研究者情報

マスター

アカウント（マスター）

• 氏名

  前仲　勝実(マエナカ　カツミ), マエナカ　カツミ

所属（マスター）

• 薬学研究院　創薬科学部門　生体機能科学分野

所属（マスター）

• 薬学研究院　創薬科学部門　生体機能科学分野

researchmap

プロフィール情報

学位

• 博士(工学)（東京大学）

プロフィール情報

• 姓

  前仲, マエナカ

• 名

  勝実, カツミ

• ID各種

  200901070791806654

対象リソース

http://convallaria.pharm.hokudai.ac.jp/bunshi/

業績リスト

研究キーワード

• X線結晶構造解析   蛋白質間相互作用   免疫制御   蛋白質   細胞表面受容体   表面プラズモン共鳴   NMR解析   ペア型レセプター   分子認識   主要組織適合性抗原   ファージディスプレイ   LILR受容体   NMR   免疫系レセプター   翻訳   MILL   ペア型受容体   相互作用解析   CD160   NK細胞   巻き戻し   Ig-like receptor   T細胞レセプター   強直性脊椎炎   HIV   MHC様分子   セレノシステイン   KIR   リボソーム   伸長因子   蛋白工学   分子免疫学   構造生物学   Protein Engineering   Molecular Immunology   Structural Biology   

研究分野

• ライフサイエンス / 免疫学
• ライフサイエンス / 生物物理学
• ライフサイエンス / 構造生物化学

経歴

• 2016年02月 - 現在 オックスフォード大学 客員教授
• 2010年04月 - 現在 北海道大学 教授
• 2002年 - 2010年 九州大学 助教授、准教授
• 2000年 - 2002年 国立遺伝学研究所 助手
• 1997年 - 1999年 オックスフォード大学 ヒューマンフロンティアサイエンスプログラム博士研究員
• 1996年 - 1997年 日本学術振興会特別研究員

学歴

• 1992年 - 1996年   東京大学   大学院工学系研究科   化学生命工学専攻
• 1987年 - 1991年   東京大学   工学部   工業化学科

委員歴

• 2018年06月 - 現在   日本生物物理学会   代議員
• 2017年05月 - 現在   日本ケミカルバイオロジー学会   幹事
• 2017年04月 - 現在   日本薬学会   代議員
• 2015年08月 - 現在   International Structural Genomics Organization (ISGO)   Advisory Board Member
• 2014年06月 - 現在   日本生化学会   評議員
• 2011年04月 - 現在   日本蛋白質科学会   理事、執行役員
• 2010年06月 - 2021年06月   Frontiers in Virology   Associate Editor, Review Editor
• 2014年06月 - 2020年06月   Journal of Biochemistry   Editor, Associate Editor
• 2015年03月 - 2017年03月   日本生物物理学会北海道支部   支部長
• 2002年 - 2003年   日本生物物理学会   編集委員   日本生物物理学会

受賞

• 2015年06月 北海道大学 北海道大学総長賞研究奨励賞
   

  受賞者: 前仲 勝実
• 2014年06月 北海道大学 北海道大学総長賞教育奨励賞
   

  受賞者: 前仲 勝実
• 2008年12月 日本免疫学会 日本免疫学会研究奨励賞
   生体防御に関わる細胞表面受容体の分子認識機構 

  受賞者: 前仲 勝実

論文

• Protocol to identify the ligand binding site of Mincle using NMR spectroscopy.

  Atsushi Furukawa, Hiroyuki Kumeta, Takashi Saitoh, Katsumi Maenaka

  STAR protocols 5 2 102996 - 102996 2024年04月03日 

  Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor that can be targeted for inducing potent adjuvant effects. Mincle can recognize trehalose dimycolate and related glycolipids. Here, we present a protocol to identify the ligand binding mode of Mincle. We describe steps for preparing labeled Mincle ectodomain, data acquisition, and analysis of nuclear magnetic resonance experiments using non-detergent sulfobetaine-195. This protocol can be applied to other protein-ligand interactions that have aggregation problems for complex formation. For complete details on the use and execution of this protocol, please refer to Furukawa et al.1.
• A triggering structure of SARS-CoV-2 BA.2.86 spike upon ACE2 binding for receptor-binding domain up

  Takao Hashiguchi, Hisano Yajima, Yuki Anraku, Yu Kaku, Kanako Kimura, Arnon Plianchaisuk, Kaho Okumura, Yoshiko Nakada-Nakura, Shunsuke Kita, Jiei Sasaki, Hiromi Sumita, Jumpei Ito, Katsumi Maenaka, Kei Sato

  2024年03月22日
• Rational in silico design identifies two mutations that restore UT28K SARS-CoV-2 monoclonal antibody activity against Omicron BA.1.

