研究者詳細 - 佐藤　ちひろ

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

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

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

Involvement of the α2,8-Polysialyltransferases II/STX and IV/PST in the Biosynthesis of Polysialic Acid Chains on the O-Linked Glycoproteins in Rainbow Trout Ovary

Polysialic acid on flagellasialin is involved in sperm motility

Miyata Shinji, Sato Chihiro, Kumita Hironobu, Toriyama Masaru, Kitajima Ken

ZOOLOGICAL SCIENCE 23 巻 ( 12 ) 頁： 1171 - 1171 2006年12月

Asahina Shinji, Sato Chihiro, Matsuno Midori

The journal of biochemistry 140 巻 ( 5 ) 頁： 687 - 701 2006年11月

Involvement of the alpha2,8-polysialyltransferases II/STX and IV/PST in the biosynthesis of polysialic acid chains on the O-linked glycoproteins in rainbow trout ovary.

Asahina S, Sato C, Matsuno M, Matsuda T, Colley K, Kitajima K

Journal of biochemistry 140 巻 ( 5 ) 頁： 687 - 701 2006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：Journal of Biochemistry

Polysialoglycoprotein (PSGP) in salmonid fish egg is a unique glycoprotein bearing α2,8-linked polysialic acid (polySia) on its O-linked glycans. Biosynthesis of the polySia chains is developmentally regulated and only occurs at later stage of oogenesis. Two α2,8-polysialyltransferases (α2,8-polySTs), PST (ST8Sia IV) and STX (ST8Sia II), responsible for the biosynthesis of polySia on N-glycans of glycoproteins, are known in mammals. However, nothing has been known about which α2,8-polySTs are involved in the biosynthesis of polySia on O-linked glycans in any glycoproteins. We thus sought to identify cDNA encoding the α2,8-polyST involved in polysialylation of PSGP. A clone for PST orthologue, rtPST, and two clones for the STX orthologue, rtSTX-ov and rtSTX-em, were identified in rainbow trout. The deduced amino acid sequence of rtPST shows a high identity (72-77%) to other vertebrate PSTs, while that of rtSTX-ov shows 92% identity with rtSTX-em and a significant identity (63-76%) to other vertebrate STXs. The rtPST exhibited the in vivo α2,8-polyST activity, although its in vitro activity was low. However, the rtSTXs showed no in vivo and very low in vitro activities. Interestingly, co-existence of rtPST and rSTX-ov in the reaction mixture synergistically enhanced the α2,8-polyST activity. During oogenesis, rtPST was constantly expressed, while the expression of rtSTX-ov was not increased until polySia chain is abundantly biosynthesized in the later stage. rtSTX-em was not expressed in ovary. These results suggest that the enhanced expression of rtSTX-ov under the co-expression with rtPST may be important for the biosynthesis of polySia on O-linked glycans of PSGP. © 2006 The Japanese Biochemical Society.

DOI： 10.1093/jb/mvj200

Oral ingestion of mannose alters the expression level of deaminoneuraminic acid (KDN) in mouse organs.

Go S, Sato C, Furuhata K, Kitajima K

Glycoconjugate journal 23 巻 ( 5-6 ) 頁： 411 - 21 2006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：Glycoconjugate Journal

Deaminoneuraminic acid (KDN) is a unique member of the sialic acid family. We previously demonstrated that free KDN is synthesized de novo from mannose as its precursor sugar in trout testis, and that the amount of intracellular KDN increases in mouse B16 melanoma cells cultured in mannose-rich media [Angata et al. (1999) J. Biol. Chem. 274, 22949-56; Angata et al. (1999) Biochem. Biophys. Res. Commun. 261, 326-31]. In the present study, we first demonstrated a mannose-induced increase in intracellular KDN in various cultured mouse and human cell lines. These results led us to examine whether KDN expression in mouse organs is altered by exogenously administered mannose. Under normal feeding conditions, intracellular free KDN was present at very low levels (19-48 pmol/mg protein) in liver, spleen, and lung, and was not detected in kidney or brain. Oral ingestion of mannose, both short-term (90 min) and long-term (2 wk), resulted in an increase of intracellular KDN up to 60-81 pmol/mg protein in spleen and lung and 6.9-18 pmol/mg protein in kidney and brain; however, no change was observed in liver. The level of KDN in organs appears not to be determined only by the KDN 9-phosphate synthase activity, but might also be affected by other enzymes that utilize mannose 6-phosphate as a substrate as well as the enzymes that breakdown KDN, like KDN-pyruvate lyase. In blood, the detectable amount of free KDN did not change on oral ingestion of mannose. These findings indicate that mannose in the diet affects KDN metabolism in various organs, and provide clues to the mechanism of altered KDN expression in some tumor cells and aged organs. © Springer Science + Business Media, LLC 2006.

DOI： 10.1007/s10719-006-6734-z