity to sOGT or ncOGT are significant different.
These results provides further insights into sugar donor
recognition mechanism of OGT, it should be also used in
proteomic substrate analysis of sOGT or ncOGT in various cells.
Glycoconj J (2013) 30:281–461
isomeric complexity. Somewhat ironically, a mass measuring instrument, operating in a sequential manner, can be
exploited to resolve structural details better than any current chromatographic system, and thereby provides a different type of resolution, based on ion fragments and their
relationships. With these tools we now focus directly of
the valance epitopes that drive glycomic function. Given
the awareness that these are responsible for most biological interactions, it seems that strategies focused on finding
and assigning these substructures would provide a more
direct focus on the actual components defining progression
in numerous diseases and in developmental processes.
Fortunately, these substructures are pendant through relatively labile, N-acetyl-hexosaminyl linkages, a lability
and subsequent rupture that allows intact substructure
(composition) to be observed as a B-type fragment at earlier
stages of disassembly. These tactics open new doors to epitope
structural detail.
098: Keynote Lecture: The Structural Epitopes of
Glycomic Function
Vernon Reinhold, David Ashline, Andrew Hanneman
and Hailong Zhang; Glycomics Center, University of
New Hampshire Durham, NH 03824, United States
vnr@unh.edu
Although glycans manifest their activity in a multitude of
ways, including modulation of a protein’s conformation and
solubility, or serving as conjugate receptors, activators, or
blocking agents. It is abundantly clear, that a large portion of
function resides within properly positioned valence epitopes
on the exposed surfaces of lipids and proteins. Protocols to
isolate and confirm such structures are lacking, and contemporary analytical applications rarely match that needed for a
comprehensive understanding. Accurate mass compositions,
chromatographic separations and profiles annotated with
undefined cartoons fail to meet the qualitative needs of
bioengineers aspiring to design blocking analogs or vaccines to control a disease process. Additionally, an absence
of these critical features misses the mark when trying to
follow disease progression and metabolic outcome. Such
conjugates possess a plethora of isomers, both structural
and stereo, details which embody the major obstacles to an
understanding of sequence. The literature abounds with
chromatographic and MS strategies to unravel carbohydrate
structural details and the approaches are largely variations in
chromatography, MS ionization and activation. Operationally, the pursuit and expectation that all components of
structure can be assigned in a one or two dimensional MS
approach may be short-sighted, especially when considering
the diverse bond energies and the need to unravel stereo and
099: Experimental and theoretical approaches to
understand the conformational differences in isomeric
glycan pairs
Yoshiki Yamaguchi1, Kana Matsumoto1, Kenji Hirose2,
Wataru Nishima2, Suyong Re2, and Yuji Sugita2; 1Structural Glycobiology Team and 3Theoretical Molecular Science Laboratory, RIKEN, Wako-shi, Saitama, 351-0198,
Japan; 2Nihon Waters K.K., Osaka, Japan
yyoshiki@riken.jp
The oligosaccharide moieties have many important biological roles, including protein quality control, cell-cell
recognition and protection from proteolysis. Recent evidence suggests that a protein with a particular glycan
structure possesses a specific biological function. Further,
glycoprotein therapeutics (e.g. use of antibodies), is now
one of the most promising approaches in the fight against
disease. Analysis of glycan structure is therefore critical
not only for basic science but also for pharmaceutical
purposes.
One of the central issues in the mass analysis of glycans is
the ambiguity of structural assignments mainly due to the
heterogeneity and complexity of glycan structures. The
many possible isomeric glycan structures of the same mass
hamper accurate annotation of each peak. Although detailed
tandem mass analysis does yield the glycan structure, the
process is often time-consuming and is not suited for handling many sets of glycan mixtures such as are required in
glycomics studies. In most cases, therefore, the separation of
Glycoconj J (2013) 30:281–461
isomeric glycans has been accomplished by the use of liquid
chromatography.
In developing methods for the accurate and rapid identification of glycan structures, we have investigated in detail
the ion mobility spectrometry (IMS) separation of a set of
fluorescently biantennary N-glycans including isomeric
structures. The cross section of each glycan derives from
the IMS data and the structural characteristics are discussed
in the light of molecular dynamics (MD) simulations. To
enhance conformational sampling of the N-glycans in vacuum, we employ the replica-exchange molecular dynamics
(REMD) method. Further, we demonstrate the separation of
pyridylaminated N-glycans using an ultra-performance liquid chromatography (UPLC) system with hydrophilic interaction liquid chromatography (HILIC), thereby establishing
a system that combines ion mobility mass spectrometry and
HILIC.
100: Structure of Gal/GalNAc-specific lectin from
marine mussel Crenomytilus grayanus
Oleg V. Chernikov, Irina V. Chikalovets, Valentina I.
Molchanova, Pavel A. Lukyanov; G.B. Elyakov Pacific
Institute of Bioorganic Chemistry FEB RAS, Vladivostok,
690022, Russian Federation
chernikov@piboc.dvo.ru
333
program have not identified any conserved domain in CGL
amino acid sequence. SMART server also could not predict
in CGL any known domains, but revealed that CGL
contained three internal repeats. Multiple alignments of the
tandem-repeat amino acid sequences (which we designed as
subdomains α, β and γ) have shown their high similarity.
CD spectra of CGL and PSIPRED data showed that a characteristic feature of the structural organization of CGL is the
predominance of β-structure. 3D-model of CGL built by
PHYRE2 server was used for prediction of GalNAc binding
site. It was found that CGL contained three putative GalNAc
binding sites.
Thus, analysis of the amino acid sequence of CGL revealed
that this protein is a member of a novel lectin family which
adopts a ß-trefoil fold. However, the elucidation of the threedimensional structure of CGL and site-directed mutagenesis
studies are necessary to verify prediction of CGL fold and
sugar-binding sites.
This research was supported by the grant from the Russian
Foundation for Basic Research (no. 12-04-00862), a grant
from the RAS Presidium (no. № 12-I-П6-10) under the
project “Molecular and Cellular Biology”.
101: Bacterial cell wall peptidoglycan fragment
library/array for investigation of their protein recognition
Lectins are sugar-binding proteins which recognize specifically carbohydrate structures. Lectins are found in all taxa.
Based on the structural similarity of carbohydrate recognition domain lectins are classified into a number of structurally distinct families. In recent years, many lectins from
marine invertebrates have been identified.
Yukari Fujimoto 1 , Ning Wang 1 , Akiyoshi Hirata 2 ,
Kiyoshi Nokihara2, Koichi Fukase1; 1Department of
Chemistry, Graduate School of Science, Osaka University,
Toyonaka, 560-0043, Japan; 2HiPep Laboratories, Kyoto,
602-8158, Japan.
yukarif@chem.sci.osaka-u.ac.jp
1Previously, the Ca2+-independent Gal/GalNAc-specific lectin (CGL) with a molecular mass of 17 kDa was isolated by us
from the mussel Crenomytilus grayanus. In the present study,
the sequence of cDNA encoding CGL was determined for the
first time. The obtained cDNA sequence of 750 bp contained
an open reading frame of 450 bp encoding a polypeptide of
150 amino acid residues (GenBank ID:JQ314213). The predicted CGL amino acid sequence comprised peptides determined by Edman degradation and ESI-MS/MS earlier. The
CGL calculated molecular weight of 17025.3 Da was in
agreement with those estimated by MALDI.
Peptidoglycan is a component of bacterial cell wall
consisting of glycans (with alternating GlcNAc-β(1→4)MurNAc) and peptide chains forming a three-dimensional
mesh-like structure outside the plasma membrane. There
are many proteins that can recognize PGN. For example,
multicellular organisms including mammals have innate
immune receptor proteins such as nucleotide-binding
oligomerization domain protein 1 (Nod1) and 2 (Nod2),
peptidoglycan recognition proteins (PGRP), and lectins.
However, the comprehensive analyses of their substrate
structures have not been really conducted because of the
lack of pure chemically synthesized PGN fragments. To
analyze the various ligand/substrate-protein interactions,
the chemical synthesis of the PGN fragment library as
well as the new analysis method are essential, leading to
Results of NCBI-BLAST and WU-BLAST search revealed
that CGL amino acid sequence had not similarity with lectins
of known families. NCBI Conserved Domain Search
334
understand the protecting system against infection of
bacteria.
Here, we report the construction of a comprehensive
library of PGN with linkers and its application on microarray for recognition protein detection, based on our
recent establishment of the PGN fragment synthesis. In
order to introduce the PGN fragments to the array, a
linker with terminal amine was successfully attached to
the PGN fragments. PGN array was then built up by
attaching synthesized PGN fragments on the carboxyl
covered solid surface of a carbon platform using amide
formation. The array could be used to detect the PGN’s
receptors and recognition proteins and measure the binding properties of these proteins. For evaluation of the
binding property of the array, two series of proteins were
used, which are wheat germ agglutinin (WGA) with
fluorescence label and human PGRP-S with combination
of fluorescence labeled antibody. PGRP-S showed stronger affinity with tetrasaccharide tripeptide (GMGM3P),
which is identical with our previous report using SPR
study. Other proteins which have unknown binding properties are also tested.
References:
1) Reviews: a) Fujimoto, Y., Pradipta, A. R., Inohara, N.,
Fukase, K., Nat. Prod. Rep. 2012, 29, 568; b) Fujimoto, Y.,
Inamura, S., Kawasaki, A., Shiokawa, Z., Shimoyama, A.,
Hashimoto, T., Kusumoto, S., Fukase, K., J. Endotoxin
Research 2007, 13, 189. (Synthesized PGN fragments for
receptor search.)
2) a)Wang, N., Huang, C.-y., Hasegawa, M., Inohara, N.,
Fujimoto, Y., Fukase, K. ChemBioChem, 2013, in press; b)
Inamura, S., Fujimoto, Y., Kawasaki, A., Shiokawa, Z.,
Woelk, E., Heine, H., Lindner, B., Inohara, N., Kusumoto,
S., Fukase, K., Org. Biomol. Chem. 2006, 4, 232.
102: Structures of Pectin Related to Galectin-3 Inhibition
Guihua Tai, Xiaoge Gao, Huiting Xue, Yifa Zhou*;
School of Life Sciences, Northeast Normal University,
Changchun, 130024, PR China
taigh477@nenu.edu.cn and zhouyf383@nenu.edu.cn
Pectin has been shown to inhibit the functions of
galectin-3, a β-galactoside-binding protein associated
with cancer progression and metastasis. The structural
components of pectin that regulate its activity are unclear.
We prepared various pectins, fragments and oligosaccharides from ginseng pectin, modified citrus pectin (MCP)
Glycoconj J (2013) 30:281–461
and potato galactan, and studied their inhibitory activities
on galectin-3 using several galectin-3-mediated cell assays. A rhamnogalacturonan I-rich ginseng pectin fragment, RG-I-4, was identified as potent inhibitor of
galectin-3. Comparative studies showed that RG-I-4 had
better activity than MCP and potato galactan, which were
two well-established galectin-3 ligands. RG-I-4, MCP and
potato galactan had dissociation constants of 22.2 nM,
143 nM and 2,590 nM, respectively, consistent with
their relative activities determined by surface plasmon
resonance analysis. The structure-activity of RG-I-4, MCP
and potato galactan was further investigated by modifying
the structure through various enzymatic and chemical
methods followed by activity tests. The results showed
that β1,4-galactan side chains were pivotal to the activity.
The activity of a linear β1,4-galactan chain was proportional to its length up to 4 Gal residues. All long chains
with ≥4 Gal residues had similar activities that were
slightly better than lactose. Thus, a chain of four Gal residues
meets the basic need for maximum activity. Galectin-3
appeared to recognize terminal tetrasaccharides. RG-I-4
contained mainly short β1,4-galactan side chains, consisting
of less 4 Gal residues. However, it had 40- to 1000-fold higher
activity than a single chain. Extensive structure-activity correlation studies indicated that the high activity of RG-I-4
resulted from the cooperative action of multiple short side
chains. However, the existence of multiple side chains did
not guarantee cooperative action. Potato galactan,
containing multiple side chains, had similar activity to a
single long side chain. The inhibitory activity of pectin
should be related to the backbone, side chains and even
whole molecule conformation.
103: Analysis of Glycosphingolipids by LC-MS
Akemi Suzuki 1, Hideshi Fujiwaki 2, and Yoshikatsu
Umemura2; 1Institute of Glycoscience, Tokai University,
Hiratsuka, 259-1292, and 2Shimadzu Co., Kyoto, 6048511, Japan
akmszk@tokai-u.jp
Recent advances in mass spectrometry make our view of
glycosphingolipids (GSLs) completely different from the
previous one. LC-MS is applicable to GSL mixtures in a
small amount, and provides structural information of
carbohydrate chains and ceramides and possibly quantitative results in the presence of appropriate internal standards. However, several subjects remaining to be
carefully clarified are the establishment of analytical conditions for obtaining fragmentations useful for structural
Glycoconj J (2013) 30:281–461
characterization, the preparation of standard or isotopelabeled GSLs for quantitation, and the establishment of
required informatics including data base construction,
collection of MS spectra, and search engine creation.
We report here our resent progress in our attempt for
solving above requirements.
We have tested several matrices for neutral GSLs and
acidic GSLs (gangliosides) in the analysis of negative ion
mode. Negative ion mode provides more fragment ions
derived from carbohydrate chains and ceramides. Matrices used for LC analysis with a C30 reversed phase
column of neutral GSLs, such as ammonium formate,
formic acid, ammonium acetate, and acetic acid, give
adduct ions and the ratios of molecular ions [M - H]and adduct ions are different among molecular species of
GlcCer as the simplest neutral GSL. Ammonium bicarbonate containing solvents give lesser amount of adducts
and can be used for comparing changes of molecular
species of neutral GSLs produced by the different conditions
of cultured cells or comparing neutral GSLs of different subsets of cells isolated from living organisms. In the analysis of
gangliosides, GM1(NeuGc), GD1a and b, GT1b, and GQ1b
were detected as double-charged ions. We detected
GM1(NeuAc) as single-charged ions [M – H] − , and
GM1(NeuAc) exhibits the highest m/z value among these
gangliosides, resulting in that detection efficiency of
GM1(NeuAc) becomes very low. We found that this result
depends on pH of elution solvents which also effect
electrospray ionization. This finding helps to detect
GM1(NeuAc) in a better sensitivity. We are now compiling
MS, MS/MS, MS/MS/MS spectra of gangliosides.
104: Interactions of carbohydrates with biomolecules
Role of the CH/π interactions
Michaela Wimmerová1,2,3, Stanislav Kozmon1,2, Josef
Houser1,2, Radek Matuška2, Jaroslav Koča1,2; 1CEITEC
– Central-European Institute of Technology, Masaryk
University (MU), Kamenice 5, 625 00, Brno, Czech
Republic, 2National Centre for Biomolecular Research,
Fac of Science, MU, Kamenice 5, 625 00, Brno, Czech
Republic, 3Department of Biochemistry, Fac of Science,
MU, Kamenice 5, 625 00, Brno, Czech Republic
jkoca@ceitec.cz
Molecular recognition plays crucial role in many biological processes, such as bacteria-host identification.
Some of these recognition processes are performed by
proteins called lectins, which are able to bind
335
saccharides in a very specific way. In our study we
have focused on RSL lectin from world-wide distributed
bacteria Ralstonia solanacearum causing lethal wilt in
many agricultural crops and the AAL lectin from
Aleuria aurantia. Both above mentioned bacteria interact with hosts through their lectins, binding predominantly L-fucose. In this paper, we are interested in the
role of the CH/π interaction in binding abilities of these
two lectins.
In the RSL lectin, we have attempted for the first time to
quantify how the CH/π interaction contributes to a overall
carbohydrate—protein interaction. We have used an experimental approach, creating single and double point mutants,
combined with high level computational methods. The
structure contains three monomer units of the lectin with
six almost identical binding sites, where three of them are
intramonomeric and the other three are intermonomeric.
Experimentally measured binding affinities were compared
with computed carbohydrate-aromatic acid residue interaction energies. Experimental binding affinities for the RSL
wild type, phenylalanine and alanine mutants were −8.5,
−7.1 and −4.1 kcal.mol−1, respectively. These affinities
agree with the computed dispersion interaction energy between the carbohydrate and aromatic amino acid residues
for RSL the wild type and the phenylalanine mutant, with
respective values of −8.8 and −7.9 kcal.mol−1, excluding
the alanine mutant where the interaction energy was
−0.9 kcal.mol−1. Molecular dynamics simulations show
that discrepancy can be caused by creation of a new hydrogen bond between the α-L-Me-fucoside and RSL. Observed
results suggest that in this and similar cases the
carbohydrate-receptor interaction can be driven mainly
by a dispersion interaction. The AAL lectin contains five
different binding sites for L-fucose. However, the main
motifs of the binding sites are similar to the RSL ones.
Therefore, we have attempted to analyze the impact of
dispersion interaction to total binding potency also for
AAL.
This work was funded by the European Community’s
Seventh Framework Programme under the European
Regional Development Fund (CZ.1.05/1.1.00/02.0068)
and Capacities specific programme (286154). The project is supported within the SoMoPro programme (project No. 2SGA2747, co-funded by FP7/2007-2013 under
grant agreement No. 229603). The research is also cofunded by the South Moravian region. The authors
thank the Czech National Supercomputing Centre,
METACENTRUM, for providing computational resources
(research intent LM2010005).
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Glycoconj J (2013) 30:281–461
105: Evidence for a species-wide general O-glycosylation
system for extracellular proteins in Lactobacillus
buchneri
1
2
Julia Anzengruber , Martin Pabst , Laura
Neumann2, Friedrich Altmann2, Christina Schäffer1, and
Paul Messner 1 ; 1 Department of NanoBiotechnology,
NanoGlycobiology and 2 Department of Chemistry,
Universität für Bodenkultur Wien Vienna, A-1190,
Austria
paul.messner@boku.ac.at
While having been long overlooked, nowadays glycosylation of prokaryotic proteins is becoming increasingly
accepted. In the past decade, general protein glycosylation
systems have been demonstrated mainly in Gram-negative
pathogenic bacteria. Based on health promoting features
and general advantages of Gram-positive lactobacilli with
GRAS status for applied research, protein glycosylation in
this bacterial genus was investigated in detail in this
study.
The cell surface of the model organism Lactobacillus
buchneri CD034 was shown to be completely covered
by an oblique 2D crystalline array formed by selfassembly of the glycosylated S-layer protein SlpB. Biochemical and mass spectrometric analyses revealed that
SlpB is decorated with an O-glycan, consisting of α1,6linked D-glucose residues, attached to specific serine
residues of the S-layer protein within the sequence SA-S-S-A-S. Strikingly, a second glycoprotein of L.
buchneri CD034, a glycosyl hydrolyzing enzyme
(LbGH25B) contains this characteristic glycosylation
sequence and carries the same glycan. Subcellular fractions of strain CD034 showed that either glycoprotein is
cell-wall associated and absence of the glycan on cytosolic proteins revealed a link between protein glycosylation and secretion. Protein glycosylation analysis was
extended to strain L. buchneri NRRL B-30929 where a
similar glycosylation scenario could be detected, with
the S-layer protein SlpN and the glycosyl hydrolase
LbGH25N as major glycoproteins. All identified
lactobacillar glycoproteins are extracellular, share the
same O-glycan and the same glycosylation sequence.
These findings corroborate previous data on the protein
glycosylation of L. buchneri 41021/251 and let us propose the general L. buchneri O-glycosylation motif S-AS-S-A-S and the first example of a general O-glycosylation system in Gram-positive and beneficial microbes.
This work was supported by the Austrian Science Fund
FWF projects P21954-B20 (to C.S.) and P24305-B20
(to P.M.).
106: Sequence analysis and domain motifs in the decorin
glycosaminoglycan chain
Xue Zhao, 1,2 Bo Yang, 2 Robert J. Linhardt, 2,3,4,5
Lingyun Li2; 1College of Food Science and Technology,
Ocean University of China, Qingdao P. R. China 266003,
2
Department of Chemistry and Chemical Biology, 3Department of Chemical and Biological Engineering, 4Department
of Biology, 5Center for Biotechnology and Interdisciplinary
Studies, Rensselaer Polytechnic Institute, Troy, NY 12180,
USA.
zhaoxue@ouc.edu.cn; lil12@rpi.edu
Decorin proteoglycan is comprised of a core protein
containing a single O-linked dermatan sulfate/chondroitin sulfate glycosaminoglycan (GAG) chain. While the sequence of
the decorin core protein is determined by the gene encoding its
structure, the structure of its GAG chain is determined in the
Golgi. The recent application of modern mass spectrometry
(MS) to bikunin, a far simpler chondroitin sulfate proteoglycans, suggests that it has a single or small number of defined
sequences. On this basis, a similar approach to sequence
decorin’s much larger and more structurally complex
dermatan sulfate/chondroitin sulfate GAG chain was undertaken. This approach resulted in information on the
consistency/variability of its linkage region at the GAG
chain’s reducing end, its iduronic acid-rich domain, glucuronic
acid-rich domain, and non-reducing end. A general motif for
the porcine skin decorin GAG chain was established. A single
small decorin GAG chain was sequenced using MS/MS analysis. The data obtained in the study suggests that the decorin
GAG chain has a small or a limited number of sequences.
Fri-Cell Biology
107: Keynote Lecture: O-GlcNAcylation on Transcription
Factor Mef2c Regulates Terminal Differentiation of Skeletal Myogenesis
Jin Won Cho, Hanbyeol Kim, Jürgen Roth; Department
of Integrated OMICS for Biomedical Science, Yonsei
University, Seoul, 120-749, Korea
chojw311@yonsei.ac.kr
O-GlcNAc modification is one of the post-translational
modifications found in nucleus and cytosol. This modification which can compete with phosphorylation is dynamically regulated by O-GlcNAc transferase(OGT) and OGlcNAcase (OGA). UDP-GlcNAc, a final product of the
hexosamine biosynthesis pathway is a substrate for OGT.
More than 1,800 proteins have been identified as OGlcNAcylated proteins. O-GlcNAcylation is reported to
Glycoconj J (2013) 30:281–461
associate with the development of skeletal muscle atrophy
and with muscle metabolism. Particularly, terminal differentiation of skeletal muscle is repressed by increased OGlcNAcylation. We observed that the expression of
myogenin, a key regulator for skeletal muscle terminal
differentiation, decreased after treatment of NButGT, an
inhibitor of OGA. In addition, we confirmed that the promoter
activity of myogenin decreased after NButGT treatment. To
find the promoter region affected by O-GlcNAc, we used
shorter length promoter gene and found that at least 169 base
pair upstream region of myogenin is affected by O-GlcNAc.
We also found that Mef2c protein can be an important transcription factor regulated by O-GlcNAc via avidin-biotin
complex DNA binding assay on 169 base pair upstream
region. We performed immunoprecipitation and mass spectrometry to confirm that Mef2c is O-GlcNAcylated and to find
O-GlcNAcylated sites on Mef2c. As we expected, mutagenesis of these serine or threonine to alanine resulted in a decrease
in O-GlcNAc modification of Mef2c.
108: O-GlcNAc of p120 inhibits the interaction between
p120 and E-cadherin
Haiyang Liu, Cuifang Han, Xinling Zhang, Chuanlin Bi,
Leina Ma, Wengong Yu, Yuchao Gu*; Key Laboratory of
Marine Drugs, Chinese Ministry of Education; Department
of Molecular Biology, School of Medicine and Pharmacy,
Ocean University of China, 5 Yushan Road, Qingdao, China.
guych@126.com
Protein O-GlcNAcylation is an O-linked glycosylation involving attachment of beta-N-acetylglucosamine (GlcNAc) to
Ser/Thr residues of intracellular proteins catalyzed by OGlcNAc transferase (OGT), whose removal is catalyzed by
O-GlcNAcase (OGA). Previously, we demonstrated that OGlcNAc promotes the formation and metastasis of some types
of cancer (including breast cancer, lung cancer and colon
cancer), which was caused mainly by the decrease of cell
surface E-cadherin. However, the molecular mechanism underlying O-GlcNAc induced the disruption of E-cadherin complexes is still unclear. Recently, we found that p120-catenin
(p120), which could bind and stabilize E-cadherin by its armadillo repeat domain and thus regulates cell-to-cell adhesion,
could be O-GlcNAcylated by OGT in vivo and in vitro. By
using CoIP and pull-down assays, we demonstrated that OGlcNAcylation of the armadillo repeat domain inhibits the
binding of p120 and E-cadherin. We mapped two O-GlcNAc
sites in the armadillo repeat domain by MS, which might
interpret the role of O-GlcNAc in the regulation of the interaction between p120 and E-cadherin. Additionally, we also
found that OGT could directly bind to the armadillo repeat
domain of p120, and the presence of OGT could interfere the
337
binding of p120 to E-cadherin in vitro. Altogether, these data
demonstrated that O-GlcNAc and OGT play important roles in
the regulation of the interaction between p120 and E-cadherin
and thus E-cadherin-mediated cell-cell adhesion.
109: Ribosomal RACK1 is stabilized by O-GlcNAc
modification under hypoxic conditions and promotes
IRES-mediated translation in hepatocellular carcinoma
Yuanyuan Ruan and Jianxin Gu*; Key Laboratory of
Glycoconjugate Research Ministry of Public Health, Fudan
University
yuanyuanruan@fudan.edu.cn
Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide. RACK1 is a classical scaffold protein present in both ribosome- and
nonribosome-bound form, and our previous report also
demonstrates that ribosome-associated RACK1 promotes
the chemoresistance and growth of HCC. However, what
modulates the association between ribosome and RACK1 is
little understood. Herein, we show that ribosomal RACK1 is
modified and stabilized by O-GlcNAc glycosylation in
HCC. Moreover, hypoxia promotes the O-GlcNAc modification of RACK1, leading to the enhanced association between ribosome and RACK1 and IRES-mediated translation
under hypoxic conditions. Our data suggest that the OGlcNAc modification of RACK1 provides the basis for
translational regulation in HCC under hypoxic conditions.
110: Modification of Transforming growth factor βactivated kinase 1 (TAK1)-binding protein 2(TAB2) by
O-linked-N-cetylglucosamine(O-GlcNAc) in proinflammatory signaling
Zengxia Li1, Guanghui Han2, Junfeng Ma2, Gerald W.
Hart2; 1Department of Biochemistry and Molecular Biology,
Shanghai Medical College, Fudan University, Shanghai,
200032,China; 2 Department of Biological Chemistry,
The Johns Hopkins University School of Medicine,
Baltimore,21205,USA
gwhart@jhmi.edu
O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly
dynamic post-translational modification of serine and threonine residues regulating physiological and stress processes.
TGF-β activated kinase 1 (TAK1)- binding protein2
(TAB2), binding partner of TAK1, plays important roles in
the regulation of various pro-inflammatory signaling pathways including TNF-α and IL-1β-mediated activation of
AP-1 and NF-κB. Here, we report that the global changes
338
of O-GlcNAc affected the IL-1β and TNF-α induced IκB
degradation and NF-κB activation signaling. TAB2 is identified as a highly O-GlcNAc modified protein. TAB2
reacted with a O-GlcNAc specific antibody CTD110.6 and
the antibody reactivity was diminished by treatment of
CPNag J. TAB2 was found to be susceptible to labeling
with beta-1,4-galactosyltransferase (GalT). We demonstrate
that TAB2 can form a complex with OGT using coimmunoprecipitation approach in both directions. OGT
in vitro labeling assay showed that TAB2 is a direct
substrate of OGT. Using mass spectrometric methods,
we validated the O-GlcNAcylation of TAB2 and localized OGlcNAc sites on 3 separate sequences (GTSSLSQQTPR,
TSSTSSSVNSQTLNR, and VVVTQPNTK). Site-directed
mutagenesis of identified sites (T164, S165, S166, S168,
T348, S349, S350, T351, S352, S353, S354, T456) confirmed
these sites to be modified. We also found that the O-GlcNAc
modification of TAB2 is a dynamic process and undergoing a
rapid cycling. Currently, we are aimed to find the
unrecognized role of O-GlcNAc modification of TAB2 in
the regulation of pro-inflammatory signaling pathways.
111: Regulation of integrin αV expression by sulphated
cerebroside scaffolding in hepatocellular carcinoma cells
Wei Wu, Yi Wei Dong, Peng Cheng Shi, Xing Zhong Wu;
Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Key Lab of
Glycoconjugate Research, Ministry of Public Health, 138
Yi Xue Yuan Road, Shanghai 200032, P.R. China;
xz-wu@shmu.edu.cn
Integrin expression is very important in cell adhesion and
tumour progression. To study the impact of cerebroside
sulfation on integrin levels, we investigated the sulphated
cerebroside regulation of the integrin αV subunit expression
in comparison with cerebroside. Exogenous sulphated cerebroside, rather than cerebroside, elicited a dose- and timedependent stimulation of integrin αV subunit mRNA and
protein expression in hepatocellular carcinoma cells (HCC).
Through a comparison of treatments with cerebroside,
ManN propanyl perac, and cyclo-ManN propanyl perac,
only the cells treated with sulphated cerebroside produced
a high expression level of the integrin αV subunit. Furthermore, the stable transfection of cerebroside sulfotransferase
(CST) into the cells led to high expression of integrin αV,
and a knockdown of CST mRNA showed down-regulation
of integrin αV. This effect occurred with a corresponding
phosphorylation of the transcription factors Sp1 and Stat3.
In the cells stimulated with sulphated cerebroside, phosphorylation of Erk1/2 as well as c-Src was noted, and
inhibition of Erk1/2 activation with either U0126 or
Glycoconj J (2013) 30:281–461
PD98059 significantly suppressed Sp1 phosphorylation.
Sulphated cerebroside was positively correlated with
integrin αV expression in HCC tissues from 125 patients
and their expression levels were associated with the patient’s
disease-free survival. Herein, we demonstrated that sulfation
of cerebroside regulated integrin αV expression and cell
adhesion via Erk activation, and was important in HCC.
112: N-glycan structure and function of cancer stem cell
marker CD133
Liu Ying1, Wei Yuanyan1, Jiang Jianhai1$; 1 Key Laboratory of Glycoconjuates Research, Ministry of Public
Health and Gene Research Center, Shanghai Medical College of Fudan University, China, 200032
jianhaijiang@fudan.edu.cn.
CD133/Prominin-1, a pentaspan membrane glycoprotein, has
been widely used as a marker of normal and cancer stem cells.
Increasing studies indicate that CD133 performs a variety of
functions in cell proliferation, differentiation, metabolism and
migration. Increasing evidences indicate that the glycosylation
status of CD133, rather than the expression of CD133 protein
itself, can act as an the marker of stem cells. And, the dynamic
glycosylation of CD133 might play a critical role in its biological functions. Thus, identification of CD133 glycosylation
status, which might be different in cells at different stages of
differentiation and in different tissues, means a crucial step in
defining the potential role of CD133 in normal and cancer
stem cells. We analyzed the N-linked oligosaccharides of
CD133 by biotin acylation lectin PHA-L, PHA-E, SNA and
ConA and identified N-glycosylation site by MS. Furthermore, we showed that N-glycosylation regulated the positive
effect of CD133 on hepatoma cell growth. We revealed
CD133 could be sialylated in neural stem cells and gliomainitiating cells, and the sialyl residues attach to CD133 Nglycan terminal via a 2, 3-linkage. These findings explored the
fundamental biological aspect of CD133 glycosylation.
113: The role of beta 1,4-galactosyltransferase I in glia
activation during neuroinflammation
Aiguo Shen*; The Jiangsu Province Key Laboratory of
Neuroregeneration, Nantong University, Nantong 226001,
P.R. China
shag@ntu.edu.cn
Protein glycosylation modification exerts great importance
on the migration, recognition and inflammatory signal transduction of inflammatory cells,but there isn’t much indepth
study i this field. We found that β4GalT1 (beta 1,4-
Glycoconj J (2013) 30:281–461
galactosyltransferase I) played an important role in the CNS
inflammatory response,but its role in regulating glycosylation modification remains unclear.We have found during the
activation of glial cells,β4GalT1 regulates glial cell migration, adhesion, and the release of Inflammatory factors by
modulating the glycosylation of E-selectin, intergrin α5 and
BMP2 respectively; then we found regulation of SSeCKS
and UBE2Q on β4GalT1 location and the modulation of
CDK11p58 and LRRK1 on β4GalT1 enzymatic
activity, respectively. Our research findings can provide the
foundation to thoroughly study the role of protein glycosylation modification in inflammatory response.
114: Core fucosylation of mu heavy chains regulates the
assembly of precursor B cell receptors and its intracellular
signaling
Wenzhe Li1, Jinhua Jin1, Akihiro Kondo2, Jianguo Gu3,
and Naoyuki Taniguchi4; 1 Dalian University, Liaoning
116622, China; 2 Osaka University Graduate School of
Medicine, Suita 565-0871, Japan; 3 Tohoku Pharmaceutical
University, Sendai, Miyagi 981-8558, Japan; 4 RIKEN,
Saitama 351-0198, Japan
liwenzhe46@hotmail.com
GDP-L-Fuc:N-acetyl-b-D-glucosaminide a1,6fucosyltransferase (Fut8) catalyzes the transfer of a fucose
residue from GDP-fucose to the innermost Nacetylglucosamine (GlcNAc) residue of hybrid and complex
N-glycans via an a1,6-linkage (core fucosylation) in the
Golgi apparatus in mammals. The presence of core fucose
in N-linked glycoprotein has been shown to be important in
glycoprotein processing and recognition. Fut8 knockout
(Fut8−/−) mice showed an abnormality in pre-B cell generation. Membrane assembly of pre-BCR is a crucial checkpoint
for pre-B cell differentiation and proliferation both in humans
and in mice. The assembly of pre-BCR on the cell surface was
substantially blocked in the Fut8-knockdown pre-B cell line,
70Z/3-KD cells, and then completely restored by reintroduction of the Fut8 gene to 70Z/3-KD (70Z/3-KD-re)
cells. Moreover, loss of a1,6 fucosylation (also called core
fucosylation) of μHC was associated with the suppression of
the interaction between μHC and λ5. In contrast to Fut8+
/+
CD19+CD43- cells, the subpopulation expressing the
μHC/λ5 complex in the Fut8−/−CD19+CD43− cell fraction
was decreased. The pre-BCR mediated tyrosinephosphorylation of CD79a and activation of Btk were attenuated in Fut8-KD cells, and restored in 70Z/3-KD-re cells.
The frequency of CD19lowCD43− cells (pre-B cell enriched
fraction) was also reduced in Fut8−/− bone marrow cells, and
then the levels of IgM, IgG and IgA of 12-week-old Fut8−/−
mice sera were significantly lower than those of Fut8+/+ mice.
339
Our results suggest that the core fucosylation of μHC mediates
the assembly of pre-BCR so as to regulate pre-BCR intracellular signaling and pre-B cell proliferation. This work was
supported by National Nature Science Fundation of China
(No. 30972675 and No. 31270864).
115: O-GlcNAc Transferase Mediates the Regulation of
26S Proteasome Functionality by Endothelial Nitric
Oxide Synthase Derived Nitric Oxide
Hongtao Liu1, Shujie Yu1, Hua Zhang1, Yu Li1,2,
Yu-Guang Du2, Jian Xu1; 1Department of Medicine,
Harold Hamm Diabetes Center, University of Oklahoma
Health Sciences Center, Oklahoma City, OK 73104, USA;
2
Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China
jian-xu@ouhsc.edu
Loss of vascular homeostasis leads to morbidity and mortality in diabetics, attributable to early loss of endothelial
nitric oxide (NO) bioavailability. In contrast, early diabetes
is found to increase 26S proteasome functionality in vasculature resulting in enhanced degradation of certain proteins
key to the vascular homeostasis. We wonder whether endothelial NO synthase (eNOS)-derived NO functions as a
physiological inhibitor of the 26S proteasome in vasculature. To test this, we exposed proteasome reporter (UbG76VGFP)-expressing vascular endothelial cells to NO and monitored proteasome functionality by detecting the
polyubiquitinated-GFP reporter proteins. We also generated
UbG76V-GFP-expressing eNOS-knockout mice to determine
the role of eNOS-derived NO in whole animal. Like the
selective NO donors, NO derived from the activated eNOS
(either by pharmacological or genetic approach) induced
(poly)-UbG76V-GFP accumulation in cultured endothelial
cells, which was associated with increased O-GlcNAc
modification of the proteasome regulatory complex
(PA700/Rpt2) and reduced proteasome chymotrypsin-like
activity (p<0.05, n=5/group). Conversely, siRNA knockdown of O-GlcNAc transferase (OGT), the key enzyme for
O-GlcNAcylation, abolished the NO-elicited effects. Consistently, adenoviral overexpression of O-GlcNAcase, the
enzyme responsible for O-GlcNAc removal, mimicked the
effects of OGT knockdown. Finally, compared to the control
UbG76V-GFP mice, eNOS-deleted mice exhibited accelerated UbG76V-GFP degradation in isolated aortas, in parallel
with elevated chymotrypsin-like activity and reduced
PA700/Rpt2 O-GlcNAcylation, without changing
PA700/Rpt2 protein levels. Importantly, the changes were
enhanced in eNOS-lacking mice when rendered diabetic
with streptozotocin (p<0.05, n=10/group). In summary,
the eNOS-derived NO functions as an OGT-mediated
340
suppressive regulator of the 26S proteasome, likely through
PA700/Rpt2 O-GlcNAcylation. The lack of eNOS-derived
NO may contribute to elevated vascular 26S proteasome
functionality in early diabetes.
116: Tumorigenic potential of MEF cells transformed
with polyoma virus oncogene correlates with β-1,4galactosyltransferase V gene dosage
Chiemi Tareyanagi, Takeshi Sato, Yukiha Ishikawa and
Kiyoshi Furukawa; Laboratory of Glycobiology, Nagaoka
University of Technology, Nagaoka, Japan
furukawa@vos.nagaokaut.ac.jp
Our previous studies showed that β-1,4galactosyltransferase (β4GalT) V is a lactosylceramide
synthase and that its gene expression increases upon malignant transformation of cells. To date, there is another
lactosylceramide synthase, β4GalT VI, whose gene expression in cells appears unchanged by malignant transformation. To investigate the biological significance of the
increased gene expression of β4GalT V in tumor cells, we
introduced the anti-sense β4GalT V cDNA into B16-F10
mouse melanoma cells, and examined the tumorigenic and
metastatic potentials of the cells. The results showed that the
B16-F10 cells with decreased β4GalT V gene expression
reduce remarkably their malignant properties when compared with the control cells. This suggests that the expression level of the β4GalT V gene which finally regulates the
amounts of lactosylceramide and its derivatives is quite
important for malignant potentials of tumor cells.
In order to investigate the above study further, we first
isolated fibroblast cells from wild-type, heterozygous and
homozygous β4GalT V-knockout (B4galT5−/−) mouse embryos and transformed them with polyoma virus middle T
antigen gene. RT-PCR analysis revealed that three types of
mouse embryonic fibroblast (MEF) clones contain almost
similar numbers of the gene copies. When they were placed
into soft agarose gels, all the clones grew in them. However,
the average numbers of colonies formed were high and the
average sizes of colonies formed were large both in order of
B4galT5+/+, B4galT5+/−, and B4galT5−/− derived MEF
clones. Furthermore, the transformed MEF clones were
transplanted subcutaneously into athymic mice, and tumors
were allowed to develop for 3 weeks. The average volumes
of the tumors formed were large also in order of B4galT5+/+
, B4galT5+/−, and B4galT5−/− derived MEF clones. These
results indicate that the tumorigenic potentials of
transformed MEF clones correlate with the gene dosage of
β4GalT V, namely with the amounts of lactosylceramide
synthesized in the cells. Since lactosylceramide is the
Glycoconj J (2013) 30:281–461
precursor for a series of glycolipids, how lactosylceramide
and/or its derivatives are important for the malignat potentials in tumor cells remains to be elucidated.
117: Endoplasmic Reticulum Located ppGalNAc-T18
(GALNTL4) is Involved in Cell Survival and Apoptosis
Yan Zhang1, AiDong Shan1,2, Xing Li1, Wei Li1, AiGuo
Shen2; 1Ministry of Education Key Laboratory of Systems
Biomedicine, Shanghai Center for Systems Biomedicine,
Shanghai Jiao Tong University, Shanghai, 200240, China;
2
Department of Biochemistry, Medical College of Nantong
University, Nantong 226001, Nantong 226001, China
yanzhang2006@sjtu.edu.cn
Mucin-type O-glycosylation is initialized by members of the
UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferase
(ppGalNAc-T, EC 2.4.1.41) family. Recently, we have identified a new member, ppGalNAc-T18, also known as polypeptide GalNAc transferase-like protein 4 (GALNTL4), which
consists the vertebrate-specific Y subfamily together with
ppGalNAc-T8, -T9 and -T17. ppGalNAc-T18 localizes in the
endoplasmic reticulum (ER) and lacks classical GalNActransferase activity. However, our results have revealed that it
can selectively modulate the in vitro GalNAc-transferase activity of ppGalNAc-T2 and -T10, and it can interact with
ppGalNAc-T2 or mucin substrate. Our further study has found
that T18 was up-regulated both in apoptotic neuronal cells of
mouse brain after intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) and in apoptotic neuron-like PC12 cells after
treatment with inflammatory cytokines. Knockdown of T18
induced cells apoptosis through activating ER stress response,
and overexpression of T18 protected cells against apoptosis
induced by pathological stimuli. Our results indicated that
T18 was an essential protein for maintaining ER functions
and cells survival. These results demonstrate that T18, an ER
localized ppGalNAc-T like protein, not only could be involved
in regulating the O-glycosylation, but also participated in
maintaining ER homeostasis, it has important roles in cells
survival and apoptosis.
Physiology & Signalling
118: Functional crosstalk between cell-cell adhesion and
cell-ECM adhesion via different expression of N-glycans
Jianguo Gu; Division of Regulatory Glycobiology, Institute of Molecular Biomembrane and Glycobiology, Tohoku
Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku,
Sendai Miyagi, 981-8558, Japan
jgu@tohoku-pharm.ac.jp
Glycoconj J (2013) 30:281–461
Changes in oligosaccharide structures are associated with
numerous physiological and pathological events. The effects
of cell-cell interactions on N-glycans were investigated in
epithelial cells. The population of the bisecting GlcNAccontaining N-glycans, which formation are catalyzed by Nacetylglucosaminyltransferase III (GnT-III), was substantially increased in cells cultured under dense condition in an Ecadherin-dependent manner. In contrast, perturbation of Ecadherin-mediated adhesion abolished the up-regulation of
expression of GnT-III. Consistently, the induction of GnT-III
was not observed in embryonic fibroblasts or E-cadherindeficient cells, which was rescued by restoration of the
expression of E-cadherin. The induction was also not observed in alpha-catenin-deficient cells, but in the restored
cells with the alpha-catenin gene. Taken together, these results strongly suggest that GnT-III expression is tightly
regulated by cell-cell adhesion via the E-cadherin-catenin
complex. However, unexpectedly, GnT-III was up-regulated
by knockdown of beta-catenin or inhibition of Wnt/betacatenin signaling, resulting in an inhibition of integrinmediated cell adhesion. In fact, integrins undergo glycosylation by GnT-III inhibits cancer metastasis in vitro and in
vivo. Beta-catenin is an essential molecule both in cadherinmediated cell adhesion and in canonical Wnt signaling, the
loss of cadherin-mediated cell adhesion can promote betacatenin release and Wnt signaling, and then Wnt signaling in
turn inhibits E-cadherin-catenin-mediated cell adhesion.
Therefore, we postulate that there are positive and negative
regulation pathways for GnT-III, i.e., E-cadherin-catenin
mediated cell adhesion signaling and Wnt/beta-catenin signaling, and the intersection point is at beta-catenin. The
GnT-III expression was down-regulated in the epithelialto-mesenchymal transition (EMT) induced by TGF-beta.
Conversely, N-acetylglucosaminyltransferase V (GnT-V),
which is closely related with cell migration and cancer
metastasis, was up-regulated in the EMT. Furthermore,
overexpression of GnT-III inhibited EMT-like changes. Taken together, these results provide new insights into the
molecular mechanism of crosstalk among cell-cell adhesion
and cell-ECM adhesion during normal development, EMT
and cancer metastasis.
119: Hydrophobically modified polysaccharides as
effective sorbents of alimental cholesterol
Andriy Synytsya1, Jan Tůma1, Tomáš Taubner1, Milan
Marounek 2 , Dagmar Dušková 2 , Jana Čopíková 1 ;
1
Department of Carbohydrate Chemistry and Technology,
ICT Prague, Technická 5, 166 28 Praha 6 Dejvice, Czech
Republic; 2Institute of Animal Science, Přátelství 815, 104
00 Praha 22 Uhřiněves, Czech Republic
sinicaa@vscht.cz
341
Lowering of LDL cholesterol level in plasma of patients with
hypercholesterolemia may improve the risk of cardiovascular
diseases. Cholestyramine, a strongly basic anion-exchange
synthetic resin, is an example of effective cholesterol lowering
agent. It reduces cholesterol absorption by sequestering bile
acid conjugates and, therefore, interrupts the enterohepatic
circulation of bile acids, increases their fecal excretion and
supports compensatory oxidation of cholesterol in the liver.
However, cholestyramine also binds anionic drugs, vitamins,
and salts, and the competition for its binding sites in the
digestive tract decreases the binding capacity for bile acids.
As a result, large doses are required for the effective sorption
that may cause serious side effects for some patients.
Another way of cholesterol level lowering is the consumption of dietary fibers that can also remove bile acids from the
digestive tract. The combination of cholestyramine with
such well-tolerated polysaccharides is effective in the treatment of patients with familial hypercholesterolemia. Hydrophobic modifications may increase cholesterol lowering
effect of polysaccharides. Alkyl- or acyl-substituted polysaccharides are amphiphilic polymers with a polar sugar
backbone and non-polar hydrocarbon side chains.
Hydrophobically modified polysaccharides have a potential
value as drug carriers and sorbents for removing non-polar
compounds from aqueous media and as potential agent for
intestinal sorption of cholesterol, fats and bile acids.
N-octadecylamides of highly methylated (HM) citrus pectin
and methylesters of cellulose derivatives (monocarboxy oxidized cellulose and carboxymethyl cellulose) were obtained
by amino-de-alkoxylation with n-octadecylamine. N,Opalmitoylchitosan was prepared by reaction of chitosan with
palmitoyl chloride. This chitosan derivative was than Nmethylated (partially quarternized) with methyl iodide. Organic elemental, spectroscopic (FTIR, Raman and CP-MAS
NMR) and thermal (DSC, TG) analyses confirmed high
substitution degree of these derivatives. In vitro sorption of
sodium cholate and cholesterol by these hydrophobically
modified polysaccharides was studied in comparison with
the initial polysaccharides and cholestyramine. The Langmuir–Freundlich (Sips) model parameters were calculated
based on the experimental data. Sorption experiments with
modified polysaccharides showed that long alkyl/acyl substitution of studied polysaccharides improved their affinity
to cholesterol, while an introduction of quarternized nitrogen groups may improve the binding of cholate. It was
found that the long alkyl/acyl modified polysaccharides,
but not initial ones are able to bind bile acids and cholesterol
effectively and thus can be used as cholestyramine replacers.
However, by contrast to cholestyramine, these polysaccharide derivatives preferably adsorb cholesterol than cholate
that could be explained by their significant hydrophobicity.
342
In vivo feeding experiments on rats with high cholesterol
level diet confirmed that hydrophobically modified HM
pectin significantly altered cholesterol homeostasis in these
animals and might be considered as a clinically effective
hypocholesterolemic agent; preliminary experiments on the
alkylated cellulose derivatives achieved similar results.
This work was supported by the Czech Science Foundation
(project No 503/11/2479)
120: The mechanism underlying uptake of polysaccharides via Clathrin/Dynamin/Eps15 and Rab5 dependent
pathway
Wenfeng Liao, Dianxiu Cao, Ying Wang, Kan Ding*;
Glycochemistry & Glycobiology Lab, Shanghai Institute of
Materia Medica, Chinese Academy of Sciences, 555
ZuChong Zhi Road, Shanghai 201203, China
kding@mail.shcnc.ac.cn
Polysaccharides play multitudinous and important role in
many biological processes such as in the immune system,
fertilization, pathogenesis prevention, blood clotting, and system development. However, most glycobiologists believe that
polysaccharide can not be absorbed by cells due to its large
molecular size and the hydrophily. Here, we firstly employed
the Caco-2 model to test the in vitro permeability of several
polysaccharides extracted from plants and fungus. The results
show that the apparent permeability coefficient (Papp) values
of all the polysaccharides are more than 10×10−6 cm/s. Interestingly, molecular size of polysaccharides is not changed
significantly before and after they across the Caco-2 monolayer. Subsequently, the rat intubation surgery was employed
to study the pharmacokinetics of the polysaccharides. The
results indicates that the bioavailability of GFPBW1 (1,3linked glucan), WGE (1,4-linked glucan) and Lentinan is
69.47 %, 73.91 % and 63.89 %, respectively. In addition, the
polysaccharides even do not degrade significantly in plasma
within at least 10 h. Using FITC-labeled polysaccharides, we
found that various linked polysaccharides can internalized
into Human Intestine Mucosa Epithelial Cell (HIMC cell)
and co-localize with Clathrin, Dynamin, Rab5 and Eps15.
Blocking Clathrin-mediated endocytosis either by the selective inhibitor Pitstop-1 or using small interfering RNA can
reduce the internalization of the polysaccharides. Furthermore, silence of Dynamin-1, Dynamin-2 and Rab5 in HIMC
cells by small interfering RNA or mutant Eps15 can also
abolish polysaccharides internalization. These data suggest
that polysaccharides may enter intestine cells by a
Clathrin/Dynamin/Eps15 and Rab5-dependent pathway. This
work provides novel evidences on the absorption of
polysaccharides.
Glycoconj J (2013) 30:281–461
121: Involvement of Ganglioside GD3-raft Signaling
in the Growth Cone Behavior of Cerebellar Granule
Neurons
Kohji Kasahara; Laboratory of Biomembrane, Tokyo
Metropolitan Institute of Medical Science,Tokyo 156-8506,
Japan
kasahara-kj@igakuken.or.jp
Gangliosides are known to exist in clusters and form
microdomains containing cholesterol at the neuronal membrane called rafts and are thought to play functional roles in
neuronal differentiation. Previously we isolated ganglioside
GD3 synthase cDNA and investigated expression of GD3
synthase. To clarify GD3 raft-mediated neuronal function,
we have been investigating the association of ganglioside
GD3 with specific proteins in the nervous system.
Anti-ganglioside GD3 monoclonal antibody (R24) coimmunoprecipitated GPI-anchored neuronal cell adhesion
molecule TAG-1, src-family tyrosine kinase Lyn and Cskbinding protein Cbp from cerebellar granule neurons. TAG1 has neurite extension activity and is transiently expressed
on premigratory granule neurons in the external granule cell
layer. Antibody-mediated cross-linking of TAG-1 or GD3
induced Lyn activation and tyrosine phosphorylation of Cbp
in primary cerebellar granule neurons. R24 also coimmunoprecipitated α subunit of heterotrimeric G protein,
Go (Goα). Treatment with SDF-1, a ligand for the G
protein-coupled receptor, stimulated GTPγS binding to Go
and caused Goα translocation to the rafts, leading to the
growth cone collapse of cerebellar granule neurons. Migration of cerebellar granule neurons is known to be impaired
in TAG-1 or SDF-1-deficient mice.
Immunoblotting analysis showed that GD3, TAG-1, active form of Lyn, tyrosine phosphorylated form of Cbp
and Goα were highly accumulated in the detergentresistant membrane raft fraction prepared from the developing cerebellum. In addition, GD3, TAG-1, Lyn,
phosphorylated Cbp and Goα were also concentrated
in the growth cone fraction. These data suggest that
GD3 rafts are signaling platforms of growth cone behavior in migrating cerebellar granule neurons.
1)
2)
3)
4)
5)
6)
7)
8)
Nara et al. (1994) Proc.Natl.Acad.Sci. 91, 7952–7956
Kasahara et al. (1997) J.Biol.Chem. 272, 29947–29953
Kasahara et al. (2000) J.Biol.Chem. 275, 34701–34709
Kasahara et al. (2000) Glycoconj.J. 17, 153–162
Kasahara et al. (2002) Neurochem.Res. 27, 823–829
Yuyama et al. (2007) J.Biol.Chem. 282. 26392–26400
Yuyama et al. (2011) J.Neurochem. 116, 845–850
Sekino-Suzuki et al. (2013) J.Neurochem. 124, 514–522
Glycoconj J (2013) 30:281–461
122: Network-based identification of microRNAmodulated programmed cell death pathways in plant
lectin-induced cancer death
Leilei Fu, Jingchun Zhang, Yongxi Liu, Jinku Bao*;
School of Life Sciences, Sichuan University, Chengdu,
610064, China
baojinku@scu.edu.cn
Plant lectins, carbohydrate-binding proteins of non-immune
origin, have recently been reported to induce programmed
cell death (PCD, referring to autophagy and apoptosis) in a
variety types of cancer cells. MicroRNAs (miRNAs), small,
non-coding endogenous RNAs, ~22 nucleotides (nt) in
length, have been well characterized to play essential roles
in regulation of the PCD in cancer; Identification of
miRNA-regulated apoptotic or autophagic pathways was
carried out through a serious of elegant bioinformatics approaches, such as the human apoptotic and autophagic
protein-protein interaction (PPI) network construction, the
modification of this network into a plant lectin-induced
network, hub protein identification, targeted miRNA prediction, microarray analyses and molecular docking, and finally experimental validation. Together, these results
demonstrated that network-based identification of miRNAmodulated apoptotic or autophagic pathways in plant lectininduced cancer death may shed new light on the discovery
of plant lectins as potent apoptotic or autophagic inducers,
for future cancer drug discovery.
Society for Glycobiology: Model Systems
in Glycobiology
123: Pathways Regulating the Expression of N- and Olinked Glycans in Drosophila
343
within glycan diversity, potentially revealing nodes of regulation. Although its small size might be thought to preclude
detailed glycomic analysis, as a model system the embryonic,
larval, and adult stages of Drosophila offer unique opportunities to investigate glycan expression. Advances in the sensitivity and robustness of mass spectrometry over the last few
decades have made it possible to harvest large amounts of
information from small amounts of tissue, allowing a comprehensive analysis of the Drosophila glycome. Dominated by
high mannose structures, the N-linked glycome of the Drosophila embryo is also populated with minor glycans of significant complexity, even glycans bearing sialic acid. The Olinked glycome includes structures seen in vertebrate species
but also glycans containing novel composition and linkage.
Therefore, both the N- and O-linked glycome of the Drosophila embryo are characterized by some similarity to vertebrate
glycomes as well as some novelty, providing a fertile domain
in which to investigate the regulation of glycan expression. To
that end, we have undertaken genetic screens and reverse
genetic manipulations in order to identify key genes that control the expression of neural-specific N-linked glycans and of
O-linked glycans relevant for developmental signaling pathways. Our combined genetic and glycomic analyses have
demonstrated an important role for intercellular signaling,
mediated by toll-like receptors and facilitated by specific adaptor proteins and by specific post-translational modifications, in
regulating the organization and function of the Golgi apparatus. Furthermore, altered expression of a glycosyltransferase
that caps O-linked glycans leads to catastrophic loss of female
fertility, but only in a specific genetic background—one that
has otherwise been considered wild-type for most of the past
century. Thus, Drosophila glycobiology is revealing the importance of signaling networks, subcellular organization, and
the complex interplay of genetic background in regulating
glycan expression in animals.
Michael Tiemeyer 1 , 2 , Varshika Kotu 1 , 2 , Marion
Marshall 1,2, Nickita Mehta 1,2, Mindy Porterfield 1,3,
Tadahiro Kumagai1, Toshihiko Katoh1, Kazuhiro Aoki1;
1
Complex Carbohydrate Research Center, University of
Georgia, Athens, GA, 30602, USA; 2Department of Biochemistry and Molecular Biology, University of Georgia,
Athens, GA, 30602, USA; 3Department of Chemistry, University of Georgia, Athens, GA, 30602, USA
124: Sialoglycoconjugates in Organ Development: A
Study in Mouse and Zebrafish
Glycan expression is regulated in all multicellular organisms,
producing patterns of protein and sphingolipid glycosylation
that are specific for developmental stage or characteristic of
tissue type. Mechanisms that regulate this universal feature of
animal development remain largely unknown. With increasing
attention focused on describing cellular, tissue, and organismal
glycomes, there is hope that patterns may become recognizable
The acidic nine-carbon sugar sialic acid (Sia) terminates the
bulk of oligosaccharide chains present on cell surface components and circulating glycoproteins. For a long time Sia was
regarded as a mere carrier of negative charge. However, the
last decades disclosed that Sia is involved in virtually all areas
of vertebrate life. The activation of Sia to CMP-Sia by CMPSia synthetase (CMAS) is a prerequisite for the biosynthesis
Anja-Katharina Muenster-Kuehnel, Wiebke Schaper,
Birgit Weinhold, Rita Gerardy-Schahn; Institute for
Cellular Chemistry, Hannover Medical School, 30625
Hannover, Germany
Gerardy-Schahn.Rita@mh-hannover.de
344
of sialoglycoconjugates. Only the activated sugar can be
transferred into the Golgi apparatus and used by
sialyltransferases to build specific sialo-glycotopes. In order
to study the role of sialo-glycotopes in organogenesis, mouse
models with constitutive or tissue specific defects in the
cellular sialylation process have been generated. Importantly,
these models demonstrate that sialo-glycotopes provide indispensable information not only for proper cellular functions but
also for the shaping of anatomical structures.
Recently, we identified two Danio rerio genes with high
sequence homology to known CMAS. The isolated zebrafish
cDNAs encode proteins with high resemblance in primary
sequence and quaternary structure. However, the zebrafish
enzymes (zfCMAS1 and 2) differ in terms of substrate
specificity and subcellular localisation. Initial results that
establish zebrafish as a suited model organism to study the
spectrum of CMAS functions will be discussed.
125: Heparan sulfate Modifications in Development and
Disease
Hannes E. Bülow; Albert Einstein College of Medicine,
Department of Genetics, Bronx, NY 10461, USA
Heparan sulfates (HS) are un-branched extracellular glycosaminoglycans exhibiting substantial molecular diversity due
to multiple, non-uniformly introduced modifications including sulfations, epimerization and acetylation. HS modifications serve specific and instructive roles in neuronal
development leading to the hypothesis of a HS code that
regulates nervous system patterning by modulating
ligand/receptor interactions. By visualizing specific HS
modification patterns in the nematode Caenorhabiditis
elegans we provide direct evidence for the existence of
neuron-specific HS modifications in living animals. Our
genetic studies establish that distinct combinations of
HS modification patterns are important for development
of the nervous system. HS 3-O-sulfation is the most
enigmatic of all HS modifications due its rarity and a
largely expanded family of enzymes predicted to introduce this modification. We show that animals lacking
all HS 3-O-sulfation are superficially normal. Moreover,
the general structure of the nervous system is unaffected.
Intriguingly, we find highly specific processes during
neural development affected by lack of different HS 3-Osulfatransferase genes suggesting that HS 3-O-sulfation may
play a combinatorial role in establishing the correct neural
circuitry of metazoans. Lastly, we show that mutations in
human genes encoding HS sulfotransferase result in neurological disorders which also affect specific aspects of neural
development.
Glycoconj J (2013) 30:281–461
126: O-glycoprotein podoplanin initiates and maintains
separated blood and lymphatic vascular systems
Lijun Xia; Cardiovascular Biology Research Program,
Oklahoma Medical Research Foundation, 825 N.E. 13th
Street, Oklahoma City, Oklahoma 73104, USA. Phone:
(405) 271–7892
lijun-xia@omrf.org
Mucin-type O-glycans (O-glycans) are highly expressed in
vascular endothelial cells. However, it is not known whether
endothelial O-glycans are important for vascular development. To investigate the roles of endothelial O-glycans, we
generated mice lacking T-synthase (T-syn), a critical
glycosyltransferase for biosynthesis of core 1-derived Oglycans, in endothelial cells (EHC T-syn−/−). EHC T-syn−/−
mice exhibited disorganized and blood-filled lymphatic vessels. We found that the O-glycoprotein podoplanin was
significantly reduced in EHC T-syn−/− lymphatics, and
podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn−/− defects. In addition, we
and others have identified that interaction between
podoplanin on lymphatic endothelial cells and platelet
CLEC-2 activates SYK/SLP-76 signaling in platelets, which
induces platelet aggregation that seals initial embryonic
blood-lymphatic vascular connections during embryonic
development. Recently, we show that this interaction is also
important for preventing blood from entering the lymphatic
system at the lympho-venous junction of the thoracic duct
and subclavian vein. These results reveal an essential role of
podoplanin for the development and maintenance of separated blood and lymphatic systems.
WorkshopI: Glyco-bioinformatics
127: Recent Efforts of Global Collaborations for
Interconnecting Glycan-related Databases on the
Semantic Web
Hiromichi Sawaki1, Kiyoko F. Aoki-Kinoshita2, Issaku
Yamada 3 , Shin Kawano 4 , Shujiro Okuda 5 , Masaki
Kato6, Hisashi Narimatsu1; 1Research Center for Medical
Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki
305-8568, Japan,2Department of Bioinformatics, Faculty of
Engineering, Soka University, Hachioji, Tokyo, 192-0003
Japan, 3The Noguchi Institute, Tokyo 173-0003, Japan,
4
Database Center for Life Science (DBCLS), Tokyo, 1130032, Japan, 5RitsumeikanUniversity, Kusatsu, Shiga, 5258577, Japan, 6RIKEN Advanced Science Institute, Wako,
Saitama, 351-0106, Japan
h.narimatsu@aist.go.jp
Glycoconj J (2013) 30:281–461
Recent high-throughput technologies for life-science research has been generating large amount of data and enlarging the size and numbers of storing databases. Technologies
for building the present databases cannot keep up with the
large amounts of data. Thus, bioinformatics researchers
have turned to the Semantic Web as the next-generation of
the World Wide Web to reconstruct life science databases
such that Big Data can be handled effectively.
Also, in Japan, the Integrated Database Project has conducted
to developing infrastructures and reconstructing life-science
databases from 2010. JCGGDB project is the glycan part of
the Integrated Database Project in Japan. We have surveyed
available resources for glycol-science fields in the world for
adopting glycoscience data to the Semantic Web technologies
in 2011, and felt that international collaboration is necessary
for this purpose. Then, we have call up a meeting to
glycoscience researchers who hold databases by themselves
at 2012. ACGG-DB3 meeting has been successfully held
in Okinawa, Japan. 25 attendees from 17 research institutes in 7 countries or areas have discussed actively
glyco-informatics and the need for collaborations in a
global framework (PMID: 23271684). Based on consensus at the Okinawa meeting, concrete development was
started and the first collaboration was organization of
glycan-team at the DBCLS BioHackathon 2012 in Toyama,
Japan, which consisted of 9 researchers representing for 5
databases; CSDB, GlycomeDB, GLYCOSCIENCES.de,
JCGGDB and UniCarbKB.
In the presentation, discussion and progress at these meetings will be reported.
This work was supported by a grant from Japan Science and
Technology Agency (JST).
128: WURCS: Unique Representation of Carbohydrate
Structures for Semantic Web
Issaku Yamada1, 2;1Laboratory of Glyco-organic Chemistry,
The Noguchi Institute, Tokyo, 173-0003, JAPAN, 2JST, National Bioscience Database Center (NBDC), Tokyo, 1020081, JAPAN
issaku@noguchi.or.jp
The Web3 Unique Representation of Carbohydrate Structures (WURCS) is a new linear notation for representing
carbohydrates for Semantic Web. In the next-generation of
the Web, it is expected that a new technology will be able to
utilize various data on the Web as a huge database. In order
to use the data effectively, it is necessary to develop a
method to represent the carbohydrate structure with the
same meaning. This new linear notation can represent
345
ambiguous and incomplete carbohydrate structures as
unique character strings. Furthermore, anyone can generate
WURCS with the same character strings related to the
equivalent carbohydrate structure in glycobiology. For
example, lactose is represented by the character strings
of “[12112h|1,5]1:1,4:2[X2122h|1,5]”. By applying
WURCS in a Web, a user will be able to use a virtual
huge database.
129: UniCarbKB: Building a Glycomics Platform in the
Cloud
Matthew P Campbell1, Jingyu Zang1, Julien Mariethoz2,
Elisabeth Gasteiger2, Catherine A Hayes3, Niclas G
Karlsson3, Frederique Lisacek2, Nicolle H. Packer1;
1
Biomolecular Frontiers Research Centre, Macquarie University, Sydney, Australia; 2Swiss Institute of Bioinformatics, Geneva, Switzerland; 3Department of Biomedicine,
Gothenburg University, Sweden
matthew.campbell@mq.edu.au
The UniCarb Knowledgebase (UniCarbKB) is an effort to
develop and provide an open access bioinformatic framework for the storage of high-quality data collections on
glycoconjugates, including informative meta-data and MIRAGE compliant experimental datasets. Initial efforts have
focused on the reengineering of GlycoSuiteDB and
EUROCarbDB to provide a unified view of structural, experimental and functional information that acts as a central
hub of glycoproteomics knowledge.
This corpus of data is rich in information, but there is a
distinct growing gap in our knowledge of glycoproteins, in
terms of functional information and the extent of glycosylation heterogeneity and site occupancy. The annotation of
individual sites provides more detailed insights between
sequence and function, as well as context for the interpretation of experimental data. In general, site annotation has to
be extracted from the published literature, and can often be
transferred to closely related sequence neighbours.
Here, we describe our approach to reorganise and curate
existing data collections associated with proteins in a sitespecific manner both from the attachment and the recognition perspective. Furthermore, we showcase our efforts to
control high-quality data depositions, notable the inclusion
of the glycosylated structures characterised from over 100
glycoproteins localised in different tissues and body fluids
and its integration with the UniProt and NextProt platforms.
These entries are published in a Semantic Web compatible
format (GlycoRDF), which is now facilitating integration
with other international resources by making connections
346
between datasets. Ultimately, this will allow researchers to
query and navigate between multiple data collections and
make discoveries that were previously not possible.
UniCarbKB offers a unique approach to access a comprehensive biocurated overview of existing glycoinformation.
The initiative is driven as a community endeavour to promote data sharing in glycobiology and ensure its future
development and growth.
130: MIRAGE: Minimum Information Required for A
Glycomics Experiment
René Ranzinger, Carsten Kettner and Will S. York;
Complex Carbohydrate Research Center, University of
Georgia, United States
rene@ccrc.uga.edu
Recent technical advances in analytical glycomics and
glycoproteomics have enhanced our ability to understand the
critical roles that complex glycans play in tissue and cell
development and in the progression of disease. These techniques can produce extremely large amounts of data, and only
a small portion of this data is usually reported in publications or
databases describing the results of glycomics or
glycoproteomics experiments. Members of the glycomics community have agreed that defining a minimum set of information
regarding the techniques used to obtain these experimental
results would allow scientists to better appreciate the depth
and scope of each analysis and to reproduce the results. Therefore, an initiative to develop an international standard for the
Minimum Information Required for A Glycomics Experiment
(MIRAGE) was established at the 2nd Beilstein Symposium on
Glyco-Bioinformatics in Potsdam Germany in June, 2011. The
MIRAGE working group (http://glycomics.ccrc.uga.edu/
MIRAGE/) has recently finalized and published the glycomics
MS guidelines and is currently working on guidelines
for other experimental techniques, such as Glycan Array
analysis, LC, HPLC and CE. The aim of MIRAGE project
is not to constrain how experiments should be performed but
rather to provide guidance regarding the information that is
required to adequately describe each type of experiment. By
listing the essential information to be provided, these guidelines are of great importance not just for authors and reviewers
of publications but also for software and database developers.
Developers can profit from the MIRAGE guidelines in that
they serve as a foundation for defining technical specifications
for applications or databases that generate or consume key
information. However, the extensive set of information specified by the MIRAGE guidelines requires intelligent implementation strategies to simplify data annotation and
submission.
Glycoconj J (2013) 30:281–461
131: Informatics Tools for Glycoscience Research
Kiyoko F. Aoki-Kinoshita; Soka University, Faculty of
Engineering, Dept. of Bioinformatics, Hachioji, Tokyo,
192-8577, Japan
kkiyoko@soka.ac.jp
Advancements in glycomics technologies have enabled more high-throughput generation of data such as glycan structure characterization of specific cells and tissues, and lectinand glycan-binding affinities. In turn, the need for applicable software and tools has increased, and many software and
web resources are being developed to allow such data to be
analyzed efficiently and effectively.
The RINGS (Resource for INformatics of Glycomes at
Soka) web resource at http://www.rings.t.soka.ac.jp provides a number of such tools. One of the first data mining
tools for analyzing glycan structure patterns is called Profile
PSTMM. This model augmented the linear Markov model
originally used for amino acid sequences to include
branched structures that had dependencies between siblings.
Thus wider-range patterns that were expected to be found in
glycan structures could be extracted rather efficiently. Another tool is the Glycan Miner Tool, which mines large
numbers of glycan structures to find common substructures
of sufficient size and diversity. There is also the Glycan
Pathway Predictor (GPP) which is based on a mathematical
model for predicting N-glycan biosynthesis for a number of
glycosyltransferases. In addition to these tools, a number of
utilities for converting between various glycan structure text
formats are also available.
Many other algorithms have also been developed, such as
for analysis of glycan biosynthesis at a systems level and
motif finding using glycan array data. Some of these have
been made available as free software as well.
132: Enhancement of the search function in JCGGDB
Toshihide Shikanai, Masako Maeda, Noriaki Fujita,
Yoshinori Suzuki, Hongling Wen, Madoka Ishizaki,
Hiromichi Sawaki, Hiroyuki Kaji, Hisashi Narimatsu;
Research Center for Medical Glycoscience (RCMG),
National Institute of Advanced Industrial Science and
Technology (AIST), Tsukuba, Japan
As entering the final year of JST/NBDC integrated database
project in 2013, we are driving the development of
JCGGDB (Japan Consortium for Glycobiology and
Glycotechnology DataBase) to achieve the integration of
Japanese databases as well as international collaboration.
Glycoconj J (2013) 30:281–461
JCGGDB was selected as a promotion program in the project, aiming to complete the integration of all the glycanrelated databases in Japan and build user-friendly search
systems. As a part of the project, we also intend a DB
integration within Asia and construct ACGG-DB (Asian
Communications of Glycobiology and Glycotechnology
DataBase) in cooperation with Asian countries.
So far we have consolidated data from various Japanese
institutes into JCGGDB and developed a couple of search
functions such as cross-search by keyword entry and integrated search by glycan structures.
In addition, “Glyco”-Keywords have been collected using textmining technique to analyze the frequency of keywords as well
as the relationship between them. Collected data is also used to
organize the synonyms and similar terms of glycan structures.
We are developing retrieval techniques which enables to display those co-occurring words as Related Words in the DB
and exchange links with related databases individually.
Working closely with Japanese and Asian institutes, we will
continuously develop base technologies for the DB integration, facilitate interactions between databases in the field of
glycoscience as well as other associated study areas, and
build bioinformatics tools to support experimental study.
Our goal is to create contents which could be easily and
intuitively understood by every user.
This work was supported by Integrated Database project in
JST/NBDC in Japan.
JCGGDB web site : http://jcggdb.jp
133: CSDB and other carbohydrate databases
Philip Toukach; N.D. Zelinsky Institute of Organic Chemistry, Leninsky prospekt 47, Moscow, 119991 Russia
netbox@toukach.ru
Nowadays, the orientation in a currently acquired volume of glyco-related information is impossible without
special features of informatics. Therefore, the progress
of glycobiology strongly depends on a presence of an
information environment including data on structures,
properties and functions of carbohydrates, as well as
on taxonomy and properties of their biological sources.
The main approach to create such environment is development of carbohydrate databases. In contrast to
genomics and proteomics, informatization of glycomics
is still suffering from incompatibility between the
existing projects. In the present mini-review I report a
comparative analysis of currently active carbohydrate
347
databases, with Russian Carbohydrate Structure Databases1 (CSDBs) emphasized.
Glyco-databases providing wide coverage are most demanded,
among them meta-database GlycomeDB, GLYCOSCIENCES,
GlycoSuiteDB, CFG Glycan Database, KEGG, JCGGDB,
GlycoBase-Dublin, UniCarbKB, Bacterial&Archaeal CSDB,
and Plant&Fungal CSDB (currently in development) and other.
Historically first carbohydrate database, CCSD (Carbbank)
pretended to have complete coverage of structures published
before 1996, when its support was ceased. Collection and
digitizing of primary data are the most time-consuming stages
of a database development, and therefore almost all modern
projects somehow use the Carbbank data.
Analysis of the distinctive features of various projects
allows to establish the criteria of database evaluation: types of
data stored, completeness of coverage, data quality, functions
provided to users, interface (usability, stability and performance), integration with other projects, and database architecture. Although the last criterion is invisible to users, it has a
strongest impact on a database usefulness, since architectural
mistakes hamper maintainability, upgradability and error control, and continuously increase the cost of a project.