  Tatsuhiko Ozawa, Yoshiki Ikeda, Liuan Chen, Rigel Suzuki, Atsushi Hoshino, Akira Noguchi, Shunsuke Kita, Yuki Anraku, Emiko Igarashi, Yumiko Saga, Noriko Inasaki, Shunta Taminishi, Jiei Sasaki, Yuhei Kirita, Hideo Fukuhara, Katsumi Maenaka, Takao Hashiguchi, Takasuke Fukuhara, Kenichi Hirabayashi, Hideki Tani, Hiroyuki Kishi, Hideki Niimi

  Structure (London, England : 1993) 32 3 263 - 272 2024年03月07日 

  SARS-CoV-2 rapidly mutates and acquires resistance to neutralizing antibodies. We report an in-silico-designed antibody that restores the neutralizing activity of a neutralizing antibody. Our previously generated antibody, UT28K, exhibited broad neutralizing activity against mutant variants; however, its efficacy against Omicron BA.1 was compromised by the mutation. Using previously determined structural information, we designed a modified-UT28K (VH T28R/N57D), UT28K-RD targeting the mutation site. In vitro and in vivo experiments demonstrated the efficacy of UT28K-RD in neutralizing Omicron BA.1. Although the experimentally determined structure partially differed from the predicted model, our study serves as a successful case of antibody design, wherein the predicted amino acid substitution enhanced the recognition of the previously elusive Omicron BA.1. We anticipate that numerous similar cases will be reported, showcasing the potential of this approach for improving protein-protein interactions. Our findings will contribute to the development of novel therapeutic strategies for highly mutable viruses, such as SARS-CoV-2.
• Virological characteristics of the SARS-CoV-2 Omicron XBB.1.5 variant.

  Tomokazu Tamura, Takashi Irie, Sayaka Deguchi, Hisano Yajima, Masumi Tsuda, Hesham Nasser, Keita Mizuma, Arnon Plianchaisuk, Saori Suzuki, Keiya Uriu, Mst Monira Begum, Ryo Shimizu, Michael Jonathan, Rigel Suzuki, Takashi Kondo, Hayato Ito, Akifumi Kamiyama, Kumiko Yoshimatsu, Maya Shofa, Rina Hashimoto, Yuki Anraku, Kanako Terakado Kimura, Shunsuke Kita, Jiei Sasaki, Kaori Sasaki-Tabata, Katsumi Maenaka, Naganori Nao, Lei Wang, Yoshitaka Oda, Terumasa Ikeda, Akatsuki Saito, Keita Matsuno, Jumpei Ito, Shinya Tanaka, Kei Sato, Takao Hashiguchi, Kazuo Takayama, Takasuke Fukuhara

  Nature communications 15 1 1176 - 1176 2024年02月08日 

  Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5.
• 2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses.

  Kentaro Uemura, Haruaki Nobori, Akihiko Sato, Shinsuke Toba, Shinji Kusakabe, Michihito Sasaki, Koshiro Tabata, Keita Matsuno, Naoyoshi Maeda, Shiori Ito, Mayu Tanaka, Yuki Anraku, Shunsuke Kita, Mayumi Ishii, Kayoko Kanamitsu, Yasuko Orba, Yoshiharu Matsuura, William W Hall, Hirofumi Sawa, Hiroshi Kida, Akira Matsuda, Katsumi Maenaka

  Proceedings of the National Academy of Sciences of the United States of America 120 42 e2304139120  2023年10月17日 

  Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are causing significant morbidity and mortality worldwide. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these or many other viruses have been approved, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral ribonucleoside analogue that exhibited antiviral activity against several positive-sense single-stranded RNA (ssRNA+) viruses, such as DENV, SARS-CoV-2, and its variants of concern, including the currently circulating Omicron subvariants. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with DENV2 or SARS-CoV-2 in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against DENV and SARS-CoV-2 but other ssRNA+ viruses.
• Beneficial effects of a new neuroprotective compound in neuronal cells and MPTP-administered mouse model of Parkinson's disease.

