JCGG Symposia

The 3rd Symposium of Japanese Consortium for Glycobiology and Glycotechnology

Integration from functional analyses of sugar chains to systems glycobiology
December 6 - 7, 2005 Tokyo Conference Center (Shinagawa)
1. Dynamic development of functional glycomics: Present state of the MEXT project

Koichi Furukawa (Nagoya University)

The MEXT “Functional Glycomics” group study is a five-years project from 2002 to 2006, and consists of 12 members and 27 applied members who have been renewed in 2005. It is composed of 3 groups entitled as follows, 1. Regulation of protein functions by carbohydrate chains. 2. Functional regulation of molecular complexes by glycosylation. 3. Molecular mechanisms for diseases based on glycosylation disorders. The aim of the 1st group is to understand the roles of carbohydrates at the molecular level in the functions of glycoproteins and also in the carbohydrate-protein interactions in various biological reactions. The aim of the 2nd group is to focus on sugar chain functions, especially GAGs’ ones exhibited when molecular complexes with protein ligands are formed. The aim of the 3rd group is to investigate the molecular mechanisms of the diseases derived from the glycosylation disorders with focus on the roles of membrane microdomains. In particular, there are outstanding progresses in the function analyses of MBP ligands, O-mannose type glycans, GPI-anchors in cytokine receptors, GAGs of proteoglycans in neurons, glycolipids in nervous systems and α1,4GlcNAc in H. pylori infection. New synthetic mechanisms of GAGs, O-glycans and GPI-anchors, and mode of carbohydrate recognition with ubiquitin ligases have also been elucidated.


2. From Glycogene (GG) Project to Structural Glycomics (SG) Project

Hisashi Narimatsu (National Institute of Advanced Industrial Science of Technology)


3. Structural studies on protein glycosylation and protein-carbohydrate interactions in the Protein 3000 project

Ryuichi Kato (Photon Factory, KEK)

Our Structural Biology Research Center in KEK-PF serves as one of the nine consortia of Protein 3000 (a national project of Japan) pursuing structural and functional analyses of post-translational modification (mainly glycosylation) and transport. The consortium consists of nine universities and five research institutes including structural biologists (X-ray crystallography, NMR and Small Angle X-ray Scattering), bioinformaticians, biochemists and cell biologists. We founded a core facility at KEK-PF, where a major part of our work, such as manipulation of recombinant DNA for gene expression, protein purification, crystallization and structure determination, is carried out. For example, we determined X-ray structures of novel glycotransferases, GlcAT-P and GlcAT-S, with and without substrates which are important for HNK-1 glycocarbohydrate. We also determined structures of the mammalian sialidase Neu2. Structural comparison between human Neu2 and influenza virus sialidases may help development of a new anti-influenza virus drug without side effect. In addition, there are many other structural and functional research projects, such as glycotransferases, glycolytic enzymes and lectins, are in progress.


4. Introduction to <Individual Type Research (PRESTO) “Metabolism and Cellular Function”>

Masahiro Nishijima (National Institute of Infectious Diseases)

As genomic structures of various organisms including human are elucidated, life science is shifting to and developing in post-genomic research for the utilization of the genomic information. The research for protein functions is being advanced by structural and proteomic analyses. In addition to the research about genes and proteins, another important theme which is the aim of this research area, is the elucidation of the dynamics and functions of the metabolites and the control of cellular functions. Metabolites contain substrates for energy metabolism, cell membrane components, physiologically active substances, etc. produce by enzymes. The metabolomic analytical techniques, in which metabolite groups are analyzed in a systematic or comprehensive method by mass spectrometry, etc., has brought research on cellular metabolism into a new era.
The research area is aimed at development of new analytical techniques which contribute to metabolomic research, identification of metabolites that prescribe specific cellular conditions, the discovery of new metabolic processes, the elucidation and regulation of cellular functions with information on alterations of metabolites, by the metabolomic analysis at state of mutation, pathology, development in microorganisms, animals, and plants, etc. Metabolic research in Japan is ranked high by international standards in various fields as lipids and carbohydrates. So, Japan can lead the world in metabolomic research. I expect the discovery of new physiologically active metabolites, the development of diagnostic methods with disease-specific metabolic markers, therapeutic drugs for metabolic diseases, the organisms which product useful metabolites efficiently.