The minimal types of data stored and processed in a glyco
database are a primary molecular structure and taxonomical
and bibliographic annotations. Many databases store analytical data, such as NMR or MS spectra. Storage of biochemical, genetic, medical and other related data is often
supported, but their coverage remains poor. Of major carbohydrate databases, KEGG lacks for taxonomical annotations, and GlycomeDB for bibliographic annotations. The
databases with stored NMR spectra provide a spectral coverage of 5– 25 % of the published data.
Higher coverage significantly increases the value of a database,
since even a negative answer to a search request presents
valuable scientific information. Restricted potential of automatization of a search for suitable publications limits the acquisition of primary data and, therefore, the coverage. Nowadays
only Bacterial CSDB reports almost complete (>75 %) structural coverage within a chosen compound class, namely glycans
from procaryotic microorganisms. Since 2005 it has accumulated ca. 10 000 structures assigned to 5 000 microorganisms in
4 000 publications.2 Newly established Plant&Fungal CSDB
aims at achieving this level of coverage in 2016, and currently
deposits re-annotated data published before 1996.
1
CSDB web-site (2012) Bacterial, plant and fungal Carbohydrate
Structure Databases, http://csdb.glycoscience.ru
2
Ph.V. Toukach (2011) Bacterial Carbohydrate Structure Database 3:
Principles and Realization J Chem Inf Model 51, 159–170.
348
To keep the coverage actual, periodic updates are needed,
which assumes one- or two-year lag between publication
and deposition of data. A universal solution for keeping the
data actual is a requirement of obligatory upload of every
published structrure to a database prior to publication with
subsequent provision of the obtained IDs to an editorial. Such
approach has been realized in genomics for long but is still
missing in glycomics. One of the reasons for that is an insufficient standardization of glycan description languages originating from high chemical variativity of carbohydrates. This
problem, as well as limited cross-project compatibility, can be
overcome by source-independent data framework, such as
RDF. Several databases, including CSDB, GlycomeDB,
UniCarbKB and other provide export of data as RDF triples
according to an experimental version of GlycanRDF ontology
formulated at Biohackathon 2012, Japan.
The process of data posting can hardly be automatized not
only at the level of publication selection but also at the level of
article interpretation. As a result, all chemical and biological
databases contain errors. These errors originate from (in
occurrence-descending order): annotators’ failures, other
imported databases, original publications, architectural inconsistencies, bugs in import and auto-annotation software.
According to our investigation,3 most records in Carbbank
contain errors, and more than one third of records contain two
or more errors. The most abundant error type is an incorrect
taxonomical annotation of a structure. Significant gaps in the
Carbbank coverage were also discovered. As most of the
modern projects use the Carbbank data, these errors are being
reproduced. Some of them can be revealed and sometimes
corrected automatically. Such control is present in a number of
databases; however, only a retrospective expert analysis of
publications can provide really high data quality. Two thirds of
CSDB budget is devoted to manual literature processing.
Database functionality is its capability to process various search
requests, combine and refine them using diverse logics and
other types of queries. E.g., “find all structures published from
2001 to 2005, that contain either an α-Gal(1–>3)KDO fragment or a monosaccharide-bound lysine or alanine, except
synthetic structures or those found in gamma-proteobactertia,
and display their 13C NMR spectra”. In contrast to a search for
bibliography, taxonomy, keywords, text fragments and similar
data, a search for structural fragments in bigger molecules (as
well as for structures or spectra resembling a specified one)
requires more meticulous programming and computational
power, making the inner database architecture critical for the
performance of such queries. In the mid-2000s, developers of
GLYCOSCIENCES.de formulated “Ten golden rules of
3
K.S. Egorova, Ph.V. Toukach (2012) Critical analysis of CCSD data
quality. J Chem Inf Model 52, 2812–2814.
Glycoconj J (2013) 30:281–461
carbohydrate database development”, which summarized
the experience of the German and Russian groups. The
key points of this document include usage of a connection
table for inward structure representation, maximal possible
indexation, minimum of free text data (which, regretfully,
are present in virtually every project), and unambiguously
controlled vocabularies for as much data types as possible.
An attempt to separate the monosaccharide vocabulary from
glyco-databases was made within MonosaccharideDB.
Nowadays it provides full coverage on monosaccharides
present in mammalian glycans.
Possibility of correct processing of structural data is directly
related to the format of both internal and user structure
descriptions. Incapabilities and inter-incompatibility of glycan description languages have been limiting the progress of
glycoinformatics for decades. The main criteria of carbohydrate language efficiency are: 1. unambiguity and uniqueness of every chemically distinct structure, including noncarbohydrate moieties; 2. support of all structural features of
carbohydrates and glycoconjugates (single and multiple repeats, cyclic and combined glycans, glycolipids, glycoproteins, non-carbohydrate and untypical constituents,
phospho- and sulfo-linkages, cyclic esters, amide and ether
linkages, etc.); 3. support of underdetermined structures at the
level of monomers and their configurations, stoichiometry,
substitution positions, and chain topology; 4. computerreadability with no need for ambiguous parsing, as in the case
of Extended JUPAC, and human-readability required for
tracking of errors that appear during human processing of data
dumps; 5. compatibility with other formats (presence of converters that help language learning and cross-database operations), e.g. monomer vocabulary widely recognized by
glycobiologists. The CSDB Linear and GlycoCT languages
possess most of these features. However, the former does not
support nested repeats and have limited aglica support, and the
latter is not human-readable and supports carbohydrate moieties only. Glycomics still lacks a standard language except the
JUPAC, which is highly imperfect.
The functionality can be extended by carbohydrate-related
services, such as conformation map simulation, spectra prediction, search for structural motifs etc. CSDB project provides a
generator of structural variety restricted by user constraints
determined from simple experiments (e.g. number of residues
per repeat and other data). This approach is a gateway to
theoretical structural elucidation and ranking based on experimental data, and can be applied to this or to any other glycan
database. Every structure in the generator output is a subject to
either database-driven averaging or fully theoretical simulation
of properties. Among these properties are the NMR spectra. As
realized in CSDB, 13C NMR simulation combines both
environment-dependent database search and statistical
Glycoconj J (2013) 30:281–461
processing, and empirical incremental prediction of chemical
shifts. To find a chemical shift in a database a twelve-step
generalization of atomic environment is applied. As well as
the other related projects (CASPER, BIOPSEL), this feature is
aimed at dramatic simplification of natural carbohydrate structural studies.
Modern quality standard in informatics implies that both
user and administrative interfaces are intuitive, welldocumented and freely accessible via Internet. Intuitiveness
includes structure input and output formats, which users
should not have to study. Standalone services for structure
input and visual editing are of extreme usefulness, as they
allow users of any database to stay within the interface
which they got used to. Cross-project integration implies
not only common interface of search requests but also
automated data interchange via API. It concerns interactions
with non-carbohydrate databases as well, at least with
NCBI Taxonomy and NCBI Pubmed. First two projects that
reported protocols for automated data exchange were
GLYCOSCIENCES.de and Bacterial CSDB, and since then
the development of glyco-related web-services has intensified.
Within the Bacterial CSDB project, we tried to develop the
database architecture and to realize it in software free of
disadvantages of other glyco-databases. Since then Bacterial
& Archaeal CSDB has been maintained and regularly
updated. In 2012, we started its expansion to plant and
fungal carbohydrates, being the pre-last step to creation of
a complete database of natural glycans, which, we hope, will
ideologically replace Carbbank.
WorkshopII : Proteoglycans
134: Keynote Lecture: Exploiting the heparan sulphate
interactome: from biology to synthetic chemistry to therapeutic applications
J. E. Turnbull; Centre for Glycobiology, Institute of
Integrative Biology, University of Liverpool, Liverpool,
L69 7ZB, UK
j.turnbull@liverpool.ac.uk
Heparan sulfates (HSs) are complex sulfated polysaccharides
present on almost all cell surfaces in multicellular organisms.
They have variant structures which represent a molecular code
that is a subset of the glycome called the ‘heparanome’. These
sulfation patterns confer the ability to interact selectively with
a wide interactome of proteins that influences many cellular
processes important in the development, regulation and repair
of tissues. Understanding the chemical biology of these enigmatic molecules is now becoming possible through a variety
349
of tools, reagents and approaches including saccharide libraries, microarray methods and novel sequencing approaches.
We are developing and exploiting semi-synthetic and synthetic chemistry strategies to produce targeted libraries for activity
screening to decode the molecular basis of the functional
diversity of HS. This has yielded new insights into this code
in a variety of biological contexts, including neural development, degeneration and repair, and cancer metastasis. This has
led to a translational pathway of biomedical applications,
including discovery of novel drug leads for Alzheimers disease and cancer metastasis, and potential interventions to
improve neural cell transplantation for nerve repair.
135: Synthesis of Heparan Sulfate Hexa- to
Dodecasaccharides as Inhibitors of the Alzheimer’s Disease
Target β-Secretase
Ralf Schwörer1, Jeremy E. Turnbull2, Peter C. Tyler1,
Olga V. Zubkova1; 1Carbohydrate Chemistry, Callaghan Innovation Research Limited, Lower Hutt, 5040, New Zealand;
2
Institute of Integrative Biology,University of Liverpool, Liverpool, L69 3BX, UK.
peter.tyler@callaghaninnovation.govt.nz
Heparan sulfates (HS) are a class of sulfated polysaccharides which function as dynamic biological regulators of the functions of diverse proteins. The structural
basis of these interactions however remains elusive, and
chemical synthesis of defined structures represents a
challenging but powerful approach for unravelling the
structure-activity relationships of their complex sulfation
patterns. HS has been shown to function as an inhibitor
of the beta-site cleaving enzyme β-secretase, a protease responsible for generating the toxic A-beta peptides that accumulate in Alzheimer’s disease (AD), with 6-O-sulfation
identified as a key requirement. Here we demonstrate a novel
generic synthetic approach to HS oligosaccharides applied to
production of a library of 16 hexa- to dodeca-saccharides,
targeted at β-secretase inhibition. Screening of this library
has provided new insights into structure-activity relationships
for optimal β-secretase inhibition, and yielded a number of
potent non-anticoagulant inhibitors with potential for development as leads for treatment of AD through lowering of Abeta peptide levels.
136: Developing an enzymatic approach to synthesize
heparin and heparin
Jian Liu; Division of Chemical Biology and Medicinal
Chemistry,Eshelman School of Pharmacy; University of
North Carolina, Chapel Hill, NC 27599
350
Heparan sulfate is a sulfated glycan that exhibits essential
physiological functions, and heparin is a clinically used
anticoagulant drug. Interrogation of the specificity of
heparan sulfate-mediated activities demands a library of
structurally defined oligosaccharides. Synthesis of heparan
sulfate using enzymes provides a promising approach because of the high regioselectivity of heparan sulfate biosynthetic enzymes. The synthesis of heparan sulfate involves 15
different enzymes, including sulfotransferases, an epimerase
and glycosyltransferases. Up to now, a number of oligosaccharides with different sulfation patterns and sizes were
synthesized. These oligosaccharides are now used to probe
the biosynthetic mechanism of heparan sulfate and heparin,
improving the synthesis of heparin drugs, and understanding
the interaction of heparan sulfate with proteins. The availability of structurally defined heparan sulfate oligosaccharides will significantly advance the ability to understand the
biology of heparan sulfate.
137: RAGE: A proteoglycan-associated receptor
Ding Xu*1, Jeffrey Young1, Kevin D. Corbett1,3, Walter
J. Chazin4, Lars C. Petersen2 and Jeffrey D. Esko1;
1
Department of Cellular and Molecular Medicine,
Glycobiology Research and Training Center, University of
California, San Diego, La Jolla, CA 92093, 2Laboratory of
Structural Biology, National Institute on Environmental
Health Sciences, National Institutes of Health, Research
Triangle Park, NC 27709, 3Ludwig Institute for Cancer
Research,4Department of Biochemistry, Center for Structural
Biology, Vanderbilt University, Nashville, TN 37232
Many cell surface receptors rely on heparan sulfate for
activity, but depend on different modes of interaction. Fibroblast growth factor (FGF) receptors form 2:2:2 complexes with heparin and FGF ligands. Based on
crystallization studies, the non-reducing end of each heparin
chain docks with one FGF and a canyon defined by the two
receptors. In contrast, vascular endothelial growth factor
(VEGF) receptors depend on heparan sulfate for activity
presumably due to interaction with a heparin-binding
coreceptor, such as neuropilin-1, or for stabilization of receptor oligomers induced by ligand binding. In this system,
interaction of the ligand with heparin is not required. Recently, we described a third model based on the receptor for
advanced glycation end products (RAGE), which also requires heparan sulfate for activity. Here we show that RAGE
undergoes a heparin-dependent, ligand-independent oligomerization, generating a stable hexamer of the RAGE extracellular domain. Site-directed mutagenesis mapped two
separate heparan sulfate-binding sites in the Ig-like V and
C1 domains of RAGE. A hydrophobic dimeric interface
Glycoconj J (2013) 30:281–461
essential for the formation of hexamer was also identified
in the V domain. Crystallization of RAGE V-C1 in the
presence of a heparin dodecasaccharide revealed a
hexameric structure in the crystal lattice, consistent with
complex composed of a trimer of dimers. A solution
structure consistent with a hexamer was also observed
by small angle X-ray scattering. Mechanistically, the
data suggests that heparan sulfate binds to a positively
charged cleft created by two subunits of the dimer,
stabilizing an otherwise weak hydrophobic dimeric interface and promoting dimer formation and assembly
into a hexamer. A monoclonal antibody targeting the
hydrophobic interface blocks receptor signaling, demonstrating that oligomerization is required for signaling.
Thus, RAGE defines a third mode of interaction in
which heparan sulfate is a subunit of the holoreceptor.
138: Receptor for Advanced Glycation End-products
(RAGE) Functions as Receptor for Sulfated Glycosaminoglycans (GAGs): Anti-RAGE Antibody or Sulfated
GAGs Inhibit Lung Metastasis of Tumor Cells
Kazuyuki Sugahara, Jun Takahashi, Shuji Mizumoto;
Hokkaido University Graduate School of Life Science,
Sapporo, Japan 001-0021
k-sugar@sci.hokudai.ac.jp
Altered expression of chondroitin sulfate (CS) and heparan
sulfate (HS) at the surfaces of tumor cells plays a key role in
malignant transformation and tumor metastasis. Previously
we demonstrated that a Lewis lung carcinoma (LLC)-derived tumor cell line with high metastatic potential had a
higher proportion of E-disaccharide units, GlcUA-GalNAc
(4,6-O-disulfate), in CS chains than low metastatic LLC
cells and that such CS chains are involved in the metastatic
process. The metastasis was markedly inhibited by the preadministration of CS-E from squid cartilage rich in E units
or by pre-incubation with a phage display antibody specific
for CS-E. However, the molecular mechanism of the inhibition remained to be investigated.
In this study the receptor molecule for CS chains
containing E-disaccharides expressed on LLC cells was
revealed to be receptor for advanced glycation end
products (RAGE), which is a member of the immunoglobulin superfamily predominantly expressed in the
lung. Interestingly, RAGE bound strongly to not only
E-disaccharide, but also HS-expressing LLC cells. Furthermore, the colonization of the lungs by LLC cells
was effectively inhibited by the blocking of CS or HS
chains at the tumor cell surface with an anti-RAGE
antibody through intravenous injections in a dose-
Glycoconj J (2013) 30:281–461
351
dependent manner. These results provide the clear evidence that RAGE is at least one of the critical receptors
for CS and HS chains expressed at the tumor cell
surface and involved in experimental lung metastasis
and that CS/HS and RAGE are potential molecular
targets in the treatment of pulmonary metastasis. Recent
advances of the work will also be discussed.
140: Role of high endothelial venule-expressed heparan
sulfate in chemokine presentation and lymphocyte homing
139: Heparan Sulfate Is Required For Prostate Cancer
Initiation And Progression
Lymphocyte homing to peripheral lymph nodes (PLNs) is
mediated by multi-step interactions between lymphocytes
and high endothelial venules (HEVs). Heparan sulfate
(HS) has been implicated in the presentation of chemokines
on the surface of HEVs during this process. However, it
remains unclear whether this cell surface presentation is
required for chemokine function in vivo. In this study,
we generated conditional knockout (cKO) mice lacking
Ext1, which encodes a glycosyltransferase essential for
HS synthesis, by crossing Ext1 flox/flox mice with
GlcNAc6ST-2-Cre transgenic mice expressing Cre
recombinase in HEVs. Immunohistochemical studies indicated that HS expression was specifically eliminated
in PLN HEVs but retained in other blood vessels in the
cKO mice. The accumulation of a major secondary
lymphoid tissue chemokine, CCL21, on HEVs was also
abrogated without affecting CCL21 mRNA levels, indicating that HS presents CCL21 on HEVs in vivo. Notably, a short-term lymphocyte homing assay indicated
that lymphocyte homing to PLNs was partially diminished in the cKO mice by 30 to 40 %, and the lymphocytes could attach to and transmigrate through HEVs
that lacked HS in vivo. The residual lymphocyte homing
to PLNs in cKO mice was completely abrogated by
pertussis toxin which inactivates Gi subunit-coupled
heterotrimeric G proteins involved in chemokine receptor signaling, suggesting that soluble chemokines
detected in the sera and PLN organ culture supernatants
of cKO mice are involved in the residual lymphocyte
homing. Furthermore, contact hypersensitivity responses
were also partially diminished in the cKO mice. These
results suggest that chemokine presentation by HS on
the surface of HEVs facilitates lymphocyte homing and
immune responses but is not absolutely required for
chemokine function.
Xuanyang Li1, Alison Nairn1, Tamas Nagy2, Fen Wang3,
Yu Yamaguchi4, Kelley Moremen1, Lianchun Wang1;
1
Complex Carbohydrate Research Center and Department
of Biochemistry and Molecular Biology, University of
Georgia, Athens, Georgia 30602, USA; 2Department of
Pathology, College of Veterinary Medicine, University of
Georgia, Athens, Georgia 30602, USA; 3Center for Cancer
and Stem Cell Biology, Institute of Biosciences and
Technology, Texas A&M Health Science Center, Houston,
TX 77030, USA; 4Sanford-Burnham Institute for Medical
Research, La Jolla, California 92037,USA
Prostate cancer (PCa) is one of the most prevalent
forms of malignancy and the second most common
cause of cancer-related death in men. The failure in
treatment of this disease is our inability to prevent and
control PCa growth and metastasis. A better understanding of the mechanisms underlying PCa pathogenesis
will greatly enhance our effort to cure this lifethreatening disease. Heparan sulfate (HS) is a linear,
sulfated polysaccharide, and expresses abundantly in
prostate and PCa tissues. Intriguingly, the HS content
and sulfation modifications appear to increase when the
prostate becomes malignance, suggesting that HS may
critically modulate PCa pathogenesis. In current study,
we specifically ablated Ext1, the enzyme that initiates
HS biosynthesis, in mouse prostate at late development
stage. The Ext1 ablation does not affect prostate development and function, instead, it protects the mice from
tumorigenesis and invasion in a spontaneous PCa mouse
model. Tissue staining showed that the Ext1 deficiency
attenuated PCa cell proliferation, increased apoptosis,
and blocked PCa stem/progenitor cell differentiation
and epithelial-mesenchymal transition. The Ext1 deficiency PCa tissues also showed significant attenuation
of fibrinosis, inflammation, MMP-9 expression and hypoxia. In summary, our studies demonstrate that HS
functions via multiple mechanisms to promote PCa tumorigenesis and invasion, and also reveal that targeting
HS may represent a novel and effect approach to cure
PCa.
Hiroto Kawashima1 and Koichiro Tsuboi1; 1Laboratory
of Microbiology and Immunology, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka 4228526, Japan
kawashih@u-shizuoka-ken.ac.jp
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Glycoconj J (2013) 30:281–461
WorkshopIII-Young Glycoscientists’
Symposium
be used for the production of glycan arrays that resemble the
spectrum of glycans present in the respiratory tract.
141: Structural glycobiology of influenza virus receptors
in respiratory tissues of humans and other animal models
142: Improved O-glycan Analysis Methods
Nan Jia1, John M. Nicholls2, Anne Dell1, Stuart M.
Haslam1;1Division of Molecular Biosciences, Imperial College London, London SW7 2AZ, UK; 2Department of Pathology, The University of Hong Kong, Queen Mary
Hospital, Pokfulam, Hong Kong SAR, China
nan.jia07@imperial.ac.uk
Influenza is a virus that infects both birds and mammals,
including pigs, ferrets, mice and humans. The seasonal flu
has been a public health concern for years and has the potential to cause global pandemics. Additionally, infected animals
are slaughtered to prevent further spreading of the disease,
leading to great economic loss. Although the exact mechanism by which the influenza becomes transmissible in human
populations and between animal reservoirs remains incompletely understood, the initial interaction between the virus
and the host cell is crucial. More specifically, it is the binding
between viral surface proteins hemagglutinin and sialic acid
residues displayed by glycoproteins and glycolipids that are
expressed on the host plasma membrane, which triggers cellular recognition, attachment and viral entry events.
Sialic acids link to their adjacent monosaccharides via α2, 3 or
α2, 6 glycosidic bonds and this linkage difference together with
the change from Neu5Ac to Neu5Gc, are key factors defining
the species barrier. The regional distributions of the two types
of the glycosidic bonds along the respiratory tract, which is the
infection site of mammalian species, have been demonstrated
mostly by lectin staining studies. To better understand the
functionality of host glycan receptors, we carried out complementary studies where structural information was extracted
from animal tissues. Samples were processed to yield purified
N- and O- linked as well as glycolipid derived glycan molecules, which were subsequently analysed by mass spectrometric instrument. Additional linkage information was further
acquired by enzymatic digestion and GC-MS experiments.
We have produced glycomic profiles of several respiratory tissues, such as lung and trachea from different animal species. The
ultimate aim of the study is to investigate how the receptor
tropism present by different hosts could affect the infectivity of
various influenza strains. Moreover, our structural data provides
fundamental information of cellular receptors, based on which
the structural heterogeneity of glycans along the respiratory tract
can be compared among different animal models and which can
Radoslaw P. Kozak1, Louise Royle1, Richard A. Gardner1,
Gerhild Zauner 2 , Emanuela Lonardi 2 , André M.
Deelder2, Daryl L. Fernandes1 and Manfred Wuhrer2;
1
Ludger Ltd. Culham Science Centre, Oxfordshire, OX14
3EB, United Kingdom; 2 Biomolecular Mass Spectrometry
Unit, Department of Parasitology, Leiden University Medical
Center, Postbus 9600, 2300 RC, Leiden, The Netherlands
rad.kozak@ludger.com
The analysis of O-glycans is essential for better understanding of
their functions in biological processes. Although many techniques for O-glycan release have been developed, in our hands,
the hydrazinolysis method has proven to be the best for producing O-glycans with free reducing termini in high yield. This
release technique allows the glycans to be labelled with a
fluorophore and analysed by fluorescence detection. Under the
hydrazinolysis release conditions, an unwanted side reaction is
observed which causes the loss of monosaccharides from the
reducing terminus of the glycans. This side reaction is known
as peeling. The reduction of this glycan degradation step is
necessary in order to obtain information on the initial oligosaccharide composition. Using bovine fetuin and bovine submaxillary gland mucin (BSM) we demonstrated that peeling
can be greatly reduced when the sample is buffer exchanged
prior to hydrazinolysis with either solutions of 0.1 %
trifluoroacetic acid (TFA) or low molarity (100, 50, 20 and
5 mM) ethylenediaminetetraacetic acid (EDTA). The
presented technique for sample preparation prior to
hydrazinolysis greatly reduces the level of undesirable cleavage products in O-glycan analysis and increases the robustness of the method.
We further evaluate the use of O-glycans obtained by
hydrazinolysis from various biological sources for the
preparation of natural O-glycan arrays. To this end, Oglycans released from BSM and porcine stomach mucin
(PSM) and human saliva are labelled with 2aminobenzamide, fractionated by hydrophilic interaction
liquid chromatography (HILIC) UPLC with fluorescence
detection, and characterized by MALDI-TOF(/TOF)-MS.
When necessary, O-glycan fractions are subjected to a
second-dimension reverse phase separation to obtain homogeneous fractions. Subsequently, O-glycans are printed
onto epoxy-activated glass slides and probed with wellcharacterized carbohydrate-binding proteins for the
Glycoconj J (2013) 30:281–461
353
detection of structural determinants as well as for the evaluation of lectin specificities.
144: NMR analyses of conformational dynamics and
interactions of oligosaccharides by employing
paramagnetic probes
143: Quick and convenient diagnostic method for
Guillain-Barré syndrome using sugar-chain immobilized
fluorescent nano-particles
Ying Zhang 1,2,3, Takumi Yamaguchi 1,2,3, Sayoko
Ya m a m o t o 1 , 3 , Yo s h i n o r i U e k u s a 1 , 3 , M a h o
Yagi-Utsumi 1,3 and Koichi Kato 1,2,3; 1Institute for
Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences and 2Department of Functional Molecular Science, The Graduate
University for Advanced Studies (SOKENDAI), 5-1
Higashiyama, Myodaiji, Okazaki, 444-8787, Japan, 3Graduate
School of Pharmaceutical Sciences, Nagoya City University, 31 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan
yzhang@ims.ac.jp
Hiroyuki Shinchi1, Nobuhiro Yuki2, Hideharu Ishida3,
Koichi Hirata 4 , Masahiro Wakao 1 , Yasuo Suda 1 ;
1
Graduate School of Science and Engineering, Kagoshima
Univ., Kagoshima, 890-0084, Japan; 2Development of Medicine, National University of Singapore, Singapore, 117599,
Singapore; 3Faculty of Applied Biological Science, Gifu Univ.,
Gifu, 501-1193, Japan; 4Dokkyo Medical Univ., Tochigi, 3210293, Japan
k9561067@kadai.jp
Guillain-Barré syndrome ranks as the most frequent cause of
acute flaccid paralyis, often being misdiagnosed as having
stroke. Patients frequently carry autoantibodies against gangliosides, which are usually detected by ELISA. However, it
takes several days to receive the assay results from the
diagnostic laboratory. Toward the rapid point of care diagnostic kit, we developed a novel method for the detection of
the anti-ganglioside antibodies in serum using sugar-chain
immobilized fluorescent nano-particles (SFNPs) containing
component sugar-chains of gangliosides.
CdTe/CdS core/shell quantum dots (QDs), possessing high
photo-stability and attractive optical properties, were used as
fluorescent nanoparticles. SFNPs were prepared by the surface ligand exchange reaction using sugar-chain ligand conjugate which was obtained by the condensation reaction of
the chemically synthesized sugar moiety of ganglioside with
our original fluorescent linker molecule. Immobilization of
sugar chain onto the nano-particle was confirmed by matrix
assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS).
The prepared SFNPs were then used for the detection of antiganglioside antibodies in sera from patients with GuillainBarré syndrome. SFNPs were mixed with an appropriate
amount of serum and left for a few hours. After centrifugation
of the mixture, fluorescent precipitates were observed only in
the Guillain-Barré syndrome patient’s serum. It was confirmed by SDS-PAGE that the precipitate contained IgG,
suggesting the specific complex of SFNP with antiganglioside antibodies. Since our developed method detects
serum anti-ganglioside antibodies in a few hours, it can be
utilized as a rapid and conventional diagnostic method.
Oligosaccharides play important physiological and pathological roles in biological systems. For better understanding
the molecular basis of the mechanisms underlying oligosaccharide functions, it is quite desirable to describe their
conformational dynamics and interaction systems at atomic
level. However, these are still the remaining challenges,
because high conformational flexibility of oligosaccharides
hinders the application of conventional analytical methods
such as X-ray crystallography.
Nuclear magnetic resonance (NMR) spectroscopy has immense potential to deal with such dynamics issues in various
ranges of spatial and time scales. For conformational characterization of flexible oligosaccharides, their structures should
not be dealt with as a single well-defined global free energy
minimum but as an ensemble of low energy conformers. To
overcome the major limitation of traditional NMR methods for
the insufficiency of conformational information of oligosaccharides provided by nuclear Overhauser effect data, we have
been developing an NMR methodology for evaluating a
dynamic ensemble of oligosaccharide conformations by
employing paramagnetic effects in conjunction with molecular
dynamics (MD) simulation. Paramagnetic effects were induced
by unpaired electron providing long-distance information on
conformations and dynamics of oligosaccharides.
By applying this approach, we have successfully characterized the 3D structure of flexible GM3 trisaccharide
(αNeu5Ac-(2-3)-βGal-(1-4)-βGlc) and branched GM2
tetrasaccharide (βGalNAc-(1-4)-[αNeu5Ac-(2-3)]-βGal-(14)-βGlc) which share the common core structure of gangliosides forming an integral part of cellular membranes. The
interbranch interactions responsible for the distinct conformational ensembles between the GM3 and GM2 oligosaccharides were elucidated by the paramagnetic-assisted NMR
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Glycoconj J (2013) 30:281–461
analyses in conjunction with MD simulations. The results
demonstrated that this methodology offers a valuable tool
for the characterization of flexible oligosaccharides conformational dynamics. Furthermore, we extended this methodology to analyze oligosaccharides interaction systems,
such as carbohydrate-carbohydrate and carbohydrateprotein interactions. For example, the weak interactions
between gangliosides were observed through the analyses
of inter-molecular paramagnetic effect offered by NMR
measurements. This methodology opens a new prospect
for conformational analyses of dynamic structures of oligosaccharides toward decoding glycocodes from the 3D
structural aspects.
indicates the occurrence of O-glycosylation. The novel
ppGalNAcT shows a pH-optimum at pH 6.0-6.5, divalent
cations are required, with manganese being optimal at
10 mM, and 37 °C as best incubation temperature. All
tested acceptor peptides were glycosylated immediately
with one but during extensive incubation also with more
GalNAc residues. Different acceptor quality of the tested
peptide substrates indicate that the amino acid sequence
plays an important role for glycosylation efficiency.
145: Expression and characterization of the first snailderived UDP-N-acetyl-α-D-galactosamine: polypeptide
N-acetylgalactosaminyltransferase
This project is supported by the Austrian Science Fund
(FWF): [P22118-B20].
Christopher Taus1, Chantal Lucini 1, Takeshi Sato 2,
Kiyoshi Furukawa2, Reingard Grabherr3 and Erika
Staudacher1; 1 Department of Chemistry, University of Natural
Resources and Life Sciences, Vienna, Austria, 2Laboratory of
Glycobiology, Nagaoka University of Technology, Nagaoka,
Japan, 3 Department of Biotechnology, University of Natural
Resources and Life Sciences, Vienna, Austria
Christopher.Taus@boku.ac.at
146: Fucosylated glycan epitopes of the malaria mosquito Anopheles gambiae
Mucin-type O-linked protein glycosylation is initiated
by the UDP-N-acetyl-α-D-galactosamine: polypeptide
N-acetylgalactosaminyltransferase (ppGalNAcT) enzyme
family. To date, several members of mammalian ppGalNAcTs,
as well as various ppGalNAcT isoforms from Caenorhabditis
elegans and Drosophila melanogaster have been identified and described. The large number of different
ppGalNAcT isoforms with varying acceptor-substrate specificities in vitro suggests that O-glycosylation in vivo is a very
complex process. In this study, we isolated and characterized
the first snail-derived ppGalNAcT from Biomphalaria
glabrata embryonic (Bge) cells.
Due to limited mollusk sequencing data, our first approach
was to create a full-length cDNA library from the Bge cell
line. Using sense and antisense degenerated primers
designed to highly conserved sequences of the ppGalNAcT
enzyme family, a corresponding fragment from snail origin
could be amplified by homology-based PCR. Within three
experimental steps we could isolate the full-length
ppGalNAcT gene. The snail glycosyltransferase has high
homology to other ppGalNAcTs and contains all domains
common to this enzyme family. Based on the amino acid
sequence, there is no N-glycosylation site, whereas Oglycosidase treatment prior to lectin blot analysis
Better insights in the glycobiology of snails will increase
the understanding of glycan biosynthesis and may also
result in new strategies for pest control, as well as a better
understanding of molecular host-parasite interactions.
Simone Kurz1, Jonas King2, Rhoel Dinglasan2, Michael
Tiemeyer3 and Iain B. H. Wilson1; 1 Department of Chemistry, University of Natural Resources and Life Sciences
(BOKU), A-1190 Vienna, Austria, 2 Johns Hopkins
Bloomberg School of Public Health, Baltimore, Maryland
21205, USA, 3 University of Georgia, Complex Carbohydrate Research Center, Athens, Georgia 30602, USA
simone.kurz@boku.ac.at
The most efficient vector of human malaria is the mosquito
Anopheles gambiae. Two fundamental steps are required for
the Plasmodium parasite development and transmission.
Both steps, binding of parasite in insect vector and in
vertebrate host cells, have been connected to the presence
of oligosaccharide structures (glycans).
By homology searching of the A. gambiae genome, we
found that the mosquito possesses three fucosyltransferases
potentially involved in N-glycan biosynthesis. All three
fucosyltransferases were cloned and expressed in Pichia
pastoris GS115 cells.
The recombinant fucosyltransferases were further purified
by Affi-Gel Blue and Ni-NTA affinity chromatography.
Various optima (pH, temperature and ion dependence) have
been determined in MALDI-TOF MS based assays. The
characterisation of all three fucosyltransferases shows that
in comparison to many other glycosyltransferases, the mosquito fucosyltransferases have no absolute requirement for
any special divalent cation cofactor. The recombinant mosquito fucosyltransferases were then used in apo-transferrin
Glycoconj J (2013) 30:281–461
remodelling experiments to create positive controls for
Western and Lectin Blot analysis.
Furthermore, immunofluorescence staining of mosquito sections with selected antibodies (anti-HRP) and lectins
(Aleuria aurantia lectin) were performed to determine if
the mosquito fucosyltransferases are also active in vivo.
The staining patterns indicate the presence of in vivo
fucosyltransferase products.
In addition, N-glycan analysis as well as Western Blot
analysis of mosquito larvae and whole protein lysates could
confirm the occurrence of fucosylated N-glycan epitopes.
These experimental data as well as forthcoming analysis of
the O-glycans are the basis for further exploration of the
glycomic potential of this organism.
147: The Ribosome-inactivating Proteins (RIPs) from elderberry (Sambucus nigra) in the Battle Against Cancer??
Chenjing Shang1, Mariusz Madej1, Winnok De Vos2, Els
J.M. Van Damme 1; 1Biochemistry and Glycobiology,
Dept. Molecular Biotechnology, Gent University, Gent,
9000, Belgium; 2Cell Systems and Imaging, Dept. Molecular Biotechnology, Gent, 9000, Belgium
chenjing.shang@ugent.be
The family of type-2 ribosome-inactivating proteins (RIPs)
groups chimeric proteins, composed of an A-chain with Nglycosidase activity and a B-chain with carbohydratebinding/lectin activity. The type-2 RIPs from Ricinus
communis and Abrus precatorius are well known as extremely toxic proteins. In the last decade it has been shown
that the cytotoxicity of type-2 RIPs relies on the binding of
the B-chain to carbohydrate structures on the cell surface, as
such facilitating the endocytosis process of the ribosomeinactivating proteins in the cell.
Elderberry (Sambucus nigra) expresses several type-2 RIPs
as well as lectins, differing from each other in their molecular structure and carbohydrate-binding properties. We studied the cytoxicity of several RIPs/lectins from elderberry
towards human cells. To this end, transformed (HeLa cervix
carcinoma) and mortal (human dermal fibroblasts) cell types
were treated with different concentrations of elderberry proteins, after which viability was scored by means of
PrestoBlue spectrophotometric assays and morphological
observation. Using confocal microscopy, the internalization
and intracellular localization of the proteins was determined.
Our findings show clear differences among the elderberry
proteins tested, depending on the lectin binding properties of
the proteins and their interaction with glycans present on the
355
cell surface. The data revealed that RIPs from elderberry are
much less toxic than the classical RIPs from Ricinus and
Abrus. In addition, fibroblasts were found to be less susceptible to some of the elderberry proteins compared to HeLa
cells. Hence, these proteins are promising candidate molecules as cancer therapeutics… Future experiments will focus
on the possible application of elderberry RIPs as a potential
tool in the battle against cancer.
148: Biological activity of the tandem-repeat type
Galectin-8 is related to the length of the linker region
Myriam Friedel 1 , Sabine André 2 , Hans-Joachim
Gabius2, Reinhard Schwartz-Albiez1; 1German Cancer
Research Center (DKFZ) Heidelberg, Germany; 2LudwigMaximilian Universität Munich, Germany
myriam.friedel@dkfz.de; r.s-albiez@dkfz-heidelberg.de
The tandem-repeat type Galectin-8 (Gal-8) has two carbohydrate recognition domains (CRD) which are connected by
a linker peptide of defined length (Gal-8S: 34aa, Gal-8L:
76aa). Several different functions of Gal-8, like its role in
angiogenesis and a pro-apoptotic effect on lymphoctes, have
been described so far. But knowledge about the influence of
the linker region on the biological activity is sparse. We
investigated the functional role of human recombinant
Galectin-8 (hGal-8) with regard to its two different
isoforms. We report that the human short, but not the long
linker isoform may play a regulatory role in angiogenesis. In
an in vitro angiogenesis assay (tube formation assay), hGal8S in contrast to hGal-8L was able to enhance angiogenesis
of human endothelial cells (HUVEC) on a feeder layer of
human fibroblasts in combination with VEGF. In comparison,
hGal-1 and hGal-3 only displayed small positive effects in this
angiogenesis assay. With regard to its effect on lymphocytes,
we could now show that hGal-8L in contrast to hGal-8S exerts
a pronounced pro-apoptotic influence on human B leukemic
cells of pre-B-cell origin (REH and Nalm-6). The different
functional effects could at least partly be due to the fact that
these isoforms vary in their ability to multi- or dimerize in
solution. Using microscale thermophoresis measurement to
quantitatively measure interactions of two molecules in solution, we were able to show that hGal-8L has a significantly
stronger ability to dimerize in solution than hGal-8S. These
observations may point to the fact that the length of the linker
between the two CRDs influences the biological action of
hGal-8. It has to be elucidated whether these findings can also
be applied for other tandem-repeat galectins like for example
human Galectin-9.
This work is funded by the “Deutsche José Carreras
Leukämie-Stiftung”.
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149: GD1a activates MAPK pathway via calcium signaling
to suppress NOS2 expression in mouse osteosarcoma cells
in an EGFR-independent manner
Xiao-Yan Yang, Lan Zhang, Xiao Zhang, Sadako
Yamagata and Tatsuya Yamagata; Laboratory of Tumor
Biology and Glycobiology, Shenyang Pharmaceutical University, 103 WenHua Rd, Shenyang, 110016 China
yangxiaoyan.syphu@gmail.com
Inducible nitric oxide synthase (NOS2) has been shown
to increase during inflammation and tumor progression.
Our previous work revealed that NOS2 was responsible
for the malignant nature of murine osteosarcoma FBJLL and that NOS2 expression was enhanced by silencing ERK1 that mediated the suppression of NOS2 by
ganglioside GD1a cells (Cao T, et al., Ganglioside
GD1a suppression of NOS2 expression via ERK1
pathway in mouse osteosarcoma FBJ cells. J Cell
Biochem, 110, 1165–1174, 2010). In the present work,
we will show that GD1a in FBJ-LL cells suppresses
NOS2 expression through calcium signaling that
activatesgrowth factor receptor-bound protein 2 (GRB2)
followed by MAPK pathway activation. In untreated
cells, NOS2 expression was suppressed through MAPK
pathway that is activated by EGFR, whose signal is
mediated by GRB2, but in GD1a-activated cells, the
GD1a signal did not include EGFR activation. When
we investigated molecules involved in MAPK pathway,
we found that silencing MEK2, but not MEK1, augmented NOS2 expression in untreated FBJ cells and
impaired the GD1a signal. Furthermore, silencing
(GRB2) resulted in an increase in NOS2 in untreated
cells and abrogated the GD1a signal. In addition, EGF
suppressed NOS2 expression through activation of
EGFR, GRB2, and MEK2 in FBJ-LL cells. However,
the GD1a suppression of NOS2 was not cancelled by
treating cells with the EGFR inhibitor, AG 1478, or by
silencing EGFR; though these treatments cancelled the
effects of EGF on NOS2 suppression. An anti-GRB2
antibody precipitated phosphorylated EGFR in the sample from EGF-activated cells, but not in the sample
from GD1a-treated cells. These results indicated that,
in FBJ-LL cells, GD1a stimulated an unidentified signaling molecule to stimulate calcium influx that activated the GRB2/MEK2/ERK1 pathway and could suppress
NOS2 without EGFR involvement. In contrast, in
untreated cells, EGFR mediated the activation of
GRB2/MEK2/ERK1, which then suppressed NOS2 expression. We have also shown that in murine Lewis
lung carcinoma cells GD1a suppressed NOS2 expression
via EGFR-independent signal pathways.
Glycoconj J (2013) 30:281–461
150: Characterization of Cell Surface Glycosylation in
Ovarian Cancer Leads to Identification of Dysregulation
in MGAT3 and ST6GAL-1 Gene Expression
Merrina Anugraham1, Sheri Nixdorf2, Francis JACOB3,
Viola Heinzelmann-Schwarz 3 , Nicolle H. Packer 1 ;
1
Macquarie
University, Sydney, 2109, Australia, 2University of New South
Wales, Sydney, 2052, Australia, 3University Hospital Basel,
Basel, 4031, Switzerland
merrina.anugraham@mq.edu.au
Ovarian cancer is the fifth most common cause of cancer
worldwide with the highest mortality rate among all
gynaecological cancers. Following diagnosis, the standard therapy includes maximal cytoreductive surgery and platinumbased chemotherapy of which more than 80 % of patients
undergo relapse due to chemotherapy resistance. This poor
prognosis is primarily attributed to difficulties in detecting the
disease at an early stage, lack of noticeable early symptoms and
inadequate screening methods. The consequences of aberrant
glycosylation are now widely established in cancer, as
evidenced by alterations in glycan structures. However, the
extent to which these glycosylation changes are influenced by
aberrant epigenetic regulation of gene expression is less extensively studied in ovarian cancer and remains crucial in understanding the development and progression of this disease.
To address this effect, we first analysed the cell surface
glycosylation of human ovarian surface epithelial (HOSE
6.3 and HOSE 17.1) and ovarian cancer cell lines
(SKOV 3, IGROV1, A2780 and OVCAR 3). Membrane
glycoproteins were enriched using Triton-X114 membrane partitioning. The N-linked glycans were released
by PNGase F, converted to alditols and analyzed using
nano-liquid chromatography on porous graphitized carbon and electrospray ionization mass spectrometry. Glycan structures were characterized based on their
molecular masses and MS/MS fragmentation patterns.
We identified characteristic glycan features that were
unique to the ovarian cancer cell lines, namely the
‘bisecting GlcNAc’ type N-linked structures and increased levels of sialylated oligosaccharides, predominantly with an alpha-2,6 sialic acid linkage. These Nglycan changes were then verified by examining their
gene transcript levels (MGAT3 and ST6GAL-1) using
RT-PCR. We further evaluated the potential epigenetic
influence on the MGAT3 coding gene by treating the cell
lines with 5-Azacytidine (5-Aza), a DNA methylation
inhibitor. For the first time, we provide evidence to support
the role of DNA hypomethylation of MGAT3 gene which lead
to the observation of the unique ‘bisecting GlcNAc’ type Nglycans in serous ovarian cancer cells.
Glycoconj J (2013) 30:281–461
It is envisaged that the specific N-glycans detected by mass
spectrometry and their complex association with epigenetic
programming in ovarian cancer could potentially be used for
the development of novel anti-glycan drug targets and
clinical diagnostic tools.
151: Accumulation of β1,6-N-acetylglucosaminebranched N-glycans Promoted PTPRT’s Dimerization
and Decreased its Catalytic Activity
Jingjing Qi1, Na Li1, Kun Fan1, Zengxia Li1,2,3, Liying
Wang1,2,3, Xiliang Zha1,2,3*;
1
Department of Biochemistry and Molecular Biology, Shanghai
Medical College, Fudan University, Shanghai 200032, China;
2
Key Laboratory of Glycoconjugate Research, Ministry of
Health, Shanghai 200032, China; 3Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai, 200032, China.
xlzha@shmu.edu.cn (Xiliang Zha)
Receptor-like protein tyrosine phosphatases (RPTPs) are type I
transmembrane proteins whose catalytic activities are regulated
by dimerization. The intrinsic mechanisms involved in
dimerizing processes remain obscure. In this study, we identified receptor protein tyrosine phosphatase rho (PTPRT) as a
novel substrate of GnT-V. Addition of β1,6 GlcNAc branches
promoted PTPRT’s dimerization through galectin-3 binding
manner. Increased dimerization subsequently decreased
PTPRT’s catalytic activity, trigging nuclear export of
phorsphorylated Y705 signal transducer and activator of transcription 3 (STAT3). Collectively, our results provide new
evidence that aberrant regulation of PTPRT and STAT3 compromise an important part in GnT-V mediated metastasis.
152: Profiling the O-GlcNAcylation of the Kinome using
Kinase Microarrary
Guanghui Han1, Johnathan Neiswinger2, Heng Zhu2,
Gerald W. Hart1; 1The Johns Hopkins University, Department
of Biological Chemistry; 2The Center for High-Throughput
Biology, Johns Hopkins University School of Medicine
ghan6@jhmi.edu
Mutations and dysregulation of kinases play causal roles in
human disease development, signaling, and metabolism. Understanding the function of kinases is of an outstanding interest
for biomarker discovery and also necessary for the development of agonists and antagonists for the use in disease therapy.
O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a
post-translational modification known to regulate different
aspects of a proteins function including localization, activity,
and stability. Like phosphorylation, O-GlcNAcylation, which
357
modifies serine and threonine residues on nuclear and cytoplasmic proteins, is a ubiquitous, reversible process that regulates numerous cellular processes. Recent evidence indicates
that site-specific crosstalk between O-GlcNAcylation and
phosphorylation and the O-GlcNAcylation of kinases play an
important role in regulating cell signaling. Hence, it is very
important to investigate the O-GlcNAcylation of the kinome.
Previously, we identified 42 kinases that are substrates for OGlcNAcylation using an in vitro OGT assay with [H3] radiolabel on a functional kinase array. However, using [H3] has serve
senstitive limiations suggesting a more sensitive approach is
needed to probe large libraries of proteins. Herein, using a novel
kinase microarray coupled with an in vitro OGT labeling assay with
immuno fluorophore detection technique, we report a simple,
yet sensitive, strategy by which this method can be used to
profile the entire kinome for O-GlcNAcylated proteins.
153: Glycomic Investigation of Neuromuscular Junction
Disorders by Mass Spectrometry
Qiushi Chen1, Juliane S. Müller2, Steven H. Laval2,
Hanns Lochmüller 2 , Stuart Haslam 1 , Anne Dell 1 ;
1
Division of Molecular Bioscience, Imperial College London,
South Kensington Campus, London SW7 2AZ, UK; 2Institute
of Human Genetics, Newcastle University, Newcastle upon
Tyne NE1 3BZ, UK.
q.chen11@imperial.ac.uk
Glutamine-fructose-6-phosphate transaminase 1 is the first
and rate-limiting enzyme of the hexosamine biosynthetic
pathway. It catalyzes the reaction of glutamine and fructose6-phosphate to produce glutamate and glucosamine-6phosphate, supplying the substrate for uridine diphosphate
N-acetylglucosamine (UDP-GlcNAc) synthesis. It is universally acknowledged that the supply of UDP-GlcNAc is critical
for the biosynthesis of several glycoconjugates and has an
influence on branching of N-glycans. It is reported that
GFPT1 mutations result in congenital myasthenic syndrome
(CMS) which is a type of neuromuscular junction disorders in
which the synapses that form between motoneurons and skeletal muscle fibers that transmit the impulse resulting in muscle
contraction have impaired function. Patients from two European countries (Germany and Spain) have been found to have
CMS, due to mutations in the GFPT1 gene. In vitro cultured
myoblasts from affected patients were glycomically investigated using highly sensitive Mass Spectrometry. The result
showed that in the patients the tetra-antennary glycans are
substantially reduced compared with those glycans in controls.
The reduction in tetra-antennary glycans in patients was compensated for by longer polylacNAc antennae. We conclude that
GFPT1 mutations in CMS patients have an influence on tetraantennary N-glycan biosynthesis.
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154: Pseudomonas aeruginosa changes the N-glycan
profile of the sputum proteins from cystic fibrosis patients
Vignesh Venkatakrishnan1, Morten Thaysen-Andersen1,
Helena Nevalainen1, Nicolle H. PACKER1; 1Department
of Chemistry and Biomolecular Sciences, Macquarie
University, Sydney, 2109, Australia
vignesh.venkatakrishnan@mq.edu.au
Cystic fibrosis (CF) is a prevalent autosomal recessive disease
characterized by chronic infection and inflammation in the
lungs. The damaged lungs continue to secrete mucus which is
the primary site of microbial infection and Pseudomonas
aeruginosa is one of the major infecting pathogens among
CF patients. Alteration in glycosylation of mucins (heavily Oglycosylated proteins) has been shown to play a role in bacterial
binding to the mucous in the lungs of CF patients. However,
there has been little reported on the N-glycosylation of the
protein complement of CF sputum. We have characterized the
N-glycans from sputum collected from CF, and other non CF,
patients with lung infection to determine any correlation between the N-glycan structures and the microorganisms which
commonly cause CF-specific lung infection. Sputum samples
were reduced, alkylated and the N-glycans were released enzymatically using PNGase F enzyme and analysed on porous
graphitized carbon (PGC)-LC-ESI-MS/MS.
The major difference in the N-glycans of CF sputum, compared to non-CF, sputum was the presence of unusual paucimannose glycans. To test whether this was due to pathogenic infection, different clinical P.aeruginosa strains isolated
from CF sputum samples were inoculated into non-CF derived sputum and incubated at 37 °C for 3 days. All the
isolated P.aeruginosa strains trimmed the complex N-glycans in the non-CF sputum to pauci-mannose structures
similar to those seen in CF sputum. However, other P.
aeruginosa cultured laboratory strains such as PAO1 and
AES1-R did not affect the N-glycan structures. A Biolog
plate assay determined that the isolated bacteria did not
use these types of trimmed sugars (sialic acid, Nacetylglucosamine and galactose) as a carbon source for
their growth. These data suggests that Pseudomonas may
be modifying the N-glycan structures on proteins to promote
bacterial adhesion in CF associated lung infection.
155: ManLAM of virulent Mycobacterium tuberculosis
induces IL-10-producing Breg cells
Chun-Hui Yuan1, Xiao-Lei Tang2, Qin Pan3, Xiao-Lian
Zhang*; Wuhan University School of Medicine, Department
of Immunology, Donghu Road 185, Wuhan 430071, China.
zhangxiaolian@whu.edu.cn
Glycoconj J (2013) 30:281–461
Mannose-capped Lipoarabinomannan (ManLAM) as a
kind of lipoglycan which is the main component of
Mycobacterium.tuberculosis (M. tb) cell wall, and of
importance, putatively referred as an immune suppressor
that facilitate M.tuberculosis escape. However the relationship between ManLAM and IL-10-producing Breg
cells remains elusive. Here we found that ManLAM
purified from Virulent M.tb H37Rv induced IL-10producing Breg cells both in vivo and in vitro and that
these Breg cells contained both CD5- and CD5+ B subset. We next detected these Breg cells’ cytokine profile,
and found that they mainly secreted IL-4, moreover,
these IL-10-producing Breg cells induced by ManLAM
negatively regulated IL-4 secretion of CD4+ T cells in
co-culture assay, and this special function could be
suppressed by IL-10 antibody. Together these results
suggest that, IL-10-producing Breg cells induced by
ManLAM belong to ‘Be2’ subtype and this is the first
report demonstrating that ManLAM from Virulent M.tb
H37Rv induces CD5+ and CD5− IL-10-producing Breg
cells which might act as a new immune suppressor
facilitating virulent M. tb immune escape.
Poster Session I-Glycoproteomics
156: A Novel Approach to Enriching Azido Labeled
Glycopeptides from Metabolically Labeled Cells
Sheng Wang, Xiu Zhang, Wenxian Xie, Xia Zou,
Yan Zhang; Ministry of Education Key Laboratory of
Systems Biomedicine, Shanghai Center for Systems
Biomedicine, Shanghai Jiao Tong University, Shanghai, P.R.
China, 200240
yanzhang2006@sjtu.edu.cn
Protein glycosylation is usually difficult to analyze due to
the low proportions of glycopeptides and the heterogeneity
in the structure of glycans. But recently, an azide-labeling
strategy has emerged as a promising approach to help decipher the myths of glycosylation. In this study, we have
synthesized disulfide- and terminal alkyne- modified magnetic silica particles (DA-MSPs) and developed a novel
method for enrichment of azido labeled glycopeptides based
on bioorthogonal chemistry. In our results, the adsorption
capacity of these particles is calculated to be over
4 pmol/mg, and the enrichment recovery rate is over 40 %,
and specific enrichment can be realized when the ration of
interference BSA peptides to targeting standard glycopeptide is 100:1. Further, Jurkat cells were cultured with
Ac4GalNAz and the membrane and cytoplasmic proteins
were collected. Metabolic labeling with Ac4GalNAz introduces a bioorthogonal chemical reporter into mucin-type O-
Glycoconj J (2013) 30:281–461
linked glycopeptides that can react with alkyne probes for
detection or enrichment. After covalently capture and reductively release azido-glycopeptides via click chemistry and
dithiothreitol treatment, the glycoproteins were isolated. The
efficiency of cell labeling and glycoprotein enrichment was
validated by fluorescence detection and lectin blot respectively. And the enriched glycoproteins were separated by 2D
lectin blotting or by on-line 2D nano-HPLC and analyzed
with LTQ-Orbitrap mass spectrometry.
157: Purification of Novel α-Galactoside-binding Lectins
from Aquatic Animals and their Distinct Cell Regulatory
Activities
Imtiaj Hasan1,2, Yasuhiro Koide1, S. M. Abe Kawsar1,3,
Ryo Matsumoto1,4, Yuki Fujii5, Yukiko Ogawa5, Shigeki
Sugawara6, Masahiro Hosono6, Kazuo Nitta6, Yasuhiro
Ozeki1; 1Graduate school of Yokohama City University, Yokohama, 236-0027 Japan; 2Department of Biochemistry and
Molecular Biology, Rajshahi University, Rajshahi-6205 Bangladesh; 3Department of Chemistry, University of Chittagong,
Chittagong-4331, Bangladesh; 4Institute for Environmental
and Gender-Specific Medicine, Juntendo University, Urayasu,
Chiba, 279-0021 Japan; 5Department of Functional Morphology and Microbiology, Faculty of Pharmaceutical Science,
Nagasaki International University, Sasebo, Nagasaki, 8593243 Japan; 6Institute of Molecular Biomembrane and
Glycobiology, Tohoku Pharmaceutical University, Sendai,
981-0905 Japan
ozeki@yokohama-cu.ac.jp
An α-galactoside-glycosphingolipid, globotoriaosyl ceramide (Gb3; Galα1-4Galβ1-4Glc-Cer) is focused to be the
trigger for cell regulations through the interaction with the
glycan-binding receptors. As glycan-binding subunit of
Vero toxin that is secreted from pathogenic bacteria, such
as O157 acts for the incorporation of the toxin into Gb3
ceramide expressing digestive cells, Gb3-binding lectins
isolated from marine invertebrates and fishes were also
reported to perform direct cytotoxic activity, up- and
down-regulation of cytokines and are the important membrane proteins on cell surface.
Two distinct cell regulative activites through Gb3 and
lectins that recognize the glycan is discussed in this presentation. MytiLec is an α-galactoside-binding lectin isolated
from Mediterranean mussel, Mytilus galloprovincialis with
a highly novel primary structure consisted of 149 amino
acids and cytotoxic activity against Burkitt lymphoma Raji
cells expressing Gb3. On the other hand, another Gb3binding lectin, SAL from Catfish (Silurus asotus) eggs that
is a member of SUEL-lectin family showed dow- regulation
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of multidrug resistance associate protein-1 (MRP1) of the
Raji cells instead of the direct cell death occurred by
MytiLec. However, the cells which are treated by SAL were
effectively killed with low concentrated anti-cancer drugs
by the deficiency of MRP1.
In addition to above studies, we found another αgalactoside-binding lectin from Sea hare (Aplysia kurodai)
eggs by the combined use of melibiosyl-agarose and
lactosyl-agarose columns. This analytical procedure showed
the presence of two different lectins in the eggs which
recognize α- and β-galactoside, respectively. Different from
MytiLec and SAL, Sea hare lectins were assumed to be
glycoproteins. We are now comparing the cell regulation
activities with these lectins against Raji cells. The discovery
of plural α-galactoside-binding lectins and the elucidation
of the distinct cell regulatory mechanisms through the
lectin-glycan interaction will provide invaluable findings
for glycan-dependent cell signaling.
158: Comparative Analysis of the surface N-linked
Glycoproteomes of HC-04 and HepG2 hepatocyte cell
lines by a highly sensitive online integrated system
Dingyin Tao, Jonas G. King, Philipp Jost, Justin Boddey
and Rhoel R. Dinglasan‡; Johns Hopkins Bloomberg
School of Public Health and the Malaria Research Institute,
Baltimore, Maryland, 21205, USA; Technical University of
Munich, Munich, Germany; Walter and Eliza Hall Institute,
Parkville, Australia, ‡Corresponding author: Rhoel R.
Dinglasan, PhD, MPH.
rdinglas@jhsph.edu
Liver cell lines are important models for understanding
hepatocyte biology as well as the pathogenesis of infectious
diseases such as malaria and hepatitis. Several critical aspects of the basic biology of these cell lines remain unknown, including the composition of their plasmalemma
surface proteome and extracellular glycocalyx, and how
these cell surface molecules interact with pathogens and
respond to their environment. Here, we focused on analyzing the surface glycoproteins of two important human liver
cell lines: HC-04, which has been shown to support human
malaria liver stage development and HepG2, a widely-used
cell line that will not support infection with human malaria
parasites. Although the determinants of susceptibility to
malarial infection are unknown, we hypothesize that a differential response to culture conditions are likely to modulate
surface protein expression density and repertoire, which may
influence susceptibility. The HC-04 and HepG2 surface glycoproteins were labeled and enriched by DIBO-AHA pull-downs
prior to SDS-PAGE separation and in-gel digestion. To
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overcome technical limitations due to the small amount of
surface glycoproteins, we developed a highly sensitive online
integrated system, and in a proof of principle study, analyzed
the surface N-linked glycoproteomes of the HC-04 and HepG2
cell lines in response to titrated concentrations of hepatocyte
growth factor. The online system is composed of a (i) weak
anion exchange (WAX) column, (ii) a PNGase F immobilized
enzymatic reactor (PIMER) and a Polaris chip, which was used
for glycopeptide enrichment by Electrostatic Repulsion-hydrophilic Interaction Chromatography (ERLIC) followed by
deglycosylation, and (iii) peptide analysis by RPLC-MS/MS.
In comparison with the conventional offline method, the total
analysis time was shortened from days to several hours, and the
sample amount for each analysis was reduced to 1~2 micrograms. Furthermore, the contamination risk was reduced since
all preparation steps were online after in-gel digestion. In
addition, we captured aglycosylated peptide fragments during
ERLIC enrichment, which could be used for glycoprotein
quantification analysis by the label-free method developed in
our previous work. The differentially expressed glycoproteins
identified between the HC-04 and HepG2 cell lines provide
insight into the impact of growth conditions on surface
glycoproteomes of these important hepatocyte lines.
159: Characterization of O-Glycans of Ovomucin Isolated
from Hen Egg White Based on the One-pot Procedure by
Electrospray Ionization Mass Spectrometry
Weibo Xing, Chengjian Wang, Xiaohua Li, Zhongfu
Wang, Linjuan Huang; Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western
China, Life Science College, Northwest University, Xi’an
710069, China
huanglj@nwu.edu.cn
Mucin-type glycans of glycoproteins are extensively believed
to play important roles in many biological processes, such as
immune responses, protection of the gastrointestinal tractus,
and egg-sperm interaction. Detailed investigations of the
structure diversity of the O-glycans from various glycoproteins can expand our still very limited knowledge of Oglycosylated proteins and facilitate the research on the
structure-function correlations of mucin-type glycans. This
study is focused on the comprehensive characterization of
the O-glycans released from a bioactive glycoprotein,
ovomucin, which possesses anti-viral, anti-bacterial and antitumor bioactivities and is supposed to have potential applicability to medicine, nutriology and health care. Ovomucin was
isolated from chicken egg white, and then its O-glycans were
released with Carlson’s reductive β-elimination and the onepot procedure we reported previously (the procedure allows
simultaneous O-glycan release and 1-phenyl-3-methyl-5-
Glycoconj J (2013) 30:281–461
pyrazolone (PMP) derivatization), separately. After comprehensive analysis of the obtained glycan samples by
electrospray ionization mass spectrometry (ESI-MS) and
tandem mass spectrometry (MS/MS), we found 14 kinds
of hen egg ovomucin O-glycans, of which 5 are neutral
and 9 are sialylated or sulfated. Additionally, it was also
found that the Carlson’s method could induce some Nglycans to cleave from ovomucin along with the O-glycans, whereas the one-pot procedure could only release the
O-glycans from ovomucin, showing the superiority of the
one-pot method over the conventional reductive β-elimination in releasing specificity for O-glycans. These analysis
results could provide a basis for the further investigations
on the structure-function correlations of ovomucin as well
as its deep exploitation.
160: Multivalent Hydrazide Functionalized Magnetic
Nanoparticles for Glycopeptides Enrichment and
Identification
Qi C. Cao1, 2,3, Wan T. Ying3*, Xiao H, Qian1, 3; 1School
of Pharmacy, Pharmaceutical University, Shenyang 110016,
China;2The affiliated Hospital of Liaoning University of
Traditional Chinese Medicine, Shenyang 110032,China;
3
State Key Laboratory of Proteomics, Beijing Proteome
Research Center, Beijing Institute of Radiation Medicine,
Beijing 102206, China;* Correspondence Author
qianxh1@163.com (Xiao H, Qian);proteomics@126.com
(Wan T. Ying)
Abstract: Among the common approaches for global glycopeptides enrichment and identification, hydrazide chemistry is well recognized. However, the mainly commercial
hydrazide functionalized products are composed of single
layer hydrazide functional group, therefore, they can only
provide limited affinity to the glycopeptides for highly complex proteomic samples. In this work, we introduced a novel
multivalent hydrazide functionalized magnetic nanoparticle
coated with hydrazide modified non-cross linked polymer
chains. Such material was synthesized via surface initiated
atom transfer radical polymerization (SI-ATRP) technique.
The density of hydrazide groups on the surface of these
nanoparticles were 10 folds higher than that of conventional
single layer magnetic particles. The multivalent hydrazide
functionalized particles were demonstrated to enrich glycopeptides with high sensitivity from a peptide mixture of
Bovine Fetuin and Bovine Serum Albumin at a mass ratio
of 1:100. The recovery of glycopeptide was investigated to
be 73 %, and the glycopeptides binding capacity was proven
to be 25 μg/mg. Finally, the novel hydrazide functionalized
particles were applied to enrich N-linked glycosylated peptides from 50 μg mouse liver tissues. In total, 116 unique
Glycoconj J (2013) 30:281–461
glycopeptides belonged to 91 different glycoproteins have
been identified, and the result further demonstrated the
multivalent particles’ potential for selective glycopeptide
isolation in complex proteomics samples.
161: In-depth profiling of the sialylated N-glycoproteome
of human plasma using optimized strategy based on metal
oxide enrichment
Wantao Ying, Xinyuan Zhao, Xiaohong Qian; State Key
Laboratory of Proteomics, Beijing Proteome Research
Center, Beijing Institute of Radiation Medicine, Beijing
102206, China
yingwtll@yahoo.com.cn
Glycosylation is an important post-translational modification of proteins and plays a crucial role in both cellular
functions and secretory pathways. Sialic acids (SAs), a
family of 9 carbon-containing acidic monosaccharides,
often terminate the glycan structures of cell surface molecules and secreted glycoproteins and perform an important role in many biological processes. Hence, in-depth
profiling of the sialylated glycoproteome may improve
our knowledge of this modification and its effects on
protein functions. Here, we systematically investigated
different strategies to enrich the SA proteins in human
plasma using a newly developed technology that utilizes
titanium dioxide for sialylated N-glycoproteome profiling by mass spectrometry. Our results showed that using
a combination of a filter-aided sample preparation
(FASP) method, TiO2 chromatography, multiple enzyme
digestion and two-dimensional reversed-phase peptide
fractionation led to in-depth profiling of the SA proteome. In total, 982 glycosylation sites in 413 proteins
were identified, among which 37.8 % were newly identified, which established the largest database of sialic
acid-containing proteins from human plasma.
162: Multiplex Profiling of Glycoproteins Using Novel
Bead-based Lectin Array
Hong Li, Hong Wang, Wei Zhang, Liming Wei, Hongxiu
Yu, Pengyuan Yang; Fudan University, Shanghai, 200032,
P.R. China
pyyang@fudan.edu.cn
Lectin array is becoming important in profiling targeted
glycan/glycoprotein, but weak interaction between lectin and
glycan causes low sensitivity of the approach. This study aims
to develop a bead-based lectin array for improving the sensitivity of glycosylation profiling. Lectins are chemically
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coupled to fluorescent dye coated microbeads, and glycanlectin recognition is carried out three-dimensionally. The
performance of this platform was evaluated, and the
limit of detection of lectin Ricinus communis agglutinin
120 (RCA120) was 50 pg/mL (1 pM) of asialofetuin,
providing the bead based lectin microarray with the
highest sensitivity among the reported lectin microarrays.
Furthermore, multiplexed assay was performed, which
allowed the simultaneous detection of multiple carbohydrate epitopes in a single reaction vessel. The glycosylation patterns of hepatocellular carcinoma associated
immunoglobulin G were analyzed, and increased (α-1, 6) core
fucosylation and (α-2, 6) sialylation patterns were observed,
which may provide significant clinical evidence for disease
diagnosis.
163: Optimal sample choice for the analysis of protein
antennary fucosylation as screening tool for HNF1A-MODY
Novokmet M 1 , Thanabalasingham G 2 , Kattla J 3 ,
Huffman J5, Rudan I5,6 Wright AF4, Campbell H4,
Owen K2, Rudd PM3, McCarthy MI2, Lauc G1,7; 1Genos
Ltd, Glycobiology Laboratory, Zagreb, Hondlova 2, Croatia,
mnovokmet@genos.hr, 2Oxford Centre for Diabetes, Endocrinology & Metabolism, University of Oxford, Oxford, UK
3
National Institute for Bioprocessing Research and Training, Dublin-Oxford Glycobiology Lab.,Conway Institute,
University College Dublin, Belfield, Dublin 4, Ireland, 4
MRC Human Genetics Unit; Institute of Genetics and
Molecular Medicine, Western GeneralHospital, Edinburgh, UK, 5 Centre for Population Health Sciences,
The University of Edinburgh Medical School, 6 University of Split School of Medicine, Šoltanska 2, Split,
Croatia, 7 University of Zagreb, Faculty of Pharmacy and
Biochemistry, Zagreb, Croatia
Maturity-onset diabetes of the young (MODY) is a dominantly inherited form of non-insulin dependent diabetes
caused by mutations in several genes. A subtype of MODY
is caused by mutations in HNF1A, a nuclear transcription
factor which appears to be one of the key regulators of
metabolic genes. Recently we showed that deleterious coding mutations in HNF1A have profound effects and that
antennary fucosylation of plasma proteins is significantly
decreased in HNF1A-MODY patients. The proportion of
HPLC peak DG9 in the sum of DG8 and DG9 (HAFU
index) roughly indicates the level of antennary fucosylation
of triantennary glycans in plasma. Low values of this index
appeared to be very indicative of HNF1A-MODY. HNF1AMODY patients could be nearly completely separated from
Type 1 diabetes, Type 2 diabetes, GCK-MODY and general
population on the basis of the HAFU index with Receiver-
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Operator Characteristic (ROC) curves approaching 90 %
specificity at 90 % sensitivity. In order to optimize and
simplify collecting and shipping procedure for samples,
we compared the analysis of standard plasma samples,
dried blood spots that were generated from whole blood
(venous blood) and dried blood spots generated from
capillary blood by finger prick test. All analyzed sample
types displayed only small differences in HPLC peaks
DG8 and DG9 and preserved the information value of
HAFU index.
164: Recent Technical Advances in Mass Spectrometrybased Glycoproteomic Applications
Kay-Hooi Khoo, Sz-Wei Wu, Chu-Wei Kuo, Shui-Hua
Wang; Institute of Biological Chemistry, Academia Sinica,
Taipei, Taiwan
kkhoo@gate.sinica.edu.tw
Despite intensive efforts over the last few years, mass spectrometry (MS)-based glycoproteomics remain difficult with
many unsolved technical problems. We and others have
advocated direct analysis of intact glycopeptides instead of
deglycosylated peptides or trimmed down glycopeptides in
order to answer the biological relevance of site-specific
protein glycosylation. We are particularly interested in asking if a specific glycotopes of interest is restricted in distribution to only a few sites or a few glycoproteins, from a
global glycoproteomic perspectives through a shotgun analysis. Over the years, we have worked on mAb-based affinity
capture and other biophysical enrichment methods to target
specific subsets of glycopeptides including the sulfated glycopeptides, investigated various modes of glycopeptide sequencing, and automated data analysis through development
of software suites. We aim to determine if terminal
glycotopes mapping can be more or equally effective at
such a shotgun glycoproteomic level compared with
glycomics. Through applications to single isolated glycoproteins, we demonstrated that novel glycosylation sites
could be identified when data analysis is not constrained
by inputs of predicted sites. On many occasions,
unpredicted proteolytic cleavages produces peptide cores
not matching anticipated peptide pools and therefore
cannot be identified unless de novo sequenced. In general, either a software-driven two-tiers MS2/MS3 on ion
trap or a combined HCD/ETD coupled with database
search provides the most objective, non-biased high
throughput analysis, which can be used in complementary. Likewise, a combination of two-step orthogonal
enrichment is often more effective in producing the
desirable glycopeptide pools. Analysis of sulfated
sialylated glycopeptides remain daunting, which can be
Glycoconj J (2013) 30:281–461
facilitated by chemical derivatization. We highlight here
several aspects of technical advances and progress we
have made over the last few years in glycoproteomics,
exemplified by a diverse range of applications including
one on endogenous EGFR from several lines of primary
lung cancer cells.
165: Development of glyco-biomarker for liver disease
Makoto Ocho 1 , Akira Togaychi 1 , Hiroyuki Kaji 1 ,
Atsushi Kuno1, Etsuko Iio2, Maki Sogabe1, Yasuhito
Tanaka2, Yuzuru Ikehara1, Masashi Mizokami3, and
Hisashi Narimatsu 1 ; 1 Research Centre for Medical
Glycoscience (RCMG), National Institute of Advanced
Industrial Science And Technology (AIST), Tsukuba,
Ibaraki 305-8568, Japan; 2Department of Virology and
Liver, Nagoya City University Medical School, Nagoya,
467-8601, Japan; 3Research Centre of Japan, Hepatitis
and Immunology, Kohnodai Hospital, International Medical
Centre, Ichikawa, 272-8516, Japan
makoto.ochou@aist.go.jp
Identification of new glyco-biomarkers predicting the
liver disease progression and the presence of hepatocellular carcinoma (HCC) has been attempted. An accurate
method for monitoring the progression of liver fibrosis,
predicting liver cirrhosis (LC) and detecting HCC is
urgently needed. We previously proposed a large number
of candidate glycoproteins for monitoring liver disease
progression which were identified by comprehensive
analysis of serum samples.
In this study, we found liver disease-associated glycan
profile on a candidate protein, which pointed out the
presence of markedly developed HCC or progressed LC
in the patients with HCV-/HBV- infected chronic liver
disease. In order to clarify the relationship between the
presence of the candidate molecule with aberrant glycosylation and the prevalence of progressed liver diseases,
we analyzed this candidate marker by a clinical validation study. As a result, the increased amount of the
candidate molecule with the aberrant glycosylation was
more relevant to poor outcome of LC than hepatic
carcinogenesis of the patients. While our result suggests
that the biomarker is feasible to use in individualization
for poor prognostic patients with a risk of LC progression, further clinical studies would yield more precious
information.
This work was supported by a grant from New Energy and
Industrial Technology Development Organization (NEDO)
in Japan.