  Izumi Kato, Yudai Ogawa, Fumika Yakushiji, Jiro Ogura, Masaki Kobayashi, Naoya Shindo, Satoshi Ichikawa, Katsumi Maenaka, Masahiro Sakaitani

  Chemical communications (Cambridge, England) 59 82 12306 - 12309 2023年09月27日 

  A new compound, a derivative of 3,4,5-trimethoxy-N-phenyl benzamide bearing an 8''-methylimidazopyridine moiety, is found to demonstrate neuroprotective effects by preventing cell death caused by oxidative stress. The compound possesses high solubility and metabolic stability, and inhibits MPTP-induced effects in vivo, indicating high potential as a therapeutic drug for Parkinson's disease.
• Protein kinase Cβ is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages.

  Tsunehito Higashi, Haruka Handa, Yosuke Mai, Katsumi Maenaka, Takashi Tadokoro

  Journal of pharmacological sciences 153 1 22 - 25 2023年09月 

  Cigarette smoking is a risk factor for respiratory infection caused by immune cell dysfunction. Cigarette smoke is divided into tar and gas phases. Although the gas phase induces cell death in various cell types, the mechanism for gas phase-induced cell death remains to be clarified. In this study, we have examined the effects of cigarette smoke gas phase on J774 macrophages. Cigarette smoke gas phase and cytotoxic factors in the gas phase induced protein kinase C (PKC)-dependent ferroptosis. Pharmacological studies using isoform-specific PKC inhibitors have revealed that PKCβ is involved in cigarette smoke gas phase-induced ferroptosis in J774 macrophages.
• Structural delineation and computational design of SARS-CoV-2-neutralizing antibodies against Omicron subvariants.

  Saya Moriyama, Yuki Anraku, Shunta Taminishi, Yu Adachi, Daisuke Kuroda, Shunsuke Kita, Yusuke Higuchi, Yuhei Kirita, Ryutaro Kotaki, Keisuke Tonouchi, Kohei Yumoto, Tateki Suzuki, Taiyou Someya, Hideo Fukuhara, Yudai Kuroda, Tsukasa Yamamoto, Taishi Onodera, Shuetsu Fukushi, Ken Maeda, Fukumi Nakamura-Uchiyama, Takao Hashiguchi, Atsushi Hoshino, Katsumi Maenaka, Yoshimasa Takahashi

  Nature communications 14 1 4198 - 4198 2023年07月14日 

  SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with high mutation-resilience under escape mutation screening. A computationally designed antibody based on one of the Y489-binding antibodies, NIV-10/FD03, was able to bind XBB with any 486 mutation and neutralized XBB.1.5. The structural basis for the mutation-resilience of this Y489-binding antibody group may provide important insights into the design of therapeutics resistant to viral escape.
• Structural basis for plastic glycolipid recognition of the C-type lectin Mincle

  Atsushi Furukawa, Yusuke Shuchi, Jiaqi Wang, Pablo Adrian Guillen-Poza, Shigenari Ishizuka, Misuzu Kagoshima, Risa Ikeno, Hiroyuki Kumeta, Sho Yamasaki, Takanori Matsumaru, Takashi Saitoh, Katsumi Maenaka

  Structure 31 9 1077 - 1085 2023年06月 

  Mincle (macrophage-inducible C-type lectin, CLEC4E) is a C-type lectin immune-stimulatory receptor for cord factor, trehalose dimycolate (TDM), which serves as a potent component of adjuvants. The recognition of glycolipids by Mincle, especially their lipid parts, is poorly understood. Here, we performed nuclear magnetic resonance analysis, revealing that titration of trehalose harboring a linear short acyl chain showed a chemical shift perturbation of hydrophobic residues next to the Ca-binding site. Notably, there were split signals for Tyr201 upon complex formation, indicating two binding modes for the acyl chain. In addition, most Mincle residues close to the Ca-binding site showed no observable signals, suggesting their mobility on an ∼ ms scale even after complex formation. Mutagenesis study supported two putative lipid-binding modes for branched acyl-chain TDM binding. These results provide novel insights into the plastic-binding modes of Mincle toward a wide range of glycol- and glycerol-lipids, important for rational adjuvant development.
• Virological characteristics of the SARS-CoV-2 XBB variant derived from recombination of two Omicron subvariants.

