2021/12/04 更新

写真a

ナツカ シュンジ
長束 俊治
NATSUKA Shunji
所属
教育研究院 自然科学系 地球・生物科学系列 教授
自然科学研究科 生命・食料科学専攻 教授
理学部 理学科 教授
職名
教授
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外部リンク

学位

  • 博士(医学) ( 1992年3月   大阪大学 )

  • 理学修士 ( 1988年3月   大阪大学 )

  • 理学士 ( 1986年3月   大阪大学 )

研究キーワード

  • データベース

  • 生化学

  • 糖鎖生物学

  • 糖質化学

  • 発生生物学

  • ゼブラフィッシュ

  • グライコミクス

  • 進化

  • 糖鎖

  • 糖転移酵素

  • 系統発生

  • 糖鎖シーケンサー

研究分野

  • ライフサイエンス / 機能生物化学

  • ライフサイエンス / 構造生物化学

経歴(researchmap)

  • 新潟大学   理学部生物学科   教授

    2009年9月 - 現在

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  • 大阪大学   大学院理学研究科化学専攻   准教授

    2004年3月 - 2009年8月

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  • 京都工芸繊維大学   応用生物学科   助教授

    2003年10月 - 2004年2月

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  • 京都工芸繊維大学   応用生物学科   講師

    1999年3月 - 2003年9月

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  • ミシガン大学   ハワードヒューズ医学研究所   研究員

    1992年10月 - 1994年9月

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  • 大阪大学   理学部化学科   助手

    1992年4月 - 1999年2月

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経歴

  • 新潟大学   理学部 理学科   教授

    2017年4月 - 現在

  • 新潟大学   自然科学研究科 生命・食料科学専攻   教授

    2009年9月 - 現在

  • 新潟大学   自然科学研究科 生命・食料科学専攻   教授

    2009年9月 - 現在

  • 新潟大学   分子細胞科学   教授

    2009年9月 - 2017年3月

学歴

  • 大阪大学   医学研究科   生理学専攻

    1988年4月 - 1992年3月

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    国名: 日本国

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  • 大阪大学   理学研究科   有機化学専攻

    1986年4月 - 1988年3月

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    国名: 日本国

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  • 大阪大学   理学部   化学科

    1982年4月 - 1986年3月

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    国名: 日本国

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所属学協会

 

論文

  • Toward robust N-glycomics of various tissue samples that may contain glycans with unknown or unexpected structures 査読

    Noriko Suzuki, Tatsuya Abe, Ken Hanzawa, Shunji Natsuka

    Scientific Reports   11   6334   2021年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Sequential modification of glycans by linkage-specific alkylamidation of sialic acids and permethylation 査読

    Tatsuya Abe, Akihiko Kameyama, Shunji Natsuka, Noriko Suzuki

    Analytical Biochemistry   606   113861   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Characterisation of N-glycans in the epithelial-like tissue of the rat cochlea. 査読 国際誌

    Yoriko Nonomura, Seishiro Sawamura, Ken Hanzawa, Takashi Nishikaze, Sadanori Sekiya, Taiga Higuchi, Fumiaki Nin, Satoru Uetsuka, Hidenori Inohara, Shujiro Okuda, Eiji Miyoshi, Arata Horii, Sugata Takahashi, Shunji Natsuka, Hiroshi Hibino

    Scientific reports   9 ( 1 )   1551 - 1551   2019年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Membrane proteins (such as ion channels, transporters, and receptors) and secreted proteins are essential for cellular activities. N-linked glycosylation is involved in stability and function of these proteins and occurs at Asn residues. In several organs, profiles of N-glycans have been determined by comprehensive analyses. Nevertheless, the cochlea of the mammalian inner ear, a tiny organ mediating hearing, has yet to be examined. Here, we focused on the stria vascularis, an epithelial-like tissue in the cochlea, and characterised N-glycans by liquid chromatography with mass spectrometry. This hypervascular tissue not only expresses several ion transporters and channels to control the electrochemical balance in the cochlea but also harbours different transporters and receptors that maintain structure and activity of the organ. Seventy-nine N-linked glycans were identified in the rat stria vascularis. Among these, in 55 glycans, the complete structures were determined; in the other 24 species, partial glycosidic linkage patterns and full profiles of the monosaccharide composition were identified. In the process of characterisation, several sialylated glycans were subjected sequentially to two different alkylamidation reactions; this derivatisation helped to distinguish α2,3-linkage and α2,6-linkage sialyl isomers with mass spectrometry. These data should accelerate elucidation of the molecular architecture of the cochlea.

    DOI: 10.1038/s41598-018-38079-0

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  • Quantitative LC-MS and MS/MS analysis of sialylated glycans modified by linkage-specific alkylamidation 査読

    Analytical Biochemistry   567   117 - 127   2019年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Preparation of a molecular library of branched β-glucan oligosaccharides derived from laminarin 査読

    Journal of Applied Glycoscience   65 ( 4 )   45 - 49   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Structures and developmental alterations of N-glycans of zebrafish embryos 査読

    Ken Hanzawa, Noriko Suzuki, Shunji Natsuka

    GLYCOBIOLOGY   27 ( 3 )   228 - 245   2017年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS INC  

    Zebrafish is a model organism suitable for studying vertebrate development. We analyzed the N-glycan structures of zebrafish embryos and their alterations during zebrafish embryogenesis to obtain basic data for studying the roles of N-glycosylation. Multiple modes of high-performance liquid chromatography and multistage mass spectrometry were used for structural analysis of N-glycans. The N-glycans from deyolked embryos at 36 hours postfertilization, a mid-pharyngula stage, contained relatively higher amounts of complex-and hybrid-type glycans with LacNAc (Gal beta 1-4GlcNAc) and/or sialyl LacNAc without additional beta 1,4-Gal, which are commonly found in mammalian tissues, as well as abundant oligomannose-type glycans. Some of the complex-and hybrid-type glycans possessed various extended LacNAc structures, such as Gal beta 1-4LacNAc, LacNAc-repeat or unique (+/- dHex)-GalNAc alpha 1-GlcNAc beta 1-LacNAc. In contrast, the yolk of the embryo contains predominant oligomannose-type glycans and complex-type glycans with Gal beta 1-4(Siaa2-3) Gal beta 1-4(Fuca1-3) GlcNAc antennae. N-Glycan profiles obtained from deyolked embryos at different stages showed stage-dependent variation of complex-and hybrid-type glycans. At gastrula and early segmentation stages, complex-and hybrid-type glycans were minor components, and their antenna structures were mainly sialyl LacdiNAc (Sia alpha 2-6GalNAc beta 1-4GlcNAc). From the mid-segmentation to pharyngula stages, those with LacNAc and/or alpha 2,6-sialyl LacNAc antenna structures increased remarkably, and those with alpha 2,3-sialyl LacNAc antenna, core alpha 1,6-Fuc and bisecting GlcNAc modifications increased gradually. These results suggest the presence of mechanisms for regulating the antenna structures of complex/ hybrid N-glycan biosynthesis in the phylotypic stage of vertebrate development.

    DOI: 10.1093/glycob/cww124

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  • Improved Method for Drawing of a Glycan Map, and the First Page of Glycan Atlas, Which Is a Compilation of Glycan Maps for a Whole Organism 査読

    Shunji Natsuka, Mayumi Masuda, Wataru Sumiyoshi, Shin-ichi Nakakita

    PLOS ONE   9 ( 7 )   e102219   2014年7月

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

    Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC) is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA-) glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.

    DOI: 10.1371/journal.pone.0102219

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  • Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography 査読

    Satsuki Itoh, Yoko Hiruta, Noritaka Hashii, Naho Fujita, Toru Natsuga, Toshiaki Hattori, Aya Bando, Yuko Sekimoto, Kazuyoshi Miyata, Hiroshi Namekawa, Kazunori Mabuchi, Toru Sakai, Hirotoshi Shimahashi, Kenzo Kawai, Hikaru Yoden, Sadatoshi Koyama, Susanne Odgaard Herr, Shunji Natsuka, Teruhide Yamaguchi, Nana Kawasaki

    BIOLOGICALS   41 ( 6 )   355 - 363   2013年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia. (C) 2013 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.biologicals.2013.06.002

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  • Hypothetical Roles of Glycan Diversity in Evolution 査読

    Shunji Natsuka

    TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY   25 ( 143 )   125 - 131   2013年5月

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    記述言語:英語   出版者・発行元:GAKUSHIN PUBL CO  

    DOI: 10.4052/tigg.25.125

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  • One-Step Purification Method for Pyridylamino Glycans 査読

    Shunji Natsuka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   76 ( 10 )   1982 - 1983   2012年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Pyridylamino derivatization is suitable for the micro-analysis of glycans but there is a problem in that by-products of the labeling reaction and fluorescent substances from samples occasionally interfere with the detection of pyridylamino glycans. We have reported a three-step purification method (S. Natsuka et FEBS J., 278, 452-460 (2011)). That method gives high purity and high yield for various glycans, but it is rather complicated. In this study I checked the efficiency of a one-step method with a spin column for the purification of pyridylamino glycans and found that it was excellent in respect of throughput. High-throughput analysis of N-glycans is desirable in glycomics.

    DOI: 10.1271/bbb.120388

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  • A Convenient Purification Method for Pyridylamino Monosaccharides 査読

    Shunji Natsuka

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   75 ( 7 )   1405 - 1407   2011年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Pyridylamino derivatization is suitable for the micro-analysis of sugars, but there is a problem in that by-products of the labeling reaction and fluorescent substances from samples occasionally interfere with the detection of pyridylamino sugars. Especially, interference by them is serious in monosaccharide analysis. I have developed a convenient purification method for pyridylamino monosaccharides by the use of a spin column with boronate-conjugated resin.

    DOI: 10.1271/bbb.110233

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  • A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals 査読

    Akira Harazono, Tetsu Kobayashi, Nana Kawasaki, Satsuki Itoh, Minoru Tada, Noritaka Hashii, Akiko Ishii, Teruyo Arato, Shigehiro Yanagihara, Yuki Yagi, Akiko Koga, Yuriko Tsuda, Mikiko Kimura, Masashi Sakita, Satoshi Kitamura, Hideto Yamaguchi, Hisashi Mimura, Yoshimi Murata, Yasuki Hamazume, Takayuki Sato, Shunji Natsuka, Kazuaki Kakehi, Mitsuhiro Kinoshita, Sakie Watanabe, Teruhide Yamaguchi

    BIOLOGICALS   39 ( 3 )   171 - 180   2011年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

    The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7 N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAECPAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix.
    Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study. (C) 2011 The International Alliance for Biologicals. Published by Elsevier Ltd. All rights reserved.

