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Systems Glycobiology Research Group,
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JCGG Symposia

The 4th Symposium of Japanese Consortium for Glycobiology and Glycotechnology

Integration from functional analyses of sugar chains to systems glycobiology
October 23 - 24, 2006 Tokyo Conference Center (Shinagawa)
Summary
S-1
Molecular Mechanisms of Membrane Trafficking and Protein Sorting

Akihiko Nakano

The secretory pathway is the place where a variety of glycoproteins and glycolipis are synthesized and modified. We have been studying molecular mechanisms underlying protein transport and sorting events during membrane traffic between secretory organelles. Yeast Saccharomyces cerevisiae has been our model system and we apply biochemical, genetic and live imaging approaches to address these problems. In this symposium, I will talk about two recent topics from my research group: 1) COPII vesicle formation from the endoplasmic reticulum and 2) protein trafficking between the cisternae of the Golgi apparatus.

 

1. Heterogeneity of lipid rafts

Toshihide Kobayashi

Lipid rafts are defined as sphingolipid- and cholesterol- rich clusters in the membrane. Details of the lipid distribution of lipid rafts are not well characterized, mainly because of a lack of appropriate probes. Ganglioside GM1-specific protein, cholera toxin, has long been the only lipid probe of lipid rafts. Recently it was shown that earthworm toxin, lysenin, specifically recognizes sphingomyelin-rich membrane domains. Binding of lysenin to sphingomyelin is accompanied by the oligomerization of the toxin that leads to pore formation in the target membrane. In this study, we generated a truncated lysenin mutant that does not oligomerize and thus is non-toxic. Using this mutant lysenin, we showed that plasma membrane sphingomyelin-rich domains are spatially distinct from ganglioside GM1-rich membrane domains in Jurkat T cells. Like T-cell receptor (TCR) activation and cross-linking of GM1, cross-linking of sphingomyelin induces calcium influx and ERK phosphorylation in the cell. However, unlike CD3 or GM1, cross-linking of sphingomyelin did not induce significant protein tyrosine phosphorylation. Combination of lysenin and sphingomyelinase treatment suggested the involvement of G-protein coupled receptor in sphingomyelin-mediated signal transduction. These results thus suggest that the sphingomyelin-rich domain provides a functional signal cascade platform that is distinct from those provided by TCR or GM1. Our study therefore elucidates the spatial and functional heterogeneity of lipid rafts.

 

2. Lipid rafts formation by glucosylated lipids and neuronal functions

Yoshio Hirabayashi

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 a mouse monoclonal antibody, termed DIM21 preferentially reacting to PtdGlc. DIM21 immunostaining of murine CNS tissues showed a stage- and cell type-specific localization of DIM21 antigen during development, with high expression in radial glia/astroglia. DIM21 immunostained cultured hippocampal astroglia in a punctate fashion. To characterize the structure of PtdGlc, we isolated DIM21 antigen from embryonic day 21 rat brains. We purified two previously unrecognized glycolipids, PGX-1 and PGX-2. Structural analyses with 600 MHz 1H-NMR, FT-ICR mass spectrometry, and GC revealed that PGX-1 is phosphatidyl-β-D-(6-O-acetyl) glucopyranoside and PGX-2 is phosphatidyl β-D-glucopyranoside. Surprisingly both glycolipids were composed exclusively of C18:0 at the C1 and C20:0 at the C2 position of the glycerol backbone. This saturated fatty acyl chain composition comprising a single molecular species rarely occurs in known mammalian lipids and provides a molecular basis of why PtdGlc resides in raft-like lipid microdomains.

 

3. Glycolipid-Mediated Axon Guidance

Hiroyuki Kamiguchi

Neuronal networks are formed by axons that have elongated and reached their appropriate targets. The tip of an elongating axon, called the growth cone, explores guidance cues in the surrounding environment and migrates along the correct path. In collaboration with the Hirabayashi Research Unit of RIKEN-BSI, we have discovered a novel glycolipid that is likely to function as an axon guidance molecule. This glycolipid is synthesized by GFAP-positive glial cells and released into the extracellular space as the lyso-form after hydrolysis. In the chick developing spinal cord, this glycolipid is distributed predominantly in the dorsomedial and ventral regions, suggesting that it acts as a repulsive cue against TrkA-positive nociceptive axons of dorsal root ganglion neurons. In vitro, the lyso-form of this glycolipid repels TrkA-positive axons at a concentration as low as 100 pM near the growth cone. The repulsive effect of this glycolipid is dependent on neuronal cell types. We have also identified a part of intracellular signaling components that mediate the repulsive guidance of growth cones. Our work would provide the first example of lipid-mediated axon guidance.

