研究者詳細 - 佐藤　ちひろ

Membrane microdomains from early gastrula embryos of medaka, Oryzias latipes, are a platform of E-cadherin- and carbohydrate-mediated cell-cell interactions during epiboly.

Adachi T, Sato C, Kishi Y, Totani K, Murata T, Usui T, Kitajima K

Glycoconjugate journal 26 巻 ( 3 ) 頁： 285 - 99 2009年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Glycoconjugate Journal

Formation of membrane microdomain is critical for cell migration (epiboly) during gastrulation of medaka fish [Adachi et al. (Biochem. Biophys. Res. Commun. 358:848-853, 2007)]. In this study, we characterized membrane microdomain from gastrula embryos to understand its roles in epiboly. A cell adhesion molecule (E-cadherin), its associated protein (β-catenin), transducer proteins (PLCγ, cSrc), and a cytoskeleton protein (β-actin) were enriched in the membrane microdomain. LeX- containing glycolipids and glycoproteins (LeX-gp) were exclusively enriched in the membrane microdomain. Interestingly, the isolated membrane microdomain had the ability to bind to each other in the presence of Ca 2+. This membrane microdomain binding was achieved through the E-cadherin homophilic and the LeX-glycan-mediated interactions. E-cadherin and LeX-gp were co-localized on the same membrane microdomain, suggesting that these two interactions are operative at the same time. Thus, the membrane microdomain functions as a platform of the E-cadherin- and LeX-glycan-mediated cell adhesion and signal transduction. © 2008 Springer Science+Business Media, LLC.

DOI： 10.1007/s10719-008-9184-y

3P-066 シアル酸結合免疫グロブリン様レクチン(シグレック)-7によるシアロ糖鎖認識の生化学的および分子ドッキング解析(蛋白質-機能(反応機構,生物活性など),第47回日本生物物理学会年会)

生物物理 49 巻 ( supplement ) 頁： S162 2009年

Direct binding of polysialic acid to a brain-derived neurotrophic factor depends on the degree of polymerization.

Kanato Y, Kitajima K, Sato C

Glycobiology 18 巻 ( 12 ) 頁： 1044 - 53 2008年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Glycobiology

Polysialic acid (polySia) is the homopolymer of sialic acid and negatively regulates neuronal cell-cell and cell-extracellular matrix interactions through steric and repulsive hindrance due to its bulky polyanionic structure. Whether polySia also functions as a positive regulator in the nervous system through binding to specific ligands is not known. In the present study, we demonstrated that a brain-derived neurotrophic factor (BDNF) dimer binds directly to polySia to form a large complex with an M<inf>r</inf> greater than 2000 kDa under physiologic conditions. Although somewhat affected by the linkage and type of sialic acid components in the polySia, the complex formation is highly dependent on the polySia chain length. The minimum degree of polymerization required for the complex formation is 12. This is the first study to demonstrate the biologic significance of the degree of polySia polymerization in eukaryotes. Similar large polySia complexes form with other neurotrophic factors such as nerve growth factor, neurotrophin-3, and neurotrophin-4. Furthermore, the BDNF, after making a complex with polySia, can bind to the BDNF receptors, TrkB and p75NTR. The complex formation of BDNF with polySia upregulates growth or/and survival of neuroblastoma cells. These findings suggest that polySia functions as a reservoir of BDNF and other neurotrophic factors and may serve to regulate their local concentrations on the cell surface. © The Author 2008. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/glycob/cwn084

Development of sensitive chemical and immunochemical methods for detecting sulfated sialic acids and their application to glycoconjugates from sea urchin sperm and eggs.

ポリシアル酸構造による細胞シグナルの調節機構

佐藤ちひろ

北島健蛋白質核酸酵素 53 頁： 1577 - 1583 2008年

ポドシアル酸構造による細胞シグナルの調節機構.

佐藤ちひろ

蛋白質核酸酵素 53 頁： 1577 - 1583 2008年

Yamakawa N, Sato C, Miyata S, Maehashi E, Toriyama M, Sato N, Furuhata K, Kitajima K

Biochimie 89 巻 ( 11 ) 頁： 1396 - 408 2007年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochimie

Sulfated sialic acid (SiaS) is a unique sialic acid (Sia) derivative in which an additional anionic group is attached to a carboxylated monosaccharide. Very little is known about the occurrence and biologic function of SiaS, due to the limitations of analytical methods to detect it in minute amounts. In this study, to develop methods and probes for detecting and pursuing the functions of SiaS, we developed sensitive chemical and immunochemical detection methods. First, we synthesized as model compounds 4-methylumbelliferyl glycosides of 8-O- and 9-O-sulfated Sia consisting of N-acetylneuraminic acid (Neu5Ac), N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (Kdn). Second, we applied fluorometric high performance liquid chromatography (HPLC) analysis to these synthetic glycosides. After acid hydrolysis of the samples, the liberated SiaS were labeled with a fluorescent reagent, 1,2-diamino-4,5-methylenedioxybenzene, and analyzed on fluorometric HPLC. We established an optimal elution condition for successful separation of 8-O- and 9-O-sulfated Neu5Ac, Neu5Gc, and Kdn on HPLC. Third, we generated a monoclonal antibody (mAb) 2C4 against SiaS using sea urchin egg components as the immunogen. mAb.2C4 recognizes both 8-O-sulfated Neu5Ac (Neu5Ac8S) and Neu5Gc8S, whereas the previously prepared mAb.3G9 only recognizes Neu5Ac8S. Finally, using the fluorometric HPLC and monoclonal antibodies, we demonstrated that glycoconjugates from sea urchin sperm exclusively contained Neu5Ac8S, whereas those from eggs contained Neu5Gc8S. Furthermore, we clarified the quantitative differences in the SiaS content in eggs and sperm from two different species of sea urchins. Immunostaining using mAb.2C4 showed that Neu5Gc8S is localized in the cortical granules in unfertilized eggs, whereas it is localized in the outer surface of the fertilization layer as well as in the inner surface of fertilized eggs. Thus, 8-O-sulfation is dependent on the species, gametic cell-type, site-localization of the eggs, and glycoconjugates. © 2007 Elsevier Masson SAS. All rights reserved.

DOI： 10.1016/j.biochi.2007.04.010

The rainbow trout CMP-sialic acid synthetase utilises a nuclear localization signal different from that identified in the mouse enzyme Open Access

Tiralongo, J; Fujita, A; Sato, C; Kitajima, K; Lehmann, F; Oschlies, M; Gerardy-Schahn, R; Münster-Kühnel, AK

GLYCOBIOLOGY 17 巻 ( 9 ) 頁： 945 - 954 2007年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Glycobiology

The terminal sugar sialic acid (Sia) plays a pivotal role in cell-cell interaction and recognition. A prerequisite for the biosynthesis of sialoglycoconjugates is the activation of Sia to cytidine monophosphate-Sia (CMP-Sia), by CMP-Sia synthetases (CMP-Sia-syn). CMP-Sia-syn are conserved from bacteria to man, and have been found to reside in the nucleus of all vertebrate species analysed to date. We previously cloned the CMP-Sia-syn from rainbow trout (rt) and identified three clusters of basic amino acids (BC) that might act as nuclear localization signals (NLS). Here, we utilised chimeric proteins and rt CMP-Sia-syn mutants in which putative NLS sequences were deleted, to identify the nuclear transport signal. Divergent from the mouse enzyme, where the crucial NLS is part of the enzyme's active site, in the rt CMP-Sia-syn the NLS and active site are disparate. The crucial NLS in the fish enzyme is bipartite and the functionality depends on a free N-terminus. Comparative analysis of all putative rt NLS in mouse and fish cells identified a second inferior motif (rtBC5-6), which was functional only in fish cells suggesting some differences in transport mechanism or folding variabilities in fish. Moreover, based on computational analyses of putative CMP-Sia-syn from distant deuterostomian organisms it was concluded that CMP-Sia-syn nuclear localization is a relatively recent invention, originating in echinoderms. In summary, our data describing structural differences in the NLS of vertebrate CMP-Sia-syn, and the independence of Sia activation from the subcellular localization of the enzyme, provide supporting evidence that nuclear localization is linked to a second yet unknown function. © The Author 2007. Published by Oxford University Press. All rights reserved.

DOI： 10.1093/glycob/cwm064

Membrane microdomain formation is crucial in epiboly during gastrulation of medaka.

Adachi T, Sato C, Kitajima K

Biochemical and biophysical research communications 358 巻 ( 3 ) 頁： 848 - 53 2007年7月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Membrane microdomain (microdomain) was isolated from early gastrula embryos. The isolated microdomain was characterized by enrichment of cholesterol and sphingomyelin, and by the presence of huge glycoproteins containing Lewis X structure. Importance of the microdomain in the progress of epiboly was assessed using methyl β-cyclodextrin (MBCD) and C2-ceramide that disrupt microdomains through different mechanisms. Both reagents efficiently disrupted the microdomain structure and concomitantly impaired epiboly. Interestingly, when embryos pretreated with MBCD, a cholesterol-binding molecule, were exogenously supplemented with cholesterol, the embryos underwent not only reconstitution of the microdomain, but also complete restoration to the normal epiboly. Thus, normal or impaired development is reversibly controlled by the cholesterol-dependent formation or disruption of microdomains. The most typical phenotype of the microdomain-disrupted embryos is detachment of cells from the blastoderm, suggesting that a major contribution of microdomains to epiboly is cell adhesion of blastodermal cells. © 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.04.197

Hypoxia-enhanced expression of free deaminoneuraminic acid in human cancer cells.