  Tomokazu Tamura, Jumpei Ito, Keiya Uriu, Jiri Zahradnik, Izumi Kida, Yuki Anraku, Hesham Nasser, Maya Shofa, Yoshitaka Oda, Spyros Lytras, Naganori Nao, Yukari Itakura, Sayaka Deguchi, Rigel Suzuki, Lei Wang, Mst Monira Begum, Shunsuke Kita, Hisano Yajima, Jiei Sasaki, Kaori Sasaki-Tabata, Ryo Shimizu, Masumi Tsuda, Yusuke Kosugi, Shigeru Fujita, Lin Pan, Daniel Sauter, Kumiko Yoshimatsu, Saori Suzuki, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Yuki Yamamoto, Tetsuharu Nagamoto, Gideon Schreiber, Katsumi Maenaka, Takao Hashiguchi, Terumasa Ikeda, Takasuke Fukuhara, Akatsuki Saito, Shinya Tanaka, Keita Matsuno, Kazuo Takayama, Kei Sato

  Nature communications 14 1 2800 - 2800 2023年05月16日 

  In late 2022, SARS-CoV-2 Omicron subvariants have become highly diversified, and XBB is spreading rapidly around the world. Our phylogenetic analyses suggested that XBB emerged through the recombination of two cocirculating BA.2 lineages, BJ.1 and BM.1.1.1 (a progeny of BA.2.75), during the summer of 2022. XBB.1 is the variant most profoundly resistant to BA.2/5 breakthrough infection sera to date and is more fusogenic than BA.2.75. The recombination breakpoint is located in the receptor-binding domain of spike, and each region of the recombinant spike confers immune evasion and increases fusogenicity. We further provide the structural basis for the interaction between XBB.1 spike and human ACE2. Finally, the intrinsic pathogenicity of XBB.1 in male hamsters is comparable to or even lower than that of BA.2.75. Our multiscale investigation provides evidence suggesting that XBB is the first observed SARS-CoV-2 variant to increase its fitness through recombination rather than substitutions.
• An Electron-Deficient CpE Iridium(III) Catalyst: Synthesis, Characterization, and Application to Ether-Directed C-H Amidation.

  Eiki Tomita, Masahiro Kojima, Yuki Nagashima, Ken Tanaka, Haruki Sugiyama, Yasutomo Segawa, Atsushi Furukawa, Katsumi Maenaka, Satoshi Maeda, Tatsuhiko Yoshino, Shigeki Matsunaga

  Angewandte Chemie (International ed. in English) 62 21 e202301259  2023年03月14日 

  The synthesis, characterization, and catalytic performance of an iridium(III) catalyst with an electron-deficient cyclopentadienyl ligand ([CpEIrI2]2) are reported. The [CpEIrI2]2 catalyst was synthesized by complexation of a precursor of the CpE ligand with [Ir(cod)OAc]2, followed by oxidation, desilylation, and removal of the COD ligand. The electron-deficient [CpEIrI2]2 catalyst enabled C-H amidation reactions assisted by a weakly coordinating ether directing group. Experimental mechanistic studies and DFT calculations suggested that the high catalytic performance of [CpEIrI2]2 is due to its electron-deficient nature, which accelerates both C-H activation and Ir(V)-nitrenoid formation.
• Ready-to-Use-Type Lyophilized Lipid Nanoparticle Formulation for the Postencapsulation of Messenger RNA.

