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研究者情報

マスター

アカウント（マスター）

  • 氏名

    尾瀬　農之(オセ　トヨユキ), オセ　トヨユキ

所属（マスター）

  • 先端生命科学研究院

所属（マスター）

  • 先端生命科学研究院

独自項目

syllabus

  • 2021, 大学院共通授業科目（一般科目）：複合領域, Inter-Graduate School Classes(General Subject):Inter-Disciplinary Sciences, 修士課程, 大学院共通科目, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • 2021, ソフトマター科学概論, Soft Matter Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • 2021, 生命融合科学概論, Transdisciplinary Life Science, 修士課程, 生命科学院, （生命医薬科学概論） 有機合成化学，天然物化学，神経，免疫，蛋白質，脂質，遺伝子解析，RNA，バイオイメージング解析，薬剤吸収，薬物送達，痛み，立体構造 （生命融合科学概論/ソフトマター科学概論） 生命融合科学，生命情報分子科学，生命物質科学，細胞機能科学，生命機能制御科学，ソフトマター科学，ソフトマター材料科学，ソフトマター生命分子科学，ソフトマター生体物理学，ソフトマター医科学，SDGs （生命システム科学概論） 細胞増殖，細胞極性，細胞分化，形態形成，遺伝子発現，植物免疫，神経回路，動物行動学，能科学，生殖機構，発生，内分泌，ホルモン，オムニバス，現代生命科学，知的財産
  • 2021, 生命情報分子科学特論, Bioinformation and Molecular Sciences, 修士課程, 生命科学院, 生体高分子，蛋白質，核酸，構造，Ｘ線回折，結晶
  • 2021, 分子生物学概論, Molecular Biology, 学士課程, 理学部, 真核生物，原核生物，細胞，ゲノム，タンパク質，核酸，進化，化学結合，熱力学，自由エネルギー
  • 2021, 化学Ⅱ, Chemistry II, 学士課程, 全学教育, 有機化合物、官能基、分子構造、化学的性質、化学反応、機能性有機物、生体関連有機物質
  • 2021, 実験生物科学, Experimental Biological Science, 学士課程, 理学部, 高分子機能学，実験技術，生命科学，物質科学，情報科学，融合科学
  • 2021, 生体高分子学実験Ⅰ, Laboratory Work on Bio-macromolecular Science I, 学士課程, 理学部, 物理学実験，化学分析実験，有機合成実験，生化学実験, 分子生物学実験，細胞生物学実験, 生物物理学実験
  • 2021, 高分子機能学基礎実験, Fundamental Laboratory Work on Bio-macromolecular Science, 学士課程, 理学部, 基礎実験手法，物理学実験，化学分析実験，有機合成実験，生化学実験，安全教育，法令

researchmap

プロフィール情報

学位

  • 博士（理学）（北海道大学）

プロフィール情報

  • 姓

    尾瀬

  • 名

    農之

  • ID各種

    201301042707881513

対象リソース

業績リスト

研究キーワード

  • 生物物理   蛋白質工学   免疫   蛋白質   NMR   中性子   シグナル伝達   結晶構造解析   構造解析   構造生物学   蛋白質化学   分子間相互作用   細胞分子生物学   

研究分野

  • ライフサイエンス / 構造生物化学

経歴

  • 2018年10月 - 2022年03月 科学技術振興機構 さきがけ 「量子技術を適用した生命科学基盤の創出」領域 研究者
  • 2022年01月 北海道大学 大学院先端生命科学研究院 教授
  • 2017年06月 - 2021年12月 北海道大学 大学院先端生命科学研究院 准教授
  • 2010年04月 - 2017年05月 北海道大学 大学院薬学研究院 准教授
  • 2008年04月 - 2010年03月 北海道大学 大学院先端生命科学研究院 特任助教
  • 2005年12月 - 2008年03月 HFSP long term fellow, University of Oxford Wellcome Trust Centre for Human Genetics
  • 2005年04月 - 2005年11月 JSPS SPD, 九州大学 生体防御医学研究所
  • 2004年04月 - 2005年03月 九州大学 生体防御医学研究所 博士研究員

学歴

  • 2000年04月 - 2003年03月   日本学術振興会 特別研究員(DC1)
  • 2000年04月 - 2003年03月   北海道大学   大学院理学研究科   生物科学専攻（博士後期課程）
  • 1998年04月 - 2000年03月   北海道大学   大学院理学研究院   生物科学専攻（博士前期課程）
  • 1995年04月 - 1998年03月   北海道大学   理学部   高分子機能学
  • 1994年04月 - 1995年03月   北海道大学   教養部   理II系

論文

  • Wataru Saburi, Takayoshi Tagami, Takuya Usui, Jian Yu, Toyoyuki Ose, Min Yao, Haruhide Mori
    Food Bioscience 61 104516 - 104516 2024年10月
  • Yuto Sasaki, Kodai Saitoh, Kota Kagohashi, Toyoyuki Ose, Shoya Kawahara, Yuichi Kitai, Ryuta Muromoto, Yuichi Sekine, Michiko Ichii, Akihiko Yoshimura, Kenji Oritani, Jun-ichi Kashiwakura, Tadashi Matsuda
    The Journal of Immunology 2023年07月07日 
    Abstract Signal-transducing adaptor protein-2 (STAP-2) is an adaptor protein that contains pleckstrin and Src homology 2–like domains, as well as a proline-rich region in its C-terminal region. Our previous study demonstrated that STAP-2 positively regulates TCR signaling by associating with TCR-proximal CD3ζ ITAMs and the lymphocyte-specific protein tyrosine kinase. In this study, we identify the STAP-2 interacting regions of CD3ζ ITAMs and show that the STAP-2–derived synthetic peptide (iSP2) directly interacts with the ITAM sequence and blocks the interactions between STAP-2 and CD3ζ ITAMs. Cell-penetrating iSP2 was delivered into human and murine T cells. iSP2 suppressed cell proliferation and TCR-induced IL-2 production. Importantly, iSP2 treatment suppressed TCR-mediated activation of naive CD4+ T cells and decreased immune responses in CD4+ T cell–mediated experimental autoimmune encephalomyelitis. It is likely that iSP2 is a novel immunomodulatory tool that modulates STAP-2-mediated activation of TCR signaling and represses the progression of autoimmune diseases.
  • Hang Wang, Xiaomei Sun, Wataru Saburi, Saki Hashiguchi, Jian Yu, Toyoyuki Ose, Haruhide Mori, Min Yao
    Acta crystallographica. Section D, Structural biology 79 Pt 7 585 - 595 2023年07月01日 
    Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of D-mannose and D-glucose, has recently been characterized to have potential for D-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME-D-glucitol and RsME(D254A)-D-glucitol]. RsME possesses the (α/α)6-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loopα7-α8). The RsME-D-glucitol structure showed that loopα7-α8 moves towards D-glucitol and closes the active pocket. Trp251 and Asp254 in loopα7-α8 are only conserved in MEs and interact with D-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)-D-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loopα7-α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.
  • Masato Ishizaka, Minghao Chen, Shun Narai, Yoshikazu Tanaka, Toyoyuki Ose, Masaki Horitani, Min Yao
    International Journal of Molecular Sciences 24 1 833 - 833 2023年01月03日 
    Iron–sulfur (Fe–S) clusters are essential cofactors for enzyme activity. These Fe–S clusters are present in structurally diverse forms, including [4Fe–4S] and [3Fe–4S]. Type-identification of the Fe–S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe–4S] and [3Fe–4S] clusters in particular is challenging because of their rapid transformation in response to oxidation–reduction events. In this study, we focused on the relationship between the Fe–S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe–4S]-TtuA, prepared [3Fe–4S]-TtuA by oxidizing [4Fe–4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe–S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe–4S]-TtuA spontaneously transforms into [4Fe–4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe–4S]-TtuA was inactive [3Fe–4S]-TtuA, its activity recovered to a significant level compared to [4Fe–4S]-TtuA after one hour, corresponding to an increase of [4Fe–4S]-TtuA in the solution. Our findings reveal that [3Fe–4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe–S cluster type used by the tRNA-thiolation enzyme.
  • Hajime Wakui, Yasuhiro Yokoi, Chieko Horidome, Toyoyuki Ose, Min Yao, Yoshikazu Tanaka, Hiroshi Hinou, Shin-Ichiro Nishimura
    RSC Chemical Biology 2023年 
    We unveil for the first time that pancreatic cancer cells (PANC-1) and secreted exosomes express MUC1 bearing cancer-relevant dynamic epitopes recognized specifically by an anti-MUC1 antibody (SN-131).
  • Taiga Maemoto, Yuichi Kitai, Haruka Shoji, Runa Takahashi, Shunsuke Yamada, Shiho Takei, Daiki Ito, Ryuta Muromoto, Jun-ichi Kashiwakura, Haruka Handa, Ari Hashimoto, Shigeru Hashimoto, Toyoyuki Ose, Kenji Oritani, Tadashi Matsuda
    Journal of Biological Chemistry, in press 299 1 102724 - 102724 2022年12月 
    Signal-transducing adaptor family member-2 (STAP-2) is an adaptor protein that regulates various intracellular signals. We previously demonstrated that STAP-2 binds to epidermal growth factor receptor (EGFR) and facilitates its stability and activation of EGFR signaling in prostate cancer cells. Inhibition of this interaction may be a promising direction for cancer treatment. Here, we found that 2D5 peptide, a STAP-2-derived peptide, blocked STAP-2-EGFR interactions and suppressed EGFR-mediated proliferation in several cancer cell lines. 2D5 peptide inhibited tumor growth of human prostate cancer cell line DU145 and human lung cancer cell line A549 in murine xenograft models. Additionally, we determined that EGFR signaling and its stability were decreased by 2D5 peptide treatment during EGF stimulation. In conclusion, our study shows that 2D5 peptide is a novel anti-cancer peptide that inhibits STAP-2-mediated activation of EGFR signaling and suppresses prostate and lung cancer progression.
  • Mizuho Kajikawa, Mizuki Hata, Maho Ishimura, Nanae Imaizumi, Minako Kimura, Kei Miyano, Toyoyuki Ose, Daisuke Asai, Satoshi Ishido, Taisei Kanamoto
    The Biochemical journal 479 20 2261 - 2278 2022年10月28日 
    Kaposi's sarcoma-associated herpesvirus (KSHV) is a carcinogenic virus that latently infects B cells and causes malignant tumors in immunocompromised patients. KSHV utilizes two viral E3 ubiquitin ligases, K3 and K5, in KSHV-infected cells to mediate the polyubiquitination-dependent down-regulation of several host membrane proteins involved in the immune system. Although K3 and K5 are members of the same family and have similar structural topologies, K3 and K5 have different substrate specificities. Hence, K5 may have a different substrate recognition mode than K3; however, the molecular basis of substrate recognition remains unclear. Here, we investigated the reason why human CD8α, which is known not to be a substrate for both K3 and K5, is not recognized by them, to obtain an understanding for molecular basis of substrate specificity. CD8α forms a disulfide-linked homodimer under experimental conditions to evaluate the viral ligase-mediated down-regulation. It is known that two interchain disulfide linkages in the stalk region between each CD8α monomer (Cys164-Cys164 and Cys181-Cys181) mediate homodimerization. When the interchain disulfide linkage of Cys181-Cys181 was eliminated, CD8α was down-regulated by K5 with a functional RING variant (RINGv) domain via polyubiquitination at the cytoplasmic tail. Aspartic acid, located at the stalk/transmembrane interface of CD8α, was essential for K5-mediated down-regulation of the CD8α mutant without a Cys181-Cys181 linkage. These results suggest that disulfide linkage near the stalk/transmembrane interface critically inhibits substrate targeting by K5. Accessibility to the extracellular juxtamembrane stalk region of membrane proteins may be important for substrate recognition by the viral ubiquitin ligase K5.
  • Kohei Yumoto, Tomoaki Arisaka, Kazuma Okada, Kyosuke Aoki, Toyoyuki Ose, Tatsunori Masatani, Makoto Sugiyama, Naoto Ito, Hideo Fukuhara, Katsumi Maenaka
    Viruses 13 11 2021年11月19日 
    Rabies has almost a 100% case-fatality rate and kills more than 59,000 people annually around the world. There is no established treatment for rabies. The rabies virus (RABV) expresses only the glycoprotein (RABVG) at the viral surface, and it is the target for the neutralizing antibodies. We previously established mouse monoclonal antibodies, 15-13 and 12-22, which showed neutralizing activity against the RABV, targeting the sequential and conformational epitopes on the RABVG, respectively. However, the molecular basis for the neutralizing activity of these antibodies is not yet fully understood. In this study, we evaluated the binding characteristics of the Fab fragments of the 15-13 and 12-22 antibodies. The recombinant RABVG protein, in prefusion form for the binding analysis, was prepared by the silkworm-baculovirus expression system. Biolayer interferometry (BLI) analysis indicated that the 15-13 Fab interacts with the RABVG, with a KD value at the nM level, and that the 12-22 Fab has a weaker binding affinity (KD ~ μM) with the RABVG compared to the 15-13 Fab. Furthermore, we determined the amino acid sequences of both the antibodies and the designed single-chain Fv fragments (scFvs) of the 15-13 and 12-22 antibodies as another potential biopharmaceutical for targeting rabies. The 15-13 and 12-22 scFvs were successfully prepared by the refolding method and were shown to interact with the RABVG at the nM level and the μM level of the KD, respectively. These binding characteristics were similar to that of each Fab. On the other hand, differential scanning fluorometry (DSF) revealed that the thermal stability of these scFvs decreases compared to their Fabs. While the improvement of the stability of scFvs will still be required, these results provide insights into the neutralizing activity and the potential therapeutic use of antibody fragments for RABV infection.
  • Aoi Sugiyama, Tomo Nomai, Xinxin Jiang, Miku Minami, Min Yao, Katsumi Maenaka, Naoto Ito, Paul R Gooley, Gregory W Moseley, Toyoyuki Ose
    Biochemical and biophysical research communications 529 2 507 - 512 2020年08月20日 [査読有り][通常論文]
     