 

4. Metabolic regulation of amyloid β peptide and Alzheimer's disease

Takaomi Saido

The conversion of what has been interpreted as "normal brain aging" to Alzheimer's disease (AD) via a transition state, i.e. mild cognitive impairment, appears to be a continuous process caused primarily by aging-dependent accumulation of amyloid β peptide (Aβ) in the brain. This notion gives us a hope that, by manipulating the Aβ levels in the brain, we may be able not only to prevent and cure the disease but also to partially control some very significant aspects of brain aging. Aβ is constantly produced from its precursor and immediately catabolized under normal conditions, whereas dysmetabolism of Aβ seems to lead to pathological deposition upon aging. We have focused our attention on elucidation of the unresolved mechanism of Aβ catabolism in the brain and identified the major Aβ-degrading enzyme as neprilysin, a type II membrane bound neutral endopeptidase. Reverse genetic studies demonstrated that reduction of the enzyme activity, which can be caused by aging, results in elevation of steady-state Aβ levels. Neprilysin can degrade in vivo both monomeric and oligomeric forms of Aβ, that cause impairment of neuronal plasticity and cognitive function. In search for pharmacological means to manipulate neprilysin activity, we discovered that somatastatin regulates brain Aβ levels via modulation of neprilysin activity, indicating that somatostatin receptor subtype(s), i.e. those robustly expressed in the brain, will be a therapeutic target. We also found that calpain, calcium-activated neutral protease, is involved in dendritic degeneration. These findings suggest that AD is one of the typical proteolytic disorders and that pharmacological modulation of proteolytic processes is likely to be a realistic strategy to prevent and treat the disease.

 

5. Pathophysiology of glycosyltransferase processing by Alzheimer's β-secretase

Yasuhiro Hashimoto

Alzheimer's β-secretase (BACE1) cleaves α2,6-sialyltranferase to downregulate α2,6-sialylation of glycoproteins. BACE1 also cleaves amyloid precursor protein (APP) to initiate production of a neurotoxic peptide Aβ, which induces neuronal cell death and ends up to cause Alzheimer's disease. Here we report that overexpression of α2,6-sialyltransferase in Neuro2a cells enhanced α2,6 sialylation of endogenous APP and increased the extracellular levels of its metabolites including Aβ, suggesting that sialyltaion of APP enhances its metabolic turnover and promotes pathogenic processes of the disease. We have recently developed a sandwich ELISA system for measuring the level of secreted sialyltransferase as a marker of BACE1 activity. We hope to apply the system for the early diagnosis of the disease. The system would be also useful for screening BACE1 inhibitors as therapeutics of Alzheimer's diasease.

 

6. Glycoprotein glycans : Organic synthasis and functional analysis

Yukishige Ito

Our research has been directed to the synthesis and functions of Asn-linked high-mannose-type oligosaccharide, [1]. Currently, a systematic study is in progress to 1) comprehensively prepare N-linked glycans and 2) synthesize glycoprotein having homogeneous glycan chain [2,3]. In glycoprotein quality control system, a variety of proteins icluding chaperones (calnexin, calreticulin), glycosidases (glucosidases, mannosidases), glucosyltransferase (UGGT), lectins (EDEM and its homologues), cargo receptors (VIP 36, ERGIC-53), PNGase, and ubiquitin ligases (Fbs) play important roles. Although most of them are considered to recognize high-mannose-type glycans, their specificieties are yet to be clarified. Our effort toward comprehensive synthesis of ER-related high-mannose-type glycans and thier application to the analyses of catbohydrate-protein interactions involved in protein quality control will be discussed. Particular focus will be placed on, the quantitative analyses of carbohydrate-calreticulin and -Fbs1 interactions and substrate specificity of UGGT and glucosidase II.