  Hiroki Tanaka, Shinya Hagiwara, Daiki Shirane, Takuma Yamakawa, Yuka Sato, Chika Matsumoto, Kota Ishizaki, Miho Hishinuma, Katsuyuki Chida, Kasumi Sasaki, Etsuo Yonemochi, Keisuke Ueda, Kenjirou Higashi, Kunikazu Moribe, Takashi Tadokoro, Katsumi Maenaka, Sakura Taneichi, Yuta Nakai, Kota Tange, Yu Sakurai, Hidetaka Akita

  ACS nano 17 3 2588 - 2601 2023年01月31日 

  Based on the clinical success of an in vitro transcribed mRNA (IVT-mRNA) that is encapsulated in lipid nanoparticles (mRNA-LNPs), there is a growing demand by researchers to test whether their own biological findings might be applicable for use in mRNA-based therapeutics. However, the equipment and/or know-how required for manufacturing such nanoparticles is often inaccessible. To encourage more innovation in mRNA therapeutics, a simple method for preparing mRNA-LNPs is prerequisite. In this study, we report on a method for encapsulating IVT-mRNA into LNPs by rehydrating a Ready-to-Use empty freeze-dried LNP (LNPs(RtoU)) formulation with IVT-mRNA solution followed by heating. The resulting mRNA-LNPs(RtoU) had a similar intraparticle structure compared to the mRNA-LNPs prepared by conventional microfluidic mixing. In vivo genome editing, a promising application of these types of mRNA-LNPs, was accomplished using the LNPs(RtoU) containing co-encapsulated Cas9-mRNA and a small guide RNA.
• Corrigendum: Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance(Front. Immunol., (2022), 13, (1008220), 10.3389/fimmu.2022.1008220)

  Mariko Shirane, Nobuyo Yawata, Daisuke Motooka, Kensuke Shibata, Seik Soon Khor, Yosuke Omae, Toshikatsu Kaburaki, Ryoji Yanai, Hisashi Mashimo, Satoshi Yamana, Takako Ito, Akira Hayashida, Yasuo Mori, Akihiko Numata, Yusuke Murakami, Kohta Fujiwara, Nobuyuki Ohguro, Mayumi Hosogai, Masato Akiyama, Eiichi Hasegawa, Michael Paley, Atsunobu Takeda, Katsumi Maenaka, Koichi Akashi, Wayne M. Yokoyama, Katsushi Tokunaga, Makoto Yawata, Koh Hei Sonoda

  Frontiers in Immunology 13 1124440 - 1124440 2023年01月05日 

  In the published article, there were errors. In the original article, there was an error in the description of the Reverse primer sequence in Materials and methods. A correction has been made to Materials and methods, “Deep amplicon sequencing of CMV-UL40 genomic DNA”. This sentence previously stated: “The following forward and reverse primers were used:Forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3′ and Reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACA GCTGGAACACGACGCATA-3’.”The corrected sentence appears below: “The following forward and reverse primers were used: Forward, 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCAACAGTCGGCAGAATGAAC-3′ and Reverse, 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGAC AGCTGGAACACGAGCGGACATA-3’.”In the original article, there was another error in the description of the concentration of the anti-HLA-E antibody in Materials and methods. A correction has been made to Materials and methods, “Immunohistochemistry analysis”. This sentence previously stated: “After antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1 h at room temperature to prevent nonspecific binding and stained with 10 mL/mL anti-HLA-E antibody [MEM-E/02] (Abcam; Cambridge, UK) or IgG from mouse serum (Sigma-Aldrich) overnight at 4°C.” The corrected sentence appears below: “After antigen retrieval with boiling citrate buffer (pH 6.0), the sections were incubated with 5% skim milk for 1 h at room temperature to prevent nonspecific binding and stained with 10 mg/mL anti-HLA-E antibody [MEM-E/02] (Abcam; Cambridge, UK) or IgG from mouse serum (Sigma-Aldrich) overnight at 4°C.” The authors apologize for these errors and state that these do not change the scientific conclusions of the article in any way. The original article has been updated.
• Structural delineation of SARS-CoV-2 broadly neutralizing antibody to Omicron subvariants including BA.4/BA.5 and BA.2.75

  Saya Moriyama, Yuki Anraku, Shunta Taminishi, Yu Adachi, Daisuke Kuroda, Shunsuke Kita, Yusuke Higuchi, Ryutaro Kotaki, Keisuke Tonouchi, Kohei Yumoto, Tateki Suzuki, Taiyou Someya, Hideo Fukuhara, Yudai Kuroda, Tsukasa Yamamoto, Taishi Onodera, Shuetsu Fukushi, Ken Maeda, Fukumi Nakamura-Uchiyama, Takao Hashiguchi, Atsushi Hoshino, Katsumi Maenaka, Yoshimasa Takahashi