    Lyssavirus P protein is a multifunctional protein that interacts with numerous host-cell proteins. The C-terminal domain (CTD) of P is important for inhibition of JAK-STAT signaling enabling the virus to evade host immunity. Several regions on the surface of rabies virus P are reported to interact with host factors. Among them, an extended, discrete hydrophobic patch of P CTD is notable. Although structures of P CTD of two strains of rabies virus, and of mokola virus have been solved, the structure of P CTD for Duvenhage virus, which is functionally divergent from these species for immune evasion function, is not known. Here, we analyze the structures of P CTD of Duvenhage and of a distinct rabies virus strain to gain further insight on the nature and potential function of the hydrophobic surface. Molecular contacts in crystals suggest that the hydrophobic patch is important to intermolecular interactions with other proteins, which differ between the lyssavirus species.
  • Yuma Nagano, Aoi Sugiyama, Madoka Kimoto, Takuya Wakahara, Yasuyo Noguchi, Xinxin Jiang, Shinya Saijo, Nobutaka Shimizu, Nana Yabuno, Min Yao, Paul R Gooley, Gregory W Moseley, Takashi Tadokoro, Katsumi Maenaka, Toyoyuki Ose
    Journal of virology 94 17 2020年08月17日 [査読有り][通常論文]
     
    Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV P gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3.IMPORTANCE To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.
  • Hiroki Kusaka, Shunsuke Kita, Takashi Tadokoro, Kouki Yoshida, Yoshiyuki Kasai, Harumi Niiyama, Yukari Fujimoto, Shinya Hanashima, Michio Murata, Shigeru Sugiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka
    Protein expression and purification 172 105631 - 105631 2020年08月 [査読有り][通常論文]
     
    CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
  • Hajime Wakui, Yoshikazu Tanaka, Toyoyuki Ose, Isamu Matsumoto, Koji Kato, Yao Min, Taro Tachibana, Masaharu Sato, Kentaro Naruchi, Fayna Garcia Martin, Hiroshi Hinou, Shin Ichiro Nishimura
    Chemical Science 11 19 4999 - 5006 2020年05月21日 [査読有り][通常論文]
     
    © The Royal Society of Chemistry 2020. Aberrantly truncated immatureO-glycosylation in proteins occurs in essentially all types of epithelial cancer cells, which was demonstrated to be a common feature of most adenocarcinomas and strongly associated with cancer proliferation and metastasis. Although extensive efforts have been made toward the development of anticancer antibodies targeting MUC1, one of the most studied mucins having cancer-relevant immatureO-glycans, no anti-MUC1 antibody recognises carbohydrates and the proximal MUC1 peptide region, concurrently. Here we present a general strategy that allows for the creation of antibodies interacting specifically with glycopeptidic neoepitopes by using homogeneous synthetic MUC1 glycopeptides designed for the streamlined process of immunization, antibody screening, three-dimensional structure analysis, epitope mapping and biochemical analysis. The X-ray crystal structure of the anti-MUC1 monoclonal antibody SN-101 complexed with the antigenic glycopeptide provides for the first time evidence that SN-101 recognises specifically the essential epitope by forming multiple hydrogen bonds both with the proximal peptide and GalNAc linked to the threonine residue, concurrently. Remarkably, the structure of the MUC1 glycopeptide in complex with SN-101 is identical to its solution NMR structure, an extended conformation induced by site-specific glycosylation. We demonstrate that this method accelerates dramatically the development of a new class of designated antibodies targeting a variety of “dynamic neoepitopes” elaborated by disease-specificO-glycosylation in the immunodominant mucin domains and mucin-like sequences found in intrinsically disordered regions of many proteins.
  • Kengo Hirao, Sophie Andrews, Kimiko Kuroki, Hiroki Kusaka, Takashi Tadokoro, Shunsuke Kita, Toyoyuki Ose, Sarah L Rowland-Jones, Katsumi Maenaka
    iScience 23 1 100758 - 100758 2020年01月24日 [査読有り][通常論文]
     
    The human immunodeficiency virus (HIV) accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2-infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The di-leucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2-mediated endocytosis for CD3. The highly conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of simian immunodeficiency virus (SIV) Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide insights into the distinct pathogenesis of HIV-2 infection.
  • Takashi Tadokoro, Mst Lubna Jahan, Yuri Ito, Maino Tahara, Surui Chen, Atsutoshi Imai, Natsumi Sugimura, Koki Yoshida, Mizuki Saito, Toyoyuki Ose, Takao Hashiguchi, Makoto Takeda, Hideo Fukuhara, Katsumi Maenaka
    The FEBS journal 287 1 145 - 159 2020年01月 [査読有り][通常論文]
     
    The measles virus (MV) is a major cause of childhood morbidity and mortality worldwide. We previously established a mouse monoclonal antibody, 2F4, which shows high neutralizing titers against eight different genotypes of MV. However, the molecular basis for the neutralizing activity of the 2F4 antibody remains incompletely understood. Here, we have evaluated the binding characteristics of a Fab fragment of the 2F4 antibody. Using the MV infectious assay, we demonstrated that 2F4 Fab inhibits viral entry via either of two cellular receptors, SLAM and Nectin4. Surface plasmon resonance (SPR) analysis of recombinant proteins indicated that 2F4 Fab interacts with MV hemagglutinin (MV-H) with a KD value at the nm level. Furthermore, we designed a single-chain Fv fragment of 2F4 antibody as another potential biopharmaceutical to target measles. The stable 2F4 scFv was successfully prepared by the refolding method and shown to interact with MV-H at the μm level. Like 2F4 Fab, scFv inhibited receptor binding and viral entry. This indicates that 2F4 mAb uses the receptor-binding site and/or a neighboring region as an epitope with high affinity. These results provide insight into the neutralizing activity and potential therapeutic use of antibody fragments for MV infection.
  • Kimiko Kuroki, Haruki Matsubara, Ryo Kanda, Naoyuki Miyashita, Mitsunori Shiroishi, Yuko Fukunaga, Jun Kamishikiryo, Atsushi Fukunaga, Hideo Fukuhara, Kaoru Hirose, Joan S Hunt, Yuji Sugita, Shunsuke Kita, Toyoyuki Ose, Katsumi Maenaka
    Journal of immunology (Baltimore, Md. : 1950) 203 12 3386 - 3394 2019年12月15日 [査読有り][通常論文]
     
    Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the β2-microglobulin (β2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized β2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with β2m, thus accounting for β2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.
  • Hossain MA, Larrous F, Rawlinson SM, Zhan J, Sethi A, Ibrahim Y, Aloi M, Lieu KG, Mok YF, Griffin MDW, Ito N, Ose T, Bourhy H, Moseley GW, Gooley PR
    Cell reports 29 7 1934 - 1945.e8 2019年11月 [査読有り][通常論文]
  • Hideo Fukuhara, Yuri Ito, Miyuki Sako, Mizuho Kajikawa, Koki Yoshida, Fumio Seki, Mwila Hilton Mwaba, Takao Hashiguchi, Masa-Aki Higashibata, Toyoyuki Ose, Kimiko Kuroki, Makoto Takeda, Katsumi Maenaka
    Viruses 11 8 2019年08月19日 [査読有り][通常論文]
     
    Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion.
  • Zhan J, Hossain MA, Sethi A, Ose T, Moseley GW, Gooley PR
    Biomolecular NMR assignments 13 1 5 - 8 2019年04月 [査読有り][通常論文]
  • Li L, Adachi M, Yu J, Kato K, Shinoda A, Ostermann A, Schrader TE, Ose T, Yao M
    Acta crystallographica. Section F, Structural biology communications 75 Pt 3 193 - 196 2019年03月 [査読有り][通常論文]
  • Narumi Shioi, Takashi Tadokoro, Seijiro Shioi, Yuki Okabe, Haruki Matsubara, Shunsuke Kita, Toyoyuki Ose, Kimiko Kuroki, Shigeyuki Terada, Katsumi Maenaka
    The Journal of biological chemistry 294 4 1250 - 1256 2019年01月25日 [査読有り][通常論文]
     
    Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal β-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.
  • Mizuho Kajikawa, Toyoyuki Ose, Yuko Fukunaga, Yuki Okabe, Naoki Matsumoto, Kento Yonezawa, Nobutaka Shimizu, Simon Kollnberger, Masanori Kasahara, Katsumi Maenaka
    Nature communications 9 1 4330 - 4330 2018年10月18日 [査読有り][通常論文]
     
    The MILL family, composed of MILL1 and MILL2, is a group of nonclassical MHC class I molecules that occur in some orders of mammals. It has been reported that mouse MILL2 is involved in wound healing; however, the molecular mechanisms remain unknown. Here, we determine the crystal structure of MILL2 at 2.15 Å resolution, revealing an organization similar to classical MHC class I. However, the α1-α2 domains are not tightly fixed on the α3-β2m domains, indicating unusual interdomain flexibility. The groove between the two helices in the α1-α2 domains is too narrow to permit ligand binding. Notably, an unusual basic patch on the α3 domain is involved in the binding to heparan sulfate which is essential for MILL2 interactions with fibroblasts. These findings suggest that MILL2 has a unique structural architecture and physiological role, with binding to heparan sulfate proteoglycans on fibroblasts possibly regulating cellular recruitment in biological events.
  • Yoichiro Fujioka, Shinya Nishide, Toyoyuki Ose, Tadaki Suzuki, Izumi Kato, Hideo Fukuhara, Mari Fujioka, Kosui Horiuchi, Aya O Satoh, Prabha Nepal, Sayaka Kashiwagi, Jing Wang, Mika Horiguchi, Yuko Sato, Sarad Paudel, Asuka Nanbo, Tadaaki Miyazaki, Hideki Hasegawa, Katsumi Maenaka, Yusuke Ohba
    Cell host & microbe 23 6 809 - 818 2018年06月13日 [査読有り][通常論文]
     
    Influenza A virus (IAV) infection is initiated by the attachment of the viral glycoprotein hemagglutinin (HA) to sialic acid on the host cell surface. However, the sialic acid-containing receptor crucial for IAV infection has remained unidentified. Here, we show that HA binds to the voltage-dependent Ca2+ channel Cav1.2 to trigger intracellular Ca2+ oscillations and subsequent IAV entry and replication. IAV entry was inhibited by Ca2+ channel blockers (CCBs) or by knockdown of Cav1.2. The CCB diltiazem also inhibited virus replication in vivo. Reintroduction of wild-type but not the glycosylation-deficient mutants of Cav1.2 restored Ca2+ oscillations and virus infection in Cav1.2-depleted cells, demonstrating the significance of Cav1.2 sialylation. Taken together, we identify Cav1.2 as a sialylated host cell surface receptor that binds HA and is critical for IAV entry.
  • Sasaki M, Anindita PD, Ito N, Sugiyama M, Carr M, Fukuhara H, Ose T, Maenaka K, Takada A, Hall WW, Orba Y, Sawa H
    The Journal of infectious diseases 217 11 1740 - 1749 2018年05月 [査読有り][通常論文]
     
    Rabies virus (RABV) is the causative agent of fatal neurological disease. Cellular attachment is the initial and essential step for viral infections. Although extensive studies have demonstrated that RABV uses various target cell molecules to mediate infection, no specific molecule has been identified as an attachment factor for RABV infection. Here we demonstrate that cellular heparan sulfate (HS) supports RABV adhesion and subsequent entry into target cells. Enzymatic removal of HS reduced cellular susceptibility to RABV infection, and heparin, a highly sulfated form of HS, blocked viral adhesion and infection. The direct binding between RABV glycoprotein and heparin was demonstrated, and this interaction was shown to require HS N- and 6-O-sulfation. We also revealed that basic amino acids in the ectodomain of RABV glycoprotein serve as major determinants for the RABV-HS interaction. Collectively, our study highlights a previously undescribed role of HS as an attachment factor for RABV infection.
  • Tanzawa T, Kato K, Girodat D, Ose T, Kumakura Y, Wieden HJ, Uchiumi T, Tanaka I, Yao M
    Nucleic acids research 46 6 3232 - 3244 2018年04月 [査読有り][通常論文]
  • 澤田 光平, 南 篤志, 久米田 博之, 斎尾 智英, 松丸 尊紀, 及川 英秋, 前仲 勝実, 尾瀬 農之
    天然有機化合物討論会講演要旨集 60 229-234  天然有機化合物討論会実行委員会 2018年 
    【背景】 ポリエーテル化合物は，海産微細藻由来のブレベトキシン，放線菌由来のモネンシン，ラサロシド，キジマイシンなどに代表されるように様々な生物が多種多様な骨格を持つポリエーテルを生産している。これら天然物は連続したエーテル環を分子骨格に持ち，生理活性において重要な役割を果たしている。例えば，ポリエーテル骨格による金属イオンのキレーション機構が知られており2, 3, エーテル環上の酸素原子が特定の金属イオンをキレートして電荷を包み込み，外側に疎水性領域の炭素骨格を向ける。これによって細胞内外のイオンの透過性が増加し，抗菌活性などを示す（イオノフォア）。ポリエーテル骨格としては，テトラヒドロフラン（THF）やテトラヒドロピラン（THP）のようなエーテル環が数珠玉状（モネンシンなど），もしくは梯子状（ブレベトキシンなど）に連結されたものである。こうしたポリエーテル系天然物の生合成経路，特に多くの不斉点を有するエーテル環の構築機構は，有機化学的にも非常に興味が持たれてきた。ポリエーテル骨格の構築機構は1983年に提唱されたCane-Celmer-Westley (CCW)モデル「ポリエン-ポリエポキシド仮説」（1）において，環化機構が統一的に説明された。このモデルでは，鎖状ポリオレフィン前駆体がエポキシ化され，生成したポリエポキシドが位置選択的なエポキシド開環反応によりポリエーテル骨格が構築される。 Scheme 1. Monensin B biosynthesis pathway catalyzed by MonBI and MonBII oligomer 私達は，Monensin生合成をモデルケースとしてポリエーテル骨格構築機構の解明に取り組んできた。多様性を決定づけるエーテル環の導入は，エポキシド加水分解酵素ホモログである環化酵素が担うことが，最近は広く知られてる。Monensinの場合，その骨格を構築するために3 回の5-exo 環化反応が必要である (Scheme 1)。しかしながら，モネンシン生合成遺伝子クラスター中には、環化酵素と相同性を持つ遺伝子がmonBI, monBII の2つしか存在しないため、この2つの環化酵素がどのように3 回の環化反応を触媒するかを解明することが，複雑なポリエーテル骨格構築メカニズムを一般化することと同義であると考えた。組換え発現させたMonBI，
  • Furukawa A, Kakita K, Yamada T, Ishizuka M, Sakamoto J, Hatori N, Maeda N, Ohsaka F, Saitoh T, Nomura T, Kuroki K, Nambu H, Arase H, Matsunaga S, Anada M, Ose T, Hashimoto S, Maenaka K
    The Journal of biological chemistry 292 51 21128 - 21136 2017年12月 [査読有り][通常論文]
     
    Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor alpha (PILR alpha) on immune cells. PILR alpha belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)- like family, members of which bind SA. PILR alpha is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILR alpha complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILR alpha binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILR alpha. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILR alpha complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILR alpha and for the rational design of herpes simplex virus-1 entry inhibitors.
  • Naoyoshi Maeda, Atsushi Furukawa, Kosuke Kakita, Masahiro Anada, Shunichi Hashimoto, Shigeki Matsunaga, Kimiko Kuroki, Toyoyuki Ose, Akihisa Kato, Jun Arii, Yasushi Kawaguchi, Hisashi Arase, Katsumi Maenaka
    BIOLOGICAL & PHARMACEUTICAL BULLETIN 39 11 1897 - 1902 2016年11月 [査読有り][通常論文]
     
    Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILR alpha) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRa were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.
  • Ami Takahashi, Kimiko Kuroki, Yuki Okabe, Yoshiyuki Kasai, Naoki Matsumoto, Chisato Yamada, Toshiyuki Takai, Toyoyuki Ose, Shigeyuki Kon, Tadashi Matsuda, Katsumi Maenaka
    HUMAN IMMUNOLOGY 77 9 754 - 759 2016年09月 [査読有り][通常論文]
     
    HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and beta 2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1 month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B. (C) 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
  • Yasuko Hirata, Hilde Brems, Mayu Suzuki, Mitsuhiro Kanamori, Masahiro Okada, Rimpei Morita, Isabel Llano-Rivas, Toyoyuki Ose, Ludwine Messiaen, Eric Legius, Akihiko Yoshimura
    JOURNAL OF BIOLOGICAL CHEMISTRY 291 7 3124 - 3134 2016年02月 [査読有り][通常論文]
     
    Constitutional heterozygous loss-of-function mutations in the SPRED1 gene cause a phenotype known as Legius syndrome, which consists of symptoms of multiple cafe-au-lait macules, axillary freckling, learning disabilities, and macrocephaly. Legius syndrome resembles a mild neurofibromatosis type 1 (NF1) phenotype. It has been demonstrated that SPRED1 functions as a negative regulator of the Ras-ERK pathway and interacts with neurofibromin, the NF1 gene product. However, the molecular details of this interaction and the effects of the mutations identified in Legius syndrome and NF1 on this interaction have not yet been investigated. In this study, using a yeast two-hybrid system and an immunoprecipitation assay in HEK293 cells, we found that the SPRED1 EVH1 domain interacts with the N-terminal 16 amino acids and the C-terminal 20 amino acids of the GTPase-activating protein (GAP)-related domain (GRD) of neurofibromin, which form two crossing -helix coils outside the GAP domain. These regions have been shown to be dispensable for GAP activity and are not present in p120(GAP). Several mutations in these N- and C-terminal regions of the GRD in NF1 patients and pathogenic missense mutations in the EVH1 domain of SPRED1 in Legius syndrome reduced the binding affinity between the EVH1 domain and the GRD. EVH1 domain mutations with reduced binding to the GRD also disrupted the ERK suppression activity of SPRED1. These data clearly demonstrate that SPRED1 inhibits the Ras-ERK pathway by recruiting neurofibromin to Ras through the EVH1-GRD interaction, and this study also provides molecular basis for the pathogenic mutations of NF1 and Legius syndrome.
  • Toyoyuki Ose, Azusa Oikawa, Yukiko Nakamura, Katsumi Maenaka, Yuya Higuchi, Yuki Satoh, Shiho Fujiwara, Makoto Demura, Teruo Sone, Masakatsu Kamiya
    JOURNAL OF BIOMOLECULAR NMR 63 2 229 - 235 2015年10月 [査読有り][通常論文]
  • Shunsuke Kita, Haruki Matsubara, Yoshiyuki Kasai, Takaharu Tamaoki, Yuki Okabe, Hideo Fukuhara, Jun Kamishikiryo, Elena Krayukhina, Susumu Uchiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka
    EUROPEAN JOURNAL OF IMMUNOLOGY 45 6 1605 - 1613 2015年06月 [査読有り][通常論文]
     
    Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended -sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.
  • Monika Srivastava, Guowen Duan, Nadia J. Kershaw, Vicki Athanasopoulos, Janet H. C. Yeo, Toyoyuki Ose, Desheng Hu, Simon H. J. Brown, Slobodan Jergic, Hardip R. Patel, Alvin Pratama, Sashika Richards, Anil Verma, E. Yvonne Jones, Vigo Heissmeyer, Thomas Preiss, Nicholas E. Dixon, Mark M. W. Chong, Jeffrey J. Babon, Carola G. Vinuesa
    NATURE COMMUNICATIONS 6 6253  2015年02月 [査読有り][通常論文]
     
    Roquin is an RNA-binding protein that prevents autoimmunity and inflammation via repression of bound target mRNAs such as inducible costimulator (Icos). When Roquin is absent or mutated (Roquin(san)), Icos is overexpressed in T cells. Here we show that Roquin enhances Dicer-mediated processing of pre-miR-146a. Roquin also directly binds Argonaute2, a central component of the RNA-induced silencing complex, and miR-146a, a microRNA that targets Icos mRNA. In the absence of functional Roquin, miR-146a accumulates in T cells. Its accumulation is not due to increased transcription or processing, rather due to enhanced stability of mature miR-146a. This is associated with decreased 3' end uridylation of the miRNA. Crystallographic studies reveal that Roquin contains a unique HEPN domain and identify the structural basis of the 'san' mutation and Roquin's ability to bind multiple RNAs. Roquin emerges as a protein that can bind Ago2, miRNAs and target mRNAs, to control homeostasis of both RNA species.
  • Teruo Sone, Yumiko Haraguchi, Aki Kuwahara, Toyoyuki Ose, Megumi Takano, Ayumi Abe, Michiko Tanaka, Isao Tanaka, Kozo Asano
    PROTEIN AND PEPTIDE LETTERS 22 1 63 - 72 2015年 [査読有り][通常論文]
     
    Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.
  • Aiping Zheng, Jian Yu, Reo Yamamoto, Toyoyuki Ose, Isao Tanaka, Min Yao
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 70 Pt 12 3090 - 3098 2014年12月 [査読有り][通常論文]
     
    eIF5B and eIF1A are two translation-initiation factors that are universally conserved among all kingdoms. They show a unique interaction in eukaryotes which is important for ribosomal subunit joining. Here, the structures of two isolated forms of yeast eIF5B and of the eIF5B-eIF1A complex (eIF1A and eIF5B do not contain the respective N-terminal domains) are reported. The eIF5B-eIF1A structure shows that the C-terminal tail of eIF1A binds to eIF5B domain IV, while the core domain of eIF1A is invisible in the electron-density map. Although the individual domains in all structures of eIF5B or archaeal IF5B (aIF5B) are similar, their domain arrangements are significantly different, indicating high structural flexibility, which is advantageous for conformational change during ribosomal subunit joining. Based on these structures, models of eIF5B, eIF1A and tRNA(i)(Met) on the 80S ribosome were built. The models suggest that the interaction between the eIF1A C-terminal tail and eIF5B helps tRNA(i)(Met) to bind to eIF5B domain IV, thus preventing tRNA(i)(Met) dissociation, stabilizing the interface for subunit joining and providing a checkpoint for correct ribosome assembly.
  • Kimiko Kuroki, Jing Wang, Toyoyuki Ose, Munechika Yamaguchi, Shigekazu Tabata, Nobuo Maita, Seiko Nakamura, Mizuho Kajikawa, Amane Kogure, Takeshi Satoh, Hisashi Arase, Katsumi Maenaka
    Proc. Natl. Acad. Sci. USA 111 24 8877 - 8882 2014年06月 [査読有り][通常論文]
     
    Paired Ig-like type 2 receptor a (PILR alpha) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILR alpha. Furthermore, we determined the crystal structures of PILR alpha and its complex with an sTn and its attached peptide region. The structures show that PILR alpha exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.
  • Yuki Satoh, Shinsuke Miki, Toyoyuki Ose, Azusa Oikawa, Katsumi Maenaka, Ryouhei Terauchi, Kozo Asano, Teruo Sone
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 4 680 - 686 2014年04月 [査読有り][通常論文]
     
    The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.
  • Atsushi Minami, Toyoyuki Ose, Kyohei Sato, Azusa Oikawa, Kimiko Kuroki, Katsumi Maenaka, Hiroki Oguri, Hideaki Oikawa
    ACS CHEMICAL BIOLOGY 9 2 562 - 569 2014年02月 [査読有り][通常論文]
     
    Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 X 10(-5) M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.
  • Atsuko Ibusuki, Kazuhiro Kawai, Shigeru Yoshida, Youhei Uchida, Ayano Nitahara-Takeuchi, Kimiko Kuroki, Mizuho Kajikawa, Toyoyuki Ose, Katsumi Maenaka, Masanori Kasahara and Takuro Kanekura
    J. Invest. Dermotol. 134 2 396 - 404 2014年02月 [査読有り][通常論文]
     
    Murine epidermal gamma delta T cells, known as dendritic epidermal T cells (DETCs), survey tissue stress through the invariant T-cell receptor (TCR) and non-clonotypic receptors such as NKG2D. NKG2D signaling via the DAP10-phosphatidylinositol 3-kinase (PI3K) pathway directly stimulates cytotoxicity in natural killer (NK) cells and costimulates CD8(+) T cells to augment TCR signals. In activated murine NK cells, NKG2D signals also via the DAP12-Syk/ZAP70 pathway that triggers both cytotoxicity and cytokine production. It remains controversial whether NKG2D on DETCs is a primary activating receptor or functions only as a costimulatory receptor, and signaling pathways initiated by NKG2D ligation in DETCs have not been analyzed. We show that stimulation of short-term DETC lines with recombinant NKG2D ligands triggers degranulation (exocytosis of cytotoxic granules) via the PI3K-dependent signaling pathway, but does not induce cytokine production or Syk/ZAP70 activation. Coengagement of TCR or Syk/ZAP70 signaling was not crucial for DETC-mediated killing of NKG2D ligand-expressing target cells. Thus, NKG2D can function as a coactivating stress receptor that directly triggers cytotoxicity in DETCs, at least after priming, via the PI3K-dependent, Syk/ZAP70-independent signaling pathway.
  • Ryo Kanda, Yoichi Sutoh, Jun Kasamatsu, Katsumi Maenaka, Masanori Kasahara, Toyoyuki Ose
    PLOS ONE 9 1 e85875  2014年01月 [査読有り][通常論文]
     
    Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to gamma delta T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.
  • Orapin Ariyawutthiphan, Toyoyuki Ose, Atsushi Minami, Sandip Shinde, Muneya Tsuda, Yong-Gui Gao, Min Yao, Hideaki Oikawa, Isao Tanaka
    Acta Crystallographica Section D: Biological Crystallography 69 12 2584  2013年12月 [査読有り][通常論文]
  • Atsushi Furukawa, Jun Kamishikiryo, Daiki Mori, Kenji Toyonaga, Yuki Okabe, Aya Toji, Ryo Kanda, Yasunobu Miyake, Toyoyuki Ose, Sho Yamasaki, Katsumi Maenaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 110 43 17438 - 17443 2013年10月 [査読有り][通常論文]
     
    Mincle [macrophage inducible Ca2+-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca2+-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.
  • Yuichi Yagita, Nozomi Kuse, Kimiko Kuroki, Hiroyuki Gatanaga, Jonathan M. Carlson, Takayuki Chikata, Zabrina L. Brumme, Hayato Murakoshi, Tomohiro Akahoshi, Nico Pfeifer, Simon Mallal, Mina John, Toyoyuki Ose, Haruki Matsubara, Ryo Kanda, Yuko Fukunaga, Kazutaka Honda, Yuka Kawashima, Yasuo Ariumi, Shinichi Oka, Katsumi Maenaka, Masafumi Takiguchi
    JOURNAL OF VIROLOGY 87 4 2253 - 2263 2013年02月 [査読有り][通常論文]
     
    Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.
  • Orapin Ariyawutthiphan, Toyoyuki Ose, Atsushi Minami, Sandip Sinde, Muneya Tsuda, Yong-Gui Gao, Min Yao, Hideaki Oikawa, Isao Tanaka
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 68 1558 - 1569 2012年11月 [査読有り][通常論文]
     
    In the typical isoprenoid-biosynthesis pathway, condensation of the universal C-5-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C-10-C-20). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.
  • Ryuta Arai, Masumi Tsuda, Takuya Watanabe, Toyoyuki Ose, Chikashi Obuse, Katsumi Maenaka, Akio Minami, Yusuke Ohba
    EUROPEAN JOURNAL OF CANCER 48 15 2417 - 2430 2012年10月 [査読有り][通常論文]
     
    Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo. (C) 2011 Elsevier Ltd. All rights reserved.
  • Takao Hashiguchi, Toyoyuki Ose, Marie Kubota, Nobuo Maita, Jun Kamishikiryo, Katsumi Maenaka, Yusuke Yanagi
    PROTEIN AND PEPTIDE LETTERS 19 4 468 - 473 2012年04月 [査読有り][通常論文]
     
    Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MV-H), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 angstrom to a final resolution of 3.15 angstrom. These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.
  • Akiko Kawaguchi, Toyoyuki Ose, Min Yao, Isao Tanaka
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS 67 1516 - 1518 2011年12月 [査読有り][通常論文]
     
    Many functions have been reported for the eukaryotic ribosomal protein L30e. L30e makes several inter-subunit and intra-subunit interactions with protein or RNA components of the 80S ribosome. Yeast L30e has been shown to bind to its own transcript to autoregulate expression at both the transcriptional and the translational levels. Furthermore, it has been reported that mammalian L30e is a component of the selenocysteine-incorporation machinery by binding to the selenocysteine-insertion sequence on mRNA. As high-resolution crystal structures of mammalian L30e are not available, the purification, crystallization and X-ray structure analysis of human L30e are presented here.
  • Kyohei Sato, Atsushi Minami, Toyoyuki Ose, Hiroki Oguri, Hideaki Oikawa
    TETRAHEDRON LETTERS 52 41 5277 - 5280 2011年10月 [査読有り][通常論文]
     
    Enzymatic epoxide-opening cascade is one of the key biosynthetic processes for constructing structurally diverse polyethers. Here we report the first in vitro analysis of the cyclization catalyzed by two epoxide hydrolases, MonBI and MonBII involved in monensin biosynthesis, using simple epoxy-alcohols and the unprecedented synergistic effect on the epoxide-opening activity between these epoxide hydrolases. (C) 2011 Elsevier Ltd. All rights reserved.
  • Takashi Saitoh, Mayumi Igura, Yusuke Miyazaki, Toyoyuki Ose, Nobuo Maita, Daisuke Kohda
    BIOCHEMISTRY 50 24 5487 - 5496 2011年06月 [査読有り][通常論文]
     
    Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The Tom20 protein, residing on the mitochondrial surface, recognizes the N-terminal presequences of precursor proteins. We previously determined the crystal structures of the Tom20 presequence complex. The successful crystallization involved tethering the presequence to Tom20 through an intermolecular disulfide bond with an optimized linker. In this work, we assessed the tethering method. The intermolecular disulfide bond was cleaved in crystal with a reducing agent. The pose (i.e., conformation and position) of the presequence was identical to the previously determined pose. In another experiment, a longer linker than the optimized length was used for the tethering. The perturbation of the tether changed the pose slightly, but the interaction mode was preserved. These results argue against the forced interaction of the presequence by its covalent attachment to Tom20. Second, as an alternative method referred to as "molecular stiffening", we introduced a disulfide bond within the presequence peptide to restrict the freedom of the peptide in the unbound states. One presequence analogue exhibited over 100-fold higher affinity than its linear counterpart and generated cocrystals with Tom20. One of the two crystallographic snapshots revealed a known pose previously determined by the tethering method, and the other snapshot depicted a new pose. These results confirmed and extended the dynamic, multiple bound state model of the Tom20-presequence interactions and also demonstrated the validity of the molecular tethering and stiffening techniques in studies of transient protein-peptide interactions.
  • Orapin Ariyawutthiphan, Toyoyuki Ose, Muneya Tsuda, YongGui Gao, Min Yao, Atsushi Minami, Hideaki Oikawa, Isao Tanaka
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 67 417 - 420 2011年03月 [査読有り][通常論文]
     
    The biosynthetic pathway of the off-flavour terpenoid alcohol 2-methylisoborneol (2-MIB) requires geranyl pyrophosphate methyltransferase (GPPMT) to methylate GPP before the cyclization reaction. GPPMT is the first example of an S-adenosyl-l-methionine-dependent methyltransferase that acts on general intermediates such as geranyl pyrophosphate and farnesyl pyrophosphate in isoprenoid biosynthetic pathways. In this study, recombinant GPPMT was overproduced, purified and crystallized in the absence and presence of cofactor, cofactor analogue and substrate. Well diffracting crystals of apo GPPMT containing one molecule in the asymmetric unit were obtained and the structure of this form was solved by the molecular-replacement method. Two crystal forms of the tertiary complex with GPP and sinefungin were also obtained. Structure analysis of these crystals is currently under way in order to understand the enzyme reaction mechanism.
  • Takao Hashiguchi, Toyoyuki Ose, Marie Kubota, Nobuo Maita, Jun Kamishikiryo, Katsumi Maenaka, Yusuke Yanagi
    NATURE STRUCTURAL & MOLECULAR BIOLOGY 18 2 135 - U191 2011年02月 [査読有り][通常論文]
     
    Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a beta-sheet of the membrane-distal ectodomain of SLAM using the side of its beta-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their beta-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.
  • Gai Zuoqi, Kitagawa Yumie, Yao Min, Ose Toyoyuki, Tanaka Yoshikazu, Tanaka Isao
    生物物理 51 S119 - S120 一般社団法人 日本生物物理学会 2011年
  • Toyoyuki Ose, Kimiko Kuroki, Masaaki Matsushima, Katsumi Maenaka, Izumi Kumagai
    JOURNAL OF BIOCHEMISTRY 146 5 651 - 657 2009年11月 [査読有り][通常論文]
     
    In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel -sheet, designated as a platform, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)(5). A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)(3) was bound in the ABC subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these platform residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.
  • Masaaki Sokabe, Toyoyuki Ose, Akiyoshi Nakamura, Keita Tokunaga, Osamu Nureki, Min Yao, Isao Tanaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 106 27 11028 - 11033 2009年07月 [査読有り][通常論文]
     
    Alanyl-tRNA synthetase (AlaRS) catalyzes synthesis of Ala-tRNA(Ala) and hydrolysis of mis-acylated Ser- and Gly-tRNA(Ala) at 2 different catalytic sites. Here, we describe the monomer structures of C-terminal truncated archaeal AlaRS, with both activation and editing domains in the apo form, in complex with an Ala-AMP analog, and in a high-resolution lysine-methylated form. The structures show docking of the editing domain to the activation domain opposite from the predicted tRNA-binding surface. Thus, the editing site is positioned > 35 angstrom from the activation site, prompting us to model 2 different tRNA complexes: one binding tRNA at the activation site, and the other binding tRNA at the editing site. Interestingly, a gel-shift assay also implies the presence of 2 types of tRNA complex with different mobility. These results suggest that tRNA translocation via a canonical CCA flipping is unlikely to occur in AlaRS. The structure also demonstrated the binding of zinc in the editing site, in which the specific coordination of zinc would be facilitated by a conserved GGQ motif, implying that the editing mechanism may not be the same as in ThrRS. As Asn-194 in eubacterial AlaRS important for Ser misactivation is replaced by Thr-213 in archaeal AlaRS, a different Ser accommodation mechanism is proposed.
  • Takashi Saitoh, Mayumi Igura, Takayuki Obita, Toyoyuki Ose, Rieko Kojima, Katsumi Maenaka, Toshiya Endo, Daisuke Kohda
    EMBO JOURNAL 26 22 4777 - 4787 2007年11月 [査読有り][通常論文]
     
    Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20 presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR N-15 relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.
  • Kaori Sasaki, Toyoyuki Ose, Naoaki Okamoto, Katsumi Maenaka, Taku Tanaka, Hisao Masai, Mihoko Saito, Tsuyoshi Shirai, Daisuke Kohda
    EMBO JOURNAL 26 10 2584 - 2593 2007年05月 [査読有り][通常論文]
     
    In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3' end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3'-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3'-OH is essential for the 3'-terminal recognition. In contrast, the direct interaction with 3'-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3' end. Thus, the N-terminal domain of PriA recognizes the 3'end base in a base-non-selective manner, in addition to the deoxyribose and 5'-side phosphodiester group, of the 3''-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.
  • Toyoyuki Ose, Nicolas Soler, Linda Rasubala, Kimiko Kuroki, Daisuke Kohda, Dominique Fourmy, Satoko Yoshizawa, Katsumi Maenaka
    STRUCTURE 15 5 577 - 586 2007年05月 [査読有り][通常論文]
     
    Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SeIB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SeIB-C). SeIB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SeIB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SeIB-RNA interaction is sequence independent, possibly reflecting SeIB-tRNA/-rRNA recognitions. Based on these data, the dynamic SeIB-ribosome-mRNA-tRNA interactions will be discussed.
  • Mitsunori Shiroishi, Mizuho Kajikawa, Kimiko Kuroki, Toyoyuki Ose, Daisuke Kohda, Katsumi Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 281 28 19536 - 19544 2006年07月 [査読有り][通常論文]
     
    Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and F alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.
  • M Shiroishi, K Kuroki, T Ose, L Rasubala, Shiratori, I, H Arase, K Tsumoto, Kumagai, I, D Kohda, K Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 281 15 10439 - 10447 2006年04月 [査読有り][通常論文]
     
    HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1: 2 (HLA-G dimer: receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.
  • Sasaki K, Ose T, Tanaka T, Mizukoshi T, Ishigaki T, Maenaka K, Masai H, Kohda D
    Biochimica et biophysica acta 1764 157 - 160 1 2006年01月 [査読有り][通常論文]
  • M Igura, T Ose, T Obita, C Sato, K Maenaka, T Endo, D Kohda
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 514 - 517 2005年05月 [査読有り][通常論文]
  • L Rasubala, D Fourmy, T Ose, D Kohda, K Maenaka, S Yoshizawa
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 296 - 298 2005年03月 [査読有り][通常論文]
  • M Kitamura, T Ose, M Okuyama, H Watanabe, M Yao, H Mori, A Kimura, Tanaka, I
    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 178 - 179 2005年02月 [査読有り][通常論文]
  • S Yoshizawa, L Rasubala, T Ose, D Kohda, D Fourmy, K Maenaka
    NATURE STRUCTURAL & MOLECULAR BIOLOGY 12 2 198 - 203 2005年02月 [査読有り][通常論文]
     
    In bacteria, incorporation of selenocysteine, the 21(st) amino acid, into proteins requires elongation factor SelB, which has the unusual property of binding to both transfer RNA (tRNA) and mRNA. SelB binds to an mRNA hairpin formed by the selenocysteine insertion sequence (SECIS) with extremely high specificity, the molecular basis of which has been unknown. We have determined the crystal structure of the mRNA-binding domain of SelB in complex with SECIS RNA at a resolution of 2.3 Angstrom. This is the first example of a complex between an RNA and a winged-helix (WH) domain, a motif found in many DNA-binding proteins and recently discovered in RNA-binding proteins. Notably, RNA binding does not induce a major conformational change in the WH motif. The structure reveals a new mode of RNA recognition with a geometry that allows the complex to wrap around the small ribosomal subunit.
  • S Shioi, T Ose, K Maenaka, M Shiroishi, Y Abe, D Kohda, T Katayama, T Ueda
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 326 4 766 - 776 2005年01月 [査読有り][通常論文]
     
    PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-Angstrom-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed. (C) 2004 Published by Elsevier Inc.
  • T Ose, T Fujie, M Yao, N Watanabe, Tanaka, I
    PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS 57 3 635 - 638 2004年11月 [査読有り][通常論文]
  • A Fujino, T Ose, M Yao, T Tokiwano, M Honma, N Watanabe, Tanaka, I
    JOURNAL OF MOLECULAR BIOLOGY 341 4 999 - 1013 2004年08月 [査読有り][通常論文]
     
    1-Aminocyclopropane-1-carboxylate deaminase (ACCD) is a pyridoxal 5'-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield alpha-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity. (C) 2004 Elsevier Ltd. All rights reserved.
  • T Ose, K Watanabe, M Yao, M Honma, H Oikawa, Tanaka, I
    ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 60 1187 - 1197 2004年07月 [査読有り][通常論文]
     
    Macrophomate synthase (MPS) is an enzyme that catalyzes an extraordinarily complex conversion reaction, including two decarboxylations, two carbon - carbon bond formations and a dehydration, to form the benzoate analogue macrophomate from a 2-pyrone derivative and oxalacetate. Of these reactions, the two carbon - carbon bond formations are especially noteworthy because previous experiments have indicated that they proceed via a Diels - Alder reaction, one of the most widely used reactions in organic synthesis. The structural evidence that MPS catalyzes an intermolecular Diels - Alder reaction has been reported recently [Ose et al. ( 2003), Nature ( London), 422, 185 - 189]. Interestingly, the tertiary structure as well as the quaternary structure of MPS are similar to those of 2-dehydro-3-deoxygalactarate (DDG) aldolase, a carbon - carbon bond-forming enzyme that catalyzes the reversible reaction of aldol condensation/cleavage. Here, the structure of MPS is described in detail and compared with that of DDG aldolase. Both enzymes have a (beta/alpha)(8)-barrel fold and are classified as belonging to the enolase superfamily based on their reaction strategy. The basic principles for carbon - carbon bond formation used by both MPS and DDG aldolase are the same with regard to trapping the enolate substrate and inducing subsequent reaction. The major differences in the active sites between these two enzymes are the recognition mechanisms of the second substrates, 2-pyrone and DDG, respectively.
  • 尾瀬 農之
    生物物理 44 S20  一般社団法人 日本生物物理学会 2004年
  • T Ose, A Fujino, M Yao, N Watanabe, M Honma, Tanaka, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 278 42 41069 - 41076 2003年10月 [査読有り][通常論文]
     
    The pyridoxal 5'-phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1) covalent-bonded external aldimine with the coenzyme in the K51T form and 2) the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys(51) residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.
  • T Ose, K Watanabe, T Mie, M Honma, H Watanabe, M Yao, H Oikawa, Tanaka, I
    NATURE 422 6928 185 - 189 2003年03月 [査読有り][通常論文]
     
    The Diels-Alder reaction, which forms a six-membered ring from an alkene (dienophile) and a 1,3-diene, is synthetically very useful for construction of cyclic products with high regio- and stereoselectivity under mild conditions(1). It has been applied to the synthesis of complex pharmaceutical and biologically active compounds(2). Although evidence(3-7) on natural Diels-Alderases has been accumulated in the biosynthesis of secondary metabolites(8), there has been no report on the structural details of the natural Diels-Alderases. The function and catalytic mechanism of the natural Diels-Alderase are of great interest owing to the diversity of molecular skeletons in natural Diels-Alder adducts(8). Here we present the 1.70 Angstrom resolution crystal structure of the natural Diels-Alderase, fungal macrophomate synthase (MPS)(3), in complex with pyruvate. The active site of the enzyme is large and hydrophobic, contributing amino acid residues that can hydrogen-bond to the substrate 2-pyrone. These data provide information on the catalytic mechanism of MPS, and suggest that the reaction proceeds via a large-scale structural reorganization of the product.
  • 藤野 愛子, 尾瀬 農之, 姚 閔, 本間 守, 田中 勲
    生物物理 41 S100  一般社団法人 日本生物物理学会 2001年
  • M Yao, T Ose, H Sugimoto, A Horiuchi, A Nakagawa, S Wakatsuki, D Yokoi, T Murakami, M Honma, Tanaka, I
    JOURNAL OF BIOLOGICAL CHEMISTRY 275 44 34557 - 34565 2000年11月 [査読有り][通常論文]
     
    The pyridoxal 5'-phosphate (PLP)-dependent enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) catalyzes a reaction that involves a ring opening of cyclopropanoid amino acid, yielding alpha -ketobutyrate and ammonia. Unlike other PUP-dependent enzymes, this enzyme has no alpha -hydrogen atom in the substrate. Thus, a unique mechanism for the bond cleavage is expected. The crystal structure of ACCD from Hansenula saturnus has been determined at 2.0 Angstrom resolution by the multiple wavelength anomalous diffraction method using mercury atoms as anomalous scatterers. The model was built on the electron density map, which was obtained by the density averaging of multiple crystal forms. The final model was refined to an R-factor of 22.5% and an R-free-factor of 26.8%. The ACCD folds into two domains, each of which has an open twisted alpha/beta structure similar to the beta -subunit of tryptophan synthase, However, in ACCD, unlike in other members of the beta family of PLP-dependent enzymes, PLP is buried deep in the molecule, The structure provides the first view of the catalytic center of the cyclopropane ring opening.
  • M Honma, T Murakami, T Ose, H Matsui, Tanaka, I
    BIOCHEMISTRY AND MOLECULAR BIOLOGY OF VITAMIN B6 AND PQQ-DEPENDENT PROTEINS 171 - 174 2000年 [査読有り][通常論文]
     
    1-Aminocyclopropane-1-carboxylate (ACC) deaminase was isolated from fungi Hansenula saturnus and Penicillium citricum, and their cDNA were analyzed. Amino acid sequences of the enzymes from H. saturnus and P. citrinum showed 61% and 52% of similarity respectively to that of a bacterial enzyme. Furthermore, the alignment of these sequences indicates their similarity to the tryptophan synthase beta -subunit. This became clearly evident when crystallographical analysis of ACC deaminase from H. saturnus was conducted. From the three-dimensional figure, three residues found near the PLP were chosen for preparation of the mutant enzymes.
  • 姚 閔, 杉本 宏, 尾瀬 農之, 堀内 篤, 中川 敦史, 田中 勲, 若槻 荘市, 横井 大輔, 邑上 農隆, 本間 守
    日本結晶学会誌 40 48 - 48 The Crystallographic Society of Japan 1998年