5. HGPI pilot study

Yoshinao Wada
(Osaka Medical Center and Research Institute for Maternal and Child Health)

MS will be the central method of glycan analysis for its high sensitivity, throughput and capability of structure elucidation. HGPI, an activity of HUPO, has conducted a pilot study on the MS analysis of glycans, in order to recognize a variety of MS methods currently used in different laboratories and their capability. Each 1 mg of 3 different preparations of human transferrin (Tf) and IgG (total 6 samples) were sent to 26 laboratories worldwide, and the data replied by 20 labs were compared especially focusing on the sialylated glycans in Tf and galactosylation in IgG. For reference, the standard glycan analysis of reductive amination followed by chromatography and fluorescence detection was performed in some laboratories. MS of glycans especially those of permethylated ones was comparable with the conventional chromatographic method. In addition, MS of glycopeptides gave valuable information, allowing site-specific analysis of Tf and delineating differential galactosylation between two difference IgG subclasses. The results will be submitted for publication by all the members involved in this study as the authors.


6. Crystal structure of pp-GalNAc-T10

Tomomi Kubota (National Institute of Advanced Industrial Science of Technology)

Pp-GalNAc-Ts are initiation enzymes to synthesize mucin type O-glycans, which transfer GalNAc from UDP-GalNAc to Ser or Thr residues of substrate proteins. To the present, 18 human isozymes have been identified. They are classified into some types according to catalytic specificities. The combination of expression pattern of all enzymes in tissues/cells and their substrate preferences will produce a large variety of O-glycosylated proteins. T10 is an unique isozyme, which has strong activity toward glyco-peptides, while it has little activity toward naked peptides. We crystallized a soluble truncated form of the enzyme expressed by Pichia pastoris. The crystal diffracted X-rays up to 2.5 Å resolution with PF-AR-NW12 beam line. The crystal structure was solved by molecular replacement method using the catalytic domain of T1 as a search model. The relative position of the lectin domain to the catalytic domain in T10 was significantly different from that in T1. From electron density map, UDP, GalNAc and Mn2+ were clearly identified, which indicated the breakage of glycoside bond between UDP and GalNAc. By comparing with the crystal structure of T1 which did not contain any substrates, it was suggested that two flexible loops undergo conformational changes concomitantly with the substrate binding.


7. NMR Structural Glycomics

Koichi Kato (Nagoya City University)

We have been developing NMR techniques for structural analyses at atomic resolution of glycoproteins in solution. In this methodology, the glycans and/or polypeptides of glycoproteins are uniformly or selectively labeled with stable isotopes (13C, 15N, and 2H) by metabolic or enzymatic manners. We demonstrate our strategy using the Fc portion of immunoglobulin G as a model system. On the basis of NMR data, structure and dynamics of the carbohydrate moieties as well as the polypeptide chains of Fc in solution will be discussed. Accumulating evidence shows that carbohydrate moieties contribute to folding, transport, and degradation of glycoproteins in cells via interactions with a variety of intracellular lectins. To gain insights into the mechanisms of the recognition of glycoproteins in those systems, we applied the stable-isotope-assisted NMR techniques to analyses of sugar binding of the intracellular lectins, which include the cargo receptor VIP36, and the ubiquitin ligase SCFFbs1. On inspection of NMR spectral data, the amino acid and the sugar residues involved in the carbohydrate-protein interactions were identified. To perform NMR analyses, glycoforms of target proteins have to be described in advance. The multi-dimensional HPLC mapping technique combined with mass spectrometric methods is powerful for determination of covalent structures of target glycoproteins. We have made a web application “GALAXY” (http://www.glycoanalysis.info/) based on the HPLC map to facilitate identification N-linked oligosaccharides expressed on proteins.