  2022年12月21日 

  Abstract SARS-CoV-2 Omicron subvariants have evolved to evade receptor-binding site (RBS) antibodies that exist in diverse individuals as public antibody clones. We rationally selected RBS antibodies resilient to mutations in emerging Omicron subvariants. Y489 was identified as a site of virus vulnerability and a common footprint of broadly neutralizing antibodies against the subvariants. Multiple Y489-binding antibodies were encoded by public clonotypes and additionally recognized F486, potentially accounting for the emergence of Omicron subvariants harboring the F486V mutation. However, a subclass of antibodies broadly neutralized BA.4/BA.5 variants via hydrophobic binding sites of rare clonotypes along with extremely high mutation-resilience under escape mutation screening. The structural basis for mutation-resilience of this antibody group may inform the design of therapeutics resistant to viral escape.
• Dihydromaniwamycin E, a Heat-Shock Metabolite from Thermotolerant Streptomyces sp. JA74, Exhibiting Antiviral Activity against Influenza and SARS-CoV-2 Viruses.

  Shun Saito, Kayo Funayama, Wataru Kato, Mayu Okuda, Meiko Kawamoto, Teruhiko Matsubara, Toshinori Sato, Akihiko Sato, Satoko Otsuguro, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Katsumi Maenaka, Kazutoshi Shindo, Masaya Imoto, Midori A Arai

  Journal of natural products 85 11 2583 - 2591 2022年11月25日 

  Dihydromaniwamycin E (1), a new maniwamycin derivative featuring an azoxy moiety, has been isolated from the culture extract of thermotolerant Streptomyces sp. JA74 along with the known analogue maniwamycin E (2). Compound 1 is produced only by cultivation of strain JA74 at 45 °C, and this type of compound has been previously designated a "heat shock metabolite (HSM)" by our research group. Compound 2 is detected as a production-enhanced metabolite at high temperature. Structures of 1 and 2 are elucidated by NMR and MS spectroscopic analyses. The absolute structure of 1 is determined after the total synthesis of four stereoisomers. Though the absolute structure of 2 has been proposed to be the same as the structure of maniwamycin D, the NMR and the optical rotation value of 2 are in agreement with those of maniwamycin E. Therefore, this study proposes a structural revision of maniwamycins D and E. Compounds 1 and 2 show inhibitory activity against the influenza (H1N1) virus infection of MDCK cells, demonstrating IC50 values of 25.7 and 63.2 μM, respectively. Notably, 1 and 2 display antiviral activity against SARS-CoV-2, the causative agent of COVID-19, when used to infect 293TA and VeroE6T cells, with 1 and 2 showing IC50 values (for infection of 293TA cells) of 19.7 and 9.7 μM, respectively. The two compounds do not exhibit cytotoxicity in these cell lines at those IC50 concentrations.
• Virological characteristics of the SARS-CoV-2 Omicron BA.2.75 variant.

  Akatsuki Saito, Tomokazu Tamura, Jiri Zahradnik, Sayaka Deguchi, Koshiro Tabata, Yuki Anraku, Izumi Kimura, Jumpei Ito, Daichi Yamasoba, Hesham Nasser, Mako Toyoda, Kayoko Nagata, Keiya Uriu, Yusuke Kosugi, Shigeru Fujita, Maya Shofa, Mst Monira Begum, Ryo Shimizu, Yoshitaka Oda, Rigel Suzuki, Hayato Ito, Naganori Nao, Lei Wang, Masumi Tsuda, Kumiko Yoshimatsu, Jin Kuramochi, Shunsuke Kita, Kaori Sasaki-Tabata, Hideo Fukuhara, Katsumi Maenaka, Yuki Yamamoto, Tetsuharu Nagamoto, Hiroyuki Asakura, Mami Nagashima, Kenji Sadamasu, Kazuhisa Yoshimura, Takamasa Ueno, Gideon Schreiber, Akifumi Takaori-Kondo, Kotaro Shirakawa, Hirofumi Sawa, Takashi Irie, Takao Hashiguchi, Kazuo Takayama, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, Kei Sato