 

7. Glycosylation of an ADAMTS protease essential for cell migration in C. elegans

Kiyoji Nishiwaki

In the nematode C. elegans, the gonad acquires two U-shaped arms by the directed migration of its distal tip cells (DTCs), which are located at the tip of the growing gonad arms. A member of the ADAMTS family protease, MIG-17, regulates directional migration of DTCs: MIG-17 is synthesized and secreted from the muscle cells of the body wall, and diffuses to the gonad, where it is required for DTC migration. mig-23, cogc-1 and cogc-3 mutants show misdirected migration of DTCs as do mig-17 mutants and are defective in gonadal localization of MIG-17. mig-23, cogc-1 and cogc-3 genes were found to encode homologs of Golgi NDPase (enzyme involved in protein glycosylation), COG-1 and COG-3 (two of the eight subunits of Conserved Oligomeric Golgi complex required for vesicle trafficking), respectively. The glycosylation and function of MIG-17 were defective in mig-23, cogc-1 and cogc-3 mutants. Furthermore, the protein levels of MIG-23 were significantly reduced in cogc-1 and cogc-3 mutants. These results support the model whereby the COG complex regulates the localization or stability of the Golgi enzyme MIG-23, and thereby affects the glycosylation and function of MIG-17/ADAMTS, which is critical for directional control of DTC migration.

 

8. Aberrant migration of the precursors of the vagus motor neurons in the mutant zebrafish embryos defective in the protein modification by fucosylation

Hitoshi Okamoto

The vagus nuclei are important as the autonomic center for the maintenance of homeostasis. In mammals, two motor nuclei, the nucleus ambiguous and the dorsal motor nucleus of vagus innervate muscles of branchial arches and parasynpathetic ganglia for control of visceral organs, respectively. Aberrant positioning of these nuclei is implicated in the etiology of the sudden infant death syndrome (SIDS) with severe respiratory disorder. Therefore, control of precursor cell migration into the right positions to establish the stereotyped arrangements of the nuclei may be crucially important for the vagus motor neurons to exert their normal functions. However the genetic program regulating this process is almost unknown.
The zebrafish embryo also has two vagus motor nuclei, the dorso-laterally and medially located nuclei (dmX and mmX). The dmX precursors are born near the floor plate, migrate dorso-laterally and then are accumulated at the defined position. In the towhead mutant embryos, ectopic neurons are distributed between bilateral dmX resulting in obliteration of the distinctive nuclear structure. Time-lapse imaging of hindbrain explants revealed that precursors of dmX neurons aberrantly migrate in dorsal direction and fail to stop at the right position in the mutant embryos. Positional cloning identified TOWHEAD as a GDP-mannose 4,6 dehydratase (GMDS), a key enzyme in the fucosylation pathway. Fucosylation of glycans is catalyzed by distinct fucosyltransferases with a requirement for the sugar nucleotide substrate GDP-fucose. The GMDS works at the first step in the de novo synthesis of GDP-fucose by which GDP-mannose is converted into GDP-fucose. Examination of distribution of various types of glycans by using biotin-conjugated lectins revealed that the amount of fucosylated glycans is remarkably and specifically reduced in the mutant embryos. Expression of fucosylated glycans was recovered in the mutant embryos by injection of wild-type GMDS mRNA, where precursor cell migration and accumulation were also recovered into the right positions. Together, our findings identify important roles of the fucosylation pathway in the regulation of migration and accumulation of vagus motor neuron precursors.

 

9. Expression analysis of glycochain-related genes using correlation index-based DNA microarray

Yasusada Kozutsumi

Changes in the expression of glyco-chains have influence on many biological events. To understand the biological role in the glyco-chain expression, the systematic study on the overall regulation of genes involved is very important. For this purpose, we created human and mouse DNA microarrays containing genes involved in expression of glyco-chains, such as glycosyltransferases, glycosidases, nucleotide sugar-modification enzymes and other components related to carbohydrates. Applications of these DNA microarrays are open to other scientists inside and outside of RIKEN as a national resource. Furthermore we established a new technology of DNA microarray, which enables us to directly find candidate genes involved in individual glyco-chain expressions. Using the new technology, we determined a glyco-gene responsible for the expression of GL7, which is a monoclonal antibody specific for the mouse germinal center upon T-dependent antigen immunization. These data suggest that the new technology of DNA microarray is a powerful tool for analysis on gene expression involved in regulation of the glyco-chain expression.