8. Analytical Systems for the Binding Interaction of Structurally Defined Oligosaccharides with Proteins/Cells: Use of Surface Plasmon Resonance (SPR) or Gold Nano-Particles (GNP)

Yasuo Suda (Kagoshima University)

We developed an advanced method for the immobilization of oligosaccharides onto a gold-coated chip for surface plasmon resonance (SPR). Linker compounds containing intra-molecular cyclic disulfide were first designed. Oligosaccharides were then reacted with linker compounds using an optimized reductive amination reaction to afford ligand-conjugates. An attachment of the solution of each ligand-conjugate with a gold coated SPR chip formed spontaneous S (in the ligand-conjugate) - Au (chip) binding to prepare oligosaccharide immobilized chips (Sugar chips). Using sugar chips, oligosaccharide - protein interactions were systematically evaluated without labeling of protein. Sugar chips were further applied for SPR/MS (MALDI-TOF/MS) tandem analysis toward the identification of unknown target.
To establish an on-site analytical tool, the above immobilization method was applied to gold nano particle (GNP). For example, a ligand-conjugate containing α-D-glucopyranoside was reacted with GNP, followed by dialysis to prepare α-D-glucopyranoside immobilized GNP (Glcα-GNP). The color of Glcα-GNP colloid solution (in PBS) was purple, showing plasmon absorption at 520 nm. When Glcα-GNP was incubated with Con A, the colloid solution became colorless as nano-particles were quickly aggregated. The change was detected visually. The aggregate was dissolved by adding excess D-glucose, and the Con A was quantitatively recovered, offering a quick purification of protein.


9. Production of Single Chain Antibodies against a Variety of Carbohydrate Epitopes

Yoko Fujita-Yamaguchi (Tokai University)

Phage-display technology has been applied to obtain human single chain antibodies (scFvs) against carbohydrate antigens. To establish methodologies as a first step, phage-displayed antibodies (phage Abs) that recognized mannose residues were isolated. Phage Ab library representing a large repertoire was prepared by CDR and VL/VH shuffling methods with unique vector constructs. The library was subjected to four rounds of panning against neoglycolipid synthesized from mannotriose (M3) and dipalmitoylphosphatidylethanolamine (DPPE) by reductive amination. Of 672 clones screened by ELISA using M3-DPPE as an antigen, 25 positive clones with different amino acid sequences were isolated as candidates for phage Abs against M3 residues. TLC-overlay assays and surface plasmon resonance analyses revealed that phage Abs thus far analyzed bound to neoglycolipids bearing mannose residues at non-reducing termini. In addition, selective binding of the phage Abs to RNase B carrying high mannose type oligosaccharides but not to fetuin carrying complex type and O-linked oligosaccharides was confirmed. Characterization of scFvs expressed from respective phages demonstrated affinity and specificity for non-reducing terminal mannose residues. These results demonstrate the usefulness of this strategy in constructing human scFvs against various carbohydrate antigens. Isolation and characterization of clones against Lewis-related carbohydrates, T antigen, and gangliosides are now in progress.


10. Regulation of carbohydrate-mediated cell-cell interactions involved in leukocyte homing by fucosyltransferases and 6-O-sulfotransferases

Reiji Kannagi (Aichi Cancer Center)

Selectins mediate routine homing of lymphocytes and inflammatory mobilization of leukocytes. Two kinds of carbohydrate ligands for selectins are noted on leukocytes. One is the conventional non-sulfated carbohydrate ligand, sialyl Lewisx, involved in the recruitment of leukocytes to inflammatory lesions. Sialyl Lewisx is constitutively expressed on granulocytes and monocytes, and these cells are ready to infiltrate the extravascular area whenever a sufficient amount of selectins is expressed on the vascular bed. The other is the sulfated form of the ligand, sialyl 6-sulfo Lewisx, which has an extra sulfate residue attached to the C-6 position of GlcNAc. This determinant is expressed on high-endothelial venules of lymphoid tissues and cell lineage-specifically expressed on the restricted subsets of resting leukocytes. The sulfated ligand mediates routine homing of naïve T cells to peripheral lymph nodes and also routine migration of gut- or skin-homing of specific subsets of helper memory T cells. It is noteworthy that sialyl 6-sulfo Lewisx is preferentially involved in the routine homing process of various subsets of helper T-lymphocytes under non-inflammatory conditions. Fucosyltransferases and 6-O-sulfotransferases figure heavily in the regulation of expression of these carbohydrate determinants, which will be the main topic of this presentation.