Glycoconj J (2013) 30:281–461
166: Development of novel marker for cholangiocarcinoma
glyco-diagnosis
Atsushi Matsuda1, Atsushi Kuno1, Hideki Matsuzaki1,
Toru Kawamoto 2 , Tatsuro Irimura 3 , Toshihide
shikanai1, Yasuni Nakanuma4, Masakazu Yamamoto2,
Nobuhiro Ohkohchi 5 , Yuzuru Ikehara 1 , Junichi
Shoda 6 , Jun Hirabayashi 1 , Hisashi Narimatsu 1 ;
1
Research Center for Medical Glycoscience (RCMG),
National Institute of Advanced Industrial Science and
Technology (AIST), Tsukuba, 305-8568, Japan; 2Department
of Surgery, Institute of Gastroenterology, Tokyo Women’s
Medical University, Tokyo, 162-8666, Japan; 3Laboratory of
Cancer Biology and Molecullar Immunology, Guraduate
School of Pharmeceutical Sciences, The University of
Tokyo, Tokyo, 113-0033, Japan; 4Department of Human
Pathology, Graduate School of Medical Science, Kanazawa
University, Kanazawa, 920-1192, Japan; 5Department of
Surgery, Graduate School of Comprehensive Human
Science, University of Tsukuba, Tsukuba, 305-8575,
Japan; 6Field of Basic Sports Medicine, Sports Medicine,
Graduate School of Comprehensive Human Science,
University of Tsukuba, Tsukuba, 305-8575, Japan.
atsushi-matsuda@aist.go.jp
Cholangiocarcinoma (CC) is an aggressive malignant tumor
arising from the epithelial lining of the biliary tract. Since
the definitive CC diagnosis relies on invasive methods such
as biliary cytology and biopsy, a noninvasive assay with
high diagnostic accuracy has been keenly required. We
previously reported that Wisteria floribunda agglutinin
(WFA) is the best probe lectin which reliably distinguishes
between the non-cancerous and cancerous region in tissue
sections of CC by the lectin microarray and histochemical
analysis. Here our aim is the development of a novel glycoprotein marker for reliable CC diagnosis by WFA-based
glycoproteomics approach. We identified several WFA
binding glycoproteins by immunohistochemistry and mass
spectrometry analysis. Then, we verified these WFApositive glycoproteins as the diagnostic marker candidates
for CC using bile and serum specimens. Sialyl-MUC1,
recognized with a specific monoclonal antibody, MY.1E12,
was the primary candidate identified as one of the WFApositive glycoproteins. A WFA-coated MY.1E12 sandwich
ELISA was constructed for validation with bile and serum,
and this marker could distinguish CC from other patients in
bile specimens (Matsuda A., et al., Hepatology, 2010). L1
cell adhesion molecule (L1CAM) was the second candidate
by the WFA-assisted glycoproteomics from surgical CC
tissue sections. We confirmed that WFA-positive L1CAM
expressed specifically in the CC tissues and bile, and the
amount was significantly different between the bile specimens of CC and benign bile duct disease. The combined use
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of WFA-sialyl MUC1 and WFA-L1CAM sufficiently
improved the accuracy of CC (overall accuracy = 0.84,
AUC = 0.93) in the biliary diagnosis. As the ultimate
goal of this study is the serological diagnosis, we are
performing further verification of these markers using
serum by immunodetection and lectin microarray analysis. This combination assay using WFA-sialyl MUC1
and WFA-L1CAM will possibly be a useful serological
test with enhanced reliability.
This work was supported a grant from New Energy and
Industrial Technology Development Organization (NEDO)
in Japan.
167: Glycoproteomics of serum proteins in hepatitis B
and hepatitis C induced liver cirrhosis
Gautam Mondal 1 , Yogesh K Chawla 2 , Hirokazu
Ya g i 3 , K o i c h i K a t o 4 , B i s h n u P C h a t t e r j e e 1 † ;
1
Department of Natural Sciences, West Bengal University
of Technology, Salt Lake, Kolkata-700064, India, 2Department of Hepatology, Post Graduate Institute of Medical
Education and Research, Chandigarh -160012, India,
3
Graduate School of Pharmaceutical Sciences, Nagoya City
University, 3-1 Tanabe-diri, mizuho-ku, Nagoya 4678603, Japan,
4
Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences,
Okazaki Institute for Integrative Biosciences, 5-1 Higashiyama,
Myodaiji, Okazaki-444-8787, Japan
cbishnup@gmail.com
Glycosylation is one of the most important events of post
translational modification in many serum proteins which can
provide information about the onset and progression of
disease. Both hepatitis B virus (HBV) and hepatitis B virus
(HCV) cause acute and chronic liver infection and most
chronically infected individuals remain asymptomatic for
many years. Although biopsy is the gold standard method
for both hepatitis B virus induced liver cirrhosis (HBV-LC)
and C virus induced liver cirrhosis (HCV-LC), it has several
disadvantages. Therefore, a non-invasive, simple test would
be highly desirable. During inflammation and other related
disorders altered glycosylation patterns are observed in several acute phase proteins besides their alteration in expressions. Our aim was to monitor the changes in the expression
level and glycosylation pattern of serum glycoproteins of
hepatitis patients which could be used as serological
markers for diagnosis. Alpha-1 acid glycoprotein (AGP)
was isolated from the albumin depleted sera of HBV-LC
and HCV-LC patients by affinity chromatography on
mononclonal anti-AGP column. The unbound fraction by
2D gel electrophoresis gave several protein spots, which
364
were identified by MALDI-TOF-MS. Database search of
MS-MS spectra identified six significantly altered serum
glycoproteins: haptoglobin alpha 2 (Hp-α2) chain, haptoglobin beta 2 (Hp-β) chain, apolipoprotein A-1(ApoA-1),
alpha-1 antitypsin (AAT), vitamin D binding protein
(BTDBP) and immunoglobulin heavy chain (IGHA) respectively. This result was further validated by immunoblot
using monoclonal antibody indicating possible increased
expression of AAT and decreased level of Hp-α2 and
Hp-β chain in both hepatitis patients with respect to control.
Lectin blots showed decreased level of sialic acid and increased of fucosylation of serum glycoproteins which was
assessed by Maackia amurensis agglutinin (MAA) and Lens
culinaris agglutinin (LCA) respectively. The alteration of
glycosylation in AGP from two groups of hepatitis patients
was determined by HPLC mapping method followed by
mass spectrometry and GALAXY database search. Nglycans released from AGP by hydrazinolysis were labeled
with 2-aminopyridine and separated by three successive
HPLC columns, viz., DEAE, ODS and amide silica. We
found decrease or absence of sialic acid in HBV-LC and
increased fucosylation of neutral N-glycans in both hepatitis patents. Thus glycoproteomics could be helpful to
search for novel glyco-markers in diagnosis of hepatitis
virus induced liver cirrhosis.
168: N-glycan Analysis of Immunoglobulin G by
Enzymatic Release with Remove-iT Endo S and LC-MS
Xiaofeng Shi; Elizabeth McLeod; Paula Magnelli; Alicia
Beliek; Ellen Guthrie; New England Biolabs Inc., Ipswich,
MA, 01938, USA
shi@neb.com
A growing number of monoclonal IgG antibodies are currently being developed as therapeutic agents. Critical for
their structure and biological activity is the N-glycan moiety
attached to the asparagine 297 residue in the constant domain. There are many variables in cell culture that can
greatly influence the heterogeneity of the glycans on IgG.
Therefore, it is critical to monitor the glycosylation profiles
of these biotherapeutics in the production process. A platform that includes efficient and complete glycan release, fast
workup, and compatibility with proteomics workflow for
IgG glycan analysis is highly sought. Here we attempt to
address these questions by using a novel endoglycosidase
with a fused chitin-binding domain.
Remove-iT Endo S (also known as Endoglycosidase S) was
cloned and purified from Streptococcus pyogenes and
overexpressed as a fusion to the chitin binding domain in E.
coli. Glycerol free IgG samples were enzymatically
Glycoconj J (2013) 30:281–461
deglycosylated under native conditions using Remove-iT Endo
S and PNGase F Glycerol Free. The deglycosylated IgG samples were then analyzed by SDS-PAGE gel shift analysis to
estimate the degree of deglycosylation. In addition, the glycans
released from IgG following deglycosylation by both enzymes
were analyzed by nanoLC-TOF MS for glycan profiling.
The results show that Remove-iT Endo S has a high specificity for removing the N-glycan moiety of IgG after the first Nacetylglucosamine (GlcNAc) residue, leaving only the
GlcNAc with or without a core fucose residue, whereas
PNGase F cleaves between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from N-linked glycoproteins. Remove-iT Endo S
is a more robust enzyme for the method described herein as it
completely removes the sugar residues from IgG. Conversely,
the PNGase F glycerol free digest does not result in a complete
digestion under native conditions. Remove-iT enzymes can be
easily eliminated from the digestion buffer by chitin magnetic
beads, immediately followed by proteomics workflow.
169: Simplified Quantitative Glycomics Using the Stable
Isotope Label Girard’s Reagent P by Electrospray
Ionization Mass Spectrometry
Chengjian Wang 1, Zhiyu Wu1, Jiangbei Yuan1, Bo
Wang1, Ping Zhang2, Ying Zhang1, Zhongfu Wang1,
Linjuan Huang1; 1Educational Ministry Key Laboratory
of Resource Biology and Biotechnology in Western China,
Life Science College, Northwest University, Xi’an 710069,
China; 2Chemistry and Chemical Engineering School,
Xianyang Normal University, Xianyang 712000, China
huanglj@nwu.edu.cn
Glycosylation alteration of proteins has proved to be associated with many diseases, such as cancer, type 2 diabetes,
and congenital disorders of glycosylation (CDG). To monitor the changes in the abundance and structure of the glycan
moieties of various glycoproteins, quantitative glycomics
based on stable isotope labeling coupled with mass spectrometric analysis represents an emerging and promising technique. However, this technique is undermined by the timeconsuming post-processing steps and the complexity of the
mass spectra of isotope-labeled glycans arising from the
presence of multiple metal ion adduct peaks, which result
in a decrease of detection sensitivity and an increase of
difficulties in data interpretation. In this study, a facile
strategy for the relative quantitative analysis of N-glycans
using the stable isotope label Girard’s reagent P (GP) has
been developed. This procedure features efficient labeling of
reducing glycans with protium- or deuterium-substituted
(d0/d5-) GP under non-reductive conditions and subsequent
Glycoconj J (2013) 30:281–461
direct detection by mass spectrometry (MS) without any
post-processing steps. The obtained intensity ratio between
the MS peaks of the d0- and d5-GP derivatives of each
glycan allows rapid and high-throughput relative quantitative
analysis. More importantly, the GP-derivatives exhibit only
[M]+ type ion peaks in their MS profiles, excluding the interference from multiple complex ion adduct peaks such as [M+
H]+, [M+Na]+ and [M+K]+ and simplifying exceedingly the
interpretation of their MS data. We have demonstrated the
reliability and high sensitivity of the method using lactose as a
model glycan by electrospray ionization mass spectrometry
(ESI-MS). The relative quantitative analysis of the N-glycans
released from bovine pancreas ribonuclease B and chicken
egg white albumin revealed the good linearity and high
reproducibility of the procedure within a 100-fold molar
ratio range. The excellent applicability of the novel method was validated via the comparative analysis of the Nglycans released from bovine and porcine immunoglobulin
G (IgG) by ESI-MS as well as of those from mouse and
rat serum by ESI-MS and hydrophilic interaction liquid
chromatography coupling with mass spectrometry
(HILIC-MS).
170: A Robust Method for the Relative Quantitative
Analysis of N-Glycan
Weiqian Cao, Wei Zhang, Jiangming Huang, Pengyuan
Yang*; Fudan University, Shanghai, 200032, China
pyyang@fudan.edu.cn
With the development of glycoproteomics, relative quantification of glycosylation has received considerable research
attention, which is important for discovering glycosylation
changes and finding potential biomarker. High throughput
glycan quantitation was developed to investigate quantitative glycan changes in structure type, composition, linkage
and conformation of glycans from different biological samples. Similar to protein quantitation, MS based isotopic label
is also a powerful tool for glycan quantitation.
In this reach, we developed a novel relative quantitation
method for glycans. The novel sodium borohydride assisted
enzymatic 18O labeling added the reducing end of all released glycans with 3 Da. After this label, the mass gap
increased to 3 Da, and the partial overlap of isotope envelopes was largely reduced. Moreover, the glycan became
more stable, and the labeled 18O would never exchange with
16
O again in normal water. We further tested the stability
and overlap of this label, which showed good results. By
this label reaction, we optimized the enzymatic glycan 18O
labeling, and made the method more suitable for quantitative glycomics by mass spectrometry.
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171: An efficient approach to prepare boronic acid functional
core-shell polymer nanoparticles for enrichment of
glycoproteins via RAFT media precipitation polymerization
Jianxi Liu1,2, Kaiguang Yang1, Yanyan Qu1,2, Lihua
Zhang1*, Zhen Liang1 and Yukui Zhang1; 1Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian
116023, China;2 Graduate School of the Chinese Academy
of Sciences, Beijing100039, China.
lihuazhang@dicp.ac.cn
Glycosylation is one of the most common post-translational
modifications of proteins, and plays an important role in a
variety of biological activities. Therefore, the global profiling
of N-linked glycosylationsites is a prerequisite to study the
biological functions of glycoproteins. Due to low abundance
and poor ionization capacity, the selective enrichment of glycoproteins or glycopeptides before MS analysis is imperative.
Herein, we developed a novel approach to prepare hydrophilic core–shell polymer nanoparticles. The hydrophilic
core was formed by distillation precipitation polymerization
(PP) method using methacrylic acid (MAA) as the monomer, and N,N-methylenebisacrylamide (MBAA) as the
cross-linker. The functional shell was prepared by reversible
addition/fragmentation chain-transfer (RAFT) with 3acrylamidophenylboronic acid (APBA) and MBAA, instead
of by traditional free radical polymerization (TFRP). The
new strategy presented here could obviously simplify the
preparation procedure. The obtained boronate affinity polymer nanoparticles, poly(MBAA-co-MAA)@(MBAA-coAPBA), showed good stimuli-response towards cis-diol
containing molecules, and the maximum adsorption
capacity for horseradish peroxidase (HRP) was
76.25 mg/g. The results demonstrated that poly(MBAAco-MAA)@(MBAA-co-APBA) nanoparticles prepared by
RAFT media PP might be promising in profiling the glycosylation site occupancy and corresponding glycan heterogeneity for glycoproteome analysis.
172: An integrated platform for quantitative
glycoproteome analysis
Yejing Weng1,2, Yanyan Qu1,2, Lihua Zhang1*, Huiming
Yuan1, Hao Jiang1,2, Zhigang Sui1,2 and Yukui Zhang1;
1
Key Laboratory of Separation Science for Analytical
Chemistry, National Chromatographic R. & A. Center,
Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian 116023, China; 2Graduate School of the
Chinese Academy of Sciences, Beijing 100049, China.
lihuazhang@dicp.ac.cn
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Protein N-glycosylation is one of the most prevalent and
complex post-translational modifications, which plays an important role in various biological process, especially in cancer
progression and immune responses. Therefore, the quantitative profiling of N-linked glycosylation sites occupancy is of
important biological and clinical significances. However, for
quantitative glycoproteome analysis, traditional offline protocols often suffer from drawbacks such as long analysis time,
sample loss and manual manipulation.
Herein, a novel integrated platform for quantitative
glycoproteome analysis was established, by which glycopeptides was first captured by a click chemistry maltose based
hydrophilic interaction chromatography (HILIC) column, online solvent exchange by a nitrogen assisted HILIC-RPLC
interface, deglycosylated by a hydrophilic PNGase F
immobilized enzymatic reactor (IMER), trapped on a C18
column, labeled by isotope dimethyl reagents, and analyzed
by MALDI-TOF MS/MS. To evaluate the performance of
such an integrated platform, the digests of IgG with the
coexistence of 50 times (mass ratio) BSA were analyzed,
and 15 fmol deglycosylated glycopeptide from IgG was selectively detected with high signal to noise ratio. Furthermore,
the quantitative accuracy and precision were demonstrated by
comparing the signal intensity ratio of light labeling
deglycosylate glycopeptide (m/z 1186) with heavy labeling
deglycosylated glycopeptide (m/z 1190). The relative standard
deviation (RSD) triplicated analysis was 5.7 %, and the experimental ratio of both peptides was kept as 1:1, in accordance with the theoretic value. All these results demonstrated
that the integrated platform was of great promising for both
qualitative and quantitative N-linked glycosylation sites profiling. The further on-line hyphenation of such a platform with
ESI-MS/MS is undergoing, to achieve high-throughput analyses of biological samples.
173: Boronic acid functionalized graphene oxide composites for specific recognition of N-linked glycopeptides
Jiang Bo 1,2 , Yang Kaiguang 1, Liang Zhen 1 , Zhang
Lihua1*, Zhang Yukui1; 1National Chromatographic R.
& A. Center, Key Laboratory of Separation Science for
Analytical Chemistry, Dalian Institute of Chemical Physics,
Chinese Academy of Science, Dalian 116023, China. 2State
Key Laboratory of Fine Chemicals, Dalian University of
Technology, Dalian 116012, China.
lihuazhang@dicp.ac.cn
Glycosylation is one of the most important post-translational
modifications in proteomes. Because of its high efficiency
and high sensitivity, MS technology shows immense potential for the detailed structure characterization of protein
Glycoconj J (2013) 30:281–461
glycosylation. Unfortunately, due to the inherent low abundance of glycopeptides and the microheterogeneity of each
glycosylation site, it remains a great challenge for the comprehensive understanding of glycoproteome by MS based
strategy. Therefore, the specific enrichment and purification
of glycopeptides is essential prior to MS analysis.
Herein, for the first time, we developed a facile method to
prepare novel boronic acid functionalized graphene oxide
composites, GO/PEI/Au/MPB, and investigated the performance on the selective enrichment of N-linked glycopeptides.
Due to the high hydrophilicity of such composites, even the
mass ratio of BSA to ASF was increased to 100:1, the signal
of glycopeptides was still high. Our experimental results
further demonstrated the robustness and reliability of such
material to capture glycopeptides from complex samples.
174: Gold nanoparticles modified hydrophilic polymer
monoliths functionalized with boronate acid for specific
capture of glycoproteins
Ci Wu1,2, Yu Liang1, Qun Zhao1,2, Yanyan Qu1,2, Zhen
Liang1, Lihua Zhang1*, and Yukui Zhang1; 1Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R. & A. Center, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences, Dalian
116023, China; 2University of Chinese Academy of Sciences, Beijing 100049, China.
lihuazhang@dicp.ac.cn
Glycoproteome plays a crucial role in numerous biological
processes. However, the main bottleneck for glycoproteome
study is the low abundance of glycoproteins in complex
samples. Recently, various boronate affinity monoliths have
been springing up for glycoproteins enrichment. However,
monoliths applied in boronate affinity chromatography are
still meeting some challenges. One is the nonspecific adsorption of proteins, which could reduce the enrichment
selectivity and the recovery of glycoproteins. The other is
the limited enrichment capacity owing to the low surface
area of monoliths. Therefore, the development of hydrophilic boronate affinity monolithic columns with high surface
area is significant for the enrichment of glycoproteins with
high efficiency and selectivity.
In this study, a novel kind of hydrophilic boronate affinity
monoliths was successfully synthesized by co-assembling 4mercaptophenylboronic acid (MPBA) with 2mercaptoethylanine (MPA) on a gold nanoparticles modified
hydrophilic monolithic column, prepared by the thermal initiated polymerization of poly (ethylene glycol) diacrylate and
glycidyl methacrylate. The matrix showed the low nonspecific
Glycoconj J (2013) 30:281–461
adsorption of proteins because of good hydrophilicity. Gold
nanoparticles modified on such matrix could obviously increase the surface area. With MPBA and MPA co-assembled,
such a new affinity monolithic column could specifically capture horseradish peroxidase (HRP) from the mixture of HRP
and BSA, even at a ratio of 1:1000 (m/m). The binding capacity
of ovalbumin reached 0.39 mg•g−1, higher than that obtained
on other boronate affinity monoliths reported. In addition, high
recovery of glycoproteins on the prepared boronate affinity
monolith (85 %) was achieved. Moreover, such a column was
successfully employed to specifically capture glycoproteins
from egg white, demonstrating the great promising for
glycoproteome research.
175: Preparation of amide functionalized hydrophilic
monolith by in situ photo-polymerization for highly
selective enrichment of glycopeptides
Hao Jiang1,2, Huiming Yuan1, Yu Liang1, Yanyan Qu1,2,
Qi Wu1,2, Lihua Zhang1*, Yukui Zhang1; 1National
Chromatographic Research and Analysis Center, Key
Laboratory of Separation Science for Analytical Chemistry,
Dalian Institute of Chemical Physics, Chinese Academy of
Science, 457 Zhongshan Road, Dalian 116023, China,
2
Graduate School of Chinese Academy of Sciences,
Beijing 100039, China
lihuazhang@dicp.ac.cn
For glycoproteome analysis, glycopeptide enrichment is an
indispensable step prior to MS analysis. Recently, various
methods have been developed, such as lectins, boronic acid
affinity, hydrazide chemistry and hydrophilic interaction chromatography (HILIC), among which HILIC based methods
have been drawn much attention due to low bias, good reproducibility and mild operation conditions. However, since the
surface property on traditional HILIC materials is not homogenous, it is necessary to develop novel materials to improve
the selectivity of glycopeptide enrichment.
In this study, a novel kind of amide functionalized monolith
was synthesized by the in situ photo-copolymerization of
acrylamide, methylene diacrylamide and N-vinyl-2pyrrolidone in a UV transparent capillary. The performance
of such a monolithic column was evaluated by the enrichment of glycopeptides from the tryptic digests of immunoglobulin G (IgG). With the captured glycopeptides analyzed
by MALDI-TOF MS, 16 glycopeptides from IgG digests
could be identified, demonstrating the high efficiency for
glycopeptide enrichment. Furthermore, the selectivity of
glycopeptide enrichment was also investigated by mixing
the digests of bovine serum albumin and IgG with the ratio
of 10000:1 (m/m), and five glycopeptides were
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unambiguously identified, further indicating the high selectivity and good specificity of such material. Moreover, the
monolithic column was also applied for the glycosylation
sites profiling of 6 μg proteins extracted from human serum
(HS), corresponding to 100 nL original HS, and 78 unique
glycosylation sites were identified, indicating its great potential for glycoproteome analysis.
176: Tandem dimethyl and solid-phase 18O stable isotope
labeling for N-glycoproteome quantitation
Yanyan Qu1,2, Yejing Weng1,2, Kaiguang Yang1, Lihua
Zhang1, Zhen Liang1, Yukui Zhang1; 1Key Laboratory of
Separation Science for Analytical Chemistry, National
Chromatographic R. & A. Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023,
China; 2University of Chinese Academy of Sciences,
Beijing 100049, China.
lihuazhang@dicp.ac.cn
N-glycosylation is one of the most common and complex
posttranslational modification of proteins. Besides the global mapping of N-linked glycosylation sites, recently much
attention has been paid on N-glycosylation occupancy quantitation at specific sites.
To date, a variety of chemical labeling approaches, i.e.,
dimethyl labeling, isotope-coded affinity tags, and tandem
18
O stable isotope labeling (TOSIL), have been used for the
relative quantitation of protein glycosylation. In contrast to
other strategies, TOSIL, which incorperates three 18O atoms
into the heavy labeled glycopeptides during tryptic and Nglycosidase F (PNGase F) mediated hydrolysis, could obviously improve the distinguishment of glyco- and nonglycopeptides, and also the isotope distribution overlapping between differently labeled glycopeptides. However, the false
positive rate of the quantification results might be increased
by the limited 18O-labeling efficiency, back exchange of 18O
and interference of residual active trypsin that incorporates
18
O into peptides during PNGase F deglycosylation.
Herein, we presented a new tandem dimethyl and solidphase 18O stable isotope labeling strategy (TDSOSIL), by
which glycopeptide pairs were first labeled with light/heavy
dimethyl after selectively enrichment, and then labeled with
light/heavy oxygen atoms during deglycosylation by
PNGase F immobilized enzymatic reactor (PNGase F
IMER), generating enlarged 6, 10 or 12 Da difference between the glycopeptide pairs. It was demonstrated that the
developed PNGase F mediated solid-phase 18O labeling
could dramatically minimize the back exchange of 18O
labeled peptides, and overcome the spontaneous
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nonenzymatic 18O labeling. In combination with the highefficient dimethyl labeling, the accuracy of N-glycosylation
quantitication and qualitation was improved. By such strategy,
146 N-glycosites were quantified with equal amount syngeneic mouse hepatocarcinoma ascites (HCA) cell digest labeled
with light/heavy isotope, and 97 % of which had the H/L
ratios in the range of 0.50–2.00 with RSD <30 %, indicating
the great promise of TDSOSIL strategy for N- glycosylation
quantitation. This strategy is now being applied for the differential glycoproteome analysis of highly and lowly metastatic
typed HCA cell lines, the result of which will provide important technical support for the revealment of metastatic mechanism and the further discovery of potencial biomarker for
desease diagnosis and drug therapy.
Poster Session I-Glyco(bio)technology
177: Analysis of highly complex glycan mixture derived
from human fluid by MALDI-TOF/TOF, nano-LC/Chip
Q-TOF and UPLC-Triple Quadruple Mass Spectrometry
Su Hee Kim1, Bum Jin Kim1, Jae Han Kim2 and Hyun
Joo An1*; 1Graduate School of Analytical Science and
Technology, Chungnam National University, Daejeon,
Korea, 2Department of Food and Nutrition, Chungnam
National University, Daejeon, Korea
sugar@cnu.ac.kr
Depending on the ionization methods and the type of mass
analyzer, various mass spectrometric analyses have been
developed. Due to their unique analytical advantages, appropriate mass spectrometric method has to be chosen practically depending on the purpose. While MALDI-TOF
enables the quick view of whole glycan profile in the samples, nanoLC/Chip Q-TOF can provide the insight of isomers which MALDI-TOF is intrinsically ignored. UPLCTriple Quadruple offers fast and quantitative analysis comparing with nano-LC/Chip Q-TOF, however, relatively low
mass accuracy and resolution are disadvantages. Although
the use of appropriate MS is crucial for the efficient analysis
of complex glycan mixtures, data comparability between
each MS has to be evaluated prior to the MS data interpretation and comparison. In this study, we assess the qualitative and quantitative comparability of each mass
spectrometry using human milk oligosaccharides (HMO).
HMO is a highly complex glycan mixture with a similar
structure to the O-glycan. Extraction of free oligosaccharides was performed by following steps including the removal of lipids by Folch method and ethanol protein
precipitation. To get rid of αand βisomers, free oligosaccharides were reduced to alditols. Extracted free oligosaccharides
were further purified by solid phase extraction enriched 20 %
Glycoconj J (2013) 30:281–461
and 40 % (in 0.05 % TFA) ACN fraction. Through an efficient
analytical method, we found 64 free oligosaccharides by
MALDI-TOF MS and 110 free oligosaccharides from nanoLC/Chip Q-TOF MS. The validity of the quantitative data was
confirmed by analysis using the UPLC-Triple Quadrupole MS
with multiple reaction monitoring (MRM) mode. Following
this analytical method we have wide application various dairy
streams, such as goat milk, unprocessed milk and low-fat
bovine milk. In the dairy industry, this study can be applied
as an analytical platform to composition profile and isomer
specific glycan quantify free oligosaccharides using suitability
Mass Spectrometry.
178: SimianTools: A software suite for the interpretation
of high throughput glycomics mass spectrometry data
René Ranzinger, Ki Tae Myoung, Mindy Porterfield,
Michael Tiemeyer and William S. York; Complex Carbohydrate Research Center, University of Georgia, Athens,
GA, USA
rene@ccrc.uga.edu
Over the last decades the approaches for analyzing and
identifying glycan structures using mass spectrometry have
evolved from the study of single purified glycans towards
the analysis of complex mixtures. However, most interpretation of the resulting datasets is still done manually. But
comprehensive analysis of complex biological samples, producing several thousand mass spectra, consumes significant
amount of time which consequently reduces the number of
samples that can be analyzed. Therefore, development of
high-throughput glycomics analysis software is crucial to
efficiently tackle the large amount of data produced.
SimGlycan® is a commercial software system which facilitates the interpretation of MS based glycomics data. However, the output of this product is not amenable to highthroughput studies designed to compare glycomics changes
across many samples. Therefore we have developed a suite
of programs that build on the SimGlycan® functionality and
extend the usefulness of this tool.
SimGlycanDatabaseBot is a program that enables the generation of customized SimGlycan® databases which can
subsequently be used for the interpretation of experimental
data. The large, publicly available glycan databases that
contribute to the database utilized by SimGlycan® contain
many redundancies and partial erroneous structures that can
result in invalid annotations. Our software allows the user to
create a database by importing own structures or by
selecting a subset of GlycomeDB structures to be
imported into the new database. SimianTools is a program we have developed to enhance the evaluation of
Glycoconj J (2013) 30:281–461
results generated by SimGlycan®. After importing annotations generated by SimGlycan®, SimianTools generates
graphical glycan representations and adds several statistical measures and specific identifiers to assist users in
the evaluation of the total dataset. The program also
supports side by side comparisons of results from several different analyses, making it a useful tool to find
changes in glycan expression. All results can be
exported in Excel format to allow post-processing of
the information using third-party programs.
Poster Session I-Structural & Chemical
Glycobiology and Glycomics
179: Cell-Selective Metabolic Glycan Labeling Based on
Ligand-Targeted Liposomes
Ran Xie, Senlian Hong, Lianshun Feng, Jie Rong, and
Xing Chen; Department of Chemical Biology, College of
Chemistry and Molecular Engineering, Peking University,
Beijing 100871, China
xingchen@gmail.com
Cell-surface glycans play key roles in mediating various
molecular recognition events; aberrant glycosylation is implicated in disease progression. Therefore, probing the dynamic changes of glycan biosynthesis and structures is of
great importance for augmenting our understanding of
glycobiology and improving disease diagnosis and therapeutics. The metabolic glycan labeling was an appealing
approach to incorporate specially designed carbohydrate
analogs into the glycans, which enables the detection and
imaging of the glycans in cells and living animals. However,
one major bottleneck of this method is cell-type selectivity.
Herein, we present the cell-specific metabolic glycan labeling using ligand-targeted liposomes to deliver unnatural
sugars to target cells in a cell-surface receptor dependent
manner. In this strategy, sugar analogs are encapsulated in
ligand-targeted liposomes. The ligands bind to specific cellsurface receptors that are only expressed or up-regulated in
target cells, which mediate the intracellular delivery of
azidosugars via endocytosis. The delivered azidosugars are
metabolically incorporated into cell-surface glycans and
detected or imaged using a bioorthogonal reaction. The
application of this strategy includes the facile introduction of myriad alternative ligands, as well as the cellspecific or tissue-specific imaging and detection of
glycosylation in vivo.
Acknowledgements
We thank Prof. P. Wu for providing the initial batch of
BTTAA ligand. This work was supported by the National
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Natural Science Foundation of China (No.21172013,
No. 91127034) and the National Basic Research Program of China (973 Program) (No. 2009CB930303, No.
2012CB917303).
180: Detection and Isolation of Dendritic Cells Using
Lewis X-functionalized Magnetic Nanoparticles
Sara H. Rouhanifard 1 , Ran Xie 2 , Guoxin Zhang 3 ,
Xiaoming Sun3, Xing Chen2 and Peng Wu1; 1Department
of Chemical Biology, College of Chemistry and Molecular
Engineering, Peking University, Beijing 100871, P. R.
China, 2Department of Biochemistry Albert Einstein
College of Medicine, Yeshiva University, 1300 Morris
Park Ave, Bronx, NY 10461, 3State Key Laboratory of
Chemical Resource Engineering, P.O. Box 98, Beijing
University of ChemicalTechnology, Beijing 100029,
P. R. China
xingchen@gmail.com
Dendritic cells (DCs) are professional antigen-presenting
cells that serve as messengers between innate and adaptive
immunity. Due to their unique roles, DCs are under active
investigations as the target for antigen delivery in vaccination against human immunodeficiency virus (HIV), cancer
and autoimmune diseases. However, there’s yet no perfect strategy for a cost-effective, high-selective and timesaving detection and isolation of DCs from a complex
cell population.
Adhesion molecules such as DC-specific intracellular adhesion molecule-3 grabbing non-integrin (DC-SIGN) on the
surface of DCs play a critical role in establishing contact
between the activated DCs and the naïve T cells through
interactions with intercellular adhesion molecule-3 on T
cells. DC-SIGN is a member of the C-type lectin receptors
and can recognize antigens bearing mannose-rich or
fucosylated glycans, as well as Lewis X (LeX). Here, we
present the fabrication of magnetic nanoparticles coated with
multivalent LeX glycans using the Cu (I)-catalyzed azidealkyne cycloaddition. The resulting nanoparticles are selective and biocompatible, serving as a highly efficient tool for
DC capture detection and enrichment.
Acknowledgements
This work was supported partially by a DuPont Young
Professor Award and the National Institutes of Health to
P.W. (R01GM093282), the National Natural Science Foundation of China to X.C. (No. 21172013, and No. 91127034)
and the National Basic Research Program of China (973
Program) to X.C. (No. 2009CB930303, and No.
2012CB917303).
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181: Functional Similarities and Differences between
New Lectin Systems in Human Organism: Protein
Hormone and Probiotic Bacterial
Mikhail V. Lakhtin, Vladimir M. Lakhtin, Andrey V.
Aleshkin, Stanislav S. Afanasiev, Vladimir A. Aleshkin;
Department of Medical Biotechnology, G.N. Gabrichevsky
Research Institute for Epidemiology & Microbiology,
Moscow, 125212, Russia
Lakhtinv@yandex.ru
New lectin systems (LS) as well as Lectins-Glycoconjugates
(L-GC) relationships on solid surfaces are of increasing
interest for glycol(bio)technology. The aim was to find
functional similarities and differences between new hierarchic LS in human organism on the examples of protein
hormone (EPO) and probiotic bacterial L (PBL) isolated
from the cultural fluids of industrial probiotic lactobacilli
and bifidobacteria of human origin.
Human erythrocytes and yeasts were used in agglutination
assays. Pseudopolysaccharides (biotinylated or not) as GC
were used (www.lectinity.com). Commercial human recombinant EPO preparations or PBL containing concentrates
separated by IEF-PAG were blotted on Immobillon P and
visualized using Streptavidin-Peroxidase or immune sandwich labeled with peroxidase followed by chemiluminescent registration in the presence of chemiluminescent
peroxidase substrate in optimal conditions of live imagination in the Dark Room of BioChemi System (UVP, Calif.).
Proteins were visualized by fluorescent dye SYPRO Ruby
protein blot stain (Bio-Rad).
1. Both LS described and L-GC/GC-L interactions were
adaptive to the test systems (soluble surroundings and solid
phase used). L specificity as well as ranging GC depended
on the choice of testing: agglutination (activation) or adhesion (stabilization), cell type (human or microbial), natural
or artificial, heterogenic or homogenic. GC ranging
depended on non-aggregated or aggregated L state. In the
latter case EPO specificity to sialylated GC was appeared. 2.
In case of PB molecular protein associates on the blot, at
least three different layers (mask, protein and subprotein)
were detected; LS mosaics within protein distributions were
registered as strain and GC dependent. 3. Similarities of GC
recognition by both LS types were established (α-L-FucPAA, LacNAc-PAA). 4. Simultaneous recognition of GC
system and discrimination of simple antigenic GC by LS are
demonstrated.
Results are useful for further understanding LS and LGC multilayer assembling and disassembling on solid
surfaces.
Glycoconj J (2013) 30:281–461
182: Neolectins with tailored valency as tools in Glycobiology
Julie Arnaud 1, Julie Claudinon 2, Kevin Tröndle 2,
Annabelle Varrot 1 , Winfried Römer 2 , Aymeric
Audfray 1 & Anne Imberty 1; 1CERMAV-CNRS, Grenoble, 38041, France; 2BIOSS Centre for Biological Signalling
Studies, Freiburg, 79104, Germany
imberty@cermav.cnrs.fr
The ability of lectins to specifically recognize glycoconjugates
on the surface of cells makes them a useful tool in biomedical
diagnosis associated with change of glycosylation (inflammation, cancer…) Also, lectins are useful research tools for studying membrane trafficking since lectin-glycosphingolipid
interactions can lead to membrane bending and endocytosis.
However, only a limited number of lectins are available, limiting their use in biotechnology and research.
RSL is a fucose-binding lectin from the bacterium Ralstonia
solanacearum that adopts a ß-propeller fold formed by trimer
association and presenting six binding sites. RSL has been
chosen as the paradigm for designing neolectins since it is
easy to produce, has a high affinity for fucose and present six
symmetrical binding sites. Alteration of the symmetry of the
ß-propeller architecture is used to produce neoRSLs (nRSLs)
with controlled valency in order to improve diversity in the
development of technological tools and to understand the
endocytosis mechanism. Several mutants have been produced
in order to modify the valency of RSL.
The R17A neo-RSL stochiometry has been reduced to three
residues per ß-propeller, while neither the affinity for monosaccharides nor the avidity to surfaces is altered. However,
when the neolectin is tested on giant unilamellar vesicle, it
shows that is not capable of forming membrane invaginations contrary to the wild type protein RSL, demonstrating a
strong effect of the degree of multivalency on membrane
dynamics and internalization. Neolectins with modified
valency therefore appears as tools of choice for studying
membrane dynamics and intracellular trafficking.
183: Chitosan Coupling Makes Bacterial Biofilm
Susceptible to Antibiotics
Amin Zhang, Haibo Mu, Jinyou Duan*; College of
science, Northwest A&F University, Yangling 712100,
Shaanxi, China
jduan@nwsuaf.edu.cn
Bacterial biofilms are prevalent in natural, industrial and hospital settings and are inherently resistant to both antimicrobial
agents and host defenses. Herein we demonstrate that covalent
Glycoconj J (2013) 30:281–461
coupling of antibiotics to cationic polymers (chitosan) can
efficiently break down established biofilms and prevent biofilm
formation, and as a result, make biofilm bacteria more susceptible to antibiotics. This conjugation is suitable to treat biofilms
formed by a broad spectrum of gram-positive, but not gramnegative organisms. Mechanistic insight demonstrates chitosan
conjugation renders antibiotics access into biofilms, thereby
available to interact with biofilm bacteria. It appears that the
amount of antibiotics, the specialized molecular size and highly
positive charge in the conjugate are the key for maintenance of
high anti-biofilm efficiency. Thus, this work represents an
innovative strategy that antibiotics covalently linked to carbohydrate carriers can overcome antibiotic resistance to
biofilms, and might provide a comprehensive solution to
combat biofilms in industrial and medical settings.
184: Metrology for the characterization of Biomolecular
Interfaces for Diagnostic Devices (BioSurf)
Véronique Blanchard 1 , Aryaline Kamalakumar 1 ,
Rudolf Tauber1, Wolfgang Unger2 and Paul Dietrich2;
1
Institute of Clinical Chemistry and Pathobiochemistry,
Charité Medical University, Augustenburger Platz 1,
13353 Berlin, Germany, 2 BAM -Bundesanstalt für
Materialforschung und -prüfung, Unter den Eichen 44-46,
12203 Berlin, Germany
veronique.blanchard@charite.de
In-vitro diagnostics are vital for the drive for cost effective
healthcare, point of care monitoring and personalized medicine.
The diagnosis and management of medical conditions is becoming increasingly reliant upon the detection and measurement of biochemical markers, or targets. BioSurf is a european
project that aims at developing measurement methods for the
research, development and quality control of biomolecular interfaces in diagnostic devices, and establish accurate, traceable
and comparable methods to determine the amount of biomolecular probe immobilized at both planar and nanoparticle interfaces. It investigates novel approaches for the measurement
of biointerfacial structures that can be correlated with activity
and binding efficiency and methods to measure and predict the
activity of immobilized probes. With the goal of supporting
high throughput and multiplexed diagnostic methods, BioSurf
is also assessing the capabilities of emerging techniques capable of detecting many targets simultaneously. TOF-SIMS uses
a pulsed primary ion beam to desorb and ionize species from a
sample surface. This technique allows determine the elemental
and molecular species on a surface or in depth and to visualize
the distribution of individual species on the surface.
The quality, reproducibility and shelf life of biomolecular
interfaces will be presented with the above-mentioned
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techniques using high-mannose N-glycans and Concavalin
A as a example.
185: High-throughput substrate profiling of the UDPGalNAc: polypeptide N-acetylgalactosaminyltransferase
using human proteome microarrays
Wenxian Xie, Xing Li, Zhijue Xu, Jing Wang, Shumin
Zhou, Shengce Tao and Yan Zhang; Ministry of Education
Key Laboratory of Systems Biomedicine, Shanghai Center for
Systems Biomedicine, Shanghai Jiao Tong University,
Shanghai, 200240, China
yanzhang2006@sjtu.edu.cn
Mucin type O-glycosylation is arguably the most abundant and
complex type of protein glycosylation. A large family of UDPGalNAc: polypeptide N-acetylgalactosaminyltransferase
(ppGalNAc-T, EC 2.4.1.41) control and regulate the initial step
in the biosynthesis of mucin-type O-glycan. To date, there are
15 members of human ppGalNAc-T family having the GalNAc
transferase activity in vitro. They display tissue-specific expression and the specificity substrate activities. Several ppGalNAcT isoforms have been reported to be important for many cellular
and developmental processes under physiological or pathological conditions by modifying specific target proteins. However,
by now we have poor knowledge on understanding how many
proteins will be GalNAc O-glycosylated and how individual
ppGalNAc-T selects the target protein and how to decide the OGalNAc modification site. To find more comrades, we developed a strategy to identify potential substrates of ppGalNAc-Ts
globally by taken advantage of the high-throughput capability
of protein microarray and the high specificity of click chemistry. We took ppGalNAc-T2 as an example and successfully
identified 226 candidates as potential substrates. Gene ontology
analysis showed that membrane-associated proteins were highly enriched. Intriguingly, other categories of protein, such as
transcriptional factors and cytoskeleton related proteins were
also significantly enriched. Among these potential substrates,
17 selected proteins were successfully glycosylated by
ppGalNAc-T2 in vitro, and also validated by HPA lectin blotting. The in vitro O-GalNAc modified sites of p53 were successfully identified by mass spectromentry. Moreover, the overexpressed p53 in U2OS cells could be found O-GalNAc
glycosylated, which is detected by HPA and VVA Lectin
blotting. In this study, we have systematically screened potential substrates of ppGalNAc-T2 on proteome-wide and found
some interesting potential substrates located in nucleus and
cytoplasm. Our strategy could be easily applied for other
ppGalNAc-Ts, and this will greatly facilitate the construction
of the complete ppGalNAc-T and substrates network for all
human ppGalNAc-Ts, and eventually help us to unveil the
mysterious function of ppGalNAc-T systematically.
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186: An Extra Fragment within the GH Module of an
Endo-β-agarase from Flammeovirga sp. MY04 is
Involved in the Degradation of Agarose
Wen-Jun Han 1 , 2 , Jing-Yan Gu 1 , Hui-Hui Liu 1 ,
Fu-Chuan Li2, ξ, Zhi-Hong Wu1, Yue-Zhong Li1, ξ; 1
State Key Laboratory of Microbial Technology, the School
of Life Science, Shandong University, Jinan 250100, P. R.
China; 2 National Glycoengineering Research Center, the
School of Life Science, Shandong University, Jinan 250100,
P. R. China
fuchuanli@sdu.edu.cn; lilab@sdu.edu.cn
Agarase hydrolyzes agarose into a series of oligosaccharides
with repeating disaccharide units. The glycoside hydrolase
(GH) module of agarase is known to be responsible for its
catalytic activity. However, variations in the composition of
the GH module and its effects on enzymatic functions have
been minimally elucidated. The agaG4 gene, cloned from the
genome of the agarolytic Flammeovirga strain MY04, encodes
a 503-amino acid protein AgaG4. Compared with elucidated
agarases, AgaG4 contains an extra peptide (N246-G302) within
its GH module. Heterologously expressed AgaG4 (rAgaG4)
was determined to be an endo-type β-agarase. The protein
degraded agarose into neoagarotetraose and neoagarohexaose
at a final molar ratio of 1.5: 1. Neoagarooctaose was the
smallest substrate for rAgaG4, whereas neoagarotetraose was
the minimal degradation product. Removing the extra fragment
from the GH module led to the inability of the mutant (rAgaG4T57) to degrade neoagarooctaose, and the final degradation
products of agarose by the truncated protein were
neoagarotetraose, neoagarohexaose and neoagarooctaose at a
final molar ratio of 2.7: 2.8: 1. The optimal temperature for
agarose degradation also decreased to 40 °C for this mutant.
Bioinformatic analysis suggested that tyrosine 276 within the
extra fragment was a candidate active site residue for the
enzymatic activity. Site-swapping experiments of Y276 to 19
various other amino acids demonstrated that the characteristics
of this residue were crucial for the AgaG4 degradation of
agarose and the cleavage pattern of substrate.
187: Ribosome-Inactivating Proteins (RIPs) in Apple
(Malus sp.)
Chenjing Shang, Willy J. Peumans, Els J.M. Van
Damme; Lab: Biochemistry and Glycobiology, Dept.
Molecular Biotechnology, Gent University, Gent, 9000,
Belgium
chenjing.shang@ugent.be
Ribosome-inactivating proteins (RIPs) are a group of plant
proteins, which possess highly specific N-glycosidase
Glycoconj J (2013) 30:281–461
activity and are capable of catalytically inactivating eukaryotic ribosomes through the removal of a specific adenine
residue from a highly conserved loop of the large rRNA.
The family of RIPs is classically divided into type-1 RIPs,
which consist only of an N-glycosidase domain and type-2
RIPs in which the RNA N-glycosidase domain is linked to a
C-terminal carbohydrate-binding domain.
Until now there are no reports of ribosome-inactivating
proteins in edible fruits. Recent searches in the fully completed genome sequences of different edible plant species
revealed the occurrence of RIP related sequences. Furthermore, transcriptome databases available for apple (Malus
sp.) provided evidence for the expression of these RIP
related sequences. We cloned the RIP related sequences
from apple, and the corresponding proteins were expressed
and purified using different eukaryotic expression systems.
The ribosome-inactivating proteins have been characterized
for what concerns their molecular structure and RNA Nglycosidase activity. In addition, the carbohydrate-binding
properties of the type 2 RIP were analyzed in detail using
glycan arrays. Future experiments will focus on the physiological role of apple RIPs in the plant and their possible
involvement in plant defense.
188: Recombinant human butyrylcholinesterase
produced in plants: the synthesis of biologically active
multi-sialylated glyco-forms
1
Jeannine D. Schneider, 1Alexandra Castilho, 2Laura
Neumann, 2Friedrich Altmann, 1Andreas Loos, 3Latha
Kannan, 3 Tsafrir S. Mor, and 1 Herta Steinkellner
1
Institute of Applied Genetics and Cell Biology, University
of Natural Resources and Life Sciences, Vienna, Austria;
2
Department of Chemistry, University of Natural Resources
and Life Sciences, Vienna, Austria;3 The Biodesign Institute, Arizona State University, Tempe, Arizona, USA
jeannine.schneider@boku.ac.at
Protein-based drugs can often not be produced satisfactorily by the established cell-based production platforms.
This is not the least because of a complex N-glycosylation pattern. An example is human serum protein
butyrylcholinesterase (BChE), which is a heavily
sialylated protein that is being considered as a potential
bioscavenger for organophosphate nerve agents. Sufficient amounts of active enzyme cannot be obtained
from current sources. Plants are being recognized as a
platform for the expression of recombinant proteins with
complex N-glycosylation. Importantly, glycoengineering
demonstrates the enormous plasticity of plant cells to
tolerate modifications towards human-like structures
Glycoconj J (2013) 30:281–461
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Nicotiana benthamiana, a tobacco-related plant species, was
used as a host for the transient expression of recombinant
human BChE (rBChE). The protein was collected from the
extracellular space (apoplast) 4 days post infiltration (dpi) of
the respective cDNA constructs and subjected to N-glycan
analysis. While rBChE expressed in wild-type plants carries
mainly complex structures with plant-specific N-glycans
(i.e. core á1,3-fucose and â1,2-xylose), the enzyme derived
from the glycosylation mutant ΔXTFT exhibits human-type
GnGn structures. Co-expression of BChE with six mammalian
genes required for in planta protein sialylation (i.e. GNE,
NANS, CMAS, CST, GalT, ST) resulted in the generation of
a rBChE that carries di-sialylated structures. Moreover, addition of GnT-IV (which carries out branching) to the infiltration
mix produced rBChE decorated with tri-sialylated N-glycans.
Notably, these structures were achieved by transient expression
using multigene vectors within 4 dpi. Surprisingly, rBChE
purified from total cellular extracts also exhibits significant
amounts of endoplasmatic reticulum-typical oligomannosidic
structures. These structures might arise from incorrect subcellular targeting of rBChE, as indicated by subcellular
localisation experiments. Finally, in vitro activity assays
exhibited biological activity of the various rBChE glycoforms.
In summary, our results establish a proof of principle study
for the in planta generation of rBChE with a di- and
multisialylated glycosylation pattern, a prerequisite for
clinical applicability. We present a highly flexible and
time-efficient procedure for the generation of complex Nglycan structures on pharmaceutically interesting proteins
that allows advanced structure-function studies, potentially
leading to drugs/vaccines with enhanced efficacies.
mammalian mucin-type O-glycosylation pathway does not
exists at all. In this project, we focus on the generation of
customized O-glycan structures on recombinant proteins
produced in plants. By transient expression of human erythropoietin fused to an IgG heavy chain fragment (EPO-Fc) in
leaves of Nicotiana benthamiana plants together with mammalian glycosyltransferases we were able to produce the
cancer-associated Tn, T and STn antigens as well as the
di-siaylated core 1 structure on the single O-glycosylation
site of EPO-Fc. For the generation of sialylated O-glycans,
we introduced an entire mammalian biosynthetic pathway
into N. benthamiana, comprising the coordinated expression
of the genes for (i) biosynthesis, (ii) activation, (iii) transport, and (iv) transfer of Neu5Ac to O-linked GalNAc. The
formation of the different O-linked-glycans on EPO-Fc
was confirmed by LC-ESI-MS. Notably, engineering of
the O-glycosylation pathway did not interfere with the
formation of N-linked glycans on EPO-Fc and by combining our approaches for customized N- and O-glycan
engineering we were able to produce multi-sialylated
EPO-Fc in planta. In summary, our studies demonstrate
that N. benthamiana are amenable to N- and O-glycosylation engineering and are a valuable platform to
produce glycoproteins with defined glycans for therapeutic use and structure-function studies.
189: Engineering of sialylated mucin-type O-glycosylation
in plants
Castilho, A., Neumann, L., Daskalova, S., Mason, H.S.,
Steinkellner, H., Altmann, F., Strasser, R. (2012) Engineering of Sialylated Mucin-type O-Glycosylation in Plants. The
Journal of Biological Chemistry 287, 36518–36526.
Martina Dicker 1 , Alexandra Castilho 1 , Laura
Neumann 2 , Daniel Maresch 2 , Herta Steinkellner 1
Friedrich Altmann2, Richard Strasser1; 1Department of
Applied Genetics and Cell Biology, University of Natural
Resources and Life Sciences, Vienna, 1190, Austria;
2
Department of Chemistry, University of Natural Resources
and Life Sciences, Vienna, 1190, Austria;
martina.dicker@boku.ac.at
Current mammalian cell-based expression systems for recombinant pharmaceutical proteins typically produce a mixture of heterogenous glycoforms that are neither identical to
human glycans nor optimized for enhanced efficacy. In
terms of glycosylation, plants offer certain advantages compared to other organisms as the N-glycosylation pathway of
plants can easily be modified towards the generation of
homogenous human-type N-glycans and a typical
References:
Castilho, A., Neumann, L., Gattinger, P., Strasser, R.,
Vorauer-Uhl, K., Sterovsky, T., Altmann, F., Steinkellner,
H. (2013) Generation of Biologically Active MultiSialylated Recombinant Human EPOFc in Plants. PLoS
ONE 8: e54836.
190: Effects of Chitosan Oligosaccharides on Improving
Cold Resistance in Crops
Meng Y. Wang12, Wen X. Wang2, Heng. Yin2, Li S X.
Huang12, Xiao M. Zhao2, Yu G. Du2; 1University of
Chinese Academy of Sciences, Beijing, 100049, China;
2
Dalian Institute of Chemical Physics, Chinese Academy
of Sciences, Dalian, 116023, China
duyg@dicp.ac.cn
Chitosan oligosaccharides (COS) has the high solubility,
which make them especially attractive in a number of useful
applications. The effects of COS on growth, antibacterial
properties and drought resistance of crops have been
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investigated. Recently, it has been demonstrated that COS
has the effect on improving cold resistance in wheat. In test
field, the wheat seedlings treated with COS suffered less
damage after late spring coldness compared to the control.
The samples of COS treament group in test field show a
increased activity of multiple enzymes and the content of the
total chlorophyll up to 10 %. In the lab research, wheat
seedlings suffered zero degrees Celsius temperature stress,
and the COS treament group show a lower growth in electric
conductivity and MDA level to control, while total chlorophyll, water solutble sugar (WSS) and reducing sugar increased. But no significant changes in the concentration of
proline among different treatments has been detected. By
accumulating the secondary metabolites production, COS
enhances the resistance ability of wheat seedlings to low
temperature stress. The concentration of chitosan oligosaccharides at 75 mg/L show the most effective results. The obtained
results prove that COS could promote photosynthesis and
metabolism and provide safety for low temperature cold damage in crops. Moreover, COS is friendly to environment, and
the crops treated with the COS have no side effects to human.
191: Heterochitooligosaccharides: Enzymatic Preparation
and Analysis by MALTI-TOF MS
Yong Zhao, Jianping Zhang, Yuguang Du; Dalian Institute of Chemical Physics, Chinese Academy of Science,
Dalian, 116023, China.
zy1805@dicp.ac.cn
Oligosaccharides derived from chitin and chitosan exhibit
various biological functions and have been attracting more
and more attentions. They can be used as elicitors in agriculture, immune regulator in animal science, and to decrease
cholesterol in healthy food and so on.
These useful oligosaccharides have been obtained by acid or
enzymatic hydrolysis of chitin and chitosan followed by
chromatographic separation. However, oligosaccharides
between chitin and chitosan oligosaccharides—so called
by heterochitooligosaccharides are still unknown to us.
In our study, we developed a method by which
heterochitooligosaccharides with average degree of acetylation 25 %, 35 % and 50 % could be prepared. They
were derived from water soluble chitin with different
degree of acetylation by an endo-type chitosanase. They
were further analyzed by MALTI-TOF MS. It was believed that deeper studies are necessary to understand
the relationship between their structure and unique biological activities and other application aspects.
Glycoconj J (2013) 30:281–461
192: Sulfonyl Hydrazine-functionalized Polymer as a
Specific Capturer of Reducing Glycans from Complex
Samples for High-throughput Analysis by Electrospray
Ionization Mass Spectrometry
Chengjian Wang, Xiaohua Li, Jiangbei Yuan, Zhongfu
Wang, Linjuan Huang; Educational Ministry Key Laboratory of Resource Biology and Biotechnology in
Western China, Life Science College, Northwest University, Xi’an 710069, China
huanglj@nwu.edu.cn
Glycosylation represents one of the most common posttranslational modifications of proteins and is involved
in various important biological processes, such as molecular recognition, cell adhesion, and signal transduction. Qualitative and quantitative analysis of structurally
diverse glycans of various glycoproteins can provide a
basis for the investigations into their complex structurefunction relationships. However, the glycan analysis is
usually troubled with tedious sample processing steps
prior to detection, including deproteination, desalting
and removal of some other impurities, which result in
a considerable sample loss and increase the difficulties
of quantitative analysis. Herein we report a facile and
versatile procedure for the specific and quantitative
capture of reducing glycans from complex samples
using sulfonyl hydrazine-functionalized polystyrene
(SHPS) beads. This method allows efficient covalent
conjugation of the aldehyde group of reducing glycans
with the active sulfonyl hydrazide group of the SHPS
beads under anhydrous conditions, resulting in the formation of a sulfonyl hydrazone bond. The unreactive
non-glycan components in the samples, such as proteins, salts and some other impurities, can be completely removed in one step by washing the glycan-coated
SHPS beads repeatedly. The regeneration of the reducing glycans can be performed via a mild hydrolysis of
the sulfonyl hydrazone bond on the washed SHPS
beads based on reversible hydrazide chemistry. The
suggested procedure is compatible with all of the current techniques for the derivatization or detection of
reducing glycans. We have obtained the fundamental
data of this method, including the optimized reaction
conditions, the glycan recovery ratio, the quantitative
capability and the reproducibility. Additionally, the
method was successfully applied to the glycan analysis
of a series of complex biological samples, such as
milk, human blood plasma and fetal bovine serum,
demonstrating the good applicability of this novel
procedure.
Glycoconj J (2013) 30:281–461
193: Production of human saposin B with mannose-6phosphate-type N-glycans in the methylotrophic yeast
Ogataea minuta
Toru Watanabe1, Ikuo Kawashima2,3, Yuki Takaoka1,
Akihiko K ameyama 1 , Seiji Matsuda 4 , H itoshi
Sakuraba3,5 and Yasunori Chiba1,3; 1 Bioproduction Research Institute, National Institute of Advanced Industrial
Science and Technology (AIST), Ibaraki 305-8566, Japan;
2
Molecular Medicine Project, Tokyo Metropolitan Institute
of Medical Science, Tokyo 156-8506, Japan; Departments
of 3Clinical Genetics and 5Analytical Biochemistry, Meiji
Pharmaceutical University, Tokyo 204-8588, Japan;
4
Division of Anatomy and Embryology, Department of
Integrated Basic Medical Science, Ehime University
School of Medicine, Ehime 791-0295, Japan.
y-chiba@aist.go.jp
Saposin B is a small and non-enzymatic proteinaceous
cofactor that mediates degradation of sulfatide by arylsulfatase
A and globotriaosylceramide by α-galactosidase A in lysosomes, where saposin B extracts the targeted sphingolipids
from membranes and forms a soluble protein-lipid complex
that is recognized by the enzymes. The inherited defect
of saposin B accumulates sphingolipids predominantly
sulfatide in the lysosome, which leads to a metachromatic
leukodystrophy-like disease, saposin B deficiency.
The lysosomal dysfunction of sphingolipidoses is a consequence of a deficiency of lysosomal enzymes and their
activators and results in the accumulation of sphingolipids.
Recently, enzyme replacement therapy with recombinant
enzymes has been developed for some sphingolipidoses,
including Fabry disease (GLA deficiency). Here we report
the production of a recombinant human saposin B (SapB)
using the methylotrophic yeast Ogataea minuta, which is a
cofactor that mediates the degradation of sulfatide and
globotriaosylceramide in lysosomes. By overexpressing
SapB gene and OmMNN4 under the control of the alcohol
oxidase promoter in O. minuta, we succeeded in producing a
large amount of SapB containing more than 95 % of
mannosylphosphorylated N-glycans. PNGase F treatment
suggested that phosphorylated O-glycan is absent in the
SapB. The recombinant SapB activated the degradation
of globotriaosylceramide by GLA in vitro and was well
incorporated into cultured human fibroblasts via mannose 6-phosphate receptors. The incorporation of SapB
with mannose-6-phosphate glycan was dose-dependent;
however, non-glycosylated SapB was not incorporated,
even at a concentration of 20 μg/ml. The recombinant
SapB holds promise as a replacement therapy for SapB
deficiency and as an enzyme activation therapy for
Fabry disease.
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194: Natural Oligosaccharides Library Preparation
Wang wenxia 1 , Li Shuguang 1 , Tuo Yaqin 1 , Guo
Zhimou1, Yin Heng1, Shi Bo2, Du Yuguang1,*; 1Dalian
Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, Liaoning 116023, China; 2Feed research Institute Chinese Academy of Agriculture Sciences, Beijing,
100081, China
duyg@dicp.ac.cn
Oligosaccharides from natural products have promising bioactive ability in a wide range of diseases prevention and
treatment through the alternative complement pathway;
however, the study of oligosaccharide is technically challenging as no effective sequencing tool and obtained method. In this article, 60 kinds of polysaccharide from natural
products were degraded by chemical or enzymatic method.
They include marine polysaccharides like carrange, Chinese
herbal polysaccharides like Poria cocos polysaccharide, fungal polysaccharides like Agaricus brasiliensis polysaccharide and other plant polysaccharides like Abelmoschus
manihot medic polysaccharide. The distribution of degraded
oligosaccharides was further analyzed through high performance liquid chromatography, hydrophobic interaction liquid chromatography or fluorophore-assisted carbohydrate
electrophoresis method. Afterward according to the linkage
type of monosaccharide unit, oligosaccharide composition
and monomer were isolated through preparative high performance liquid chromatography. Finally a natural oligosaccharide library including more than 1,000 kinds of
oligosaccharide component and monomer was established.
The library offer an opportunity to identify new active
oligosaccharide functional subunits and revealing oligosaccharide structure-activity relationship, therefore, the library
have value for novel carbohydrate-based medicines discovery and development.
195: Differences and Similarities Between New Probiotic
Bifidobacterial and Lactobacillus Lectin Systems
Interacting to Glycoconjugates
Mikhail V. Lakhtin, Vladimir M. Lakhtin, Andrey V.
Aleshkin, Stanislav S. Afanasiev, Vladimir A. Aleshkin;
Department of Medical Biotechnology, G.N. Gabrichevsky
Research Institute for Epidemiology & Microbiology,
Moscow, 125212, Russia
Lakhtinv@yandex.ru
New Lectins-Glycoconjugates (L-GC) relationships on
membranes are of importance for glycol(bio)technology.
The aim was to describe functional similarities and differences between probiotic bacterial L (PBL) on the examples
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of L identified in cultural fluids of industrial lactobacillus
(LL) and bifidobacterial (LB) strains.
Pseudopolysaccharides, artificial antigens and peptidoglycans
(biotinylated or not) were used as GC (www.lectinity.com).
Lectin systems (LS) of strains of Lactobacillus helveticus,
L.amylovorus, Bifidobacterium adolescentis, B.angulatum,
B.bifidum, B.gallinarum, B.longum and B.pseudocatenulatum
were investigated. PBL containing crude concentrates were
separated by IEF-PAG in gradients pH 2–6 or 4–8,
electroblotted on Immobillon P and visualized using GCbiotin followed by Streptavidin-Peroxidase treatments.
Chemiluminescence of activated highly sensitive peroxidase
substrate in optimized conditions of live imagination kinetic
regimes was registered using Dark Room of BioChemi System (UVP, Calif.). Protein distribution was supported by fluorescent dye SYPRO Ruby protein blot stain (Bio-Rad)
visualization.
Different LS recognizing α–D-mannan (Man: phosphorylated or not), α-L-fucan (Fuc), peptidoglycans (MDP),
asialylated mammalian gut mucin-like (GalNAc); antigens
Тn, blood group A (Adi) and/or Forssman (Fs) were identified among bacterial acidic proteins. The found LS were
able to discriminate GalNAc-containing GC (GalNAc,
LacNAc, Adi and Fs). Different L mosaics in tracks were
involved in simultaneous recognition of different GC types
(for example, Fs+MDP, Fuc+Adi). LS (as major 1-2 or
major+minor components) were characterized as genera,
species and strains fundamental features.
Results indicate important for biotechnology significant differences between LB and LL in GC specificities in identical
testing conditions. The data support our findings on PBL
interactions to eukaryotic and prokaryotic cells, synergistic
antimicrobial action of LB and LL against fungal and Gram
positive pathogens.
196: Enzymatic synthesis of N-acetylneuraminc acid
derivatives of 5-bromo-4-chloroindolyl 4,7-di-Omethyl-β-D-galactopyranoside to determine α-2,3- and
α-2,6-specific neuraminidase activity
Juana Elizabeth Reyes Martinez, Robert Sardzik, Josef
Voglmeir, Sabine L Flitsch*; Manchester Institute of Biotechnology, School of Chemistry, The University of Manchester, Manchester M1 7DN, U.K. Corresponding author:
Tel: +44(0)161-306-5172
juana.reyesmartinez@postgrad.manchester.ac.uk.
Sialic acid (e.g. N-acetylneuraminic acid (Neu5Ac)) plays a
significant role in many biological and pathogenic processes
Glycoconj J (2013) 30:281–461
such as cell-cell recognition, signal transduction, viral entry
into the host cell and immune response. Neu5Ac can most
commonly be found on glycoproteins as a terminal sugar
linked to β-galactose residues with either α-2,3- or α-2,6linkage. Influenza viruses which bind Neu5Ac are probably
the most studied pathogens due to the easy spread among
humans causing a great number of hospitalizations and
economic losses each year. The linkage recognition is an
important aspect in the invasive process of those viruses.
The majority of methods used to determine the linkage
selectivity of neuraminidases require expensive reagents,
specialised equipments and well trained technicians. Using
a one-step enzymatic approach we synthesized α-2,3- and
α-2,6-Neu5Ac modified 5-bromo-4-chloro-3-indolyl-β-Dgalactopyranosides as novel substrates for probing neuraminidase activity and specificity. A colorimetric method
based on an indigo-derivative formation has been
established. Upon incubation of these substrates with an
active neuraminidase an indolyl compound is released in
the presence of β-galactosidase which quickly undergoes
oxidation to form 5,5′-dibromo-4,4′-dichloro-indigo. The
sialidase activity can be easily detected by blue coloration
of the sample. Spectrophotometric techniques can be used
for quantification.
The enzymatic syntheses were performed using trans-sialidase
from Trypanozoma cruzi (TcTS) for α-2,3- linkage formation
and sialyltransferase from Photobacterium damsela (ST6Pd)
for α-2,6-linked Neu5Ac. The Neu5Ac addition to 5-bromo4-chloro-indolyl-β-D-galactopyranoside was confirmed by
MALDI-ToF MS and HPLC. The two novel substrates were
successfully used for hydrolytic activity evaluation of 3 bacterial sialidases (S. typhimurium neuraminidase, C.
perfringens neuraminidase, A. ureafaciens neuraminidase).
In addition we also confirmed the presence of neuraminidase
activity and specificity of B. pumilus and A. aurescens towards α-2,3 linkage sialoglycoconjugates.
197: The Study on Oligochitosan Inducing Cotton
Resistance against Verticillium dahliae kleb
Xiao M. Zhao12, Heng. Yin12, Wen X. Wang12, Meng Y.
Wang123, Yu G. Du12; 1Liaoning Provincial Key Laboratory of Carbohydrates Dalian, 116023, China, 2Dalian Institute of Chemical Physics, Chinese Academy of Sciences,
Dalian, 116023, China; 3University of Chinese Academy of
Sciences, Beijing, 100049, China
zhaoxm@dicp.ac.cn
Verticillium wilt of cotton is difficult to control by chemical
pesticide. Oligochitosan can induce cotton resistance against
verticillium dahliae kleb. Oligochitosan can promote root
Glycoconj J (2013) 30:281–461
growth of cotton, improve resistance of cotton root cap
cell against toxin secreted by verticillium dahliae kleb,
activate resistance genes expression. Oligosaccharides
also increase accumulation of lignin on cotton root
cells.
198: Advance in Application of Oligochitosan on Crops
Xiao M. Zhao12, Heng. Yin12, Wen X. Wang12, Meng Y.
Wang123, Yu G. Du12; 1Liaoning Provincial Key Laboratory of Carbohydrates Dalian, 116023, China, 2Dalian Institute of Chemical Physics, Chinese Academy of Sciences,
Dalian, 116023, China;3University of Chinese Academy of
Sciences, Beijing, 100049, China
zhaoxm@dicp.ac.cn
Oligochitosan was prepared from enzymatic hydrolysis of
chitosan (the degree of N-acetylation is below 5 %) and
separated with membrane in our Laboratory. Oligochitosan
can induce defense responses of plant. Our recent research
results showed that oligochitosan could activate plant cold
resistance,thus Improve plant fruit-set rate.low concentrations of oligochitosan promoted the germination of seeds
and growth of crops. Oligochitosan can increase crop yield
and quality.
199: Systematic Analysis of Drug Glycosylation Critical
Quality Attributes (GCQAs)
Louise Royle, Radoslaw P. Kozak, Li Phing Liew, Robert
J. Emery and Daryl L. Fernandes; Ludger Ltd. Culham
Science Centre, Oxfordshire, OX14 3EB, United Kingdom
rad.kozak@ludger.com
Glycosylation can have a significant effect on the clinical
safety and efficacy of biopharmaceuticals. Issues with
glycans have caused great financial, legal and regulatory
problems for those companies who have not dealt effectively with their product’s glycosylation. Regulatory
authorities are now tightening the requirements for biopharmaceutical companies to characterise, control and
compare the glycosylation of their therapeutics. However, measurement and control of drug glycans can be
difficult to achieve due to their complexity and heterogeneity. Consequently, changes in glycosylation are the
major cause of batch variability for most glycoprotein
therapeutics.
At Ludger we use a systematic approach to greatly reduce
the risks of suffering from problems with glycosylation.
This system aligns with current and emerging regulatory
377
guidelines from FDA, EMA and ICH and has three broad
steps:
1. GCQAs. Specification of Glycosylation Critical Quality Attributes (GCQAs) (i.e. those glycosylation parameters that most influence the drug product’s safety and
efficacy profiles).
2. Glycoprofiling. Implemention of appropriate, affordable glycoprofiling modules to measure the GCQAs
throughout the drug’s life cycle.
3. Interpretation and Corrective Action. Interpretation
of the glycoprofiling data and taking appropriate action
if the product falls out of specification (OOS) or trends
towards OOS.
This poster focuses on the glycoprofiling modules of step two.
A wide range of analytical techniques are available for
glycan structure analysis and profiling—but it can be
difficult to select the most appropriate of these modules
for a particular drug. The types of glycoprofiling analyses required may also change at different points during
a drug’s life cycle. For example a wide range of complementary methods may be required for initial characterisation of glycosylation and identification of the
GCQAs, but once they have been identified a simpler,
faster method may be sufficient for quality control. To
reduce regulatory problems it is crucial that reliable,
robust, validated methods are used. We present here
some straightforward strategies that drug developers
and biomanufacturers can use for detection and quantification of common biopharmaceutical GCQAs such as
sialylation, core fucosylation, antennary composition,
lactosamine extensions, alpha-galactose and N-glycolylsialic acid. The emphasis is on methods that allow
compliance with emerging drug regulations.
200: Metabolic Engineering of Escherichia Coli for
Mannose Production
Hailong Cao1, Yuguang Du1; 1Dalian Institute of Chemical Physics, Zhongshan Road 457, Dalian, 116023, China
duyg@dicp.ac.cn
A whole-cell catalyst for the conversion of fructose to
mannose was developed in Escherichia coli BL21(DE3).
The gene of NAD-dependent mannitol-1-dehydrogenase
(MTD) which catalyzes the conversion of mannitol to
mannose was synthesized and cloned into pET23b for
optimal expression in Escherichia coli BL21 (DE3). The
mannitol dehydrogenase (MDH) from Leuconostoc
mesenteroides was co-expressed with MTD in E.coli
BL21 (DE3). Using the created recombinant E. coli strain
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Glycoconj J (2013) 30:281–461
as a whole-cell catalyst, a high conversion rate of fructose to mannose was achieved.
202: Enhancement of N-glycan Isomer Separation for
Glycomic Characterization of Biotherapeutics and
Biosimilars
201: Efficient Adhesion-based Plasma Membrane
Isolation for Cell Surface N-glycan Analysis
Min Kyung So, Myung Jin Oh, Serenus Hua and Hyun Joo
An*; GraduateSchool of Analytical Science Technology,
ChungnamNationalUniversity, Daejeon, Korea
hjan@cnu.ac.kr, Tel:82-42-821-8547,Fax:82-42-821-8541
Ji-Young Mun, Kyung Jin Lee, Hoon Seo, Ohsuk Kwon
and Doo-Byoung Oh*; Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience and
Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu,
Daejeon 305-806, South Korea
dboh@kribb.re.kr, Tel: 82-42-860-4457
Glycans, as attached to proteins and lipids on the cell
surface, play crucial roles in various physiological phenomena such as cell-cell communication, differentiation
and development. However, analysis of cell surface
glycans requires difficult and laborious processes for
the isolation of plasma membrane from other cellular
components. Especially, efficient removal of endoplasmic reticulum (ER) and Golgi membranes are required
since the high amount of high-mannose type glycans
mainly located in ER has hampered the analysis of
complex-type glycans on the cell surface. In the present
study, we employed the simple adhesion-based method
for the isolation of plasma membrane to analyze the Nglycans coating on the cell surface efficiently. The purity of the isolated plasma membranes was evaluated by
fluorescence imaging using organelle-specific probes
and optimal isolation conditions were established to
minimize the contamination of ER and Golgi fractions.