    DOI: 10.1016/j.biologicals.2011.04.002

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  • β-galactosidases from jack bean and Streptococcus have different cleaving abilities towards fucose-containing sugars. 査読

    Takeuchi T, Sugiura K, Nishiyama K, Takahashi H, Natsugari H, Arata Y, Natsuka S, Kasai K

    Biological & pharmaceutical bulletin   34 ( 4 )   567 - 569   2011年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1248/bpb.34.567

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  • Structural analysis of N-glycans of the planarian Dugesia japonica 査読

    Shunji Natsuka, Yukiko Hirohata, Shin-ichi Nakakita, Wataru Sumiyoshi, Sumihiro Hase

    FEBS JOURNAL   278 ( 3 )   452 - 460   2011年2月

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

    To investigate the relationship between phylogeny and glycan structures, we analyzed the structure of planarian N-glycans. The planarian Dugesia japonica, a member of the flatworm family, is a lower metazoan. N-glycans were prepared from whole worms by hydrazinolysis, followed by tagging with the fluorophore 2-aminopyridine at their reducing end. The labeled N-glycans were purified, and separated by three HPLC steps. By comparison with standard pyridylaminated N-glycans, it was shown that the N-glycans of planarian include high mannose-type and pauci-mannose-type glycans. However, many of the major N-glycans from planarians have novel structures, as their elution positions did not match those of the standard glycans. The results of mass spectrometry and sugar component analyses indicated that these glycans include methyl mannoses, and that the most probable linkage was 3-O-methylation. Furthermore, the methyl residues on the most abundant glycan may be attached to the non-reducing-end mannose, as the glycans were resistant to alpha-mannosidase digestion. These results indicate that methylated high-mannose-type glycans are the most abundant structure in planarians.

    DOI: 10.1111/j.1742-4658.2010.07966.x

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  • N-Glycosylation patterns of hemolymph glycoproteins from Biomphalaria glabrata strains expressing different susceptibility to Schistosoma mansoni infection 査読

    Tobias Lehr, Sandra Frank, Shunji Natsuka, Hildegard Geyer, Knut Beuerlein, Michael J. Doenhoff, Sumihiro Hase, Rudolf Geyer

    EXPERIMENTAL PARASITOLOGY   126 ( 4 )   592 - 602   2010年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Comparative analyses of the N-glycosylation pattern of hemolymph glycoproteins from Biomphalaria glabrata strains Puerto Rico (BgPR) and Salvador (BgBS-90), differing in their susceptibility towards Schistosoma mansoni infection, were performed by Western blotting, enzyme-linked immunosorbent assays, two-dimensional high-performance liquid chromatography and mass spectrometry. Obtained data demonstrated an enhanced expression of serologically cross-reacting, fucosylated carbohydrate epitopes by the highly susceptible BgPR-strain in comparison to the resistant BgBS-90-strain. In particular, glycoproteins of BgPR snails exhibited larger amounts of glycans with (beta 1-2)-linked xylose or terminal Fuc(alpha 1-3)GalNAc(beta 1-4)[+/- Fuc(alpha 1 -3)]GlcNAc(beta 1-)-units which are known to mediate cross-reactivity with schistosomal glycoconjugates. This finding could be corroborated by immunohistochemical studies showing again an enhanced expression of such carbohydrate epitopes in BgPR tissue. Hence, our results provide evidence for a correlation of B. glabrata susceptibility towards S. mansoni infection and the expression of carbohydrate determinants shared by the parasite and its intermediate host. (C) 2010 Elsevier Inc. All rights reserved.

    DOI: 10.1016/j.exppara.2010.06.031

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  • Involvement of murine β-1,4-galactosyltransferase V in lactosylceramide biosynthesis. 査読

    Kumagai T, Sato T, Natsuka S, Kobayashi Y, Zhou D, Shinkai T, Hayakawa S, Furukawa K

    Glycoconjugate journal   27 ( 7-9 )   685 - 695   2010年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1007/s10719-010-9313-2

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  • The och1 Mutant of Schizosaccharomyces pombe Produces Galactosylated Core Structures of N-Linked Oligosaccharides 査読

    Takao Ohashi, Yuka Ikeda, Naotaka Tanaka, Shin-ichi Nakakita, Shunji Natsuka, Yuko Giga-Hama, Kaoru Takegawa

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   73 ( 2 )   407 - 414   2009年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Unlike the budding yeast Saccharomyces cerevisiae, the fission yeast Schizosaccharomyces pombe synthesizes large outer chains on the N-linked oligosaccharides that consist mainly Of D-Gal and D-Man residues. The fission yeast och1(+) gene product has alpha 1,6-mannosyltransferase activity, and Och1p is the key enzyme in the initiation of outer chain elongation. Although the in vitro substrate specificity of S. pombe Och1p has been reported (Yoko-o et al., FEBS Lett., 489, 75-80 (2001)), the structure of the N-linked oligosaccharides of och1 Delta cells has not been investigated. In this study, we report a structural analysis of S. pombe N-linked oligosaccharides. Lectin blot analysis indicated that galactose residues were attached to the cell surface glycoproteins of the och1 Delta cells. We conducted a structural analysis of pyridylaminated N-linked oligosaccharides prepared from galactomannoproteins by HPLC and H-1 NMR. These analyses revealed that the N-linked oligosaccharides of the och1 Delta cells displayed heterogeneity in the glycan consisting of Hex(11-15)GlcNAc(2). The structural heterogeneity arose mainly from the addition of alpha 1,2- and alpha 1,3-Gal residues to the Man(9)GlcNAc(2) core structure.

    DOI: 10.1271/bbb.80712

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  • Biochemical characterization of plasma-derived tissue factor pathway inhibitor: post-translational modification of free, full-length form with particular reference to the sugar chain 査読

    Y. Mori, T. Hamuro, T. Nakashima, T. Hamamoto, S. Natsuka, S. Hase, S. Iwanaga

    JOURNAL OF THROMBOSIS AND HAEMOSTASIS   7 ( 1 )   111 - 120   2009年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    Background: Tissue factor pathway inhibitor (TFPI) is a physiological protease inhibitor that inhibits the initial reactions of the extrinsic blood coagulation pathway. Most TFPI in human plasma is associated with lipoproteins; however, the most functionally active form is thought to be the free, full-length form (f-pTFPI). Cell culture derived TFPI and recombinant TFPI (rTFPI) exhibit variations in their respective anticoagulant activity, which may be caused by post-translational modifications, such as the frequent differences in sugar chain structures among recombinant proteins. Sugar chain structures in rTFPI expressed in Chinese hamster ovary (CHO) cells have been reported previously, but those of plasma TFPI have not been. Objectives: To purify f-pTFPI and analyze the sugar chain structures. Results and conclusion: f-pTFPI was purified to homogeneity from blood plasma using a combination of anion-exchange, heparin affinity, immunoaffinity, and reversed-phase chromatographies, resulting in a yield of 76%. f-pTFPI showed a partially phosphorylated glycoprotein comprising a total of 276 amino acids by peptide mapping. The sugar chain structures were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion of the pyridylamino sugar chains and the following results were obtained. (Sialyl) Gal beta 1-3GalNAc was linked to Thr(175), partially to Thr(14) and Ser(174); sialyl complex-type sugar chains to Asn(117) and Asn(167), whereas Asn(228) was not glycosylated. Neuraminidase-resistant acidic sugar chains including sulfated sugar chains were not observed significantly. The protease inhibitory activities of f-pTFPI towards activated factor (F) X and tissue factor-activated FVII complex were identical to those of full-length rTFPI expressed in CHO cells.

    DOI: 10.1111/j.1538-7836.2008.03222.x

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  • Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo 査読

    Kazunobu Moriguchi, Tatsuya Takemoto, Takafumi Aoki, Shin-ichi Nakakita, Shunji Natsuka, Sumihiro Hase

    JOURNAL OF BIOCHEMISTRY   142 ( 2 )   213 - 227   2007年8月

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

    We previously reported that zebrafish alpha 1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta 1-4(Fuc alpha 1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha 1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta 1-4)(0,1,2)-{Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-2Man alpha 1-6[Gal beta 1-4(Fuc alpha 1-3)GlcNA beta 1-2Man alpha 1-3]}Man beta 1-4GlcNAc, and (Gal beta 1-4)(0,1)-{Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-2Man alpha 1-6[Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-2Man alpha 1-3]} Man beta 1-4GIcNAc beta 1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).

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  • Structure determination of a sulfated N-glycans, candidate for a precursor of the selectin ligand in bovine lung 査読

    Tomonori Murakami, Shunji Natsuka, Shin-ichi Nakakita, Sumihiro Hase

    GLYCOCONJUGATE JOURNAL   24 ( 4-5 )   195 - 206   2007年7月

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

    To clarify the structure of non-sialic acid anionic residue on N-glycans in the mammalian tissues, we have isolated sialidase-resistant anionic residue on N-glycans from bovine lung. Analyses by partial acid hydrolysis and glycosidase digestions combined with a two-dimensional HPLC mapping method revealed that the major sialidase-resistant anionic N-glycan had a fucosylbianntenary core structure. The anionic residue was identified as a sulfate ester by methanolysis, anion-exchange chromatography, and mass spectrometry. The linkage position of the sulfate ester was the 6-position of the GlcNAc residue on the Man alpha 1-6 branch. This conclusion was based on the results of glycosidase digestions followed by two-dimensional HPLC mapping. Furthermore, the disialylated form of this sulfated glycan was dominant, and no asialo form was detected. The structure of the major anionic N-glycan prepared from bovine lung and having a sulfate was proposed to be the pyridylamino derivative of Sia alpha 2-3G alpha l beta 1-4(HSO3-6)GlcNAc beta 1-2Man alpha 1-6(Sia alpha 2-3Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc.

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  • Gas-phase pyridylamination of saccharides: Development and applications 査読

    Shin-Ichi Nakakita, Wataru Sumiyoshi, Nobumitsu Miyanishi, Shunji Natsuka, Sumihiro Hase, Jun Hirabayashi

    ANALYTICAL CHEMISTRY   79 ( 7 )   2674 - 2679   2007年4月

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

    Pyridylamination is a versatile method for fluorescence labeling of oligosaccharides. The technique affords sensitive detection of saccharides with reducing termini and high-resolution separation by high-performance liquid chromatography. The conventional method, based on a liquid-phase reaction, has been extensively used in various aspects of glycobiology and glycotechnology. Unfortunately, the necessity for removing excess 2-aminopyridine makes the technique both laborious and time-consuming. Furthermore, removal of excess reagent can result in a significant loss of short saccharide components. In the present paper, we report an alternative methodology based on a "gas-phase" reaction, in which dried saccharides are reacted with vaporized 2-aminopyridine. The resultant Schiff base was also reduced in the gas phase within the same reaction microtube using a purpose-built device. The newly developed procedure was applied to both monosaccharide (GlcNAc) and oligosaccharides (isomalto-oligosaccharides) at quantitative yields with no requirement to remove excess reagent. The acid-labile sialyl linkages of alpha 2-6-disialobiantennary oligosaccharides proved to be fully stable during the procedure. The developed method was also successfully applied to profiling N-linked oligosaccharides liberated from glycoproteins by hydrazinolysis and, thus, should contribute to various fields of glycomics.