Go S, Sato C, Yin J, Kannagi R, Kitajima K

Biochemical and biophysical research communications 357 巻 ( 2 ) 頁： 537 - 42 2007年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

Hypoxia is a common characteristic of locally advanced tumors and cancer cells of high invasiveness and metastasis. We first demonstrate that hypoxic culture of human cancer HeLa, LS174T, and Caco-2 cells enhances the de novo synthesis of free sialic acids including deaminoneuraminic acid (Kdn), a unique sialic acid. Hypoxic cultures enhance expression of the N-acetylneuraminic acid (Neu5Ac) 9-phosphate synthetase (NPS) and phosphomannoisomerase (PMI) mRNAs and their enzymatic activities. In addition, incorporation of mannose (Man) into the cells is also enhanced. The elevated NPS activity facilitates the synthesis of Kdn as well as Neu5Ac, a typical sialic acid in human. The increased PMI activity, together with the enhanced Man-incorporation ability, leads to the increase of mannose 6-phosphate, which is a substrate of NPS, and subsequently results in the increased expression of Kdn, but not Neu5Ac. Kdn may thus be a target for diagnosis of cancers. © 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.03.181

Identification of the nuclear export signals that regulate the intracellular localization of the mouse CMP-sialic acid synthetase.

Fujita A, Sato C, Kitajima K

Biochemical and biophysical research communications 355 巻 ( 1 ) 頁： 174 - 80 2007年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

The CMP-sialic acid synthetase (CSS) catalyzes the activation of sialic acid (Sia) to CMP-Sia which is a donor substrate of sialyltransferases. The vertebrate CSSs are usually localized in nucleus due to the nuclear localization signal (NLS) on the molecule. In this study, we first point out that a small, but significant population of the mouse CMP-sialic acid synthetase (mCSS) is also present in cytoplasm, though mostly in nucleus. As a mechanism for the localization in cytoplasm, we first identified two nuclear export signals (NESs) in mCSS, based on the localization studies of the potential NES-deleted mCSS mutants as well as the potential NES-tagged eGFP proteins. These two NESs are conserved among mammalian and fish CSSs, but not present in the bacterial or insect CSS. These results suggest that the intracellular localization of vertebrate CSSs is regulated by not only the NLS, but also the NES sequences. © 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.01.139

Identification of an Inflammation-inducible Serum Protein Recognized by Anti-disialic Acid Antibodies as Carbonic Anhydrase II Open Access

YASUKAWA Zenta, SATO Chihiro, KITAJIMA Ken

Journal of Biochemistry 141 巻 ( 3 ) 頁： 429 - 441 2007年3月

Glycobiology of polysialic acids on sea urchin gametes Open Access

Miyata S., Sato C., Kitajima K.

Trends in Glycoscience and Glycotechnology 19 巻 ( 106 ) 頁： 85 - 98 2007年3月

記述言語：日本語掲載種別：研究論文（学術雑誌）出版者・発行元：Trends in Glycoscience and Glycotechnology

Naturally occurring polysialic acids (polySias), a unique homopolymer of sialic acid (Sia) residues, have a large structural diversity, primarily arising from the diversities in Sia components as well as in the intersialyl linkages. The α2,8-linked polySia on the neural cell adhesion molecules (NCAM) is the most extensively studied and recognized as a regulator of cell-cell interactions during development and differentiation in vertebrate brain. However, little is known about the occurrence and functions of polySia other than the α2,8-linked polySia on NCAM. Recently, it has been shown that polySia occurs in nature more frequently than previously recognized, as analytical methods to detect polySia structures have been greatly improved. Using these methods, two different types of polySia, α2,8- and α2,9-linked polySia, have been discovered on the sea urchin sperm, although the α2,5O glycolyl-linked polySia was already known in sea urchin egg. This is the first example of the co-localization of polySia with different intersialyl linkages in the same cell. The α2,9-linked polySia-containing glycoprotein of sea urchin sperm flagella was identified and named "flagellasialin". The α2,9-linked polySia on flagellasialin is suggested as a regulator of intracellular Ca 2+ and sperm motility. In addition, the α2,50 glycolyl-linked polySia has recently been shown to increase intracellular pH and potentiate acrosome reaction of sea urchin sperm. These results indicate that these polySia structures in sea urchin gametes have new functions other than the negative regulator for the NCAM-mediated cell adhesion. In this review, we summarize the recent advances in structural and functional studies of polySia in sea urchin gametes. © 2007 FCCA.

DOI： 10.4052/tigg.19.85

Complex formation of 70-kDa heat shock protein with acidic glycolipids and phospholipids.

CiNii Research

Harada Y, Sato C, Kitajima K

Biochemical and biophysical research communications 353 巻 ( 3 ) 頁： 655 - 60 2007年2月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：Biochemical and Biophysical Research Communications

A new property of a heat-inducible heat shock protein (Hsp) 70.1 that it forms a complex with acidic lipids was first demonstrated. Based on the behaviors of the complexes on the native PAGE, the acidic lipid/Hsp70.1 complexes are categorized into two groups. The first group is the sulfatide-induced large-sized complex, which stays on the gel top on the native PAGE. Only the N-terminal ATPase domain is responsible for the complex formation. The second group is the ganglioside-induced complex, which is diffused in the resolution gel on the native PAGE. Both the N-terminal ATPase and the C-terminal peptide-binding domains are involved in the complex formation. No complex is formed by neutral glyco- and phospholipids. The complex formation with the acidic glyco- and phospholipids implicates the various functions of Hsp70 on the membrane surfaces. © 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.12.068