Cell surface N-glycans of Chinese hamster ovary (CHO)
cells obtained by this adhesion-based plasma membrane
isolation method were analyzed using matrix-assisted
laser desorption/ionization time-of-flight and highperformance liquid chromatography, which were compared to N-glycan profiles derived from the corresponding total cell fractions. Complex-type N-glycans capped
with terminal sialic acids were observed as major
populations in plasma membrane fractions whereas
high-mannose type glycans mainly detected in the profiles
of total cells. Moreover, when compared with widely used
plasma membrane isolation methods based on ultracentrifugation or cell compartment kit, our adhesion-based method
showed the highest efficiency to detect complex-type N-glycans with the smallest amount of high-mannose type
glycans. All these results indicated that the adhesionbased plasma isolation provides easy, rapid and efficient
preparation method for the analysis of cell surface glycans, which will contribute to elucidating the biological
implications of their changes in cell-cell communication,
differentiation and development.
Glycosylation is largely influenced by the quality, safety,
and potency of biopharmaceutical products.
Isomer separation for proper characterization of glycans in
biotherapeutics, such as EPO (erythropoietin) or mAbs is particularly important because different isomers lead to different
biological functions. PGC (porous graphitized carbon) is widely
employed for effective isomer-specific separation and enrichment of glycans based on polarity, size, and three-dimensional
structure. PNGase F treatment releases N-glycans as aldehydes.
However, PGC separates aldehyde sugars into α and β
anomers, increasing the complexity of isomer-specific separations. Here, we aimed to optimize glycan reducing conditions to
get rid of α and β anomers by reducing aldehydes to alditols
using sodium borohydride. Glycans released from ribonuclease
B (high–mannose type), IgG (complex type), fetuin (highly
sialylated) and EPO were used to optimize reducing conditions
for all glycan types. After releasing N-glycans using PNGase F,
released glycans were purified by SPE (solid phase extraction)
and reduced by sodium borohydride. Reduced glycans were
purified and enriched by SPE. Reduced N-glycans were easily
identified by MALDI/TOF MS due to the two Da mass increase
resulting from conversion of the reducing terminal to an alditol.
To obtain isomer-specific information, glycans were analyzed
before and after reduction using chip-based PGC nano-LC/MS.
We found less peaks in chromatograms after reducing Nglycans,suggesting the removal of α and β anomers. For
example,in common N-glycan[Hex]5[HexNAc]4[Fuc][NeuAc]2,
four chromatographic peaks were observed before reducing.
After reduction, only two chromatographic peaks were observed, both at different retention times from the previous four
peaks. Isomer-specific MS/MS also revealed different fragmentation patterns associated with each chromatographic
peak. There are a number of biosynthetic explanations for
the different isomers; for example, the fucose may be attached
to either the N-glycan core or one of the antennae; or, the sialic
acids may be either alpha-2,3 or alpha-2,6-linked.
We successfully applied and demonstrated the feasibility of
our reducing method with various biopharmaceuticals products including EPO or mAbs. This analytical platform will
provide a powerful analytical tool for isomer-specific separation of glycans on PGC and be globally used in the
biopharmaceutical field.
Glycoconj J (2013) 30:281–461
203: Gas1 Engineering for Selection of a Yeast Strain
with Improved Protein Secretion
Hoon Seo1, 2, Dong Hyeun Hwang1, Ji-Young Mun1, Jin
Young Gil1, Kyung Jin Lee1, Ohsuk Kwon1, Sang Ki
Rhee2 and Doo-Byung Oh1; 1Systems & Synthetic Biology Research Center, Korea Research Institute of Bioscience
and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu,
Daejeon, South Korea, 2Department of Medicinal Biotechnology, Soon Chun Hyang University, Asan, 336-745,
Korea
dboh@kribb.re.kr
Gas1 protein is a beta(1,3)-glucanosyltransglycosylase playing
an essential role in the assembly of cell wall as localized on the
yeast surface through a glycosylphsphatidylinositol (GPI) anchor. When GAS1 gene was disrupted in several yeasts including Saccharomyces cerevisiae and Pichia pastoris, the resulting
mutant strains were reported to exhibit hypersensitivity to cell
wall-perturbing reagents and temperature-sensitive phenotype
together with increased capability of protein secretion due to
the loosed cell wall structure. In the present study, functional
complementation of cell wall-defective phenotype of GAS1deletion mutant using recombinat expression of Gas1 protein
was employed to generate a screening system for a strain with
improved capability of protein secretion. We constructed the
expression vectors encoding fusion proteins with N-terminal
secretory protein of interest linked to Gas1 proteins without
signal sequence. After these vectors were transformed into
GAS1-deletion mutant, the growths of the resulting
transformants were tested on the agar plate containing cell
wall-perturbing reagent. Only the strains expressing Gas1 proteins fused to well secreted proteins showed restored growth
phenotype under cell-wall stress condition. Currently, we are
exploring the possiblity that this system can be used to enrich
yeasts with improved secretion capability after genome-wide
random mutagenesis, which would lead to the development of
Gas1-based screening system for super secretory yeast.
204: Rapid oligosaccharides sequencing by exoenzymatic
digestion on the basis of microfluidic device
Benwei Zhu1, 2, Runtao Zhong1, 3, Bingcheng Lin1, 3,
Yuguang Du*1, 3; 1Dalian Institute of Chemical Physics,
Chinese Academy of Sciences, Dalian, 116023, China;
2
University of Chinese Academy of Sciences, Beijing,
100049, China; 3Research Center of Lab on a Chip, Dalian,
China
duyg@dicp.ac.cn
Glycosylation is the most significant procedure of the posttranslational modifications. The structural analysis of
379
oligosaccharides attached on the surface of the glycoprotein is
essential for understanding the vital process and developing
efficacious drugs as well as antibodies. Exoglycosidase digestion coupled with matrix-assisted laser desportion/ionization
mass spectrometry (MALDI-MS), all through the way, has
been demonstrated to be an effective method for oligosaccharides sequencing. But the major disadvantage of the sequential
digestion is that it requires the repeated isolation, which not
only waste much more time and efforts, but the incubation of
enzyme and oligosaccharides mixture takes a dozen hours.
To address the circumstances mentioned above, we report a
novel method for oligosaccharides sequencing, which combines enzymatic digestion with microfluidic devices. The
mixture of digestion reaction is compartmentalized into
aqueous picoliter-volume droplets, dispersed in inert carrier
oil. Each droplet is an enzymatic reaction vesssel with
highly efficient hydrolysis (~37 °C) due to rapid mixing
and enhanced mass transfer within microdroplets, as well
as high uniformity of droplet size and temperature. After
enzymatic digestion, the products are recovered and characterized by matrix-assisted laser desportion/ionization mass
spectrometry (MALDI-MS). By performing microfluidic
droplet-based reaction, the time required for glycosidase
digestion could be shortened to several hours, which is very
important for oligosaccharides sequencing and analysis.
205: Inactivation of the gene rmla remodels Burkholderia
kururiensis exopolyssacharide reducing ability to
promote rice growth and colonization
Letícia Hallack, F1; Priscila A.V. Oliveira1; Luciana L.
Penha1; Bruna G. Coutinho2; Daniel Passos da Silva2;
Jose Osvaldo Previato1; Lucia Mendonça-Previato1;
1
Federal University of Rio de Janeiro, Rio de Janeiro,
Brazil; 2 International Centre for Genetic Engineering &
Biotechnology, Trieste, Italy
lehallack@gmail.com; luciamp@biof.ufrj.br
Burkholderia kururiensis is a diazotrophic endophytic bacterium isolated from internal tissues of several plants. Interaction
studies showed that B. kururiensis is able to promote growth
and increase grain yield of rice plants. The molecular mechanisms involved in the endophytic bacteria-grass interactions are
unknown, but a specific relationship between bacterium
exopolysaccharide (EPS), capsular polysaccharide (CPS) or
lipopolysaccharide (LPS) and host plant has been proposed.
B. kururiensis M130 produces large amounts of acidic EPS.
Structural studies of this polymer showed the presence of a
linear pentasaccharide repeats units with the following structure: –>4)-alpha-D-Glcp-(1–>2)-alpha-L-Rhap-(1–>4)-alpha-
380
D-GlcpA-(1–>3)-beta-L-Rhap[2OAc]-(1–>4)-beta-DGlcp-(1–>.
The monosaccharide rhamnose (Rha) is commonly found in
bacterial EPS that interact with plants. Some studies have
shown the direct involvement of L-rhamnose in the interaction processes. Four enzymes (RmlA-D) are present in the
dTDP-L-rhamnose biosynthetic pathway, and D-glucose-1phosphate thymidylyltransferase (RmlA) catalyzes the first
step of dTTP and D-glucose-1-phophate (D-Glc-1-P) to
dTDP-D-glucose (dTDP-D-Glc) and PPi
In this study we performed a functional knockout in the
biosynthesis of L-rhamnose. In order to investigate the
function of the EPS in the rice x bacteria interaction, the
B. kururiensis rmla gene was interrupted, generating the
mutant rmla. Structural analysis of EPS of the mutant strain
showed the presence of a new EPS composed of
tetrasaccharide repeat units containing two residues of Drhamnose, instead L-rhamnose, and two residues of heptose.
Gnobiotic experiments with the rice were done to comparatively evaluate the endophytic colonization ability of the
mutant strain. It was observed a three-fold reduction on the
capacity to promote plant growth, also the ability to colonize
roots and aerial parts of the rice plant was reduced.
These data demonstrate the involvement of EPS in the
endophytic colonization of the B. kururiensis and rice,
adding further insight into the possible molecular mechanisms involved in this interaction.
206: Refining the specificities of anti-I and anti-i antibodies using neoglycolipid-based array technologies and
applications in the immuno-sequencing of poly-Nacetyllactosamine backbones
Chao Gao1, Angelina S. Palma1,2, Wengang Chai1, Yan
Liu1, Robert A. Childs1, Ten Feizi1; 1Glycosciences Laboratory, Department of Medicine, Imperial College London,
Lodon, W12 0NN, UK; 2REQUIMTE,Departamento de
Química, Universidade Nova de Lisboa, 2829-516
Caparica, Portugal
c.gao11@imperial.ac.uk
Monoclonal autoantibodies in sera of patients with cold agglutinin syndrome recognize developmentally-regulated carbohydrate antigens termed I and i on erythrocytes and other
cell types. Their characterization and demonstration of their
power as sequence-specific reagents ante-dated similar work
with hybridoma-derived antibodies generated to oncodevelopmental antigens. Anti-I and anti-i recognize branched
and linear sequences of poly-N-acetyllactosamine type,
Glycoconj J (2013) 30:281–461
respectively, and they are unique reagents for detection of
un-substituted forms of these sequences. This presentation
will be concerned with neoglycolipid (NGL)-based arrays to
define more precisely the sizes of the I and i antigens and
apply these antibodies as immuno-sequencing reagents.
In the original studies of Ten Feizi and colleagues, the specificities of several anti-I and -i antibodies were elucidated in a
succession of analyses that in essence involved inhibition of
binding, using as inhibitors structurally-defined synthetic or
natural oligosaccharides, and also glycolipids. Methodologies
included quantitative inhibition of precipitation or
haemagglutination, and inhibition of binding in radioimmunoassays. These antibodies proved to be invaluable in monitoring
changes that occur in the branching patterns of carbohydrates in
the course of embryonic development, cell differentiation and
malignancy. Among the antibodies, anti-I Ma has been studied
in greatest detail. On the branched poly-N-acetyllactosamine
chains, it recognizes the trisaccharide domain Galβ14GlcNAcβ1-6R (where R is Gal or GalNAc). Anti-I Ma is
the prototype of one group of anti-I. A second group recognize
mainly the Galβ1-4GlcNAcβ1-3 domains of branched chains,
with individual antibodies differing in their additional requirements for all or part of the Galβ1-4GlcNAcβ1-6 branch. These
differences are advantageous in the use of these antibodies as
tools for analysis of backbone sequences of glycans.
The NGL-based microarray system enables antigenic analyses of a series of oligosaccharides at low fmol levels, and
represents a substantial miniaturization relative to the mmol
of oligosaccharides required for inhibition studies. We now
observe that for optimal binding, anti-I Ma requires a longer
sequence than tetrasaccharide (Galβ1-4GlcNAcβ1-6Galβ14GlcNAc). We will describe results of a re-evaluation of
anti-I/i specificities using macro and microarrays and application of the antibodies, in conjunction with glycosidase
treatments and mass spectrometry, as immuno-sequencing
reagents to characterize novel carbohydrate antigens on Oglycans and glycolipids.
207: Lectin microarray assists development of the
glycodiagnostic systems for direct measurement
Atsushi Kuno1, Takashi Sato, Atsushi Matsuda, Yuzuru
Ikehara, Hisashi Narimatsu; 1Research Center for Medical Glycoscience (RCMG), National Institute of Advanced
Industrial Science and Technology (AIST), Tsukuba, Ibaraki
305-8568, Japan
atsu-kuno@aist.go.jp
“Glycodiagnosis” is a new paradigm of clinical diagnosis
based on the quantitation of glyco-alteration well reflecting
Glycoconj J (2013) 30:281–461
381
This work was supported by a grant from New Energy and
Industrial Technology Development Organization (NEDO)
of Japan.
oncogenesis. Thus, DSA has been used for an essential tool
in glycobiology. DSA is a dimer composed of two subunits
linked by disulfide bonds. It was reported that an affinitypurified DSA fraction was a mixture of three different
isolectins which arise from AA, AB and BB combination
of the both subunits. The A- and B-subunits had similar
but not identical Mr values (32,000 and 28,000, respectively). At present, cDNA of DSA had not been isolated and
its molecular structure remains unknown. In order to isolate
the DSA gene, we analyzed the N-terminal and the internal
amino acid sequences from the isolated B subunit of DSA
(DSA-B). Based on the sequences, the degenarate primers
were designed and RACE was conducted. The isolated
lectin cDNA (dsa-b) contained an open reading frame
encoding 279 amino acids, including peptides that were
sequenced. The deduced sequence consisted of two distinct
motifs: (1) a Cys-rich carbohydrate binding domain composed of four conserved chitin-binding domains; (2) an
extensin-like domain of 37 residues containing 4 SerPro46 motifs inserted between the second and third chitinbinding domains. Although each chitin-binding domain
contained 8 Cys residues at the conserved positions, only
the second chitin-binding domain contained an extra Cys
residue, which may participate in dimerization through an
intra-disulfide bridge formation. The molecular mass of the
native DSA-B was determined as 68,821 Da by MALDITOF MS. The molecular mass of the isolated subunit of
DSA-B was 37,593 Da and that of the deglycosylated form
was 26,320 Da, as pyridilethylated derivatives. This correlated with the estimated molecular weight of the deduced
sequence. Transgenic Arabidopsis plants overexpressing
dsa-b possessed hemagglutinating activity. The recombinant
DSA was produced as a homo-dimeric glycoprotein with
similar molecular mass compared to native one. Moreover,
N-terminus of the purified recombinant DSA was identical
with that of the native DSA-B. These findings confirmed that
the cDNA encoded DSA-B.
208: Molecular cloning and functional expression of lectin from Datura stramonium seeds
209: Unique gangliosides synthesis by sialyltransferases
from marine bacteria
Suguru Oguri, Kaori Tanaka, Keisuke Nishimoto,
Takahiro Murakami, Hikaru Sakamoto; 1Department of
Bioproduction, Fuculty of Bioindustry, Tokyo University of
Agriculture, Hokkaido, 099-2422, Japan
S-oguri@bioindustry.nodai.ac.jp
Hisashi Kamimiya1, Yusuke Suzuki1, Anila Mathew1,
Shinobu Watarai2, Toshiki Mine3, Takeshi Yamamoto3,
and Yasunori Kushi1; 1College of Science and Technology,
Nihon University, Tokyo, 101-8308, Japan; 2Graduate
school of Life and Environmental Science, Osaka Prefecture
University, Osaka, 599-8531, Japan; 3Product Science Division, Japan Tobacco Inc., Kanagawa, 227-8512, Japan.
h.kamimiya@gmail.com
disease progression. To detect such changes in glycosylation,
all of the currently available glycomics techniques (e.g., mass
spectrometry, liquid chromatography, capillary electrophoresis) require at least 3 h of sample preparation before analysis.
Alternative technologies based on a lectin–antibody sandwich
assay had been proposed for detection of proteins bearing
disease-specific glyco-alterations. Most of them detect the
changes in fucosylation of N-linked glycans, which are wellknown to be associated with liver disease. However, the
fucose-binding lectins force us the essential of preliminary
enrichment of the target protein, and thus are inappropriate in
practical use for serological diagnosis. In this study, we present
a new methodology with two technical advances to overcome
these problems in glycodiagnosis associated with clinical implementation: (1) We adopted a microarray-based method with
a unique subtraction process for easy selection of the most
robust lectin. In fact, the method efficiently led us to WFA to
establish the direct measurement system. (2) We selected
M2BP as a potential glycobiomarker that shows a fibrosisrelated glyco-alteration which we proved for the first time. The
diagnostic utility of M2BP is greatly owing to a favorable
density and orientation of the disease-related glycan on the
homomultimer resembling a “sweet-doughnut”. These characteristic structures contribute to a major increase of the avidity
of M2BP for the plated WFA in ELISA. The resulting glycan–
lectin interaction (i.e., WFA-M2BP interaction), which is remarkably strong and specific, made it possible to develop the
rapid (17 min) and highly sensitive assay and realized “on-site
diagnosis”. This unique methodological approach is applicable
to develop other glycodiagnostic tools, thus it will revolutionize the use of glycodiagnosis in clinical medicine and potentially provide a framework for the development of a new
generation of biomarker assays.
The Datura stramonium agglutinin (DSA) is a chitin-binding
lectin purified from Jimson weed (D. stramonium) seeds. DSA
binds selectively to the branch structure on complex-type Nglycans containing N-acetyllactosaminyl structures. It has been
used to analyze structural changes of sugar chains during
Sialyltransferases (ST) are enzymes that transfer Nacetylneuraminic acid (NeuAc) from the common
382
donor substrate cytidine 5′-monophospho-NeuAc
(CMP-NeuAc), to acceptor substrates. Many STs and
the genes encoding them have been obtained from
various sources including mammalian, bacterial and
viral sources. Recently, we had discovered new bacterial STs from marine bacteria belonging to genus
Photobacterium and closely related to genus Vibrio,
both of which are gram-negative bacteria. One of the
authors have previously cloned these STs and enzymatically characterized them using fluorescencelabeled oligosaccharides, and they were found to be
β-galactoside α2-3 or α2-6ST, which catalyzes the
incorporation of NeuAc from CMP-NeuAc into the
terminal galactose residue in carbohydrate chain at
position 3 or 6.
To further characterize these STs, we tested ganglioside
synthesis by STs using various glycosphongolipids
(GSLs) as substrates. Bacterial STs showed affinity to
neolacto- and lacto-series GSLs, particularly in
neolactotetraosyl ceramide (PG, nLc 4 Cer, Galβ14GlcNAcβ1-3Galβ1-4Glcβ1-1’Cer). Gangliosides synthesized from nLc 4 Cer by α2-3 and α2-6ST were
structurally characterized by several analytical and immunological methods, and they were identified as
IV 3 αNeuAc-nLc 4 Cer (S2-3PG) and IV 6 αNeuAcnLc4Cer (S2-6PG). In addition, these STs also showed
affinity to asialoganglio-series GSLs. As a results,
GA1 (Galβ1-3GalNAcβ1-4Galβ1-4Glcβ1-1’Cer) abd
GA2 (GalNAcβ1-4Galβ1-4Glcβ1-1’Cer) were found
to be good substrates for these STs. Ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b (NeuAcα2-3Galβ1-3GalNAcβ1-4Galβ14Glcβ1-1’Cer). On the other hand, when enzyme reaction by α2-6STs were performed using substrates GA1
and GA2, very unique gangliosides were generated. By
using several analytical procedures, the generated
gansgliosides were identified as NeuAcα2-6GA2 (S26GA2) and NeuAcα2-6GA1 (S2-6GA1), respectively.
Furthermore, the above synthesized ganglioside, S26GA2, showed binding activity to the influenza A
virus {A/panama/2007/99 (H3N2)} at a similar level
to purified S2-3PG and S2-6PG from mammalian
sources. These results suggest that bacterial STs have
unique features, including substrate specificities restricted not only to neolacto-series, but also to
asialoganglio-series GSLs, and catalytic potentials for
unique ganglioside synthesis. This demonstrates that
effective in vitro ganglioside synthesis could be a
valuable tool for selectively synthesizing NeuAc modifications. We are currently preparing transformed
mammalian expression systems using STs gene from
marine bacteria.
Glycoconj J (2013) 30:281–461
210: Characterisation and manipulation of N-glycans in
insect cells
Rhiannon Stanton1, Dieter Palmberger1 and Iain B. H.
Wilson 1 ; 1 Department für Chemie, Universität für
Bodenkultur, Vienna, 1190, Austria.
rhiannon.stanton@boku.ac.at
Anti-carbohydrate antibodies are present when foreign glycans are detected by the immune system and thus be a
problem if recombinant glycoproteins, such as antibodies,
are used as therapeutic agents. It is also of importance that
the recombinant antibodies have the correct glycosylation in
order to prevent clearance from the serum. By screening for
the glycans from insect cells, anti-carbohydrate antibodies,
lectins and their binding partners can be detected.
Using glycans from natural sources can be problematic because of the low amounts generated. However, even though
only small quantities are extracted, these can still be used in
glycomic experiments. This work demonstrates how glycans
isolated from natural sources (in this case glycans from
the Trichoplusia ni insect cell line High Five) can be
used in glycan microarray screens. First, N-glycans can
be isolated from the glycopeptides from the cells, labelled and purified prior to characterisation by a variety
of HPLC and MS methods. Labelled N-glycans were
then applied to glass slides prior to detection by lectins
and anti-horseradish peroxidase.
As T. ni cells tend to be an excellent choice for expression of
antibodies, engineering of these cells is a pre-requisite for
humanisation of the glycans and avoidance of potential
immune responses. The recently-described SweetBac technology can be used to produce modified antibodies with
altered glycosylation; in particular, introduction of a nematode N-acetylglucosaminyltransferase II and a mammalian
galactosyltransferase results in a high proportion of
biantennary N-glycans on recombinant antibodies derived
from T. ni cells.
211: Generation of Complete Series α-2,8-sialic Acid
Oligosaccharides from Polysialic Acid Produced by
Escherichia coli CCTCCM208088
Li Zhu 1, Xiao-bei Zhan1,2,*, Hong-Tao Zhang2, JiangRong Wu2, Zhi-Yong Zheng2, Chi-Chung Lin2; 1 Jiangsu
Rayguang Biotech Co. Ltd., Wuxi, Jiangsu 214125, China; 2
Key Laboratory of Carbohydrate Chemistry and Biotechnology of Ministry of Education, School of Biotechnology,
Jiangnan University, Wuxi, Jiangsu 214122, China;
xbzhan@yahoo.com (X.-B. Zhan)
Glycoconj J (2013) 30:281–461
Polysialic acid (PSA) mainly exists in cell surface
serves to modulate the distance between cells and involved in a number of plasticity-related responses in
adult central nervous system. However, PSA-signal protein recognition is the key step for PSA playing its role.
To exploration the minimal oligosaccharide for signal
protein, it is necessary to acquiring complete series of
α-2,8-sialic acid oligosaccharides. In this work, PSA
was treated with 0.1 M HoAc at 40 °C, 60 °C and
80 °C for 30, 60, 90, 120, 160 and 190 min respectively and then adjusted all solution to neutral with 0.5 M
NH4HCO3. All the hydrolysis products were desalted
further with G10 column. Separation step were done
using HPLC with evaporative light—scattering detector
(ELSD) and Click mail column. The optimal condition
for acquiring complete series sialic acid oligosaccharides
from 3 to 15 mer is at 60 °C for 160 min.
212: SPRi-based Lectin Array Chip Powerful Glycoprotein
Analysis Tool
John Zhang 1, Yue Yuan 1, Trevor Johnston 1, Christopher
Lausted 3, Bridget Sexauer 1, Andrew Weber 1, Hong Liu
2
, Jingsong Zhu 2; 1 Plexera LLC, 401 Terry Avenue North,
Seattle, WA 98109, USA; 2 National Center for Nanoscience
and Technology, Haidian, Beijing, 100190, China; 3 Institute
of SystemBiology, 401 Terry Avenue North, Seattle, WA
98109, USA
Cro@plexera.com
Lectins are well-characterized, highly-specific sugarbinding proteins that can be produced economically
for a wide range of applications. Microarrays of
these sugar-binders allow multiplexed high-throughput
protein analysis in small sample volume. The
carbohydrate-specific binding proteins serve many different biological functions and play a key role in cells
and protein recognition pathways. They also are involved in immune system response by recognizing
carbohydrates that are found exclusively on pathogens
or alternatively formed carbohydrates on cancer cells.
Their ability to bind to soluble extracellular and
intercellular glycoproteins allows them to be excellent
glycoprotein screening agents. Our first SPRi-based
Lectin microarray chip contains 41 of the most common lectins targeting seven classes of typical sugar
motifs. This high-throughput glycoprotein biomarker
discovery platform termed Lectin-NanoChip™ is able
to identify and monitor glycosylation changes in a
different types of samples rapidly. Surface Plasmon
Resonance Imaging (SPRi) also offers label free detection of the glycan and its respective sugar subtypes.
383
Additionally, the affinity of different glycan can be
determined via binding constant determination supplied
by our analysis tool. Therefore, lectin array combining
with SPRi technology is a great platform for glycoproteins characterization. The Lectin-NanoChip™ can be
applied to a vast glycoform characterization fields that
include but not limited to these following: diseaserelevant glycol-biomarkers discovery, cell surface
glycome profiling, pathogen recognition, bacterial tropism identification, cancer stem cell markers detection
and altered glycan structure classification.
213: A high throughput screening of ligands to multiple
targets by Surface Plasmon Resonance Imaging (SPRi)
Platform, PlexArray®
John Zhang 1, Christopher Lausted 3, Trevor Johnston 1,
Yue Yuan 1, Bridget Sexauer 1, Andrew Weber 1, Hong
Liu 2, Jingsong Zhu 2; 1 Plexera LLC, 401 Terry Avenue
North, Seattle, WA 98109, USA; 2 National Center for
Nanoscience and Technology, Haidian, Beijing, 100190,
China; 3 Institute of SystemBiology, 401 Terry Avenue
North, Seattle, WA 98109, USA
Cro@plexera.com
Researchers have been seeking new and improved
methods for multiplex, label-free and real-time monitoring of bio-molecular interactions. Surface Plasmon Resonance Imaging (SPRi) is a versatile method for
observing such interactions. This system enables the
measurement of a wide range of targets that includes
kinases, peptides, DNA, antibodies and adhesive molecules in both single and multiple assay formats.
Plexera’s unique platform combines array with SPRi
technology to monitor high-throughput interactions up
to five thousand molecules. The powerful platform can
be applied for high-throughput, label-free, fast biomarker screening, personalized diagnostics development,
drug discovery,and various other proteomic studies.
Our SPRi array platform has become a powerful tool
for studies on multiplexing interactions of many different biomolecues. So far, cytokines, lectins, peptides,
DNA, kinases and even expressing proteins on cells
have been successfully validated by this real-time bioassay. The tool can be applied to many different biological functional studies, such as epitope mapping,
cytokine and glycan profiling, as well as active kinase
interactions. Most importantly, it is able to select targets for small molecule binders of interesting. Thus, we
can accelerate the speed for discovering novel biomarkers dramatically.
384
Glycoconj J (2013) 30:281–461
214: Synthesis of Thioglycoside Analogues of KRN7000
Xiangming Zhu1,2, Ravindra T. Dere2, Junyan Jiang1;
1
College of Chemistry and Life Sciences, Zhejiang Normal
University, Jinhua 321004, China; 2Centre for Synthesis and
Chemical Biology, School of Chemistry and Chemical Biology, University College Dublin, Dublin 4, Ireland
xiangming.zhu@ucd.ie
α-Galactosylceramide, KRN7000, has been utilized in studies for the treatment of many diseases including cancer,
diabetes, malaria, and hepatitis B, and in most cases
HO
HO
O
OH
O
HN
HO
HO
impressive activities have been observed.1 This compound
can stimulate NKT cells to release a variety of cytokines in
vitro and in vivo, which are recognized subsequently by
other cells of the immune system and may have a widespread influence on immune responses. Nevertheless, the
efficacy of KRN7000 has been limited because it stimulates
production of a mixture of TH1 and TH2 cytokines, which
are antagonistic to each other. Therefore, many efforts have
been devoted in the past decade to synthesize its analogues
with the hope of developing novel lead compounds with
better cytokine-inducing selectivities and appropriate potency as immunostimulatory agents.2
O
OH
O
(CH2)24CH3
OH
O
(CH2)13CH3
OH
KRN7000
HN
HO
HO
R
OH
S
R'
OH
R, R' = Hydrocarbon chain
Thioglycoside analogues
We present here the first total synthesis of thioglycoside
analogue of KRN7000,3 and convenient synthesis of
other thioglycoside analogues of α-galactosylceramide
carrying the truncated sphingoid chain.4 A great advantage of the used synthetic strategy is that the anomeric
stereochemistry was introduced at an early stage in the
synthesis by use of a galactosyl thiol building block,
and the strategy would also be applicable to other α-Sgalactosylceramides of interest. Also, the synthesis efficiency was greatly increased in the synthesis of the
truncated analogues by employing Mitsunobu reaction
to retrieve an unwanted diastereoisomer.
The subsequent bioassay demonstrated that the
thioglycoside analogue of KRN7000 possesses similar
potency to KRN7000 in human NKT cell activation,
and stimulates cytokine release in the same manner as
well. 5 As such, the thioglycoside analogue may be
superior to KRN7000 as a parent compound for developing immune stimulants for humans in view of its
bioactivity, stability, ease of synthesis, and flexibility
for making other analogues.
Acknowledgement: This work was supported by Research
Frontier Program of Science Foundation Ireland and the
Natural Science Foundation of Zhejiang Province
(R4110195).
215: Polysaccharide isolated from Lonicera japonica Thunb,
a novel inhibitor of amyloid beta peptide aggregation
Peipei Wang, Jianping Fang, Qin Liu, Kan Ding*;
Glycochemistry & Glycobiology Lab, Shanghai Institute
of Materia Medica, Chinese Academy of Sciences,
Shanghai,201203,China
kding@mail.shcnc.ac.cn
The Alzheimer’s disease (AD) is becoming more prevalent in
ageing populations worldwide, yet there are currently no
effective treatments. Compelling evidence indicates that aggregates of amyloid-beta (Aβ) peptides plays a central role in
the in the development of AD due to a cascade of harmful
event that cause cellular dysfunction and neuronal death.
Therefore, inhibition Aβ accumulation is thought to be an
effective strategy for the prevention and/or treatment of patients with AD. Recent studies reported that several polysaccharides from Chinese medicinal herbs had a neuroprotective
effect against the toxicity of Aβ peptides, such as these from
Antrodia camphorate, Nerium indicum and Lycium
barbarum. However, little is known about the molecular
mechanism underlying their bioactivity. In the present study,
a homogeneous polysaccharide, named as LJW0F2, with an
average molecular weight of 37.1 kDa was purified from
Lonicera japonica Thunb, by anion-exchange chromatography and gel permeation chromatography. Using monosaccharides composition analysis, methylation analysis, IR and
NMR spectroscopy, LJW0F2 was elucidated to an alphaD-(1→4)-glucan with an alpha-(1→4) linked branch attached
Glycoconj J (2013) 30:281–461
to C-6 position. Its inhibitory effect on Aβ42 aggregation was
monitored by fluorescence spectroscopic analysis with
thioflavin T (ThT) and the morphological changes were investigated by atomic force microscopy (AFM). The results showed
that LJW0F2 could significantly inhibit Aβ42 aggregation in a
dose-dependent way, where LJW0F2 inhibited more than 90 %
of aggregation at the concentration of 100 μg/ml. AFM showed
that regular fibril aggregates formed by Aβ42 significantly
decreased in the presence of polysaccharide LJW0F2. To the
best of our knowledge, this is the first report showing that the
exogenous plant-derived polysaccharides can directly target
Aβ42 and block Aβ42 aggregation, which maybe a potentially
therapeutic agent for Alzheimer’s disease.
216: Structural Characterization of polysaccharides
from Lonicera japonica Thunb and their protective
effects on islet β cells
Peipei Wang, L ina L u, Qin L iu, K an Ding*;
(Glycochemistry & Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences,
Shanghai,201203,China)
kding@mail.shcnc.ac.cn
Abstract: Flos lonicerae is the flower of Lonicera japonica
Thunb and has been commonly used as traditional Chinese
medicine with heat-clearing and detoxifying effects. Polysaccharides isolated from those flowers of Lonicera japonica
Thunb have been shown many biological activities, e.g.,
antibacterial, antiviral, antioxidant and anti-inflammatory.
However, only few polysaccharides with those bioactivities
were homogeneous and their structure activity relationships
(SAR) were still unknown. To further exploit and utilize of this
medicinal herb, the fine structure of polysaccharides from those
flowers of Lonicera japonica Thunb will be necessarily investigated. In the present study, two homogeneous polysaccharides, LJW2F1 and LJW2F2 were isolated from those flowers
of Lonicera japonica Thunb with hot water extraction and
further purified by ion-exchange chromatography and gel permeation chromatography, respectively. Using monosaccharide
analysis, methylation analysis, IR and NMR spectroscopy, their
structures were evaluated. LJW2F1 had a very complex structure, containing galactose, rhamnose, arabinose, xylose, glucose and galacturonic acid. LJW2F2 was elucidated to be a
linear poly-(α1→4)-a-D-galactopyranosyluronic acid with an
average molecular weight of 7.2 kDa. Bioactivity test indicated
that the acid pectic polysaccharides, LJW2F1 and LJW2F2
could remarkably protect islet β cell-line INS-1 from high
glucose induced cell apoptosis. Furthermore, the protective
effect of LJW2F2 was more significant than that of LJW2F1.
These results suggested that the content of uronic acid had
significant effect on this bioactivity of polysaccharides. This
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is the first report of the fine structure of polysaccharides from
those flowers of Lonicera japonica Thunb. However, their
protective mechanism of INS-1 cells needs further study.
217: Preparation and Antitumor Activity of Sulfated
Polysaccharides
Qun Dong, Xinyan Ni, Kan Ding; Glycochemistry and
Glycobiology Lab, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, Shanghai, 201203, China
qdong@mail.shcnc.ac.cn (Q. Dong); kding@mail.shcnc.ac.cn
(K. Ding)
Sulfated polysaccharides play critical roles in various biological events in a variety of organisms, especially in higher
and lower animals and in algae. They can be obtained either
by isolation from biological tissues or by chemical modification of non-sulfated polysaccharides. Sulfated polysaccharides have been reported to show different biological
activities, such as antiviral, antitumor, anti-angiogenesis,
immune activation/inhibition, and antioxidation, etc. Although the antitumor activity of some sulfated polysaccharides has been reported in different pharmacological models,
the mechanism and the structure-activity relation remains
largely unknown. Sulfated polysaccharides can inhibit tumor growth via several different pathways: directly inducing
apoptosis of cancer cells, inhibiting tumor angiogenesis, or
activating immune response against cancer. In this study we
prepared different types of sulfated polysaccharides by
chemical modification of non-sulfated polysaccharides, including Aga-S from agarose, Dex-S from dextran, ALGH-S
from alginate, CFAA2-S from galactomannan, CFBB2-S
from homogalacturonan, CRAA2-S from amylose, and
CRAB1-2-S from amylopectin. Then their antitumor activity
was examined against different cancer cell lines, and their
immunomodulatory activity was also tested on NF-κB
activation/inhibition experiments. The results showed that sulfated amylopectin exhibited a strong NF-κB activation, while
sulfated alginate exhibited NF-κB inhibition. According to in
vitro antitumor tests, the sulfated polysaccharides showed different degree of inhibition to different cancer cells. For PANC-1
pancreatic cancer cells, significant inhibition activity was observed in the order: CFAA3-S>CFBB2-S >CRAA2-S >ALGHS> Dex-S. Aga-S showed no inhibition on PANC-1 but it can
inhibit other pancreatic cancer cell lines, such as BXPC-3 and
ASPC-1. All these sulfated polysaccharides showed no significant inhibition against proliferation of SMMC 7721 liver cancer cells. The results indicated that the immunomodulatory and
antitumor activity of sulfated polysaccharides are strongly dependent on the types and structure of polysaccharides,while the
action of the polysaccharides showed cell specificity response.
Further study is still urgently needed for understanding the
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Glycoconj J (2013) 30:281–461
complex relationship between the structure of sulfated polysaccharides and their antitumor activity.
218: Immunological Effects and Chemical Structure of
Antrodia cinnamomea Polysaccharides
Feng-Ling Yang1, Shih-Hao Zhan1, Yu-Liang Yang2,
Kuo-Feng Hua3, Jung-Feng Hsieh4, and Shih-Hsiung
Wu1; 1Institute of Biological Chemistry, Academia Sinica, Taipei,
Taiwan; 2Agricultural Biotechnology Research Center, Academia
Sinica, Taipei, Taiwan; 3 Department of Biotechnology and Animal Science, National Ilan University, Ilan, Taiwan; 4 Department
of Food Science, Fu Jen Catholic University, Taipei, Taiwan
yang111@gate.sinica.edu.tw
Antrodia cinnamomea is used as traditional Chinese medicine. Water-soluble polysaccharide of mycelia of A.
cinnamomea was separated by size-exclusion chromatography and assayed the cytokine stimulation.
The molecular weight of the biological active polysaccharide of A. cinnamomea is around 70 to several
hundred kilodaltons. The major sugar component is
mannose (86 %), and galactose as minor (14 %). The
sugar linkages include 1,2; 1,3; 1,6; 1,2,6-, and terminal
mannosyl-linkages and a 1,6-galactosidic linkage. This
polysaccharide can stimulate tumor necrosis factor-α
(TNF-α) and interleukin-6 (IL-6) in dendrite cells. The
proposed structure of Antrodia cinnamomea polysaccharide is:
D-Manp
6
2)-D-Manp-(1
6)-D-Galp-(1
6)-D-Manp-(1
219: Structural Characterization of Two Homogalacturonans
from the Recycled Residue of Green Tea and Anticomplementary Activity of Their Sulfated Derivatives
Huijun Wang 1,2, Songshan Shi1,2, Bin Bao1,2, Chao
Zhu1,2, Shunchun Wang1,2,*; 1Institute of Chinese Materia
Medica, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China; 2Shanghai R&D Center for
Standardization of Traditional Chinese Medicines, Shanghai201203, China
shunchunwang@126.com
The residue of green tea, which was commonly discarded by tea
polyphenols manufacturer, was collected. Two homogenous
water-soluble polysaccharides (TPSR4-2B and TPSR4-2C) were
obtained from the recycled residue of green tea. Their average
molecular weights were estimated to be 41 kDa and 28 kDa,
respectively. A combination of composition, methylation and
configuration analysis, as well as NMR spectroscopy, indicated
that both TPSR4-2B and TPSR4-2C were poly-(1-4)-α-Dgalactopyranosyluronic acid in which 30.5±0.3 % and 28.3±
0.5 % of uronic acid existed as methyl ester respectively. Two
sulfated derivatives (Sul-R4-2B and Sul-R4-2C) at C-2 and/or C3 of GalpA from TPSR4-2B and TPSR4-2C were prepared after
sulfation with 2:1 of chlorosulfonic acid and pyridine. The anticomplementary assay showed that Sul-R4-2B and Sul-R4-2C
demonstrated a stronger inhibitory effect (CH50: 5.4±0.6 μg/mL
for Sul-R4-2B; CH50: 5.2±0.4 μg/mL for Sul-R4-2C) on the
complement activation through the classic pathway, compared to
3)-D-Manp-(1
2)-D-Manp-(1
that of heparin (CH50: 8.9±0.3 μg/mL). In contrast, native
TPSR4-2B and TPSR4-2C had no such effects. These
results suggested that the sulfated derivatives of
homogalacturonans from green tea residue might be considered as a promising candidate of an anti-complement
agent in the treatment of many diseases with characteristic of excessive activation of complement system, such
as system lupus erythematosus, rheumatoid arthritis, and
acute respiratory distress syndrome.
220: Top-down synthesis of a library of high mannosetype glycans: preparation of various high mannose glycans from a tetradeca-saccharide precursor by glycosidase digestions
Akira Seko1, Ichiro Matsuo2, Yoichi Takeda1, Akihiko
Koizumi1, Masakazu Hachisu1, Maki Takatani1, Junichi
Aikawa3, and Yukishige Ito1,3; 1JST, ERATO, Wako, 3510198, Japan; 2Gunma Univ, Kiryu, 376-8515, Japan; 3RIKEN
ASI, Wako, 351-0198, Japan
seko@glt.jst.go.jp
High mannose-type glycans play important roles in assisting
in proper folding of nascent glycoproteins in the ER quality
control (QC) system. The glycans serve as a signal
reflecting folding states of attached polypeptide chains. To
clarify molecular interaction of the glycans with various
QC-associated proteins, it is necessary to provide a series
Glycoconj J (2013) 30:281–461
of structurally definite high mannose-type glycans. Here we
show top-down synthesis of a library of high mannose-type
glycans, which features trimming of the common precursor,
a synthetic tetradeca-saccharide precursor by various glycosidase digestions.
This method is based on chemical synthesis of a tetradecasaccharide precursor that consists of Man9-oligosaccharide,
capped with sugar residues at the three non-reducing termini,
and fluorescent BODIPY residue at the reducing terminal. The
capping sugars were chosen to be α1,3-linked Glc, β1,4-linked
Gal, and β1,6-linked GlcNAc at the termini of A-, B-, and Carms, respectively. These capping enabled selective cleavage of
five α1,2-linked Man residues by Aspergillus saitoi α1,2mannosidase (α1,2-Man’ase). In order to cleave these capping
selectively, we examined several glycosidases, and found that
the α-Glc, β-Gal, and β-GlcNAc capping were selectively
removed by rat liver α-glucosidase-II (G-II), Aspergillus oryzae
β-galactosidase (β-Gal’ase), and jack bean β-Nacetylhexosaminidase (β-HexNAc’ase), respectively. For the
trimming of single α1,2-linked Man on the A-arm, which
contains two sequential α1,2-linked Man residues, we used
Golgi-resident, Branchiostoma floridae endo-α-mannosidase
(endoMan’ase) under the presence of α1,3-linked Glc residue.
This enzyme could be produced in E. coli cells in a high yield.
Simply by chenging the order of glycosidase digestions, we
could separately prepare glycan isoforms. For example, M8A
was prepared by the sequential digestions of β-HexNAc’ase,
endoMan’ase, and β-Gal’ase, while M8B was prepared by
those of β-Gal’ase, α1,2-Man’ase, β-HexNAc’ase, and G-II.
So far, thirty-one high mannose-type glycans have been successfully prepared from the common precursor. The library of
high mannose-type glycans will be applied to various studies
for binding specificities of lectins and substrate specificities of
glycosidases and glycosyltransferases involved in the QC system in the ER.
221: The Presence of Immunoglobulin Specific for Nlinked Glycan without Terminal Galactose in Rheumatoid Arthritis
Xiukun Xu,Yanping Wang, Yunsen Li*; Laboratory of Cellular and Molecular Tumor Immunology, Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and
Medical Sciences, Soochow University, Suzhou 215123, China
yunsenli@suda.edu.cn
Previous studies have reported that altered N-glycosylation
occurs in rheumatoid arthritis (RA), particularly the reduction in galactose residues in IgG. To further observe the Nglycans in RA disease, we showed profiling approaches to
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N-glycan of total immunoglobulin from RA and normal
serum, employing electrospray ionization mass spectrometry. Through comparing the N-glycans from 25 RA patients
and 21 healthy controls, we identified about 55 N-glycans
and found that the most significant change seen in RA was
decreased levels of mono-galactosyl bi-antennary Nglycans, which is also consistent with the previous reports
focusing on the N-glycan in IgG. For instance, many
articles have proved many anti-antibodies specific for
IgG0 are presence in RA serum, so our hypothesis was
that there may exist some antibodies or immonogloblins
that are specific for redundant N-glycans without terminal galactose. Therefore, high performance liquid chromatograph (HPLC) with both ultraviolet and fluorescence
detector was used for further detection of co-culture of
total immunoglobulin and standard N-glycans with PA
labeled, and our result confirmed our former hypothesis.
We speculate that our preliminary results will provide a
promising method both for studies of RA mechanisms
and diagnosis.
222: Preparation and characterization of guluronic acid
oligosaccharides degraded by a rapid microwave irradiation method
Ting Hua, Chunxia Lia,b, Xia Zhaoa,b*, Guangsheng Lia,
Guangli Yua,b, Huashi Guana,b; aKey Laboratory of Marine Drugs, Ministry of Education, School of Medicine and
Pharmacy, Ocean University of China, Qingdao 266003,
C h i n a b S h an do ng P r o vi n cia l K ey la bo r a t or y of
Glycoscience and Glycoengineering, Ocean University of
China, Qingdao 266003, China
Guluronic acid oligosaccharides (GOS) with degree of polymerization (DP) ranging from 1 to 10 were prepared by a rapid
an d non -acid ic m icro wa ve d egra dation m etho d.
Polyguluronic acid, fractionated from alginate hydrolysate,
was dissolved in dilute ammonia water at a concentration of
20 mg/mL (pH=5) and then hydrolyzed under microwave
irradiation (1600 W) at 130ºC for 15 min to produce GOS
mixture. The GOS mixture was separated by a Bio-Gel P6
column and ten fractions were obtained. Each GOS fraction
was further characterized by electrospray ionization mass
spectrometry, 1H-NMR, 13C-NMR, and 2D-NMR spectroscopy techniques. The data showed that the GOS fractions were
saturated oligoguluronates with general molecular formula
C6nH8n+2O6n+1 (n=1−10). This microwave degradation method was not only convenient, less time consuming, and
environment-friendly, but also produced GOS with high yield
(71 %) and eliminating a desalting procedure compared to
conventional acid hydrolysis method.
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223: Aberrant Fucosylation of Glycosphingolipids in
Human Hepatocellular Carcinoma Tissues
Yanping Wang,Jian Zhu, Yunsen Li*; Laboratory of Cellular
and Molecular Tumor Immunology, Jiangsu Key Laboratory of
Infection and Immunity, Institutes of Biology and Medical
Sciences, Soochow University, Suzhou 215123, China
yunsenli@suda.edu.cn
Glycosylation promoting or inhibiting tumor cell invasion
and metastasis is of crucial importance in current cancer
research. Tumor-associated carbohydrate antigens are
predominantly expressed on the tumor cell surface.
Glycosphingolipids (GSLs) are members of this family. In
this study, we performed glycosphingolipidomic assays on
neutral GSLs obtained from solid hepatocellular carcinoma
(HCC) tissues and paired peritumoral tissues by linear ion
trap-electrospray ionization mass spectrometry. The expression of a group of fucosylated neutral GSLs was found to be
higher in the tumor tissues, as their proportion of total
cellular GSLs in the tumor tissue was 3.3-fold higher than
that in the peritumoral tissue (P<0.01). Moreover, we finished the qualitative analysis of these fucosylated GSLs by
comparing theirMS fragments with standardsand/or
reference data. Seven types of fucosylated GSLs were
identified, includingFucα2Galβ3GlcNAcβ3Galβ4Glcβ1
C e r ( Ty p e I H a n t i g e n ) , F u c α 2 G a l β 4 G l c N A c β 3
Galβ 4 Glcβ 1 Cer(Type II H antigen),Fucα3(Galβ 4 )
G l c N A c β 3 G a l β 4 G lc β 1 C e r ( L e x ) , F u c α 4 ( G a l β 3 )
GlcNAcβ 3 Galβ 4 Glcβ 1 Cer(Le a ),Fucα 2 Galβ 3 (Fucα 4 )
GlcNAcβ 3 Galβ 4 Glcβ 1 Cer(Le b ), Fucα 2 Galβ 4 (Fucα 3 )
GlcNAcβ 3 Galβ 4 Glcβ 1 Cer(Le y ),and Fucα2Galβ3
GalNAcβ3Galα4GlcNAcβ3Galβ4-Glcβ1Cer(Globo H).
And these aberrant GSLs mainly contained terminal
Fucα2Gal- topology. This result may lead to improved immunotherapy of HCC and contribute to the understanding of the
role of aberrant fucosylated GSLs in development and
progress of HCC in following studies.
224: Structural analysis of extracellular polysaccharide
from micro-algae Dunaliela tertiolecta
Bon Geun Gooa, Yong Il Parkb , Andriy Synytsya c ,
Choul-Gyun Leed, Jae Kweon Parka; a Department of
Pharmaceutical Science, Gachon University, Yeonsu-gu,
Yeonsu-dong, 406-799 Incheon, Republic of Korea b Department of Biotechnology, The Catholic University of Korea,
Bucheon 420-743, Republic of Korea c Department of Carbohydrate Chemistry and Technology, ICT Prague, Technická 5,
166 28 Praha 6 Dejvice, Czech Republic d Department of
Biological Engineering, Inha University, Incheon 402-751,
Republic of Korea
Glycoconj J (2013) 30:281–461
Extracellular polysaccharide (EPS) was isolated from
defatted micro-algae Dunaliela tertiolecta and defined as
linear (1 -> 4)-a-D-glucan based on monosaccharide composition, enzymatic and spectroscopic analyses. Optimization and characterization of acidic and enzymatic hydrolyses
of EPS have been performed for its potential use as a
renewable biorefinery material. The hydrolytic methods
were improved to assess the effect of substrate specificity,
reaction time, pH, ionic strength and temperature on efficiency of glucose production. EPS was effectively
converted into glucose within one-step enzymatic or acidic
hydrolysis under optimized conditions. Over 90 % recovery
of glucose was achieved for both hydrolytic approaches.
High potential production of EPS and high yield conversion
of this substrate to glucose may allow further exploration of
microalga D. tertiolecta as a potential biomass producer for
biotechnological and industrial exploitation of bioethanol.
225: A Novel Lectin from Mussel Mytilus trossulus
Induces Cytokines Production
Aleksandra S. Kondrashina, Irina V. Chikalovets, Oleg
V. Chernikov; G.B. Elyakov Pacific Institute of Bioorganic
Chemistry, Far Eastern Branch of the Russian Academy
of Sciences, Vladivostok, 690022, Russian Federation
kondrashina.alex@gmail.com
Lectins are proteins or glycoproteins with specific binding
affinity for carbohydrate moiety of glycoproteins or glycolipids on cell surface. Many lectins also possess various
biological activities in vitro and in vivo, and some lectins
bind to specific carbohydrate receptors on cells, which can
activate the receptors and thereby induce intracellular signalling cascades leading to alterations in cellular behaviour.
If the target cells are involved in the innate immune system,
the lectin binding can result in specific cellular responses,
including cytokine secretion.
The lectin MTL (Mytilus trossulus lectin) was purified from
the mantle of bay mussel Mytilus trossulus by affinity chromatography on PSM-agarose and following gel filtration.
The purified lectin was homogeneous on SDS-PAGE with
apparent molecular weight of 18 kDa and 17 kDa on
MALDI.
MTL agglutinated all human blood types. The
hemagglutinating activity of MTL was independent of the
divalent cation Ca2+. Significant MTL activity was observed
between pH 9–10 and up to 60 °C. In hemagglutination
inhibition assays, N-acetyl-D-galactosamine and D-galactose
were the most potent inhibitors among the monosaccharides
tested. Among the glycoproteins, PSM and fetuin were
Glycoconj J (2013) 30:281–461
inhibitors as well. Isoelectric point of the protein was determined by capillary isoelectric focusing to be 6.09 ± 0.01.
MTL have been investigated for its in vitro effect on the
cytokine profile (IFN-γ, TNF-α, IL-10, IL-4) of unstimulated
or stimulated with LPS whole human blood cells. MTL at
high concentrations (80 μg/mL) enhanced the synthesis of
proinflammatory cytokines in stimulated and unstimulated
cells but at low concentration (5 μg/mL) possesses
immunomodulating action, reducing the IL-10 overexpression
in stimulated cells. MTL didn’t render any influence on production of IL-4. The obtained data allow to assume MTL role
as the factor stimulating production of analogs cytokines in an
organism of a mollusk. Besides, in different physiological and
pathological conditions lectin can render both inhibiting and
stimulating action for maintenance of an immune cellular
homeostasis and inflammation regulation.
This research was supported by the grant from the Russian
Foundation for Basic Research (no. 12-04-31208\12), a
grant from the RAS Presidium (no. № 12-I-П6-10) under
the project “Molecular and Cellular Biology”.
226: Synergistic effects of ginseng pectic polysaccharides
on inhibition of L-929 cell migration
Yuying Fan, Guihua Tai, Yifa Zhou*; School of Life
Sciences, Northeast Normal University, 130024, Changchun, Jilin Province, China
zhouyf383@nenu.edu.cn; fanyy033@nenu.edu.cn
The structures of ginseng pectic polysaccharides related to
the cell migration inhibitory effects in our previous study,
but the underlying mechanisms are less known. In present
study, rhamnogalacturonan I (RG-I) -rich pectins prepared
from ginseng pectin were tested for their effects on L-929
cell migration. The combination of homogalacturonan (HG)
and RG-I-rich pectins exerted stronger effect than either
HG- or RG-I-rich pectin alone. Further studies revealed that
the effects of HG- and RG-I-rich pectins were dependent on
pretreatment of the cells using pectin samples. The
pretreatment caused alterations in cell morphologies such
as cell size and shape, focal adhesion, and the organization
of actin filaments, suggesting that HG and RG-I pectins exert
synergistic effects on cell migration and they probably act by
different way. Morphological data and quantitative cell adhesion and spreading assays showed that HG- and RG-I-rich
pectin treatment decreased cell adhesion and cell spreading on
the substratum, suggesting that HG- and RG-I-rich pectins
might exert their effects on cell migration via decreasing cell
adhesion and cell spreading. The experimental results that L929 cells expressed little galectin-3 (Gal-3) and that lactose, an
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inhibitor of Gal-3, did not block the activities of HG- and RGI-rich pectins, implicated that cell migration inhibited by
pectin did not correlate to Gal-3.
227: Anti-HRP epitopes in lower eukaryotes
Stefan Mereiter, Alba Hykollari, Birgit Schiller,
Katharina Paschinger, Shi Yan and Iain Wilson; Department of Chemistry, University of Natural Sciences and
Applied Life Sciences, A-1190 Vienna, Austria
stefan.mereiter@boku.ac.at
HRP (horseradish peroxidase) is a well characterized plant
protein carrying N-glycans with beta-1-2-xylose and alpha1-3-fucose on its core region. Those Anti-HRP antibodies
which are specifically binding this N-glycan epitope are also
cross-reacting to similar structures and are therefore a standard tool for analyzing N-glycans in other organisms. It
already has been shown that anti-HRP is capable of crossreacting with neurons in Drosophila melanogaster due to its
core-fucose. We have now shown that HRP-epitopes are as
present in other lower eukaryotes, due to the presence of
either a beta-1-2-xylose, in the case of the unicellular parasites Acanthamoeba and Trichomonas, or an alpha-1-3-fucose in the N-glycan core region as in Dictyostelium
discoideum and Caenorhabditis elegans. A mix of Western
blotting, HPLC and mass spectrometric approaches have
been used to define the presence of these glycan modifications in these organisms. Even though these glycan structures are evolutionary widespread their function has not yet
been elucidated.
228: Generation, Structural Validation and Utilization of
A Large Library of Diverse Xyloglucan-derived Oligosaccharides for Analytical Purposes
Sami T. Tuomivaara1,2, Utku Avci1, Michael G. Hahn1,3,
and William S. York1,2; 1Complex Carbohydrate Research
Center, 2Department of Biochemistry and Molecular Biology, 3Department of Plant Sciences,University of Georgia,
Athens, GA 30602.
stuomiva@uga.edu
Xyloglucans are among the most structurally complex polysaccharides in the plant cell wall and exhibit structural
diversity at all levels of biological hierarchy. Xyloglucan
structure consists of a backbone of β(1,4)-linked D glucopyranosyl residues with frequent and regularly situated
side-chains. Nearly twenty side-chain structures with
lengths up to four glycosyl residues have been described
to date. The biological and functional significance of this
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diversity and complexity is largely unknown. The physical
and chemical recalcitrance of plant cell walls creates additional hurdles for the extraction and analysis of its components. Consequently, xyloglucan analysis poses many
challenges that are common in the field of glycobiology,
including the generation and purification of homogenous
preparations for functional assays from complex matrices.
Well-defined oligosaccharides are indispensable tools for
functional analyses and would greatly advance the analytical possibilities in xyloglucan analysis, and in understanding many aspects of xyloglucan biology, including
synthesis, turnover, localization, and interactions with other
components in the plant cell wall. We have undertaken a
task to generate, mostly by enzymatic hydrolyses, a large
library of xyloglucan-derived oligosaccharides that can be
used as standards in various chromatographic and mass
spectrometry applications as well as in validating substrate
specificities of xyloglucan-active glycosyl transferases and
glycosyl hydrolases, and defining the epitopes recognized by
xyloglucan-binding monoclonal antibodies. We show that it is
possible to generate diverse library of oligosaccharides and to
purify them in preparative amounts for analytical purposes. We
then demonstrate several analytical applications for this oligosaccharides collection. First, we utilize the oligosaccharides to
analyze the epitopes of nearly thirty monoclonal antibodies
that were generated against xyloglucan polymers from various
plant sources. The characterized monoclonal antibodies show
clear differences in their binding specificities towards the
oligosaccharides and thus are useful probes of xyloglucan
structure in various experimental applications, including
glycome profiling and in muro immunofluorescence experiments. Additionally, we utilize the oligosaccharides to explore
mass spectrometric, chromatographic and spectrophotometric
methods as quantitative tools in oligosaccharide analysis.
Glycoconj J (2013) 30:281–461
form mechanically and chemically resistant domains (rafts) in
the cellular membrane and shield hepatocytes against detergent effect of bile acids.
The aim of this study was to determine changes in topography of ganglioside GM1 (GM1) in cholestatic rat livers, as
well as to assess the relationship between heme oxygenase-1
activity and ganglioside content, density and localization in
cholestatic conditions.
Wistar female rats were intraperitonealy pretreated with
hemin (HMOX-inductor) orSn-mesoporphyrin (HMOX-inhibitor) followed by bile duct ligation (BDL, 5 days) or
sham operation. Serum markers of cholestasis, liver histology, total lipid sialic acid (total gangliosides), amount of aand b- series of gangliosides, GM1 topography and density
were determined. Expression of key enzymes in gangliosides synthesis, were evaluated (GalT2, Sia2).
BDL, as well as HMOX inhibition, resulted in severe histopathological cholestatic injury compared to controls - increased
area of ductular proliferation (P<0.001) and amount of bile
infarcts (P<0.05). Compared to it, activation of HMOX decreased the area and intensity of ductular proliferation (P<
0.05). Total sialic acid content was higher in BDL and after
HMOX activation (P<0.001). BDL led to 2.4 fold increase of
b-series of gangliosides. High concentrations of bile acids as
well as modulation of HMOX activity led to redistribution of
GM1 within the hepatocyte, exactly from the cytoplasm to the
sinusoidal part of hepatocyte membrane (P<0.001; <0.05 respectively). Moreover, HMOX inhibition resulted in a significant increase in GalT2 expression compared to controls (P<
0.01), while HMOX induction had an opposite effect (P<0.05).
Supported by GAUK 516912, SVV-2011-262513.
229: Changes in content, density and localization of
gangliosides in obstructive cholestatic rat livers and the
influence of activity of heme oxygenase-1
Smid Vaclav 1,2 , Petr Tomas 1 , Vitek Libor 1,2 , Smid
Frantisek1, Muchova Lucie1; 1Institute of Medical Biochemistry and Laboratory Diagnostics, 24th Department of
Internal Medicine – Gastroenterology and Hepatology, General Teaching Hospital and First Medical Faculty, Charles
University in Prague, 121 08, Czech Republic
venca.smid@gmail.com
Accumulation of bile acids in cholestasis is linked to liver
alternation through mechanisms comprising oxidative stress,
bile induced apoptosis as well as inflammatory cell-mediated
liver cell necrosis. While induction of heme oxygenase
(HMOX) protects liver against these stressors, gangliosides
230: Mass Spectrometry-based glycan profiling for characterization of liver tissue from wild type and genetically
modified mice mimicking hepatocellular carcinoma
Tianhua Wang1, Lyn Chiin Sim1, Jianxin Huo1, Muriel
Bardor1, 2 and Kong-Peng Lam1; 1Bioprocessing Technology Institute, Agency for Science, Technology and Research
(A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore
138668. 2Université de Rouen, Laboratoire Glycobiologie et
Matrice Extracellulaire Végétale EA 4358, Institut de
Recherche et d’Innovation Biomédicale (IRIB), Faculté des
Sciences, 76821 Mont-Saint-Aignan Cédex, France.
wang_tianhua@bti.a-star.edu.sg
Liver cancer (hepatocellular carcinoma, HCC) is among the
leading cancers affecting people in South-East Asia, China,
Glycoconj J (2013) 30:281–461
Japan and Korea. In Singapore, it is the 4th most frequently
occurring and third most fatal cancers in males. If untreated,
most patients do not survive beyond 6 months after diagnosis. Surgery is the only treatment that allows for a reasonable
survival beyond 5 years. However, most liver cancers are
diagnosed at advanced stages when it is usually too late for
surgery. As a consequence of late detection, the general
prognosis for liver cancers is poor. Glycosylation changes
are a universal feature of malignant transformation and
tumor progression, possible readouts for changes in the
expression of genes during oncogenic transformation and
progression which may underlie alterations of intracellular
signaling occurring as cells gradually lost control in proliferation, adhesion and apoptosis.
A genetically knockout mice were generated and found to be
more prone to develop chemically induced HCC. In this study,
we profiled the N- and O-linked glycans extracted from the
liver tissue from this knockout mouse and compared it to that
of the wild type mouse so as to help understand the glycosylation changes occurring in HCC which may lead to better
appreciation of the onset of HCC. For that, we have developed
in the institute a glycomics platform which allows us to do
glycan profiling starting from tissue. With optimized sample
preparation protocol and MALDI-TOF MS analysis, we are
able to profile the N-linked glycan structures of the liver
tissues from both wild type and genetically knock-out mice.
Some of the ions identified correspond to possible glycan
structures not reported in previous literature. The more abundant species among them were subjected to tandem MS analysis for further structural elucidation while attempts for
comparative liver glycomics are being made to pinpoint salient differences in the glycan profiles between the wide-type
and knock-out mice.
231: Betel nut lectin: quantum dot nanoconjugate in the
identification of serum glycoproteins
Urmimala Chatterjee1, Partha P Bose2, Shuvendu
Singha1, Gautam Mondal1, Bishnu P Chattejee1*;
1
Department of Natural Sciences, West Bengal University of Technology, Salt Lake, Kolkata-700064, India
2
Department of Biotechnology, National Institute of
Pharmaceutical Education and Research, Hajipur, Bihar844101, India
*cbishnup@gmail.com
Betel nut (Areca catechu) extract was found to contain
two lectins of different carbohydrate specificities. One
bound fetuin while the other recognized asialofeutin.
The former was purified by affinity chromatography
on fetuin-Sepharose column. The homogeneity of the
391
lectin was judged by 10 % SDS-PAGE and showed
molecular mass of approximately 75 kDa. The lectin
was Ca2+ dependent and was active from pH 6.5 to
7.0 and stable upto 25 °C. It precipited N-linked serum
glycoproteins. The lectin agglutinated mammalian erythrocytes including human irrespective of any blood group
system. The heamagglutination titer was found to be
more with pronase treated human erythrocytes.
Heamagglutination-inhibition assay was performed with
serum glycoproteins viz., transferrin, fetuin, alpha 2HS
glycoprotein, ceruloplasmin, lactoferrin, and immunoglobulins. Of them transferrin and immunoglobulins
were found to be better inhibitor requiring 0.1 mg/ml.
Betel nut lectin was conjugated with quantum–dot
nanoparticles, which was tested by TEM, XRD and
DLS. The QD-lectin conjugate detected high level of
expression of transferrin in alcoholic liver cirrhosis patients’ sera than those of normal individuals.
232: Characterization of novel glycophosphosphingolipids
found in insect parasite protozoa
Tatia na C . G arcez 1 , Ma rilza Corrêa 1 , Jorick
Vanbeselaere 2 , Bernadette Coddeville 2 , Emmanuel
Maes2, Yann Guérardel2, Lucia Mendonça Previato1,
Jose Osvaldo Previato1; 1Laboratório de Glicobiologia,
Instituto de Biofísica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro, Av Carlos Chagas Filho 373,
21941 902-Rio de Janeiro, RJ, Brasil. 2 Unité de
Glycobiologie Structurale et Fonctionnelle, Université de
Lille 1, 59 655- Villeneuve d’Ascq, France
luciamp@biof.ufrj.br
The protozoan flagellates of genus Crithidia are monoxenous
trypanosomatids parasite able to colonize the gut epithelium
of their insect hosts. Lipoarabinogalactan is the predominant
cell surface glycoconjugate of Crithidia species so far
studied. However, recent studies by our group have
found that Crithidia deanei and Crithidia desouzai produce lipofucoglucan instead of lipoarabinogalactan.
Although fucosylated glycoinositolphospholipids (GIPLs)
or fucosylated polysaccharides have never been described
in protozoan parasites of the Trypanosomatidae family, it
was observed that L-Fucose can replace D-Arabinose in
lipophosphoglycan (LPG) when Leishmania major is cultured with 50 mM of L-Fucose. To gain insights into the
biological significance of fucosylated glycotopes in
trypanosomatid parasites, we began the structural analysis
of a highly fucosylated GIPLs isolated from C. deanei.
To establish the structure of this glycophosphosphingolipid,
it was isolated by hot aqueous phenol extraction and
392
purified by hydrophobic chromatography. Monosaccharide
composition of purified GIPLs determined by gas chromatography–mass spectrometry showed that the glycan structure is composed of fucose (Fuc), glucose (Glc), mannose
(Man), galactose (Gal), N-acetyl-glucosamine (GlcNAc)
and glucosamine (GlcN) in a ratio of 5:4:2:1:1:1, respectively. The sphingolipid anchor is linked through an inositol
phosphate bond to the glycan domain. The compositional
analysis revealed that the sphingolipid anchor consist of C21 phytosphingosine N-acylated with 2-hydroxystearic acid
or lignoceric acid, as the major component; with smaller
amounts of C-22 phytosphingosine N-acylated with
2-hydroxysteric acid or lignoceric acid; and C-20
phytosphingosine N-acylated 2-hydroxysteric acid or
lignoceric acid. NMR spectroscopy and Electrospray Mass
Spectrometry analysis showed that the glycan of C. deanei
GIPLs is a branched structure, with a backbone containing
the following monosaccharide sequence: βGal-βGlcNAcβGlc-βGlc-βGlc-βGlc-αMan-αMan-αGlcN. The side
chains are composed of αFuc-αFuc-αFuc or αFuc.
Taken together, the results suggest that the C. deanei GIPLS
could be useful in studies related with the biological significance of fucosylated epitote in the trypanosomatid x insect
host interactions.
233: Chemoenzymatic synthesis of mono- and difluorinated Thomsen-Friedenreich (T) antigens and
their sialylated derivatives
Jun Yan1, Xi Chen2, Fengshan Wang1 and Hongzhi
Cao1; 1National Glycoengineering Research Center, School
of Pharmaceutical Science, Shandong Universtiy, Jinan,
250012, China; 2Department of Chemistry, University of
California, Davis, California, 95616, USA
hzcao@sdu.edu.cn
The Thomsen–Friedenreich antigen (TF or T-antigen, Galβ1–
3GalNAcαSer/Thr) is one of the most common tumorassociated carbohydrate antigens (TACAs). Recently, TMUC1 glycopeptide analogs containing one or two fluorine
substituents on the sugar were synthesized. They have been
conjugated to a tetanus toxoid carrier protein and the conjugate vaccines elicited strong and specific immune responses in
mice. These “foreign” fluorinated TACA based vaccines not
only provided enhanced immunogenicity and metabolic stability but also improved bioavailability. However, the chemical synthesis of fluorinated T-antigens and other Galβ1–3GalNAc-containing O-glycans is challenging. A highly efficient two-step one-pot two-enzyme protocol for the preparation of fluorinated T-antigens was developed by adding two
enzymes sequentially to accommodate their distinct pH
Glycoconj J (2013) 30:281–461
preferences. The substrate promiscuity of a recombinant
galactokinase (EcGalK), a novel Bifidobacterium infantis Dgalactosyl-β-1–3-N-acetyl-D-hexosamine phosphorylase
(BiGalHexNAcP), and an α-2–3-sialyltransferase from
Pasteurella multocida (PmST1) allows the high-yield
chemoenzymatic synthesis of fluorinated T- and ST-antigens.
In addition, the high expression levels in the E. coli expression
systems of these enzymes and a Neisseria meningitidis CMPsialic acid synthetase (NmCSS) permit their application in
large-scale synthesis.
234: Crystal structure of anti-polysialic antibody single
chain Fv fragment in complex with alpha2-8 linked
oligosialic acids
Masamichi Nagae1, Akemi Ikeda1, Shinya Hanashima1,
Ken Kitajima2, Chihiro Sato2, and Yoshiki Yamaguchi1;
1
Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN Global Research Cluster, RIKENMax Planck Joint Research Center, RIKEN, 2-1 Hirosawa,
Wako, Saitama 351-0198, Japan; 2Bioscience and Biotechnology Center and the Graduate School of Bioagricultural
Sciences, Nagoya University, Nagoya 464-8601, Japan
mnagae@riken.jp
Polysialic acid is a linear homopolymer typically of α28 linked sialic acids on gangliosides and glycoproteins. Cell
surface polysialic acid plays important roles in cell adhesion
and differentiation events in a manner that is often dependent
on the degree of polymerization (DP). Anti-oligo/polysialic
acid antibodies have DP-dependent antigenic specificity, and
such antibodies are widely utilized in biological studies for the
detection and differentiation of oligo/polysialic acids. Although a murine monoclonal antibody mAb735 has a preference for long polysialic acid, the atomic details of polysialic
acid recognition mechanism of this antibody still remain
unclear. Here we report the crystal structure of mAb735 single
chain Fv fragment (scFv735) in complex with octa-sialic
acid at 1.8 Å resolution. In the asymmetric unit, two
scFv735 molecules cooperatively grab one octa-sialic acid.
In each complex, all the complementarity determining regions except for L3 interact with three sequential sialic
acid residues out of eight residues. A striking feature is
that ordered water molecules bridge the gap between
antibody and ligand. The dihedral angles of the three sialic
acid residues interacting with scFv735 are not uniform,
indicating that mAb735 does not favor the previously
suggested helical conformation. Importantly, both reducing
and non-reducing ends of the bound ligand are completely
exposed to solvent. We suggest that mAb735 gains its
apparent high affinity for long polysialic acid chain by
recognizing every three sialic acid unit within it.
Glycoconj J (2013) 30:281–461
235: Synthesis of Mycobacterium Peptidoglycan (PGN)
Fragments Containing N-Glycolylmuramic Acid
Qianqian Wang1, Fukase Matsuo1, Yukari Fujimoto1,
Koichi Fukase1; 1Graduate School of Science, Osaka
University, Osaka, 560-0043, Japan;
yukarif@chem.sci.osaka-u.ac.jp
Peptidoglycan (PGN) is a glycoconjugate that constitutes
bacterial cell wall, which has been known as a potent
immunopotentiator and an adjuvant for antibody production. PGN consists of polysaccharide chains, which are
cross-linked with peptides. Our group has synthesized series of PGN partial structures and revealed the ligand
structures of several PGN-binding proteins including the
innate immune receptor proteins Nod1 and Nod2; Nod1
recognizes diaminopimelic acid (DAP)-containing peptides, whereas Nod2 recognizes the MurNAc-L-Ala-DGln. The polysaccharide of PGN in many bacteria is a
β(1-4) glycan composed of alternating Nacetylglucosamine (GlcNAc) and N-acetylmuramic acid
(MurNAc), while Mycobacterium PGN contains Nacetylglucosamine and a mixture of N-glycolylmuramic
acid (MurGlyc) and N-acetylmuramic acid (MurNAc). Because biological activities of PGN fragments containing
MurNGlyc have not been elucidated well, we started the
synthetic study of PGN fragments having MurNGlyc. Mycobacterium PGN has also meso-DAP as an amino acid at
the branched position, and we also developed the efficient
synthetic method for it.
The synthesis of orthogonally protected (2S,6R)-DAP was
first examined. Because it is a key intermediate and
crucial moiety for Nod1 receptor recognition, an efficient
and highly stereoselective synthesis for meso-DAP was
developed by using chemoenzymatic method. It is advantageous for the short reaction steps and mild conditions
comparing with previously reported methods. DAPcontaining tripeptide (L-Ala-D-iso-Gln-meso-DAP) and
tetrapeptide (L-Ala-D-iso-Gln-meso-DAP-D-Ala)) were
obtained efficiently with the newly developed method.
We then synthesized a series of the fragment structures
of Mycobacterium PGN containing one of the following
saccharide structures; MurNAc, MurNGlyc, 1,6-anhydroMurNAc or 1,6-anhydro-MurNGlyc. For the introduction
of the glycolic group, we examined the selective
amidation reaction for shorter reaction steps.