11. Relating Sugar Chains' Structures and Localization with their Biological Functions

Taro Kinoshita (Osaka University)

Structures of sugar chains are modified during and after biosynthesis in connection with their intra and extra cellular locations. Some sugar chains are enriched in particular domains of cellular membranes. Dynamics of sugar chains, including structural changes, localization and mode of distribution, are closely related to their biological functions. In this research program, we will clarify relationships between dynamics and biological functions of sugar chains. We also plan to translate results from these research into practical methods to regulate biological functions of sugar chains.


12. Function of heparan sulfate-6-O-sulfation : Studies of mice with deficienct heparan sulfate 6-O-sulfotransferase-1 or -2 (HS6ST-1, -2)

Hiroko Habuchi (Aichi Medical University)

HS interacts with numerous HB-GFs, and regulates their signalings. These interactions are dependent on specific structures characterized by the sulfation pattern and uronic acid isomer. The 6-O-sulfation is catalyzed by three isoforms of HS6ST (HS6ST-1, -2, -3) and one spliced form of HS6ST-2. To elucidate an in vivo role of each HS6ST isoform, we generated HS6ST-1- and HS6ST-2-deficient mice (6ST1-KO and 6ST2-KO, respectively). 6ST1-KO mice mostly died between the E15.5 and the perinatal stage, but a few were viable with retarded rates of growth. A marked reduction of GlcNAc(6SO4) and GlcNSO3(6SO4) residues was observed in the HSs of various organs including liver, kidney, lung, and placenta, but the reduction of IdoA(2SO4)-GlcNSO3(6SO4) residues was modest. Fetal microvessels in the labyrinthine zone of the placenta was reduced to about 50%, which may be caused by the dramatic reduction of f the VEGF-A transcript and corresponding protein. Furthermore, abnormal lung morphology was often observed. On the other hand, 6ST-2 KO mice appear to be healthy, but 6-O-sulfation of heparin were decreased to about 50%. Taken together, 6ST-1 plays critical roles in normal mouse development, and 6ST-2 may function in pathological processes such as inflammations and allergy mediated by mast cells.


13. Developmental study for clinical application and basic study with collectins

Nobutaka Wakamiya (Asahikawa Medical College)

Collectins are C-type lectin family, which have collagen-like domains and carbohydrate recognition domains. We focus on two collectins of MBL and CL-P1. We made the plan to use MBL as anti-viral drugs for some viral infections. We have already established the expression system of MBL and improved it and selected good MBL expression clones for individual virus. Now we are studying on efficacy of each MBL in animal level. CL-P1 expresses on the endothelial cell and has functions as scavenger receptors for oxidized low density lipoprotein (ox-LDL), micro-organisms. To find the in vivo important roles of CL-P1, we investigated the role of CL-P1 on zebrafish or rat. After cloned zebrafish CL-P1 (zCL-P1) gene was transfected into the CHO cell, the expressed zCL-P1 bound to the ox-LDL, and microorganism. Immunohistochemistry and in situ hybridization studies showed that zCL-P1 expressed in the vasculature (endothelium) and zCL-P1 as well as VEGF mRNA expressed in the early stage of the post fertilization detected by RT-PCR. Gene knockdown (by using morpholino oligonucleotide) study demonstrated the failure of the vascular development which might be followed by the body trunks formation collapse. This phenotype was rescued by the insertion of the zCL-P1 mRNA. These findings indicate the CL-P1 is involved in the vascular and body development during embryogenesis. We have confirmed that ischemia/reperfusion induces CL-P1 delayed expression in the endothelium on the rat carotid artery while early expression of LOX-1. The studies obtained from both models proposed the novel linkage between angiogenesis and scavenger receptors.


