Chikusa ku, Japan


Chikusa ku, Japan
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Nishio M.,Nagoya City University | Nishio M.,Okazaki Institute for Integrative Bioscience | Nishio M.,Japan Institute for Molecular Science | Kamiya Y.,Nagoya City University | And 16 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2010

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 1:1 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

Kamiya Y.,Japan Institute for Molecular Science | Kamiya Y.,Nagoya City University | Yagi-Utsumi M.,Japan Institute for Molecular Science | Yagi-Utsumi M.,Nagoya City University | And 5 more authors.
Current Pharmaceutical Design | Year: 2011

The sugar chains covalently modifying proteins and lipids are recognized by a variety of proteins, thereby mediating a broad range of physiological and pathological events on cell surfaces as well as in cells. Hence, these carbohydrate-protein interaction systems could be potential therapeutic targets for various diseases, including viral infections, autoimmune diseases and neurodegenerative disorders. Cumulative crystallographic data of lectins complexed with their cognate carbohydrate ligands have elucidated the sugar recognition modes of these proteins, offering a structural basis for the design of drugs targeting carbohydrate-lectin interaction systems. In particular, structural and functional studies of animal L-type lectins, which possess a carbohydrate recognition domain with a structural resemblance to that of leguminous lectins such as concanavalin A, have demonstrated the molecular mechanisms underlying their distinct roles in sorting and trafficking of glycoproteins in cells, exemplifying the structure-based engineering that manipulates the sugar-binding properties of lectins. Furthermore, structural basis has been provided for the functional interplay between the L-type lectin ERGIC-53 and the EF-hand Ca2+-binding protein MCFD2 in the intracellular transport of the coagulation factors V and VIII. This article also deals with pathological carbohydrate-protein interactions involving ganglioside clusters on cell surfaces, particularly focusing on the interaction between amyloid β(Aβ) and GM1 ganglioside. This interaction triggers conformational transition and consequent aggregation of Aβ, and therefore, is considered to be a key step in Alzheimer's disease. The recently reported structural information of the Aβ-GM1 interaction is presented, underscoring the significance of assemblages of glycoconjugates as therapeutic targets. © 2011 Bentham Science Publishers.

Kato K.,Japan Institute for Molecular Science | Kato K.,Nagoya City University | Kato K.,Ochanomizu University | Kato K.,GLYENCE Co. | And 2 more authors.
Progress in Nuclear Magnetic Resonance Spectroscopy | Year: 2010

A paper published in Progress in Nuclear Magnetic Resonance Spectroscopy informs about stable-isotope-assisted NMR approaches to glycoproteins using immunoglobulin G (IgG) as a model system. The paper describes about the application of the stable-isotope-assisted NMR approach to structural analyses of glycoprotein glycans using IgG as a model system. IgG is described as a multi-domain glycoprotein with a molecular mass of 150 kDa, functioning as the major class of antibodies in the immune system. Three-dimensional structures have been determined by X-ray crystallographic analyses and are available for intact IgG molecules and for a proteolytic fragment Fc. Two methods are employed for stable-isotope-labeling of IgG-Fc glycans. These two methods include metabolic labeling through biosynthetic pathways of production vehicles and in vitro enzymatic attachment of isotopically labeled monosaccharide(s) onto the non-reducing end of the Fc glycans.

Miyagawa S.,Osaka University | Maeda A.,Osaka University | Kawamura T.,Osaka University | Ueno T.,Osaka University | And 6 more authors.
Glycobiology | Year: 2014

After producing α1-3-galactosyltransferase knockout (GKO) pigs, most of the organs of these pigs showed less antigenicity to the human body. However, wild-type adult pig islets (API) that originally contained negligible levels of α-galactosidase now showed a clear antigenicity to human serum. In this study, N-glycans were isolated from both APIs and human islets. Their structures were then analyzed by a mapping technique based on their high-performance liquid chromatography elution positions and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric data. Both preparations contained substantial amounts of high-mannose structures. The N-glycans from human islets were separated into 17 neutral, 8 mono-sialyl and 4 di-sialyl glycans, and the API glycans were comprised of 11 neutral, 8 mono-sialyl, 3 di-sialyl, 2 mono-sulfated, 3 mono-sialyl-mono-sulfated and 1 di-sulfated glycans. Among them, the API preparation contained one neutral, five mono-sialyl glycans and six sulfated glycans that were not detected in human islets. The structures of 9 of these 12 could be clearly determined. In addition, a study of the sulfate-depleted API suggests that sulfate residues could be antigenic to humans. The data herein will be helpful for future studies of the antigenicity associated with API. © The Author 2013.

Hiroshima University and Glyence Co. | Date: 2012-03-22

The object was to provide a method for distinguishing between species within the genus Staphylococcus; binding affinities between many types of lectins and bacteria belonging to the genus Staphylococcus were examined; and lectins of Tachylectin-2, LEL, KAA1, BCL11, CBA, HAA, HPA, STL, proBCA1, proBCA2, ULL, DSA, PWM, UDA, WFL, hypninA3, BCL11d, CFA1, CFA2, CLA, MPA1, MPA2, AC-avranin, algCSA, BML11b, BML11c, etc. were selected. Further, it was found that these lectins could be used to distinguish between species within the genus Staphylococcus.