The compounds will be used for the evaluation of their
immunostimulatory activities via Nod1 or Nod2 receptors and
also other PGN recognition proteins. The present study will
contribute to the understanding of immune-responses in Mycobacterium-related diseases.
393
236: Typing of Blood-Group Antigens of Neutral Oligosaccharides by Negative-Ion Electrospray Ionization
Tandem Mass Spectrometry
Hongtao Zhang 1, Shuang Zhuang2 , Guanjun Tao2 ,
Yibing Zhang 3 , Barbara Mulloy 3 , Xiaobei Zhan 1* ,
Wengang Chai3*; 1The Key Laboratory of Carbohydrate
Chemistry and Biotechnology of Ministry of Education,
School of Biotechnology, Jiangnan University, 1800 Lihu
Avenue, Wuxi 214122, China; 2Testing and Analysis Centre, State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi
214122, China; 3Glycosciences Laboratory, Department
of Medicine, Imperial College London, Hammersmith
Campus, Du Cane Road, London W12 0NN, United
Kingdom
w.chai@imperial.ac.uk; xbzhan@yahoo.com
Blood group antigens, such as those containing fucose
and bearing the ABO(H)- and Lewis-type determinants
expressed on the carbohydrate chains of glycoproteins
and glycolipids, and also on unconjugated free oligosaccharides in human milk and other secretions, are
associated with various biological functions. We have
previously shown the utility of negative-ion electrospay
ionization tandem mass spectrometry with collisioninduced dissociation (ESI-CID-MS/MS) for typing of
Lewis (Le) determinants, e.g. Lea, Lex, Leb, and Ley
on neutral and sialylated oligosaccharide chains. In the
present report we extended the strategy to characterization of blood-group A-, B- and H-determinants on
type 1 and type 2, and also on type 4 globoside chains
to provide a high sensitivity method for typing of all
the major blood-group antigens, including the A, B, H,
Lea, Lex, Leb, and Ley determinants, present in oligosaccharides. Using the principles established we identified two minor unknown oligosaccharide components
present in the products of enzymatic synthesis by bacterial fermentation. We also demonstrated that the
unique fragmentations derived from the D- and 0,2Atype cleavages observed in ESI-CID-MS/MS, which
are important for assigning blood-group and chain
types, only occur under the negative-ion conditions for
reducing sugars but not for reduced alditols or under
positive-ion conditions.
394
Glycoconj J (2013) 30:281–461
237: Conformational properties of alpha- and betaglycolipids as Mycoplasma cytoplasmic-membrane
components
1
1
1
Mengfei Yuan , Ryoko Matsunaga , Hirofumi Dohi ,
Sachie Matsuda 2 , Kazuhiro Matsuda 2 , Yoshihiro
Nishida1; 1Advanced Integration Science, Graduate School
of Chiba University, Matsudo-648, 271-8510, Japan; 2M.
Biotechnology, Fukazawa 2-1-3-1103, Setagaya, Tokyo,
158-0081, Japan.
Ynishida@faculty.chiba-u.jp
Pathogenic Mycoplasmas such as M. pneumoniae and
M. fermentans utilize either alpha or beta-glycosyl
glycerolipids as constituents of cytoplasmic membrane
together with phospholipids. These glycolipids are made
of saturated fatty acid like C16 and C18 and, therefore,
may be discriminated from ubiquitous beta-galactolipids
in plant chloroplast thylakoid-membrane. In the present
study, we examined 3D structures of synthetic alphaand beta-glycolipids having defined fatty acids (C2 or
C16) with proton NMR spectroscopy and compared the
.
results with those of phosphatidylcholine having C16
fatty acid (DPPC).
Our NMR analysis was based on the complete assignment of
any diastereomeric protons at the glycerol sn-1 and 3 positions
as well as at the sugar C-6 position. Time-averaged distributions of three staggered conformations (gg, gt, tg) were estimated from vicinal coupling constants of diastereomeric
protons for these acyclic positions. The analysis showed that
the acyclic glycerol moiety in beta-galactolipids keeps a
conformational property very close to that of DPPC; the polar
head-group takes three staggered conformations randomly
(gg = g t = tg), while the hydrophobic tail group takes gt and
gg conformations out of the possible three conformers. The
observed unique property was little affected by glycerol acyl
groups (C2 and C16) and solvents (D2O, CD3OD, CDCl3).
Conformations at the sugar C6 position were changed significantly by solvents and also by glycerol acyl group to some
extents. Conformations of alpha-glycolipids (C16) showed a
similar tendency to those of beta-glycolipids and DPPC, while
the time-averaged populations changed slightly by substituent
groups at the sugar C-6 position.
Glycoconj J (2013) 30:281–461
238: Mass spectrometry based sulfoglycomics of lymphoid tissues
Shin-Yi Yu1, Chu-Wen Cheng2, Ming-Yi Ho2, Kay-Hooi
Khoo2, Reiji Kannagi1; 1Institute of Biomedical Sciences
and 2Institute of Biological Chemistry, Academia Sinica,
Nankang, Taipei 115, Taiwan
Sulfate modification on carbohydrate modifies the
physico-chemical properties of carrier, and alters its
cognate recognition by specific lectins, which is involved in normal physiological process and diseases
such as chronic inflammation. Additionally, precise positioning of sulfate on these glycans can determine
whether they serve as ligands for one or more glycan
binding proteins. One of the better-studied system is the
homing of lymphocytes to secondary lymphoid organs,
which is dependent on L-selectin correctly recognizing
specific sulfated glycotopes presented on sialomucins
lining the high endothelial venules of peripheral lymph
nodes and the mucosa-associated lymphoid tissue. Few
sulfated glycotopes were characterized due to limited
availability of monoclonal antibodies. Moreover, antibody detection cannot provide information on the structural and conformational context of the underlying
glycan carriers, which may dictate their actual in vivo
physiological functions.
Advanced mass spectrometry offers a precise and high
sensitivity mapping of the glycomic repertoire of a
particular tissue or cell at any one pathophysiological
stage. Due to low abundance and multiple negatively
charges of sulfate, the sulfated glycome is often refractory to MS-based glycomic mapping. Previously, we
have established a viable workflow for sulfoglycomic
analysis based on optimized sample preparation, detection by MALDI-MS mapping and MS/MS sequencing.
To progress further, nano-ESI-MS and MS/MS are now
shown to afford a more comprehensive MS/MS analyses
and detection of multiply sulfated glycans without losing the extra sulfates during MS ionization. A series of
diagnostic fragment ions for precise location of sulfates
was identified based on either MALDI-MS/MS or HCDMS/MS analysis of a panel of chemically synthesized
sulfo-glycan standards. In recent applications to peripheral lymph nodes from different sulfotransferase knockout mice, we have successfully identified the location of
sulfate, their carriers, as well as discovering novel sulfated glycotopes. These findings attest to the practical
utility of our sulfoglycomic approach, which will be
presented here.
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239: Total cellular glycomics: a glycomic approach to
describe cells and streamline the discovery of cellular
biomarkers
Yasuro Shinohara, Naoki Fujitani, Jun-ichi Furukawa,
Jinhua Piao; Graduate School of Advanced Life Science
and Frontier Research Center for Post-Genome Science and
Technology, Hokkaido University, Sapporo 001-0021, Japan
yshinohara@sci.hokudai.ac.jp
Cell surfaces are coated with a variety of intricately arranged
glycoconjugates such as glycoproteins, glycolipids and proteoglycans. Therefore, elucidating the expression profiles of
glycans derived from various classes of glycoconjugates is
important to understand cellular glycosylation homeostasis
and systems biology glycomics. We have established a series
of methodologies for the analysis of N- and O-glycans derived
from glycoproteins, glycosphingolipid glycans, glycosaminoglycans, and free oligosaccharides using mass spectrometry
and liquid chromatography. Procedures to analyze each class
of glycan were then combined to visualize the entire complement of sugars in the cellular glycome, so-called total cellular
glycomics. As an example, analysis of total cellular glycomics
analysis of the wild-type CHO cell line and its lectin-resistant
mutant (Lec 1 and Lec 8) was performed. Perturbation of a
glycan synthetic pathway(s) can cause unexpected glycan expression profiles, which may be difficult to predict without the
use of glycomics. When this technique was applied to various
human cells including embryonic stem cells, induced pluripotent stem cells and various cells derived from normal and
carcinoma cells, total cellular glycomes were found to be highly
cell type-specific, demonstrating their utility as unique cellular
descriptors. Total cellular glycomics can streamline the discovery of cellular biomarkers as demonstrated by the identification
of known pluripotency biomarkers as well as novel candidate
biomarkers.
References
1. Furukawa, J., Fujitani, N., Araki, K., Takegawa, Y.,
Kodama, K., and Shinohara, Y. (2011) Anal. Chem. 83,
9060–9067
2. Fujitani, N., Takegawa, Y., Ishibashi, Y., Araki, K.,
Furukawa, J., Mitsutake, S., Igarashi, Y., Ito, M., and
Shinohara, Y. (2011) J. Biol. Chem. 286, 41669-41679
3. Takegawa, Y., Araki, K., Fujitani, N., Furukawa, J.,
Sugiyama, H., Sakai, H., and Shinohara, Y. (2011) Anal.
Chem. 83, 9443-9449
4. Fujitani, N., Furukawa, J., Araki, K., Fujioka, T.,
Takegawa, Y., Piao, J., Nishioka, T., Tamura, T., Nikaido,
T., Ito, M., Nakamura, Y., and Shinohara, Y. (2013) Proc.
Natl. Acad. Sci. USA, 110, 2105-2110
396
240: Structural Analysis of a Heteropolysaccharide from
Saccharina Japonica by ESI -CID-MS/MS
Weihua Jin 1,2,3 , Jing Wang 1,3 , Wenjing Zhang 1,2 ,
Quanbin Zhang1,3; 1Institute of Oceanology, Chinese
Academy of Science, Qingdao 266071, China; 2University
of Chinese Academy of Sciences, Beijing 100049, China;
3
Nantong Marine Science and Technology R & D Center,
IOCAS, Jiangsu 226006, China
qbzhang@qdio.ac.cn
A fucoidan extracted from Saccharina japonica was fractionated by anion exchange chromatography. The most
complex fraction F0.5 was degraded by dilute sulphuric acid
and then separated by use of an activated carbon column.
Fraction Y1 was fractionated by anion exchange and gel
filtration chromatography while Fraction Y2 was fractionated by gel filtration chromatography. The fractions were
determined by ESI-MS and analyzed by ESI-CID-MS/MS.
It was concluded that F0.5 had a backbone of alternating 4linked GlcA and 2-linked Man with the first Man residue
from the nonreducing end accidentally sulfated at C6. In
addition, F0.5 had a 3-linked glucuronan, in accordance
with a previous report by NMR. Some other structural
characteristics included GlcA 1→3 Man 1→4 GlcA, Man
1→3 GlcA 1→4 GlcA, Fuc 1→4 GlcA and Fuc 1→3 Fuc.
Finally, it was shown that fucose was sulfated at C2 or C4
while galactose was sulfated at C2, C4 or C6.
Glycoconj J (2013) 30:281–461
the closely related RetS and LadS sensor kinases to switch
between acute and chronicinfections. These sensor proteins
antagonistically mediate biofilm formation through communication with a central TCS, GacA/GacS. Recently, it was
shown that RetS modulates the GacS sensor activity by
forming RetS/GacS heterodimers. LadS and RetS are hybrid
sensors with a signalling domain consisting of a 7transmembrane (7TMR) region and a periplasmic sensor
domain (diverse intracellular signalling module extracellular
2, DISMED2). The 2.65 Å resolution crystal structure of
RetS DISMED2, called RetSp, reveals three distinct oligomeric states capable of domain swapping. The RetSp structure also displays two putative ligand binding sites. One is
equivalent to the analogous site in the structurally-related
carbohydrate carbohydrate binding module (CBM) but the
second site is located at a dimer interface. These observations highlight the modular architecture and assembly of the
RetSp fold and give clues on how homodimerization of
RetS could be modulated upon ligand binding to control
formation of a RetS/GacS heterodimer. Modelling the
DISMED2 of LadS reveals conservation of only one ligand
binding site, suggesting a distinct mechanism underlying the
activity of this sensor kinase.
Poster Session II-Biosynthesis &
Metabolism of Glycoconjugates
242: N-glycosylation of β-1,3-glucanosyltransferases is
vital for cell wall synthesis in Aspergillus fumigatus
241: The Pseudomonas Aeruginosa RetS periplasmic
sensor domain reveals an intriguing CBM-like fold
Florence Vincent 1, Adam Round2, Aline Reynaud1,
Christophe Bordi3, Alain Filloux4 and Yves Bourne1;
1
Architecture et Fonction des Macromolécules Biologiques,
UMR6098, CNRS et Universités Aix-Marseille I et II, 163
avenue de Luminy, 13288 Marseille, France; 2 Institut de
Biologie Structurale J.P. Ebel 41, rue Jules Horowitz 38027
Grenoble Cedex 1, France; 3Laboratoire d’Ingénierie des
Systèmes Macromoléculaires, UPR9027, CNRS-IMM,
Université de la Méditerranée, 31 Chemin Joseph Aiguier,
13402 Marseille cedex 20, France; 4Imperial College London, Division of Cell and Molecular Biology, Centre for
Molecular Microbiology and Infection, South Kensington
Campus, Flowers Building, London SW7 2AZ, UK.
florence.vincent@afmb.univ-mrs.fr
Bacterial two-component regulatory systems (TCSs) sense
environmental stimuli to adapt the lifestyle of microbial
populations. For many TCSs the stimulus is a ligand of
unknown chemical nature. Pseudomonas aeruginosa utilizes
Zhao Wan and Jin Cheng; State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, CHINA
jinc@sun.im.ac.cn
Gel1p and Gel2p have been described as β-1,3glucanosyltranferase working with β-1,3-glucan synthase
(Fks1p) to synthesize β-1,3-glucan chains in Aspergillus
fumigatus. All of them are detected to possess N-glycan
chains.
α-mannosidaseI(Cwh41) has been found as the key enzyme to initiate synthesis of N-glycan processing pathway and deletion of cwh41 gene can cause a defective
N-glycan processing of the proteins and a temperaturesensitive deficiency of cell wall integrity (CWI) in
A.fumigatus.
According to these, We hypothesized that loss of natural
N-glycosylation (e.g. in Δcwh41) would impair the cell
wall integrity of A.fumigatus,and some enzyme which
Glycoconj J (2013) 30:281–461
related to cell wall synthesis such as Gel1p,Gel2p and
Fks1p could not work as usual. To verify this prediction, Δgel1Δcwh41 and Δgel2Δcwh41 double disruptions were constructed, and the phenotypes were observed.
The single Δcwh41 mutant and two double mutants in
A.fumigatus showed the similar growth condition and much
more sensitive than the Δgel1 or Δgel2 single mutant strains.
Then N-glycosylation-site-mutant revertants of different mutants were also constructed by site-directed mutagenesis,named
Δgel1 Gel1-NM ,Δgel2 Gel2-NM ,Δgel1Δcwh41 Gel1-NM and
Δgel2Δcwh41Gel2-NM. According to the comparison results
we found that the revertants without N-glycosylation-sites
displayed similar growth condition as the corresponding knock
out strains revealing that the N-glycosylation is a necessary
process of A.fumigatus life.
243: One-pot three enzymes catalyzed synthesis of UDPsugars and their derivatives
Jun Liua,b, Yang Zoua,b, Mengyang Xuea,b, Xueer Zhaoa,b,
Junkai Dongb, Wenjun Wangc, Jie Shenc, Peng George
Wanga, Min Chena,b*; a National Glycoengineering Research Center, Jinan, Shandong 250100, People’s Republic
of China; b The State Key Laboratory of Microbial Technology and School of Life Sciences, Jinan, Shandong 250100,
People’s Republic of China; c College of Pharmacy Nankai
University, Tianjin 300071, People’s Republic of China
chenmin@sdu.edu.cn
Monosaccharide nucleotides play key roles in the biological
systems. The availability of structure variety of nucleotide
sugars is particularly important for the characterization study on glycoconjugates and glycosyltransfersases. Enzymatic
synthesis of these nucleotide sugars displays more advantages than chemical method. Therefore, finding enzymes
with highly active and broad substrates specificity become
a key factor to obtain these nucleotide sugars. In this study, a
promiscuous USP (AtUSP) was cloned from Arabidopsis
thaliana; a UTP-glucose-1-phosphate uridylyltransferase
(GalUSpe4) and a galactokinase (GalKSpe4) were cloned
from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-sugars and their derivatives in
an efficient one-pot reaction system. The properties of the
one-pot reaction were annotated. GalKSpe4 had relaxed
activity towards galactose derivatives with modifications
on the C-6, 4- or 2-positions. Additionally, GalKSpe4 can
also tolerate glucose while glucose derivatives with modifications on the C-6, 4- or 2-positions were unacceptable.
More interestingly,GalKSpe4 can phosphorylate Lmannose in moderate yield (43 %), while other L-sugars
such as L-Gal cannot be recognized by this enzyme. AtUSP
397
has different tolerance for C4 and C2 derivative of Gal-1-P
compared to GalUSpe4. This provides abundant information for better understanding of the enzymatic synthesis of
nucleotide sugars.
244: Snail glycosylation—a continuous challenge
Chantal Lucini 1 , Christopher Taus 1 , Reingard
Grabherr 2 and Erika Staudacher1; 1 Department of
Chemistry, University of Natural Resources and Life Sciences, Vienna, Austria; 2 Department of Biotechnology,
University of Natural Resources and Life Sciences, Vienna,
Austria
Erika.staudacher@boku.ac.at
Glycosylation abilities of snails deserve attention, because
snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites. In
analogy to many other host-pathogen relations, the glycosylation of snail proteins may likewise contribute to these
host-parasite interactions. Furthermore due to the large
heterogeneity of structures and their unusual modifications the class of gastropods is a fascinating target for
investigation.
In previous studies we have successfully analyzed the N- and
O-glycan patterns of a number of snail species (Gutternigg et
al. (2007) Glycoconjugate J. 24, 475–489, Stepan et al. (2012)
Glycoconjugate J. 29, 189–198). We found structural elements
of mammals, plants, insects, nematodes and trematodes in snail
N-glycans and small O-glycans with galactoses and mannoses,
sometimes modified by methyl groups and fucoses, linked to a
protein bound N-acetylgalactosamine residue. Going now for
the corresponding glycosyltransferases is an extra challenge as
only few sequencing data exist from mollusk origin. Starting
with cDNA libraries of several snail species (Arion lusitanicus,
Achatina fulica, Biomphalaria glabrata) we could identify a
fucosyltransferase involved in N-glycosylation and enzymes involved in O-glycosylation, a T-synthase and a
polypeptide N-acetylgalactosaminyltransferase (for more
information about the latter one see the presentation of
Ch. Taus). The cloning strategy, expression and characterization of these enzymes are presented here. All these
novel glycosyltransferases show decent homology to
previously described enzymes with similar specificity
and fit well into the phylogenetic tree.
This is the first detailed description of glycosyltransferases
from the phylum of mollusks.
This work was supported by the Austrian Science Fund
(FWF) [grant number P22118-B20].
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Glycoconj J (2013) 30:281–461
245: Identification and characterization of endo-β-Nacetylglucosaminidase from methylotrophic yeast
Ogataea minuta
246: Expression of Poly-N-acetyllactosamine on the
Surface of Human Hepatocarcinoma Cells and Its Enzymatic Mechanism
Satoshi Murakami1, Yuki Takaoka1, Hisashi Ashida2,
Kenji Yamamoto3, Hisashi Narimatsu4 and Yasunori
Chiba1*; 1Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology
(AIST), Tsukuba 305-8566, Japan; 2Department of Science
and Technology on Food Safety, Faculty of BiologyOriented Science and Technology, Kinki University,
Kinokawa 649-6493, Japan; 3 Research Institute for
Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi 921-8836, Japan; 4Research Center for
Medical Glycoscience, AIST, Tsukuba 305-8566, Japan
y-chiba@aist.go.jp
Hao Qiu1, Wei P. Wang1, Mei F. Jin2, Mei H. Miao2, Hui
L. Chen3; 1Department of Biochemistry and Molecular
Biology, Medical School of Soochow University, Suzhou
215123, China; 2Affiliated Children’s Hospital, Soochow
University, Suzhou 215006, China; 3Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Shanghai 200032, China.
Endo-β-N-acetylglucosaminidase (EC 3.2.1.96) catalyzes
the digestion of N,N′-diacetylchitobiose core of
asparagine-linked oligosaccharides on glycopeptides and
glycoproteins. In 4 yeast strains, Ogataea minuta, Candida
parapolymorpha, Pichia anomala, and Zygosaccharomyces
rouxii, we identified endo-β-N-acetylglucosaminidase homologous sequences by database searches, and also a corresponding enzyme activity was confirmed in crude cell
extract obtained from each strain. The O. minuta endo-βN-acetylglucosaminidase (Endo-Om) -encoding gene was
directly amplified from O. minuta genomic DNA and sequenced. The Endo-Om-encoding gene contained a 2319-bp
open reading frame; the deduced amino acid sequence indicated that the putative protein belonged to glycoside hydrolase family 85. The gene was introduced into O. minuta, and
the recombinant Endo-Om was overexpressed and purified.
When the enzyme assay was performed using an agalactobiantennary oligosaccharide as a substrate, Endo-Om
exhibited both hydrolysis and transglycosylation activities.
Endo-Om exhibited hydrolytic activity for high-mannose,
hybrid, biantennary, and (2,6)-branched triantennary Nlinked oligosaccharides, but not for tetraantennary, (2,4)branched triantennary, biantennary with bisecting GlcNAc,
and core-fucosylated biantennary N-linked oligosaccharides. Endo-Om also was able to hydrolyze N-glycans attached to RNase B and human transferrin under both
denaturing and non-denaturing conditions. Thus, the present
study reports the detection and characterization of a novel
yeast endo-β-N-acetylglucosaminidase.
By using FITC-labeled tomato lectin (Lycopersicon esculentum)
as the probe, it was discovered that poly-N-acetyllactosamine
(PolyLacNAc, [Galβ1,4Gnβ1,3]n) was moderately expressed
on the cell surface of H7721 human hepatocarcinoma cell line.
After the treatment of 20 ng/ml EGF (epidermal growth factor)
for 72 h, the expression of PolyLacNAc was highly increased.
Oppositely, it was significantly decreased after the treatment of
10−5 M of ATRA (all-trans retinoic acid) for 72 h. To elucidate
the enzymatic mechanism of the altered expression of
PolyLacNAc, five glycosyltransferases related to the synthesis
of PolyLacNAc were studied, including β6/β4-Nacetylglucosaminyltransferase-V/IVb (GnT-V/IVb), the
branching enzymes in N-glycan synthesis; β4galactosyltranferase-5 (β4GalT-5), the main enzyme
transferring the galactose residue at the outside of the
β6 branched N-acetylglucosamine; as well as β3-Nacetylglucosaminyltransferase-2 (β3GnT-2) and β4galactosyltransferase-1(β4GalT-1), two enzymes participated
in the synthesis of [Galβ1,4Gnβ1,3]n in H7721 cells (this cell
line does not express β3GnT-8, which is considered as the
main enzyme for PolyLacNAc synthesis in other carcinoma
cells such as column cancer). It was found by RT-PCR method
that the expressions of GnT-V and GnT-IVb were elevated
about 3 or 2 fold after the treatment of EGF, while they were
declined apparently after treated with ATRA. β4GalT-5 was
also up- or down-regulated by the treatment of EGF or ATRA
respectively as GnT-IVb. The expression of β3GnT-2, the
rate-limiting enzyme in PolyLacNAc synthesis, was approximately doubled after EGF and decreased to very low level
after ATRA, while the expression of β4GalT-1 was changed
slightly with no statistic significance after both EGF and
ATRA treatment. Two other glycosyltransferases, β2-Nacetylglucosaminyltransferase-I (GnT-I) and β3-Nacetylglucosaminyltransferase-5 (β3GnT-5) selected as the
Glycoconj J (2013) 30:281–461
negative controls, were almost unchanged after the treatment
of EGF and ATRA. Among the four glycosyltransferases with
altered expressions, GnT-V was most elevated by EGF,
while GnT-V and β3GnT-2 were most declined by
ATRA. However, which one is most important as a
leading enzyme in the regulation of PolyLacNAc synthesis, will be further investigated.
247: Essentiality and a colorimetric assay of mycobacterial MurA
Liming Xu1, Dongting Wu1, Qi Zheng1, Liang Liu1 and
Yufang Ma1, 2; 1Department of Biochemistry and Molecular
Biology, Dalian Medical University, Dalian 116044, China;
2
Liaoning Provincial Core Lab of Glycobiology and
Glycoengineering, Dalian Medical University, Dalian
116044, China
yufang_ma@hotmail.com
The cell wall core of mycobacteria consists of mycolic acid,
arabinogalactan, and peptidoglycan. The peptidoglycan is a
structural basis for sustaining the morphous and osmotic
pressure of mycobacteria. The pepetidoglycan is a reticular
molecule that is the connection of N-acetylglucosamine and
N-acetylmuramate in turn, then crosslink with oligopeptide.
U D P - N - ac e t y l m u r a m a t e i s a s u ga r d on o r o f N acetylmuramate. The formation of UDP-N-acetylmuramate
from UDP-N-acetylglucosamine is catalyzed by two enzymes (MurA and MurB) and MurA, UDP-Nacetylglucosamine enolpyruvyle transferase catalyzes the
first reaction. By catalysis of MurA enzyme, the
enolpyruvyle group is transferred from phosphoenolpyruvate to the 3′-OH of UDP-N-acetylglucosamine to form
UDP-N-acetylenolpyruvylglucosamine. The metabolic pathway of UDP-N-acetylmuramate does not exist in mammalian cells. Therefore, MurA may be a potential target to
develop anti-tuberculosis drugs.
In this study, M. semgmatis murA gene knockout strain
was constructed by DNA homologous recombination.
The changed growth rate, cell morphology and structure
of M. semgmatis murA gene knockout strain clarified
the essentiality of murA gene for mycobacterial growth.
Therefore, it strongly supports that murA gene is a
potential drug target.
399
To investigate the catalytic mechanism of both M. smegmatis
MurA and M. tuberculosis MurA, expression vectors were
constructed respectively and MurA proteins were
overexpressed. A colorimetric assay of MurA enzyme was
established and the analyses of MurA kinetics are in progress.
This work was supported by the National Basic Research Program of China (2012CB518803) and Research
Fund for the Doctoral Program of Higher Education
(20112105110002).
248: Roles of N-acetylglucosaminyltransferase III in
Epithelial-to-mesenchymal Transition Induced by
TGF-β1
Qingsong Xu1, Jianguo Gu2* and Yuguang Du1*; 1Dalian
Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China; 2Division of Regulatory
Glycobiology, Institute of Molecular Biomembrane and
Glycobiology, Tohoku Pharmaceutical University, Sendai
Miyagi, 981-8558, Japan
The epithelial-to-mesenchymal transition (EMT) plays crucial
roles in the embryonic development, wound healing, tissue
repair and cancer progression. In the present study, we demonstrate that transforming growth factor β (TGF-β1) downregulated expression of N-acetylglucosaminyltransferase III
(GnT-III) during EMT. The treatment with TGF-β1 resulted
in a decrease in E-cadherin expression and GnT-III expression
as well as its product, the bisected N-glycans, which was
confirmed by E4-PHA lectin blot in human MCF-10A and
mouse GE11 cells. To understand roles of GnT-III expression
in EMT, the MCF-10A cell was stably transfected with GnTIII. Of particular interesting, overexpression of GnT-III partially, but did inhibit EMT induced by TGF-β1, which were
confirmed by cell morphological changes of phase-contrast,
immunochemical staining patterns of E-cadherin and actin. In
addition, GnT-III modified E-cadherin, which contributed to
prolong E-cadherin turnover on cell surface examined by
biotinylation and pulse-chase experiments. Consistently,
GnT-III expression inhibited β-catenin translocation
from cell-cell contact into cytoplasm and nucleus. Furthermore, the transwell assay showed that GnT-III expression suppresses TGF-β1-induced cell motility. Taken
together, these observations are the first to clearly
400
demonstrate that GnT-III plays important roles in EMT,
and explain a molecular mechanism for inhibitory effects of GnT-III on cancer metastasis.
249: M. tuberculosis Rv1096, a novel peptidoglycan
deacetylase
Shufeng Yang 1, 2, Jian Kang2, Wenli Zhang2, Yufang
Ma2; 1 Department of Biochemistry and Molecular Biology,
Dalian Medical University, Dlian, 116044, China; 2 Department of Microbiology, Dalian Medical University, Dlian,
116044, China
yufang_ma@hotmail.com
Tuberculosis is a major chronic infectious disease in the
world and caused by pathogen Mycobacterium tuberculosis.
The bacteria in alveolar macrophages of lung are not
digested by lysosome, therefore, they remain dormant for
months, years, and even decades without increasing in number and without making the person sick. Once the bacteria
are reactivated in the people with weakened immune
systems, the people are at a greater risk for developing
tuberculosis.
The cell wall of M. tuberculosis contains unusual polysaccharides, lipids and proteins. The modification of cell wall
may prevent the fusion of the phagosome with the lysosome
of macrophages. The evidences from Streptococcus pneumonia and Listeria monocytogenes showed that
deacetylated peptidoglycan was resistant to degradation of
lysosome. Bioinformatic analysis revealed that M. tuberculosis Rv1096, a cell wall protein, was homologous to peptidoglycan deacetylases from S. pneumonia and L.
monocytogenes.
Glycoconj J (2013) 30:281–461
250: Construction and characterization of M. smegmatis
glmM gene knockdown strain
Jian Kang1, Liming Xu1, Yi Xin2, 3 Yufang Ma1, 3;
1
Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian 116044, China; 2Department of Biotechnology, Dalian Medical University, Dalian
116044, China; 3 Liaoning Provincial Core Lab of
Glycobiology and Glycoengineering, Dalian Medical University, Dalian 116044, China
yufang_ma@hotmail.com
Due to the essential roles of M. tuberculosis cell wall,
the enzymes involved in the biosynthesis of the cell
wall are potential targets for new anti-tuberculosis
drugs. The cell wall core of M. tuberculosis consists
of mycolic acid, arabinogalactan, and peptidoglycan and
a disaccharide linker (L-Rha-D-GlcNAc-P) connects
mycolated-arabinogalactan to peptidoglycan. UDPGlcNAc is a glycosyl donor of the linker and also a
precursor of peptidoglycan. The phosphoglucosamine
mutase (GlmM) which catalyses the second step in
UDP-GlcNAc biosynthetic pathway. Our previous results from M. smegmatis glmM gene knockout strain
confirmed that glmM gene was essential for the growth
of mycobacteria. Therefore, GlmM could be a potential
target of anti-tuberculosis drugs.
To gain insights into the functions of glmM gene in
mycobacteria, a tetracycline inducible M. smegmatis glmM
gene knockdown strain was constructed by using antisense
RNA technology. The growth, morphology, biofilm formation and antimicrobial susceptibility of the glmM gene
knockdown strain were detected to elucidate the biological
roles of GlmM protein.
Here, we constructed two types of expression vectors:
pColdII-Rv1096 and pVV2-Rv1096. The Rv1096 protein
was expressed from E. coli ER2566/pColdII-Rv1096 and
M. smegmatis mc2155/pVV2-Rv1096 and was purified. The
peptidoglycan was prepared from M. smegmatis mc2155
and incubated with the purified Rv1096 protein. The acetyl
group released by enzyme reaction was measured by using a
acetic acid detection kit. In addition, M. smegmatis
mc2155/pVV2-Rv1096 showed resistance to lysozyme
compared to wild type M. smegmatis mc2155 in the exponential growth phase. Therefore, we conclude that Rv1096
is a novel peptidoglycan deacetylase. The characterization
of Rv1096 is under way in our laboratory.
We found that after induction with tetracycline, the expression of GlmM protein in the glmM gene knockdown strain
was significantly decreased, resulting in a decline of cell
growth. The morphology of the glmM gene knockdown
strain exhibited a wrinkled cellular surface and an enlarged
shape. Furthermore, insufficient GlmM protein reduced the
biofilm formation and increased the sensitivity to isoniazid
and ethambutol. In addition, we also performed twodimensional gel electrophoresis for both wild-type M.
smegmatis and the glmM gene knockdown strain and found
some different protein spots between them. These spots will
be identified by MS/MS and confirmed by qRT-PCR or
Western blot.
This work was supported by the National Basic Research
Program of China (2012CB518803) and Research Fund for
the Doctoral Program of Higher Education (20112105110002).
This work was supported by the National Basic Research
Program of China (2012CB518803) and National Natural
Science Foundation of China (30970067).
Glycoconj J (2013) 30:281–461
251: UDP-N-acetylglucosamine pyrophosphorylase is essential for cell wall biogenesis in Aspergillus fumigatus
Ting Du, Cheng Jin; State Key Laboratory of
Mycology,Institute of Microbiology, Chinese Academy of
Sciences, Beijing, 100101, China
duting2008@163.com
As an important cell wall composition, chitin is synthesized
from uridine diphosphate-N- acetylglucosamine (UDPGlcNAc). UDP-GlcNAc is also a sugar donor for the synthesis
of glycoproteins, lipopolysacharide and GPI anchors. UDP-Nacetylglucosamine pyrophosphorylase (UAP1), a member of
the large family of nucleotide diphosphate sugar
pyrophosphorylases, catalyses the last step in eukaryotic biosynthesis of UDP-GlcNAc, converting UTP and GlcNAc-1P to
the sugar nucleotide.
In this study, Afuap1, a gene encoding UAP1p, was identified in the opportunistic pathogen Aspergillus fumigatus. A
conditional inactivation mutant was constructed because
loss of uap1 was lethal. Under the suppression condition
the mutant showed increased sensitivity to the cell wall
interference and abnormal conidial germination. Our results
suggested that suppression of the uap1 gene in A. fumigatus
led to retarded growth and cell wall defect. Further analysis
demonstrated that down-regulation of the uap1 gene
resulted in a reduction of α-glucan and chitin in cell wall.
Although the amounts of glycoprotein and β-glucan in
mutant strain were similar with the wild-type ones, GlcNAc
released from these proteins was decreased by 23 % and
mannose was increased by 59 %.
These results indicate that the uap1 is essential for cell wall
synthesis in A. fumigatus. The insights of the influence of
this gene to cell wall will aid the future exploitation of this
genetically validated enzyme as a potential target for the
discovery of novel antifungal compounds.
252: GDP-Mannose transport in Cryptococcus neoformans
is mediated by two distinct proteins and is dispensable for
viability
Zhuo A. Wang, Matthew Williams, and Tamara L.
Doering; Department of Molecular Microbiology, Washington
University School of Medicine, St. Louis, MO 63110, USA
zhuowang@borcim.wustl.edu, doering@borcim.wustl.edu
401
Cryptococcus neoformans is an opportunistic pathogen responsible for cryptococcal meningoencephalitis. Every year this
disease kills over 600,000 people who are immunocompromised due to AIDS or other conditions. The fungal pathogen
is surrounded by a polysaccharide capsule, which is its major
virulence factor. The capsule is composed primarily of two
polysaccharides, glucuronoxylomannan (GXM) and
glucuronoxylomannogalactan (GXMGal); mannose comprises
over half of the capsule mass. C. neoformans also extensively
utilizes mannose in cell wall synthesis and glycosylation of
proteins and lipids.
GDP-mannose (GDP-Man) is produced in the cytosol by the
sequential actions of phosphomannose isomerase,
phosphomannomutase, and GDP-Man pyrophosphorylase.
However, most of the glycan synthetic reactions for which
this compound serves as the donor occur in the Golgi
complex. This highly charged compound thus requires specific nucleotide sugar transporters to convey it to the site of
these biosynthetic reactions, similar to the case for many
other nucleotide sugars. Transport of GDP-Man is of particular interest in the context of a microbial pathogen, however, since mammalian cells lack this capability.
We previously identified two GDP-mannose transporters in
C. neoformans, which we termed Gmt1 and Gmt2. Biochemical studies of each protein expressed in Saccharomyces cerevisiae showed that both are functional, with similar
kinetics and substrate specificities. Surprisingly, microarray
experiments indicated that the genes encoding Gmt1 and
Gmt2 are transcribed with distinct patterns of expression in
response to variations in growth conditions. To investigate
potential functional differences between these two transporters, we first generated a double mutant strain. We were
surprised that this strain was viable, since the single gene
encoding a GDP-Man transporter in S. cerevisiae is essential. We next compared cell growth, colony morphology,
protein glycosylation, and capsule phenotypes of the Gmt
single and double mutants. In all of these studies, the gmt1
mutant showed significant phenotypic differences from
gmt2 mutant, suggesting the two proteins play different
roles in cryptococcal biology. This hypothesis was
supported by nonidentical subcellular localization of the
Gmts, as defined by immunofluorescence microscopy. We
also found that the double mutant exhibited severe defects
in capsule synthesis and protein glycosylation, and was
completely avirulent in mouse models of cryptococcal
infection.
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Glycoconj J (2013) 30:281–461
253: The insect CMP-sialic acid synthetases: New
features in the enzyme activity and intracellular
localization
254: Purification and Characterization of a Fucoidan
Degrading Enzyme from Sphingomonas paucimobilis
PF-1
Di Wu1,2,3, Akiko Fujita1, Kayo Hamaguchi1,3, Anne
Harduin-Lepers4, Philippe Delannoy4, Vladislav M.
Panin5, Chihiro Sato1,2,3, Ken Kitajima1,2,3; 1Bioscience
& Biotechnology Center, Nagoya University; 2Program for
Leading Graduate Schools IGER, Nagoya University;
3
Graduate School of Bioagricultural Sciences, Nagoya University, 464-8601, Japan; 4Univ of Lille, Villeneuve d’Ascq,
France; 5Texas A&M Univ., Texsas, US.
kitajima@agr.nagoya-u.ac.jp.
Woo Jung Kim, Doo Jin Choi, Jisun Lee, Chang Won
Lee, Seul Lee, Ji Won Choi, Yong II Park; Department of
Biotechnology and The Research Centre for Biopharmaceutical Lead Molecule (GRRC), The Catholic University of
Korea, Bucheon, Gyeonggi-do, 420-743, Korea
yongil382@catholic.ac.kr
Sialic acid (Sia) is a family of nine-carbon sugars with a
carboxyl group, consisting of N-acetylneuraminic acid
(Neu5Ac), N-glycolylneuraminic acid (Neu5Gc),
deaminoneuraminic acid (Kdn), and their modified forms.
Although many studies have revealed the importance of
sialylation in prokaryotes and higher animals, sialylation in
invertebrates has not been well studied. The objective of this
study is to gain insights into sialylation in insects, focusing on
the CMP-Sia synthetases (CSSs), which catalyze the synthesis
of donor substrates for sialyltransferases. The following results have revealed new features of insect CSSs in their
activity and intracellular localizaion: (i) The CSS cDNAs were
cloned from Tribolium castaneum (red flour beetle), Aedes
aegypti (yellow fever mosquito), and Drosophilla
melanogaster (fluit fly). Amino acid sequence alignment of
these CSSs showed that they share high sequence identities
with the vertebrate CSSs in the N-terminal catalytic domain,
while they lack the extra C-terminal domain common to
vertebrate CSSs; (ii) The bacterially expressed recombinant
insect CSSs had no in vitro activity to Neu5Ac, Neu5Gc, or
Kdn. In contrast, the in vivo activity was detected with these
insect CSSs using a CSS-impaired CHO mutant cell line
LEC29.Lec32, which is negative in the surface expression of
polysialic acid (polySia). The transfectant LEC29.Lec32 cells
with the insect CSS cDNA plasmids were changed to be
polySia-positive; (iii) To determine the intracellular localization of insect CSSs, the Azami-Green fluorescent proteinconjugated CSSs (AG-CSSs) were expressed in CHO cells.
As previously reported, Drosophila CSS was localized in the
Golgi. In contrast, Aedes CSS and Tribolium CSS were
localized in cytosol and nucleus. These results indicate
that the insect CSSs are functional in the enzyme activity
at least in mammalian cells. Surprisingly, intracellular
localization of CSS appears not to be common among
insect species. Underling mechanisms for regulating the
intracellular localization in insect cells remain to be
elucidated.
A fucoidan degrading enzyme activity has been produced
from Sphingomonas paucimobilis PF-1 (KCTC 11130BP)
using Miyeokgui fucoidan (MF), isolated from Korean
Undaria pinnatifida sporophyll, as a sole carbon source of
culture medium. The enzyme was purified to an apparent
homogeneity mainly by sonic disruption of cells, ammonium sulfate precipitation, DEAE-Sepharose column chromatography and chromatofocusing, with a final recovery of
3.2 % and a specific activity of 2.142 U/mg protein. The
purified enzyme (tentatively named FNase S) appeared as a
single band on Native PAGE gels with a molecular mass of
approximately 130 kDa. However, the SDS-PAGE gels gave
3 separate protein with molecular masses of approx. 130
(designated as S1), 70 (S2) and 60 (S3) kDa, respectively,
suggesting a multi-protein complex nature of this enzyme.
The first 10 N-terminal amino acid sequences of S1
(SXPEAASLPG), S2 (SPQFDVVXIG), and S3
(SLQFDVVVIG) showed high homology (over 80–90 identity) with the N-terminal regions of ATPase, core domain
(EGF 27867), carbohydrate kinase, PfkB family protein
(YP003854323) and dihydrolipoyl dehydrogenase
(ZP08017938), suggesting that these three proteins may
delineate a new family of glycoside hydrolases. The optimum conditions for enzyme activity on MF were pH 6.0–
7.0 and 40–45 °C. The activity was stable within pH 5.5–8.0
and most stable at 40–45 °C for 1 day at pH 6.0. The
enzyme was activated by Mn2+ and Na+ at the concentration
of 1 mM. The enzyme was specific (almost equally active)
towards MF, commercial fucoidan (Sigma) and alginate, but
exhibited very low activity towards heparin, starch, laminarin and dextran. Apparent Km, Vmax and Kcat values for
MF were 1.7 mM, 0.62 μmol/ml · min, and 0.38 S-1,
respectively. The purified FNase S depolymerized MF into
at least more than 7 distinct low-molecular weight
galactofuco-oligosaccharides, ranging from 318 to
3,312 Da, with no production of monosaccharides,
suggesting that this enzyme is an endo-acting fucoidanase
and may be an attractive material for not only industrial
applications utilizing low-molecular weight galactofucooligosaccharides but also structural analysis of fucoidan.
Glycoconj J (2013) 30:281–461
255: Fluorescence-based assays for plant glycopolymer
biosynthesis
Irina Ivanova, Stephan Goetz, Sergey A. Nepogodiev,
Robert A. Field*; Department of Biologycal Chemistry,
John Innes Centre, Norwich, NR4 7UH, UK
irina.ivanova@jic.ac.uk
Glycosylphosphatidylinositol (GPI) anchors are simple glycolipids that are involved in the post- translational attachment
of arabinogalactan proteins to the plasma membrane of plant
cells. Despite their importance in plants, little work has been
done to elaborate the structural or and biosynthetic pathway
leading to GPI anchorts in plants. To our knowledge, the only
plant GPI structure that has been determined is from Pyrus
communis. Consequently, the biosynthetic pathway for the
assembly of plant GPI anchors and their attachment to
arabinogalactan proteins has only been predicted on the basis
of bioinformatics analysis. Therefore, the aim of this project is
to assess the biosynthetic pathway using non-radioactive
methodology
The initial goal of this project is the establishment of
fluorescence-based methodologies which will include the
incorporation of fluorophore into glycoside acceptors or
the post-biotransformation attachment of fluorescent labels
to glycosides using “click chemistry”. As with any new
methodology it will be necessary to validate and benchmark
against the established radiochemical assays, which have
long been used to determine the structure and biosynthetic
pathways of GPI anchors in Trypanosoma brucei. These
new fluorescence-based methodologies will then be used
to investigate aspects of plant GPI anchors biosynthesis,
building on ongoing work in the Field group on plant cell
wall biosynthesis.
256: Ubiquitous occurrence and biosynthesis of sulfated
sialic acids in mammals
Thanyaluck Phitak1, Hiroshi Fujita2, Chihiro Sato1,2,
Ken Kitajima1,2; 1Bioscience & Biotechnology Center;
and 2Graduate School of Bioagricultural Sciences, Nagoya
University, Nagoya 464-8601; 3Department of Pharmaceutical Sciences, Kitasato University, Tokyo 108-8641,
Japan
kitajima@agr.nagoya-u.ac.jp
It is well known that sialic acids (Sias) show structural
diversity in which various modifications occur on three
fundamental Sia species, i.e., N-acetylneuraminic acid
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(Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and
deaminoneuraminic acid (Kdn). We previously found sulfated
Sia (SiaS) residues in glycolipids and glycoproteins of sea
urchin gametes, and demonstrated that they are involved in
sperm-egg interaction at fertilization. In mammals the presence of SiaS has been demonstrated in bovine gastric glycolipids and human and mouse tissue homogenates. However,
nothing has been known about the biosynthesis and biological
functions of SiaS. We have thus sought to understand biological significance and biosynthetic pathway of SiaS residues. In
this study, we first developed chemical and immunochemical
detection methods of SiaS: The DMB labeling-based fluorometric high-performance liquid chromatography (HPLC)
analysis, and immunochemical detection using two monoclonal antibodies: mAb.3G9 specific for the 8-O-sulfated
Neu5Ac (Neu5Ac8S) residue, and mAb.2C4 specific for the
Neu5Ac8S/Neu5Gc8S residues. Immunohistochemistry and
Western blotting showed that SiaS was present in various
human and mouse tissues. On the fluorometric HPLC, not
only NeuAc8S/9S and Neu5Gc8S/9S, but also Kdn8S/Kdn9S
were detected in certain mouse and rat tissues. These results
indicate that various SiaS residues ubiquitously occur in
mammals. To understand the biosynthesis of the SiaS, we
are now searching for the sialate sulfotransferase (SulT-Sia)
by direct purification from sea urchin gametes, PCR cloning
based on the motif structures of sulfotransferases, and expression cloning using mAb.3G9. Preliminary results showed the
presence of a critical gene for expression of cell surface
Neu5Ac8S residues.
257: Systematic identification of in vivo target proteins
specific for a glycosyltransferases isozyme, β1,4galactosyltransferase-I
Daisuke Sugahara1, Hiroyuki Kaji1, Kazushi Sugihara2,
Masahide Asano2, Hisashi Narimatsu1; 1Research Center
for Medical Glycoscience (RCMG), National Institute of
Advanced Industrial Science and Technology (AIST),
Tsukuba, Ibaraki 305-8568, Japan; 2Division of Transgenic
Animal Science, Advanced Science Research Center,
Kanazawa University, Kanazawa, Ishikawa 920-8640,
Japan.
d-sugahara@ks.kyorin-u.ac.jp
Recently, as a major focused issue in the glycobiology study
field, clarification of the biological role of glycans is receiving specific attention. Model organisms containing deletion
or mutation in a glycosyltransferase-gene exhibit various
physiological abnormalities, suggesting that a specific glycan motif on certain glycoproteins control the molecular
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function of the glycoproteins in vivo. In order to determine
proteins responsible for the abnormalities and also their
underlying molecular mechanisms, it is highly desirable to
identify the target proteins for the glycosyltransferase responsible for the biosynthesis of the glycan motif. However,
presence and co-expression of other isozymes interfere with
the identification of the target proteins specific for the isozyme. The fact that isozymes could produce one particular
glycan motif under the physiological condition complicates
the situation.
Here, we present a systematic approach to identify the in
vivo target proteins of a particular glycosyltransferase isozyme on a proteomic scale. To identify the target proteins
specific for β1,4-galactosyltransferase-I (β4GalT-I) under
the co-expression of other β4GalT isozymes, we determined and compared presence of Galβ1,4-terminated glycans on individual proteins of wild-type and β4GalT-I-/mice. By utilizing a lectin-mediated affinity capture of
glycopeptides carrying Galβ1,4-terminated glycans,
isotope-coding glycosylated site specific tagging (IGOT)
and shotgun LC/MS analysis, we achieved comprehensive
identification of proteins carrying Galβ1,4-terminated glycans in mouse liver. Glycoproteins that were present in the
wild-type mice, but not in the β4GalT-I−/− mice, were
identified as plausible candidates for the β4GalT-I-specific
target proteins, because the β4GalT-I-specific target proteins
is assumed not to be β1,4-galactosylated in the β4GalT-I−/−
mice. Among 1,176 proteins identified in this study, 181
proteins were identified as the target proteins. Bioinformatic
analysis of the identified proteins offered some unique information on the molecular characteristics of the target proteins.
Our approach is a powerful means to move forward the
elucidation of the biological role of glycans and the
understanding of the mechanism that controls assembly of a
particular glycan motif on specific proteins in vivo.
Glycoconj J (2013) 30:281–461
GalT-I, distributed in the Golgi apparatus, exercises the
functions of glycosyltransferase. Long-chain β1,4-GalT-I,
located located on the cell membrane,acts as a cell adhesion molecule playing an important role in cell adhesion,
sperm-egg recognition, neurite growth, tumor migration
process, etc. TGF-β1 is one of the transforming growth
factor-β superfamily members, two-way function to promote and inhibit of cell proliferation, and will display
different biological roles for the different target cells, as
well as different functional states of the same target cells.
In the study, we investigate the function of TGF-β1, β1,4galactosyltransferaseIin embryo implantation and the correlation of them on other implantation factors expression.
Significantly, The expression of total β 1, 4-GalT-I reached
the peak in estrogen role for the concentration of 10−4
ug/ul, time for 12 h by RT-PCR in RL95-2 cells (analog
implantation of endometrial cells)(P<0.05); The expression
of total β 1, 4-GalT-I reached the peak in estrogen role for
the concentration of 10−4 ug/ul, time for 12 h by RT-PCR
in RL95-2 cells(P<0.05). After RL95-2 cells were treated
with β1,4-GalT-I gene over-expression plasmid transfection, the expression of the total β1, 4-GalT I gene and
long-chain β1,4-GalT-I gene were significantly
increased(P<0.05); But the expression of the TGF-β1 gene
was not changed obviously. After RL95-2 cells were treated with β1,4-GalT-I gene interference plasmid,the expression of the total β1,4-GalT-I gene and long-chain β1,4GalT-I expression were significantly decreased(P<0.05);
But the expression of the TGF-β1 gene was not changed
obviously. We hypothesize hormones might regulate the
implantation of the embryo through the β1,4-GalT-I and
β1,4-GalT-I might not affect the expression of TGF-β1.
(This work was supported by National Natural Scientific
Grant No.30970646,PR China)
259: Glycosylation of protodioscin by Arthrobacter sp.
258: Function study of TGFβ1、β1,4- galactosyltransferaseIin embryo implantation and the correlation
of them on other implantation factors expression
Yunpeng Xie1, Ting Gong, lin Zhang, Cheng Zhang,
Ying Kong *; 1Department of Biochemistry and Molecular
Biology, Dalian Medical University . Dalian,116044,China .
kongying@dlmedu.edu.cn
The mammalian embryo implantation is an extremely complex process. The blastocyst invasive and the establishment
of endometrial receptivity are keys to ensure the successful
implantation of the embryo. β1, 4 - galactosyltransferase
enzyme I (β1,4-GalT-I) is divided into two phenotypes,
which are short-chain and long-chain. Short-chain β1,4-
Jingyuan Liu1, 2, Li Lu1, Liping Kang1, 2, Heshui Yu1, 2,
Yang Zhao1, Chenqi Xiong1, Liyan Yu3, Baiping Ma1*;
1
Beijing Institute of Radiation Medicine, Beijing 100850,
China; 2 Tianjin University of Traditional Chinese Medcine,
Tianjin 300193, China; 3Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking
Union Medical College, Beijing 100050, China
mabaiping@sina.com
Steroidal saponins are known as a main kind of constituents in traditional Chinese medicine with activities
such as inhibiting platelet-aggregation, anti-dementia,
antibacterial and cytotoxicity. It was reported that sugar
Glycoconj J (2013) 30:281–461
chains play important roles on bioactivities of steroidal
saponins. Therefore, studies on glycosylation of steroidal saponins, which are seldom reported, should be an
important part in medical research. A strain of
Arthrobacter sp. was found possessing glycosylation
activity on steroidal saponins. Protodioscin, a steroidal
saponin from Dioscorea nipponica Makino, could be
converted to the corresponding fructosylation product
by Arthrobacter sp. in this study. The biotransformation
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mixture was isolated by column chromatography on
MCI and ODS silica-gel after being extracted by
water-saturated n-butanol. One product was purified
and its structure was elucidated by spectroscopic analysis of HR-ESI-MS, 1H NMR, 13C NMR, HSQC, 1H-1H
COSY and HMBC and the acid hydrolysis. A fructosyl
was added to the substrate at the position of C6-OH of
26-O-β-D-glucopyranosyl. This study provided a new
way to the glycosylation of steroidal saponins.
Fig. 1 Biotransformation of protodioscin by Arthrobacter sp.
260: Rational design of synthetic pathway in Escherichia
coli for direct N-acetylneuraminic acid production from
glucose
Junhua Kang, Pengfei Gu, Qian Wang, Qingsheng Qi*;
State Key Laboratory of Microbial Technology, National
Glycoengineering Research Center, Shandong University,
Jinan, 250100 P. R. China
* Corresponding author: Tel & Fax: +86-531-88365628;
qiqingsheng@sdu.edu.cn
N-acetylneuraminic acid (NeuAc) recently drew much
attention owing to its applications in many aspects. In
this study, we achieved recombinant Escherichia coli
strains for the production of NeuAc from glucose.
Genes involved in NeuAc synthetic pathway were
overexpressed to construct a basic strain for next genetic manipulation. Mutation and overexpression of glmS
gene improved NeuAc production from 0.12 g l−1 to
0.26 g l−1. Afterward, deletion of GlcNAc degradation
genes in E. coli resulted in 0.30 g l−1 NeuAc accumulation. Genes encoding acetate kinase, pyruvate oxidase
and lactate dehydrogenase were eliminated to block
pyruvate metabolic bypass, which enhanced the metabolic flux to NeuAc synthesis. The engineered E. coli
produced 1.36 g l−1 NeuAc. Finally, nanATEK encoding
enzymes of NeuAc catabolic pathway were eliminated
together, which resulted in 1.62 g l−1 NeuAc production. In a 5 l fermentor study, after 4 days cultivation
7.85 g l−1 NeuAc was obtained. This process offered an
efficient method to produce NeuAc in E. coli with
glucose as carbon source.
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Glycoconj J (2013) 30:281–461
Figure. schematic presentation of NeuAc metabolic pathway in engineerd E. coli. The enzymes and substrates are described
in the context. Dotted line means feedback inhibition. X indicated the genes were knocked out.
Poster Session II- Carbohydrate & Disease
261: Expression of chondroitin sulphate sulphation motifs and SLRPs in articular cartilage of Kashin-Beck
disease
Jinghong Chen1, Wei Wang1, Mingxiu Luo1, Siyuan Li1,
Zengtie Zhang1, Boquan Yu, Clare E Hughes2, Bruce
Caterson2, Junling Cao1*; 1Institute of Endemic Diseases,
Xi’an Jiaotong University College of Medicine; Key Laboratory of Environment and Genes related to Diseases (Xi’an
Jiaotong University), Ministry of Education, Xi’an 710061,
Shaanxi, P.R. China. 2Connective Tissue Biology Laboratories, Division of Pathophysiology and Repair, School of Biosciences, Cardiff University, Cardiff, Wales, UK CF10 3AX
jixiang46@163.com
Objective: To clarify the expression changes of these Proteoglycans incuding aggrecan and SLRPs and association
with the catabolism of the proteoglycans in KBD cartilages.
Methods: Samples of articular cartilage were divided into
2 groups: control children (5 samples from 5 cases), and
children with KBD (5 samples from 5 cases). The
morphology and pathology of hand joint cartilage of
KBD children were examined by light microscopy and
the expression of proteoglycans was determined by histochemical staining. A panel of well-characterized antibodies that recognize glycosaminoglycan or protein
(linear or neoepitope) sequences was used to detect
proteoglycans, such as aggrecan and glycosaminoglycan
or the small leucine-rich proteoglycans (SLRPs) family
proteins biglycan, decorin, karatocan in cartilages using
immunohistochemistry.
Results: There was lesser amounts of toluidine blue staining
and aggrecan in the extracellular matrix of the articular
cartilage of KBD. Similarly, increased positive staining of
2B6 in KBD child cartilages was observed throughout the
depth of cartilage, while increased staining of 3B3(+) from
middle to deep zone of articular cartilag, suggesting that
Glycoconj J (2013) 30:281–461
6-sulphated and 4- sulphated N-acetyl galactosamine residues expression is increased in KBD cartilages. In contrast, There was a reduced intensity of 1B5 and 3B3(+)
staining seen in the deep zone of KBD child cartilages,
suggesting a decrease proteoglycans such as aggrecan or
reduce the 0-sulphation patterns along CS/DS stubs.
Interestingly, there was increased 7D4 and 5D4 staining
from middle to deep zones, suggesting an initiation of
an osteoarthritis-like lesion. Expression of keratocan and
biglycan was prominent increased in the superficial and
upper middle zone of the KBD cartilage samples, while
expression of decorin was decreased which was only
detected in the superficial zone or upper middle zone
of the KBD cartilage samples, indicating additional molecular
pathways that might compromise the integrity of the
collagen matrix.
Conclusion: The study provides a characterization of glycosaminoglycan epitopes associated with the proteoglycans
of cartilage, illustrating disrupt CS-GAG metabolism in
ECM of the articular cartilages in KBD patients. And, the
SLRPs family proteins biglycan, decorin and keratocan may
play a role in the development of KBD, possibly contribute
to cartilage potentially degradation in KBD.
* Joint corresponding authors: Prof. Junling Cao, Email:
caojl@mail.xjtu.edu.cn
262: Sulfatide/miR-223/Sp1/integrin αV Pathway Stimulates Migration of Hepatocellular Carcinoma Cells
Yi Wei Dong, Qianqian Cai, Rong Wang, Xing Zhong
Wu; Department of Biochemistry and Molecular Biology,
Shanghai Medical College, Fudan University, Key Lab of
Glycoconjugate Research, Ministry of Public Health, 138 Yi
Xue Yuan Road, Shanghai 200032, P.R. China;
xz-wu@shmu.edu.cn
Hepatocellular carcinoma (HCC) is a common cause of
cancer-related deaths worldwide, The roles of miR-223
and its regulation in HCC metastasis remain elusive. We
here show distinct down-regulation of miR-223 in cancerous tissues verse matched adjacent non-tumor tissues in 57
patients with HCC. In highly metastatic MHCC97H cells
miR-223 was expressed at a lower level than low metastatic
MHCC97L cells. Ectopic expression of miR-223 had a
negative effect on cell migration. Further experiments identified that Sp1 was a direct target of miR-223.and selectively
stimulated the mRNA synthesis from integrin αV subunit
gene whose promoter contains functional recognition sites
for Sp1. Sustained expression of miR-223 suppressed the
level of Sp1 and integrin αV expression. An inverse
407
correlation between miR-223 and integrin αV was observed
in HCC samples from 57 patients. Intriguingly, expression
of miR-223 was suppressed by sulfatide, leading to Sp1 and
integrin αV expression. The suppression of miR-223 by
sulfatide was associated with reduced histone H3 and
C/EBPα recruitment to the promoter of miR-223 gene. In
vivo tumorigenicity and metastasis assays, miR-223 was
proved to effectively inhibit HCC growth and metastasis in
nude mice. These data support differential expression of
miR-223 in HCC and demonstrate sulfatide/miR223/Sp1/integrin αV pathway in HCC, which might be
broadly employed by the intrinsic cell fate determining cell
motility to promptly stimulate metastasis.
263: Fucoidan targeted to CTGF and inhibited glucose
induced rat messangial cell proliferation
Jiejie Hao1,2, Xin Liu1,2, Xiaoliang Zhao1,2, Guangli
Yu1,2*; 1Key Laboratory of Marine Drugs, Ministry of
Education, Ocean University of China, Qingdao 266003, China, 2Shandong Provincial Key Laboratory of Glycoscience and
Glycotechnology, Ocean University of China, Qingdao
266003, China
glyu@ouc.edu.cn
Fucoidans, which were complex sulfated polysaccharides
extracted from brown algae and some marine invertebrates,
have been extensively investigated because of their various biological activities, e.g. anticoagulant, antiviral,
antitumor and antidiabetes activities, and these activities
differ according to the sulfate content, monosaccharide
composition, glycosidic linkage and molecular mass. In
the present study, we purified 12 fucoidans from different
brown alge and showed that the fucoidan (FV2) from
Fucus vesiculous could specifically interact with connective tissue growth factor (CTGF) by glycoarray technology. And the binding strength of fucoidan to CTGF not
only depended on the backbone structure and positions of
sulfate groups but also the branching sugar residues. We found
that the repeated [→4Fuc2Sα1→3Fuc2S/4Sα1→] backbone
of fucoidan was essential for binding to CTGF, and the binding strength was positively correlated with the molecular
weight (120 to 380kD), and was enhanced by some hybrid
xlyose and galactose residues. Furtherly, the effect of FV2 on
diabetic kidney disease was investigated through mesangial
cell model. Results showed that FV2 could significantly inhibit the high glucose induced HBZY-1 messangial cell proliferation and extracellular matrix (ECM) production. The
secretory levels of collagen IVand fibronectin 1 were distinctly reduced by FV2 as compared with high glucose induced
model group (1.17±0.12 vs. 2.19±0.16, and 1.23±0.15 vs.
2.37±0.21, respectively).
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264: Mechanism study underlying the role of microRNAs
in sulfated polysaccharide WSS25-inhibited angiogenesis
Fei Xiao, Kan Ding *; Glycochemistry & Glycobiology
Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Shanghai 201203,
China; To whom correspondence should be addressed: Kan
Ding, Glycochemistry & Glycobiology Lab, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555
Zu Chong Zhi Road, Shanghai 201203, China, Tel.: +86 21
50806928; Fax: +86 21 50806928;
kding@mail.shcnc.ac.cn
WSS25 is a α-1,4-linked sulfated polysaccharide which inhibits angiogenesis. However, the mechanism underlying
the regulation of angiogenesis by WSS25 is not well understood. Using microRNA (miRNA) microarray analysis, 25
miRNAs are found to be upregulated and 12 downregulated
by WSS25 in human microvascular endothelial cells
(HMEC-1), including microRNA-210 (miR-210) and
microRNA-885-3p (miR-885-3p). Interestingly, Dicer, a
key enzyme for miRNA biosynthesis, is downregulated by
WSS25 in HMEC-1 cells. Further studies indicate that
HMEC-1 cell tube formation and miR-210 are suppressed
while Ephrin-A3 is enhanced by the silencing of Dicer. In
contrast, HMEC-1 cell tube formation and miR-210 are
induced while Ephrin-A3 is suppressed by Dicer
overexpression. Moreover, miR-210 is downregulated while
Ephrin-A3 is upregulated by WSS25 in HMEC-1 cells.
HMEC-1 cell migration and tube formation are arrested,
while Ephrin-A3 expression is augmented by anti-miR210. In addition, HMEC-1 cell tube formation is significantly attenuated or augmented when Ephrin-A3 is
overexpressed or silenced, respectively. Nevertheless, the
tube formation blocked by WSS25 is partially rescued by
manipulation of Dicer, miR-210, and Ephrin-A3. The above
data indicates that WSS25 inhibits angiogenesis via suppression of Dicer, leading to downregulation of miR-210
and upregulation of Ephrin-A3. We also show that miR-8853p dramatically suppresses angiogenesis in vitro and in vivo.
MiR-885-3p inhibits Smad1/5/8 phosphorylation and
downregulates DNA-binding protein inhibitor ID-1 (Id1), a
proangiogenic factor, by targeting bone morphogenetic protein receptor, type IA (BMPR1A), leading to impaired angiogenesis. Overexpression or silencing of BMPR1A affects
angiogenesis in a Smad/Id1-dependent manner. Furthermore, miR-885-3p impairs the growth of HT-29 colon cancer cell xenografts in nude mice by suppressing
angiogenesis through disruption of BMPR1A and Smad/Id1
signaling. These results support a novel role for miR-885-3p
in tumor angiogenesis by targeting BMPR1A, which regulates a proangiogenic factor. In summary, our research provides the first demonstration concerning the role of Dicer
Glycoconj J (2013) 30:281–461
and miRNAs probed by polysaccharide in mediating angiogenesis. These findings add to new evidences that WSS25
not only modulates angiogenesis factors, but also influences
miRNAs function. This study advances our understanding
of the actions of WSS25 in angiogenesis and even provides
further insight into potential WSS25-based new drug development in terms of miRNAs.
265: Stereoselective synthesis and biological evaluation
of 2-deoxy-α-glycosides and oligosaccharides
Guofang Yang, Qingbing Wang, Jiafen Zhou, Xiaosheng
Luo, Jianbo Zhang* and Jie Tang; Department of
Chemistry, East China Normal University, Shanghai
200241, China
jbzhang@chem.ecnu.edu.cn
2-Deoxy glycosides and oligosaccharides has recently received considerable attention because of many natural products composed of 2-deoxysugars are known to exhibit
antimicrobial, anticancer, and antibiotic activities. For example, 2, 6-dideoxy hexopyranoses are common structural
units of antitumor agents or antibiotics, such as
chromomycin (1) and olivomycin (2). The synthesis of 2deoxy glycosides; however, can be difficult to achieve due
to the lack of directing group at the C-2 position and the acid
lability of the resulting 2-deoxy glycosides. Thioglycoside,
activated oxygen derivatives, glycosyl halides, and glycals
have been used as donors in the formation of either α- or β2-deoxyglycosides. These glycosyl donors work well in the
glycosylations with excellent anomeric selectivities in indirect strategy, which involves the introduction of a temporary
directing group such as halides, phenylseleno, and sulfenyl
derivatives at C-2, and the reductive removal of the group
after the glycosylation step has been completed. Obviously,
additional steps are needed and toxic reagents were often
applied in the removal step. Compared with the indirect
strategy, using 2-deoxyglycopyranosyl donors in a direct
way for the formation of 2-deoxyglycosyl linkages would
be more efficient and desirable. Early works in this area
employ glycosyl halides, glycosyl phosphites, and
trichloroacetimidates as donors for the formation of βselective glycosylation in the presence of mild activators.
On the other hand, α-2-deoxyglycosides have been obtained
under acid-catalyzed activation of glycals, anomeric
ethers, anomeric esters, glycosyl halides, thioglycosides
and glycosyl ortho-alkynylbenzoates. Despite a variety of
methods available, the direct synthesis of 2deoxyglycopyranosides from 2-deoxyglycosyl donors,
with high stereoselectivity, still remains a challenge because the above methodologies often demand specified
donors, promotors or harsh reaction conditions. An
Glycoconj J (2013) 30:281–461
efficient and mild method for stereoselective synthesis of
2-deoxy-containing glycosides and oligosaccharides has
been developed in our group. Stable and convenient
donors, 2-deoxy glucosyl and galactosyl acetates were
reacted with phenols, alcohols, thioalcohols and glycosyl
acceptors in the presence of catalytic TMSOTf at 0 °C
with excellent α-selectivity (4:1 to above 19:1) and high
yields (up to 99 %). Trisaccharides were obtained in onepot synthesis. The structure and stereochemistry of all
the glycosidation products were characterized by high
resolution NMR and MS data. Preliminary biological
evaluation revealed some of the oligosaccharides have
good anticancer bioactivity against human cancer cells
lines (K562, A549, MM7721).
The authors gratefully thank financial supports from the
Natural Science Foundation of Shanghai (11ZR1410400).
266: GM3 but not GM2 interaction with tetraspanin CD82
leads to inhibition of EGFR-phoshorylation –dependent
colon cancer cell sw620 motility
Xiaohua Huang1#, 2, Ying Li1#, Jianing Zhang1, Yuefei
Xu1, Yuxiang Tian1, Keli Ma1*; 1 Department of Biochemistry and Molecular Biology, Dalian Medical University,
Dalian 116044, China; 2 Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical
University,Dalian 116044, China
1*
Corresponding author: makeli666@yahoo.com.cn
Tel: +86-411-86110311(O), 86-15998663363(mobile),
Fax: +86-411-86110309
1 #These authors contributed equally to this work
*This work was supported by Grant from the National
Program on Key Basic Research Project 973
It has been documented that the metastasis suppressor
CD82/KAI with gangliosides can exert synergistic inhibitory effect on cell mobility and migration. However, the
precise mechanism has not been fully elucidated. In the
present study, we want to known whether the EGFR signaling is involved in the mechanism by which CD82 and
gangliosides synergistically inhibit the motility and migration of SW620 cells. SW620 cell is colon cancer cells with a
high lymph node metastatic potential and a low expression
of CD82, and a maior ganglioside composition GM2. By
alteration of the ganglioside composition with P4 (D-1threo-1-phenyl-2-palmi-Toylamino-3- pyrro-idino-1propanol, an specific inhibitor of glucosylceramide synthesis), and/or addation of exogenous gangliosides, and/or
overexpression of CD82 by transferring of CD82 cDNA,
we found that (i) GM3 alone suppressed the EGF- stimulated cell motility and migration but GM2 didn’t; CD82 alone
409
dramatically inhibited the EGF- stimulated cell motility and
migration; GM3 can enhance the inhibitory effect of CD82
on cell mobility and migration but GM2 didn’t. These results suggested that, in this cell line, GM3, but not GM2
with CD82 synergistically inhibit the cell motility and
migration.(ii) GM3 alone suppressed the EGF- stimulated
phosphorylation of EGFR at the Tyr845 and Tyr1173;
Overexpression of CD82 alone inhibited the EGF- stimulated phosphorylation of EGFR at the Tyr 1045. Addation of
GM3 as well as overexpression of CD82 inhibited the EGFstimulated phosphorylation of EGFR at the Tyr845, Tyr1173
and Tyr 1045. These results suggested that the mechanism
for GM3 inhibiting cell migration and phosphorylation of
EGFR is different from that of CD82. (iii) GM3 alone
suppressed the EGF- stimulated phosphorylation of Akt at
Ser473 and Thr308; Overexpression of CD82 alone
inhibited the EGF- stimulated phosphorylation of ERK;
these demonsdrated that CD82 combined with GM3 can
reduced EGF- stimulated activity of PI-3K/AKT and ERK
signaling pathway.
Taken together, all the exparimental data suggested that the
synergistic inhibitory effect of GM3 and CD82 on cell
mobility and migration involve the inhibition of phosphorylation of EGFR and the activity of PI-3K/AKT and ERK
signaling pathway.
267: Modular Synthesis Methodology for the Generation
of Heparan Sulfate Oligosaccharides
Ralf Schwörer1, Jeremy E. Turnbull2, Peter C. Tyler1,
Olga V. Zubkova1; 1Carbohydrate Chemistry, Callaghan
Innovation Research Limited, Lower Hutt, 5040, New
Zealand; 2Institute of Integrative Biology,University of
Liverpool, Liverpool, L69 3BX, UK.
ralf.schwoerer@callaghaninnovation.govt.nz
Heparan sulfate (HS) is a linear polysaccharide with highly
diverse functionality. Its disaccharide repeating unit consists
of of D-glucosamine and D-glucuronic acid or L-iduronic
acid and can be variously O- or N-sulfated or N-acetylated.
HS is found as a proteoglycan on cell surfaces and in the
extracellular matrix and mediates many important biological
processes [1,2].
We have developed methodologies for the assembly of
heparan sulfate oligosaccharides. The control of protecting
groups enables selective sulfation of the oligosaccharide
targets, as well as the selective functionalisation of the
amino groups. The key element of our strategy is to establish the synthetically more difficult α-linkage first (85–95 %
yield, α) to generate a set of disaccharide building blocks.
410
The anomeric configuration was confirmed by x-ray crystal
structure analysis.
The use of Fmoc at th e n on- red uc ing a nd pmethoxyphenyl at the reducing end gives rapid access to
donors as well as acceptors for oligosaccharide assembly.
Chain elongation is achieved by glycosidation of disaccharide trichloroacetimidates onto the non-reducing end of
the oligosaccharide chain, again in excellent yields with
high stereoselectivity (78–97 % yield, β). The uronic acid
functionalities are introduced late in the synthesis by
oxidation on the fully assembled oligosaccharide.
Here we report the synthesis of defined hexa- to
dodecasaccharides containing GlcNAc6S-UA or
GlcNAc6S-UA2S disaccharides with the uronic acids
being either D-glucuronic or L-iduronic, both with and
without the uronic acid 2-O-sulfates as it may be beneficial for bioavailability and reduction of off-target
effects to keep down the numbers of sulfate residues.
In vitro activity assays using FRET peptides identified
several lead compounds as effective BACE1 inhibitors
(β-site APP cleaving enzyme 1).
[1] Bishop, J.R. et al., 2007, Nature 446, 1030–1037.
[2] Turnbull, J.et al., 2001, Trends Cell Biol 11, 75–82.