  Cell host & microbe 30 11 1540 - 1555 2022年11月09日 [査読無し]
   

  The SARS-CoV-2 Omicron BA.2.75 variant emerged in May 2022. BA.2.75 is a BA.2 descendant but is phylogenetically distinct from BA.5, the currently predominant BA.2 descendant. Here, we show that BA.2.75 has a greater effective reproduction number and different immunogenicity profile than BA.5. We determined the sensitivity of BA.2.75 to vaccinee and convalescent sera as well as a panel of clinically available antiviral drugs and antibodies. Antiviral drugs largely retained potency, but antibody sensitivity varied depending on several key BA.2.75-specific substitutions. The BA.2.75 spike exhibited a profoundly higher affinity for its human receptor, ACE2. Additionally, the fusogenicity, growth efficiency in human alveolar epithelial cells, and intrinsic pathogenicity in hamsters of BA.2.75 were greater than those of BA.2. Our multilevel investigations suggest that BA.2.75 acquired virological properties independent of BA.5, and the potential risk of BA.2.75 to global health is greater than that of BA.5.
• Genome-wide CRISPR screens identify CD48 defining susceptibility to NK cytotoxicity in peripheral T-cell lymphomas.

  Masahiro Chiba, Joji Shimono, Takashi Ishio, Norio Takei, Kohei Kasahara, Reiki Ogasawara, Takahide Ara, Hideki Goto, Koh Izumiyama, Satoko Otsuguro, Liyanage P Perera, Hiroo Hasegawa, Michiyuki Maeda, Satoshi Hashino, Katsumi Maenaka, Takanori Teshima, Thomas A Waldmann, Yibin Yang, Masao Nakagawa

  Blood 140 18 1951 - 1963 2022年11月03日 

  Adult T-cell leukemia/lymphoma (ATLL) is one of the aggressive peripheral T-cell neoplasms with a poor prognosis. Accumulating evidence demonstrates that escape from adaptive immunity is a hallmark of ATLL pathogenesis. However, the mechanisms by which ATLL cells evade natural killer (NK)-cell-mediated immunity have been poorly understood. Here we show that CD48 expression in ATLL cells determines the sensitivity for NK-cell-mediated cytotoxicity against ATLL cells. We performed unbiased genome-wide clustered regularly interspaced short palindromic repeat (CRISPR) screening using 2 ATLL-derived cell lines and discovered CD48 as one of the best-enriched genes whose knockout conferred resistance to YT1-NK cell line-mediated cytotoxicity. The ability of CD48-knockout ATLL cells to evade NK-cell effector function was confirmed using human primary NK cells with reduced interferon-γ (IFNγ) induction and degranulation. We found that primary ATLL cells had reduced CD48 expression along with disease progression. Furthermore, other subgroups among aggressive peripheral T-cell lymphomas (PTCLs) also expressed lower concentrations of CD48 than normal T cells, suggesting that CD48 is a key molecule in malignant T-cell evasion of NK-cell surveillance. Thus, this study demonstrates that CD48 expression is likely critical for malignant T-cell lymphoma cell regulation of NK-cell-mediated immunity and provides a rationale for future evaluation of CD48 as a molecular biomarker in NK-cell-associated immunotherapies.
• High-Throughput Screening Assay Identifies Berberine and Mubritinib as Neuroprotection Drugs for Spinal Cord Injury via Blood-Spinal Cord Barrier Protection.

  Yuki Suzuki, Shinsuke Nakagawa, Takeshi Endo, Akihito Sotome, Rufei Yuan, Tsuyoshi Asano, Satoko Otsuguro, Katsumi Maenaka, Norimasa Iwasaki, Ken Kadoya

  Neurotherapeutics : the journal of the American Society for Experimental NeuroTherapeutics 19 6 1976 - 1991 2022年10月 