14. Role of membrane-bound lectins in the regulation of humoral immune responses

Takeshi Tsubata (Tokyo Medical and Dental University)

CD22/Siglec2 and CD72 are inhibitory co-receptors that negatively regulate signaling through the B cell antigen receptor (BCR) by activating the SHP-1 phosphatase. CD22 is a member of the Siglec family that recognizes α2,6 sialic acid. CD72 contains a C-type lectin-like domain, but whether it recognizes a glycan ligand is not yet known. We demonstrated that these membrane-bound lectins function as a molecular switch that determines whether antigen-stimulated B cells undergo apoptosis or activation. Moreover, we demonstrated that CD22 does not negatively regulate BCR signaling in memory B cells, resulting in augmented BCR signaling. Augmented BCR signaling in memory B cells appears to be involved in rapid B cell activation and antibody production in memory responses, which is crucial for host defense by vaccination. Taken together, membrane-bound lectins and probably their glycan ligands play important roles in the regulation of humoral immune responses.


15. Production of oligosaccharides by utilizing phosphorolytic enzymes

Motomitsu Kitamoto (National Food Research Institute)

Phosphorolytic enzymes that act on carbohydrates are interesting enzymes not only in terms of their basic properties but also in regard to their various applications. However, they have not been studied in as much detail as other types of enzymes such as the hydrolases and the synthases. Phosphorolytic enzymes have attracted attention because of their application in the synthesis of carbohydrate chains, exploiting their strict regio-specificities. In addition, a number of new phosphorolytic enzymes have been discovered recently. In this presentation, the applications of the phosphorolytic enzymes are described in an overview of the research that we have performed on this interesting class of enzymes. The possibility of the production of biologically functional sugar chains at a cost applicable for food ingredient will be discussed.


16. Application of endoglycosidases to glycotechnology

Kenji Yamamoto (Kyoto University)

Endoglycosidases which release sugar chains from various glycoconjugates are becoming indispensable tools for elucidating the functions and structures of sugar chains in glycoconjugates. Endo-β-N-acetylglucosaminidase (Endo-β-GlcNAc-ase) is an unique endoglycosidase that hydrolyzes N, N'-diacetylchitobiosyl linkages in oligosaccharides bound to asparaginyl residues of various glycoproteins, and leaves one N-acetylglucosamine (GlcNAc) residue on the protein. A novel Endo-β-GlcNAc-ase of Mucor hiemalis, named as Endo-M, could cleave the complex type of oligosaccharide, unlike other microbial Endo-β-GlcNAc-ases. This enzyme showed transglycosylation activity and could transfer the oligosaccharides from glycoprotein to an acceptors with a GlcNAc residue. We developed a procedure for the synthesis of glycopeptides combining chemical and enzymatic methods using transglycosylation activity of Endo-M and chemo-enzymatically synthesized various bioactive glycopeptides such as glycosylated Peptide T and calcitonin. We could also add sialooligosaccharide to the glutamine residues of Substance P neuropeptide and yeast α-mating factor using the transglycosylation activity of Endo-M. The conversion of high-mannose type of oligosaccharide in bovine RNase B to complex type of oligosaccharide was successfully completed and sialoglycopolymer with a chitosan backbone as a potent inhibitor of human influenza virus hemagglutination was also prepared by use of Endo-M. Other endoglycosidases from various sources will be discussed.


17. Elucidation of the Roles of Glycans using RNAi Technology

Shoko Nishihara (Soka University)

Glycans on proteins and lipids are developmentally regulated and play an important role for cell-cell recognition. The glycosylation is performed in the Golgi apparatus by glycosyltransferases, which transfer various sugars from sugar-nucleotides to acceptor substrates. Our molecular evolutionary study showed that a prototype of glycosyltransferases was conserved between mammals and Drosophila, which is well established as a model for genetic analysis. RNA interference (RNAi) is becoming an efficient reverse genetic tool for studying gene function in model organisms. To clarify the basic physiological functions of glycans, we established Drosophila inducible RNAi system and applied it to glycosyltransferases.
If the silencing of each gene was induced ubiquitously in the fly, many RNAi mutant flies showed lethality to prove essential roles of glycans in development. Tissue-specific silencing induced various malformed phenotypes in the flies, by which genes were classified into some groups. These clusters might reflect the biosynthetic pathways and distinct functions of the various glycans. We will present a new approach to the comprehensive analysis of glycan function in development.