268: Lowered level of core fucosylation is a possible noninvasive predictive marker for chronic obstructive pulmonary disease (COPD)
Congxiao Gao1, 2, Koichiro Kamio3, Fumi Ota1, 2, Takeo
Ishii3, Kozui Kida3, Naoyuki Taniguchi1, 2; 1RIKEN Alliance Laboratory, The Institute of Scientific and Industrial
Research, Osaka University, Japan; 2Disease Glycomics
Team, RIKEN Advanced Science Institute, Japan; 3Dept.
of Internal Medicine, Division of Pulmonary Medicine,
Infectious Diseases and Oncology, and Respiratory Care
Clinic, Nippon Medical School Tokyo, Japan
cxgao@sanken.osaka-u.ac.jp, tani52@wd5.so-net.ne.jp
Deficiency in core fucosylation by the genetic disruption of
α 1, 6- fucosyltransferase (Fut8) leads to lethal abnormalities and the development of emphysematous lesions in the
lung (Ref. 1). The physiological relevance of core
fucosylation in the pathogenesis of COPD was herein investigated using cigarette smoke (CS) exposed heterozygous knockout mice (Fut8+/−)(Ref. 2). A marked decrease
in FUT8 activity and elevated matrix metalloproteinase
(MMP)-9 activities were observed at an early stage of exposure. Emphysema developed after a three-month-CS-
Glycoconj J (2013) 30:281–461
exposure, which is half the time required for wild type mice.
Moreover, we investigated whether reduced Fut8 levels
leads to increased inflammatory response in human COPD
and the disease progression among symptomatic current or
ex-smokers with stable COPD or at risk outpatients (n=226)
(Ref. 3). Although FUT8 activity increased with age among
the at risk patients, this association was not clearly observed
in the case of COPD patients. A faster annual decline of the
forced expiratory volume in 1 s (FEV1) was significantly
associated with lower FUT8 activity. Patients with lower
FUT8 activity experienced exacerbations more frequently.
These data suggest that reduced FUT8 activity is associated
with the progression of COPD and serum FUT8 activity is a
minimally invasive predictive biomarker for progression
and exacerbation of COPD.
Ref. 1 Wang X et al. Proc Natl Acad Sci. USA (1995) 102,
15791–15796
Ref. 2 Gao C et al. J Biol Chem. (2012) 287:16699–16708
Ref. 3 Kamio K et al. Biochem. Biophys. Res. Commun.
(2012), 424, 112–117
269: Expression of β-1, 4 galactosyltransferase V in rat
Schwann cells in vivo and in vitro*
Meijuan Yan, Chun Cheng, Aiguo Shen**; The Jiangsu
Key Laboratory of Neuroregeneration, Nantong University, Nantong, Jiangsu Province 226001, P. R. China
shag@ntu.edu.cn
Glycosylation is one of the most important post-translational
modifications. It is clear that the single step of β-1, 4galactosylation is performed by a family of β-1, 4galactosyltransferases (β-1, 4-GalTs), and that each member
of this family may play a distinct role in different tissues and
cells. In the present study, Real-time PCR revealed that the
β-1, 4-GalT V mRNA reached peaks at 2w after sciatic
nerve crush and 1w after sciatic nerve transection. Combined in situ hybridization for β1, 4-GalT V mRNA and
immunohistochemistry for S-100 showed that β1, 4-GalT V
mRNAs were mainly located in Schwann cells after sciatic
nerve injury. In conclusion, β1, 4-GalT V might play important roles in the regeneration and degeneration of the
injuried sciatic nerve. In other pathology, such as inflammation, we found that LPS administration affects β-1, 4-GalT
*[Acknowledgements] This work was supported by National Natural
Science Foundation of Jiangsu Province(BK2011188), Post-doctoral
Project in Jiangsu Province(1201028C), Doctor Fund of Nantong
University(03080532)
Glycoconj J (2013) 30:281–461
V mRNA expression in sciatic nerve in a time- and dosedependent manner, and β-1, 4-GalT V mRNA expressed
mainly in Schwann cells. These results indicated that β-1, 4GalT-V play an important role in the inflammation reaction induced by intraperitoneal injection of LPS. Similarly, we found that β-1, 4-GalT-V in Schwann cells and
the binding with RCA-I on the Schwann cell surface in
vitro were affected in a time- and concentration dependent manner in response to LPS stimulation, and the
trend of the binding with RCA-I on the cell surface
was similar to the trend of β-1, 4-GalT V. All these
results suggest that β-1, 4-GalT V and Gal β14GlcNAc containing glycan structure plays an important
role in inflammation. In addition, β-1, 4-GalT V production and overall lectin binding were drastically
suppressed by U0126 (ERK inhibitor), SB203580 (p38
inhibitor), or SP600125 (SAPK/JNK inhibitor), which
indicated that Schwann cells regulated expression of β1, 4-GalT V and galactosylation of membrane glycoproteins after LPS stimulation were via ERK, SAPK/JNK,
and P38MAP kinase signal pathway.
411
inhibition of gastric cancer cells by ICC, Western blot,
FACS. We found a significant higher expression of LeY,
CA724 and H. pylori infection copy in gastric cancer tissues
as compared to those in the gastric ulcers and chronic
gastritis (p<0.05). The significant correlation (R-0.86161,
R-0.7420, R-0.7146) between LeY, CA724 and H. pylori
(p<0.01) was shown in the gastric cancer tissues. Furthermore, the decreased expression of LeY and CA724 in antiLeY antibody or celecoxib treated cells was corresponding
to the lower infection of H. pylori and CA724 (p<0.01), and
the cancer cell growth was also inhibited. Conclusion:
Gastric cancer has a significantly positive correlation of H.
pylori infection, LeY and CA724. The study suggests that H.
pylori infection triggered gastric cancer may be more efficiently blocked by the coinhibition of LeY and CA724.
271: Gu-4 improves suvival in experimental sepsis by
inhibiting the release and proinflammatory activity of
HMGB1
HuiTing Zhou1, Qing Li2, ZhongJun Li2, ZhiHui Zhao1;
JiangSu Province Key Laboratory for Molecular and
Medical Biotechnology, College of Life Science, Nanjing
Normal University, Jiangsu Province, China; 2State Key
Laboratory of Natural and Biomimetic of Pharmaceutical
Sciences, Peking University, Beijing, China.
zhaozhihui_1964@yahoo.com.cn
1
270: Helicobacter Pylori Infection Leads to Gastric
Cancer with the coexpression of Lewis Y and CA724
Faisal Aziz1, Xuesong Yang1, Xiaoqi Wang2, Yan Qiu1;
1
Department of Biochemistry and Molecular Biology,
Dalian Medical University, Liaoning Provincial Core Lab
of Glycobiology and Glycoengineering, Dalian 116044,
China; 2Department of Dermatology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611, USA
yanqiu63@126.com
Helicobacter pylori (H. pylori), the gram-negative bacteria
pathogen, is known as a major causative agent for the
induction of chronic gastritis and gastroduodenal ulcer disease as well as gastric adenocarcinoma. This particular risk
factor is ranked as a class I carcinogen by the International
Research Agency on Cancer (IRAC). Although more than
50 % of the world population is colonized by H. pylori, only
a few suffer from active diseases. Lewis Y (LeY) carried by
the glycoproteins and glycolipids on the cell membrane is a
difucosylated oligosaccharide which is highly expressed in
70–90 % of human epithelial cancers. CA724 is a sensitive
and specific carbohydrate antigen of gastric cancer in Chinese population. To explore the potential correlation of H.
pylori infection, LeY and CA724, a total of over 200 gastric
patient's tissues and serums were evaluated by IHC and
ELISA. Gastric primary cell culture from the surgical tissues
of the gastric cancer patients was also developed to study the
effect of anti-LeY antibody and celecoxib drug on the
High mobility group box 1(HMGB1) was first recognized as
a nuclear protein that increases the chromatin remodeling
and regulates transcription of many genes. During the past
two decades, HMGB1 has been identified as an important
“late” proinflammatory mediator due to its unique secretion
pattern and lethal effects in sepsis. Therefore, preventing the
active release and inhibiting the proinflammatory activity of
HMGB1 become alternative ways for the treatment of sepsis. Here,we report the therapeutic effects of Gu-4, a lactosyl
derivative, in sepsis and the underlying mechanism that
related with HMGB1.
On an experimental rat model of sepsis caused by cecal
ligation and puncture (CLP), in vivo treatment of Gu-4
prominently attenuated lung ingjury and improved the
survival of the animals, which was closely related with
the decrease of serum HMGB1 level. Data from our in
vitro experiments showed that lipopolysaccharide (LPS)
induced HMGB1 cytoplasmic translocation and release
in RAW264.7 macrophages was significantly suppressed
by Gu-4 treatment. Moreover, HMGB1-induced production of proinflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-
412
1β), was significantly decreased by Gu-4 treatment.
Further studies by flow cytometry and Western blot
analysis demonstrated that Gu-4 could effectively inhibit
HMGB1-stimulated activation of CD11b and NF-κB
signaling pathways.
Taken together, our results suggested that Gu-4 could exert
therapeutic effects in experimental sepsis model, and the
underlying mechanisms of which might lie in: 1) Gu-4
inhibits LPS-induced HMGB1 release; 2) Gu-4 suppresses
proinflammatory activity of HMGB1.
272: Effect of dietary Chitooligosaccharides on the Reproduction and Embryo development in the mouse
Lun X. Wang1, Yu G. Du1, *; 1Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian 116023, PR
China.
[Objective] The effect of Chitooligosaccharides (COS)
on the reproductive capacity of animals has not been
reported yet. The current study was to test the effect of
dietary COS on the litter size and embryonic development in the mouse. [Method] The Kunming female and
male mouse at 1 month after birth were fed with or
without COS in the diet, which included COS group
(300 mg/kg), SPM group (100 mg/kg COS, 100 mg/kg
Jerusalem artichoke powder, and 100 mg/kg Polysaccharide yeast),and Control group (basal diet). The female
was caged with male mice (1:1) when they were sexually mature. Litter size in successive two generations
and embryonic development including total number of
embryos and litter weight were recorded. [Result] In the
first parity, both SPM supplement and COS supplement
improved (P<0.05) total litter size compared with that
in Control group (13.40 ±3.00/13.50 ±1.83/11.33 ±3.11,
SPM group/COS group/Control group). In the second
parity, SPM supplement significantly improved total litter size compared with that in Control group (13.75±
2.45/11.56±2.96). COS supplement improved one more
litter compared with Control group (12.56±1.51/11.56±
2.96), although there was no significant difference between them. In addition, COS supplement improved
(P < 0.05) both total numbers of embryos (15.83 ±
1.94/11.71 ± 0.95) and total numbers of live embryos
(15.00±1.67/10.86±1.35) compared with Control group.
COS supplement improved (P< 0.05) both litter fetus
weight (19.33 ± 2.43/14.94 ± 2.39) and litter placental
weight(1.79 ± 0.33/1.36 ± 0.19) compared with Control
group. [Conclusion] These results indicated that dietary
COS can markedly improve the reproductive capacity
and embryo development in the mouse.
Glycoconj J (2013) 30:281–461
273: The elevated expression of chondroitin sulphated
proteoglycans in human hepatocellular carcinoma
Xiaoli Jia 1 , Shuangsuo Dang 1 , Siyuan Li 2 , Bruce
Caterson3, Clare E. Hughes3 & Junling Cao4; 1The
Department of Infectious Diseases, 2The Department of
Anaesthesia, The Second Hospital of Xi’an Jiaotong
University, Xi’an, 710004, China; 3The Department of
Pathophysiology and Repair, School of Biosciences, Cardiff
University, CF10 3AX, UK; 4The Institute of Endemic
Diseases, Medical School of Xi’an Jiaotong University,
Xi’an, Shaanxi, 710061,China
dang212@126.com
Introduction: In previous studies we found that there
was an increase in chondroitin sulphated/dermatan
sulphated glycosaminoglycan (CS-GAG) content in rat
hepatocellular carcinoma (HCC) tissue, which is associated with higher CS proteoglycan (PG) expression in the
tumor [Jia et al. World J Gastroenterol. 2012, 18(30):
3962–76]. However, the alteration of specific CSPG contents in human HCC is still largely unknown. In this
study, we investigated the expression of CSPGs in human
HCC tissues.
Materials & Methods: Twelve HCC and five normal adult
human liver tissues were collected. Histological and immunohistochemical staining was used to investigate the expression and distribution patterns of CS/DS GAG and different
CSPGs including aggrecan, versican, biglycan and decorin
in the tissues. PCR and Western Blotting were used to
measure the gene and protein levels of these CSPG members in the control and tumor tissues.
Results: Toluidine blue staining indicated a higher
sulphated GAG content in the HCC tissues when compared
with the normal liver tissues, suggesting higher PG expression in the tumor. Our further immunohistochemical
staining results showed an elevated CSPG expression including aggrecan, versican, biglycan and decorin in the
HCC tissues when compared with the control group, although their positive staining patterns were different. Interestingly, there was no positive staining for lumican and
keratocan in liver tissues from both HCC and control
groups. Quantitative PCR and Western blotting results demonstrated that there was a significant increase in aggrecan,
versican, biglycan and decorin gene and protein expression
in HCC tissues when compared with the normal liver tissues
(p≤0.05).
Conclusion: HCC liver tissues showed an altered expression and distribution patterns in these CSPGs in
Glycoconj J (2013) 30:281–461
comparison with the normal liver tissues. There was an
elevated expression in CS/DS GAG contents in HCC
tissues, which was associated with higher CSPG levels
in the tumor. These results demonstrate that CSPGs play
an important role in the onset and progression of HCC,
and may provide potential therapeutic targets and clinical
biomarkers for this commonly prevalent tumor in humans.
274: Binding Specificity of the H1N1 Swine-Origin Influenza A Virus (S-OIV)
Laura Neumann 1 , Florian Krammer 2 , Miranda de
Graaf 3 , Cornelis H. Hokke 4 , Friedrich Altmann 1 ;
1
University of Natural Resources and Life Sciences, Vienna,
1190, Austria; 2Icahn School of Medicine at Mount Sinai,
New York, 10029, USA; 3ErasmusMC, Rotterdam, 3015GE,
The Netherlands; 4Leids Universitair Medisch Centrum
(LUMC), Leiden, 2333ZA, The Netherlands
Friedrich.Altmann@boku.ac.at
Influenza A viruses are known to attack their hosts by
an initial binding to a glycoconjugate containing receptor. It is widely known that the crucial element of the
glycoconjugate for Influenza A virus binding is Nacetylneuraminic acid (sialic acid). Depending on the
species origin hemagglutinins differ in their prevalence
for sialic acids in different linkages (e.g. á2,6-linkage
found in the human nasopharyngeal tract, á2,3-linked
sialic acid found in avian tissues and á2,6- as well as
á2,3-linked sialic acid in porcine tissues).
In this study we investigated the binding specificity of
the recombinantly expressed H1 hemagglutinin from
the H1N1 Influenz A virus (California/04/09) and several other hemagglutinins as controls to N-glycans isolated from mammalian tissue of the nasopharyngeal
tract.
The H1 hemagglutinin (A/California/04/09) and as a control
several other hemagglutinins, originating from different pandemic viruses, were recombinantly expressed in insect cells
to obtain a soluble functionally active trimer.
To investigate the binding specificity we chose two different
approaches.
In the first set of experiments N-glycans from different tissues
were isolated, labeled with anthranilic acid or 2aminobenzamide for fluorescent detection, separated by a
multidimensional HPLC approach and printed on NHSactivated glass slides to produce glycan arrays. Theses glycan
413
arrays were used to investigate distinct binding of the different
hemagglutinins originating from several pandemic virus strains.
Data was analysed with the free software Multi Experiment
Viewer (MeV). Cluster analysis showed distinct binding
patterns of different viruses according to their sialic acid
linkage prevalence.
In a second approach affinity chromatography was
employed to investigate retention of glycans to immobilized
hemagglutinins. To this end, therefore we coupled the
recombinant protein to NHS-activated sepharose and
monitored retention of fluorescently labelled glycans
by fluorescent detection, fractionation and subsequent
Matrix Assisted Laser Desorption Ionisation-Time of
Flight-Mass spectrometry (MALDI-TOF-MS) analysis.
275: Consistent Gene Regulations of Toll-Like Receptors
in Cultured Cancer Cells by Carbohydrate-Specific
RP215 Monoclonal Antibody and Anti-Antigen Receptors
Yiting Tang1, Hao Zhang1, Gregory Lee1,2; 1UBC Center
for Reproductive Health, University of British Columbia,
Vancouver, V6H 3 N1, Canada; 2Department of Pathology,
Shantou University Medical College, Shantou, 515041, China
cyglee@yahoo.com
RP215 is a monoclonal antibody generated against a
carbohydrate-associated epitope of glycoproteins designated
as CA215 which consist mainly of immunoglobulin superfamily proteins expressed by cancer cells. Since
RP215 was shown to induce apoptosis and inhibit tumor
growth in nude mouse models, effects of RP215 and
antibodies against immunoglobulins as well as T-cell receptors (anti-antigen receptors) were investigated on the
gene regulations of cultured ovarian as well as cervical
cancer cells. By using semi-quantitative RT-PCR,
changes in expressions of a number of selected genes
involved in proliferation, protein synthesis, cell cycle
regulations as well as the innate immunity were analyzed upon
various antibody treatments of cultured cancer cells. RP215
and anti-antigen receptors were found to regulate similarly a
number of genes (n=12) including NFκB-1, IgG, P21, Cyclin
D1, ribosomal P1 and c-fos. Among toll-like receptor genes
(TLR-2, -3, -4, -6, -7 and -9), the expressions of TLR-3, TLR4 and TLR-9 were highly detected. RP215 and anti-antigen
receptors were found to up-regulate TLR-3, whereas
those of TLR-4 and TLR-9 were down-regulated. Based
on these observations, it can be concluded that apoptosis of cultured cancer cells was induced similarly by
either RP215, anti-human IgG or anti-T cell receptors
through the regulations of the same set of genes with
414
Glycoconj J (2013) 30:281–461
few exceptions. The innate immunity of cancer cells can
also be affected by RP215, anti-human IgG or anti-T
cell receptors through the unidirectional mediations of
certain toll-like receptors. Therefore, the anti-cancer
therapy of RP215 Mab is in part, directly related to
surface bound immunoglobulins and T-cell receptors,
the expressions of which may be involved in the growth
and proliferation of cancer cells. The linkage relationship of toll-like receptors in gene regulations of cancerous immunoglobulins as well as NFκB-1 transcription
factor and others was first demonstrated in this study.
In view of the fact that fungi and bacterial infections are
becoming more common and these species are more resistant to the treatment, we have made an attempt to find new
substances active against those pathogens. Synthesized by
us glycosides consists of diosgenin and D-glucosamine residue. The carbohydrate chain constitutes a hydrophilic part,
while appropriate sapogenin is a hydrophobic fragment in
this kind of glycosides. Our synthetic strategy is based on
the preparation of glycosyl donors, coupling of the respective donors with diosgenin, deprotection of the NH2 and
OH groups and finally obtaining of N-acyl and ureido
derivatives (Fig. 1).
276: Synthesis of N-acyl and Ureido Derivatives of
Diosgenyl Glycoside with Potent Antifungal and
Antibacterial Activity
In biological set of experiments we have investigated the
antibacterial and antifungal effect of some N-acyl and
ureido derivatives. Minimum inhibitory concentration
(MIC) was determined for reference strains of the following bacteria: B. subtilis ATCC 6633, E. faecalis
ATCC 29212, R. equi ATCC 6939, S. aureus ATCC
25923, S. epidermidis PCM 2118, Escherichia coli ATCC
25922, K. pneumoniae ATCC 700603, P. mirabilis PCM
543, P. vulgaris PCM 2668, P. aeruginosa ATCC 9027
and the following fungi: C. albicans ATCC 10231, C.
tropicalis PCM 2681, C. lipolytica PCM 2680. Synthesized saponins exhibit various degrees of activity against
fungi and bacteria, e.g. some of them turned out active
against Candida species.
Daria GRZYWACZ1, Henryk MYSZKA1, Malgorzata
DAWGUL2, Wojciech KAMYSZ2; 1Faculty of Chemistry,
University of Gdansk, Gdansk, 80-952, Poland; 2Faculty of
Pharmacy, Medical University of Gdansk, Gdamsk, 80-416,
Poland
E-mail: plaskiew@chem.univ.gda.pl
Saponins are natural glycosides which possess a wide range of
pharmacological properties. They are used in traditional medicine as an antidiabetes and antihyperglycemia agents, medical material to treat malaria, helminthes infections and snake
bites. Some of diosgenyl glycosides exhibit a wide spectrum
of biological activities including antifungal, antibacterial and
anticancer properties.
Fig. 1.
This research was part-financed by the European Union
within the European Regional Development Fund - grant
UDA-POIG.01.01.02-14-102/09-03.
Glycoconj J (2013) 30:281–461
277: Conformational Studies of the Furanosides, Important Components of Bacterial Glycans
Dominik Walczak 1 , Daria Grzywacz 1 , Andrzej
Nowacki1, Beata Liberek1; 1Facult of Chemistry, University of Gdańsk, Gdansk, 80-952, Poland
domelek@chem.univ.gda.pl
It is difficult to overstate the importance of the furanose ring
in biology. These moieties are found as constituents of
nucleic acids, bacterial, parasitic, and fungal cell wall polysaccharides, as well as other natural products. Importantly,
oligosaccharides involving furanosyl constituents are present in various microorganisms whereas these are absent in
the mammals glycans. This fact suggests that the enzymes
involved in the metabolism of such sugars in bacteria, fungi
and protozoa would constitute a good target for the design
of new drugs.
The conformational preferences of oligosaccharides composed
of furanose moieties are not well understood. This is due both
to a lack of experimental data on oligofuranosides and the
415
significant flexibility of the five-membered ring. To understand
the conformational preferences of oligofuranosides it is
necessary to well understand the conformational preferences of the single furanose ring. By understanding
the conformational preferences of these smaller components, it is hoped that an understanding of the secondary
and tertiary structure of the large biomolecules will also
be attained.
Conformations of furanosides in solution are hard identified
by NMR techniques because these are equilibrating rapidly
and averaging of coupling constants occurs. However, when
a furanoid ring is conformationally restricted, e.g. by a rigid
second skeleton, it is possible to predict its conformation.
Therefore, to study conformations of a furanoid ring we
synthesized series of furanoses and furanosides having bicyclic structures with the five-membered rings fused at
carbons C3-C4, C2-C3, and C1-C2, respectively (Fig. 1).
The NMR studies of the synthesized compounds point that
spectra of each groups of furanoses and furanosides are very
characteristic and indicative of one specific conformation or
configuration (Fig. 1).
Fig. 1
278: The Inhibitory Effect of Marine Oligosaccharide
Sulfate OMS against Influenza a H1N1 Virus
Wei Wang1,2, Shi-Xin Wang1, Chun-Xia Li1,2, Guang-Li
Yu1,2, Hua-Shi Guan1,2; 1 Key Laboratory of Marine
Drugs, Ministry of Education, Ocean University of China,
Qingdao 266003, PR China; 2 Shandong Provincial Key
Laboratory of Glycoscience & Glycoengineering, Ocean
University of China, Qingdao 266003, PR China
wwwakin@ouc.edu.cn
Marine oligosaccharide sulfate OMS is a low molecular
weight compound which obtained from alginate polysaccharide by acid degradation, fractionation and sulfated modification. The results of the cytopathic effect (CPE) inhibition
assay and MTT assay showed that OMS with the average
molecular weight of 3~5 KD could significantly inhibit the
replication of influenza A H1N1 virus in MDCK cells. The
cytotoxicity of OMS is very low, and the selectivity index (SI)
of OMS in vitro is more than 15.0. Moreover, OMS could
obviously inhibit the activity of influenza A H1N1 virus
neuraminidase, and the 50 % inhibition concentration (IC50)
is less than 50 μg/ml. Furthermore, OMS could significantly
alleviate the lung inflammation of BALB/C mice caused by
the infection of influenza A virus (A/PR/8/34) at the dose of
40 mg/kg/day, and the inhibition rate of lung index was more
than 33.9 %, which is comparable with the effect of positive
drug oseltamivir phosphate. Compared to the model group,
OMS could obviously decrease the lung viral load in virus
infected mice (P<0.01), reduce the death rate of mice (death
prevention rate >40 %), and prolong the survival time of mice.
OMS could also enhance the production of interferon-γ in
spleen 4 days post infection. In conclusion, the low molecular
weight compound OMS can significantly inhibit the activity
of influenza A virus neuraminidase and possess good antiH1N1 virus effects in vitro and in vivo, which suggest that this
compound merits further investigation as a potential antiinfluenza A virus drug in the future.
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Glycoconj J (2013) 30:281–461
279: Biological Activity of Diosgenyl 2-Amino-2Deoxy-β-D-Glucopyranoside Hydrochloride and its
N,N-Dialkyl Derivatives
ATCC 10231, Candida tropicalis PCM 2681, Candida
lipolytica PCM 2680, Aspergillus niger ATCC 16404)
was studied.
Henryk Myszka 1 , Daria Grzywacz 1 , Malgorzata
Dawgul2, Wojciech Kamysz2; 1Faculty of Chemistry, University of Gdansk, Gdansk, 80-952, Poland; 2Faculty of
Pharmacy, Medical University of Gdansk, Gdansk, 80-416,
Poland,
myszka@chem.univ.gda.pl
Gram negative bacteria turned out to be resistant to the
compounds at tested concentrations (0.5−1,024 mg/L),
while the growth of gram positive strains and fungi was
inhibited at concentrations 0.5−128 mg/L. Strains of Candida sp. were the most susceptible to saponins. Therefore
MIC assay was performed for clinical isolates (C. glabrata,
C. krusei, C. tropicalis, C. parapsilosis). The test was
carried out also for conventional antifungal agents
(amphotericin B, clotrimazole, fluconazole, itraconazole,
natamycin, nystatin).
Diosgenyl glycosides are steroid saponins isolated from
a variety of plants, for example Costus, Dioscorea,
Paris, Solanum, Trillium, Yucca. Some of them exhibit
a wide spectrum of biological activities including antifungal, antibacterial and anticancer properties. The carbohydrate residue is covalently attached to the
diosgenin backbone. Usually, in natural diosgenyl glycosides the first sugar connected to diosgenin is β-Dglucopyranose.
We have synthesized a diosgenyl glycosides containing Dglucosamine derivatives as a carbohydrate residue. Some of
them were tested for their antifungal and antibacterial
activity. (Fig. 1)
Antimicrobial activity against reference strains of bacteria and fungi (Bacillus subtilis ATCC 6633, Enterococcus faecalis ATCC 29212, Rhodococcus equi ATCC
6939, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis PCM 2118, Escherichia coli ATCC
25922, Klebsiella pneumoniae ATCC 700603, Proteus
mirabilis PCM 543 Proteus vulgaris PCM 2668 i Pseudomonas aeruginosa ATCC 9027; Candida albicans
Fig. 1
Among clinical strains of C. krusei and C. tropicalis we
have identified numerous isolates resistant to tested compounds at applied concentrations (0.025−512 μg/mL). The
saponins presented very strong activity towards clinical isolates of C. glabrata and C. parapsilosis comparable or
stronger than conventional antimicrobials.
Afterwards the most active compounds were tested
according to their hemolytic activity. Obtained glycosides
were did not exhibit hemolytic activity towards human
erythrocytes while applied at their microbiogically active
concentrations. Results of presented study suggest potential application of saponins as future antifungal
agents.
Part-financed by the European Union within the European
Regional Development Fund—UDA-POIG.01.01.02-14102/09-03.
Glycoconj J (2013) 30:281–461
280: Synthesis of Phosphono and Phosphate Derivatives
of Hydroxyimino-D-alditols as New Potential Antifungal
Agents
Magdalena Cyman 1 , Henryk Myszka 1 , Karolina
Kwiatkowska2, Slawomir Milewski2, Beata LIBEREK1;
1
Faculty of Chemistry, University of Gdansk, Sobieskiego 18,
80-952 Gdansk, Poland; 2Department of Pharmaceutical Technology and Biochemistry, Gdansk University of Technology,
Narutowicza 11/12, 80-233 Gdansk, Poland
cyman@chem.univ.gda.pl
In search for new effective antifungals we focus on two enzymes involved in biosynthesis of the fungal cell wall. The first
enzyme is glucosamine-6-phosphate synthase (GlmS), which
catalyzes transformation of D-fructose-6-phosphate (Fru-6P)
to D-glucosamine-6-phosphate (GlcN-6P) in the chitin biosynthesis pathway. The second enzyme is phosphomannose isomerase (PMI) reported to play a crucial role in biosynthesis of
many mannosylated structures, including cell wall components
of fungi. PMI is aldose-ketose isomerase and catalyzes
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reversible isomerization of D-manno-6-phosphate (Man-6P)
to D-fructose-6-phosphate (Fru-6P). Both enzymes are proposed as the targets for antifungal chemotherapy and a search
for their selective inhibitors is continued.
Mechanisms of the reactions catalyzed by both enzymes are
known and similar. The reaction performed by GlmS is
believed to proceed through the formation of an imine
intermediate 1, whereas the reaction catalyzed by PMI proceeds via a cis-endiol intermediate 2 (Fig.1).
In search of mimetics of intermediates 1 and 2 we synthesize
phosphono and phosphate derivatives of the hydroxyiminoD-glucitols (3a-c–6a-c). Similarity in the structures of the
planed compounds to intermediates 1 and 2 allow us to
assume that they can be the potential inhibitors of the both
enzymes. Dimethyl and diethyl ester analogs (3b,c–6b,c) will
have more lipophilic character, which make them easier to
penetrate through the cytoplasmic cell membrane. It was
proved that similar esters are hydrolyzed inside a cell. Here,
the completed stages of our syntheses are presented.
Fig.1
281: Convergent Approach to Glycoalkaloids: Syntheses
of Solasonine and 25(R)-Solanine
Ming Li*, Yannan Peng, Pengwen Chen, Peng Wang;
Key Laboratory of Marine Medicine, Education Ministry of
China, School of Medicine and Pharmacy, Ocean University
of China, Qingdao, 266003, Shandong, China
lmsnouc@ouc.edu.cn
Steriodal glycoalkaloids are rich in Solanaceae family including important agricutural crop plants such as potato,
tomato and eggplant. According to the aglycon skeleton,
they are classified into two basic types: the spirosolans and
solanidans, which contain oxa-azaspirodecane and
indolizidine subunit, respectively. The studies have shown
that glycoalkaloids display various biological activities such
as antitumor, antiheptatoxicity and molluscicide. A cream
containing solansodine glycosides mainly composed by
solamargine and solasonine has been marketed as
“Curaderm” for solar keratosis. So far the syntheses of
glycoalkaloids have been sporatically documented. Herein,
we take solasonine and 25(R)-solanine as a representative of
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spirosolan and solanidan glycosides, respectively, to illustrate our convergent syntheses of glycoalkaloids.
Our approaches to solasonine and 25(R)-solanine constitute
preparation of aglycons of solansodine and 25(R)solanidine, construction of solatriosyl imidate and their
stereselective couplings followed by removal of protective
groups. Solansodine and 25(R)-solanidine were achieved in
6 and 11 steps, respectively, by using the common intermediate of 3-O-tert-butyldiphenylsilyl-16-O-acetyl-26-azido22-oxo-cholestane derived from the industrial diosgenin.
The key steps involved E, F-ring openings of diosgenin
and highly efficient reduction of azide by catalytic
hydrogenolysis as well as reductive aminations. Acyl
protected solatriosyl imidate was prepared from Dgalactose, D-glucose and L-rhamnose following protection,
deprotection and functional group manipulations as usual.
Due to solatriosyl imidate bearing 1,2-linkage at the reducing end, thereby devoid of neighboring group assistance
during glycosylation, its 1,2-trans stereselective couplings
with aglycons are challenging. After extensive screenings of
reaction conditions, the glycosylations of imidate with aglycons were realized under the promotion of HB(C6F5)4 in a
mixed solvent of PhCF3/t-BuCN/CH2Cl2 and affored the desired glycoalkaloids with high efficiency and stereoselectivity,
which were globally deprotected under Zemplén conditions to
lead to solansonine and 25(R)-solanine. The synthetic
glycoalkaloids were characterized by 1D and 2D NMR spectroscopy and the data are fully indentical with the reported.
The developed method might be applied to the syntheses of
other active glycoalkaloids and their analogues, which should
facilitate in-depth investigations on structure-activity relationship of glycoalkaloids and their mode of action in detail.
Financial support from National Science Funding of China
(21002095) is greatly appreciated.
282: Pectic polysaccharides from Apocynum venetum L.
promote proliferation of beta-cells
Chengxin Sun, Xinzhi Li, Yuying Fan, Yifa Zhou; School
of Life Sciences, Northeast Normal University, Changchun,
130024, China
zhouyf383@nenu.edu.cn
Diabetes mellitus is a chronic and insidious disease affecting
much more people in the world. The blood glucose of
patients could be controlled in a normal level under the
treatment of commercial agents. However, all the hypoglycemic drugs have shown some side effects. It is reported
that, due to oxidative stress, beta-cell dysfunction or reduction occurs both in type 1 and type 2 diabetes mellitus. Thus,
Glycoconj J (2013) 30:281–461
it is more worthy of finding new hypoglycemic agents
which can improve or promote the proliferation of betacells, especially with the natural original. Polysaccharides
are well documented to have antioxidant capacities. We
have scanned 10 kinds of polysaccharides from different
hypoglycemic herbs. A water-soluble polysaccharide
(ALP) was extracted from the leaves of Apocynum venetum.
ALP could specifically promote the pancreatic beta-cells
proliferation (INS-1 and RIN-m5F), but no effect on human
colon carcinoma cells (HT29 and HCT116). Afterwards,
ALP was further fractionated into ALPAP and other two
fractions, using a combination of ion-exchange and gel
filtration chromatography. Beta-cells proliferation assay
showed that ALPAP presented stronger promotion than the
other two fractions. HPLC analysis results indicated that
ALPAP was homogenous with molecular weight of
18 kDa. The structural features of ALPAP were elucidated
by periodate oxidation, partial acid hydrolysis, IR spectra
and 13C NMR spectroscopy. The results indicated that
ALPAP is a homogalacturonan (HG) -rich pectin with
RG-I domain. ALPAP stimulated beta-cells growth in a
bell-shaped dose–response curve from 50 μg/ml to
5 mg/ml. After 1.0 mg/ml of ALPAP treatment for
48 h, the cell proliferation rate could reach up to
70 % compared with control. Flow cytometry data
showed that ALPAP could elevate mitochondrial membrane potential of beta-cells, but no influence on cell
cycle. All the results indicated that HG-rich pectic
ALPAP was the main active fraction to stimulate betacells proliferation, which is probably mediated through
enhancement on mitochondrial function.
283: Intracellular Localization and Phosphorylation of
Galectin-4 is Controlled by Src Family Kinases
Hiroko Ideo1, Ikue Hoshi1, Katsuko Yamashita1, and
Masaru Sakamoto; 1Mol. Biosci .of Medi, Sasaki Institute;
2
Dept. of Gynecology, Kyoundo hospital, Sasaki foundation, Tokyo, 101-0062, Japan.
ideo@po.kyoundo.jp
Galectins are members of a family of lectins defined by
their affinity for β-galactoside–containing saccharides and
are involved in regulating diverse biological phenomena .
Galectin-4 is expressed abundantly in the epithelium of
the alimentary tract and has 2 carbohydrate-recognition
domains. Galectin-4 is a cytosolic protein that lacks a
signal sequence but is externalized and binds to 3-Osulfated glycoconjugates extracellularly. There is abundant evidence for the extracellular roles played by
galectins as modulators of cell adhesion and signaling,
although the secretion mechanism is not yet understood.
Galectin-4 is also localized on the cell surface and binds
Glycoconj J (2013) 30:281–461
to sulfated glycosphingolipids and CEA, thereby modulating the adhesion of the cells. However, the mechanism
of subcellular localization and externalization of galectin4 has not yet been determined. It has been reported that
galectin-4 recruits apical glycoproteins, including MUC-1,
CEA, etc., in DRMs, because these glycoproteins are
depleted in DRMs isolated from galectin-4-knockdown
HT-29 5M12 cells. This implies that galectin-4 plays an
important role in trafficking by functioning as the carrier
of these proteins.
A preliminary experiment using pervanadate (PV)
showed that galectin-4 is tyrosine-phosphorylated in
cells and suggested that Src kinases are involved. Cell
transfection with galectin-4 and active Src plasmids
showed that galectin-4 can be tyrosine phosphorylated
by members of the Src kinase family. The C-terminal
peptide YVQI of galectin-4 was found to play an important role in its tyrosine phosphorylation, and the SH2
domains of Src and SHP2 were found to bind to this
peptide. Immunofluorescence analysis showed that the
sub-membrane area of Src-activated cells was intensely
stained by anti-galectin-4, indicating that Src kinase
activity is important for subcellular localization of
galectin-4. Furthermore, MUC1 derived from NUGC-4
cells was observed to bind to galectin-4, and externalization of the bound molecules from the cell to the
medium increased in the hyperphosphorylated condition.
These results suggest that localization of galectin-4 can
be regulated by signaling molecules and that it may
function intracellularly as an adaptor protein serving to
modulate the trafficking of glycoproteins
284: Epigenetic modulation of HeLa cell membrane
N-glycome by epigenetic inhibitors and reversibility
of inhibition effects in a drug-free environment
Tomislav Horvat1, Darko Barišić1, Martina Deželjin3,
Ana Mužinić4, Maja Herak Bosnar3, Gordan, Lauc2,4,
Vlatka Zoldoš1; 1 University of Zagreb, Faculty of Science,
Horvatovac 102a, Zagreb, Croatia; 2 University of Zagreb,
Faculty of Pharmacy and Biochemistry, Zagreb, Croatia; 3
Rudjer Boskovic Institute, Division of Molecular Medicine,
Bijenicka 54, Zagreb, Croatia; 4 Genos Ltd, Glycobiology
Laboratory, Planinska 1, Zagreb, Croatia
vzoldos@biol.pmf.hr
Deregulation of glycosylation occurs in a wide range of multifactorial diseases and aberrant glyco-phenotypes have been
associated with specific pathophysiological states. Glycogenes are one of the groups of cancer-associated genes since
changes in glycan structures are hallmarks of many cancers.
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Cancer-specific changes in glycan biosynthetic pathways are
resulting from aberrant expressions of glycosyltransferases
and glycosidases, which is often a result of epigenetic changes
including DNA methylation and/or histone modifications
(acetylation, methylation or phosphorylation). The aberrant
gene expression pattern can be potentially restored using
various epigenetic inhibitors. To test potential therapeutic
usefulness of DNA methylation inhibitors, zebularine and 5aza-2-deoxycytidine, and histone deacetylation inhibitors,
trichostatin A and Na-butyrate, in inducing a reversal of
undesired glyco-phenotypes, we developed an HPLC- based
method for the analysis of glycan structures from HeLa cells
embedded in polyacrylamide gels. In addition, we specifically
investigated the preservation of altered glycan profiles over a
prolonged period of time in a drug-free environment. Our
results emphasize the importance of epigenetic control in the
regulation of N-glycosylation, but also suggest the stability of
complex biosynthetic pathways responsible for the establishment of glycan profiles in human cells in culture.
285: Dissecting the impact of O-GlcNAc modification on
insulin action using different OGA inhibitors
Chin Fen Teo* and Lance Wells; Complex Carbohydrate
Research Center and the Department of Biochemistry and
Molecular Biology, the University of Georgia. 315
Riverbend Road, Athens, GA 30602, USA.
cfteo@uga.edu
O-GlcNAc modification is a ubiquitous and reversible glycosylation found on intracellular proteins. The spatial and temporal presence of O-GlcNAc is orchestrated by a pair of
cycling enzymes, O-GlcNAc transferase (OGT) and β-Nacetylglucosaminidase (OGA), in response to a variety of
cellular and environmental stimuli. Given that UDPGlcNAc, the end product of the hexosamine biosynthetic
pathway (HBP), is an obligatory donor substrate of OGT, OGlcNAc is posed as an effector of excessive glucose flux
through the HBP which in turn can lead to the development
of insulin resistance, a hallmark of type 2 diabetes. Toward
this end, previous studies have utilized PUGNAc, the first
reported OGA inhibitor, albeit that can inhibit lysosomal
hexosaminidases as well, to increase global O-GlcNAc levels
and observed a correlation of elevated O-GlcNAc with the
development of insulin resistance. This notion has been recently challenged by results from the Vocadlo’s group, in
which elevated global O-GlcNAc levels using a more selective OGA inhibitor, NButGT, did not lead to insulin resistance
in cell culture or in vivo. In this study, we evaluated the impact
of the O-GlcNAc modification on the pro-survival action of
insulin under serum-deprivation induced apoptosis using three
different OGA inhibitors, GlcNAcstatin-g (GNSg, another
420
OGA selective inhibitor), thiamet-G (TMG, a derivative of
NButGT), and PUGNAc. We found that while PUGNAc inhibits the protective action of insulin, neither GNSg nor TMG
negates the anti-apoptotic action of insulin. To address
whether inhibition of the lysosomal hexosaminidase activity
via PUGNAc, which would elevate global GM2 ganglioside
levels, leads to its unique characteristic in blocking insulin
action, we also examined the pro-survival role of insulin in the
presence of a selective lysosomal hexosaminidase inhibitor,
INJ2. We established that neither INJ2 alone nor the combination of OGA selective inhibitors with INJ2 mimic the
inhibitory effect of PUGNAc. These results strongly suggest
that the defect in insulin action upon PUGNAc treatment does
not derive from its inhibition of OGA or lysososomal hexosaminidases, and that there is a third, yet unknown, target of
PUGNAc that is the likely culprit in inhibiting the protective
effect of insulin from apoptosis.
286: Structure and Immunological Activities of
Mannogalactoglucans from Lentinus edodes Fruiting
Bodies
Iteku B. Jeff1,2, Tao Zhang1, Yifa Zhou1; 1 School of
Life Sciences, Northeast Normal University, 130024,
Changchun, PR China; 2 Department of Biology, Kinshasa
University, P.O. Box 190 Kinshasa, Democratic Republic
of Congo
jeffitekubekomo@gmail.com (Iteku B. Jeff), zhouyf383@
nenu.edu.cn (Yifa Zhou)
Lentinusedodes importance is attributed to both its nutritional value and medical application. Polysaccharides are the
most important bioactive components in Lentinusedodes,
with data from studies often focus on (1→3)-β-Dglucans.To investigate total active components of
Lentinusedodes, we extracted L. edodeswith cold and hot
water as well as alkali to obtain the total polysaccharides.
Among them, besides (1→3)-β-D-glucans and(1→6)-β-Dglucans, there are a series of mannogalactoglucans.
Themannogalactoglucanswere obtained from WPLE (the
hot water extracted polysaccharide), after freeze-thawing process and fractionation by size-exclusion chromatography into
fourheteroglucansfractions: S-WPLE-II-a, S-WPLE-II-b, SWPLE-III-a and S-WPLE-III-b,with Mw (7.5–2,000 kDa).
Chemical and spectroscopic, including 2D NMR, studies
indicated that themannogalactoglucanscontained(1→6)-,
(1→4)- and (1→3)-Glcp, (1→6)-Galp, (1→3,6)- and
(1→2,4)-Manp residues. But among four fractions, the ratios
of each linkage are different. All these polysaccharides
exhibited antitumor activities against S-180, HCT-116 and
HT-29 cancer cells in vitro at a dose-dependent manner. Interestingly, highest Mw fractions presented stronger antitumor
Glycoconj J (2013) 30:281–461
activitiesthanthe native polymer, while the antitumor activities
of lower Mw fractions were lower than the native one.
Suggesting that the antitumor activities of these polysaccharides are related with the monosaccharide content, Mw
andglycosidic linkage. The polysaccharides also showed selectively higher antitumor activity against suspended cells
than adherent ones. Our results provided essential data for a
better understanding of L. edodes polysaccharides. Our investigations also provided helpful information for careful
selection of appropriateglucans is essential if we wish to
investigate the effects of glucans clinically.
287: Plasma membrane-associated sialidase NEU3
confers neoplastic potential on colon cancer cells by
regulating Wnt/β-catenin signaling
Kohta Takahashi1, Masahiro Hosono2, Keiko Hata1,
Tadashi Wada1, Kazunori Yamaguchi3, Kazuo Nitta2,
and Taeko Miyagi1; Divisions of 1Cancar Glycosylation
Research, and 2Cell Recognition Study, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, 981-8558, and 3 Division of
Molecular and Cellular Oncology, Miyagi Cancer Center
Research Institute, Natori, 981-1293, Japan.
tmiyagi@tohoku-pharm.ac.jp
The plasma membrane-associated sialidase NEU3 is a key
enzyme for degradation of gangliosides, components of cell
surface membranes. We previously demonstrated NEU3 to be
markedly up-regulated in various human cancers including
colon cancer, associated with augmented malignant properties
including increased cell migration, invasiveness and cell
survival. Here, we have found a molecular link between
Wnt/β-catenin signaling and NEU3 expression in colon cancer cells by analyzing cancer stem-like characteristics and
tumor initiating capability. NEU3-silencing in HT29 and
HCT116 colon cancer cells resulted in significant decrease
in clonogenic growth, chemoresistance to oxaliplatin,
clonogenicity on soft agar and in vivo tumor growth, along
with down-regulation of stemness genes, Nanog, Oct4, and
Sox2, and Wnt–related genes Lgr5, Wnt 1 and Wnt3a. Analyses further revealed that NEU3 enhanced phosphorylation of
the Wnt receptor LRP6 and consequently β-catenin activation
by accelerating complex formation with LRP6 and recruitment of GSK3β and Axin, whereas its silencing exerted the
opposite effects. As the null-activation mutant of NEU3,
N88D and Y370C, could not accelerated the LRP6 phosphorylation, indicating that the glycolipid changes as results of
catalytic reaction of NEU3 likely need for the acceleration.
Indeed, analysis of lipids extraction of the cells by the ThinLayer Chromatography (TLC), lactosylceramide (Lac-Cer)
was decreased in NEU3-silenced HT29 cells, and exogenous
Glycoconj J (2013) 30:281–461
addition of the Lac-Cer accelerated the LRP6 phosphorylation. Under sphere-forming condition, when stemness genes
were up-regulated, endogenous NEU3 expression was significantly increased, whereas NEU3 silencing suppressed
sphere-formation. In fact, NEU3-silenced spheroids demonstrated reduced in vivo tumor formation in NOD-SCID mice,
with down-regulation of stemness-and Wnt- related genes.
Interestingly, microarray analyses in NEU3-silenced HT29
revealed that NEU3 affects Cripto-1 expression, that is primary target of Wnt/β-catenin signaling and directly regulated by
Nanog and Oct4 in embryonic stem cells and colon carcinoma
cells. These results suggest that NEU3 regulates maintenance
of stem-like characteristics and tumorigenic potential of colon
cancer cells via Wnt/β-catenin signaling.
288: Impairment of lipid metabolism in GD3 synthase
knockout mice
Shuting Ji1, Yuhsuke Ohmi1, Yuki Ohkawa1, Keiko
Furukawa1,2 and Koichi Furukawa1; 1Department of
Biochemistry II, Nagoya University Graduate School of
Medicine, Nagoya, 466-0065, Japan; and 2Department of
Biomedical Sciences, Chubu University College of Life and
Health Science, Nagoya, 487-8501, Japan
koichi@med.nagoya-u.ac.jp
The expression patterns and levels of acidic glycosphingolipids,
gangliosides are different in tissues and organs, and high expression in central nervous system is observed. Although GD3
syntahse KO mice lack b-series gangliosides in whole body,
there is no severe phenotype during the development and
generation. When we analyzed serum samples from mice at
the age of 15 weeks and 30 weeks, extremely low levels of
leptin were observed in GD3 synthase KO mice compared with
wild type mice.
Although the blood levels of leptin were significantly low,
body weights of GD3 synthase KO mice were normal. Futher,
when they were fed with high fat high sucrose diet, wild type
and GD3 synthase KO mice showed similar obesity trend, and
there were no significant differences in body weights. Then
we analyzed histology of epididymal adipose tissues to assess
the amount of total body fat and brown adipose tissues which
relate with the burning of body fat. Consequently, white
adipose tissues appeared normal, while the number of cells
of brown adipose tissues were significantly increased in GD3
synthase KO mice. When high fat high sucrose was given, a
futher rise of the cell number was observed. In addition,
expression levels of UCP-1 gene increased in GD3 synthase
KO mice at 10 weeks after birth, and the higher body temperature was observed. These results suggested that brown adipocytes of GD3 synthase KO mice are over-working. On the
421
other hand, the gene expression of leptin was at the similar
levels between GD3 synthase KO and wild type mice, although the protein levels of leptin showed significantly higher
in GD3 synthase KO mice than wild type mice. In addition,
immunohistochemistry of leptin revealed strong staining both
in white and brown adipose tissues of GD3 synthase KO mice,
suggesting that the secretion of leptin from adipose tissues
were impaired in GD3 synthase KO mice.
In future, primary culture adipocytes of stromal vascular
cells isolated from epididymal adipose tissues and brown
adipose tissues will be used to clarify the molecular mechanisms for the morphological abnormalities and increased
activity of brown adipose tissues, and the abnormal leptin
secretion from white adipose tissues.
289: Analysis of polySia-NCAM biosynthesized by a
mutated polysialyltransferase ST8SIA2/STX found in a
schizophrenic patient
Masaya Hane1,2,3, Ken Kitajima1,2,3, Chihiro Sato1,2,3;
1
Department of Bioengineering Sciences, Graduate School of
Bioagricultural Sciences, Nagoya University; 2Bioscience and
Biotechnology Center, Nagoya University; 3Program for
Leading Graduate Schools IGER, Nagoya University
hane.masaya@d.mbox.nagoya-u.ac.jp
Schizophrenia is a severe psychiatric disorder that affects
approximately 1 % of the population worldwide. Causes are
known to be multiple, genetic and environmental factors.
However, the overall mechanism leading to schizophrenia
remains unclear. A number of reports have shown the relationships between impaired expression of polysialic acid
(polySia) and schizophrenia. PolySia is a homopolymer of
sialic acid with a degree of polymerization of 8–400, and has
anti-adhesive effects on cell-cell interactions due to its bulky
polyanionic nature when present on neural cell adhesion molecule (NCAM). Recently, we demonstrated that polySia functions not only as an anti-cell adhesion molecule, but also as a
reservoir scaffold for brain-derived neurotrophic factor
(BDNF), a neurotransmitter (Dopamine) and fibroblast
growth factor 2 (FGF2), which are biologically active molecules in neurogenesis and neural function. In 2006, Arai and
his colleagues reported single nucleotide polymorphisms
(SNPs) in the promoter region of ST8SIA2/STX in schizophrenic patients. We focused on a SNP of the ST8SIA2/STX
gene, SNP7 (421G>A) on exon 4, which has been identified
in a schizophrenic patient in their report because structural
features of the polySia-NCAM in any case of the schizophrenia patients have not been clarified. Therefore, our objective is
to gain an insight into the relationship between structure and
function of polySia-NCAM biosynthesized by
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ST8SIA2/STX. In this study, we analyzed the enzymatic
activity of the mutated ST8SIA2/STX toward membranebound and secreted forms of NCAM, and chemically evaluated the polySia-NCAM biosynthesized by the mutated
ST8SIA2/STX. In addition, we investigated the ability of
polySia to bind BDNF and FGF2, using a surface plasmon
resonance (SPR)-based method. We showed that polySiaNCAM biosynthesized by the mutated ST8SIA/STX
contained less polySia with shorter chain length and that it
exhibited impaired reservoir function for BDNF and FGF2 as
compared with that synthesized by wild-type ST8SIA2/STX.
Our findings suggest that the quantity and quality of polySia
on NCAM is important for normal neuronal functioning.
290: Novel Glyco-biomarker for Epithelial Ovarian
Cancer Selected by Glycoproteomic Approaches
Maki Sogabe 1, Hirofumi Nozaki1, Tomomi Kubota1,
Atsushi Kuno 1 , Hiroyuki Kaji 1 , Akira Togayachi 1 ,
Hayao Nakanishi 2 , Toru Nakanishi 3 , Nao Suzuki 4 ,
Kazushige Kiguchi4, Mikio Mikami5, Yuzuru Ikehara1,
Hisashi Narimatsu 1 ; 1 Research Center for Medical
Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, 305-8568,
Japan; 2Division of Oncological Pathology, Aichi Cancer
Center Research Institute, Nagoya, 464-8681, Japan; 3Department of Gynecologic Oncology, Aichi Cancer Center Hospital, Nagoya, 464-8681, Japan; 4Department of Obstetrics and
Gynecology, St. Marianna University School of Medicine,
Kawasaki, 216-8511, Japan; 5Department of Obstetrics and
Gynecology, School of Medicine, Tokai University, Isehara,
259-1193, Japan;
h.narimatsu@aist.go.jp
Epithelial ovarian cancer (EOC), is the leading causes of
death among gynecological malignancies, and its incidence
rate tends to increase is Japan. The indications of EOC are
asymptomatic, so more that two-third of EOC cases are
diagnosed in the advanced stages, thus, whose five-year
survival rate is less than 30 %.
Lectin microarray analysis of conditioned media from ovarian, gastric, and colon cancer cell line showed that AAL
signals were high in general. To select tumor biomarker candidates exhaustively, AAL column were used to collect glycopeptides for the analysis. In addition to the conditioned
media, peritoneal washing (PW) from EOC and gastric cancer
patients were subjected to IGOT-LC/MS analysis and more
than 300 glycoproteins were found from EOC samples.
Validation study was performed with the lectin-agarose catch
followed by Western Blotting analysis using the antibody for
Glycoconj J (2013) 30:281–461
the candidates. Several glycoproteins were found in the lectin
bound fraction of the PW from EOC patients. One of the
candidate glycoprotein was validated for detail.
Sandwich ELISA system with the lectin and antibody for
the glycoprotein was established. The ascites fluid from
EOC showed higher ELISA signals than those from benign,
including chocolate cyst and uterine endometrial carcinoma.
Immunohistochemical study showed that both the lectin and
antibody for the glycoprotein stained same cancerous part of
the tissue from EOC patients.
Glycoproteomics tools, lectin microarray and IGOT-LC/MS,
enable to find a novel EOC biomarker candidate, which is
capable to distinguish EOC from other gynecological disease.
This work was supported by a grant from New Energy and
Industrial Technology Development Organization (NEDO)
in Japan.
291: Identification of glycoproteins carrying the sialyl
lewis x antigen in human lung adenocarcinoma cells
Songlin Jiang1, Naoki Ikeda2 and Koichi Honke1,3; 1Kochi System Glycobiology Center, Kochi University Medical
School, Nankoku, Kochi 783-8505, Japan; 2Dept. Surgery,
Kinki-Chuo Chest Medical Center, Sakai, Osaka 591-8555,
Japan; 3Dept. Biochemistry, Kochi University Medical
School, Nankoku, Kochi 783-8505, Japan
The interaction of the sialyl lewis x antigen on cancer
cells with E-selectin on the endothelial cells initiates
the cell adhesion process of cancer hematogenous metastasis. Therefore, expression of the sialyl lewis x
antigen on cancer cells is thought to enhance cancer
metastasis. Because sulfation and sialylation occur at
the same position of the same precursor sugar chains,
overexpression of a relevant sulfotransferase, Gal3ST-2
suppresses the expression of sialyl lewis x in human
lung adenocarinoma-derived ABC-1 cells, and reduces
the interaction with E-selectin and the hematogeneous
metastasis in a nude mouse model. Since the two major
sialyl lewis x-positive protein bands turn negative parallel to the above phenomena, we hypothesize that
these proteins may be involved in the metastasis mechanism. In the present study, we have purified these
glycoproteins and identified them by mass spectrometrybased proteomics analysis. Effects of siRNA knock down of
these glycoproteins on expression of sialyl lewis x antigen and
cancer metastasis by interaction with E-selectin are under
study. Identified glycoproteins could serve as a new biomarker
for clinical diagnosis of lung adenocarcinoma.
Glycoconj J (2013) 30:281–461
423
292: Silencing of GnT-V Potentiates the Antitumor
Activity of Gemcitabine against Bladder Cancer Cells
293: Therapeutic effects of glycosaminoglycans on
elastase-induced emphysema in mice
Jianhui Fan1, Ying Tang2, Jingna He1, Shujing Wang1,
Lifen Wang2, Jianing Zhang1*; 1Department of Biochemistry, Institute of Glycobiology, Dalian Medical University,
Dalian, 116044, China; 2Department of Pathology, Second
Affiliated Hospital of Dalian Medical University, Dalian
116023,China.
jnzhang@dlmedu.edu.cn
Satoshi Kobayashi1, Reiko Fujinawa1, Shiho
Kobayashi 1 , Congxiao Gao 1 , Fumi Ota 1 , Shinobu
Kitazume 1 , Takashi Angata 2 , Keiichi Yoshida 1 ,
Takayuki Yoshida3, Tomoko Betsuyaku4, and Naoyuki
Taniguchi1; 1Disease Glycomics Team; 2Glycan Recognition Team, Systems Glycobiology Research Group, Global
Research Cluster, RIKEN-Max Planck Joint Research Center, RIKEN, Wako, 351-0198, Japan; 3First Department of
Medicine, Hokkaido University School of Medicine, Sapporo,
060-8638, Japan; 4Division, Pulmonary Medicine, Keio
University School of Medicine, Tokyo, 160-8582, Japan
skobayashi@riken.jp
Gemcitabine is a deoxycytidine analog used for the treatment of a wide range of solid tumors and has been widely
accepted as the first-line treatment for patients with advanced bladder cancer. Its sensitivity is however often reduced due to the development of bladder cancer. Nacetylglucosaminyl-transferase V (GnT-V) is an enzyme
that catalyses β1-6 branching of N-Glycans during synthesis of glycoproteins. Previous studies have shown that GnTV was associated with tumorigenesis and metastasis. The
functional roles of GnT-V in acquired resistance to
gemcitabine remain unknown in bladder cancer. Our present
study aimed to investigate the relationship between GnT-V
expression and gemcitabine sensitivity in bladder cancer
cells. GnT-V expression was studied by immunohistochemistry in 99 surgically resected bladder cancers, and the
staining intensity was evaluated. High GnT-V expression
in high grade tumor cells was found in 20 of 38 GnT-V
overexpression cases (p<0.05), and was positively correlated with pathological grade and lymph vascular space. Consistently, GnT-V overexpression was examined in multiple
human bladder cancer cell lines by RT-PCR and western
blot assay. Furthermore, GnT-V expression was significantly
downregulated by lentivirus induced RNAi in EJ bladder
cancer cells and result in increased gemcitabine sensitivity
with time and dose dependent way in vitro. More importantly, cellular administration of the GnT-V specific RNAi
in EJ cells significantly inhibited the growth tumor cells
implant solid tumor in nude mice and sensitized the tumors
to gemcitabine treatment in vivo. These findings suggest that
silencing GnT-V expression using lentivirus RNAi could
potentially be an effective strategy to enhance the antitumor
activity of gemcitabine in bladder cancer cells.
This work was supported by grants from the National Program on Key Basic Research Project (973 Program) (NO.
2012CB822103) and National Science Fund Committee
(NSFC) (NO. 31000372, 31170774)
Our previous studies indicated alpha 1,6 fucosyltransferase
(Fut8) KO mice develop emphysematous changes due to the
activation of matrix metalloproteinases (MMPs) by the
dysregulation of transforming growth factor-beta signaling.
There are increasing evidences to indicate that integrity and
balance of extracellular matrix components are essential for
normal lung function and the response to injury including
emphysema. The inhibitors against MMPs are promising for
the treatment of emphysema, but none of the suitable inhibitors are available at present because of the lack of
specificity and the strong side effects.
Glycosaminoglycans (GAGs) are distributed in the bronchial walls and in airway secretions. They play a key role in
many biological functions. We found that one of the GAGs,
keratan sulfate (KS)-disaccharide (L4) inhibited MMP-9
activity in vitro. Thus, we determined whether exogenous
GAGs, including L4 and hyaluronan (HA)-oligosaccharide,
prevented or corrected the early stage of elastase-induced
pulmonary emphysema in vivo. We intratracheally administered KS-I isolated from bovine cornea, L4, HA-tetramer
(HA4) and -dodecamer (HA12) into C57BL/6 mice. Next
day, we treated with porcine pancreatic elastase by
intratracheal administration to induce emphysema. On the
following day, we measured the accumulation of inflammatory cells and the production of tumor necrosis factor-alpha
(TNF-α) and the activity of MMP-9 in the bronchoalveolar
lavage (BAL) fluid. We found that KS-I significantly attenuated the accumulation of total inflammatory cells and the
production of TNF-α in BAL fluid and the MMP-9 activity
was suppressed in all groups except HA12-treated group as
compared with the PBS-treated group. Furthermore, we also
evaluated the progression of emphysema with microcomputed X-ray tomography (micro-CT) by calculation
the percentage of the emphysema area to the total lung area.
The analysis of micro-CT images showed that L4 and KS-I
significantly suppressed the emphysema progression at 7
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Glycoconj J (2013) 30:281–461
and 14 days after elastase administration as compared with
the PBS-treated group. These results suggested that these
GAGs, especially L4 and KS-I, might be effective against
the lung inflammation and the early stage of emphysema
formation.
for detail investigation of childhood-ALL associated
sialoglycoconjugate(s) present on the lymphoblasts. Further, due to high specificity of IgGA to fetuin/GM2, it may be
possible to develop a simple alternative diagnostic approach
(fetuin/GM2 based ELISA) for childhood-ALL.
294: Detection of disease associated sialoglycoconjugate
specific IgG in the sera of children with acute lymphoblastic leukemia
295: Potential importance of Maackia amurensis agglutinin in non-small cell lung cancer
a*
b
b
Sujata Ghosh , Sarika Kapoor , Ram Marwaha ;
a
Department of Experimental Medicine and Biotechnology,
b
Department of Pediatrics, Postgraduate Institute of Medical
Education and Research, Chandigarh,160012, India.
suassi1@gmail.com
Acute lymphoblastic leukemia (ALL) is the most common
malignancy in children. With existing treatment protocols,
virtually all patients achieve remission and~70 % are eventually cured. However, patients in remission may have residual
leukemic cells, which cannot be identified by the routine
methods. Thus, the need of the hour is to develop clinically
useful strategies to determine the treatment outcome and
predict relapse. Change in glycosylation pattern especially
aberrant sialylation in the cellular glycoconjugates is a prominent biomolecular alteration in cancer cells. The presence of
disease associated glycoconjugate specific antibodies in the
sera of cancer patients has been reported earlier. In the present
study, we report the presence of significantly higher level of
fetuin (taken as a broad spectrum sialoglycoconjugate) specific IgG (IgGA) in the serum samples of children having ALL as
compared to other non-neoplastic disorders. IgGA level was
found to be significantly higher as compared to the
fetuin specific IgM and IgA level in the sera of ALLpatients. A remarkable reduction in the level of IgGA
was detected during therapy up to 1 year. To further
confirm the sialoglycoconjugate specificity of IgGA, different gangliosides [known to contain NeuAcα(2→3)Gal
unit] were used and maximum specificity of IgGA was
found for GM2. IgGA from the pooled sera of ALLpatients was isolated by sequential chromatography on
Protein A-Sepharose CL-4B and Fetuin- Sepharose CL4B. The purified IgGA showed strong membrane positivity
with CEM-CM3 cells as well as the lymphoblasts of ALL–
patients. Further, the %age of IgGA positive cells was more
than 70 % in case of ALL-patients. A ~66 kDa band in the
membrane fraction of CEM-CM3 cells as well as PBMCs of
ALL-patients was found to interact specifically with IgGA as
well as Maackia amurensis agglutinin [specific for
NeuAcα(2→3)Gal/GalNAc], the intensity of which was
drastically reduced in presence of GM2. Taken together,
IgGA may have the potential to serve as a unique probe
Rakhee Chhetraa, Sangeeta Mehtab, Radhika Srinivasanc,
Suresh C. Sharmab, Digambar Beherad & Sujata Ghosha;
a
Department of Experimental Medicine & Biotechnology,
b
Department of Radiotherapy, cDepartment of Cytology &
Gynaecological Pathology, Department of Pulmonary
Medicine, Post Graduate Institute of Medical Education &
Research,Chandigarh,160012, India
Malignant transformation is associated with alterations in cell
surface carbohydrate architecture, which can be detected by
lectins. Among various types of sugar residues, sialic acids are
extremely important, since increased sialylation in the cellular
glycoconjugates has been found to be associated with tumour
progression and metastasis. Altered sialylation in cellular
glycans was reported in case of non-small cell lung cancer
(NSCLC). Although, drugs for treatment of NSCLC have
been available for many years, but the use of such regimens
are plagued by serious side effects because of their nonspecific action on normal healthy cells. Thus, medical science
is in need of prospective studies to develop newer targeted
approaches for treatment of this life threatening disease. In this
context, lectins are the potentially important biomolecules.
Maackia amurensis agglutinin [specific for NeuNAcα (2-3)
Gal/GalNAc] is gaining recognition as an important lectin,
which can specifically distinguish cancer cells from normal
cells and thereby it seems to have diagnostic potential in
cancer of different origin. In the present study, we report that
this lectin interacted strongly with human NSCLC cell lines as
well as the tissue biopsies and cells obtained from fine needle
aspirations of NSCLC patients, indicating the binding of this
lectin to specific sialylated glycans on NSCLC cells. We have
observed that this lectin selectively induced apoptosis in
NSCLC cell lines but not in normal lung fibroblast cell line.
The inhibition of Maackia amurensis agglutinin induced apoptosis in presence of GM2 and IgGMAA indicated the importance of sugar binding epitope as well as antibody binding
epitope of the lectin in inducing apoptosis in NSCLC cells.
Our observation was further authenticated by studying this
lectin induced apoptosis in the cells obtained by the fine
needle aspiration of the tumour of NSCLC patients. Moreover,
this lectin induced activation of apoptotic pathway in NSCLC
cells was found to be accompanied by downregulation
of Bcl-XL, upregulation of Bax, release of cytochrome c
Glycoconj J (2013) 30:281–461
and activation of procaspase-3. Further, a synergistic
effect of this lectin on paclitaxel induced cytotoxicity
in NSCLC cell lines was also noted. Collectively our
results have indicated the potentiality of Maackia
amurensis agglutinin in NSCLC.
296: N-Glycans of IgG as a complementary biomarker to
CA125 for ovarian cancer diagnosis
Yifan Qian1, Yisheng Wang2, Xingwang Zhang1, Shifang
Ren1, Congjian Xu2, Jianxin Gu1; 1Key Laboratory of
Glycoconjugate Research Ministry of Public Health; Department of Biochemistry and Molecular Biology, Shanghai
Medical College, Fudan University, Shanghai, P.R. China,
200032; 2Obstetrics and Gynecology Hospital, Department
of Obstetrics and Gynecology of Shanghai Medical School,
Fudan University, Shanghai, P.R. China, 200032
11211010023@fudan.edu.cn
Ovarian cancer (OC) is the most lethal malignancy of all
gynecological cancers among women, causing more deaths
of the female reproductive system. In general, elevated CA125 level in blood is the most robust serum biomarker for
OC detection. However, CA125 can give a false positive
response in benign conditions due to its low sensitivity and
specificity. The aim of this work was to find additional
markers for the OC diagnosis which would complement
the use of CA125 by analyzing the alteration of glycan of
IgG in human blood. N-glycans were released from serum
IgG by PNGase F digestion, purified in a solid phase
extraction, permethylated und subsequently analyzed by
MALDI-QIT-TOF MS. 31 ovarian cancer patients and
22 age-matched benign tumor patients were enrolled in
this study. Statistical analyses were carried out using the
SPSS 17.0 software. Statistical analyses show statistic
significance difference between malignant and benign
patients by combining the level of serum IgGassociated N-glycans with CA125. The results indicate
that combinations of changes in the IgG glycans with
CA125 might improve both sensitivity and specificity
for acute ovarian cancer diagnosis.
297: Hydrolysis of secreted sialoglycoproteins in ex vivo
and biochemical models of bacterial vaginosis
Warren G. Lewis1, Lloyd S. Robinson2, Amanda L.
Lewis2,3; Departments of 1Medicine, 2Molecular Microbiology, and 3Obstetrics and Gynecology, Center for Women’s
Infectious Disease Research, Washington University School
of Medicine, St. Louis, MO, 63110 USA
geelewis@gmail.com, lewis@borcim.wustl.edu
425
Bacterial vaginosis (BV) is a common polymicrobial imbalance of the vaginal flora associated with a wide variety of
obstetric and gynecologic complications including serious
infections and preterm birth. As evidenced by high recurrence rates following treatment, interventions for BV are still
lacking. Several hydrolytic activities, including glycosidases and proteases, have been previously correlated with
BV and have been hypothesized to degrade host
sialoglycoproteins that participate in mucosal immune functions. Sialidase activity is most predictive of BV status and
correlates strongly with adverse health outcomes. Here we
combine clinical specimens with biochemical approaches to
investigate secretory immunoglobulin A (SIgA) as a substrate of BV-associated glycosidases and proteases. We
show that BV clinical specimens hydrolyze sialic acid from
SIgA, but not in the presence of the sialidase inhibitor
dehydro-deoxy-sialic acid. The collective action of BVassociated glycosidases exposes underlying mannose residues of SIgA, most apparent on the heavily N-glycosylated
secretory component of the antibody. Terminal sialic acid
residues on SIgA protect underlying carbohydrate residues
from exposure and hydrolysis by exoglycosidases (galactosidase and hexosaminidase). It is known that both IgG and
SIgA are present in the human reproductive tract. We show
that the IgG heavy chain is more susceptible to proteolysis
than its IgA counterpart. Gentle partial deglycosylation of
the SIgA secretory component enhanced susceptibility to
proteolysis. Together, these data support a model of BV in
which SIgA is subject to stepwise exodeglycosylation
and enhanced proteolysis, likely compromising the ability
of the reproductive mucosa to neutralize and eliminate
pathogens.
Poster
298: Inhibiting activities of chitooligosaccharides on the
growth of two different tumors
Haige Wu1,2, Ziang Yao2, Yuguang Du1; 1 Dalian Institute
of Chemical Physics, Chinese Academy of Science, Dalian
116023, China; 2 Bioengineering College Dalian University,
Dalian 116622, China
duyg@dicp.ac.cn
Anti-tumor activity of chitooligosaccharides (COS) has
been reported previously, but its mechanism is not clear. In
this research, two different animal models, Kunming mice
bearing ascites tumor (S180) and hairless mice bearing
human breast cancer (MCF-7) are used, tumor bearing mice
are administered with different dose of COS, the growth of
tumors are monitored. After administered with COS for
426
28 days, mice are sacrificed, tumor tissue are taken out and
weighed, Elisa method is used to detect the contents of cytokines correlated with immunological regulation and angiogenesis in the serum of Kunming mice or hairless mice. The
results show that COS can decrease the contents of VEGF
(vascular endothelial growth factor) and VEGFR (vascular
endothelial growth factor receptors) in both Kunming and
hairless mice. In Kunming mice, COS can increase the thymus
and spleen index, the contents of IL-2 and IL-6, therefore
display immunological regulation activity. COS could inhibit
the growth of tumor both in Kunming and hairless mice, but
the inhibiting rate on S180 tumor in Kunming mice (maximum 65 %) is greater than that of on human breast cancer in
hairless mice (maximum 47 %). Maybe this is because that in
hairless mice COS couldn’t display its immunological regulation activity. All the results indicate that COS have antitumor activity, and its anti-tumor activity is correlated with its
anti-angiogenic and immunological regulation activities.
299: Ginseng pectin potentiates TRAIL-induced apoptosis
through upregulation of death receptors
Hairong Cheng, Qi Sun, Guihua Tai, Yifa Zhou; School
of Life Sciences, Northeast Normal University, 130024,
Changchun, Jilin Province, China
chenghr893@nenu.edu.cn
Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), is a potent cancer cell-specific apoptosisinducing agent with little to no effect on normal tissues.
However, acquired resistance of cancer cells to TRAIL is a
roadblock, allowing them to evade the pro-apoptotic effects
of TRAIL. Ginseng pectin exerts significant anti-tumor effect and potentiates anti-tumor effect of a series of chemotherapeutic drugs. In the present report, we investigated the
effect of ginseng pectin on cancer cell apoptosis induced by
TRAIL. We found that two homogenous homogalacturonan
with small percentage of type-I rhamnogalacturonan domains (WGPA-3-RG and WGPA-4-RG) and two Heattreated homogenous homogalacturonan (WGPA-3-HG and
WGPA-4-HG) not only potentiated TRAIL-induced apoptosis in HCT116 colon cancer cells but also sensitized TRAILresistant colon cancer cells HT-29 to the cytokine. At a mechanistic level, ginseng pectin downregulated cell survival proteins, including Bcl-2, Mcl-1 and Bcl-XL, and upregulated
pro-apoptotic protein Bax and the expression of TRAIL death
receptors DR5. Gene silencing of DR5 by short hairpin RNA
reduced the apoptosis induced by combination treatment of
Ginseng pectin and TRAIL. Induction of DR5 by ginseng
pectin was independent of p53 and Bax, but was dependent
on production of reactive oxygen species (ROS), and sequestering of ROS abolished both upregulation of the receptor and
Glycoconj J (2013) 30:281–461
potentiation of TRAIL-induced apoptosis. Taken together, our
results provide the first mechanistic evidence that ginseng
pectin treatment render cancer cells more sensitive to the
cytotoxic activities of TRAIL, suggesting that Ginseng pectin
can be given in combination with TRAIL, especially for those
tumors that develop resistance to TRAIL.