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  • Comparative biochemical study of N-linked glycans from skin of a squid, Todarodes pacificus 査読

    Shunji Natsuka, Miwa Ishida, Akira Ichikawa, Koji Ikura, Sumihiro Hase

    JOURNAL OF BIOCHEMISTRY   140 ( 1 )   87 - 93   2006年7月

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

    For comparative biochemical interest, we analyzed the structures of N-glycans in a squid belonging to the Lophotrochozoa, one of the protostome clades. N-Glycans were prepared from squid skin by hydrazinolysis and re-N-acetylation followed by fluorescent tagging with 2-aminopyridine. The labeled N-glycans were purified, and their structures were determined by the two-dimensional HPLC mapping method combined with glycosidase digestions and mass spectrometry. We found that high mannose-type glycans, paucimannose-type glycans and complex-type glycans with a type-1 structure (Ga1 beta 1-3GlcNAc) were dominant in squid skin. The complex-type glycans detected in the squid were similar to those in vertebrates, but have not yet been found in the Ecdysozoa, which is another protostome clade. However, paucimannose-type glycans are commonly found in the Ecdysozoa. Thus, the N-glycan structures of the squid belonging to the Lophotrochozoa have features common to those in vertebrates and the Ecdysozoa including insects and nematodes.

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  • EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming 査読

    K Hirao, Y Natsuka, T Tamura, Wada, I, D Morito, S Natsuka, P Romero, B Sleno, LO Tremblay, A Herscovics, K Nagata, N Hosokawa

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 14 )   9650 - 9658   2006年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Quality control in the endoplasmic reticulum ensures that only properly folded proteins are retained in the cell through mechanisms that recognize and discard misfolded or unassembled proteins in a process called endoplasmic reticulum-associated degradation (ERAD). We previously cloned EDEM ( ER degradation-enhancing alpha-mannosidase- like protein) and showed that it accelerates ERAD of misfolded glycoproteins. We now cloned mouse EDEM3, a soluble homolog of EDEM. EDEM3 consists of 931 amino acids and has all the signature motifs of Class I alpha-mannosidases ( glycosyl hydrolase family 47) in its N-terminal domain and a protease-associated motif in its C-terminal region. EDEM3 accelerates glycoprotein ERAD in transfected HEK293 cells, as shown by increased degradation of misfolded alpha 1-antitrypsin variant ( null ( Hong Kong)) and of TCR alpha. Overexpression of EDEM3 also greatly stimulates mannose trimming not only from misfolded alpha 1-AT null ( Hong Kong) but also from total glycoproteins, in contrast to EDEM, which has no apparent alpha 1,2-mannosidase activity. Furthermore, overexpression of the E147Q EDEM3 mutant, which has the mutation in one of the conserved acidic residues essential for enzyme activity of alpha 1,2-mannosidases, abolishes the stimulation of mannose trimming and greatly decreases the stimulation of ERAD by EDEM3. These results show that EDEM3 has alpha 1,2-mannosidase activity in vivo, suggesting that the mechanism whereby EDEM3 accelerates glycoprotein ERAD is different from that of EDEM.

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  • Structural diversity of cytosolic free oligosaccharides in the human hepatoma cell line, HepG2 査読

    K Yanagida, S Natsuka, S Hase

    GLYCOBIOLOGY   16 ( 4 )   294 - 304   2006年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS INC  

    It is thought that free oligosaccharides in the cytosol are an outcome of quality control of glycoproteins by endoplasmic reticulum-associated degradation (ERAD). Although considerable amounts of free oligosaccharides accumulate in the cytosol, where they presumably have some function, detailed analyses of their structures have not yet been carried out. We isolated 21 oligosaccharides from the cytosolic fraction of HepG2 cells and analyzed their structures by the two-dimensional high-performance liquid chromatography (HPLC) sugar-mapping method. Sixteen novel oligosaccharides were identified in the cytosol in this study. All had a single N-acetylglucosamine at their reducing-end cores and could be expressed as (Man)(n) (GlcNAc)(1). No free oligosaccharide with N,N'-diacetylchitobiose was detected in the cytosolic fraction of HepG2 cells. This suggested that endo-beta-N-acetylglucosaminidase was a key enzyme in the production of cytosolic free oligosaccharides. The 21 oligosaccharides were classified into three series-series 1: oligosaccharides processed from Man alpha 1-2Man alpha 1-6 (Man alpha 1-2Man alpha 1-3)Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3) Man beta 1-4GlcNAc (M9A') and Man alpha 1-2Man alpha 1-6(Man alpha 1-3) Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc (M8A') by digestion with cytosolic alpha-mannosidase; series 2: oligosaccharides processed with Golgi alpha-mannosidases in addition to endoplasmic reticulum (ER) and cytosolic alpha-mannosidases; and series 3: glucosylated oligosaccharides produced from Glc(1)Man(9)GlcNAc(1) by hydrolysis with cytosolic alpha-mannosidase. The presence of the series "2" oligosaccharides suggests that some of the misfolded glycoproteins had been processed in pre-cis-Golgi vesicles and/or the Golgi apparatus. When the cells were treated with swainsonine to inhibit cytosolic alpha-mannosidase, the amounts of M9A' and M8A' increased remarkably, suggesting that these oligosaccharides were translocated into the cytosol. Four oligosaccharides of series "2" also increased. In contrast, there were obvious reductions in Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNAc (M5B'), the end product from M9A' by digestion with cytosolic alpha-mannosidase, and Man alpha 1-6(Man alpha 1- 2Man alpha 1-3)Man beta 1-4GlcNAc, derived from series "2" oligosaccharides by digestion with cytosolic alpha-mannosidase. Our data suggest that (1) some of the cytosolic oligosaccharides had been processed with Golgi alpha-mannosidases, (2) the major oligosaccharides translocated from the ER were M9A' and M8A', and (3) M5B' and Glc(1)M5B' were maintained at relatively high concentrations in the cytosol.

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  • Alteration of brain type N-glycans in neurological mutant mouse brain 査読

    S Nakakita, S Natsuka, J Okamoto, K Ikenaka, S Hase

    JOURNAL OF BIOCHEMISTRY   138 ( 3 )   277 - 283   2005年9月

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

    We have previously detected two brain-specific and development-dependent N-glycans [H. Shimizu, K. Ochiai, K. Ikenaka, K. Mikoshiba, and S. Hase (1993) J. Biochem. 114, 334-338]. In the present study we attempted to analyze specific N-glycans detected in neurological mutant mice. N-glycans in cerebrum and cerebellum obtained from 3-week-old neurological mutant mice (jimpy, staggerer, and shiverer) were compared with those obtained from normal mice. N-glycans liberated from the cerebrum and cerebellum by hydrazinolysis-N-acetylation were pyridylamino, and pyridylamino derivatives of N-glycans thus obtained were separated into neutral and five acidic fractions by anion exchange chromatography. PA-N-glycans in each fraction were compared with those obtained from normal mice by reversed-phase HPLC, and the following results were obtained. The ratio of the two brain-type N-glycans, Man alpha 1-3(wGlcNAc beta 1-2Man alpha 1-6)(GlcNAc beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNac)(BA-1) to GlcNAc beta Man alpha 1-3(GlcNAc beta 1-2Man alpha 1-6)(GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fucal-6)GlcNAc (BA-2), was higher in staggerer mice than other mutant mice and normal mice. Sia-Gal-BA-2, triantennary N-glycans, and bisected biantennary N-glycans were found in the cerebellum of shiverer and staggerer mice but not in normal or jimpy mice. High-mannose type N-glycans were not altered in mutant mice brains. The amounts of disialylbiantennary N-glycans and disialylfucosylbiantennary N-glycans were lower in jimpy mouse cerebellum than in normal mouse cerebellum, but were higher in shiverer mouse. Some alterations of N-glycans specific to mutations were successfully identified, suggesting that expression of component(s) of the N-glycan biosynthetic pathway was specifically affected in neurological mutations.

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  • Comparative biochemical view of N-glycans 査読

    S Natsuka

    TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY   17 ( 97 )   229 - 236   2005年9月

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    記述言語:英語   出版者・発行元:GAKUSHIN PUBL CO  

    N-Linked glycans are common in eukaryotes, as they are attached to many different proteins. They are classified into three categories termed high mannose, paucimannose, and complex (include hybrid) types. Each phylogenetic group of living organisms has characteristic glycans. In most cases their full significance is unknown, although several functions may be known. For example high mannose-type glycans take part in quality control during the synthesis of proteins. In this review, N-linked glycans are looked at from the viewpoint of their phylogeny and ontogeny.

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  • Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans 査読

    S Natsuka, M Kawaguchi, Y Wada, A Ichikawa, K Ikura, S Hase

    JOURNAL OF BIOCHEMISTRY   138 ( 2 )   209 - 213   2005年8月

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

    Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agaroseresin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (Ga1NAc alpha 1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.

    DOI: 10.1093/jb/mvi117

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  • Expression of complex-type N-glycans in developmental periods of zebrafish embryo 査読

    T Takemoto, S Natsuka, S Nakakita, S Hase

    GLYCOCONJUGATE JOURNAL   22 ( 1-2 )   21 - 26   2005年2月

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

    As a first step to elucidate a role of N-glycans in development of vertebrates, we analyzed structures of the glycans expressed in early stages of zebrafish embryo. N-glycans were prepared from zebrafish embryos at several developmental stages followed by tagging with a fluorophore, 2-aminopyridine. The labeled glycans were analyzed by two modes of HPLC's. The comparison of the elution profiles of HPLC's unveil the change of the oligosaccharide structure during the development. These peaks were merely detected during 4 - 7 h after fertilization, however, increased from 12 h, and at 15 h a fairly amount of them was appeared. Structure analysis revealed that they were bianntenary complex-type N-glycans with or without fucose and/or bisecting N-acetylglucosamine residues. These results suggest that the complex-type N-glycans are concerned in some developmental event from segmentation period downward in zebrafish.

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  • In vitro modification of betaine-homocysteine S-methyltransferase by tissue-type transglutaminase 査読

    A Ichikawa, Y Ohashi, S Terada, S Natsuka, K Ikura

    INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY   36 ( 10 )   1981 - 1992   2004年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PERGAMON-ELSEVIER SCIENCE LTD  

    Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. To elucidate the physiological roles of transglutaminase at the molecular level, we need to identify its physiological protein substrates and clarify the relationship between transglutaminase modification of protein substrates and biological responses. Here we examined whether betaine-homocysteine S-methyltransferase (BHMT: EC 2.1.1.5) can be a substrate of tissue-type transglutaminase by in vitro experiments using porcine liver BHMT and guinea pig liver transglutaminase. Guinea pig liver transglutaminase incorporated 5-(biotinamido) pentylamine and [3 H] histamine into BHMT in a time-dependent manner. Putrescine and spermidine also seemed to be incorporated into BHMT by transglutaminase. In the absence of the primary amines, BHMT subunits were cross-linked intra- and intermolecularly. BHMT activity was decreased significantly through the cross-linking by transglutaminase. Histamine incorporation slightly reduced the BHMT activity. Peptide fragments of BHMT containing the glutamine residues reactive for transglutaminase reaction were isolated through biotin labelling, proteinase digestion, biotin-avidin a affinity separation, and reverse phase HPLC. The results of amino acid sequence analyses of these peptides and sequence homology alignment with other mammalian liver BHMT subunits showed that these reactive glutamine residues were located in the region near the carboxyl terminal of porcine BHMT subunit. These results suggested that the liver BHMT can be modified by tissue-type transglutaminase and its activity is regulated repressively by the modification, especially by the cross-linking. This regulatory reaction might be involved in the regulation of homocysteine metabolism in the liver. (C) 2004 Elsevier Ltd. All rights reserved.