18. Strategic analysis of carbohydrate functions through gene knockouts

Kazuya Momura (Kyushu University)

The nematode Caenorhabditis elegans is an ideal model organism for functional genomics. Human genome and the nematode genome have many glycome-related genes in common. In this project, we select human glycome-related genes systematically and study functions of the genes by using the nematode. RNAi and deletion mutagenesis is systematically used for this purpose, aiming of the complete functional analysis of glycome-related genes and associated gene networks. For instance, we found 145 human glycosyltransferase orthologs in the nematode genome, and especially severe phenotypes were observed in 20% of their gene knockout experiments, confirming the importance of glycome-related genes in cell division, neural circuit formation and morphogenesis. In addition to DNA microarray analysis of mutant animals, proteome analysis is being used extensively in this study, and revealing the gene networks of carbohydrate related proteins will throw light on the hidden roles of sugar chains in multicellular organisms including ourselves.


19. Modulation of angiogenesis in zebrafish embryos by sphingolipids

Toshiro Okazaki (Tottori University)


20. Regulation of sphingolipid-signaling by neutral ceramidase

Makoto Ito (Kyushu University)

Neutral ceramidase is a type II integral membrane protein which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell-surface ceramide which increased in amount after the treatment of cells with bacterial sphingomyelinase or doxorubicin, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate (S1P). On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as S1P were generated in the cell-free conditioned medium of ceramidase transfectants, compared to mock transfectants. A S1P receptor, S1P1, was internalized much faster by the treatment of S1P1-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Interestingly, knockdown of the enzyme by anti-sense morpholino oligonucleotide inhibited the S1P-mediated angiogenesis during zebrafish embryogenesis. These results clearly indicate that the enzyme is involved in ceramide metabolism at the plasma membrane and in extra milieu, which regulates the S1P level and consequently S1P-mediated signaling.


21. New roles of brain glucosylated lipids

Yoshio Hirabayashi (RIKEN)

Glycosphingolipids and cholesterol form lipid microdomains in the plasma membranes. The microdomains were isolated biochemically as detergent-insoluble membrane (DIM) by sucrose density-gradient centrifugation of nonionic detergent lysate. We previously reported the presence of phosphatidylglucoside (PtdGlc)-based lipid microdomain distinct from the sphingolipids-based domains in the plasma membranes of HL60 cells. In order to understand the biological roles of these microdomains, we have isolated 50 clones of mouse monoclonal antibodies (MAbs) against the DIM fractions from HL60 and PC12 cells. We found that one of MAbs termed DIM21 preferentially reacted to PtdGlc. Immunohistochemical studies with DIM21 revealed that the antigen was strongly expressed in the fetal mouse brain in which the neuronal cells actively proliferate and migrate. Our observation indicates that PtdGlc might play a pivotal role in the development and differentiation of CNS.
PtdGlc has a mass number identical to phosphatidylinositol. The TLC migration pattern is almost identical to a major phospholipid PC (Phosphatidylcholine). These are major reasons why the presence PtdGlc has been overlooked until now. We recently have isolated the glycolipid to a pure form fetal rat brains and determined it's complete configuration using 600MHz NMR. This is the first case of the presence of PtdGlc in the central nervous system. We suggest here that PtdGlc is a novel surface marker for the radial glia cells.


22. Functions of glycoconjugates in the membrane microdomains in pathophysiological processes

Koichi Honke (Kochi University Medical School)

The plasma membranes include various microdomains in which special glycolipids and glycoproteins are associated, serving as platforms for cell adhesion and signal transduction. In this study, we will elucidate biological roles of glycoconjugates in these microdomains in development, differentiation, cancer, virus infection and immunity by production of antibodies against microdomain structures and development of probes to observe membrane microdomains in living cells.


23. Synthetic study of glycoconjugates for elucidation of their biological functions

Koichi Fukase (Osaka University)