300: Chitosan Oligosaccharides Protect Human Monocytes U937 from LPS-induced Inflammatory Damage
through Blockade p38 MAPK Phosphorylation and Increasing O-GlcNAcylation of Proteins
Yu Li1,2, Qiang Peng1,2, Qingsong Xu1, Yuguang Du1;
1
Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian, 116023, China; 2Graduate School of
Chinese Academy of Sciences, Beijing 110864, China
articles1805@gmail.com
To investigate the effects of Chitosan oligosaccharides(COS)
with different degree of polymerization(DP) on LPS-induced
inflammation, U937 human monocytes were cultured with
DP2-8(COS-A) and DP7-15(COS-B) respectively, which were
prepared by our group. The results showed that both kinds of
COS demonstrated obviously anti-inflammatory effects against
LPS-induced over-expression of TNF-α and IL-8. Signal transduction studies indicated COS-A and COS-B efficiently downregulated LPS-induced the phosphorylation of p38 MAPK. In
several documented instances, phosphorylation and OGlcNAc modification are reciprocal, occurring at the
same or adjacent hydroxyl moieties. In this study, we also
find COS-A and COS -B could significantly increase OGlcNAc modification of proteins in LPS-induced U937
monocytes. Finally, we postulate that COS-A and COS-B
may have anti-inflammatory effects via suppression of the
expression levels of TNF-α and IL-8, regulated by p38
MAPK pathways and O-GlcNAcylation of proteins.
301: The Creation of Clostridium difficile Glycoconjugate
Vaccines
Yuening Jiao, Zuchao Ma, Doug Hodgins, Brittany
Pequegnat, Lisa Bertolo, Luis Arroyo, Mario A.
Monteiro; Department of Chemistry, University of Guelph,
Guelph, ON, N1G 2W1, Canada
jiaoy@uoguelph.ca
Clostridium difficile is the most common cause of antimicrobial-associated diarrhea in humans and may cause death.
The bacterium expresses three phosphorylated polysaccharides, which we named PSI, PSII and PSIII. Due to the fact
that PS-I is not expressed in great amount in vitro, we
Glycoconj J (2013) 30:281–461
developed a glycoconjugate containing a synthetic PSI
pentasaccharide repeating unit carrying a linker at the reducing end, α-L-Rhap-(1→3)-β- D -Glcp-(1→4)-[α-LRhap-(1→3)]-α-D-Glcp-(1→2)-α-D-Glcp-(1→O(CH2)5NH2
by a linear synthesis strategy from four monosaccharide building blocks. The synthesized PSI pentasaccharide was conjugated to a subunit of C. difficile exotoxin B yielding a
potential dual C. difficile vaccine. More significantly, sera
from healthy horses were shown here to contain natural antiPSI IgG antibodies that recognized the synthetic PSI
pentasaccharide.
427
activation of MMP-2. Consequently, the activation of
the two MMPs triggered by GnT-V intensified the invasive potential. A quantitative analysis using clinical tissues revealed a relatively strong correlation between
GnT-V overexpression and MT1-MMP upregulation. In
this study, we report for the first time that GnT-V directs
cancer progression by modulating MMPs in cancer.
303: Role of bacterial surface factors in adherence of
enteric bacteria to plant glycolipid
Yannick Rossez1, Ashleigh Holmes1, Louise Isager Ahl2,
Henriette Lodberg Pedersen2, Louise Birse1, William
GT Willats2, Ian Toth1 and Nicola Holden1; 1Cellular
and Molecular Sciences, James Hutton Institute, Dundee,
Scotland, United Kingdom, 2Department of Plant Biology
and Biotechnology, University of Copenhagen, Denmark.
Yannick.Rossez@gmail.com
Poster Session II-Cell Biology
302: N-Acetylglucosaminyltransferase V triggers
overexpression of MT1-MMP and reinforces the
invasive/metastatic potential of cancer cells
Juhee Lee, Jeong Gu Kang, Kyoungjin Song, Seong
Kook Jeon, Sejeong Oh, Yong-Sam Kim and JeongHeon Ko; Cancer Biomarkers Development Research Center, KRIBB, Daejeon 305-806, Korea
kang@kribb.re.kr, Tel:82-42-860-4135
N-Acetylglucosaminyltransferase V (GnT-V) is an enzyme that catalyzes the formation of a β1,6-Nacetylglucosamine (GlcNAc) side chain to a core
mannosyl residue in N-linked glycoproteins. Besides its
direct function of producing aberrant glycoproteins, it
promotes cancer progression by its involvement in the
stimulation of oncoproteins. Herein, we report that GnTV guided the transcriptional activation of membrane-type
matrix metalloproteinase-1 (MT1-MMP) in cancer cells.
The activated MT1-MMP expression had dual effects on
cancer progression. It not only promoted proteolytic
activity for cancer cells per se, but also led to the
It is generally recognized that bacterial attachment to plant
or animal surfaces constitutes the first step in colonisation.
This mechanism is mediated by interactions between adherence factors that are expressed on the bacterial cell surface,
such as fimbriae or flagella, and host cell surface glycans.
For example, E. coli type 1 fimbriae are well characterised
structures for their interaction with mammalian epithelial
cells and are known to recognise specific glycans present
on host cell surfaces, such as α-D-mannose. In planta,
recent papers have demonstrated the implication of curli
fibers and flagella from Enteropathogenic E coli and Salmonella species respectively. However the molecular recognition pattern implicated in adhesion of human pathogen
bacteria to plant tissues has yet to be described.
In this work plant glycan arrays have been used to investigate specific interactions with bacterial adherence factors.
We found in this study a specific interaction between FliC,
the flagella main structural subunit, and ionic plant membrane lipids. The work provides an improved understanding
about the molecular mechanisms underpinning human
foodborne pathogens with plants.
304: HSF1 and Sp1 are involved in regulation of FUT4
gene expression and cell proliferation in breast cancer cells
Xuesong Yang1, Shuai Liu1, Qiu Yan1; 1Department of
Biochemistry and Molecular Biology, Dalian Medical University, Liaoning Provincial Core Lab of Glycobiology and
Glycoengineering, Dalian, 116044,People’s Republic of
China
yxmsnow@126.com
428
Lewis Y (LeY) is a carbohydrate tumor-asssociated antigen.
The majority of cancer cells derived from epithelial tissue
express LeY type difucosylated oligosaccharide.
Fucosyltransferase IV (FUT4) is an essential enzyme that
catalyzes the synthesis of LeY oligosaccharide. In our previous study, we found that FUT4 was associated with the cell
proliferation. However, despite the important role of FUT4 in
cancer proliferation and apoptosis, little is known about the
mechanisms of its transcription regulation. In this study, we
investigated the transcriptional regulation of human FUT4 in
human breast cancer. We compared the transcriptional regulation of the human FUT4 gene in human breast cancer cells
(MCF-7 and MDA-MB-231) by promoter/luciferase analyses.
Using a series of promoter deletion constructs, we indentified
that a potential regulatory site that is located between 0.8 and
1.6 kb of the FUT4 promoter. As shown by EMSA and ChIP
analysis, HSF1 and SP1 were required for promoter activity.
In addition, we explored the role of HSF1 and SP1 on cell
proliferation. We found that ERK1/2 MAPK and PI3K/Akt
signaling pathways regulate the expression of FUT4, which
play a role in the cell proliferation, via HSF1 and SP1. These
results suggest that FUT4 is a target gene for HSF1 and SP1
that is required for cell cycle progression in breast cancer
epithelial cells.
Key word: Fucosyltransferase IV; breast cancer; HSF1; Sp1;
transccription regualtion
305: Effects of Cell Surface α2,3-Sialic Acid on Osteogenesis
Lan Xu, Zhi Jiang, Shiliang Wu; Department of Biochemistry and Molecular Biology School of Medicine, Soochow
University, Suzhou, 215007.
A cell culture model of osteoblast differentiation was applied in our study of the effect of sialic acid on the osteogenesis by using the pre-osteoblast of MC3T3-E1 subclone
14 cells. Following the treatment of different concentrations
of α2,3-neuraminidase, which specifically removed the
α2,3-sialic acid from cell surface, a significant decrease of
α2,3-sialic acid was detected with fluorescein isothiocyanate (FITC)-labeled Maackia amurensis lectin (MAL-II) by
flow cytometry analysis. von Kossa staining showed that the
bone mineralization decreased in MC3T3-E1 subclone 14
cells after the treatment of α2,3-neuraminidase for 2 weeks.
However α2,3-neuraminidase did not affect the formation of
osteoblasts in MC3T3-E1 subclone 14 cells, which was demonstrated by positive alkaline phosphatase (ALP)-staining.
Characteristic biological markers and osteoblast-like cellrelated factors of osteoblastic cells were also examined. Both
RT-PCR and Western blot analysis demonstrated that the
expression of bone sialoprotein (BSP), osteoprotegerin
Glycoconj J (2013) 30:281–461
(OPG), and vitamin D receptor (VDR) were significantly
decreased when α2,3-sialic acid expression decreased on the
cell surface, while the expression of osteocalcin (OC) and
osteopontin (OPN) remained unchanged. We propose a
hypothesis that α2,3-sialic acid affects bone mineralization but not osteogenic differentiation.
306: Regulation of the cell surface expression level of
AMPA-type glutamate receptor by N-linked glycans
Jyoji Morise, Yusuke Takeuchi, Shogo Oka; Human
Health Sci., Grad. Sch. of Med., Kyoto Univ., Kyoto, Japan
shogo@hs.med.kyoto-u.ac.jp
AMPA-type glutamate receptors (AMPAR) are hetero or
homotetrameric complexes composed of various combinations of four subunits (GluA1-4). AMPAR mediate
most of the fast excitatory synaptic transmission in the
nervous system. Their cell surface expression level and
abundance in postsynaptic region modulate synaptic
plasticity. So far, we found that a unique trisaccharide
(HSO3-3GlcAβ1-3Galβ1-4GlcNAc), named human natural killer-1 (HNK-1) carbohydrate specifically
expressed on N-linked glycans of GluA2, regulated cell
surface expression level of AMPAR and spine maturation process. Since the particular carbohydrate possesses
such functions to AMPAR, it is speculated that other Nlinked glycans expressed on AMPAR also have a potential to regulate functions of AMPAR. In this study, to
elucidate the role of N-glycans on GluA1 and GluA2
(major subunits of AMPAR), we generated a series of
GluA1 and GluA2 mutants of which Asn residue in the
consensus sequence (N-X-S/T) was mutated to Ser, and
investigated cell surface expression levels of these mutants by using a cell surface biotinylation assay. As a
result, cell surface expression levels of GluA1N63S,
N363S and GluA2N370S were decreased compared with
those of wild-type (WT) GluA1 and GluA2, and these
mutants were mainly distributed in ER. The cell surface
expression level of GluA1N363S was increased by coexpression of GluA2WT but not GluA1N63S whereas
that of GluA2WT was unchanged regardless of coexpression of GluA1 mutants, suggesting that N-linked
glycans at N63 and N363 on GluA1 had different roles
in trafficking from ER to cell surface. In contrast, coexpression of GluA1WT had little effect on the cell
surface expression level of GluA2N370S but rather
GluA1WT was likely to accumulate in ER, suggesting
that the trafficking of GluA1 was controlled by N-linked
glycan at N370 on GluA2. These results show that the
cell surface expression level of AMPAR was regulated
by N-linked glycans in a different manner.
Glycoconj J (2013) 30:281–461
307: Synthesis and
glycoglycerolipids
antiviral
429
activity
of amino-
Jun zhang, Chunxia Li, Huashi Guan; Key Laboratory of
Marine Drugs, Ministry of Education, School of medicine
and pharmacy, Ocean University of China, Qingdao
266003, P. R. China
lchunxia@ouc.edu.cn
Various Glycoglycerolipids have been isolated from algae,
which have shown many bioactivities, such as tumor growth
and DNA polymerase inhibition, fatty acid synthase inhibiton,
glucose-lowing effect and anti-inflammatory action et al.
Aminoglycoglycerolipid, 1,2-dipalmitoyl-3-(N-palmitoyl-6′amino- 6′-deoxy-α-D-glucosyl)-sn-glycerol, which was
isolated from an algal species showed high activity
against the enzyme Myt1-kinase. Due to the unique structure and activity, we had synthesized its 22 analogues by
altering the length of acyl chains and glycoside residues
for bioactivity screening.
All the glycoglycerolipids were evaluated for anti-influenza
A virus (IAV) activity by the cytopathic effects (CPE) inhibition assay. Compared with ribavirin, some compounds
showed better protection from virus-induced cell death.
The results indicated that the glycosyl residue and long acyl
chain played critical roles in improving the inhibition of IAV
multiplication in MDCK cells. Further structure–activity
relationship study is underway.
308: T synthase Glycosylates and Activates c-MET in
Hepatocellular Carcinoma Cells
Min-Chuan Huang1, Chiung-Hui Liu1, Yao-Ming Wu2,
John Huang2; 1Graduate Institute of Anatomy and Cell
Biology, National Taiwan University College of Medicine,
Taipei 100, Taiwan, 2Department of Surgery, National
Taiwan University Hospital, Taipei 100, Taiwan
mchuang@ntu.edu.tw
Altered glycosylation has been recognized as a hallmark
of cancer. T synthase, also named core 1 β1,3galactosyltransferase (C1GALT1), controls the formation
of mucin-type O-glycans. Although these carbohydrates
play crucial roles in a variety of cancers, the expression
and function of T synthase in hepatocellular carcinoma
(HCC) have never been studied. Here we showed that T
synthase mRNA and protein were frequently
overexpressed in HCC tumors compared with non-tumor
liver tissues. T synthase expression significantly correlated with increased histologic grade and metastasis of
HCC. Knockdown of T synthase suppressed HCC cell
growth and caused G1 arrest of cell cycle in vitro and
d e c r e a s e d t u m o r g r o w t h i n v i v o . C o n v e r s e l y,
overexpression of T synthase in Sk-Hep1 cells enhanced
cell growth in vitro and in vivo. Interestingly, we found
that T synthase regulated hepatocyte growth factor
(HGF)-mediated phosphorylation of c-MET in HCC cells,
and the expression level of phospho-c-MET was significantly associated with that of T synthase in primary HCC
tissues. Mechanistic investigation showed that c-MET was
decorated with O-glycans revealed by Vicia villosa agglutinin (VVA) and peanut agglutinin (PNA) binding. Moreover, T synthase modified the O-glycosylation and HGFinduced dimerization and activation of c-MET. Together,
our results suggest that T synthase is frequently
overexpressed in hepatocellular carcinoma and activates
HGF signaling via modulation of O-glycosylation and
dimerization of c-MET. These findings provide novel insights into the role of O-glycosylation in the pathogenesis
of HCC.
309: Dietary of Chitooligosaccharides Supplementation
Enhances the Reproductive Performance of Sows
Lun X. Wang1, Yu G. Du1*; 1Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, 116023, PR
China;
[Objective] The swine breeding efficiency in China is
relatively low compared with that from other developed
country such as America. The aim of this study was to
investigate the effect of dietary chitooligosaccharides
(COS) on the reproductive performance of sows. [Method] The pregnant sows (Landrace×Large White Sows)
after mating were assigned randomly into four groups in
corn-soybean-based diets with or without COS, including COS group, Jerusalem artichoke powder (JAP)
group, Several polysaccharides mixed (SPM) group
(containing COS),and the Control group (basal diet).
[Result] SPM supplement improved (P<0.05) total piglets born and born alive compared with that in Control
group (12.3 ± 0.5/10.5 ± 0.6, 11.7 ± 0.4/9.8 ± 0.5). COS
supplement improved one more piglets born and born
alive compared with Control group (11.7±0.5/10.5±0.6,
11.0 ± 0.4/9.8 ± 0.5), although there was no significant
difference between them. Meanwhile, both COS supplement and SPM supplement improved (P < 0.05) born
litter weight compared with Control group (17.5 ±
0.8/18.4 ± 0.6/14.0 ± 0.9). In addition, COS group improved (P<0.05) born individual weight compared with
Control group (1.7 ± 0.05/1.5± 0.04). [Conclusion] The
results indicated that dietary COS can markedly improve
the reproductive capacity of sows.
430
310: Human recombinant Intelectin-1 expressed in a
heart capillary endothelial cell line displays unique
carbohydrate binding specificity
Jin Kyu Lee, Jonathan Viola, Kiara Swanier and J.
Michael Pierce; Department of Biochemistry and Molecular Biology and Complex Carbohydrate Research Center,
University of Georgia, Athens, Georgia 30605
jinkyu@uga.edu
Since Intelectins (Omnetins) were first identified as homologs of
the Xenopus laevis oocyte cortical granule lectin (XL35), members of the family of intelectins have been reported from many
eukaryotes, including ascidians, fishes, frogs and humans. In
addition to its function in formation of the fertilization, which
serves as a block to sperm and microbes, intelectins have been
studied as a mediator during pathogen surveillance. The infection
by several pathogens induces a dramatic increase in the expression of Intelectin transcripts in intestinal and bronchial epithelial
cells, mediated by IL-13. Reports have suggested that very low
levels of recombinant int-1 could be secreted from transfected
cells. Since we have shown that Int-1 is constitutively expressed
in human heart endothelial cells (and not in most types of endothelial cells), we attempted to express cDNAs of human Int-1 and
-2 in stably transfected H5V cells, a mouse heart capillary endothelial cultured line, to produce significant levels of secreted proteins. The recombinantly expressed Int-1 was secreted into the
media. MS analysis determined that Int-1 was not a GPI-anchored
protein in H5V cells. There have been some reports that Int-1
binds specifically to galactofuranose coupled to agarose; however,
H5V-secreted, recombinant Int-1 bound to native agarose resin
and was eluted by EDTA, similar to our report for XL35, but also
by 100 mM ribose. The eluted protein fraction showed a single
band in silver staining after SDS-PAGE resulting in a yield of X
mg of purified Int-1/Y ml of starting culture media. Glycan
binding array analysis by Core H of the CFG showed binding
of Int-1 was to the synthetic glycan: GlcNAcβ1-4Galβ14GlcNAcβ-Sp8, which has not been reported in humans. The
binding specificity for XL35 on the same array was clearly
Galα1-3GalNAc, by contrast. Although Int-1 binds tightly to
agarose and with low affinity to the synthetic glycan above in a
calcium-dependent manner, a high affinity glycan ligand in
humans or human pathogens has not yet been identified.
311: GOLPH3 regulates integrin-mediated cell migration via up-regulation of sialylation of β1 integrin
Tomoya Isaji, Wei Gu, Tomohiko Fukuda and Jianguo Gu;
Division of Regulatory Glycobiology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai Miyagi, 9818558, Japan
tfukuda@tohoku-pharm.ac.jpisaji@tohoku-pharm.ac.jp
Glycoconj J (2013) 30:281–461
Recently, a Golgi protein, GOLPH3 was identified as a
new oncogene that is commonly amplified in human
cancers. An ortholog of GOLPH3, VPS74p has been
reported to be essential for glycosyltransferase activation
of yeast. To investigate whether the expression of
GOLPH3 was involved in the cell migration and Nglycosylation in mammalian cells, we performed a lossof-functional study. Cell migration on fibronectin or laminin was suppressed in GOLPH3 knockdown (KD) cells,
and the suppression was restored by re-introduction of
GOLPH3 gene. The cell migration was abolished in the
presence of neutralized anti-β1 integrin antibody. Interestingly, N-glycosylation status of β1 integrin obtained
from KD cells was apparently different from those in
control cells, while it was normalized in the restored cells.
HPLC and LC/MS analysis showed that the sialylation
level of N-glycans was specifically decreased in KD cells,
and the aberrant N-glycosylation was significantly rescued in the restored cells. To explore the specific effect
on sialylation by GOLPH3 expression, we examined the
interactions between a glycosyltransferase and GOLPH3,
and found GOPLH3 specifically associated with α2,3sialyltransferase IV, but not β1,4-galactosyltransferase I.
Furthermore, the cytoplasmic tail of α2,3-sialyltransferase
IV was shown to be important for the association. Taken
together, these results suggest that GOLPH3 regulates Nglycosylation status of β1integrin and it’s biological functions, which may give a new insight for the functions of
GOLPH3 in cancer.
312: Large protein complexes retained in the ER are
dislocated by non-COPII vesicles and degraded by selective
autophagy
Valerie Le Fourn1,4,*, Sujin Park2,*, Insook Jang3,*,
Katarina Gaplovska-Kysela1,5, Bruno Guhl1, Yangsin
Lee2, Jin Won Cho2,3, Christian Zuber1, Jürgen Roth1,2;
1
Division of Cell and Molecular Pathology, Department of
Pathology, University of Zurich, CH-8091 Zurich, Switzerland, 2Department of Integrated OMICS for Biomedical
Science, WCU Program, Yonsei University Graduate
School, Seoul 120-749, Korea, 3Department of Systems
Biology, Yonsei University, Seoul 120-749, Korea, 4Present
address: Selexis SA, CH-1228 Plan-les-Ouates/Geneva,
Switzerland, 5Present address: Department of Genetics, Comenius University, SR-84215 Bratislava, Slovak Republic
* These authors contributed equally to this study.
jurgen.roth@yonsei.ac.kr, Tel:82-2-2123-7673, Fax:82-2312-5657
Multisubunit protein complexes are assembled in the endoplasmic reticulum (ER). Existing pools of single subunits
Glycoconj J (2013) 30:281–461
431
and assembly intermediates ensure the efficient and rapid
formation of complete complexes. While being kinetically
beneficial, surplus components must be eliminated to prevent potentially harmful accumulation in the ER. Surplus
single chains are cleared by the ubiquitin–proteasome system. However, the fate of not secreted assembly intermediates of multisubunit proteins remains elusive. Here we show
by high-resolution double-label confocal immunofluorescence and immunogold electron microscopy that naturally
occurring surplus fibrinogen Aα–γ assembly intermediates
in HepG2 cells are dislocated together with EDEM1 from
the ER to the cytoplasm in ER-derived vesicles not corresponding to COPII-coated vesicles originating from the
transitional ER. This route corresponds to the novel ER exit
path we have previously identified for EDEM1 (Zuber et al.
Proc Natl Acad Sci USA 104:4407–4412, 2007). In the
cytoplasm, detergent-insoluble aggregates of fibrinogen
Aα–γ dimers develop that are targeted by the selective
autophagy cargo receptors p62/SQSTM1 and NBR1. These
aggregates are degraded by selective autophagy as directly
demonstrated by high-resolution microscopy as well as
biochemical analysis and inhibition of autophagy by
siRNA and kinase inhibitors. Our findings demonstrate that
different pathways exist in parallel for ER-to-cytoplasm
dislocation and subsequent proteolytic degradation of large
luminal protein complexes and of surplus luminal singlechain proteins. This implies that ER-associated protein
degradation (ERAD) has a broader function in ER
proteostasis and is not limited to the elimination of
misfolded glycoproteins.
oligosaccharides of glycoproteins, its complex mode of interaction with substrates has become clear only recently. However, still very little is known about the turnover and
degradation mechanism of EDEM1 and how this relates to
the fate of its substrates. We already reported that EDEM1
becomes rapidly degraded and that this occurs by basal
autophagy. Here, we provide detailed insight into the mechanism by which EDEM1 becomes degraded. After its dislocation to the cytosol, EDEM1 is apparently making complexes
with the selective autophagy receptors p62, NBR1 and Alfy.
We observed co-distribution of EDEM1 and selective
autophagy receptors by double or triple confocal laser scanning immunofluorescence. By quantifying the relationship of
EDEM1 and the selective autophagy receptors as visualized
by confocal laser scanning immunofluorescence, dramatical
changes were observed in HepG2 cells following inhibition of
autophagy by wortmannin treatment. These changes were
fully reversible upon wortmannin wash-out. In addition, we
observed its ubiquitination after dislocation to the cytosol.
This demonstrates that the ERAD component EDEM1 itself
undergoes ERAD involving selective autophagy.
313: Selective autophagy receptors are involved in
degradation of EDEM1
314: GnT-V attenuates TGF-β-induced EMT and metastatic potential in lung cancer
Insook Jang, Sujin Park, Jin Won Cho and Jürgen Roth;
Department of Biology, Yonsei University, Seoul, 120-749,
South Korea
o-glcnac@yonsei.ac.kr
Na Li1, Haineng Xu4, Xijun Liu1, Kun Fan1,2,3, Jingjing
Qi1,2,3, Liying Wang1,2,3, Zengxia Li1,2,3, Xiliang Zha1,2,3;
1
Department of Biochemistry and Molecular Biology,
Shanghai Medical College, Fudan University, Shanghai,200032, China, 2Key Laboratory of Glycoconjugate Research, Ministry of Health, Shanghai 200032, China, 3Key
Laboratory of Molecular Medicine, Ministry of Education,
Shanghai, 200032,China, 4China Laboratory of Molecular
Cell Biology, Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, 200031,China
lizengxia@gmail.com; xlzha@shmu.edu
Misfolded and mis-assembled glycoproteins are retained in
the endoplasmic reticulum (ER) where they are exposed to
the protein folding machinery and protein quality control.
The UPR (Unfoled Protein Response) is activated in response to an accumulation of unfolded or misfolded proteins
in the lumen of the endoplasmic reticulum. Eventually,
misfolded and mis-assembled glycoproteins are targeted
for degradation by a process called ER-associated protein
degradation (ERAD). EDEM1 is an ERAD component that
recognizes misfolded luminal glycoproteins and is routing
them for dislocation to the cytosol. This is classically
followed by their degradation. Although EDEM1 was initially
proposed to be lectin–like and to react with Man8GlcNAc2
Acknowledgment
This work was supported by the World Class University
program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-2008-000-10086-0) and a grant from the
National Research Foundation of Korea by the Ministry of
Education, Science and Technology (2010-0027736). I.J. is
a fellowship awardee of the Brain Korea 21 program.
N-acetylglucosaminyltransferase V (GnT-V), a key enzyme catalyzing β1, 6-N-acetylglucosamine branching
on asparagine-linked oligosaccharides of cell proteins,
is overexpressed and as a metastasis-promoting
oncoprotein in invasion/metastatic colon and breast
432
cancers. However, here, we found that GnT-V expression
levels were strongly reduced during EMT of human
alveolar basal epithelial cells, in small cell lung cancers
and non-small cell lung cancers (NSCLC) except squamous cell carcinomas. Further study showed that suppression of β1-6GlcNAc glycosylation by swainsonine
and knockdown of GnT-V expression by stably
transfected with GnT-V shRNA in A549 cell line advanced EMT-like changes, cell-ECM adhesion, cell migration and invasion potential induced by TGF-β,
whereas additional expression of GnT-V reduced
TGF-β-induced-EMT, invasion and metastasis. Furthermore, we found that down regulation of GnT-V expression promoted metastasis formation by enhancing
TGF-β/Smad signaling, actin reorganization and
TGF-β/FAK signaling, which facilitates an EMT-like
switch, cells spreading, cellular adhesion to extracellular
matrix proteins, thereby allowing efficient cell migration
and invasion of metastatic lung cancer cells. Taken together, our findings reveal a novel mechanism of GnT-V
as a suppressor of both EMT and metastasis in human
lung cancer cells via TGF-β/Smad, TGF-β/FAK signalings inhibition that suppresses lung cancer metastasis.
315: Glycan Profiling and Quantitation of Cancer Cell
Exosomes and their Origin Cell Lines for Biomarker
Discovery
Seunghyup Jeong1, Serenus Hua1, Do-Young Choi2,
Pyong-Gon Moon 3 , Rudolf Grimm 1,4 , Kwang Pyo
Kim2, Moon Chang Baek3, and Hyun Joo An1*; 1Graduate
School of Analytical Science and Technology, Chungnam
National University, Daejeon 305-764, Korea, 2Department
of molecular biotechnology, Konkuk University, Seoul 143701, Korea, 3School of Medicine, Kyungpook National
University, Daegu 700-842, Korea, 4Agilent technologies,
Santa Clara, California, USA
shjeong0512@gmail.com
Exosomes (circulating microvesicles, extracellular
microvesicles, or microparticles) are small membraneenclosed vesicles in body fluids such as blood, urine and
ascites that are secreted by various cell types, including
tumor cells. These vesicles play an important role as mediators in extracellular communication. They are composed of
phospholipid bilayer membrane, cellular proteins, DNA,
and RNA derived from their origin cells. It is also known
that exosomes are involved in tumor metastasis, angiogenesis, and antitumor immunity in cancer cells. These biological functions are probably due to the glycosylation on their
membrane proteins. Thus, the study of glycosylation of
exosomes will be another potential source of new
Glycoconj J (2013) 30:281–461
biomarker. However, there is a little study about the glycosylation of exosomes. Here, we targeted and analyzed Nglycans of exosomes derived from five cancer cell lines
(A549, PC9, PC9/ZD, MCF-7, and MDA-MB231) using
nano-LC/MS. We also have compared glycans of exosomes
with glycans on their origin cell membrane to examine
glycosylation correlation between origin cells and exosomes
on cancer cell lines. Additionally, we have compared anticancer drug resistant cell line PC9/ZD (Gefitinib resistant)
and untreated PC9 in terms of origin cells and exosomes
glycan profiling via isomer separation. Exosomes and cell
membranes were prepared from three lung cancer cell lines:
A549, PC9, and PC9/ZD. And two breast cancer cell lines:
MCF-7 and MDA-MB231. These two types of samples
were isolated by ultracentrifugation. Glycans were directly
released by PNGase F, and then enriched by graphitized
carbon solid phase extraction. Nano-LC/chip Q-TOF MS
was used for overall glycan profiling and quantitation. We
successfully release and profile N-glycans from exosomes.
Origin cells and exosomes of lung cancer contain highmannose glycans in abundance. Although, exosomes
have less high-mannose glycans compared with origin cells.
Both origin cells and exosomes, isomer separation of
sialylated glycans are different between PC9 and PC9/ZD.
On breast cancer exosomes, two different metastatic samples represent quite different profiling. This is the first study
of comprehensive glycan profiling of exosomes using mass
spectrometry.
316: Isolation and Identification of Hyaluronandegrading Bacterial Strain and Anti-obesity Effects of
Low-molecular Weight Hyaluronan
Chang Won Lee1†, Joo Woong Park2†, Woon Seop Shin3,
Doo Jin Choi1, Jisun Lee1, Seul Lee1, Ji Won Choi1,
Young Hwan Kim2, and Yong II Park1*; 1Department of
Biotechnology, The Catholic University of Korea, Bucheon,
Gyeonggi-do 420-743, Korea, 2Biostream Technologies,
Yongin-si, Gyeonggi-do 446-930, Korea, 3The Kwandong University of Korea, Gangneung-si, Gangwon-do 210-701, Korea
yongil382@catholic.ac.kr
Hyaluronan (HA), also called hyaluronic acid or hyaluronate,
is an anionic, nonsulfated glycosaminoglycan distributed
widely throughout connective, epithelial, and neural tissues.
Hyaluronan, a core component of the extracellular matrix,
comprises a repeating disaccharide of N-acetylglucosamine
and D-glucuronic acid. A marine bacterial strain that showed
confluent growth on a minimal medium containing of
hyaluronan as the sole carbon source was isolated and identified based on the 16S rDNA sequence analysis as a strain of
Vibrio splindidus, and thus named Vibrio splindidus BST-398.
Glycoconj J (2013) 30:281–461
433
Low-molecular weight HA (LMW-HA) was produced from
the high-molecular weight HA (HMW-HA, 1,922 kDa) after
the reaction with a portion of culture filtrate as a crude enzyme
preparation for 4 h at 30 °C. The molecular weight of the
reaction product (LMW-HA) was estimated to be approximately 43.5 kDa by size exclusion HPLC (SEC). The antiadipogenic effect of LMW-HA was evaluated using 3T3-L1
preadipocyte cells. Both LMW-HA and HMW-HA did not
show any detectable level of cytotoxicity on 3T3-L1
preadipocyte cells at up to 200 μg/ml. Previously, we showed
that LMW-HA causes a significant reduction in intracellular
lipid drops by 53 % when measured by Oil Red O staining
while the HMW-HA showed no effect. The mRNA expression of PPAR-γ and aP2 in 3T3-L1 preadipocyte cells
maintained in adipocyte-induction media with 200 μg/ml
LMW-HA significantly and dose-dependently decreased by
56 % and 55 %, respectively, whereas HMW-HA treatment
resulted in only negligible effect (5 % and 5 % reduction,
respectively). PPAR-γ and aP2 are known as the major
adipocyte differentiation specific markers. In the present
study, we attempted to further confirm these observations
by measuring intracellular triglyceride (TG) levels and the
levels of differentiation marker proteins like PPAR-γ and
aP2. When measured by TG Colorimetric Assay Kit
(sigma), the TG level in LMW-HA treated cells significantly, dose-dependently decreased by 53 % at 200 μg/ml.
Western blot analysis also showed dose-dependent decreases in the protein levels of PPAR-γ and aP2 in
LMW-HA treated cells. Taken collectively, these results
clearly suggested that the LMW-HA may have an antiobesity effect by inhibition of the differentiation of
preadipocyte cells whereas the higher molecular weight
HA (HMW-HA) have no anti-obesity effect.
†
These two authors contributed equally to this work.
amino acids. People believed that full OGA exists both in
cytosol and nucleus, primarily to the cytoplasm. There were
two opposite reports about the subcellular localization of
OGA isoforms by different research groups. The first group
figured out that full OGA localizes in cytoplasm, whereas
variant OGA exists within nucleus in 2001. However, recently the other group reported that full OGA localized
diffusely throughout the nucleus and cytoplasm, whereas
variant OGA resides with lipid droplets in cytosol. These
two finding motivates our laboratory to commence this
study as to nucleocytoplasmic shuttling regulation of two
isoforms.
We also focus on the various post-translational modifications on OGAs. Full OGA should weigh 103 kDa, however,
it weighs approximately 130 kDa. So we speculate that
various modifications might be related to OGA’s subcellular
distribution as well.
To verify the nucleocytoplasmic regulation, we first
overexpressed FLAG tagged-OGA isoforms in HeLa cells
to trace the subcellular localization of them. Then we made
three C-terminal domain deletion mutants to know the critical domain for its localization. And we also check modification occurred on OGAs by using immune-precipitation
and mass spectrometry. Consequentially we can get schematic drawing as to OGA’s nucleocytoplasmic shuttling,
and we expect that this research makes it easy to approach
O-GlcNAc modification.
Acknowledgment
This work was supported by the World Class University
program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-2008-000-10086-0) and a grant from the
National Research Foundation of Korea by the Ministry of
Education, Science and Technology (2010-0027736).
317: The Identification of Nucleocytoplasmic Shuttling
Mechnism of O-GlcNAcase and Its PTMs
1
1, 2 1
Yeon Jung Kim , Jin Won Cho ; Department of Biology, Yonsei University, Seoul, 120-749, Korea, 2Department of integrated OMICS for Biomedical Science, WCU
Program of Graduate School,Yonsei University, Seoul, 120749, Korea
scientist@yonsei.ac.kr
O-GlcNAcase (OGA) is the enzyme which catalyzes the
removal of O-GlcNAc moiety on its substrates. This enzyme
has two isoforms, full OGA and variant OGA that has
alternative stop codon within intron which is not splicedout. As a result, variant OGA lacks C-terminal HAT (histone
acetyl transferase) domain and it contains additional 15
318: The Connection between Increased O-GlcNAcylation
by stortage of Glucose and Cancer Metastasis
Eunah Kim1, Jin Won Cho1,2; 1Department of Integrated
OMICS for Biomedical Sciences, Graduate School, Yonsei
University, Seoul 120-749, Korea, 2Department of Biology,
Yonsei University, Seoul 120-749, Korea
bioniceunah@hanmail.net, Tel:82-2-2123-7673
Most cancer cells need more glucose than normal cell.
because they produce energy by fermentation, even though
they exist in the presence of oxygen. Cancer cell can escape
a poor environment like hypoxia and undernutrition condition. Considering this fact, It is deducible that hypoglycemic
434
condition affect cancer metastasis. But the research about
cancer metastasis has been limited to hypoxia condition
until now. Interestingly, it was reported that O-GlcNAc
modification on protein was significantly increased under
hypoglycemic condition.
O-GlcNAcylation is one of the post-translational protein
modification and it appears to be involved in many
different cellular activities. And it was thought that OGlcNAcylation is also related with EMT(Epithelial–mesenchymal transition).
In our study, we want to examine whether hypoglycemic
condition can induce cancer metastasis and it is related to
increased O-GlcNAc modification or not. So we focused on
EMT to visualize cancer metastasis. We found that the
amount of several EMT marker were changed under hypoglycemic condition in cancer cell. And one of them was
O-GlcNAcylated.
These findings tell us that hypoglycemic condition can be
closely connected with cancer metastasis via OGlcNAclation. So we suggest possibilities that hypoglycemic condition induced EMT in cancer cell and it related with
increased O-GlcNAc modification.
Acknowledgment
This work was Supported by the World Class University
program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-2008-000-10086-0) and a grant from the
National Research Foundation of Korea by the Ministry of
Education, Science and Technology (2010-0027736).
319: Structural and functional analysis of the Alg1
glycosyltransferase involved in N-glycosylation of endoplasmic reticulum
Sha Xu 1,2, Sheng-Tao Li1,2, Xiao-Dong Gao 1,2*; 1School
of Biotechnology, Key Laboratory of Glycobiology and
Biotechnology, Ministry of Education, Wuxi 214122, China,
2
State Key Laboratory of Food Science and Technology,
Jiangnan University, Wuxi 214122, China
xus1984@gmail.com, xdgao@jiangnan.edu.cn
ALG1 encodes an essential beta-1,4-mannosyltransferas that
adds the first mannose moiety to the growing dolichallinked oligosaccharide (DLO) precursor on the cytosolic
face of the endoplasmic reticulum (ER). Alg1 protein physically interacts with Alg2 and Alg11 manosyltransferases,
which together catalyze the five mannose additions on the
ER cytosolic face. To further understanding of how these
Glycoconj J (2013) 30:281–461
mannosyltransferases function, we have undertaken a structural and functional study on eukaryotic Alg1 proteins. The
Saccharomyces cerevisiae Alg1p contains 449 amino acids
(51.9 kDa) with a hydrophobic region near the N-terminal.
Sequence analysis reveals its structural homology to the
heterodimeric Alg13/14 UDP-GlcNAc transferase, which
produces GlcNAc2-PP-dolichol intermediate before the reaction of adding first mannose by Alg1. Guided by the
structural information of Alg1p, our molecular biological
studies demonstrated that N-terminal hydrophobic region of
Alg1 works as a transmembrane domain and is required for
its ER membrane localization. Deletion of this domain
clearly stabilized the Alg1 protein indicating its possibility
to works as a target for protein degradation. Like the
Alg13/14 complex, scAlg1p possesses a second membrane
association site, which contributes to an integral interaction
with ER membrane. Interestingly, human Alg1p seems
lack such domain, might be due to the evolutional
necessary. In the presentation, we also will discuss the
protein regions of Alg1p required for the interaction
with other mannosyltransferases.
* Corresponding Author
320: Nuclear localization of O-GlcNAc transferase is
regulated by its own O-GlcNAc modification
Hyeon Gyu Seo, Ryum Joo Hwan and Jin Won Cho;
Department of Systems Biology, Yonsei University,
Seodaemun-gu, 120-749, Seoul, Korea
hyeongyuseo@gmail.com
It has been reported that one of the downstream molecules generated from glucose is uridine diphosphate-Nacetly glucosamine(UDP-GlcNAc) via the hexoamine
biopsynthetic pathway (HBP). The dynamic cycle of
addition and removal of O-linked-N-acetlyglucosamine
(O-GlcNAc) to Ser/Thr residues is involved in regulating
nuclear and cytoplasmic proteins. Nucleocytoplasmic OGlcNAc transferase (ncOGT) adds a single GlcNAc onto
hydroxyl groups of serine and threonine residues. Interestingly, O-GlcNAc glycosylation occurs in ncOGT itself
as well and several putative sites have been reported
mainly within TPR domain. However, the mechanism
by which nuclear translocation of O-GlcNAc transferase
is not clear. Here, we identified specific nuclear localization signals (NLS) in O-GlcNAc transferase that is
required for nuclear transport. The three amino acid
domain in NLS inserts a non-diffusible protein to the
nucleus autonomously. Also, we show that ncOGT binds
importin α proteins, and the association between
importin α proteins and ncOGT is interfered by O-
Glycoconj J (2013) 30:281–461
435
GlcNAcylation on TPR domain. This ongoing effort
would give us clear understanding of the key enzyme
of O-GlcNAc metabolism.
we will concentrate on demonstrating how the ATP synthase
β is modified with O-GlcNAc and what the functional roles of
O-GlcNAcylation on ATP synthase β are.
321: Functional relationship between ATP synthase beta
subunit and its O-GlcNAc modification
322: A sialidase exists on the surface of PNA-nonreactive
thymocytes and remove sialic acid specifically from
probably CD5
Joo-hwan Ryum1, Jin-won Cho1; 1Glycobiology Laboratory, Yonsei Univ., Seoul, 120-749, Republic of Korea
chojw311@yonsei.ac.kr
O-GlcNAcylation is the addition of β-N-acetylglucosamine(OGlcNAc) to serine or threonine residues of nuclear and cytoplasmic proteins. It is highly abundant on myriad proteins,
and cycles on proteins with a timescale similar to protein
phosphorylation, and has surprisingly extensive cross talk
with phosphorylation, where is serves as a nutrient/stress
sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation
underlie the etiology of insulin resistance and glucose
toxicity in diabetes, and this type of modification plays
a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also
regulated by O-GlcNAcylation.
Mitochondrial membrane ATP synthase(F1F0 ATP synthase
complex) produces ATP from ADP in the presence of a
proton gradient across the membrane which is generated
by electron transport complexes of the respiratory chain.
Subunits α and β form the catalytic core in F1, and rotation
of the central stalk agaist the surrounding α(3)β(3) subunits
leads to hydrolysis of ATP in three separate catalytic sites on
the β subunits. It is also known that ATP synthase β has
several phosphorylation site, and phosphorylation affects its
complex formation, and catalytic activity.
To verify O-GlcNAc modifications in Drosophila SL2 cell,
We performed immunoblotting with CTD 110.6 antibody. OGlcNAcylated proteins in Drosophila SL2 cell were analyzed
using two-dimensional gel electrophoresis and MALDI-TOFMS, and ATP synthase β subunit was identified as a novel OGlcNAcylated protein in Drosophila SL2 cell. In order to
check the O-GlcNAcylation of ATP synthase β, immunoblotting was performed with antibody of this protein after SWGA
lectin precipitation. Also, immunoblotting was performed
with ATP synthase β antibody and CTD 110.6 antibody after
immunoprecipitation with CTD 110.6 antibody and ATP
synthase β antibody, respectively. ATP synthase β subunit is
encoded in the nucleus, synthesized in the cytosol and finally
transported into the mitochondria. Interestingly, we found that
the O-GlcNAcylation of ATP synthase β subunit is increased
by nucleocytoplasmic O-GlcNAc transferase(ncOGT). Hence
Shigeko Kijimoto-Ochiai1,2, Toshiaki Koda2; 1Life Space
COSMOS, Sapporo, 060-0003 Japan, 2Graduate School of
Life Science and Creative research Institution, Hokkaido
University, Sapporo 001-0021, Japan
sko@cris.hokudai.ac.jp
In the thymus, T-cell progenitors progress through a series
of developmental stages from double-negative, doublepositive to single-positive T-cells. Mouse thymocytes that
were unadsorbed on a PNA-coated dish (PNAunad T) include
mainly sialylated mature (single positive) T cells and some
poorly glycosylated immature (double negative) T cells,
while double-positive immature cells adsorbed on the dish.
We prepared PNAunad T and analyzed the cells by flow
cytometory after staining with PNA-FITC. When the cells
were incubated in PBS alone, the cells were stained more
intensively with PNA-FITC than before incubation,
suggesting that some sialic acids were removed from the
cells during incubation. Thus, PNAunad T includes cells
having a sialidase on their surface. We therefore tried to
identify the glycoprotein from which sialic acids were removed. We found two bands by Western-blot analysis,
around 62 and 70 kDa, that were stained more strongly by
PNA after incubating the PNAunad T in PBS alone than
those without incubation, while we detected two new bands,
97 and 100 kDa, after incubating the cells with a soluble
cytosolic fraction from the thymus. We conclude that sialic
acids on PNAunad T were removed by a sialidase existing on
the cell surface during incubation, not by a soluble sialidase
released from dead cells. After removal of sialic acids from
the cell surface by exogenous sialidase, apoptotic cell death
was observed by FITC-annexin histogram analysis. We also
showed that the 62- and 70-kDa bands corresponded to the
bands detected with anti-CD5 antibody. CD5 is a cell surface glycoprotein expressed on T and B cells. In the thymus,
CD5 is expressed in double-positive and single positive
thymocytes and reported to tuning the interaction of the
TCR and self-peptide:MHC complex. Really if the 62- and
70-kDa bands are CD5, this sialidase on the cell surface
seems to have an important role in the thymus. [Acknowledgement] This study was started at Institute of Immunology when S.K-O. was there and supported technically by Dr.
C. Iwabuchi and Ms. T. Matsumoto-Mizuno for flow
cytometory analysis.
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323: During cell culturing the signal transduction in cells
is affected by the decrease of glucose concentration in the
media
Ho-Joong Seo1, Hanbyeol Kim2, Ji Young Yoon1 and Jin
Won Cho; 1Department of Integrated OMICS for Biomedical Sciences, Graduate School, Yonsei University,
Seoul 120-749, Korea, 2Department of Biology, Yonsei
University, Seoul 120-149, Korea
existstar@hanmail.net
The only glycosylation to be found in both nucleus and
cytosol, O-linked beta-Nacetylglucosamine(O-GlcNAc) is
one of the post-translational modifications. UDP-GlcNAc,
the sugar donor of O-GlcNAc, is mainly synthesized from
glucose by Hexosamine Biosynthetic Pathway, which is sensitive to glucose concentration. We confirmed through our
study, that a decrease of glucose in media can be observed
during cell culturing. Along with this change, OGlcNAcylation level also appeared to decrease. A comparison
was made between a glucose-concentration-maintained sample and a non-maintained sample, and a difference in insulin
sensitivity, indicated by phosphor-Akt, was observed. GSK3beta, a downstream target protein, was also found to be less
phosphorylated under glucose maintained condition meaning
the level of activated Akt decreased. These findings are evidences supporting the occurrence of the crosstalk between OGlcNAcylation and O-phosphorylation on Akt. From these
results we can also hypothesize that the signal transduction
can be influences by the natural change of glucose concentration in the media during cell culturing.
324: Self-Evolving Oxidative Stress With Identifiable
Pre- and Postmitochondrial Phases in PC12 Cells
Ganlin Zhang1,2, Xiaoxin Zhu3, Morin Christophe1,
Ping Li 4 , Xiaomin Wang 2 , Guowang Yang 2 , Dulce
Papy-Garcia1; 1Laboratoire CRRET, EAC CNRS 7149,
Faculté des Sciences et Technologie, Université Paris Est
Créteil, France, 2Beijing Hospital of Traditional Chinese Medicine, affiliated with Capital Medical University, Beijing, People’s Republic of China, 3Institute of Chinese Materia Medica,
China Academy of Chinese Medical Sciences, Beijing, People’s Republic of China, 4Beijing Institute of Chinese Medicine, Beijing, People’s Republic of China
Abstract: During the neurodegenerative process in several
brain diseases, oxidative stress is known to play important
roles in disease severity and evolution. Although early events
of stress, such as increased lipid peroxidation and decreased
superoxide dismutase, are known to characterize early onsets
of these diseases, little is known about the events that
Glycoconj J (2013) 30:281–461
participate in maintaining the chronic evolving phase influencing the disease pro- gression in neurons. Here, we used differentiated PC12 cells to identify premitochondrial and
postmitochondrial events occurring during the oxidative stress
cascade leading to apoptosis. Our data indicate that an acute
and strong oxidative impulse (500 lM H2O2, 30 min) can
induce, in this model, a 24-hr self-evolving stress, which
advances from a premitochondrial phase characterized
by lysosomes and cathepsin B and D translocations to
cytosol and early mitochondrial membrane hyperpolarization.
This phase lasts for about 5 hr and is followed by a
postmitochondrial phase distinguished by mitochondrial
membrane depolarization, reactive oxygen species increase,
caspase-9 and caspase-3 activations, and apoptosis. Inhibition
of cathepsins B and D suggests that cells can be protected at
the premitochondrial phase of stress evolution and that new
cathepsins regulators, such as glycosaminoglycans mimetics,
can be considered as new therapeutic prototypes for
neurodegeneration. Insofar as early oxidative stress markers
have been related to the early onset of neurodegeneration,
strategies protecting cells at the premitochondrial phase of
oxidative stress may have important therapeutic applications.
Key words: neurodegeneration; lysosome; cathepsin;
apoptosis; glycosaminoglycans
325: HBV infection downregulates miR-122 contributes
to Sorafenib resistance by targeting GALNT10 in hepatocellular carcinoma
Qian Wu1, Jiejie Xu1,2,*, Jianxin Gu1; 1Department of
Biochemistry and Molecular Biology, 2Key Laboratory of
Medical Molecular Virology, Ministry of Education and
Health, Shanghai Medical College, Fudan University,
Shanghai, China
jjxufdu@fudan.edu.cn
The pathological relevance and significance of microRNAs
(miRNAs) in hepatocarcinogenesis have attracted much attention in recent years; however little is known about the
underlying molecular mechanisms through which miRNAs
are involved in the development and progression of hepatocellular carcinoma (HCC). In this study, we demonstrated
that miR-122 is frequently down-regulated in HCC and that
its expression is further suppressed by hepatitis B virus X
protein (HBx). Furthermore, the reduced expression of miR122 could be rescued by over-expression of hepatocyte nuclear factor 4α (HNF4α). GALNT10 (N- acetylgalactosamine
transferase-10) was identified as the direct and functional
target of miR-122 with integrated bioinformatics analysis
and messenger RNA array assay. This regulation was further
confirmed by luciferase reporter assays. In addition, our
Glycoconj J (2013) 30:281–461
results, for the first time, showed that GALNT10 was frequently increased in HCC by way of immunohistochemical
staining assays. The increased expression of the GALNT10
significantly inhibited, whereas knockdown of the GALNT10
markedly enhanced sorafenib induced cell apoptosis and proliferation inhibition in HBx transfected hepatomca cells. Conclusion: The newly identified miR-122/GALNT10 axis
elucidates the molecular mechanism of HBV-related HCC
sorafenib resistance and represents a new potential therapeutic
target for HCC treatment.
326: HBV promotes migration and invasion of hepatoma
cells through miR-9 mediated down-regulation of
GALNT4
Yidong Liu1, Jiejie Xu1,2*, Jianxin Gu1; 1Department of
Biochemistry and Molecular Biology, 2Key Laboratory of
Medical Molecular Virology, Ministry of Education and
Health, Shanghai Medical College, Fudan University,
Shanghai, China.
jjxufdu@fudan.edu.cn
Recent evidence indicates that aberrant O-glycosylation may
play a critical role during tumorigenesis including the development of hepatocellular carcinoma (HCC). Polypeptide
GalNAc-transferases are a number of enzymes which are
responsible for the initiation of mucin-type O-linked protein
glycosylation, in which N-acetylgalactosamine is transferred
to serine and threonine amino acid residues. Here, we found
that HBV down-regulates GALNT4 expression significantly
in HCC cells. And over-expression of GALNT4 in vitro can
attenuate HBV driving migration and invasion of HCC cells.
Further studies showed that HBV activates NF-κB to upregulate miR-9, which directly targets the 3′UTR of GALNT4
mRNA to promote its degradation, thus the deregulation of
GALNT4 mediating O-glycosylation induces malignant behavior of HCC cells. Moreover, we found an inverse correlation between GALNT4 and HBV infection in HCC clinical
specimens. Taken together, this study reveals a novel regulatory circuit adopted by HBV, which may contribute in part to
migration and invasion of HCC cells. Hence, strategies that
blockade this circuit might be developed to eliminate the
metastatic potential of HCC cells conferred by HBV.
327: Galectin-9 affects phagocytosis in THP-1 cells
Chi-Shan Li1, Dan Hsu1,2, Fu-Tong Liu1,2, Huan-Yuan
Chen1,2; 1Institute of Biomedical Sciences, Academia
Sinica,Taiwan, 2Department of Dermatology, University of
California, Davis, School of Medicine, Sacramento, CA, USA
chishan@ibms.sinica.edu.tw
437
Galectin-9 (Gal9), a member of the beta-galactoside-binding
animal lectin family, is involved in various cellular biological events, including tumor cell adhesion, metastasis, and
modulation of various immune responses. Gal9 is expressed
in antigen presenting cells (APCs) including macrophages
and dendritic cells. Since the main functions of APCs are
antigen recognition, uptake and presentation to T cells, it is
possible that Gal9 affects the biological functions of APCs.
By immunoblotting and immunofluorescence staining, we
found that Gal9 is expressed in the cytoplasm of THP-1, a
human monocytic leukemia cell line. With treatment of
phorbol esters, THP-1 cells could differentiate into
macrophage-like cells. By using fluorescein-labeled ovalbumin and dextran, the ability of endocytosis was decreased in
Gal9 knocking down (Gal9KD) THP-1 derived macrophages
compared to control cells. Moreover, Fc receptor-mediated
phagocytosis of opsonised sheep red blood cells was also
reduced in Gal9KD THP-1 derived macrophages compared
to control cells. There were no differences of IL6 and IL1-β
levels between control and Gal9KD THP-1 cells when
treating cells with 1 μg/ml LPS for 24 h. Since it has been
demonstrated that Gal9 is one of the components of
phagosomes in human dendritic cells, the intracellular
Gal9 may play an essential role for antigen uptake in
human APCs.
328: Role of Galectins in Lung Cancer Progression
Po-Cheng Chiang1, Dan Hsu1, 2, Fu-Tong Liu1, 2, HuanYuan Chen1, 2; 1Institute of Biomedical Sciences, Academia Sinica, Taiwan, 2Department of Dermatology, University of California, Davis, School of Medicine, Sacramento,
CA, USA
cpc0325@ibms.sinica.edu.tw
Non small cell lung cancer (NSCLC) is one of the most
common cancers and the leading cause of cancer-related
death in many countries around the world. A large body of
experimental and clinical work supports the view that epidermal growth factor receptor (EGFR) is a relevant target
for lung cancer therapy. Somatic mutations within the tyrosine kinase domain of EGFR are the most reliable predictors
of response to EGFR tyrosine kinase inhibitors (TKI), such
as Iressa (Gefitnib), in patients with NSCLC. Deletions in
exon 19 and a point mutation L858R in exon 21 account for
over 90 % of all sensitizing mutations and result in the
constitutive activation of EGFR that promote cell proliferation and survival. Galectins are a family of evolutionaryconserved carbohydrate-binding proteins. They are distributed widely in organisms and have been implicated in many
essential functions including development, differentiation,
cell-cell adhesion, cell-matrix interaction, growth regulation
438
and apoptosis. Several members of the galectin family have
also been shown to be involved in cancer progression and
metastasis. Galectin-1 and -3 can mediate neoplastic transformation and cell cycle progression by regulating ras activity and cell-cycle regulators expression. Our Q-PCR data
indicate that galectin-1, -3 and -7 are expressed in lung
cancer cells with distinct EGFR status. Galectin-1 has been
reported to be an important factor to promote lung cancer
progression and chemoresistance. However, role of galectin3 and -7 are still unclear in lung cancer. In our preliminary
data, knockdown of galectin-3 reduced cellular ability of
migration, proliferation, colony formation and anchorageindependent cell growth in several lung cancer cells.
Overexpression of galectin-7 reduced cell proliferation,
invasion and migration in PC9 cells. Interestingly,
overexpression of galectin-7 led to Iressa resistance in
both CL100 and PC9 cells. Comparing to parental PC9
cells, higher galectin-7 level was observed in PC9-IR,
an acquired resistant cell, suggesting the correlation
between galectin-7 and Iressa sensitivity. Taken together,
galectin-3 and -7 may play an important role in mediating lung cancer progression and resistance to TKIs
therapy in lung cancer.
329: Wnt/β-catenin up-regulates hFUT8 expression in
hepatocarcinoma cell SMMC-7721
Nanyang Li♣ 1, Lijun Zhang♣ 1, Linhua Liu 1, Shujing
Wang1, Jianhui Fan1, Jianing Zhang 1*; 1 Department of
Biochemistry, Institute of Glycobiology, Dalian Medical
University, Dalian 116044, Liaoning Province, China
♣
These authors contributed equally to this work.
*Correspondence to: Jianing Zhang
jnzhang@dlmedu.edu.cn
Wnt signaling plays important role in determining cell fate
during embryogenesis and maintaining the tissue homeostasis
after birth. β-catenin is a key component of Wnt signaling and
mediates canonical Wnt pathway. The protein glycosylaiton is
widely exit of the organism and is closely related to the
disease. Fucosylation is one of the glycosylation and is catalyzed by Fucosyltransferase. Fucosyltransferase 8 (FUT8) is
only one enzyme to catalyze α1, 6-fucosylation in mammals.
Up-regulation of FUT8 has been observed in several malignant cancers including liver, ovarian, thyroid, and colorectal
cancers. However, the pathological role and the regulatory
mechanism of FUT8 in cancers remain largely unknown. In
this study, we report that FUT8 is up-regulated by Wnt signaling activator LiCl and overexpressed β-catenin in
hepatocarcinoma cell line SMMC-7721. In contrast, knocking
down β-catenin can down-regulates expression levels of
FUT8 in SMMC-7721. And then, Bioinformatic analysis
Glycoconj J (2013) 30:281–461
predicted that Fut8 promoter region contains TCF/LEF binding sites. So, we constructed hFut8 gene reporter vector
that contain hFut8 region from −1910 to +329. Reporter
assay showed that transcriptional activity of hFut8 gene
is significantly increased by Wnt-1, β-catenin in
SMMC-7721. Taken together, these data suggest that
Wnt/β-catenin signaling can up-regulates hFUT8 expression
in human hepatocarsinoma cell SMMC-7721. This work was
supported by grants from the Major State Basic Research
Development program of China (2012CB822103), and National Natural Science Foundation of China (31170774,
31000372 and 31000618).
330: Shedding of GPI-anchored proteins by a GPIcleaving enzyme
Morihisa Fujita1, Gun-Hee Lee1, Yoshiko Murakami1,
Yusuke Maeda1, and Taroh Kinoshita1,1 Research Institute for Microbial Diseases, Suita, Osaka, 565-0871, Japan
morihisa@biken.osaka-u.ac.jp
Glycosylphosphatidylinositol (GPI) anchoring of proteins
is a conserved post-translational modification in eukaryotes. It has been suggested that GPI-anchors act as functional molecules regulating trafficking and membrane
localization of the modified proteins. One of the characteristic
features of GPI-anchored proteins is that these proteins are
releasable from the cell membrane by cleaving GPI moieties.
There are several GPI-cleaving enzymes. Here, we identified a
novel GPI-cleaving enzyme. PGAP3 is required for the removal of an unsaturated fatty acid from GPI lipid at the Golgi
apparatus, which is essential for GPI fatty acid remodeling. By
homology to PGAP3 that belongs to a transmembrane hydrolase superfamily, an uncharacterized protein, designated
PGAP6a, was found in the superfamily. PGAP6a is a transmembrane protein conserved among metazoa and was mainly
localized at the cell surface. In PGAP6a overexpressing cells,
surface expressions of several GPI-anchored proteins including CD55, CD59 and Sca-1 were significantly decreased.
These results suggest that PGAP6a is a novel GPI-cleaving
enzyme at the cell surface.
331: Research on effect of expression of β1,4-GalT-I by
estrogen and progesterone and correlation with EGFR
signaling pathway
Ying Xia1, Yunpeng Xie, Cheng Zhang, Ting Gong, lin
Zhang, Ying Kong *; 1Department of Biochemistry, Dalian
Medical University. Dalian, Liaoning Province. People’s
Republic of China.
kongying@dlmedu.edu.cn
Glycoconj J (2013) 30:281–461
439
β1,4-Galactosyltransferase-I(β1,4-GalT-I) gene codes two
proteins, long form and short form: The short and some
long form play the role in glycosyltransferase, which mainly
transfer galactose from UDP-galactoside to GlcNAc, and
form Galβl,4-GlcNAc; The other parts of the long form
β1,4-GalT-I,which distribute on cell membrane surface,
play a crucial role as a cell adhesion molecule through
recognizing and adhering other extracellular matrix and
galactose of cell surface glycoprotein and glycolipid. Epidermal growth factor receptor(EGFR) is a multi-function
transmembrane protein, which widely distributes on cell
membrane surface of each human body tissues. EGFR has
protein tyrosine kinase(PTK) activity which is a important
receptor. After binding to its ligand, EGFR starts intracellular signal transduction through the cytoplasm adapter protein and enzyme cascade reaction, then regulates nuclear
transcription factor kappa B (NF-κB) gene. The enabled
NF-κB regulates intercellular adhesion molecule-I(ICAM1) transcription through MAPK/PKC and other signal pathway, sequentially controls the blastocyst implantation. In
this study, we demonstrated the female progestational hormone induced the β1,4-GalT-I and Galβl,4-GlcNAc upregulation in RL95-2 cells; Under the effect of progesterone,
the expression of β1,4-GalT-I was positively correlated with
EGFR related pathways; Inhibition of β1,4-GalT-I increased
EGFR protein. In addition, female progestational hormone
promoted the adhesion of blastocystscells in vitro in β1,4GalT-I-dependent manner. β1,4-GalT-I,which was regulated
by progesterone, induced embryo implantation via EGFR
related signal path.
gangliosides on cell migration and its correlation with activation of EGFR and cMet in Hepa1-6 cell lines whose motility
and migration in vitro can be stimulated by both EGF and
HGF. We found that (i) When infection with the CD82 gene,
decreased migration ability was observed in Hepa1-6 cells that
were induced by EGF and HGF. Transfection of the CD82
gene to Hepa1-6 inhibited EGF-stimulated phosphorylationof
EGFR at tyr1173 and contributed to the attenuation of EGFR;
while, ectopic expression of CD82 in Hepa1-6 inhibited HGFstimulated tyrosine phosphorylation of cMet at tyr1313 and
tyr1365 without affecting the expression of cMet. These
inhibitory effects were enhanced when CD82 is in conjunction with GM3 or particularly with GM2/GM3. (ii)
Reduction of CD82 expression by RNA interference and
depletion of glycosphingolipids with P4 together, significantly enhanced cell motility and increased expression of
EGFR and activation of EGFR at tyr1173 stimulated by
EGF; however, increased cell motility and activation of
cMet at tyr1313 and tyr1365 stimulated by HGF, were not
only due to the effect of decreased CD82 alone but also
on account of the effect of increased GM3. Furthermore,
CD82 selectively attenuated EGFR and cMet signaling via
phosphatidylinositol 3-kinase/Akt,but had no affect on the
activity of MAPK signaling pathway. These results suggest
that the different effects of CD82 and synergistic effects
with GM3 and GM2/GM3 on the phosphorylation and
expression of EGFR and cMet may be associated with a
mechanism by which CD82 exerts inhibitory actions on
the motility and migration of Hepa1-6 cells induced by
EGF and HGF in vitro.
(This work was supported by National Natural Scientific
Grant No.30970646,PR China)
Poster Session II-Physiology & Signalling
332: The Synergistic Inhibitory Effect of Tetraspanin
CD82 and Gangliosides on Cell Migration through
EGFR or cMet-activated Pl3K/Akt signal pathway
Ying Li1#, Xiaohua Huang1, 2#, Jianing Zhang1, Keli
Ma1*; 1 Department of Biochemistry and Molecular Biology,
Dalian Medical University, Dalian 116044, China;2 Department of Clinical Biochemistry, College of Laboratory Medicine, Dalian Medical University, Dalian 116044, China
1
*Corresponding author: makeli666@yahoo.com.cn
#
These authors contributed equally to this work
It has been proposed that the metastasis suppressor
CD82/KAI-1,which is a member of the tetraspanin superfamily, exerts its biological activity by associated with
glycosphingolipids (GSLs); however, its mechanism has not
been fully elucidated. The present study aimed to investigate
the synergistic inhibitory effect of Tetraspanin CD82 and
333: α1, 6-Fucosylation bidirectionally regulates
TGF-β/activin-mediated signaling
Wei Gu, Tomohiko Fukuda, Tomoya Isaji, Hirokazu
Hashimoto, Yuqin Wang and Jianguo Gu; Division of
Regulatory Glycobiology, Institute of Molecular
Biomembrane and Glycobiology, Tohoku Pharmaceutical
University, 4-4-1 Komatsushima, Aoba-ku, Sendai Miyagi,
981-8558, Japan
jgu@tohoku-pharm.ac.jp
It is well known that α1,6-fucosyltransferase (Fut8) and its
products, α1,6-fucosylated N-glycans, are highly expressed in
brain tissue. Recently, we reported that Fut8-knockout mice
exhibited multiple behavioral abnormalities with a
schizophrenia-like phenotype, suggesting that α1,6fucosylation plays important roles in the brain and neuron
system. In the present study, we screened several neural cell
lines, and found that PC12 cells express the highest levels of
440
Glycoconj J (2013) 30:281–461
α1,6-fucosylation. The knockdown (KD) of Fut8 promoted a
significant enhancement of neurite formation and induction of
neurofilament expression. Surprisingly, the levels of phosphoSmad2 were greatly increased in the KD cells. Finally, we
found that the activin-mediated signal pathway was essential
for these changes in KD cells. Exogenous activin, not
transforming growth factor beta (TGF-β), induced neurite outgrowth and phospho-Smad2. In addition, the α1,6-fucosylation
level on the activin receptor 2A was greatly decreased in KD
cells, whiles the total expression level was unchanged,
suggesting that α1,6-fucosylation negatively regulated
activin-mediated signaling. Furthermore, inhibition of activin
receptor-mediated signaling or restoration of Fut8 expression
rescued cell morphology and phospho-Smad2 levels, which
were enhanced in KD cells. Considering the fact that α1, 6fucosylation is important for TGF-β-mediated signaling,
the results of this study strongly suggest that Fut8 plays
a dual role in TGF-β/activin-mediated signaling.
According to HSQC and HMBC analysis, the mannuronic
acid is found to be β-1,4-linked, while guluronic acid is α1,4-linked, as in common alginate. The nuclear kappa B
(NF-κB) pathway plays a key role in the signaling transduction in many pathological events, particularly in the innate
immune response. To investigate the activity and mechanism
of action of the algal polysaccharides on immune system, the
effect of 04S2P on NF-κB signaling pathway was tested, and
the result showed that the activation rate was close to 52 %.
However, 04S2P displayed no significant activation after the
sample was treated with Affi-Prep polymyxin matrix to
remove endotoxin. In comparison, commercial alginate with
a lower molar ratio of M/G (1.9:1) demonstrated an
obvious inhibition to NF-kB even after endotoxin was
removed. Therefore, the M/G molar ratio, glycosyl sequence, and the contamination of endotoxin in alginic
acid all probably contribute to its activity on NF-κB
signaling pathway.
334: The effect of an alginic acid from Sargassum
fusiforme on NF-κB signaling pathway
335: Structure characterization of a glucuronoxylan
isolated from Cassia obtusifolia seeds and its effect on
NF-κB signaling pathway
Qifei Cong, Xinyan Ni, Qun Dong, Kan Ding;
Glycobiology and Glycochemistry Lab. Shanghai Institutes
of Materia Medica, Chinese Academy of Sciences,
Shanghai 201203, China
qdong@mail.shcnc.ac.cn (Q. Dong), kding@mail.
shcnc.ac.cn (K. Ding)
Sodium alginate is a natural polysaccharide abundant in
various brown sea weeds, which is used in pharmaceutical
industry as a suspending agent, a disintegrant, or a tablet
binder. Alginate is a linear polysaccharide containing 1,4linked β-D-mannuronic acid (M) and 1,4-linked α-Lguluronic acid (G) in various proportions dependent on the
algal species and growth conditions, arranged in either homogeneous or heterogeneous block patterns. As an effort to
find immunoregulatory polysaccharides, we try to obtain the
polysaccharide fractions and evaluate their NF-κB activation
or inhibition activity. An alginic acid polysaccharide, 04S2P,
was isolated from Sargassum fusiforme, by boiling water
extraction, DEAE-Cellulose anion-exchange chromatography, and Sephacryl S-300 HR gel permeation chromatography, as well as by acid precipitation. The average molecular
weight of 04S2P, as determined by high performance gel
permeation chromatography, was 29.2 kDa. The sugar compositions, as characterized by high performance liquid chromatography method of pre-column derivatization with 1phenyl-3-methyl-5-pyrazolone (PMP), were mannuronic acid and guluronic acid. The molar ratio of mannuronic acid to
guluronic acid was estimated to be 7.5: 1.0, according to
integral of anomeric proton resonances in 1 H NMR.
Qifei Cong, Mingsheng Shang, Xinyan Ni, Qun Dong,
Kan Ding; Glycobiology and Glycochemistry Lab.
Shanghai Institutes of Materia Medica, Chinese Academy
of Sciences, Shanghai 201203, China
qdong@mail.shcnc.ac.cn (Q. Dong), kding@mail.shcnc.
ac.cn (K. Ding)
Xylan is one of the major hemicelluloses, which generally
consists of a backbone of 1, 4-linked β-D-xylose units, in
most cases, with α-L-arabinfuranosl and 4-O-methyl-α-Dglucuronopyranosyl residues occasionally attached to O2/O-3 as side groups. In this study, a heteroxylan, designated as COB1B1S2, was isolated from the alkaline extract of
the whole seeds of C. obtusifolia, by ethanol precipitation,
DEAE-cellulose anion-exchange chromatography, and
Sephacryl S-300 gel permeation chromatography. Using
chemical and spectroscopic methods, the polysaccharide
was elucidated to be a glucuronoxylan. Its average molecular weight was estimated to be 70.4 kDa. The polysaccharide , as dete rmined by HPLC a fter p re-co lumn
derivatization with 1-phenyl-3-methyl-5-pyrazolone
(PMP), contains xylose, glucuronic acid, in the molar ratio
of 7.8: 1.0. Combined with methylation analysis and NMR
spectroscopy, COB1B1S2 was elucidated to contain a
poly-(1→4)-β-D-xylose backbone, with α-D-glucuronosyl
residues as branches, substituted exclusively at O-2 position. Interestingly, no 4-O-methyl-D-glucuronic acid was
identified, which is reported to be present in most
heteroxylans, and this characterizes COB1B1S2 as a novel
Glycoconj J (2013) 30:281–461
glucuronoxylan different from those reported previously. As
one of the most important nuclear factors, NF-κB takes part
in the signaling transduction in the innate immune system.
To investigate the biological activity of the xylans on immune system, the effect of COB1B1S2 on NF-κB signaling
pathway was tested, and the result showed that the activation rate was close to 77 %. Furthermore, the polysaccharide
remains to exhibit an activation rate of 40 % even after it
was treated with Affi-Prep polymyxin matrix to remove
possible endotoxin contamination. In conclusion, a novel
glucuronoxylan was isolated from Cassia obtusifolia seeds,
and it was shown to be capable of activating NF-κB signaling pathway, indicating a potential immunostimulatory
activity.
336: Mice lacking fucosyltransferase 8 exhibit abnormal
behavioral abnormalities associated with a schizophrenialike phenotype
Tomohiko Fukuda, Wei Gu, Tomoya Isaji, Naoyuki
Taniguchi, and Jianguo Gu; Division of Regulatory
Glycobiology, Institute of Molecular Biomembrane and
Glycobiology, Tohoku Pharmaceutical University, 4-4-1
Komatsushima, Aoba-ku, Sendai Miyagi, 981-8558, Japan,
Disease Glycomics Team, RIKEN, Advanced Science Institute, Wako 351-0198, Japan
tfukuda@tohoku-pharm.ac.jp
α1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a
fucose residue from GDP-fucose to position 6 of the innermost GlcNAc residue to form α1,6-fucose in hybrid and
complex N-linked oligosaccharides of glycoproteins. α1,6Fucosylated glycoproteins are abundantly present in brain
tissue. We investigated the physiological functions of α1,6fucose in brain tissue by using Fut8-deficient (Fut8KO) mice.
