General Education

松下 文雄

Fumio Matsushita

基本情報

所属
藤田医科大学 保健衛生学部 リハビリテーション学科 生物学 教授
学位
博士(農学)(京都大学)

J-GLOBAL ID
200901053954844786
researchmap会員ID
1000226786

論文

 18
  • Youhei Nashimoto, Fumio Matsushita, Johannes M Dijkstra, Yuta Nakamura, Hidehiko Akiyama, Jiharu Hamako, Takashi Morita, Satohiko Araki, Taei Matsui
    Toxins 14(4) 2022年3月25日  
    Bitiscetin-1 (aka bitiscetin) and bitiscetin-2 are C-type lectin-like proteins purified from the venom of Bitis arietans (puff adder). They bind to von Willebrand factor (VWF) and-at least bitiscetin-1-induce platelet agglutination via enhancement of VWF binding to platelet glycoprotein Ib (GPIb). Bitiscetin-1 and -2 bind the VWF A1 and A3 domains, respectively. The A3 domain includes the major site of VWF for binding collagen, explaining why bitiscetin-2 blocks VWF-to-collagen binding. In the present study, sequences for a novel bitiscetin protein-bitiscetin-3-were identified in cDNA constructed from the B. arietans venom gland. The deduced amino acid sequences of bitiscetin-3 subunits α and β share 79 and 80% identity with those of bitiscetin-1, respectively. Expression vectors for bitiscetin-3α and -3β were co-transfected to 293T cells, producing the heterodimer protein recombinant bitiscetin-3 (rBit-3). Functionally, purified rBit-3 (1) induced platelet agglutination involving VWF and GPIb, (2) did not compete with bitiscetin-1 for binding to VWF, (3) blocked VWF-to-collagen binding, and (4) lost its platelet agglutination inducing ability in the presence of an anti-VWF monoclonal antibody that blocked VWF-to-collagen binding. These combined results suggest that bitiscetin-3 binds to the A3 domain, as does bitiscetin-2. Except for a small N-terminal fragment of a single subunit-which differs from that of both bitiscetin-3 subunits-the sequences of bitiscetin-2 have never been determined. Therefore, by identifying and analyzing bitiscetin-3, the present study is the first to present the full-length α- and β-subunit sequences and recombinant expression of a bitiscetin-family toxin that blocks the binding of VWF to collagen.
  • Riona Hatazawa, Saori Fukuda, Kanako Kumamoto, Fumio Matsushita, Shizuko Nagao, Takayuki Murata, Koki Taniguchi, Taei Matsui, Satoshi Komoto
    The Journal of general virology 102(4) 2021年4月  査読有り
    With the recent establishment of robust reverse genetics systems for rotavirus, rotavirus is being developed as a vector to express foreign genes. However, insertion of larger sequences such as those encoding multiple foreign genes into the rotavirus genome has been challenging because the virus segments are small. In this paper, we attempted to insert multiple foreign genes into a single gene segment of rotavirus to determine whether it can efficiently express multiple exogenous genes from its genome. At first, we engineered a truncated NSP1 segment platform lacking most of the NSP1 open reading frame and including a self-cleaving 2A sequence (2A), which made it possible to generate a recombinant rotavirus stably expressing NanoLuc (Nluc) luciferase as a model foreign gene. Based on this approach, we then demonstrated the generation of a replication-competent recombinant rotavirus expressing three reporter genes (Nluc, EGFP, and mCherry) by separating them with self-cleaving 2As, indicating the capacity of rotaviruses as to the insertion of multiple foreign genes. Importantly, the inserted multiple foreign genes remained genetically stable during serial passages in cell culture, indicating the potential of rotaviruses as attractive expression vectors. The strategy described here will serve as a model for the generation of rotavirus-based vectors designed for the expression and/or delivery of multiple foreign genes.
  • Taiki Kano, Kazunao Kondo, Jiharu Hamako, Fumio Matsushita, Kazuya Sakai, Taei Matsui
    International Journal of Hematology 108(2) 1-6 2018年4月4日  査読有り
    Von Willebrand factor (VWF) is one of the plasma protein carrying ABO(H) blood group antigens, but the combining process of these antigens is not clear. In the present study, we examined whether plasma glycosyltransferase affects the blood group antigens on VWF. VWF expressing H-antigen (H-VWF) from blood group O and bovine serum albumin conjugated with H-antigen (H-BSA) were incubated with recombinant α1-3-N-acetylgalactosaminyltransferase (rA-transferase) and A-plasma with or without an additional UDP-GalNAc. Transformed antigens were detected by western blotting and ELISA, using an anti-A antibody. Both H-VWF and H-BSA acquired the A-antigen after incubation with rA-transferase and UDP-GalNAc. Incubation with A-plasma very weakly converted the H-antigen on BSA and VWF to A-antigen only in the presence of supplemented UDP-GalNAc. This conversion was enhanced on desialylation of H-VWF. These results indicate that sugar chains of plasma VWF can be modified by the external glycosyltransferase, but that plasma glycosyltransferase has no effect on the blood group antigens of VWF due to its low activity and the lack of donor sugars. Further, sialic acid residues of VWF may exert a protective effect against post-translational glycosylation. Our results clearly exclude the possibility that blood group antigens of VWF are constructed extracellularly in plasma.