 

10. NKT cell therapy using α-GalCer-pulsed DCs

Masaru Taniguchi

Human NKT cells bearing an invariant Vα24JαQ antigen receptor are activated by a specific ligand, α-Galactosylceramide (α-GalCer; KRN7000). Intravenous administration of α-GalCer-pulsed DCs induces significant activation of NKT cells and results in the complete eradication of metastatic tumor in the liver (Figure). We performed a phase I dose escalation and phase IIa studies with α-GalCer-pulsed DCs in lung cancer patients. Eleven patients were enrolled in the phase I, and 20 cases are ongoing in the phase IIa studies. No severe adverse events were observed during the phase I in any patient. In the phase IIa, dramatic increase in NKT cells was observed in >50% cases, and significant IFN-γ responses were seen in >90% cases. Concerning anti-tumor responses, no patient was found to meet the criteria for partial or complete responses, while several cases remained unchanged tumor size for more than 2 years with good QOL. Since we have not completed the phase IIa, median survival time cannot be calculated. However, the tentative median survival time by α-GalCer/DC therapy (based on 12 cases who has finished the protocols) is >20 months compared to those by conventional therapies (7.8 months). Thus, α-GalCer-pulsed DC therapy was well tolerated.

 

11. Regulation of B cell activation mediated by Siglec2/CD22

Takeshi Tsubata

Siglec2/CD22, a member of the Siglec family, negatively regulates signaling through the B cell antigen receptor (BCR) by activating the SHP-1 phosphatase, and functions as a molecular switch that determines whether antigen-stimulated B cells undergo apoptosis or activation. CD22-mediated signal inhibition is regulated by various factors such as Ig class of BCR, glycan ligands, BCR ligands etc. The cytoplasmic tail of both IgG and IgE abrogates CD22-mediated signal inhibition by perturbing association of CD22 with BCR, thereby generating efficient BCR signaling crucial for efficient antibody production. CD22-mediated signal regulation is also regulated by its glycan ligand especially when CD22 function is compromised. Further investigation is required for elucidating the role of various factors that regulate CD22 in vivo, and for developing a new strategy that controls antibody responses by regulating CD22.

 

12. Generation and analysis of siglec-1+ macrophage depletion mice

Yasunobu Miyake

Apoptotic cell death is a critical process for elimination of unnecessary cells. After cells undergo apoptosis, apoptotic cells are rapidly recognized and engulfed by phagocytes such as macrophages and dendritic cells (DCs). Rapid removal of apoptotic cells by phagocytes prevents the release of potentially toxic materials from dying cells. When apoptotic cells are injected into mice, they initially accumulate in the marginal zone of spleen. Marginal zone is located between white pulp and red pulp in the spleen, and we can find siglec-1+ macrophages in this area. To analyze the role of siglec-1+ macrophages in apoptotic cell clearance, we generated siglec-1+ macrophage depletion mice. Clearance of apoptotic cells in the marginal zone was greatly delayed in siglec-1+ macrophage depleted mice, suggesting that siglec-1+ macrophages were important for efficient clearance of apoptotic cells in the marginal zone. The apoptotic cells are also engulfed by DCs. In the spleen, there are two DC subsets, CD8+ DC and CD11b+ DC. Although CD8+ DC selectively engulfed apoptotic cells in wild type mice, the aberrant engulfment of apoptotic cells by CD11b+ DC was observed in siglec-1+ macrophage depleted mice. This result indicates that siglec-1+ macrophages regulate selective engulfment of apoptotic cells by CD8+ DC.

 

13. Clinical utility of AFP-L3% : Fucosylated AFP

Shinji Satomura

Chronic liver diseases caused by hepatitis B and C viruses is an established significant risk factor for the development of hepatocellular carcinoma (HCC). Alpha-fetoprotein (AFP) is an oncofetal glycoprotein that has been used as tumor marker for HCC. However, it is not specific for HCC, AFP is commonly elevated in patients with chronic hepatitis and cirrhosis without HCC. The Lens culinaris agglutinin-reactive-AFP (AFP-L3) has a 1-6 fucose residue appended to the N-acetylglucosamine at the reducing end has been reported to be a more specific marker for HCC. By multi-center, prospective clinical studies on a larger population of patients with HCC and related benign liver diseases in Japan and US, the determination of AFP-L3 has a significance in increasing the specificity to HCC, allowing us to use it as diagnostic markers of HCC in an early stage of evolution.

 

14. Development and application of trehalose from basic research to business

Shigeharu Fukuda

Trehalose is a non-reducing disaccharide that is widespread in nature. This sugar is very common in bacteria, mushrooms, seaweed and insects as a source of reserve energy. It has also been reported that this sugar may function to protect the tissues of some plants and animals from damage due to desiccation and freezing. Therefore, this sugar has been expected to serve for various applications, for example as a stabilizer for dried or frozenfoods, in cosmetics, and as a drug additive.
A novel enzymatic synthesis pathway of trehalose from starch was found in some bacteria isolated from soil such as strains belonging to Arthrobacter and Rizobium. It was found that two enzymes, MTSase and MTHase, were involved in trehalose synthesis from starch. By sequencing analysis, these enzyme and isoamylase genes are found to be formed trehalose-operon. Now, we produce high purity trehalose in industrial scale with combination of above two enzymes and isoamylase.