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年07月 -2026年03月 
    代表者 : 高野 義孝, 大木 進野, 尾瀬 農之
     
    本研究では、宿主特異性成立に関与する植物病原菌エフェクター（EPC1、EPC2、EPC3）の解析を起点に、エフェクターを介した植物病原菌の宿主特異性成立の分子基盤を解明することを目的とする。本年度は、まずエフェクターEPC1、EPC2、EPC3をベンサミアナタバコ葉に一過的に発現させ、その発現が同植物のPAMP誘導免疫に与える影響を調べた。その結果、EPC1、EPC2、EPC3いずれも、細菌のPAMP（flg22）が引き起こす活性酸素生成を抑制することを明らかにした。続いて、エピトープタグを付加した各EPCエフェクターをベンサミアナタバコ葉およびメロン子葉に一過的に発現させ、続いて免疫沈降解析を実施し、共沈降してくるタンパク質について、LC-MS/MS解析を実施し、標的因子候補のリスト化を完了した。また、EPC3の特定の領域について、その領域のみで機能が保持されることを明らかにし、さらにその領域を発現させた場合は、発現タンパク質が可溶化することを見出している。ウリ科作物への宿主特異性に関わるエフェクターを網羅的に同定するために、ウリ類炭疽病菌とウリ類炭疽病菌が属するオービクラクレード内の別種に対する比較オミクス解析を実施し、ウリ類炭疽病菌の分離株すべてにおいて存在し、他種では必ずしも存在しない遺伝子群、あるいは、ウリ類炭疽病菌において発現するが他種では必ずしも発現しない遺伝子群の選抜を完了した。さらに最近になり、EPC4と命名した新規のエフェクターの発見に成功している。EPC4は、EPC1、EPC2、EPC3と同様にウリ科作物への病原性には必要である一方、ベンサミアナタバコへの病原性には必須ではない。EPC4について、その一過的発現解析を実施した結果、EPC4がflg22が誘導する活性酸素生成を抑制することを見出している。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2022年06月 -2024年03月 
    代表者 : 久米田 博之, 姚 閔, 尾瀬 農之
     
    立体構造を形成しないIDPとして特に研究が進んでいる蛋白質は，高等生物シグナル伝達系や転写翻訳系に多く含まれ，他の蛋白質との相互作用時に過渡的部分構造形成をおこなうものも多い。一方で，ポリペプチド鎖の大部分がIDPで構成される酵素はこれまで報告が無い。天然に存在するポリエーテル化合物は，多様かつ強力な生物活性を示し，産業応用や細胞生物学への利用が多く見られる。これらのポリエーテル化合物は、テトラヒドロフラン環やテトラヒドロピラン環が多くの不斉点を介し連結されており，構造多様性はエーテル環の数や順序，大きさが決定している。ポリエーテル骨格の構築機構は，鎖状ポリオレフィン前駆体がエポキシ化され，引き続き位置選択的なエポキシド開環反応によりポリエーテル骨格が連鎖的に構築される。私達は，monensin生合成をモデルケースとしてポリエーテル骨格構築機構の解明に取り組んできた。Monensinの場合，その骨格を構築するために3 回の5-exo 酵素環化反応が必要である。これまでポリエーテル天然物生合成経路に存在するエポキシド開環エーテル環化酵素はIDP型の酵素であることを提唱し，構造形成機構や存在意義の解析を進めてきた。結晶構造解析，1H-15N HSQC (NMR), CD, DSC, Native-MSを組み合せ，モネンシン生合成経路中に存在する蛋白質のうち，１つは酵素活性を持たず，もう一方の構造形成を補助するシャペロン様分子であることを示すことができた。一般的にポリエーテル環化反応進行のためには，長大な基質を収納するポケットが必要であり，また，環化反応をうける基質のエポキシドおよび水酸基周辺の構造変化に対応できる柔軟な活性部位が必要である。我々のペア型酵素仮説を使用すると，酵素がこういった課題を解決するために採用している戦略を説明できる。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 尾瀬 農之, 久米田 博之, 杉田 征彦, 于 健
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 姚 閔, 尾瀬 農之
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2019年04月 -2023年03月 
    代表者 : 西村 紳一郎, 田中 良和, 比能 洋, 尾瀬 農之
     
    本研究課題は、申請者らの独創的な「動的エピトープを標的とする新薬開発」という発想と、既にその基盤が完成しており効率的でシームレスな抗体開発までを可能とする「糖鎖工学プラットフォームを活用する戦略」によって創出されたED抗体（epitope-defined antibody）を用いて、主としてがん領域におけるアンメットメディカルニーズに応え得る新たながん治療薬の開発を目的としている。 この研究課題においては多くのがん細胞表面に高発現してがんの増殖や転移の促進などに深く関与することが広く知られるムチンMUC1の細胞外タンデムリピートドメインに生成する多様な動的エピトープ（dynamic epitope）を特異的な標的とするED抗体である抗MUC1モノクローナル抗体の構造改変と低分子化によって高機能化することで新たな創薬モダリティーを創出することを具体的な目標として進めている。 2021年度（３年目）はMUC1-STを動的エピトープとするSN131のFab-MUC1-ST共結晶の構造解析に成功した。また、物理化学的・生化学的キャラクタリゼーションに加えて、すい臓がん細胞表面のMUC1との結合やSiglecなどとの相互作用などを多角的に解析した（論文投稿準備中）。SN131の改変型抗体であるSN132（動的エピトープとの結合領域を含むanti-parallel coiled-coil構造のFv-claspとリン脂質DPC膜結合性membrane-associated peptide領域を融合した分子量40K程度の全く新しいタイプの機能性抗体フラグメント）の作成法を確立した（特許出願準備中）。 また、SN131およびSN121のFv領域を、2種類のリンパ球を認識する抗体（OKT3およびL2K）のFv領域と融合させて新たに８種の二重特異性抗体の作成を進めており高活性を示す高性能な二重特異性抗体取得に向けて研究が進展している。
  • 生体分子中の窒素の特性を，中性子回折と計算化学により解明する
    日本学術振興会：科学研究費助成事業 国際共同研究加速基金(国際共同研究強化(B))
    研究期間 : 2019年02月 -2022年03月 
    代表者 : 尾瀬 農之, 齋藤 徹, 塚本 卓
     
    核酸やアミノ酸等の窒素化合物を代謝する過程においては，アミンのプロトタイプであるアンモニアが発生する。その反応性のために様々な細胞毒性を引き起こすアンモニアは，代謝や輸送をおこなう上で体内で注意して取り扱われなければならない。血液中に誤って放出されたアンモニアは，脳血液関門を容易に通過し脳障害をも引き起こす。陸上動物はふんだんに水を使用できないため，爬虫類・鳥類では尿酸として，両生類・哺乳類では尿素としてアンモニアを排出する系を備えている。これら排出回路を含め，アンモニアを扱う酵素には特別な設計がなされているおり，これの破綻は病気につながる。例えば，カルバモイルリン酸合成酵素(CPS)，シチジン３リン酸合成酵素(CTS)，グルタミン依存型アミドトランスフェラーゼ(Gat)を始めとし，アンモニアを反応中間体として利用する酵素は，求核性の高いアミンであるアンモニアが外部（細胞質）に漏れないよう，活性部位間がトンネルで結ばれている。トンネル内でアンモニアがどのように輸送されているかは，非常に興味深いトピックである。疎水性トンネルではアンモニアのまま輸送されていると予測される。また，親水性トンネルではプロトン化されていると予想されるが，アミンの性質を使用し，プロトン化と脱プロトン化を繰り返しながら輸送されているかもしれない。いずれにしても，Ｘ線結晶解析では水分子とアンモニア分子の違いを見分けることができないため，実験科学はデッドエンドになっていた。本提案では中性子線回折の利用方法をさらに進歩させ，窒素原子の持つ特徴を活かした構造生物学へと拡張する。アンモニアの分子内トンネルを使用した輸送機構は古細菌，真正細菌，真核生物に普遍的に見られる現象であるが，分子メカニズムの詳細は謎のままである。とりわけ，輸送に必要な水との協調作用を知る上では水分子と明確に区別する必要がある。
  • 生体分子中におけるアミンの量子特性を解明する
    JST：さきがけ
    研究期間 : 2018年10月 -2022年03月 
    代表者 : 尾瀬農之
  • 必要時に可逆的立体構造形成する新規ペア型エーテル環化酵素の解析と再設計による応用
    日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)
    研究期間 : 2019年04月 -2021年03月 
    代表者 : 尾瀬 農之
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 姚 閔, 尾瀬 農之, 田中 良和, 加藤 公児
     
    古細菌におけるCys-tRNA(Cys) の生合成は，2 つの酵素，SepRS とSepCysS が，それぞれ触媒する２段階の反応から成り立っている．申請者らは，この反応を推進するためには，第3のタンパク質SepCysE がSepRS とSepCysS をつないで3者複合体（transsulfursome）を形成することが必須であることを明らかにした．本研究では，そのtranssulfursome とtRNA から成る４者複合体の構造解析により，transsulfursomeが２つのリンカーを利用して動的なtRNAをプロセスする分子機構を明らかにする．そのために，H29年度に引き続き，H30年度に下記のように研究を進めた． H30年度に，transsulfursomeとtRNAの結合実験で確認しながら，各反応段階を反映するtranssulfursomeとtRNAの変異体作製を続き，精製できた第１段階反応状態を反映するtranssulfursome変異体SepRS-SepCysE(H)とtRNA複合体を結晶化し，初期結晶を得た．現在に，その結晶の条件最適化を行っている．また，transsulfursome-tRNA複合体のクライオ電子顕微鏡の測定について，当学科に既存の共用透過型電子顕微鏡（Hitachi社製 H7650）を用いて，硝酸ウランを用いたネガティブ染色法によりtranssulfursome-tRNA複合体の確認を行ったところで，tRNA結合状態のtranssulfursomeが単体より，まっすぐのような形が多かったということが分かった．さらに，放射光施設PFにて，凍結条件のスクリーニングを実施した．その結果，transsulfursome-tRNA複合体が沈殿しやすい，クライオ電子顕微鏡の測定に適切なグリッドの作製が困難であることが分かった．
  • JAK-STAT経路を不活化するため，ウイルスが採用する様々な戦略の分子機構解析
    日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 尾瀬 農之
     