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  • 脳特異的糖鎖とその生合成に関わる“脳型”ガラクトース転移酵素 査読

    中北 愼一, 長束 俊治, 池中 一裕, 長谷 純宏

    生物物理化学   48 ( 1 )   1 - 4   2004年

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  • 糖鎖生物学のモデル動物としての線虫 査読

    長束俊治

    生化学   75 ( 2 )   131 - 133   2003年2月

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    記述言語:中国語   掲載種別:研究論文(学術雑誌)  

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  • Identification of an endo-beta-N-acetylglucosaminidase gene in Caenorhabditis elegans and its expression in Escherichia coli 査読

    T Kato, K Fujita, M Takeuchi, K Kobayashi, S Natsuka, K Ikura, H Kumagai, Y Yamamoto

    GLYCOBIOLOGY   12 ( 10 )   581 - 587   2002年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS INC  

    We report the identification, molecular cloning, and characterization of an endo-beta-N-acetylglucosaminidase from the nematode Caenorhabditis elegans. A search of the C. elegans genome database revealed the existence of a gene exhibiting 34% identity to Mucor hiemalis (a fungus) endo-beta-N-acetylglucosaminidase (Endo-M). Actually, the C. elegans extract contained endo-beta-N-acetylglucosaminidase activity. The putative cDNA for the C. elegans endo-beta-N-acetylglucosaminidase (Endo-CE) was amplified by polymerase chain reaction from the Uni-ZAP XR library, cloned, and sequenced. The recombinant Endo-CE expressed in Escherichia coli exhibited substrate specificity mainly for high-mannose type oligosaccharides. Man(8)GlcNAc(2) was the best substrate for Endo-CE, and Man(3)GlcNAc(2) was also hydrolyzed. Biantennary complex type oligosaccharides were poor substrates, and triantennary complex substrates were not hydrolyzed: Its substrate specificity was similar to those of Endo-M and endo-beta-N-acetylglucosaminidase from hen oviduct. Endo-CE was confirmed to exhibit transglycosylation activity, as seen for some microbial endo-beta-N-acetylglucosaminidases. This is the first report of the molecular cloning of an endo-beta-N-acetylglucosaminidase gene from a multicellular organism, which shows the possibility of using this well-characterized nematode as a model system for elucidating the role of this enzyme.

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  • Co(II)-regulated substrate specificity of cytosolic alpha-mannosidase 査読

    M Yamagishi, T Ishimizu, S Natsuka, S Hase

    JOURNAL OF BIOCHEMISTRY   132 ( 2 )   253 - 256   2002年8月

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

    Cytosolic neutral alpha-mannosidase is a putative catabolic enzyme that produces cytosolic free oligomannosides. Activation of the enzyme by Co(II) treatment has been reported using pyridylamino, derivatives of Man(5)GlcNAc and Man(5)GleNAc(2), and p-nitrophenyl alpha-mannoside as substrates, with the Co(II)-treated enzyme releasing four alpha-mannose residues from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as an end product. When Man9GlcNAc, which is considered to be the actual substrate in the cytosol, was used as a substrate, we found that even before treatment with Co(II) the enzyme was able to cleave a single Manalpha1-2 residue from Man(9)GlcNAc to give Manalpha1-6(Manalpha1-2Manalpha1-3)Manalpha1-6(Manalpha1-2Manalpha1-2Manalpha1-3)Manbeta1-4GlcNAc as the end product. The K-m value of the Co(II)-treated enzyme for Man(9)GlcNAc was found to be 37 muM, which is one-twelfth that of the non-treated enzyme, while the values were V-max values were almost the same, indicating that the affinity of the substrate is higher with Co(II). These results indicate that Co(II) regulates the substrate specificity of the enzyme.

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  • Inhibition of transglutaminase by synthetic tyrosine melanin 査読

    K Ikura, C Otomo, S Natsuka, A Ichikawa, K Wakamatsu, S Ito, S Taoguchi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   66 ( 6 )   1412 - 1414   2002年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena. Previously, we found a high molecular mass transglutaminase-inhibitory substance produced by Streptomyces lavendulde Y-200 that appeared to be a melanin substance. Here, we report that synthetic tyrosine melanin inhibited various types of transglutaminases. Tyrosine melanin inhibited tissue-type transglutaminase in a competitive manner with a glutamine substrate, and also inhibited the cross-linking of casein catalyzed by a tissue-type transglutaminase. The melanized hemolymph of the silkworm and melanin solutions prepared from melanin precursors inhibited tissue-type transglutaminase. These results suggested that the melanin substances generally inhibit transglutaminases.

    DOI: 10.1271/bbb.66.1412

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  • Structural analysis of N-linked Glycans in Caenorhabditis elegans 査読

    S Natsuka, J Adachi, M Kawaguchi, S Nakakita, S Hase, A Ichikawa, K Ikura

    JOURNAL OF BIOCHEMISTRY   131 ( 6 )   807 - 813   2002年6月

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

    Caenorhabditis elegans is an excellent model for morphogenetic research. However, little information is available on the structure of cell-surface glycans in C. elegans, although several lines of evidence have suggested a role for these glycans in cell-cell interactions during development. In this study, we analyzed N-glycan structures. Oligosaccharides liberated by hydrazinolysis from a total membrane fraction were labeled by pyridylamination, and around 90% of the N-glycans were detected as neutral oligosaccharides. The most dominant structure was Manalpha1-6(Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc, which is commonly found in insects. Branching structures of major oligomannose-type glycans were the same as those found in mammals. Structures that had a core fucose or non-reducing end N-acetylglucosamine were also identified, but ordinary complex-type glycans with N-acetyllactosamine were not detected as major components.

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  • Method for purification of fluorescence-labeled oligosaccharides by pyridylamination 査読

    S Natsuka, J Adachi, M Kawaguchi, A Ichikawa, K Ikura

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   66 ( 5 )   1174 - 1175   2002年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    We developed a convenient method for purification of PA-oligosaccharides to remove contaminants originating from natural fluorescent materials, and excess reagents as well as by-products of tagging reactions in glycan analysis. The method, using a C18-cartridge, is simple and powerful to remove them. Several examples of experiments that showed the usefulness of this purification method are described in this report.

    DOI: 10.1271/bbb.66.1174

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  • Analysis of oligosaccharide structures of glycoproteins in polyacrylamide gel 査読

    S Nakakita, D Ama, S Natsuka, S Hase

    ANALYTICAL BIOCHEMISTRY   303 ( 2 )   206 - 209   2002年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    DOI: 10.1006/abio.2002.5583

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  • A monoclonal antibody to cyclomaltoheptaose (beta-cyclodextrin): Characterization and use for immunoassay of beta-cyclodextrin and its derivatives 査読

    K Ikura, J Fujimoto, K Kubonishi, S Natsuka, H Hashimoto, T Ito, K Fujita

    CYTOTECHNOLOGY   40 ( 1-3 )   23 - 29   2002年

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

    Cyclomaltoheptaose (beta-cyclodextrin, beta-CD) is a promising compound for application in various industrial fields because of its ability to entrap various compounds into its hydrophobic cavity. A monoclonal antibody (A7) to beta-CD was generated by using a conjugate of glucosaminylmaltosyl-beta-CD and bovine serum albumin as an antigen. The A7 monoclonal antibody was IgM/kappa and reacted with beta-CD with high specificity. The epitope recognized by the A7 monoclonal antibody seemed to be located on the secondary hydroxyl groups of the rim side of the beta-CD molecule. The dissociation constant of the complex of beta-CD and the immobilized A7 monoclonal antibody was determined to be 1.2 x 10(-4) M. A competitive ELISA using the A7 monoclonal antibody enabled determination of beta-CD and its derivatives with a detection limit of 0.05 muM. This immunoassay was useful to determine beta-CD in biological fluids such as human plasma and urine after appropriate pretreatment of the samples.

    DOI: 10.1023/A:1023997501517

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  • Co-overexpression of folding modulators improves the solubility of the recombinant guinea pig liver transglutaminase expressed in Escherichia coli 査読

    K Ikura, T Kokubu, S Natsuka, A Ichikawa, M Adachi, K Nishihara, H Yanagi, S Utsumi

    PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY   32 ( 2 )   189 - 205   2002年

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

    Transglutaminases (EC 2.3.2.13) catalyze the formation of epsilon-(gamma-glutamyl)lysine cross-links and the substitution of primary amines for the gamma-carboxamide groups of protein bound glutamine residues, and are involved in many biological phenomena. Transglutaminase reactions are also applicable in applied enzymology. Here, we established an expression system of recombinant mammalian tissue-type transglutaminase with high productivity. Overexpression of guinea pig liver transglutaminase in Escherichia coli, using a plasmid pET21-d, mostly resulted in the accumulation of insoluble and inactive enzyme protein. By the expression culture at lower temperatures (25 and 18degreesC), however, a fraction of the soluble and active enzyme protein slightly increased. Co-overexpression of a molecular chaperone system (DnaK-DnaJ-GrpE) and/or a folding catalyst (trigger factor) improved the solubility of the recombinant enzyme produced in E. coli cells. The specific activity, the affinity to the amine substrate. and the sensitivity to the calcium activation and GTP inhibition of the purified soluble recombinant enzyme were lower than those of the natural liver enzyme. These results indicated that co-overexpression of folding modulators tested improved the solubility of the overproduced recombinant mammalian tissue-type transglutaminase, but the catalytic properties of the soluble recombinant enzyme were not exactly the same as those of the natural enzyme.

    DOI: 10.1081/PB-120004130

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  • 蛍光標識による糖鎖の発現パターン解析法 査読

    長束俊治

    生物物理化学   46 ( 2 )   35 - 38   2002年

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  • Molecular cloning and expression of Caenorhabditis elegans ERp57-homologue with transglutaminase activity 査読

    S Natsuka, R Takubo, R Seki, K Ikura

    JOURNAL OF BIOCHEMISTRY   130 ( 6 )   731 - 735   2001年12月

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

    Formation of cross-linking between proteins via a gamma -glutamyl-epsilon -lysine residue is an important process in many biological phenomena including apoptosis. Formation of this linkage is catalyzed by the enzyme transglutaminase, which is widely distributed from bacteria to the animal kingdom. The simple multi-cellular organism Caenorhabditis elegans also possesses transglutaminase activity associated with apoptosis [Madi, A. et al. (1998) Eur. J. Biochem. 253, 583-590], but no gene with significant homology to vertebrate or bacterial transglutaminases has been found in the C. elegans genome sequence database. On the other hand, protein disulfide isomerases were recently recognized as a new family of transglutaminases [Chandrashekar, R. et aL (1998) Proc. Natl. Acad Sci. USA 95, 531-536]. To identify the molecule with transglutaminase activity in C. elegans, we isolated from C. elegans a gene homologous to ERp57, which encodes a protein disulfide isomerase, expressed it in recombinant form, and characterized the transglutaminase and protein disulfide isomerase activities of the resultant protein. The C. elegans ERp57 protein had both enzyme activities, and the transglutaminase activity had similar characteristics to the activity in lysate of the whole worm. These results suggested that the ERp57 homologue was one of the substances with transglutaminase activity in C. elegans.