We examined Fut8KO mice using a combination of neurological and behavioral tests. Fut8KO mice exhibited multiple
behavioral abnormalities consistent with a schizophrenia-like
phenotype. Fut8KO mice displayed increased locomotion
compared with wild-type and heterozygous mice. In particular, Fut8KO mice showed strenuous hopping behavior in a
novel environment. Novelty-induced hyperactivity has been
viewed as a preclinical model of the positive symptoms of
schizophrenia and of psychomotor agitation in particular.
Working memory performance was impaired in Fut8KO mice
as evidenced by the Y-maze tests. Deficits in social interaction
are hallmarks of schizophrenia. Furthermore, Fut8KO mice
showed prepulse inhibition (PPI) deficiency, which had been
reported in schizophrenia patient as well as mouse models.
These results suggest that reduced expression of Fut8 is a
plausible cause of schizophrenia and related disorders. Because locomotor hyperactivity is commonly associated with
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increased monoamine tone, the effect of Fut8 deficiency on
monoamine turnover was determined by HPLC. The levels of
serotonin metabolites were significantly decreased in both the
striatum and nucleus accumbens of the Fut8KO mice. Likewise, treatment with haloperidol, which is an antipsychotic
drug that antagonizes dopaminergic and serotonergic receptors significantly reduced hopping behaviors. Based on these
results, we hypothesize that the balance between dopaminergic and serotonergic signaling might be disrupted in the
Fut8KO mice.
Recently, Electrophysiologic data of hippocampal CA1 region showed the long-term potentiation was decreased in
Fut8KO mice, compared with Wild-type mice. Although the
detailed underlying molecular mechanisms remain unclear,
the present study is the first to clearly demonstrate that α1,6fucosylation plays an important role in the brain, and that it
might be related to schizophrenia-like behaviors.
337: GnT-V expression levels regulate colon cancer stem
cells and colon adenoma progression of Apcmin/+ mice
through a canonical Wnt signaling pathway
Huabei Guo1*, Heather Johnson1, Tamas Nagy2, and
Michael Pierce1*; Department of 1 Biochemistry and Molecular Biology, Complex Carbohydrate Research Center,
and 2 Pathology, College of Veterinary Medicine, University
of Georgia, Athens, GA,30606
*Address correspondence: Department of Biochemistry and
Molecular Biology and Complex Carbohydrate Research
Center, 315 Riverbend Rd. University of Georgia, Athens,
Georgia 30602; Tel: 1-706-542-1701; Fax: 1-706-542-1759;
hawkeye@uga.edu; hbguo@uga.edu
There is increasing evidence that a variety of cancers are
initiated and maintained by a small proportion of cells called
cancer stem cells (CSC). Our recent results show that deletion
of GnT-V, which catalyzes a specific modification of Nglycans with β(1,6) branching, reduced the size of the compartment of CSC in the her-2 mouse model, consequently
leading to a inhibited tumor onset. GnT-V expression is increased during the oncogenesis of human colorectal cancer,
including adenoma formation. In the present study, the regulation of colon CSC and colon adenoma development were
investigated both in vitro and in vivo. Overexpression of GnTV increased anchorage-independent cell growth and tumor
formation induced by injection of colon tumor cells into
NOD/SCID mice, indicating a regulation of cell proliferation
and tumorigenicity by GnT-V. Using Apcmin/+ mice with
different GnT-V backgrounds, we found that knockout of
GnT-V had no significant effect on intestine (colon) adenoma
formation (number of adenoma/mouse), but adenoma size
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was remarkably reduced, which was accompanied by
increased survival of ApcMin/+ mice with GnT-V deletion
(p<0.01), suggesting an inhibition in the progression of colon
adenoma caused by deletion of GnT-V. Increased expression
level of GnT-V up-regulated the population of colon
(intestine) CSC by affecting their self-renewal, and their tumorigenicity in NOD/SCID mice. Furthermore, altered nuclear translocation of β-catenin and expression of Wnt target
genes were observed after aberrant expression of GnT-V,
indicating the regulation of canonical Wnt/β-catenin
signaling by GnT-V. These results demonstrate that
GnT-V expression and its branched N-glycan products
effectively modulate Wnt signaling pathway that, in
turn, regulates the relative proportion of colon CSC, as
well as colon tumor development, thereby providing a
potential therapeutic target for the inhibition of colon
carcinoma progression.
338: Study of Chitosan oligosaccharide inducing resistance to TMV virus on Arabidopsis thaliana and its
resistance pathway
Qingshan Meng1,2, Yin Heng1, Duyu Guang1*; 1Dalian
Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023 China; 2Dalian Ocean University,
Dalian 116023 China
dyguang@gmail.com
Many recent researches have found chitosan oligosaccharide
is an effective elicitor which can induce plant to resist against
pathogen. In my paper, we use different concentrations of
chitosan oligosaccharide solution to spray plant leaves. After
pretreating leaves 24 h, 48 h and 72 h individually, TMV virus
is injected in plant leaves and incubated for different days.
Then I use western blot to detect TMV coat protein as an
indicator to quantify TMV. By using this model,I have
screened 50 ppm as an optimum concentration of chitosan
oligosaccharide to pretreat wild type Arabidopsis thaliana
24 h and incubate TMV virus for 7 days in Arabidopsis
thaliana leaves which can resist against TMV virus efficiently.
By the way we also build a platform which can be used to
research oligosaccharides inducing Arabidopsis thaliana resistance to resist against TMV virus successfully.
339: HCG to β1,4-GalTI in embryonic implantation and
the influence of role and regulation mechanism analysis
Lili Chen1, Cheng Zhang,Ting Gong, lin Zhang, Ying
Kong *; 1Department of Biochemistry and Molecular Biology, Dalian Medical University, Dalian, 116044, China.
kongying@dlmedu.edu.cn
Glycoconj J (2013) 30:281–461
The change of β 1,4 - GalTI expression level in endometrial
cancer cells (RL95-2 cells),which can be seen as implantation
period endometrial cells, as well as the adhesive ability of
embryos and endometrial was discussed after the treatment of
hCG.At the same time, some pathways have been investigated. Control RL95-2 cells β 1, 4 - GalTI expression, testing the
corresponding matrix metalloproteinase MMPs and extracellular matrix ECM expression change, analysis and utilization
of endometrial adhesion of influence, further discussed β 1, 4
- GalTI embryonic implantation and the molecular mechanism
Methods: (1)Building model in vitro implantation in villi
cancer cells (JAR cells) simulation before implantation embryo to endometrial cancer cells (RL95-2 cells) simulation
before implantation endometrial; Change the endometrial environment hCG levels, western blot, RT - PCR detection
technology of hCG β 1, 4 - GalTI expression influence;
Immune cell chemical dyeing analysis under the action of
hCG β 1, 4 - GalTI galactosyl change change; RCA - 1 Lectin
- blot technical analysis under the action of hCG Gal β 1, 4 GlcNAc glycoprotein branch form; HCG test influence related
signal path EGFR protein expression level; Respectively
through the fluorescence microscope and flow cytometry
analysis technology RL95-2 cells and JAR intercellular adhesion influence.(2)Beta-secretase 1, 4 - GalTI gene regulation
plasmid (after expression plasmid, interference plasmid), interference contrast plasmid and empty plasmid transfection
respectively to RL95-2 cells, using immunofluorescence, RT PCR and western blotting technology, immune cell chemical
dyeing technology respectively the beta testing each transfection cell 1, 4 - GalTI expression and the influence of MMPs,
ECM expression; Respectively through the fluorescence microscope and flow cytometry technique to observe β 1, 4 GalTI to in vitro implantation model RL95-2 cells and JAR
cell adhesion influence; RT - PCR detection technology of
HCG β 1, 4 - GalT I and MMPs - TIMP - ECM path
expression effect. Results: (1) endometrial cells β 1, 4 - GalT
- I expression and hCG is dose, time - effect relationship, hCG
best action conditions for 5×10-4 ug/ul, 36 h (P<0.01); (2)
under the action of hCG obviously raised EGFR, the nf-kappa
B, ICAM - 1, EGF expression (P<0.01); (3) hCG can promote
JAR cell to RL95-2 cell adhesion effect (P<0.01); (4) endometrial cells transfection β 1, 4 - GalT - I gene regulation
plasmid after express group can obviously increase MMP2,
MMP9, and significantly cut LN, TIMP - 1, interference
group can obviously cut MMP2, MMP9, and obviously increase LN, TIMP - 1 (P<0.01); (5) the endometrial cells
transfection β 1, 4 - GalT - I gene regulation plasmid after
express group can obviously improve the JAR cell to RL95-2
cell adhesion effect, interference group can make the JAR cell
to RL95-2 cell adhesion effect decrease (P<0.01); (6) RT PCR results show that obvious increase hCG MMP2, MMP9
expression, cut LN, TIMP - 1 expression (P<0.01). Conclusion: (1) hCG adjustable endometrial cells β 1, 4 - GalT - I
Glycoconj J (2013) 30:281–461
expression (present time, dose dependency relationship), promote the JAR cell to RL95-2 cell adhesion effect; (2) hCG
adjustable endometrial cells EGFR signaling pathways related
molecular expression to adjust and control the embryo implantation. (3) regulation of β 1, 4 - GalT - I express influence
embryo implantation may through the MMPs, ECM related
signal path, adjust then JAR cell to RL95-2 cell adhesion
effect. (4) hCG adjustable endometrial cells MMPs - TIMP ECM path expression to adjust and control the embryo
implantation.
(This work was supported by National Natural Ssientific
Grant No.30970646,PR China)
340: Molecular Mechanisms of Lycoris Aurea
Agglutinin-induced Apoptosis and G2/M Cell Cycle Arrest in Human Lung Adenocarcinoma A549 Cells both
in in vitro and in vivo
Chun-yang Li, Zheng shi, Jingchun Zhang, Yong-xi Liu,
Jin-ku Bao*; School of Life Sciences, Sichuan University,
Chengdu 610064, China
baojinku@scu.edu.cn
Lycoris aurea agglutinin (LAA) has been drawing rising
attention due to its remarkable bioactivities. Herein, we
report that LAA triggers G2/M phase cell cycle arrest via
up-regulating p21 expression as well as down-regulating
cdk1-cyclinA singling pathway, and induces apoptotic cell
death through inhibiting PI3K-Akt survival pathway in human lung adenocarcinoma A549 cells. While LAA has no
significant cytotoxic effect toward normal human embryonic lung fibroblast HELF cells, and moreover, LAA could
amplify the anti-neoplastic effects of cisplatin toward A549
cells. Lastly, LAA also bears anti-cancer and apoptosisinducing effects in vivo, and it could decrease the volume
and weight of subcutaneous tumor mass obviously as well
as expand lifespan of mice. These findings may provide a
new perspective for elucidating the complicated molecular
mechanisms of LAA-induced cancer cell growth-inhibition
and death, providing a new opportunity of LAA as a
potential candidate anti-neoplastic drug for future cancer
therapeutics.
341: Molecular Mechanisms of Agaricus Bisporus
Lectins-induced Apoptosis and G1 Cell Cycle Arrest in
Human Breast Adenocarcinoma MCF-7 Cells both in in
vitro and in vivo
Chun-yang Li, Yong-xi Liu, Jin-ku Bao*; School of Life
Sciences, Sichuan University, Chengdu 610064, China
baojinku@scu.edu.cn
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Mushrooms from Agaricus Bisporus are edible possessing
tonic and medicinal attributes. Herein, in silico analyses first
demonstrated that Agaricus Bisporus lectin (ABL) could
bind with specific sugar-containing receptors EGFR, inferring that ABL could suppress EGFR-mediated antiapoptotic or survival pathways, ultimately resulted in
growth-inhibition and cell death. Subsequent in vitro investigations confirmed the simulation results, showing that
ABL induced apoptotic cell death through inhibiting
EGFR-Ras-Raf-PI3K-Akt survival pathway in MCF7 cells.
Also, ABL triggered G1 phase cell-cycle arrest via downregulating cyclinD-cdk4 singling pathway. Finally, in vivo
anti-tumor effects of ABL were detected, ABL decreased
the volume and weight of subcutaneous tumor mass obviously as well as expanded lifespan meanwhile ABL could
induce apoptotic cell death in mice. Taken together, ABL
isolated from Agaricus Bisporus exerts remarkable antitumor effects both in in vitro and in vivo with noncytotoxic
fashion, making ABL as an ‘ideal’ anti-cancer candidate
drugs in future cancer therapy.
Poster Session II-Development &
Differentiation
342: The Regulatory Function of ppGalNAc-T13 in Neural Differentiation of P19 Embryonal Carcinoma Cells
Yingjiao Xu, Lei Zhang, Wenjie Pang, Wei Li, Yan
Zhang; Ministry of Education Key Laboratory of Systems
Biomedicine,Shanghai Center for Systems Biomedicine
(SCSB), Shanghai Jiao Tong University, Shanghai,
200240, China
yanzhang2006@sjtu.edu.cn
Glycosylation plays vital regulatory roles in various biological processes such as development, immunity and neural
functions. Neural cells require correct glycosylation patterns
for their viability, function and differentiation. Mucin-type
O-glycosylation is initiated by a family of UDPGalNAc:polypeptide α-N-acetylgalactosaminyltransferases
(ppGalNAc-Ts, EC 2.4.1.41) containing 19 members in
mouse. A member of this family, ppGalNAc-T13, has been
found highly and restrictively expressed in the brain and
neurons. In this study, we have explored the effect of
ppGalNAc-T13 on mouse neural differentiation. We found
that the mRNA expression of ppGalNAc-T13 was dramatically increasing during the process of mouse brain development and the maturation of primary neuronal cultures.
P19 mouse embryonal carcinoma cells can be induced by
all-trans retinoic acid (ATRA) to differentiate into neural
cells in vitro. Similarly, ppGalNAc-T13 expression was
increasing from the later period of ATRA treatment to neural
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differentiation stage of P19 cells, accompanying with
elevated O-glycosylation level. Interestingly, without
ATRA treatment, overexpressing ppGalNAc-T13 in P19
cells induced the formation of projection-like structures
and improved the O-glycosylation level as well. Inhibition of ppGalNAc-T13 expression by RNA interference
suppressed P19 cell growth during ATRA treatment and
impeded the neurite outgrowth. Moreover, we found
ppGalNAc-T13 occurred in axons of the neurons derived from
P19 cells. Together, our results suggest a critical function of
ppGalNAc-T13 in neuronal differentiation and neurite outgrowth by regulating the O-glycosylation level.
343: Characterization of an Immunologically Active
Pectin from the Fruits of Lycium ruthenicum
Qiang Peng1, Yuguang Du1, 1 Dalian Institute of Chemical
Physics, Chinese Academy of Sciences, Dalian, 116023,
China; 2 Northwest University, Xi’an, 710069, China
articles1805@gmail.com
Lycium ruthenicum Murr. belongs to the Solanaceae family
and which is widely distributed in the salinized desert of
Qinghai-Tibet Plateau. Its special physiological characteristics
of drought-resistance and salt-resistance make it an ideal plant
for preventing soil desertification and alleviating the degree of
soil salinit-alkalinity. In addition, Lycium ruthenicum is a
famous traditional Chinese herb which has been used for
treatment of heart disease, irregular menstruation and menopause. In our previous studies, we have isolated and characterized the neutral polysaccharides from Lycium ruthenicum,
which were type II arabinogalactan-proteins containing a
(1→3)-linked β-D-galactopyranosyl residues main chain,
substituted at O-6 of galatose by arabinosyl groups. In the
present study, an immunologically active pectin, named
LRGP5 (Lycium ruthenicum glycoconjugate polysaccharide
5), was firstly isolated from the fruits of Lycium ruthenicum
Murr. It contained rhamnose, arabinose, xylose, galactose and
galacturonic acid in the molar ratio of 1:2.2:0.5:1.2:4.7. Its
molecular weight was estimated to be approximately 2.25×
105 Da by HPGPC. The structure was elucidated using methylation analysis, partial acid hydrolysis, NMR and ESI-MS
analysis. Results showed that LRGP5 consisted of a (1→4)linked galacturonic acid backbone occasionally interrupted by
(1→2)-linked rhamnose. The side chains were attached to
position 4 of the rhamnose, including (1→5)-linked arabinose, (1→3)-linked galactose, (1→3, 6)-linked galactose,
(1→4)-linked galacturonic acid, (1→2)-linked rhamnose and
(1→2,4)-linked rhamnose, and the termini were arabinose and
rhamnose. The immunological assay results demonstrated that
LRGP5 could significantly promote macrophages proliferation and enhance the secretion of nitrogen monoxide in vitro.
Glycoconj J (2013) 30:281–461
344: A glycosyltransferase-related gene, Galntl5, is a
functional molecule indispensable to mature sperm
formation
Nobuyoshi Takasaki 1 , Kouichi Tachibana 1, Satoshi
Ogasawara 1 , Hideki Matsuzaki 1 , Keiji Mochida 2 ,
Kimiko Inoue 2 , Narumi Ogonuki 2 , Atsuo Ogura 2 ,
Toshiaki Noce3, Chizuru Ito4, Kiyotaka Toshimori4,
Hisashi Narimatsu 1 , 1 Research Center for Medical
Glycoscience (RCMG), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki
305-8568, Japan; 2RIKEN BioResource Center, Tsukuba,
Ibaraki 305-0074, Japan; 3Research Center of Animal Life
Science, Shiga University of Medical Science, Otsu, Shiga
520-2192, Japan; 4Department of Anatomy and Developmental Biology, Graduate School of Medicine, Chiba University, Chuo-ku, Chiba 260-8670, Japan
E-mail: nobu-takasaki@aist.go.jp
It is important for normal fertilization that functionally
mature sperm consist of canonical motility and acrosome formation. A novel glycosyltransferase-related
gene, Galntl5, expresses exclusively in differentiating
spermatids. Galntl5 belongs to the polypeptide
GalNAc-transferase (pp-GalNAc-T) gene family for its
conserved glycosyltransferase domains, whereas
GALNTL5 exhibits no in vitro glycosyltransferase activity. Moreover, in contrast to typical pp-GalNAc-Ts
positioned in the Golgi apparatus, the exogenous
GALNTL5 is retained within vesicles in the cytoplasm
and abolishes the Golgi apparatus of cultured cells. To investigate the role of Galntl5 in spermiogenesis, we established
hetero-deficient mutant male mice, which were infertile and
exhibited impaired sperm motility. In spermatozoa, the heterodeficiency attenuated glycolytic enzymes required for motility, disrupted protein loading into acrosomes and caused aberrant localization of the ubiquitin-proteasome system. These
findings suggest that GALNTL5 is a functional molecule
indispensable for mature sperm formation.
Acknowledgments
This work was supported by a grant from New Energy and
Industrial Technology Development Organization (NEDO)
in Japan.
345: Identification of Genes Involved in Protein Glycosylation in Halophilic Archaea
Hua Lu, Cheng Jin; State Key Laboratory of Mycology,
Institute of Microbiology, Chinese Academy of Sciences,
Beijing 100101, PR China
publuhua@126.com
Glycoconj J (2013) 30:281–461
Archaea express proteins that enable them to succeed in
extreme habitats, which are hostile to many other organisms.
Post-translational modifications may help archaeal proteins
overcome the challenges presented by their surroundings. In
Archaea, the cell wall consists solely of S-layer protein
lattices, which are glycosylated by both N- and O-glycans.
However, as compared with the glycosylation in eukaryotes
and bacteria, less is known of the post-translational modification in archaea.
As S-layer glycoproteins have been widely used as
reporter proteins to study post-translational modifications in archaea, in this study we identified the S-layer
glycoprotein from halophilic archaeon Haloarcula
hispanica by MALDI-TOF and confirmed its glycosylation through SDS-PAGE glycoprotein staining and
chemical deglycosylation. Then glycans were released
from purified S-layer glycoprotein and subjected to
HPLC separation and ESI-Mass. It turned out that H.
hispanica S-layer glcoprotein was decorated by neutral
disaccharide containing a glucose and a galactose, moreover, acidic oligosaccharide containing a glucose and a
unknown sulfurted or phosphated monosaccharide.
Furthermore, two putative genes that are invovled in
glycosylation were identified in H. hispanica, namely
pmt1 and pmt2. RT-PCR analysis showed that both genes
were actively transcripted during early log phase. Deletion of pmt1 led to a complete loss of glycosylation on
S-layer glycoprotein and reduced stability of the S-layer,
while the mutant lacking pmt2 was unable to modify Slayer glycoprotein with acidic oligosaccharide. Both H.
hispanica mutants showed a retarded growth at high salt
conditions as compare with the wild type. These results
suggest that glycosylation may play an important role in
maintaining intact and stable cell envelope in hypersaline
surroundings and thus ensure survival of H. hispanica in
this extreme environment. Further investigations of these
two genes are undertaken.
Poster Session II-Infection & Immunity
346: CLEC-2 was involved in the phagocytosis of microglia by interacting with its ligand podoplanin
Tao Tao1, Aiguo Shen2; 1Department of Immunology, Medical
Colledge, Nantong University, Nantong, 226001, P.R. China,
2
The Jiangsu Province Key Laboratory of Neuroregeneration,
Nantong University, Nantong 226001, P.R. China
Inflammation occurs in many neurologic diseases and is
believed to exacerbate neuronal loss. Because dead neurons
445
may both initiate and accelerate disease progression, clearing dead neurons from extracellular spaces may be critical.
It is well-kown that dead neurons are quickly removed
through phagocytosis by the microglia. It is reported that
CLEC-2 is a phagocytic activation receptor expressed
on myeloid cells and its ligand podoplanin is expressed
in apoptotic cells. In this study, we hypothesized that
the process which activated microglia engulf apoptotic
neurons may enhance by CLEC-2 interacting with its
ligand podoplanin. We found that CLEC-2 was
expressed in microglia and podoplanin was expressed
in neuron.
347: Two polysaccharides from fruit bodies of Grifola
frondosa (Fr.) S. F. Gray, induce cytokines release by
dectin-1 and toll-like receptor 2 in macrophages and
inhibit tumor growth in vivo
Jianping Fang, Ying Wang, Kan Ding*; Glycochemistry
& Glycobiology Lab, Shanghai Institute of Materia Medica,
Chinese Academy of Sciences, 555 Zu Chong Zhi Road,
Pudong, Shanghai 201203, China
kding@mail.shcnc.ac.cn
Grifola frondosa is a widely consumed edible mushroom in
Asian countries. Recently, intense researches have been
focused on isolation and bioactivities investigation of polysaccharides from this mushroom. However, only few polysaccharides isolated are homogeneous, and little is known
about the cellular mechanism of them on the immunomodulatory activities. In this study, two homogeneous polysaccharides named GFPBW1 (300 kDa) and GFPBW2
(27.3 kDa) were purified from the fruit bodies of Grifola
frondosa. Using various methods such as IR, NMR, methylation, monosaccharide composition analysis, partial acid
hydrolysis, and Smith degradation, GFPBW1 was determined to be a β-D-(1-3)-linked glucan backbone with a
single β-D-(1-6)-linked glucopyranosyl residue branched
at C-6 on every third residue. GFPBW2 possesses a backbone consisting of β-D-1,3, β-D-1,4-linked glucopyranosyl
residues, with branches attached to O-6 of β-D-1,3-linked
glucopyranosyl residues.
Bioactivity study indicated that GFPBW1 and GFPBW2 were
effective inducers of TNF-α and IL-6 secretion in murine
resident peritoneal macrophages. Using quartz crystal microbalance (QCM) analysis, we found that GFPBW1 and
GFPBW2 could bind dendritic cell-associated C-type lectin1 (dectin-1) with an affinity constant (K d) value of 2.18×
10−9 M, and 1.08×10−7 M respectively. Meanwhile, both of
them could activate Syk and enhance TNF-α production in
RAW264.7 cells overexpressing wild type but not mutant
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dectin-1. Furthermore, Syk/NF-κB signaling and cytokines
release in resident peritoneal macrophages induced by them
were significantly inhibited by a specific dectin-1 blocking
reagent, laminarin. In addition, two polysaccharides also activated the transcriptional activity of NF-κB through toll-like
receptor (TLR) 2 in the luciferase reporter HEK293/NF-κB
cells expressing TLR2 but not TLR4 and dectin-1. Moreover,
they induced antitumor activity against Sarcoma 180 allografts growth in ICR mice accompanied with increased splenic indices but not in immunodeficient nude mice. These results
indicated that both GFPBW1 and GFPBW2 were effective
immunomodulator and inhibited tumor growth mainly
through activation of macrophages and inducing secretion of
cytokines via dectin-1 and TLR2 receptors, and GFPBW1
showed a better effect than GFPBW2. Both of them might
be promising biological response modifiers as immunotherapy tools for antitumor therapies.
348: Profiling Trypanosomatid-Pattern Recognition
Receptors interaction
1
Laura Rangel, 1Laura Corvo, 2Miguel Ángel Pineda,
1
Manuel Soto, 1Pedro Bonay; 1Centro de Biología Molecular “Severo Ochoa”-Universidad Autónoma de Madrid.
Madrid 28049. Spain, 2Institute of Infection, Immunity
and Inflammation, University of Glasgow, Scotland
pbonay@cbm.uam.es
Innate immunity activation largely depends on recognition
of micro-organism structures by Pattern Recognition Receptors (PRRs). PRR downstream signaling results in production of pro- and anti-inflammatory cytokines and other
mediators that define the pathogenic curse of disease. Moreover, PRR engagement in antigen-presenting cells initiates
the activation of adaptive immunity. C-type lectins like
galectins are conserved receptors recognizing carbohydrate
structures on viruses, bacteria, parasites, and fungi. C-type
lectins such as DC-SIGN, langerin, and dectin-1 and
galectins are expressed by dendritic cell subsets and macrophages. Pathogen recognition by those proteins triggers
signaling pathways that lead to the expression of specific
cytokines which subsequently instruct adaptive T helper
immune responses. No systematic analysis of the interaction
between PRRs and pathogens has been carried to date.
Among the main human PRR systems described so far we
can find the C-type lectin and galectin families. Here we
present the binding profile of representative C-type lectins
(langerin, intelectin-1, DC-SIGN, dectin-1 and mincle) and
some galectin (1,3,4,7,8) against parasite protozoans (pathogenic and non-pathogenic) from the Kinetoplastidae family
Glycoconj J (2013) 30:281–461
(Trypanosoma cruzi and T. rangeli as well as several Leishmania species) as a first step to delineate the hypothesis that
differential recognition by innate immune system components
may lead to differential and specific responses to each parasite
and subsequent pathology. The data presented suggest
that indeed there is a differential recognition profile that
is able to distinguish pathogenic from non-pathogenic
species inside a genus and furthermore able to discriminate tissue tropism among a similar pathogenic group.
349: The functional studies of M. tuberculosis Rv0431, a
cell wall protein
Guoying Deng1, Jian Kang2, Yufang Ma2; 1 Department
of Biochemistry and Molecular Biology, Dalian Medical
University, Dlian, 116044, China; 2 Department of Microbiology, Dalian Medical University, Dlian, 116044, China
yufang_ma@hotmail.com
Once Mycobacterium tuberculosis is inhaled and exposed to
host immune cells, the immune responses including innate
immunity and adapted immunity to the pathogen occur. The
macrophages are of mostly important for innate immune
response because of their potent microbicidal activities.
However, resting macrophages fail to harm M. tuberculosis,
which allow M. tuberculosis to replicate within these cells
and evade the innate immune defenses. Many proteins were
found in M. tuberculosis cell wall, where they function to
regulate the action of macrophages or play a role in bacterial
escape from the host macrophage through apoptosis. Therefore, to characterize these cell wall associated proteins is
critical to understanding bacterial survival and immune
modulation in the host.
The proteomics studies on M. tuberculosis indicated that
Rv0431 is a cell wall protein. WGA (wheat germ agglutinin)-affinity purification for M. tuberculosis glycosylated
proteins showed that Rv0431 is a glycoprotein. However,
the composition, structure and function of sugar chain of
Rv0413 are unclear now.
In this study, we cloned Rv0431 from M. tuberculosis
H37Rv genome and constructed mycobacterial expression
vectors pVV2-Rv0431 (the histidine tag at N-terminus of
Rv0431) and pVV16-Rv0431 (the histidine tag at Cterminus of Rv0431). The vectors were electroporated to
M. smegmatic mc2155 strain respectively. The purified
Rv0431 protein will be identified by Western blot using
different primary antibodies, such as Triticum vulgaris lectin, limulus polyphemus lectin, etc. We also performed Co-
Glycoconj J (2013) 30:281–461
IP and MS/MS to find out the interacted proteins of the host
cells with Rv0431 protein.
447
efficacy of DNA vaccines. (The authors thank the Natural
Science Foundation of China for financial support, Grant
No. 30970639).
This work was supported by the National Basic Research
Program of China (2012CB518803).
351: Cryptococcus neoformans mannoproteins: cloning,
expression and characterization in Pichia pastoris
350: Combined effects of DNA vaccination and Polysaccharide isolated from fruits of Physalis alkekengi L. var.
francheti (Mast.) Makino enhances the protective immunity against systemic candidiasis in mice
Huimin Yang, Shuying Han, Danyang Zhao, Guohua
Zhang, Guiyun Wang*; School of Life Sciences, Northeast
Normal University, 5268 Renmin Street, Changchun City, Jilin
Province, 130024, China
*Corresponding author. wanggy737@nenu.edu.cn
Protective efficacy against systemic candidiasis mediated by
DNA vaccine alone or with polysaccharide was evaluated in
this study. DNA vaccine (pD-HSP90C) used was recombinant plasmid containing epitope C (LKVIRK) from heat
shock protein 90 (HSP90) of Candida.albicans (C. albican).
Polysaccharide (PPSB) was isolated from the fruits of
Physalis alkekengi, L. var. francheti (Mast.) Makino. ICR
mice were immunized intramuscularly followed by electroporation with pD-HSP90C in the absence or presence of
PPSB (20, 40, 80 μg); pcDNA3.1, PPSB, and PBS were
used as controls. This finding indicated pD-HSP90C was
able to elicit the protective immune responses against systemic candidiasis by inducing the production of IgG, IgG2b,
IgG1, IL-2, and IL-4 in sera of mice; pD-HSP90C reduced
greatly colony forming unites (CFU) of C.albican in the
kidneys of mice, which were challenged with living C.
albican cells after immunization with pD-HSP90C. The results also indicated that PPSB (40, 80 μg) significantly
enhanced specific antibody titers IgG, IgG1, IgG2b, and
concentration of IL-2 and IL-4 in sera of mice immunized
with pD-HSP90C (p<0.05 or p<0.01). More importantly, it
was found that the mice immunized with pDHSP90C/PPSB-40 μg not only had fewer CFU in the kidneys than mice immunized with pD-HSP90C, but also a
statistically significant higher survival rate over PBSinjected group (p<0.05) when the immunized mice were
challenged with living C. albican cells. However, no statistically significant difference in survival rate was observed
between pD-HSP90C-immunized group and PBS-injected
group. Therefore, pD-HSP90C could be a useful candidate
for the development of DNA vaccine against systemic candidiasis; PPSB can be considered as a promising adjuvant
eliciting both Th1 and Th2 responses to enhance the
Pedro Antônio Castelo Teixeira1, Isis Cristina Ferreira1,
Luciana Loureiro Penha 1, Diego Allonso2, Ronaldo
Mohana-Borges2, Lucia Mendonça-Previato1 and José
Osvaldo Previato 1 ; 1 Laboratório de Glicobiologia,
Universidade Federal do Rio de Janeiro, Rio de Janeiro,
Brasil, 2Laboratório de Genômica Estrutural, Universidade
Federal do Rio de Janeiro, Rio de Janeiro, Brasil
pedroacteixeira@gmail.com
The oportunistic fungal pathogen Cryptococcus neoformans
is the etiological agent of cryptococcosis, a potentially fatal
disease, especially in immunocrompromised patients, as
those with AIDS. Growth at 37 °C, melanin production
and, mainly, the polysaccharide capsule production are
the most important virulence factors of this fungus. C.
neoformans capsular polysaccharide has three components known as glucuronoxylomannan (GXM),
galactoxylomannan (GalXM) and mannoproteins (MP),
comprising around 90–95 %, 5–8 % and <1 % of the capsule
mass, respectively. Despite MPs are the minority capsule
components, and the less studied, four of these molecules
with molecular weight of 115, 98, 88 and 84 kDa were
identified, and characterized as immunoreactive antigens
and potential vaccine candidates against cryptococcosis.
Due to the involvement of MPs in the pathogenesis of this
mycosis, it is important to characterize the chemical structure and the biological function of these molecules. The first
step for these studies is to obtain a good amount of MPs.
Thus, the aim of our work was to clone, express and characterize MP98 and MP84 on Pichia pastoris yeast. A C.
neoformans mutant strain (CAP67), deficient in GXM production, was used in this study. The c-DNA was obtained
from the fungal RNA, and was used as template to PCR
reactions to amplify the encoding genes of MP98 and
MP84. The amplified genes were cloned into expression
vectors pPICZαA, to MP98, and pPICZαB to MP84, and
then transformed into P. pastoris. Expression tests were
performed to find the colonies that most express MP98
and MP84. The two MPs had a histidine tag which allowed
their purification, from the culture supernatant, by an affinity chromatography with nickel columns. Here, we demonstrated suitable protocols for C. neoformans MPs expression
and purification.
448
352: Aptamers against ManLAM inhibited virulent Mycobacterium tuberculosis H37Rv infection in mice and
rhesus monkey
Qilong Wang, Qin Pan, Min Li, Dongdong Shi, Xianru
Xia, Xiao-Lian Zhang*; State Key Laboratory of Virology,
Department of Immunology, Hubei Province Key Laboratory of Allergy and Immunology, Wuhan University School
of Medicine, Wuhan 430071, China
zhangxiaolian@whu.edu.cn
The Mycobacterium tuberculosis (M.tb) surface heavily
lipoglycans, mannose-capped lipoarabinomannan (ManLAM)
has strong immunosuppressive effects which might be one of
reasons that causes persistent infection in vivo. In this study,
aptamer “antibody” ZXL1 which specifically bound to
ManLAM of virulent M.tb H37Rv was screened out by Systematic Evolution of Ligands by EXponential enrichment
(SELEX). The selected ssDNA aptamer ZXL1 demonstrated
the highest binding affinity to the ManLAM of M.tb H37Rv
and was measured as 8.907X10−8 M of quilibrium dissociation
constant (KD) value. We found that ZXL1 significantly
blocked ManLAM or H37Rv binding to mannose receptor
(MR) and reversd ManLAM-induced immunosuppressive effects on DCs and T cells. More importantly, we demonstrated
that single injection of ZXL1 significantly protected mice and
rhesus monkeys against M.tb H37Rv infection. These results
suggested that ManLAm-aptamer can be used as a new potential drugs for treatment of M.tb infection and as a TB vaccine
immune-enhancing adjuvant.
353: Immunopotentiating and adjuvant effects of an
α-(1→4)-glucan from Isatis Indigotica immunized with
H1N1 influenza vaccine in mice
Junjie Shan, Qian Li, Ting Zhu, Peiyuan Jia, Hao Ma,
Yuxia Wang*; Beijing Institute of Pharmacology and Toxicology, Beijing, 100850, China
The root of Isatis Indigotica is a traditional Chinese herbal
medicine. The water-extract of the roots is widely used in
clinical practice for the treatment of influenza, epidemic hepatitis etc. In the previous study, we investigated an α-(1→4) linked glucan (Mw=3600 Da) from the roots and it had a
significant adjuvant activity immunized with H1N1 influenza
vaccine and hepatitis B virus protein in mice. In this study, we
further investigated the immuno-regulation effects of the αglucan on the cellular and humoral immune response in
Balb/C mice after its co-immunization with H1N1 influenza
vaccine. The results showed that the Con A-, LPS-, and
H1N1-induced splenocyte proliferation and the serum
H1N1-specific IgG, IgG1, IgG2a, and IgG2b antibody titers
Glycoconj J (2013) 30:281–461
in the immunized mice were significantly enhanced by the αglucan. The glucan also significantly promoted the production
of Th1 (IFN-γ) and Th2 (IL-4) cytokines in splenocytes from
the immunized mice. The results indicated that the α-(1→4)glucan has a strong potential to increase both cellular and
humoral immune responses, and that it may be a safe and
efficacious adjuvant candidate suitable for a wide spectrum of
prophylactic and therapeutic vaccines.
354: Matrix Metalloproteinases (MMPs) involved in degradation of glycoconjugates of extracelular matrix
(ECM) during murine subcutaneous sporotrichosis
Andreia Dantas Medeiros 1, Pedro Antonio Castelo
Teixeira 2, Lucia Mendonça-Previato 2, Jose Osvaldo
Previato 2and Christina Maeda Takiya 1; 1 Laboratório
Multidisciplinar de Pathologia, Instituto de Biofísica Carlos
Chagas Filho (IBCCF), Universidade Federal do Rio de
Janeiro (UFRJ) and 2 Laboratório de Glicobiologia,
IBCCF/UFRJ
cmtakiya@gmail.com
The ECM is a complex, interdigitating network comprised of
glycoconjugates such as glycosaminoglycans, collagen, fibronectin which serve as a scaffold for attachment, growth, differentiation and migration of cells. The MMPs are considered
the main proteases involved in proteolytic remodeling of
ECM. They participate in many physiological and pathological
mechanisms and seem to be involved in the tissues’ invasion
by infectious agents and cancer cells.
Sporothricosis is a mycosis caused by Sporothrix schenckii.
Most cases of sporotrichosis involve the skin, subcutaneous
tissue and lymphatic vessels. However, the fungus can disseminate, especially in immunocompromised patients. But, the
fungus-related mechanisms of invasion are not totally clear.
Material and Methods: C57Bl6 mice were subcutaneously
inoculated into the right footpad with 5×106 yeasts of two S.
schenckii strains isolated in the zoonotic sporotrichosis endemic area: Ss 5822 or Ss 5555-1 and with sterile saline (control
group). Mice were euthanized at 15 days-post infection. Another group of animals were either inoculated with yeasts or
saline and received daily 30 mg/Kg doxycycline subcutaneously for 4 days. Biopsies from mouse footpads were collected
for histological and immunohistochemical/morphometrical
analyses and also for gelatin zymography and in situ
zymography (ZIS). Sections were stained with hematoxylineosin (HE), Grocott’s methenamine silver (GMS) and Picrosirius. For immunohistochemistry, sections were incubated
with antibodies directed against MMP-1, MMP-9, MMP-12,
myeoloperoxidase and F4/80.
Glycoconj J (2013) 30:281–461
Results: The HE examination revealed a diffuse infiltration of
inflammatory cells widely distributed in the dermis and subcutaneous tissue in infected mice. A large number of round or
oval yeast-like fungal forms were evidenced with GMS.
Picro-sirius staining showed that the infection by the fungus
reduces collagen. Furthermore, MMP-1, MMP-9, MMP-12,
myeloperoxidase and F4/80 were increased in mice infected
by the fungus. MMPs were predominantly found in
keratinocytes and in the inflammatory cells (macrophages,
neutrophils and lymphocytes) as well as in ECM. It was also
observed higher gelatinase and collagenase activities in
S. schenckii infected mice. However, gelatin zymography
and ZIS showed that doxycycline administration was able to
inhibit the activity of MMPs, especially the collagenase.
Ours results suggest the participation of MMPs in the pathogenesis of sporotrichosis.
355: The effect of exopolysaccharides production on
swarming motility of Pseudomonas aeruginosa PAO1
Shiwei Wang1, Zhenyin Zhang2, Di Wang1, Hongsheng
Liu2, Luyan Ma1*; 1State Key Laboratory of Microbial
Resources, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100101, China; 2 College of Life Sciences, Liaoning University, Shenyang, 110136, China
449
Pseudomonas aeruginosa is an ubiquitous microorganism, and a model organism for biofilms research.
Exopolysaccharides are a key biofilm matrix component of
many bacteria including P. aeruginosa, as they contribute to
the overall biofilm architecture and stress resistance. Bacterial motilities also affect the biofilm architecture. However,
it is not clear whether the production of exopolysaccharides
can affect bacterial motilities. P. aeruginosa have three types
of motilities, flagella-mediated swimming, Type-IV Pili
(T4P)-driven twitching, and swarming that requires the production of rhamnolipids and flagella/T4P. In this study, we
found that enhances of exopolysaccharides production reduced the swarming, but not swimming and twitching motility. This suggested that the effect of exopolysaccharides
production on swarming motility was due to the change of
rhamnolipids production. Further investigation demonstrated that overproduction of Psl or/and Pel did decrease
rhamnolipids production and the reduction occurred at
post-transcriptional level. Previous reports showed that synthesis of exopolysaccharides Psl and Pel competed for sugar
precursors and Psl may share sugar precursors with
rhamnolipids synthesis pathway. Taking together, this
suggested that the synthesis of rhamnolipids competed
with exopolysaccharides Psl and Pel for the sugar
precursors. In summary, our results indicate the effect of
exopolysaccharides production on swarming motility of
P. aeruginosa by reduction of rhamnolipids synthesis.
450
Glycoconj J (2013) 30:281–461
Author Index
Aastrup T.
Abbott K.L.
Abe Kawsar S.M.
Adamová L.
Aebi M.
Afanasiev S.S.
Aguilar-Moncayo M.
Ahl L.I.
Ahn H.-J.
Aikawa J.-i.
Akiyoshi S.
Akke M.
Aleshkin A.V.
Aleshkin V.A.
Allonso D.
Altmann F.
Amin M.
An H.J.
André S.
Angata T.
Anugraham M.
Anzengruber J.
Aoki K.
Aoki-Kinoshita K.F.
Arnaud J.
Arroyo L.
Asano M.
Ashida H.
Ashline D.
Audfray A.
Aureli M.
Avci U.
Aziz F.
Baek M.C.
Bakker H.
Baksi K.
Banerjee A.
Banerjee D.K.
Bangs J.D.
Bao B.
Bao J.
Bardor M.
Barišić D.
Bartley M.
Bassi R.
Basu M.
Basu S.
Behera D.
Beliek A.
Beloqui A.
Berger M.
064
042, 089
157
053
096
181, 195
030
303
061
033, 220
042
078
181, 195
181, 195
351
051, 105, 188, 189, 274
058
090, 093, 177, 202, 315
148
293
150
105
123
127, 131
182
301
257
245
098
182
036
228
270
315
032
041
041, 058
041
038
219
122, 340, 341
230
284
022
036
077
077
295
169
015
084
Bertolo L.
Betesh L.
Betsuyaku T.
Bi C.
Bianchet M.A.
Birse L.
Biskup K.
Biswas S.
Blader I.
Blanchard V.
Block T.
Bo J.
Bo S.
Boddey J.
Bonay P.
Bordi C.
Bose P.P.
Bosnar M.H.
Bourne Y.
Bovin N.V.
Braicu E.I.
Briscoe A.C.
Bruce C.
Bülow H.E.
Burleigh B.A.
Cai Q.
Calvo J.
Campbell H.
Campbell M.P.
Cao D.
Cao H.
Cao H.
Cao J.
Cao Q.C.
Cao W.
Caradonna K.L.
Carasquillo E.A.
Carlsson M.
Castilho A.
Caterson B.
Chai W.
Chatterjee B.P.
Chatterjee U.
Chawla Y.K.
Chazin W.J.
Cheah W.Y.
Chen G.
Chen H.-Y.
Chen H.L.
Chen J.
Chen L.
Chen M.
Chen P.
301
019
293
108
058
303
084
027
004
084, 184
019
173
194
158
348
241
231
284
241
057, 059
084
025
047
125
038
262
015
163
018, 129
120
200
233
047, 075, 261, 273
160
170
038
041
078
188, 189
075, 261, 273
052, 071, 206, 236
027, 167, 231
231
167
137
018
072
327, 328
246
075, 261
339
243
281
Glycoconj J (2013) 30:281–461
Chen Q.
Chen X.
Chen X.
Chen Y.
Chen Z.
Cheng C.
Cheng C.-W.
Cheng H.
Cheng J.
Cheng L.
Chernikov O.V.
Chernyy E.S.
Chhetra R.
Chiang P.-C.
Chiba Y.
Chigorno V.
Chikalovets I.V.
Childs R.A.
Chiu H.
Cho J.W.
Choi D.-Y.
Choi D.J.
Choi J.W.
Christophe M.
Clark P.
Claudinon J.
Coddeville B.
Comunale M.A.
Cong Q.
Čopíková J.
Corbett K.D.
Corrêa M.
Corvo L.
Coutinho B.G.
Cummings R.D.
Cyman M.
da Silva D.P.
Dang L.
Dang S.
Das H.R.
Date K.
Dawgul M.
de Graaf M.
De Vos W.
Deelder A.M.
Dejima K.
Delannoy P.
Dell A.
Deng G.
Dere R.T.
DeVoe D.L.
Deželjin M.
451
153
094, 179, 180
233
020
014
269
238
299
242
016
100, 225
057
295
328
193, 245
036
100, 225
206
091
107, 312, 313, 317, 318,
320, 321, 323
315
254, 316
254, 316
324
012
182
232
019
334, 335
119
137
232
348
205
008
280
205
014
273
027
088
276, 279
274
147
142
042
253
002, 141, 153
349
214
091
284
Dicker M.
Dietrich P.
Ding K.
Ding X.
Dinglasan R.
Doering T.L.
Dohi H.
Dolezal S.
Dong J.
Dong Q.
Dong W.
Dong Y.W.
Drickamer K.
Driggers E.
Du T.
Du Y.
Du Y.-G.
Duan J.
Dušková D.
Emery R.J.
Endo T.
Eshghi S.T.
Esko J.D.
Everest-Dass A.
Fan J.
Fan K.
Fan Y.
Fang J.
Farrugia W.
Feizi T.
Feng C.
Feng L.
Fernandes D.L.
Ferreira I.C.
Field R.A.
Filloux A.
Flitsch S.L.
Fotopoulou C.
Frantisek S.
Friedel M.
Froehlich J.W.
Fu L.
Fujii Y.
Fujimoto Y.
Fujinawa R.
Fujita A.
Fujita H.
Fujita M.
Fujita N.
Fujitani N.
189
184
120, 215, 216, 217, 264,
334, 335, 347
008
146, 158
252
237
089
243
217, 334, 335
095
111, 262
001
012
040, 251
066, 068, 070, 191, 200,
204, 248, 298, 300, 343
115, 190, 197, 198, 272,
309
183
119
199
081
091
137
018
079, 292, 329
151, 314
226, 282
215, 347
022
013, 071, 206
058
179
142, 199
351
029, 030, 255
241
196
084
229
148
025
122
157
101, 235
293
253
256
330
132
239
452
Fujiwaki H.
Fukase K.
Fukuda M.
Fukuda T.
Furukawa J.-i.
Furukawa K.
Furukawa K.
Furukawa K.
Gabius H.-J.
Gale A.J.
Gao C.
Gao C.
Gao X.
Gao X.-D.
Gaplovska-Kysela K.
Garcez T.C.
Gardner R.A.
Garénaux E.
Gasteiger E.
Gengyo-Ando K.
Gerardy-Schahn R.
Ghosh A.
Ghosh S.
Gil J.Y.
Gilbert N.M.
Giomarelli B.
Goetz S.
Gong T.
Goo B.G.
Gordan L.
Grabherr R.
Grimm R.
Grzywacz D.
Gu J.
Gu J.
Gu J.-Y.
Gu P.
Gu W.
Gu Y.
Guan H.
Guang D.
Guérardel Y.
Guhl B.
Guo H.
Guo S.-J.
Guo Z.
Guthrie E.
Hachisu M.
Hagiwara Y.
Hahn M.G.
Hakomori S.-i.
Glycoconj J (2013) 30:281–461
103
092, 101, 235
045
311, 333, 336
239
116, 145
288
288
057, 148
006
206
268, 293
102
319
312
232
142
049
129
042
124
058
294, 295
203
011
058
030, 255
258, 331, 339
224
284
145, 244
090, 315
276, 277, 278
021, 083, 109, 296, 325,
326
114, 118, 248, 311, 333,
336
186
260
311, 333, 336
108
222, 278, 307
338
232
312
089, 337
016
060
168
220
085
228
076
Hallack L.F.
Hamaguchi K.
Han C.
Han C.
Han G.
Han S.
Han W.-J.
Hanashima S.
Handa K.
Hane M.
Hanneman A.
Hao J.
Harduin-Lepers A.
Harnett W.
Hart G.W.
Hasan I.
Hashii N.
Hashimoto H.
Haslam S.
Hata K.
Hayes A.J.
Hayes C.A.
He J.
Heinzelmann-Schwarz V.
Heng Y.
Henrissat B.
Hirabayashi J.
Hirano K.
Hirata A.
Hirata K.
Hirose K.
Hirschberg C.B.
Ho M.-Y.
Hodgins D.
Hokke C.H.
Holden N.
Holmes A.
Hong S.
Honke K.
Hori K.
Horvat T.
Hoshi I.
Hoshino H.
Hosono M.
Houser J.
Hsieh J.-F.
Hsieh-Wilson L.C.
Hsu D.
Hu T.
Hua K.-F.
Hua S.
Huang J.
Huang J.
205
253
093
108
110, 152
350
186
234
076
048, 289
098
263
253
087
010, 110, 152
157
088
333
141, 153
287
047
129
292
150
194, 338
029
166
046
101
143
099
038
238
301
274
303
303
094, 179
291
049
284
283
063
080, 157, 287
104
218
012
327, 328
222
218
202, 315
170
308
Glycoconj J (2013) 30:281–461
Huang L.
Huang L.S.X.
Huang M.-C.
Huang X.
Huffman J.
Hughes C.E.
Huo J.
Hwan R.J.
Hwang D.H.
Hykollari A.
Ichimiya T.
Ideo H.
Iio E.
Ikeda A.
Ikeda N.
Ikehara Y.
Imamaki R.
Imberty A.
Inoue K.
Irimura T.
Isaji T.
Ishida H.
Ishii T.
Ishikawa Y.
Ishizaki M.
Ito C.
Ito Y.
Ivanova I.
Iwabuchi K.
Iwata N.
Jacob F.
Jang I.
Jeff I.B.
Jensen P.H.
Jeon S.K.
Jeong S.
Ji S.
Jia N.
Jia P.
Jia X.
Jiang H.
Jiang J.
Jiang S.
Jiang Z.
Jianhai J.
Jiao Y.
Jin C.
Jin C.
Jin J.
Jin M.F.
Jin Q.
Jin W.
Johnson H.
453
159, 169, 192
190
308
266, 332
163
047, 075, 261, 273
230
320
203
005, 227
046
283
165
234
291
165, 166, 207, 290
085
182
344
166
311, 333, 336
143
268
116
132
344
033, 220
255
055
085
150
312, 313
286
022
302
315
288
141
353
273
172, 175
214
291
034, 305
112
301
040, 251, 345
087
114
246
069
240
337
Johnston T.
Jost P.
Ju T.
Kage-Nakadai E.
Kaiguang Y.
Kaji H.
Kamalakumar A.
Kameyama A.
Kamimiya H.
Kamio K.
Kamysz W.
Kanazawa T.
Kang J.
Kang J.
Kang J.G.
Kang L.
Kannagi R.
Kannan L.
Kano F.
Kapoor S.
Karlsson N.
Kasahara K.
Kasekarn W.
Kato K.
Kato M.
Katoh T.
Kattla J.
Kautto L.
Kawamoto T.
Kawano S.
Kawasaki N.
Kawasaki T.
Kawashima I.
Kawashima H.
Kettner C.
Khasbiullina N.R.
Khoo K.-H.
Kida K.
Kiguchi K.
Kijimoto-Ochiai S.
Kim B.J.
Kim E.
Kim H.
Kim J.
Kim J.-H.
Kim J.H.
Kim K.P.
Kim S.H.
Kim W.J.
Kim Y.-S.
Kim Y.-W.
Kim Y.H.
Kim Y.J.
212, 213
158
008
042
173
132, 165, 257, 290
184
095, 193
209
268
276, 279
049
249, 250, 349
260
302
259
238
188
063
294
087, 129
121
049
144, 167
127
123
163
018
166
127
042, 043, 088
043
193
140
130
059
164, 238
268
290
322
177
318
107, 323
093
061
177
315
177
254
302
061
316
317
454
King J.
Kinoshita N.
Kinoshita T.
Kitajima K.
Kitajima T.
Kitazume S.
Kizuka Y.
Ko J.-H.
Kobayashi S.
Kobayashi S.
Koča J.
Koda T.
Koerdt A.
Koide Y.
Koizumi A.
Kolarich D.
Kondo A.
Kondrashina A.S.
Kong Y.
Kotu V.
Kozak R.P.
Kozmon S.
Krammer F.
Kruzel E.K.
Kubota T.
Kumagai T.
Kumar S.
Kuno A.
Kuo C.-W.
Kurz S.
Kushi Y.
Kusuda Furue M.
Kwiatkowska K.
Kwon O.
Laine R.A.
Lakhtin M.V.
Lakhtin V.M.
Lam K.-P.
Lauc G.
Lausted C.
Laval S.H.
Le Fourn V.
Lebrilla C.
Lee C.-G.
Lee C.W.
Lee G.
Lee G.-H.
Lee J.
Lee J.
Lee J.-K.
Lee K.J.
Lee R.S.
Glycoconj J (2013) 30:281–461
146, 158
085
330
048, 049, 234, 253, 256,
289
096
035, 085, 293
035
302
293
293
104
322
051
157
220
018, 022
114
225
258, 331, 339
123
142, 199
104
274
038
290
123
027
165, 166, 207, 290
164
005,146
209
043
280
201, 203
086
181, 195
181, 195
230
050, 163
212, 213
153
312
093
224
254, 316
275
330
254, 316
302
089, 310
201, 203
025
Lee S.
Lee Y.
Leffler H.
Lewis A.L.
Lewis W.G.
Li C.
Li C.
Li C.-S.
Li C.-X.
Li C.-y.
Li F.-C.
Li G.
Li H.
Li J.
Li L.
Li M.
Li M.
Li N.
Li N.
Li P.
Li Q.
Li Q.
Li S.
Li S.-T.
Li W.
Li W.
Li X.
Li X.
Li X.
Li X.
Li X.
Li X.
Li Y.
Li Y.
Li Y.
Li Y.
Li Y.-Z.
Li Z.
Li Z.
Li Z.
Liang X.
Liang Y.
Liang Z.
Liao W.
Liberek B.
Libor V.
Liew L.P.
Lihua Z.
Lin B.
Lin C.-C.
Lin C.-H.
Lin W.
Linhardt R.J.
254, 316
312
078
011, 297
011, 297
061
222, 307
327
278
340, 341
186
222
162
073
106
281
352
079, 329
151, 314
324
271
353
047, 075, 261, 273
319
114
117, 342
026
064
117, 185
139
159, 192
282
091
115, 300
221, 223
266, 332
186
014
110, 151, 314
271
024, 026
174, 175
171, 174, 176
120
277, 280
229
199
173
066, 204
073, 211
028
094
106
Glycoconj J (2013) 30:281–461
Lisacek F.
Liu C.-H.
Liu F.-T.
Liu H.
Liu H.
Liu H.
Liu H.
Liu H.
Liu H.-H.
Liu J.
Liu J.
Liu J.
Liu J.
Liu L.
Liu L.
Liu L.
Liu N.
Liu Q.
Liu S.
Liu X.
Liu X.
Liu X.
Liu Y.
Liu Y.
Liu Y.
Liu Y.-x.
Loberto N.
Lochmüller H.
Logan D.
Lonardi E.
Loos A.
Lu H.
Lu H.
Lu L.
Lu L.
Lucie M.
Lucini C.
Lukyanov P.A.
Luo M.
Luo X.
Ma B.
Ma H.
Ma J.
Ma K.
Ma L.
Ma L.
Ma R.
Ma X.
Ma Y.
Ma Z.
Macauley M.S.
Madej M.
Maeda M.
455
018, 129
308
327, 328
083
108
115
212, 213
355
186
136
171
243
259
038
079, 329
247
020
215, 216
039, 304
097
263
314
052, 071, 206
122
326
340, 341
036
153
078
142
188
023
345
216
259
229
145, 244
100
075, 261
265
259
353
110
266, 332
044, 355
108
077
097
247, 249, 250, 349
301
006
147
132
Maeda Y.
Maes E.
Magnani J.L.
Magnelli P.
Malinovská L.
Maresch D.
Mariethoz J.
Marounek M.
Marshall M.
Martin-Lomas M.
Marwaha R.
Mason D.
Mathew A.
Matsuda A.
Matsuda A.
Matsuda K.
Matsuda S.
Matsuda S.
Matsumoto K.
Matsumoto R.
Matsumoto S.
Matsunaga R.
Matsuno Y.-k.
Matsuo F.
Matsuo I.
Matsuzaki H.
Maturana A.D.
Matuška R.
Mayampurath A.
McCarthy M.I.
McLeod E.
Mechref Y.
Medeiros A.D.
Mehta A.
Mehta N.
Mehta S.
Mendonça-Previato L.
Meng Q.
Mercer N.
Mereiter S.
Messner P.
Miao M.H.
Mikami M.
Milewski S.
Mine T.
Mitani S.
Miura T.
Miyagi T.
Miyoshi E.
Mizokami M.
Mizumoto S.
Mochida K.
Mohana-Borges R.
330
232
082
168
053
189
129
119
123
015
294
012
209
042
166, 207
237
193
237
099
157
043
237
095
235
033, 220
166, 344
049
104
017
163
168
017
354
019
123
295
205, 351, 354
338
065
227
051, 105
246
290
280
209
042
046
080, 287
092
165
138
344
351
456
Molchanova V.I.
Mondal G.
Monteiro M.A.
Moon P.-G.
Mor T.S.
Moremen K.
Mori H.
Morise J.
Moriwaki K.
Mu H.
Muenster-Kuehnel A.-K.
Müller J.S.
Mulloy B.
Mun J.-Y.
Murakami S.
Murakami T.
Murakami Y.
Murata D.
Mužinić A.
Myers D.D.
Myoung K.T.
Myszka H.
Nagae M.
Nagy T.
Nairn A.
Nakamura K.
Nakanishi H.
Nakanishi T.
Nakanuma Y.
Nakao H.
Narimatsu H.
Negulescu I.H.
Neiswinger J.
Nepogodiev S.A.
Neu U.
Neumann L.
Nevalainen H.
Nguyen-Khuong T.
Ni X.
Nicholls J.M.
Nilsson U.J.
Nishida Y.
Nishihara S.
Nishima W.
Nishimoto K.
Nitta K.
Nixdorf S.
Noce T.
Nokihara K.
Nomura K.
Nomura K.H.
Novokmet M.
Glycoconj J (2013) 30:281–461
100
167, 231
301
315
188
139
063
306
092
183
124
153
071, 236
201, 203
245
208
330
042
284
082
178
276, 279, 280
234
139, 337
139
063
290
290
166
043
007, 127, 132, 165, 166,
207, 245, 257, 290, 344
086
152
255
052
051, 105, 188, 189, 274
154
018
217, 334, 335
141
078
237
046
099
208
080, 157, 287
150
344
101
042
042
163
Nowacki A.
Nozaki H.
Nycholat C.M.
O'Neill E.
Ocho M.
Ogasawara S.
Ogawa H.
Ogawa K.
Ogawa Y.
Ogonuki N.
Ogura A.
Oguri S.
Oh D.-B.
Oh M.J.
Oh S.
Ohkawa Y.
Ohkohchi N.
Ohmi Y.
Oikawa M.
Oka S.
Okuda S.
Oliveira P.A.V.
Olszewski N.E.
Ono S.
Ota F.
Otsuki T.
Owen K.
Ozcan S.
Ozeki Y.
Pabst M.
Packer N.H.
Palma A.S.
Palmberger D.
Pan L.
Pan Q.
Pang W.
Panin V.M.
Papy-Garcia D.
Park II Y.
Park J.K.
Park J.W.
Park S.
Paschinger K.
Patton J.T.
Paulson J.C.
Pavel L.
Pedersen H.L.
Pei Y.
Pei Z.
Peng Q.
Peng Y.
Penha L.L.
Pequegnat B.
277
290
006
029
165
344
063, 088
085
157
344
344
208
201, 203
090, 202
302
288
166
288
085
306
127
205
067
048
268, 293
063
163
090
157
105
018, 022, 129, 150, 154
052, 071, 206
210
020
155, 352
342
253
324
316, 224, 254
224
316
312, 313
005, 056, 227
082
006
069
303
064
064
300, 343
281
205, 351
301
Glycoconj J (2013) 30:281–461
Peters E.
Petersen L.C.
Petersen R.
Peumans W.J.
Pfrengle F.
Phitak T.
Piao J.
Pierce J.M.
Pierce M.
Pineda M.Á.
Porterfield M.
Posch G.
Previato J.O.
Previato L.M.
Prinetti A.
Qasba P.
Qi J.
Qi Q.
Qian X.
Qian X.H.
Qian Y.
Qin Y.
Qiu H.
Qiu Y.
Qu M.
Qu Y.
Rademacher C.
Ramakrishnan B.
Ramsland P.A.
Rangel L.
Ranzinger R.
Re S.
Reichardt N.-C.
Reinhold V.
Reis C.A.
Reiss K.
Ren S.
Ren Y.
Rendic D.
Reyes Martinez J.E.
Reynaud A.
Rhee S.K.
Rillahan C.D.
Robinson L.S.
Römer W.
Rong J.
Rossez Y.
Roth J.
Rouhanifard S.H.
Round A.
Royle L.
Ruan Y.
457
012
137
018
187
006
256
239
310
089, 337
348
123, 178
051
205, 232, 351, 354
232
036
065
151, 314
260
161
160
021, 296
014
246
270
069
171, 172, 174,
175, 176
006
065
022
348
130, 178
099
015, 056
098
074
052
021, 296
054
005
196
241
203
006
011, 297
182
094, 179
303
107, 312, 313
180
241
142, 199
109
Rudan I.
Rudd P.M.
Ryum J.-h.
Saido T.
Sakagami H.
Sakamoto H.
Sakamoto M.
Sakuraba H.
Sardzik R.
Saroha A.
Sasaki N.
Sato C.
Sato T.
Sato T.
Sawaki H.
Schafer C.M.
Schäffer C.
Schaper W.
Schiller B.
Schneider J.D.
Schwartz-Albiez R.
Schwörer R.
Sehouli J.
Seko A.
Seo H.
Seo H.-J.
Seo H.G.
Serna S.
Sexauer B.
Shan A.
Shan J.
Shang C.
Shang M.
Sharma S.C.
Sheikh M.O.
Shen A.
Shen J.
Shi D.
Shi P.C.
Shi S.
Shi X.
Shi Z.
Shikanai T.
Shilova N.V.
Shin W.S.
Shinchi H.
Shinohara Y.
Shoda J.
Shuguang L.
Sim L.C.
Simon S.I.
Singha S.
163
163
321
085
063, 088
208
283
193
196
027
046
048, 049, 234, 253, 256,
289
116, 145
207
127, 132
004
051, 105
124
005, 227
188
148
135, 267
084
220
201, 203
323
320
056
212, 213
117
353
147, 187
335
295
004
113, 117, 269, 346
243
352
111
219
168
340
132, 166
059
316
143
239
166
194
230
082
231
458
So M.K.
Sogabe M.
Sohng J.K.
Song E.
Song K.
Sonnino S.
Soto M.
Sridhar S.
Srinivasan R.
Stanton R.
Staudacher E.
Stehle T.
Steinkellner H.
Stowell S.
Strasser R.
Stroeh L.
Suda Y.
Sugahara D.
Sugahara K.
Sugawara S.
Sugihara K.
Sugita Y.
Sui Z.
Sun C.
Sun L.
Sun Q.
Sun X.
Suzuki A.
Suzuki N.
Suzuki T.
Suzuki Y.
Suzuki Y. ...
Swanier K.
Synytsya A.
Tachibana K.
Tachida Y.
Tai G.
Takahashi J.
Takahashi K.
Takaoka Y.
Takasaki N.
Takatani M.
Takeda Y.
Takeishi Y.
Takeuchi Y.
Takiya C.M.
Tanaka K.
Tanaka K.
Tanaka Y.
Tang H.
Tang J.
Tang X.-L.
Tang Y.
Glycoconj J (2013) 30:281–461
202
165, 290
031
017
302
036
348
058
295
210
145, 244
052
188, 189
008
189
052
143
257
138
157
257
099
172
282
062
299
180
103
290
037
132
209
310
119, 224
344
085
102, 226, 299
138
080, 287
193, 245
344
220
033, 220
085
306
354
092
208
165
017
265
155
275
Tang Y.
Taniguchi N.
Tao D.
Tao G.
Tao S.
Tao T.
Tareyanagi C.
Tauber R.
Taubner T.
Taus C.
Teixeira P.A.C.
Ten F.
Teo C.F.
Thackray H.
Thanabalasingham G.
Thaysen-Andersen M.
Tian Y.
Tiemeyer M.
Togayachi A.
Togaychi A.
Tomas P.
Tong C.
Toshimori K.
Toth I.
Toukach P.
Toyoda H.
Trick M.
Tröndle K.
Troy II F.A.
Tsuboi K.
Tůma J.
Tuomivaara S.T.
Turnbull J.E.
Tyler P.C.
Uekusa Y.
Ulf Ryde U.
Umemura Y.
Unger W.
Vaclav S.
Vallas V.
Van Damme E.J.M.
van der Wel H.
Van Kuppevelt T.H.
Vanbeselaere J.
Varrot A.
Vasta G.R.
Venkatakrishnan V.
Vincent F.
Viola J.
Voglmeir J.
von Drygalski A.
Wada T.
292
003, 035, 085, 114, 268,
293, 336
158
236
016, 185
346
116
084, 184
119
145, 244
351, 354
052
285
082
163
154
266
089, 123, 146, 178
290
165
229
069
344
303
133
043
029
182
020
140
119
228
134, 135, 267
135, 267
144
078
103
184
229
022
147, 187
004
046
232
182
058
154
241
310
196
006
287
Glycoconj J (2013) 30:281–461
Wakao M.
Walczak D.
Wan Z.
Wang B.
Wang B.
Wang C.
Wang D.
Wang F.
Wang F.
Wang G.
Wang H.
Wang H.
Wang J.
Wang J.
Wang L.
Wang L.
Wang L.
Wang L.-X.
Wang L.X.
Wang M.
Wang M.Y.
Wang N.
Wang P.
Wang P.
Wang Q.
Wang Q.
Wang Q.
Wang Q.
Wang R.
Wang S.
Wang S.
Wang S.
Wang S.
Wang S.-H.
Wang S.-X.
Wang T.
Wang W.
Wang W.
Wang W.
Wang W.X.
Wang X.
Wang X.
Wang X.
Wang X.
Wang Y.
Wang Y.
Wang Y.
Wang Y.
Wang Y.
Wang Y.
Wang Y.
Wang Z.
Wang Z.A.
459
143
277
242
020
169
159, 169, 192
355
139
233
350
162
219
039
185, 240
139
151, 314
292
058
272, 309
019
190, 197, 198
101
215, 216
243, 281
235
260
265
352
262
080, 292, 329
156
219
355
164
278
230
069
243
261, 278, 246
190, 197, 198
014
039, 270
066
324
008
021, 296
097
120, 347
221, 223
333
353
159, 169, 192
252
Watanabe T.
Watarai S.
Weber A.
Wei L.
Wei L.
Weinhold B.
Wells L.
Wen H.
Weng Y.
wenxia W.
West C.M.
Willats W.G.T.
Williams M.
Wilson I.B.H.
Wimmerová M.
Wright A.F.
Wu C.
Wu D.
Wu D.
Wu H.
Wu J.-R.
Wu P.
Wu Q.
Wu Q.
Wu S.
Wu S.-H.
Wu S.-W.
Wu W.
Wu X.Z.
Wu Y.-M.
Wu Z.
Wu Z.-H.
Wuhrer M.
Wun T.
Xia L.
Xia X.
Xia Y.
Xiao F.
Xie R.
Xie W.
Xie Y.
Xin Y.
Xing W.
Xiong C.
Xu C.
Xu D.
Xu H.
Xu J.
Xu J.
Xu L.
Xu L.
Xu Q.
193
209
212, 213
069
162
124
042, 089, 285
132
172, 176
194
004
303
252
005, 056, 058, 146, 210,
087, 227
053, 104
163
174
062, 253
247
298
211
180
175
325
305, 034
218
164
111
111, 262
308
169
186
142
082
126
352
331
264
094, 179, 180
156, 185
258, 331
250
159
259
021, 296
137
314
083, 325, 326
115
034, 305
247, 250
070, 248, 300
460
Xu S.
Xu X.
Xu Y.
Xu Y.
Xu Y.-X.
Xu Z.
Xue H.
Xue M.
Xue R.
Yagi H.
Yagi-Utsumi M.
Yamada I.
Yamagata S.
Yamagata T.
Yamaguchi K.
Yamaguchi T.
Yamaguchi Y.
Yamaguchi Y.
Yamaki T.
Yamamoto K.
Yamamoto K.
Yamamoto M.
Yamamoto N.
Yamamoto S.
Yamamoto T.
Yamashita K.
Yan H.
Yan J.
Yan J.
Yan M.
Yan Q.
Yan S.
Yang B.
Yang B.
Yang F.-L.
Yang G.
Yang G.
Yang H.
Yang K.
Yang P.
Yang S.
Yang S.
Yang X.
Yang X.-Y.
Yang Y.-L.
Yao Z.
Yaqin T.
Yasue H.
Yi W.
Yin H.
Ying L.
Ying W.
Ying W.T.
Glycoconj J (2013) 30:281–461
319
221
266
342
038
185
102
243
047
167
144
127, 128
149
149
287
144
099, 234
139
085
080
245
166
085
144
209
283
097
040
233
269
039, 304
005, 056, 227
026
106
218
265
324
350
171, 176
023, 162, 170
091
249
039, 270, 304
149
218
298
194
049
012
068, 190, 197, 198
112
161
160
Yoon J.Y.
York W.S.
York W.S.
Yoshida K.
Yoshida T.
Yoshihisa A.
Young J.
Yu B.
Yu B.
Yu C.-y.
Yu G.
Yu H.
Yu L.
Yu L.
Yu S.
Yu S.
Yu S.-Y.
Yu W.
Yu X.
Yuan C.-H.
Yuan H.
Yuan J.
Yuan M.
Yuan Y.
Yuanyan W.
Yuguang D.
Yuki N.
Yukui Z.
Zang J.
Zauner G.
Zha X.
Zhan S.-H.
Zhan X.
Zhang A.
Zhang C.
Zhang G.
Zhang G.
Zhang G.
Zhang H.
Zhang H.
Zhang H.
Zhang H.
Zhang H.
Zhang J.
Zhang J.
Zhang J.
Zhang J.
Zhang J.
zhang J.
Zhang L.
Zhang L.
Zhang L.
Zhang L.
323
130
178, 228
293
293
085
137
009
261
017
222, 263, 278
014, 162, 259
024
259
079
115
238
108
069
155
172, 175
169, 192
237
212, 213
112
194
143
173
129
142
151, 314
218
236, 073, 211
183
258, 331, 339
180
324
350
014, 115
071, 073, 211, 236
091
098
275
079, 266, 292, 329, 332
122, 340
191
212, 213
265
307
079, 329
097
149
171, 172, 174, 175, 176
Glycoconj J (2013) 30:281–461
Zhang L.
Zhang L.
Zhang P.
Zhang Q.
Zhang T.
Zhang W.
Zhang W.
Zhang W.
Zhang X.
Zhang X.
Zhang X.
Zhang X.
Zhang X.
Zhang X.
Zhang X.-L.
Zhang Y.
Zhang Y.
Zhang Y.
Zhang Y.
Zhang Z.
Zhang Z.
Zhang Z.
Zhao D.
Zhao P.
Zhao Q.
Zhao W.
Zhao X.
Zhao X.
Zhao X.
Zhao X.
Zhao X.
Zhao X.
Zhao X.M.
Zhao Y.
Zhao Y.
461
258, 331, 339
342
169
240
286
162, 170
240
249
021
054
108
149
156
296
054, 155, 352
071, 236
117, 156, 185, 342
144, 169
171, 172, 174, 175, 176
041
261
355
350
054
174
040
068
106
161
222
243
263
190, 197, 198
026
054
Zhao Y.
Zhao Y.
Zhao Z.
Zhen L.
Zheng Q.
Zheng Z.-Y.
Zheng Z.-Y.
Zhimou G.
Zhong R.
Zhong Y.
Zhou F.
Zhou H.
Zhou H.
Zhou J.
Zhou S.
Zhou Y.
Zhou Y.-h.
Zhu B.
Zhu C.
Zhu H.
Zhu J.
Zhu J.
Zhu L.
Zhu M.
Zhu T.
Zhu X.
Zhu X.
Zhuang S.
Zoldoš V.
Zou X.
Zou Y.
Zuber C.
Zubkova O.V.
191
259
271
173
247
073
211
194
066, 204
014
097
025
271
265
016, 185
062, 102, 226, 282, 286,
299
034
204
219
152
212, 213
223
073, 211
014
353
214
324
236
284
156
243
312
135, 267