    今回私達は, ITC測定を用いてMeV-VがSTAT1よりSTAT2に対して特異性が高いことを明らかにした。そこで，STAT2を主な標的としてType I IFNシグナル経路を阻害していると考えた。STAT2は, Type I IFNシグナル経路において, リン酸化, ヘテロ複合体化, 核移行, 転写活性化という過程を経る。この過程の中でMeV-VがSTAT2に干渉してシグナル伝達を阻害することを考えると, メカニズムとして リン酸化阻害， ヘテロ複合体化阻害，核移行阻害，転写活性化阻害が考えられた。 リン酸化阻害について, STAT2のリン酸化前後でMeV-Vの結合親和性が変化するか検証することを考え, 大腸菌TKB1でSTAT2を調製することを試みた。しかし, STAT1と同じ精製法でSTAT2を精製することはできなかった。また, リン酸化させたSTAT2は非常に凝集しやすい性質であった。バッファーや精製条件の検討により, リン酸化STAT2の調製を試みたいと考えている。したがってMeV-VのSTAT2に対する結合がリン酸化阻害に関わっているか不明であるが, Type I IFN経路阻害メカニズムとして現在多くの研究者に受け入れられているのは, MeV-VによるSTAT1やSTAT2のリン酸化阻害である（Chambers R. et al., 2009; Audsley M. D. et al., 2013）。私達が組換え調製したpY-STAT1を使ったITCによる相互作用解析では, MeV-Vとの相互作用が観測できなかった。これは, 非リン酸化STAT1の時に露出していた相互作用面がリン酸化に伴うコンホメーション変化によって隠れてしまったものと考えられ, MeV-Vはリン酸化を受ける前のSTAT1に対して特異的に結合する。
  • JAK-STAT経路を不活化するため，ウイルスが採用する様々な戦略の分子機構解析
    科学研究費補助金（基盤C）：
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 尾瀬農之
  • ４者複合体の構造解析によるtranssulfursomeのｔＲＮＡ変換機構の解明
    科学研究費補助金（基盤B）：
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 姚閔
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 尾瀬 農之
     
    Brk (Breast tumor kinase) は細胞内に存在する非受容体型チロシンキナーゼであるが，多くの乳がん細胞や卵巣がん細胞に発現が見られる。ヒト乳がん培養細胞において、STAP-2とBrk の存在によりSTAT3およびSTAT5が活性化され，細胞増殖が誘導される。そこで，この分子メカニズムを明らかにするため，それぞれのタンパク質を高純度調製し，生化学的・構造生物学的な手法により評価した．Brkによる自身のリン酸化部位特定やSTAP-2, Brk間の相互作用解析，溶液構造解析をおこない，調節メカニズムに対する考察をおこなった。
  • アダプタータンパク質が担う，乳がん細胞における転写因子STATの活性化基盤の解明
    科学研究費補助金（基盤C）：
    研究期間 : 2014年04月 -2017年03月 
    代表者 : 尾瀬農之
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2012年04月 -2014年03月 
    代表者 : 尾瀬 農之
     
    乳がん特異的チロシンキナーゼ(Brk) は，正常乳腺組織において発現していない一方，乳がん細胞の60%以上という高頻度で発現がみられる．特にtriple negative (エストロゲン受容体・プロゲステロン受容体・HER2の3つのマーカーが確認されない) の乳がんは予後不良であるが，ヒトTNBC由来の培養細胞および腫瘍組織でBrkの発現が確認されている．ヒト乳がん培養細胞において，STAP-2とBrk の存在によりSTAT3およびSTAT5bが活性化された結果、細胞増殖が誘導されるため，それぞれのタンパク質を高純度調製し，生化学的・構造生物学的な手法により評価した．
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2011年04月 -2014年03月 
    代表者 : 曾根 輝雄, 寺内 良平, 尾瀬 農之, 尾瀬 農之
     
    いもち病はイネの最重要病害である．いもち病の防除は主に農薬と抵抗性品種を使って行われている．しかし，抵抗性品種がどのようにしてその抵抗性を発揮するかについて，まだ完全に理解されていない．本研究はイネのいもち病抵抗性遺伝子Piaの抵抗性発現のメカニズムについて，いもち病菌のAVR-Pia遺伝子との相互作用を詳細に明らかにしようとしたものである．本研究の結果，いもち病菌はイネに侵入する前からAVR-Pia遺伝子を発現しはじめること，分泌後AVR-Piaタンパクは二量体を形成し，イネのPiaタンパク質と相互作用することがわかった．これらの知見は，いもち病抵抗性を理解するのに役立つものと期待される．
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2010年 -2011年 
    代表者 : 尾瀬 農之
     
    ヒト由来SBP2, L30, EFSec, SECIS-RNA, tRNASecの調製に成功し,生化学的実験を進めてきた. L30に関して結晶構造解析及びRNAとの結合実験をおなうことができ,論文発表した.また,機能的tRNASecの調製方法を確立することができたため,アミノ酸存在下においてEFSecやSBP2との相互作用研究を進めている.野生型では等温滴定型熱量計を用いてGDPとの相互作用解析を行い, K_d=229nMでの相互作用が見られ,調製したタンパク質は正しい構造をとっていることが確認できた.また作製した変異体では,二次構造を保持していることを確認した.
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2010年 -2011年 
    代表者 : 笠原 正典, 尾瀬 農之
     
    獲得免疫系と、それを支えるリンパ球は長い間、有顎類に固有であると考えられてきた。ところが、leucine-rich repeatモジュールを組み換えることにより10^<10>を超える多様性を生み出す抗原レセプターである可変性リンパ球レセプター(variable lymphocyte receptor、以下VLR)が無顎類ヤツメウナギで発見されるに及んで、この考えは根底から覆されるに至った。先に、われわれは、VLRにAとBが存在することを示したが、最近、VLRAとBはそれぞれT、B様のリンパ球に発現され、Aは膜結合型レセプターとして、Bは抗体として機能することが示された。以上から、無顎類の段階で、細胞性免疫、液性免疫を担うリンパ球のサブセットがすでに存在することが明らかになった。今回、我々は第3のVLRを発見し、それをVLRCと命名した。本年度の研究により、1)VLRCはVLA+細胞、VLRB+細胞とは別のリンパ球サブセットに発現されていること、2)VLRCは分泌されないこと、3)VLRCは配列的には、VLRBに対してよりVLRAに類似性が高いこと、4)まれに単一リンパ球においてVLRAとVLRCの両者の組み換えが起きるが、一方のみが機能的なVLRタンパク質をコードできることなどが明らかになった。おそらく、VLRC+細胞はVLRA+細胞と同様、T細胞系の細胞と想定される。したがって、有顎類に3種のリンパ球サブセット(B、αβT、γδT細胞)が存在するように、無顎類にも3種のリンパ球サブセットが存在し、そのうちの二つはT細胞様で、一つはB細胞様であることが示唆された。研究分担者はVLRCの結晶を得て、その構造解析に成功した。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2005年 -2005年 
    代表者 : 尾瀬 農之
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2000年 -2002年 
    代表者 : 尾瀬 農之
     
    炭素-炭素結合を協奏的に形成する反応を触媒する酵素(macrophomate synthase)の立体構造解析に成功した。得られた立体構造から、このステップはDiels-Alder反応の可能性が極めて高くなった。Diels-Alder反応は、シクロヘキセンを合成する有機化学上極めて重要な反応である。この反応を触媒する酵素の立体構造は世界初のものであり、Nature誌で発表した。活性部位は巧妙に設計されており、より効率的にDiels-Alder反応を進行させるための戦略を見ることができる。基質を導入した活性部位の構造及び、変異実験や反応中間体の絶対配置に関する情報を組み合わせ、1)より活性化した基質を用いること、2)水素結合によって反応性を大きく上昇させること、3)生成物阻害を回避する構造変化を伴うこと、4)柔軟性に富んだループ領域をもっていることなどを具体的に明らかにした。この活性部位は、新たな化合物を作るための、抗体触媒を設計する上で非常に重要な情報となるものである。 三員環アミノ酸ACC(1-aminocyclopropane-1-carboxylate)を分解できる特殊な酵素ACC deaminase(yACCD;酵母Hansenula saturunus由来)の結晶構造を、分解能2.0Åで精密化した。立体構造および構造情報から予測した反応機構を、J.Biol.Chem.誌上に発表した。この反応機構を検証するために変異体を9種類作製した。活性消失が確認された変異体のうち、K51T, Y295FとACCを結合させた複合体の結晶化に成功し、反応中間体構造を得た。反応中でビタミンB6は基質と結合し、回転することで反応に最適な立体環境を提供する。活性残基Lys51はACCのメチレン側鎖からプロトンを引き抜くモデルを提唱することができた。これはビタミンB6酵素では初のものであり、進化の多様性を表すモデルと言える。論文投稿中である。また、超高度好熱古細菌P.horikoshiiにおいてACCDとアサインされた遺伝子(PH0054)がコードする蛋白質の、ACC存在下での構造解析にも成功し、大きなドメイン変化が観察できた。ACCD活性の発現は当初の予想以上に厳密な環境制御を要している。論文投稿準備中である。

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