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  • Structural analysis of the sugar chains of human urinary thrombomodulin 査読

    H Wakabayashi, S Natsuka, M Honda, M Naotsuka, Y Ito, J Kajihara, S Hase

    JOURNAL OF BIOCHEMISTRY   130 ( 4 )   543 - 552   2001年10月

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

    The sugar chains of human urinary thrombomodulin were studied. N- and O-linked sugar chains were simultaneously liberated by hydrazinolysis followed by N-acetylation and were tagged with 2-aminopyridine. Then the structures of the N- and O-linked pyridylamino (PA-) sugar chains were analyzed by two-dimensional sugar mapping combined with exoglycosidase digestion. The major N-linked sugar chains of human urinary thrombomodulin were found to be monosialo- and disialofucosylbiantennary chains, while the major O-linked sugar chain was +/- Sia alpha2-3Gal beta1-3(+/- Sia alpha2-6)GalNAc. Thrombomodulin also contained the reported structure SO4-3GlcA beta1-3Gal beta1-3(+/- Sia alpha2-6)Gal beta1-4Xyl [H. Wakabayashi, S. Natsuka, T. Mega, N. Otsuki, AL Isaji, M. Naotsuka, S. Koyama, T. Kanamori, M Sakai, and S. Hase (1999) J. Biol. Chem. 274, 5436-5442]. In addition to these sugar chains, a single Glc was linked to Ser 287.

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  • Chemical structures of C. elegans O-glycans 査読

    S Natsuka

    TRENDS IN GLYCOSCIENCE AND GLYCOTECHNOLOGY   13 ( 73 )   551 - 553   2001年9月

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

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  • High molecular weight transglutaminase inhibitor produced by a microorganism (Streptomyces lavendulae Y-200) 査読

    K Ikura, K Minami, C Otomo, H Hashimoto, S Natsuka, K Oda, K Nakanishi

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   64 ( 1 )   116 - 124   2000年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:TAYLOR & FRANCIS LTD  

    Transglutaminases catalyze the cross-linking and amine incorporation of proteins, and are implicated in various biological phenomena such as blood clotting, wound healing, apoptosis, and cell differentiation. Streptomyces lavendulae Y-200, isolated from sea, produced a substance that inhibited transglutaminases. The inhibitory substance was purified from the cultured medium by procedures of acid precipitation, deoxyribonuclease treatment, and gel filtration chromatography. The partially purified sample was dark brown. The inhibitory activity was stable under acidic, alkaline, and high temperature conditions, and resistant to the treatment with proteinases such as trypsin and Pronase. The molecular weight of the inhibitory substance was estimated to be between 10(4) and 10(5) from its permeability through ultrafilter membranes. The acid hydrolysate of the inhibitory substance contained amino acids and sugars. The inhibitory substance inhibited both calcium-dependent and calcium-independent transglutaminases in a competitive manner with a glutamine substrate. The extent of inhibition caused by the calcium-dependent transglutaminase increased with increasing calcium concentration. The results obtained here may help identify a novel regulatory substance of transglutaminase in biological systems.

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  • beta 1-4Galactosyltransferase activity of mouse brain as revealed by analysis of brain-specific complex-type N-linked sugar chains 査読

    S Nakakita, KK Menon, S Natsuka, K Ikenaka, S Hase

    JOURNAL OF BIOCHEMISTRY   126 ( 6 )   1161 - 1169   1999年12月

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

    We previously reported two brain-specific agalactobiantennary N-linked sugar chains with bisecting GlcNAc and alpha 1-6Fuc residues, (GlcNAc beta 1-2)(0 or 1)Man alpha 1-3(GlcNAc beta 1-2Man alpha 1-6)(GlcNAc beta 1-4)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-6)GlcNAc [Shimizu, H., Ochiai, K., Ikenaka, K,, Mikoshiba, K,, and Hase, S. (1993) J. Biochem, 114, 334-338], Here, the reason for the absence of Gal on the sugar chains was analyzed through the detection of other complex type sugar chains. Analysis of N-linked sugar chains revealed the absence of Sia-Gal and Gal on the GlcNAc residues of brain-specific agalactobiantennary N-linked sugar chains. We therefore investigated the substrate specificity of galactosyltransferase activities in brain using pyridylamino derivatives of agalactobiantennary sugar chains with structural variations in the bisecting GlcNAc and alpha 1-6Fuc residues as acceptor substrates, While the beta 1-4galactosyltransferases in liver and kidney could utilize all four oligosaccharides as substrates, the beta 1-4galactosyltransferase(s) in brain could not utilize the agalactobiantennary sugar chain with both bisecting GlcNAc and Fuc residues, but could utilize the other three accepters. Similar results were obtained using glycopeptides with agalactobiantennary sugar chains and bisecting GlcNAc and alpha 1-6Fuc residues as substrates. The beta 1-4galactosyltransferase activity of adult mouse brain thus appears to be responsible for producing the brain-specific sugar chains and to be different from beta 1-4galactosyltransferase-I. The agalactebiantennary sugar chain with bisecting GlcNAc and alpha 1-6Fuc residues acts as an inhibitor against "brain type" beta 1-4galactosyltransferase with a K-I value of 0.29 mM.

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  • A pyridylamination method aimed at automatic oligosaccharide analysis of N-linked sugar chains 査読

    K Yanagida, S Natsuka, S Hase

    ANALYTICAL BIOCHEMISTRY   274 ( 2 )   229 - 234   1999年10月

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

    The procedure for preparation of pyridylaminated sugar chains from glycoproteins was improved with a view to its eventual automation. Following on the coupling reaction improvement already reported [N. Kuraya and S. Hase (1992) J. Biochem. 112, 122-126], two further aspects were improved in this study. Instead of sodium bicarbonate-acetic anhydride, volatile reagents were adopted for the re-N-acetylation of hexosamine residues after hydrazinolysis to give rapid removal of excess reagents. Subsequent to the pyridylamination reaction, excess reagents were removed by cation-exchange to isolate the pyridylaminated oligosaccharides in place of gel filtration. These alterations rendered a one-pot reaction possible and resulted in a large reduction in the amount of time needed compared with other methods so far reported. The procedure was successfully applied to the detection of sugar chains from Taka-amylase A and human erythrocyte membranes. (C) 1999 Academic Press.

    DOI: 10.1006/abio.1999.4263

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  • Presence of asparagine-linked N-acetylglucosamine and chitobiose in Pyrus pyrifolia S-RNases associated with gametophytic self-incompatibility 査読

    T Ishimizu, Y Mitsukami, T Shinkawa, S Natsuka, S Hase, M Miyagi, F Sakiyama, S Norioka

    EUROPEAN JOURNAL OF BIOCHEMISTRY   263 ( 3 )   624 - 634   1999年8月

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

    S-RNases encoded by the S-locus of rosaceous and solanaceous plants discriminate between the S-alleles of pollen in gametophytic self-incompatibility reactions, but it is not clear how. We report the structures of N-glycans attached to each of the N-glycosylation sites of seven S-RNases in Pyr us pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridyiaminnted sugar chains prepared from S-4-RNase and by liquid chromatography/electrospray ionization-mass spectrometric analysis of the protease digests of reduced and S-carboxymethylated S-RNases. S-4-RNase carries various types of sugar chains, including plant-specific ones with beta 1-->2-linked xylose and alpha 1-->3-linked fucose residues. More than 70% of the total N-glycans of S-4-RNase are, however, an N-acetylglucosamine or a chitobiose (GlcNAc beta 1-->4GlcNAc), which has not been found naturally. The N-acetylglucosamine and chitobiose are mainly present at the N-glycosylation sites within the putative recognition sites of the S-RNase, suggesting that these sugar chains may interact with pollen S-product(s).

    DOI: 10.1046/j.1432-1327.1999.00499.x

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  • Molecular cloning and characterization of two zebrafish alpha(1,3)fucosyltransferase genes developmentally regulated in embryogenesis 査読

    N Kageyama, S Natsuka, S Hase

    JOURNAL OF BIOCHEMISTRY   125 ( 4 )   838 - 845   1999年4月

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

    Some alpha(1,3)fucosylated oligosaccharides serve as counter receptors to lectin-like adhesion proteins or are expressed with temporal precision during embryogenesis, and alpha(1,3)fucosyltransferase is a key enzyme in the production of these oligosaccharides. Two alpha(1,3)fucosyltransferase genes, designated zFT1 and zFT2, were cloned from zebrafish, Sequence comparisons with other genes indicated that zFT1 and zFT2 share about 30% amino acid sequence identity with human alpha(1,3)fucosyltransferases. Although the alpha(1,3)fucosyltransferases cloned so far can be classified into three types-myeloid, Lewis, and leukocyte-by virtue of their amino acid sequences, phylogenetic analysis indicated that neither zFT1 nor zFT2 belongs to any of these categories. The expression of zFT1 or zFT2 in mammalian cells induces alpha(1,3)fucosyltransferase activity to synthesize the Lewis x structure from pyridyl-aminated lacto-N-neotetraose however, lacto-N-tetraose does not serve as a substrate. Reverse transcriptase-polymerase chain reaction analysis revealed that zFT1 is transcribed during a restricted period before hatching, whereas the mRNA for zFT2 was detected only after hatching.

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  • Novel proteoglycan linkage tetrasaccharides of human urinary soluble thrombomodulin, SO4-3GlcA beta 1-3Gal beta 1-3(+/- Sia alpha 2-6)Gal beta 1-4Xyl 査読

    H Wakabayashi, S Natsuka, T Mega, N Otsuki, M Isaji, M Naotsuka, S Koyama, T Kanamori, K Sakai, S Hase

    JOURNAL OF BIOLOGICAL CHEMISTRY   274 ( 9 )   5436 - 5442   1999年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    O-linked sugar chains with xylose as a reducing end linked to human urinary soluble thrombomodulin were studied. Sugar chains were Liberated by hydrazinolysis followed by N-acetylation and tagged with 2-aminopyridine. Two fractions containing pyridylaminated Xyl as a reducing end were collected. Their structures were determined by partial acid hydrolysis, two-dimensional sugar mapping combined with exoglycosidase digestions, methylation analysis, mass spectrometry, and NMR as SO4-3GlcA beta 1-3Gal beta 1-3(+/-Sia alpha 2-6)Gal beta 1-4Xyl. These sugar chains could bind to an HNK-1 monoclonal antibody. This is believed to be the first example of a proteoglycan linkage tetrasaccharide with glucuronic acid 3-sulfate and sialic acid.