Innate immunity is a phylogenetically ancient defense system against micro-organisms. The innate immune response is activated by receptors that recognize microbial components such as bacterial peptidoglycan (PGN) and lipopolysaccharide (LPS).
LPS is a cell surface glycoconjugate of Gram-negative bacteria. The active entity of LPS is its lipophilic part designated lipid A. Various structural analogues, including radio and fluorescence labeled derivatives, were synthesized to elucidate the mechanism of immunostimulation. Recent studies have revealed that the complex of toll like receptor 4 (TLR4)/MD-2 is the receptor of LPS and lipid A. Binding study of TLR4/MD-2 with the radio-labeled lipid A clearly demonstrated that TLR4/MD-2 directly binds to lipid A.
PGN, a major component of cell wall, is an alternating β(1,4) linked N-acetylmuramyl-N-acetylglucosaminyl glycan whose residues are cross-linked by short peptides. We previously demonstrated that the minimum structure required for the immunostimulation of PGN is muramyl dipeptide (MDP). MDP and various PGN partial structures (mono-, tetra- and octasaccharides having di-, tri-, tetra- or pentapeptides) showed the activity via TLR2 independent pathway, although TLR2 was reported to be a PGN receptor. An intracellular receptor, NOD2, was then found to recognize MDP as well as these partial structures.


24. Studies on Development of Novel Glycotherapeutics to Control Infection and Symbiosis

Makoto Kiso (Gifu University)

Studies on development of novel glycotherapeutics to control infection and symbiosis have been achieved with the following research projects: (1) Development of innovative basic technology for structure analysis of glycans and its application, (2) Analysis of interaction between botulinum toxin and the small intestinal mucin glycans and its application, (3) Enzymatic synthesis of functional glycocomplex and analysis of interaction between enterobacteria and glycans, and (4) Development of innovative chemical synthetic methods and invention of functional glycan molecules.


25. Preparation of Dibranched Complex Type Oligosaccharide Libraries and Sialylglycopeptide Synthesis

Yasuhiro Kajihara (Yokohama City University)

We have found a convenient preparation method of diverse Asn-oligosaccharides on a gram scale and its application method for the synthesis of glycopeptides (Angew. Chem Int. Ed. 42 (22), 2537-2540, (2003); Chemistry Eur. J. 10 (4), 971-985 (2004)).
Our strategy for solid phase glycopeptide synthesis uses Fmoc-asparagine linked oligosaccharides of which hydroxyl groups are free. We established first time the synthesis of large glycopeptide having pure complex type sialyloligosaccharides on the peptide backbone.


26. Synthetic Approaches to Glycoprotein Quality Control System

Yukishige Ito (RIKEN)

N-Glycosylation of Asn is a prominent modification of eukaryotic proteins. It occurs co-translationally in ER and then becomes a subject of further processing by a variety of glycosidases and glycosyltransferases to produce highly diverse structures. It is becoming clear that a number of intracellular proteins are in involved in this process. For instance, endoplasmic reticulum (ER) residing molecular chaperones calnexin (CNX) and calreticulin (CRT) are considered to recognize the oligosaccharide portion (Glc1Man9GlcNAc2) of glycoproteins and assist their folding. Other major players in glycoprotein quality control are glucosyl transferase (UGGT), mannosidase-like lectin (EDEM) and cargo receptors (VIP36, ERGIC-53) and ubiquitin ligase (Fbs1). All of these proteins likely recognize precisely different oligosaccharide structures, although molecular basis of these phenomena is unclear. We achieved the first chemical synthesis of dodecasaccharide (GlcMan9GlcNAc2), which is a putative ligand of CNX and CRT. Recently, we developed the first synthetic substrate of UGGT. A systematic study is in progress to 1) comprehensively prepare N-linked glycans and library of their partial structures, that may exist in ER and play roles in protein quality control, 2) synthesize glycoprotein having homogeneous glycan chain, and analyses of interactions between synthetic glycans and various proteins involved in glycoprotein quality control.


27. Development of IgA nephropathy-like disease with deficient N-glycosylation of serum IgA in β-1,4-galactosyltransferase-I-deficient mice

Masahide Asano (Kanazawa University)

IgA nephropathy (IgAN) is the most common form of glomerulonephritis worldwide and 30-40% of patients progress to renal failure 20 years after the onset of the disease in Japan. However, pathological molecular mechanisms of IgAN remain to be elucidated. Here we report that mice deficient in β-1,4-galactosyltransferase (βGalT)-I developed human IgAN-like glomerular lesions with IgA deposition, expanded mesangial matrix, and mesangial cell proliferation. Furthermore, they showed high serum IgA levels with increased polymeric forms. These features are consistent with the pathological diagnosis of human IgAN. Thus, the β4GalT-I KO mouse is a novel pathological model for human IgAN. In humans, serum IgA1 with truncated O-glycans in its hinge region is suggested to be involved in the pathogenesis of IgAN. The polymeric and Gal-deficient IgA from the β4GalT-I KO mouse tends to accumulate in the glomeruli. Since mouse IgA does not have O-glycans in its hinge region, our results strongly suggest that aberrant N-glycans as well as O-glycans of serum IgA play a pathological role in the development of IgAN.