 

15. Towards integrated understanding of signal regulation mechanisms with glycosylation

Koichi Furukawa

Glycosphingolipids are amphopathic molecules consisting of ceramides and polymorphic sugar moiety. Carbohydrate remodeling in cultured cells and experimental animals with manipulation of glycosyltransferases has provided a number of evidences indicating that sugar chains interact with various molecules and play important roles in the regulation of signaling, sometimes leading to the decision of cell fates. Here, representative results on the signals regulated by glycolipids in cancer cells and nervous tissues will be presented, and approaches toward integrated understanding of glyco-functions will be discussed. Recent investigation using sugar remodeling in PC12, melanomas and small cell lung cancers etc revealed that disialyl-ganglioisides play roles in the enhancement of malignant properties of the tumor cells by interacting with integrins, focal adhesion kinases, and other signaling molecules probably at lipid rafts. On the other hand, expression of GM1 resulted in the suppressin of signals for PC12 differentiation and metastatic potential in Lewis lung cancer cells, indicating the significances of the individual glycosylation patterns in the control of lipid rafts. Various abnormal phenotypes found in the ganglioside-defective mutant mice are also ascribed to modulated lipid rafts in vivo. Taken together, modes of interaction between glycolipids and proteins in the membrane are key issues to be investigated.

 

16. Function of Notch signaling in somitogenesis via glycosylation

Yumiko Saga

The metameric structure of the vertebrate is based on the correctly segmented somites. Somitogenesis proceeds by sequential segmentation of the paraxial mesoderm. The periodicity is generated by a segmentation clock in the posterior presomitic mesoderm (PSM) and this temporal periodicity is then translated into the segmental units in the anterior PSM. Knockout mouse studies have shown that Notch signaling is critically important signaling pathway in somitogenesis. In addition, glycosylation of Notch receptor via Pofut1 and Lunatic fringe (L-fng) is implicated in the regulation. Recently, we succeeded in visualization of oscillation of Notch1-activity in mice, and found that the periodicity was generated by negative-feedback function of L-fng. In the anterior PSM, a transcription factor Mesp2 functions to translate information provided by segmentation clock into the segmental morphology. Notch signal is suppressed in the Mesp2 expression domain which demarcates the next segmental border. We demonstrate that the function of Mesp2 is mediated via activating L-fng. However, the detailed mechanism has remained elusive. We are studying how the Notch singling is regulated and what really happens by glycosylation.

 

17. The structure and function of a glycosidase analyzed by ultra-high resolution crystallography

Hiroaki Kato

Development of high brilliant synchrotron radiation and efficient X-ray detectors enabled us the analysis of very precise X-ray crystal structure of proteins. At an ultra-high-resolution that exceeds 1.0 Å, one can experimentally determine hydrogen atom positions in protein molecules and solvents in the crystalline state. One could also define the ionization states of amino acid residues of proteins. On the other hand, because neutron is scattered by the atomic nucleus, neutron diffraction has the advantage in the structural analysis of hydrogen atoms instead of X-ray. Therefore, precise crystallographic analyses using combination of X-ray and neutron diffractions provide us extremely accurate structural information for the architecture of the protein structure including hydrogen atoms.
Endo-polygaracturonase (EC 3.2.1.15; endoPG) is an enzyme that catalyzes the hydrolysis of polygalacturonic acid. We determined the three-dimensional structure of endoPG by high resolution X-ray diffraction. The crystals of EndoPG diffracted at least 0.62 Å on the beam line BL41XU, SPring-8. The crystals also provided the neutron diffraction data at 1.5 Å resolution on the research reactor JRR-3, Japan Atomic Energy Research Institute.
I will present architecture of endoPG structure based on the precise crystallographic analyses.