Kamiya Y.,Japan Institute for Molecular Science | Kamiya Y.,Nagoya City University | Yamamoto S.,Japan Institute for Molecular Science | Yamamoto S.,Nagoya City University | And 7 more authors.
Journal of Biomolecular NMR | Year: 2011

This report describes a novel method for overexpression of 13C-labeled oligosaccharides using genetically engineered Saccharomyces cerevisiae cells, in which a homogeneous high-mannose-type oligosaccharide accumulates because of deletions of genes encoding three enzymes involved in the processing pathway of asparagine-linked oligosaccharides in the Golgi complex. Using uniformly 13C-labeled glucose as the sole carbon source in the culture medium of these engineered yeast cells, high yields of the isotopically labeled Man 8GlcNAc 2 oligosaccharide could be successfully harvested from glycoprotein extracts of the cells. Furthermore, 13C labeling at selected positions of the sugar residues in the oligosaccharide could be achieved using a site-specific 13C-enriched glucose as the metabolic precursor, facilitating NMR spectral assignments. The 13C-labeling method presented provides the technical basis for NMR analyses of structures, dynamics, and interactions of larger, branched oligosaccharides. © 2011 Springer Science+Business Media B.V.

Mizushima T.,Nagoya City University | Yagi H.,Nagoya City University | Takemoto E.,Nagoya City University | Shibata-Koyama M.,Kyowa Hakko Kirin Co. | And 8 more authors.
Genes to Cells | Year: 2011

Removal of the fucose residue from the N-glycans of the Fc portion of immunoglobulin G (IgG) results in a dramatic enhancement of antibody-dependent cellular cytotoxicity (ADCC) through improved affinity for Fcγ receptor IIIa (FcγRIIIa). Here, we present the 2.2-Å structure of the complex formed between nonfucosylated IgG1-Fc and a soluble form of FcγRIIIa (sFcγRIIIa) with two N-glycosylation sites. The crystal structure shows that one of the two N-glycans of sFcγRIIIa mediates the interaction with nonfucosylated Fc, thereby stabilizing the complex. However, fucosylation of the Fc N-glycans inhibits this interaction, because of steric hindrance, and furthermore, negatively affects the dynamics of the receptor binding site. Our results offer a structural basis for improvement in ADCC of therapeutic antibodies by defucosylation. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

Kamiya Y.,Japan Institute for Molecular Science | Kamiya Y.,Nagoya City University | Satoh T.,Nagoya City University | Kato K.,Japan Institute for Molecular Science | And 3 more authors.
Biochimica et Biophysica Acta - General Subjects | Year: 2012

Background: N-linked oligosaccharides operate as tags for protein quality control, consigning glycoproteins to different fates, i.e. folding in the endoplasmic reticulum (ER), vesicular transport between the ER and the Golgi complex, and ER-associated degradation of glycoproteins, by interacting with a panel of intracellular lectins in the early secretory pathway. Scope of review: This review summarizes the current state of knowledge regarding the molecular and structural basis for glycoprotein-fate determination in cells that is achieved through the actions of the intracellular lectins and its partner proteins. Major conclusions: Cumulative frontal affinity chromatography (FAC) data demonstrated that the intracellular lectins exhibit distinct sugar-binding specificity profiles. The glycotopes recognized by these lectins as fate determinants are embedded in the triantennary structures of the high-mannose-type oligosaccharides and are exposed upon trimming of the outer glucose and mannose residues during the N-glycan processing pathway. Furthermore, recently emerged 3D structural data offer mechanistic insights into functional interplay between an intracellular lectin and its binding partner in the early secretory pathway. General significance: Structural biology approaches in conjunction with FAC methods provide atomic pictures of the mechanisms behind the glycoprotein-fate determination in cells. This article is a part of a Special issue entitled: Glycoproteomics. © 2011 Elsevier B.V. All rights reserved.

Kamiya Y.,Japan Institute for Molecular Science | Kamiya Y.,Nagoya City University | Satoh T.,Nagoya City University | Satoh T.,Japan Science and Technology Agency | And 4 more authors.
Current Opinion in Structural Biology | Year: 2014

Because of the complexity, heterogeneity, and flexibility of the glycans, the structural analysis of glycoproteins has been eschewed until recently, with a few prominent exceptions. This aversion may have branded structural biologists as glycophobics. However, recent technological advancements in glycoprotein expression systems, employing genetically engineered production vehicles derived from mammalian, insect, yeast, and even bacterial cells, have yielded encouraging breakthroughs. The major advance is the active control of glycoform expression of target glycoproteins based on the genetic manipulation of glycan biogenetic pathways, which was previously overlooked, abolished, or considered unmanageable. Moreover, synthetic and/or chemoenzymatic approaches now enable the preparation of glycoproteins with uniform glycoforms designed in a tailored fashion. © 2014 Elsevier Ltd.

Imamura K.,Glyence Co. | Takeuchi H.,Glyence Co. | Yabe R.,Japan National Institute of Advanced Industrial Science and Technology | Tateno H.,Japan National Institute of Advanced Industrial Science and Technology | Hirabayashi J.,Japan National Institute of Advanced Industrial Science and Technology
Journal of Biochemistry | Year: 2011

Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3′-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3′-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3′-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology. © The Author 2011.

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