    DOI: 10.1074/jbc.274.9.5436

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  • Partial purification and characterization of a novel endo-beta-mannosidase acting on N-linked sugar chains from Lilium longflorum thumb 査読

    A Sasaki, M Yamagishi, T Mega, S Norioka, S Natsuka, S Hase

    JOURNAL OF BIOCHEMISTRY   125 ( 2 )   363 - 367   1999年2月

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

    An enzyme catalyzing the hydrolysis of the Man beta 1-4GlcNAc linkage of N-Iinked sugar chains was partially purified and characterized. Endo-p-mannosidase activity was detected using pyridylaminated (PA-) Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc as the substrate in a homogenate of lily flowers (Lilium longflorum Thumb). The enzyme was partially purified by ammonium sulfate precipitation, and Q-Sepharose, Superdex 200, hydroxyapatite, Poros PE/M, Mono Q, and Superdex 200 column chromatographies. The optimum pH was 5.0 and the estimated molecular weight of the enzyme was 78,000, as determined by gel filtration. The K-m value found for Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was 1.4 mM. The enzymatic activity was not influenced by the addition of 10 mM EDTA or 2 mM Ca2+. Experiments on the hydrolysis of several PA-N-linked sugar chains revealed that the enzyme hydrolyzed Man(n)Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc-PA (n = 0-2) into a mixture of Man(n)Man alpha 1-6Man and GlcNAc beta 1-4GlcNAc-PA, indicating that it is an endoglycosidase in nature. However, the enzyme did not hydrolyze beta 1-4mannohexaose or p-nitrophenyl beta-mannopyranoside.

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  • Systematic analysis of N-linked sugar chains from whole tissue employing partial automation 査読

    Fujimoto, I, KK Menon, Y Otake, F Tanaka, H Wada, H Takahashi, S Tsuji, S Natsuka, S Nakakita, S Hase, K Ikenaka

    ANALYTICAL BIOCHEMISTRY   267 ( 2 )   336 - 343   1999年2月

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

    A partially automated technique for the isolation and characterization of N-linked sugar chains from glycoproteins of crude tissue samples is established. The N-linked sugar chains from the acetone-extracted tissues are made free by a process of hydrazinolysis and subsequently N-acetylated by GlycoPrep 1000 (Oxford Glycosystems). These free sugar chains are further converted to pyridylamino derivatives by Glyco-Tag (Takara). Characterization of these sugar chains is achieved by a combination of HPLC columns using a highly sensitive fluorescence detector at femtomole levels. Tissue sample can be successfully pyridylaminated and analyzed to give highly reproducible results with consistent yield, requiring fewer purification steps, minimum skills, and less time. Moreover, fixed tissues can also be analyzed employing this technique, giving a similar sugar chain pattern compared to normal tissue samples. Using this method we show that the pattern of N-linked sugar chains present in human sera or in one small region of brain is strikingly similar among the different individuals. However, the absence of a highlighted peak in one of the samples suggests this method can be extrapolated to identify changes, if any, associated with disorders such as inflammation or cancer. Furthermore, this two-dimensional display of sugar chains would discover the function-specific molecules as we see in proteins. (C) 1999 Academic Press.

    DOI: 10.1006/abio.1998.2968

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  • Development-dependent expression of complex-type sugar chains specific to mouse brain 査読

    S Nakakita, S Natsuka, K Ikenaka, S Hase

    JOURNAL OF BIOCHEMISTRY   123 ( 6 )   1164 - 1168   1998年6月

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

    We previously detected a fucosylagalactobiantenna with a bisecting GlcNAc residue (BA-2) and one lacking the GlcNAc residue linked to the Man alpha 1-3 residue of BA-2 (BA-1), which were enriched specifically in mouse brain [Shimizu, H., Ochiai, K., Ikenaka, K., Mikoshiba, K., and Hase, S. (1993) J. Biochem. 114, 334-338]. Pyridylamino sugar chains were prepared from mouse brains of various ages, and BA-1 and BA-2 were quantified after separation by HPLC. In cerebrum, BA-1 was scarcely expressed in newborn brain but gradually increased in amount during development, while expression of BA-2 reached a maximum 1 week after birth followed by a rapid decrease; in adult mice, the amount of BA-1 was almost the same as that of BA-2. In cerebellum, expression of BA-1 was lower than that of BA-2 at all stages. Glycoproteins with the BA-1 and BA-2 structures were enriched in the membrane fraction, and the glycoproteins solubilized were purified by lectin-affinity chromatography and gel filtration. The results indicated that BA-1 and BA-2 occurred in glycoproteins of more than 20 kDa in cerebellum, but most BA-1 and BA-2 were found in a 80-200 kDa fraction in cerebrum. These results show that the two brain-specific sugar chains are developmentally regulated and linked to the membrane-associated glycoproteins of subcellular organellas.

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  • Purification and characterization of neutral alpha-mannosidase from hen oviduct: Studies on the activation mechanism of Co2+ 査読

    K Yamashiro, H Itoh, M Yamagishi, S Natsuka, T Mega, S Hase

    JOURNAL OF BIOCHEMISTRY   122 ( 6 )   1174 - 1181   1997年12月

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

    Neutral alpha-mannosidase was purified to homogeneity from hen oviduct, The molecular mass of the enzyme was 480 kDa on gel filtration, and the 110-kDa band on SDS-PAGE in the presence of 2-mercaptoethanol indicated that it is composed of four subunits, The activated enzyme hydrolyzed both p-nitrophenyl alpha-D-mannoside and high mannose-type sugar chains. This substrate specificity is almost the same as that reported for the neutral alpha-mannosidase from Japanese quail oviduct [Oku and Hase (1991) J. Biochem. 110, 982-989]. Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNac (K-m=0.44 mM) was hydrolyzed four times faster than Man alpha 1-6(Man alpha 1-3)Man alpha 1-6(Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNac, and Man alpha 1-6(Man alpha 1-2Man alpha 1-2Man alpha 1-3)Man beta 1-4GlcNac was obtained as the end product from Man(9)GlcNAc on digestion with the activated alpha-mannosidase. The enzyme was activated 24-fold on preincubation with Co2+. The activation with other metal ions, like Mn2+, Ca2+, Fe2+, Fe3+, and Sr2+, was less than 5-fold, and Zn2+, Cu2+, and Hg2+ inhibited the enzyme activity. The optimum pHs for both the enzyme activity and activation with Co2+ were around 7. The cobalt ion contents of the purified, EDTA-treated, and Co2+-activated enzymes were 1.5, 0.0, and 3.9, respectively, per molecule, Since the Co2+-activated enzyme gradually lost its activity on incubation With EDTA and the activity was restored promptly on the addition of Co2+, the binding of Co2+ to the enzyme seems to be essential for its activation, The results obtained with protease inhibitors together with those of the SDS-PAGE before and after activation, showed that the proteolytic cleavage reported for the activation of monkey brain alpha-mannosidase seems not to be involved.

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  • Structures of the sugar chains of recombinant macrophage colony-stimulating factor produced in Chinese hamster ovary cells 査読

    Y Ushida, S Natsuka, Y Shimokawa, Z Takatsu, S Shimamura, S Hase

    JOURNAL OF BIOCHEMISTRY   122 ( 1 )   148 - 156   1997年7月

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

    The structures of the N- and O-linked sugar chains of recombinant human macrophage colony-stimulating factor (rhM-CSF) from Chinese hamster ovary (CHO) cells were studied. rhM-CSF is a homodimeric glycoprotein. Sugar composition analysis revealed that rhM-CSF contained 4.1 mol N-acetylgalactosamine, 10.3 mol N-acetylglucosamine, 5.0 mol mannose, 10.0 mol galactose, 1.4 mol fucose, and 11.8 mol sialic acid per mol of the monomer, The N- and O-linked sugar chains liberated by hydrazinolysis were N-acetylated, and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC, The structures of the PA-sugar chains were analyzed by a combination of reversed-phase and size-fractionation HPLC, and exoglycosidase digestions, from which the structures of the rhM-CSF sugar chains were estimated to be as follows: monosialo biantennary sugar chain (9 mol%), monosialo fucosylbiantennary sugar chain (10 mol%), disialo biantennary sugar chain (30 mol%), disialo fucosylbiantennary sugar chain (28 mol%), disialo triantennary sugar chain (7 mol%), trisialo triantennary sugar chain (11 mol%), and trisialo fucosyltriantennary sugar chain (5 mol%) for the N-linked sugar chains, and asialo (27 mol%), monosialo (51 mol%), and disialo (22 mol%) Gal beta 1-3GalNAc for the O-linked sugar chains, Sialic acid residues were linked to the N-linked sugar chains through an alpha 2-3 linkage.

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  • Detection of UDP-D-Xylose: alpha-D-xyloside alpha 1->3Xylosyltransferase activity in human hepatoma cell line HepG2 査読

    S Minamida, K Aoki, S Natsuka, K Omichi, K Fukase, S Kusumoto, S Hase

    JOURNAL OF BIOCHEMISTRY   120 ( 5 )   1002 - 1006   1996年11月

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

    We previously reported the detection of novel O-linked sugar chains classified as being of the glucosyl-O-serine type [Hase et al. (1988) J. Biochem. 104, 867-868]. The sugar chains are a disaccharide (Xyl alpha 1-3Glc) and a trisaccharide (Xyl alpha 1-3Xyl alpha 1-3Glc) linked to serine residues in epidermal growth factor-like domains of human and bovine blood coagulation factors, The structures of these sugar chains suggested the presence of an alpha 1-->3xylosyl-transferase for their biosynthesis, We report here on the detection of alpha 1-->3xylosyltransferase activity which catalyzes the transfer of xylose to Xyl alpha 1-SGlc in the human hepatoma cell line HepG2. We employed pyridylaminated Xyl alpha 1-3Glc as a fluorescent acceptor and UDP-D-Xyl as a donor, The reaction product was purified by reversed-phase HPLC, and the structure of the transfer product isolated was confirmed to be pyridylaminated Xyl alpha 1-3Xyl alpha 1-3Glc by Smith degradation, mass spectrometry, and alpha- and beta-xylosidase digestions, The apparent K-m value for pyridylaminated Xyl alpha 1-3Glc was 52 mM and for UDP-D-Xyl 0.28 mM, Optimum pH was 7.2, The enzyme was inactivated by addition of EDTA, and its activity was restored by addition of Mn2+ and Mg2+. These results indicate the presence of a novel enzyme which is able to transfer xylose to Xyl alpha 1-3Glc, forming Xyl alpha 1-3Xyl alpha 1-3Glc in human cells.