28. Development of the recombinant lysosomal enzyme replacement therapy targeted to brain

Koji Itoh (University of Tokushima)


29. Development of a novel glyco therapy/glyco diagnosis for cancer and viral diseases

Nobuto Koyama (Takara Bio Inc.)

Changes in oligosaccharide structure of glycoproteins are observed in cancer, inflammation, and viral diseases. The objects of our study are to elucidate the biological significance of the structure of N-linked oligosaccharides, and to develop a novel therapeutic tool or a diagnostic method for cancer and viral diseases. Our projects are summarized in 3 categories as follows.
(1) Intracellular trafficking of glycoproteins and lectins: Annexin V is an animal lectin which recognizes a bisecting GlcNAc structure. We found that annexin V interacts with a molecular chaperon Hsp47 through its bisecting GlcNAc. Secretion of HBs antigen was selectively inhibited by gene transfection of GnT-III into HB611 cells. Glycomic approach revealed that oligosaccharide structures on adhesion molecules such as cadherin and integrins were selectively modified. These results suggest that bisecting GlcNAc affects the intracellular trafficking of certain kinds of glycoproteins.
(2) Oligosaccharide and hepatic stem cells: A rat hepatic stem-like cell line, RLE was found to express high levels of bisecting GlcNAc on its cell surface, and we are developing the method for separating hepatic stem cells by using E4-PHA lectin. RLE cells showed reduced expression of bisecting GlcNAc during differentiation, which suggests that bisecting GlcNAc and differentiation are closely related in hepatocytes.
(3) Modulation of oligosaccharide structures: The structure of N-linked sugar chain is related to tumor metastasis and proliferation of cancer cells. We analyzed the structure and function of GnT-III and GnT-V promoters with the object of searching substances for modulating the expression of these genes to utilize as drugs and supplements. Knockout/knockdown of fucosyltransferase 8 gene leads to growth retardation in vivo and in vitro. We found that trypsinogens and PAR-2 receptor are responsible for the mechanism. Thus, we intend to develop a cancer therapy by modulating the expression of glycosyltransferase genes.


30. Impact of oligosaccharide modification on therapeutic antibody

Mitsuo Sato (Kyowa Hakko Kogyo Co., Ltd.)

IgG1 has two N-linked oligosaccharide chains bound to the Fc region. The oligosaccharides are the complex biantennary type, composed of a trimannosyl core structure with the presence or absence of core fucose, bisecting N-acetylglucosamine (GlcNAc) and terminal galactose, giving rise to structural heterogeneity. Antibody-dependent cellular cytotoxicity (ADCC), a lytic attack on antibody-targeted cells, has been found to be one of the critical effector functions responsible for the clinical efficacy of therapeutic antibody such as anti-CD20 IgG1 (RituxanTM) and anti-Her2/neu IgG1 (HerceptinTM). ADCC is triggered upon binding of lymphocyte receptors (FcγRs) to the antibody Fc region, depending on the amount of fucose attached to the innermost GlcNAc of N- linked oligosaccharide via α1,6-linkage, and is dramatically enhanced by the fucose reduction. Importantly, fucose-negative therapeutic antibodies are not expected to be immunogenic as their carbohydrate structures are a normal component of natural human serum IgG. Thus, the application of fucose-negative antibodies should be a powerful and elegant approach as a next generation of therapeutic antibody with improved efficacy.


31. A new function of influenza A virus neuraminidase spike
- Relation to host range, viral replication and global pandemic –

Yasuo Suzuki (University of Shizuoka)

We found that low-pH stability of influenza A virus neuraminidase spike is functionally linked to virus entry via endocytic pathway and contributes to the ability of virus replication, host range and also global pandemic.