 

18. Significance of glycosylation analysis in cell therapy and challenges to characterization of cellular therapy products based on glycosylation

Nana Kawasaki

Advances in cell processing technologies are making it possible to provide human/mammalian cells as cellular therapy products. To ensure the quality, efficacy and safety of these products, the Japanese Ministry of Health Labor and Welfare requests characterization and appropriate-quality testing of them. Glycosylation is involved in many biological events in cells, including differentiation, adhesion, and cellular function; therefore, glycosylation analysis should be crucial in the characterization of cellular therapy products. Glycan profiling and/or carbohydrate structure analysis have a potential as characteristic indexing methods for these products. A desired glycoprotein to be secreted from a cell therapy product should be quantified and well characterized by appropriate analytical techniques. Contaminants such as NeuGc should be suitably controlled with the in-process acceptance criteria. Some glyco-epitopes have the potential to be used for purity and impurity profiling of the cells. In this symposium, we would like to introduce some methods for the characterization of cellular therapy products based on the glycosylation analysis using proteomic/glycomic approaches, including liquid chromatography/mass spectrometry.

 

19. Structural Characterization of Protein and Carbohydrates Using MALDI-MSn

Koichi Tanaka

Matrices (e.g.: 2,5-dihydroxybenzoic acid (DHBA), 1,5-diaminonaphtalene (1,5-DAN), nor-harmane) have been further developed for ionizing oligosaccharide and glycopeptide samples, and for obtaining the structural information of those.

 

20. Structural characterization of glycopeptides and glycosphingolipids by mass spectrometry

Minoru Suzuki

Glycopeptides derived from Ribonuclease B, purified glycosphingolipids and amino pyridyl (PA) derivatives of oligosaccharide isomers have been successfully characterized by MALDI-QIT-TOF MS, MS/MS and MS/MS/MS. However, information about the site of oligosaccharide attachment of chemically synthesized O-linked glycopeptides could not be obtained by the method. In order to obtain information on the attachment site as well as peptide and oligosaccharide structures, we analyzed the O-linked glycopeptides by N-terminal protein ladder sequencing and MALDI-TOF mass spectrometry. As a result, structures of the O-linked glycopeptides could be characterized successfully.
Structural characterization of a mixture of globo-series glycosphingolipids, GlcCer, LacCer, Gb3Cer, Gb4Cer and Forssman antigen, has been performed by a system of ESI polarity switching ion-trap time-of-flight mass spectrometry coupled to high-performance liquid chromatography, which enables to obtain both positive- and negative-MSn mass spectra within 1 sec. The method would be useful for the structural characterization of a mixture of neutral glycosphingolipids from biological materials.

 

21. MS Imaging of glycolipid

Mitsutoshi Seto

In a conventional approach of mass spectrometry for biological specimens, analyte is extracted from tissues, separated and purified with several procedures such as chromatography. After these treatments, positional information of a target molecule in vivo is lost. However, mass imaging in microscopic resolution and identification, to say, mass microscopy, can provide discernible differences. This method is especially powerful for the research of molecular traffic which result in the spatial transportation but not the change of total amount of the target. In this presentation, we will offer our recent success mass microscope invention, which can visualize and identify molecules directly from tissues, and offer a topic of our recent data on a glycolipid, especially ganglioside, in mouse brain.

 

22. Post-translational modification analysis of a cryoglobulin derived from a myeloma patient using mass spectrometry

Masaki Yamada

An aberrant N-glycosylation on the light chain of monoclonal cryocrystalglobulin in a myelome patient was determined by gel electrophoresis and mass spectrometry. The cryocrystalglobulin was identified as IgG1 kappa type by peptide mass fingerprinting. The aberrant N-glycosylation on the kappa chain was indicated by the change in its electrophoretic mobility on SDS-PAGE after the treatment with PNGase F. The peptide sequence around the glycosylation site and the structure of the N-glycan were determined by MALDI-QIT-TOF MSn analysis. The major forms of the N-glycan were assigned to A2G2 type, and the terminal sialylation was also suggested by the MALDI-TOF mass spectrometry. The aberrant N-glycosylation might cause a critical protein misfolding of the kappa chain, and resulted in the formation of cryocrystalglobulin and skin ulcer of the myeloma patient.