  • T. Matsui, A. Hori, J. Hamako, F. Matsushita, Y. Ozeki, Y. Sakurai, M. Hayakawa, M. Matsumoto, Y. Fujimura
    JOURNAL OF THROMBOSIS AND HAEMOSTASIS 15(3) 538-548 2017年3月  査読有り
    Background Botrocetin-2 (Bot2) is a botrocetin-like protein composed of and subunits that have been cloned from the snake Bothrops jararaca. Bot2 binds specifically to von Willebrand factor (VWF), and the complex induces glycoprotein (GP) Ib-dependent platelet agglutination. Objectives To exploit Bot2's VWF-binding capacity in order to attempt to create a mutant Bot2 that binds to VWF but inhibits platelet agglutination. Methods and Results Several point mutations were introduced into Bot2 cDNA, and the recombinant protein (recombinant Bot2 [rBot2]) was purified on an anti-botrocetin column. The mutant rBot2 with either Ala at Asp70 in the subunit (Asp70Ala), or Arg115Ala and Lys117Ala, showed reduced platelet agglutination-inducing activity. rBot2 with Asp70Ala showed little binding activity towards immobilized VWF on an ELISA plate, whereas rBot2 with Arg115Ala/Lys117Ala showed reduced binding activity towards GPIb (glycocalicin) after forming a complex with VWF. rBot2 point-mutated to oppositely charged Glu at both Arg115 and Lys117 showed normal binding activity towards VWF but no platelet-agglutinating activity. Furthermore, this doubly mutated protein inhibited ristocetin-induced or high shear stress-induced platelet aggregation, and restrained thrombus formation under flow conditions. Conclusions Asp70 in the subunit of botrocetin is important for VWF binding, and Arg115 and Lys117 in the subunit are essential for interaction with GPIb. Doubly mutated rBot2, with Arg115Glu and Lys117Glu, repels GPIb and might have potential as an antithrombotic reagent that specifically blocks VWF function. This is the first report on an artificial botrocetin that can inhibit the VWF-GPIb interaction.
  • Fumio Matsushita, Toshiki Kameyama, Yuzo Kadokawa, Tohru Marunouchi
    DEVELOPMENTAL DYNAMICS 243(4) 588-600 2014年4月  査読有り
    Background: Three members of the Myt/NZF family of transcription factors are involved in many processes of vertebrate development. Several studies have reported that Myt1/NZF-2 has a regulatory function in the development of cultured oligodendrocyte progenitors or in neuronal differentiation during Xenopus primary neurogenesis. However, little is known about the proper function of Myt/NZF family proteins during mammalian nervous system development. To assess the possible function of Myt/NZF transcription factors in mammalian neuronal differentiation, we determined the comparative spatial and temporal expression patterns of all three types of Myt/NZF family genes in the embryonic mouse nervous system using quantitative reverse transcriptase polymerase chain reaction and in situ hybridization. Results: All three Myt/NZF family genes were extensively expressed in developing mouse nervous tissues, and their expression was transient. NZF-1 was expressed later in post-mitotic neurons. NZF-2 was initially expressed in neuronal cells a little earlier than NZF-3. NZF-3 was initially expressed in neuronal cells, just after proliferation was complete. Conclusion: These expression patterns suggest that the expression of NZF family genes is spatially and temporally regulated, and each Myt/NZF family gene may have a regulatory function in a specific phase during neuronal differentiation. Developmental Dynamics 243:588-600, 2014. (c) 2013 Wiley Periodicals, Inc.

MISC

 12

講演・口頭発表等

 28

担当経験のある科目(授業)

 7

共同研究・競争的資金等の研究課題

 4

その他

 2

作成した教科書、教材、参考書

 4
  • 件名
    生物学基礎実験テキスト(臨床検査学科編)
    開始年月日
    2009
    終了年月日
    2015/10
    概要
    顕微鏡操作の基礎、細胞の観察、DNAの抽出と形質転換、DNAの切断と電気泳動
  • 件名
    生物学基礎実験テキスト(放射線学科編)
    開始年月日
    2009
    終了年月日
    2018
    概要
    顕微鏡操作の基礎、細胞や筋収縮の観察、DNAの性質、DNAに対する紫外線の効果
  • 件名
    自然科学情報論演習テキスト(生命科学編)
    開始年月日
    2009
    終了年月日
    2015/11
    概要
    タンパク質のアミノ酸配列や立体構造、遺伝子の塩基配列の情報の検索・表示・比較
  • 件名
    基礎科学実験(生物学)テキスト
    開始年月日
    2019
    終了年月日
    2024