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  • The fucosyltransferase FucT-VII regulates E-selectin ligand synthesis in human T cells 査読

    RN Knibbs, RA Craig, S Natsuka, A Chang, M Cameron, JB Lowe, LM Stoolman

    JOURNAL OF CELL BIOLOGY   133 ( 4 )   911 - 920   1996年5月

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

    Selectin-ligands on T cells contribute to the recruitment of circulating cells into chronic inflammatory lesions in the skin and elsewhere, This report provides the first evidence that a single fucosyltransferase, termed FucT-VII, controls the synthesis of E-selectin ligands in human T-lymphoblasts. The FucT-IV transferase (the ELFT enzyme), in contrast, constructs lower avidity E-selectin ligands and requires enzyme levels found only in myeloid cells. Treatment of Jurkat cells with phorbol myristate acetate increased the expression of sialylated Lewis(x)-related (sLe(x)-related) epitopes and induced the synthesis of E-selectin ligands functional at physiologic levels of linear shear-stress. Northern analysis revealed a parallel increase in the steady-state level of FucT-VII mRNA, but there were no increases in the two other leukocyte-associated fucosyltransferases (FucT-IV and VI). The stable transfection of the FucT-VII gene into Jurkat cells induced high levels of the sLe(x)-related epitopes and the synthesis of E-selectin ligands which equaled or exceeded the avidity of those on circulating lymphocytes. The growth of T-lymphoblasts under conditions which induced expression of the sLe(x,a) epitopes increased the level of FucT-VII mRNA, the synthesis of sialylated-Lewis(x) structures by cell-free extracts and the synthesis of E-selectin ligands equal in avidity to those on FucT-VII transfectants. In contrast, neither the mRNA levels nor activities of the FucT-IV and VI enzymes increased in association with E-selectin ligand synthesis in T-lymphoblasts, Myeloid cell lines, unlike lymphoblasts, expressed high levels of both the FucT-VII and IV enzymes in conjunction with E-selectin ligands raising the possibility that both enzymes contributed to ligand synthesis, FucT-IV transfected Jurkat cells synthesized low avidity ligands for E-selectin but only in association with the CDw65 (VIM-2) carbohydrate epitope. Only blood neutrophils and myeloid cell lines expressed this epitope at the levels associated with E-ligand synthesis in the transfectants. In contrast, native Jurkat cells, blood monocytes, blood lymphocytes, and cultured T-lymphoblasts expressed low levels or none, We conclude that FucT-VII is a principal regulator of E-selectin ligand synthesis in human T-lymphoblasts while both FucT-VII and FucT-IV may direct ligand synthesis in some myeloid cells.

    DOI: 10.1083/jcb.133.4.911

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  • Expression of the alpha(1,3)fucosyltransferase Fuc-TVII in lymphoid aggregate high endothelial venules correlates with expression of L-selectin ligands 査読

    PL Smith, KM Gersten, B Petryniak, RJ Kelly, C Rogers, Y Natsuka, JA Alford, EP Scheidegger, S Natsuka, JB Lowe

    JOURNAL OF BIOLOGICAL CHEMISTRY   271 ( 14 )   8250 - 8259   1996年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Lymphocyte homing to lymph nodes and Peyer's patches is mediated, in part, by adhesive interactions between L-selectin expressed by lymphocytes and L-selectin ligands displayed at the surface of the cuboidal endothelial cells lining the post capillary venules within lymphoid aggregates, Candidate terminal oligosaccharide structures thought to be essential for effective L-selectin ligand activity include a sulfated derivative of the sialyl Lewis x tetrasaccharide. Cell type-specific synthesis of this oligosaccharide is presumed to require one or more alpha(1,3)fucosyltransferases, operating upon common 3'-sialylated and/or sulfated N-acetyllactosamine-type precursors, The identity of the alpha(1,3)fucosyltransferase(s) expressed in cells that bear L-selectin ligands has not been defined, We report here the molecular cloning and characterization of a murine alpha(1,3)fucosyltransferase locus whose expression pattern correlates with expression of high affinity ligands for L-selectin. In situ hybridization and immunohistochemical analyses demonstrate that this cDNA and its cognate alpha(1,3)fucosyltransferase are expressed in endothelial cells lining the high endothelial venules of peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches, These expression patterns correlate precisely with the expression pattern of L-selectin ligands identified with a chimeric L-selectin/IgM immunohistochemical probe and by the high endothelial venule-reactive monoclonal antibody MECA-79. Transcripts corresponding to this cDNA are also detected in isolated bone marrow cells, a source rich in the surface-localized ligands for E- and P-selectins. Sequence and functional analyses indicate that this murine enzyme corresponds to the human Fuc-TVII locus, These observations suggest that Fuc-TVII participates in the generation of alpha(1,3)fucosylated ligands for L-selectin and provide further evidence for a role for this enzyme in E- and P-selectin ligand expression in leukocytes.

    DOI: 10.1074/jbc.271.14.8250

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  • MOLECULAR-CLONING, EXPRESSION, CHROMOSOMAL ASSIGNMENT, AND TISSUE-SPECIFIC EXPRESSION OF A MURINE ALPHA-(1,3)-FUCOSYL-TRANSFERASE LOCUS CORRESPONDING TO THE HUMAN ELAM-1 LIGAND FUCOSYL-TRANSFERASE 査読

    KM GERSTEN, S NATSUKA, M TRINCHERA, B PETRYNIAK, RJ KELLY, N HIRAIWA, NA JENKINS, DJ GILBERT, NG COPELAND, JB LOWE

    JOURNAL OF BIOLOGICAL CHEMISTRY   270 ( 42 )   25047 - 25056   1995年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Terminal Fuc alpha 1-3GlcNAc moieties are displayed by mammalian cell surface glycoconjugates in a tissue-specific manner. These oligosaccharides participate in selectin-dependent leukocyte adhesion and have been implicated in adhesive events during murine embryogenesis. Other functions for these molecules remain to be defined, as do the tissue-specific expression patterns of the corresponding alpha-(1-3)-fucosyltransferase (alpha 1-3FT) genes. This report characterizes a murine alpha 1-3FT that shares 77% amino acid sequence identity with human ELAM ligand fucosyltransferase (ELFT, also termed Fuc-TIV). The corresponding gene maps to mouse chromosome 9 in a region of homology with the Fuc-TIV locus on human chromosome 11q. In vitro, the murine (alpha 1-3FT can efficiently fucosylate the trisaccharide Gal alpha 1-3Gal beta 1-4GlcNAc (apparent K-m of 0.71 mM) to form an unusual tetrasaccharide (Gal alpha 1-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc) described in periimplantation mouse tissues. The enzyme can also form the Lewis x determinant from Gal beta 1-4GlcNAc (K-m = 2.05 mM), and the sialyl Lewis x determinant from NeuNAc alpha 2-3Gal beta 1-4GlcNAc (K-m = 1.78 mM). However, it does not yield sialyl Lewis x determinants when expressed in a mammalian cell line that maintains sialyl Lewis x precursors. Transcripts from this gene accumulate to low levels in hematopoietic organs, but are unexpectedly abundant in epithelia that line the stomach, small intestine, colon, and epididymus. Epithelial cell-specific expression of this gene suggests function(s) in addition to, and distinct from, its proposed role in selectin ligand synthesis.

    DOI: 10.1074/jbc.270.42.25047

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  • セレクチン・リガンドの生合成に携わるフコース転移酵素 査読

    長束俊治

    生化学   67 ( 5 )   368 - 372   1995年5月

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    記述言語:日本語  

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  • ENZYMES INVOLVED IN MAMMALIAN OLIGOSACCHARIDE BIOSYNTHESIS 査読

    S NATSUKA, JB LOWE

    CURRENT OPINION IN STRUCTURAL BIOLOGY   4 ( 5 )   683 - 691   1994年10月

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

    Several new sialyltransferases, N-acetylgalactosaminyltransferase and fucosyltransferase genes have been reported in this past year. These sequences have advanced our understanding of the structural, functional and evolutionary relationships amongst the glycosyltransferases, including their roles in selectin ligand biosynthesis. Ablation of the murine N-acetylgalactosaminyltransferase I gene through gene 'knock out' technology has yielded insight into the role of this gene in the developing mouse. Novel 'O-linked' protein glycosylation events described in the past year have added to the substantial known diversity in the oligosaccharide structure and glycosyltransferase repertoire of mammalian organisms.

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  • MOLECULAR-CLONING OF A CDNA-ENCODING A NOVEL HUMAN-LEUKOCYTE ALPHA-1,3-FUCOSYL-TRANSFERASE CAPABLE OF SYNTHESIZING THE SIALYL-LEWIS-X DETERMINANT 査読

    S NATSUKA, KM GERSTEN, K ZENITA, R KANNAGI, JB LOWE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 24 )   16789 - 16794   1994年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The sialyl Lewis x determinant (NeuAc alpha 2,3Gal beta 1, 4[Fuc alpha 1,3]GlcNAc) is an essential component of leukocyte counterreceptors for E-selectin and P-selectin. The final step in sialyl Lewis x synthesis is catalyzed by alpha-1,3-fucosyltransferases acting on sialylated glycoconjugate precursors. Cultured human leukocytic cell lines express an alpha-1,3-fucosyltransferase gene termed Fuc-TIV or ELFT but do not express the other three cloned human alpha-1,3-fucosyltransferase genes to any significant degree. The physiological role of Fuc-TIV/ELFT in sialyl Lewis x biosynthesis is uncertain, however, since it can catalyze the synthesis of this determinant in some, but not all, transfected cell lines in a manner that is dependent upon the glycosylation phenotype of the host cell. We report here the molecular cloning of a cDNA encoding a new human leukocyte alpha-1,3-fucosyltransferase, termed Fuc-TVII, capable of synthesizing the sialyl Lewis x moiety. The cDNA sequence predicts a 341-amino acid-long type II transmembrane protein typical of mammalian glycosyltransferases. When expressed in mammalian cells, the Fuc-TVII cDNA directs the synthesis of cell surface sialyl Lewis x moieties but not the Lewis x, Lewis a, sialyl Lewis a, or VIM-2 determinants. Fuc-TVII can efficiently utilize alpha-2,3-sialyllactosamine in vitro to form the sialyl Lewis x tetrasaccharide but does not utilize lactosamine to form the Lewis x moiety. Northern blot analyses show that the Fuc-TVII gene is transcribed in HL-60 cells, a human promyelocytic cell line, and in YT cells, a natural killer-like cell line. Fuc-TVII represents a leukocytic alpha-1,3-fucosyltransferase that can participate in selectin ligand synthesis via its ability to catalyze the synthesis of sialyl Lewis x determinants.

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  • STRUCTURE-FUNCTION ANALYSIS OF HUMAN IL-6 RECEPTOR - DISSOCIATION OF AMINO-ACID-RESIDUES REQUIRED FOR IL-6-BINDING AND FOR IL-6 SIGNAL TRANSDUCTION THROUGH GP130 査読

    H YAWATA, K YASUKAWA, S NATSUKA, M MURAKAMI, K YAMASAKI, M HIBI, T TAGA, T KISHIMOTO

    EMBO JOURNAL   12 ( 4 )   1705 - 1712   1993年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS UNITED KINGDOM  

    Here, we report the analysis of the structure - function relationship of the extracellular region of human interleukin 6 receptor (IL-6R). Upon binding of IL-6, IL-6R becomes associated extracellularly with a non-IL-6-binding but signal tranducing molecule, gp130, and the IL-6 signal is generated. In this region, the cytokine receptor family domain, but not the immunoglobulin-like domain, was responsible both for IL-6 binding and for signal transduction through gp130. Because a soluble, extracellular portion of IL-6R (sIL-6R) could bind IL-6 and mediate IL-6 functions through gp130, amino acid substitutions were introduced into sIL-6R by site-directed mutagenesis. The results, together with the previously proposed tertiary structure model, suggested that the amino acid residues critical for IL-6 binding have a tendency to be distributed to the hinge region between the two 'barrel'-like fibronectin type III modules and to the same side of these two 'barrels'. Amino acid residues, of which substitutions barely affected the IL-6-binding but did abolish the IL-6 signalling capability of sIL-6R, were identified and found to be located mainly in the membrane proximal half of the second barrel. sIL-6R mutants carrying such substitutions lacked the capacity to associate with gp130 in the presence of IL-6.