 

23. A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells.

Hideyuki Okano

In the subventricular zone (SVZ) of the adult mammalian forebrain, neural stem cells (NSCs) reside and proliferate to generate young neurons. We screened factors that promoted the proliferation of NSCs in vitro by a recently developed proteomics technique, the Protein Chip system. In this screen, we identified a soluble carbohydrate-binding protein, Galectin-1, as a candidate. We show herein that Galectin-1 is expressed in a subset of slowly dividing SVZ astrocytes, which includes the NSCs. Intraventricular infusion of Galectin-1 resulted in increase of neural progenitors in the SVZ. The carbohydrate binding activity of Galectin-1 is required for this activity. The mutant mice of galectin-1 showed decreased neural progenitors in the SVZ. Based on these results, we propose that Galectin-1 promotes proliferation of adult NSCs. In addition, we show Integrin β1 as one of the counter receptors of Galectin-1 in the adult SVZ. In the present talk, I wish to also mention about the application of Galectin-1 to the therapeutic intervention for the damaged central nervous system.

 

24. The function of the HNK-1 carbohydrate

Shogo Oka

The HNK-1 carbohydrate is characteristically expressed on a series of cell adhesion molecules in the nervous system. The expression is spatially and temporally regulated during the development of the nervous system. The HNK-1 carbohydrate has a unique structure comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) and is sequentially biosynthesized by one of two glucuronyltransferases (GlcAT-P or GlcAT-S) and a sulfotransferase (HNK-1ST). To reveal the function of the HNK-1 carbohydrate, we generated gene-deficient mice of GlcAT-P, which is a major glucuronyltransferase compared with GlcAT-S in the nervous system. The GlcAT-P gene deficient mice exhibited reduced long-term potentiation (LTP) at the Schaffer collateral-CA1 synapses and defects in spatial memory formation. Therefore, the HNK-1 carbohydrate plays crucial roles in synaptic plasticity. However little is known about the molecular mechanisms how the HNK-1 carbohydrate controls synaptic plasticity. In this study, we produced the evidence that the HNK-1 carbohydrate is expressed on AMPA receptor, which is one of glutamate receptors involved in the synaptic plasticity. Using cultured hippocampal neurons, it was revealed that lacking of the HNK-1 carbohydrate affected the localization of AMPA receptor. These results suggest that the HNK-1 carbohydrate on AMPA receptor could be involved in the regulation of synaptic plasticity.

 

25. Protein O-mannosylation and α-dystroglycanopathies

Tamao Endo

Congenital muscular dystrophies (CMD) are a group of clinically and genetically heterogeneous conditions. Defects in glycosylation are now known to play a major role in CMD. A defect of initiation of protein O-mannosylation causes CMD with brain malformation and structural eye abnormalities, so called Walker-Warburg syndrome (WWS). α-Dystroglycan is an O-mannosyl-modified glycoprotein that is a central component of the dystrophin-glycoprotein complex. Protein O-mannosylation is catalyzed by protein O-mannosyltransferase 1 (POMT1) and its homolog, POMT2. Coexpression of POMT1 and POMT2 is required to show O-mannosylation activity. We show that POMT1 forms a complex with POMT2 by immunoprecipitation experiments. POMT1 and POMT2 were detected in the immunoprecipitates, and the precipitates possessed protein O-mannosyltransferase activity. Results indicate that POMT1 and POMT2 associate physically and functionally in vivo. Mutations found in the POMT1 gene of patients with not only typical WWS but also milder phenotypic WWS lead to great reduction of protein O-mannosyltransferase activity, although all POMT1 mutants coprecipitated with POMT2. These results indicate that the mutant POMT1s found in WWS patients could form hetero-complexes with POMT2 but that such complexes are insufficient for enzymatic activity. These findings suggest that protein O-mannosylation of α-dystroglycan is important in muscle and brain development, and that other factors in addition to protein O-mannosyl enzyme activity lead to a large spectrum of severity found in WWS patients.

 

26. Polysialic acid: Its frequent occurrence, structural diversity and functions

Ken Kitajima

Polysialic acid (PolySia) is a polymerized form of sialic acids, and is one of the most unique glycan structures on the cell surface. PolySia exhibits a large structural diversity, arising from differences in Sia species (Neu5Ac; Neu5Gc; KDN), linkages (α2,8-; α2,9; α2,8/9; α2,5Oglycolyl-), chain lengths (diSia for DP 2; oligoSia for DP 3-7; polySia for DP ≥8), and modifications (O-acetylation; O-sulfation). PolySia is first identified in the glycocalyx of neuroinvasive bacteria. In vertebrate, neural cell adhesion molecule (NCAM), a voltage-gated sodium channels, and fish egg polysialoglycoprotein (PSGP) are modified by polySia chains. Studies on NCAM showed that polySia chains are regulatory molecules in cell-cell/matrix interaction via anti-adhesive effect during neurogenesis. However, no biological functions of the polySia-containing glycoproteins other than NCAM have remained unknown. Thus, to understand biological functions of polySia in glycoproteins, we have focused on the structural diversity-function relationship of polySia. Here we show our recent results on the functional importance of the α2,8-diSia residues on CD166 in neurite outgrowth, and our new discoveries on two new polySia-containing glycoproteins, the human and mouse milk α2,8-polySia-containing glycoprotein, CD36 and the sea urchin sperm flagellar α2,9-polySia-containing glycoprotein, flagellasialin.