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  • MACROPHAGE DIFFERENTIATION-SPECIFIC EXPRESSION OF NF-IL6, A TRANSCRIPTION FACTOR FOR INTERLEUKIN-6 査読

    S NATSUKA, S AKIRA, Y NISHIO, S HASHIMOTO, T SUGITA, H ISSHIKI, T KISHIMOTO

    BLOOD   79 ( 2 )   460 - 466   1992年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:W B SAUNDERS CO  

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  • REGULATION OF EXPRESSION OF THE INTERLEUKIN-6 GENE - STRUCTURE AND FUNCTION OF THE TRANSCRIPTION FACTOR NF-IL6 査読

    S AKIRA, H ISSHIKI, T NAKAJIMA, S KINOSHITA, Y NISHIO, S NATSUKA, T KISHIMOTO

    CIBA FOUNDATION SYMPOSIA   167   47 - 67   1992年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JOHN WILEY & SONS LTD  

    The interleukin 6 (IL-6) promoter is rapidly and transiently activated by other cytokines, including IL-1 and tumour necrosis factor (TNF), as well as by phorbol esters and cyclic AMP agonists. Studies using promoter mutants suggested that an IL-1-responsive element mapped within the -180 to -123 region of the IL-6 promoter. A nuclear factor (NF-IL6) that recognized a unique sequence containing an inverted repeat, ACATTGCACAATCT, was identified within the region. Direct cloning of the human NF-IL6 revealed its similarity to C/EBP, a liver- and adipose tissue-specific transcription factor. C/EBP and NF-IL6 recognize the same nucleotide sequence, but exhibit distinct patterns of expression. NF-IL6 is expressed at a low level in normal tissues, but is rapidly and drastically induced by bacterial lipopolysaccharide (LPS) or inflammatory cytokines such as IL-1, TNF and IL-6. Recently, NF-IL6 has been shown to be identical to IL-6DBP, the DNA-binding protein which is responsible for IL-6-mediated induction of several acute-phase proteins. Evidence that NF-IL6 DNA-binding activity is increased after IL-6 stimulation without increased NF-IL6 protein synthesis demonstrates the importance of post-translational modification. There are some results indicating that phosphorylation is involved in transcriptional and binding activities of NF-IL6. Taken together, these findings indicate that NF-IL6 may be an important transcription factor on the signal transduction pathways of IL-1 and IL-6.

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  • A NUCLEAR FACTOR FOR THE IL-6 GENE (NF-IL6) 査読

    S AKIRA, H ISSHIKI, T NAKAJIMA, S KINOSHITA, Y NISHIO, S HASHIMOTO, S NATSUKA, T KISHIMOTO

    CHEMICAL IMMUNOLOGY   51   299 - 322   1992年

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    記述言語:英語   出版者・発行元:KARGER  

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  • AUGMENTATION OF HAPTOGLOBIN PRODUCTION IN HEP3B CELL-LINE BY A NUCLEAR FACTOR NF-IL6 査読

    S NATSUKA, H ISSHIKI, S AKIRA, T KISHIMOTO

    FEBS LETTERS   291 ( 1 )   58 - 62   1991年10月

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

    The nuclear factor NF-IL6 had been suggested to be responsible for the IL-6-mediated induction of several acute-phase proteins. To obtain evidence for the involvement of NF-IL6 in the induction of acute-phase proteins, we introduced the NF-IL6 gene and its truncated mutant (delNFIL6) gene into a hepatoma cell line Hep3B. Then, we examined the effect of the overproduced NF-IL6 and delNFIL6 on the expression of haptoglobin, fibrinogen and albumin. As a result, basal production as well as induction of haptoglobin by IL-6 were augmented by the expression of NF-IL6, whereas delNFIL6 blocked the production of haptoglobin, fibrinogen and albumin.

    DOI: 10.1016/0014-5793(91)81103-F

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  • INTERLEUKIN-6 RECEPTOR AND A UNIQUE MECHANISM OF ITS SIGNAL TRANSDUCTION 査読

    T TAGA, M HIBI, Y HIRATA, H YAWATA, S NATSUKA, K YASUKAWA, T TOTSUKA, K YAMASAKI, T HIRANO, T KISHIMOTO

    IMMUNOLOGICAL RECOGNITION, PTS 1 AND 2   54   713 - 722   1989年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:COLD SPRING HARBOR LABORATORY PRESS  

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  • Structures of sugar chains of a p-nitrophenyl acetate-hydrolyzing esterase from the microsomes of rat liver. 査読

    Natsuka S, Himeno M, Hase S, Ito H, Ueda T, Kato K, Ikenaka T

    Journal of biochemistry   103 ( 6 )   986 - 991   1988年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  • Identification method for twelve oligomannose-type sugar chains thought to be processing intermediates of glycoproteins. 査読

    Hase S, Natsuka S, Oku H, Ikenaka T

    Analytical biochemistry   167 ( 2 )   321 - 326   1987年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/0003-2697(87)90171-0

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  • FLUORESCENCE METHOD FOR THE STRUCTURAL-ANALYSIS OF OLIGOMANNOSE-TYPE SUGAR CHAINS BY PARTIAL ACETOLYSIS 査読

    S NATSUKA, S HASE, T IKENAKA

    ANALYTICAL BIOCHEMISTRY   167 ( 1 )   154 - 159   1987年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

    DOI: 10.1016/0003-2697(87)90146-1

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書籍等出版物

  • Glycoscience : basic science to applications : insights from the Japan consortium for glycobiology and glycotechnology (JCGG)

    Shunji Natsuka( 担当: 分担執筆 ,  範囲: Genetic modification: small fish (zebrafish and medaka))

    Springer  2019年  ( ISBN:9789811358555

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    総ページ数:xv, 405 p.   記述言語:英語

    CiNii Books

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  • 未来を創るグライコサイエンス ―我が国のロードマップ―

    長束俊治( 担当: 分担執筆 ,  範囲: 糖鎖機能解析のキーテクノロジー:遺伝子改変:小型魚類(4))

    日本糖鎖科学コンソーシアム  2018年5月 

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  • 生物薬科学実験講座,糖質Ⅱ糖タンパク質実験法

    長束 俊治, 長谷 純宏( 担当: 分担執筆 ,  範囲: 二次元HPLCマッピングと溶出の規則性,プロトン核磁気共鳴(1H-NMR))

    廣川書店  2011年 

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  • ピリジルアミノ化による糖鎖解析-糖鎖多様性の解析に向けて

    長谷 純宏, 長束 俊治, 中北 愼一, 石水 毅( 担当: 共著)

    大阪大学出版会  2009年3月  ( ISBN:4872592697

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    総ページ数:141  

    ASIN

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  • 生物薬科学実験講座,糖質Ⅰ基本実験操作法

    長束 俊治, 長谷 純宏( 担当: 分担執筆 ,  範囲: 蛍光標識による糖組成分析,蛍光標識,PA-糖鎖の逆相カラムによる分離と精製,陰イオン交換HPLCによるPA-糖鎖の分離)

    廣川書店  2009年 

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  • Experimental Glycoscience: Glycochemistry

    長束 俊治( 担当: 共著 ,  範囲: “Release of O-glycans by chemical methods”, “Separation of oligosaccharides by 2D HPLC”, “Equilibrium dialysis”)

    Springer  2008年 

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  • タンパク質修飾解析プロトコール

    長束 俊治, 長谷 純宏( 担当: 共著 ,  範囲: O-結合型糖鎖の構造解析)

    羊土社  2005年 

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  • 未来を拓く糖鎖科学

    長束 俊治( 担当: 共著 ,  範囲: 糖タンパク質よりO-結合型糖鎖の化学的切り出し,オリゴ糖の2次元HPLCによる分離,平衡透析を用いた糖鎖-タンパク質相互作用の解析)

    金芳堂  2005年 

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  • 糖鎖科学の新展開

    長束 俊治( 担当: 共著 ,  範囲: 糖鎖分析の統合オペレーティングシステムとしてのピリジルアミノ化法)

    NTS  2005年 

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  • ポストゲノムの糖鎖生物学がわかる

    長束 俊治( 担当: 共著 ,  範囲: アスパラギン結合型糖鎖の多様性形成機構)

    羊土社  2002年 

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  • Glycoanalysis Protocols

    長束 俊治, 長谷 純宏( 担当: 共著 ,  範囲: Analysis of N- and O-glycans by pyridylamination)

    Humana Press  1998年 

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  • グライコバイオロジー実験プロトコール

    長束 俊治, 長谷 純宏( 担当: 共著 ,  範囲: ピリジルアミノ(PA)化法による糖鎖の蛍光標識と分離)

    秀潤社  1996年 

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  • 糖―その多様性を探る

    長束 俊治, 長谷 純宏( 担当: 共著 ,  範囲: ピリジルアミノ化法による糖鎖の高感度分析)

    化学同人  1992年 

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MISC

  • 希少糖を含む分泌型糖ペプチド 招待 査読

    長束 俊治

    化学と生物   57 ( 1 )   5 - 6   2019年1月

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    担当区分:筆頭著者, 責任著者   記述言語:日本語   掲載種別:記事・総説・解説・論説等(学術雑誌)  

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  • N-Glycosylation Patterns of Hemolymph Glycoproteins from Biomphalaria Glabrata Strains Expressing Different Susceptibility to Schistosoma Mansoni Infection

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  • EDEM ホモログ蛋白質 の機能解析 査読

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  • FUCOSYL-TRANSFERASES IN THE SYNTHESIS OF SELECTIN LIGANDS ON T-CELL

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  • 白血球ホーミングにおけるフコース転移酵素の役割

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  • MOLECULAR-GENETICS OF MAMMALIAN FUCOSYL-TRANSFERASES

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  • STRUCTURE AND FUNCTION OF MAMMALIAN GLYCOSYLTRANSFERASE GENES

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  • REGULATION OF EXPRESSION OF THE INTERLEUKIN-6 GENE - STRUCTURE AND FUNCTION OF THE TRANSCRIPTION FACTOR NF-IL6 査読

    S AKIRA, H ISSHIKI, T NAKAJIMA, S KINOSHITA, Y NISHIO, S NATSUKA, T KISHIMOTO

    POLYFUNCTIONAL CYTOKINES : IL-6 AND LIF   167   47 - 67   1992年

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  • INTERLEUKIN-6 RECEPTOR AND A UNIQUE MECHANISM OF ITS SIGNAL TRANSDUCTION 査読

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    COLD SPRING HARBOR SYMPOSIA ON QUANTITATIVE BIOLOGY   54   713 - 722   1989年

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  • Interleukin 6 and its receptor

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    Recent Progress in Cytokine Research   265 - 285   1989年

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受賞

  • 日本糖質学会奨励賞

    2004年8月  

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  • 日本生化学会JB論文賞

    2000年10月  

    長束 俊治

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  • 分子生物学

    機関名:京都工芸繊維大学

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  • 基礎生物化学

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  • 課題研究I(生物学)

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  • 生物学基礎実習b

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  • 生命・食料科学博士特定研究Ⅱ

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