 

27. Customization of the extracellular microenvironment and an emerging concept of matriome

Kiyotoshi Sekiguchi

A hallmark of the extracelllular matrix (ECM) is its diversity of molecular composition. Individual cell types are believed to have their own customized extracellular environment with a distinct molecular composition. Such customized environment has been considered important for cells to maintain their specified functions. Uncovering the molecular composition of the customized ECM is the key to defining and reconstructing the microenvironment for in vitro manipulation of cells. We developed a transcriptome-wide strategy for identification of novel ECM proteins, based on computational screening of >60,000 mouse full-length cDNAs for secreted proteins, followed by in vitro screening for their capabilities of ECM assembly, interactions with other ECM proteins, modification with glycosaminoglycans, and cell-adhesive activities and by in vivo screening for their tissue localization. We identified 16 novel ECM proteins, of which 7 localized at the basement membrane (BM). We also screened for novel ECM proteins up-regulated in hair morphogenesis, identifying two novel BM proteins with an RGD-dependent cell-adhesive activity. We produced antibodies against these novel BM proteins and other known BM proteins and determined their tissue localization in mouse embryos. The resulting immunohistochemical images have been compiled in a database that can be viewed on the web at desired magnification without loss of resolution. The database, which we named the Basement Membrane Bodymap, provides the first bird's-eye view of the cell- and tissue-specific customization of ECM.

 

28. Formation of the tumor angiogenic microenvironment by hyaluronan-rich matrix

Naoki Itano

Overproduction and accumulation of hyaluronan in the most invasive breast cancers are well documented by extensive clinical evidence. In an effort to simulate the overproduction of hyaluronan observed in malignant human breast cancer, we generated transgenic mice that express murine hyaluronan synthase 2 (HAS2) in a Cre-mediated recombination-dependent manner. The HAS2 conditional transgenic mice were then crossed with mice of the MMTV-Neu mammary tumor model. Upon Cre-mediated recombination, the double transgenic mice exhibited overproduction of hyaluronan and its accumulation in mammary tumors. When we examined the early stages of tumor development, the incidence of mammary tumor was significantly greater and growth faster in HAS2 overexpressing mice than in the control mice. Notably, forced expression of HAS2 enhanced the recruitment of stromal cells into mammary tumors and increased intratumoral formation of microvessels compared with control mice. To reveal the molecular basis of hyaluronan-mediated neovascularization, Matrigel angiogenesis assay was performed in the presence of either hyaluronan or its aggregate containing the large chondroitin sulfate proteoglycan, versican. Administration of hyaluronan-versican aggregates, but not native hyaluronan alone, promoted stromal cell recruitment concurrently with the infiltration of endothelial cells. Taken together, these results suggest that hyaluronan-versican extracellular matrix accelerates tumor angiogenesis through stromal cell recruitment.

 

29. A role for oligosaccharide-based signaling in tumor invasion and metastasis

Michinari Hamaguchi

We will report the biological importance of oligosaccharide-based signaling in tumor invasion and metastasis by focusing on two molecules, ie Hyaluronan (HA) and SHPS-1/SIRPα. HA is emerging as an important metastatic marker in a number of human carcinomas. We have studying the role of HA with v-Src transformed cells and human tumor cell lines in which Src kinase is activated. We found, HA promotes cell locomotion in a tumor-specific manner. The v-Src kinase activates HA production via Ras and STAT3 signaling. Among 3 HAS, HAS2 is most important enzyme responsible for the tumor-specific production of HA. Thus, v-Src control the locomotion by a autocrine-like mechanism. SHPS-1/SIRPα. Was identified as a v-Src substrate. We found, however, SHPS-1/SIRPα directly binds with ConA and transduce its signal to produce MMP. We have identified several downstream signaling molecules of SHPS-1/SIRPα critical for the production of MMP. Suppression of ConA-dependent MMP production as well as those of proinflammatory cytokines by siRNA will be discussed in detail.