  Because the breakdown of the blood-brain spinal cord barrier (BBSCB) worsens many central nervous system (CNS) diseases, prevention of BBSCB breakdown has been a major therapeutic target, especially for spinal cord injury (SCI). However, effective drugs that protect BBSCB function have yet to be developed. The purpose of the current study was 1) to develop a high-throughput screening assay (HTSA) to identify candidate drugs to protect BBSCB function, 2) to identify candidate drugs from existing drugs with newly developed HTSA, and 3) to examine the therapeutic effects of candidate drugs on SCI. Our HTSA included a culture of immortalized human brain endothelial cells primed with candidate drugs, stress with H2O2, and evaluation of their viability. A combination of the resazurin-based assay with 0.45 mM H2O2 qualified as a reliable HTSA. Screening of 1,570 existing drugs identified 90 drugs as hit drugs. Through a combination of reproducibility tests, exclusion of drugs inappropriate for clinical translation, and dose dependency tests, berberine, mubritinib, and pioglitazone were identified as a candidate. An in vitro BBSCB functional test revealed that berberine and mubritinib, but not pioglitazone, protected BBSCB from oxygen-glucose deprivation and reoxygenation stress. Additionally, these two drugs minimized BBSCB breakdown 1 day after cervical SCI in mice. Furthermore, berberine and mubritinib reduced neuronal loss and improved gait performance 8 weeks after SCI. Collectively, the current study established a useful HTSA to identify potential neuroprotective drugs by maintaining BBSCB function and demonstrated the neuroprotective effect of berberine and mubritinib after SCI.
• Hydrophobic Alpha-Helical Short Peptides in Overlapping Reading Frames of the Coronavirus Genome

  Takashi Okura, Kazuya Shirato, Masatoshi Kakizaki, Satoko Sugimoto, Shutoku Matsuyama, Tomohisa Tanaka, Yohei Kume, Mina Chishiki, Takashi Ono, Kohji Moriishi, Masashi Sonoyama, Mitsuaki Hosoya, Koichi Hashimoto, Katsumi Maenaka, Makoto Takeda

  Pathogens 11 8 877 - 877 2022年08月03日 

  In this study, we show that the coronavirus (CoV) genome may encode many functional hydrophobic alpha-helical peptides (HAHPs) in overlapping reading frames of major coronaviral proteins throughout the entire viral genome. These HAHPs can theoretically be expressed from non-canonical sub-genomic (sg)RNAs that are synthesized in substantial amounts in infected cells. We selected and analyzed five and six HAHPs encoded in the S gene regions of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Middle East respiratory syndrome coronavirus (MERS-CoV), respectively. Two and three HAHPs derived from SARS-CoV-2 and MERS-CoV, respectively, specifically interacted with both the SARS-CoV-2 and MERS-CoV S proteins and inhibited their membrane fusion activity. Furthermore, one of the SARS-CoV-2 HAHPs specifically inhibited viral RNA synthesis by accumulating at the site of viral RNA synthesis. Our data show that a group of HAHPs in the coronaviral genome potentially has a regulatory role in viral propagation.
• 免疫受容体LILRA2のANGPTL6認識機構(Molecular mechanism of ANGPTL6 recognition by immune activation receptor LILRA2)

  Wang Jiaqi, Furukawa Atsushi, Yamazaki Rika, Hirayasu Kouyuki, Kadomatsu Tsuyoshi, Oike Yuichi, Arase Hisashi, Maenaka Katsumi

  生物物理 62 Suppl.1-2 S269 - S269 2022年08月
• 構造解析に向けたヒト免疫不全ウイルス2(HIV-2)エンベロープ糖タンパク質の調製(Preparation of human immunodeficiency virus type-2(HIV-2) envelope glycoprotein for structure analysis)

  Anraku Yuki, Kita Shunsuke, Fukuhara Hideo, Kawabata Haruka, Akiyama Takaki, Davis Simon, Furukawa Atsushi, de Silva Thushan I., Robinson James E., Zhao Yuguang, Jones E. Yvonne, Stuart David, Huiskonen Juha T., Rowland-Jones Sarah, Maenaka Katsumi

  生物物理 62 Suppl.1-2 S350 - S350 2022年08月
• Structure of the human galanin receptor 2 bound to galanin and Gq reveals the basis of ligand specificity and how binding affects the G-protein interface.

  Yunseok Heo, Naito Ishimoto, Ye-Eun Jeon, Ji-Hye Yun, Mio Ohki, Yuki Anraku, Mina Sasaki, Shunsuke Kita, Hideo Fukuhara, Tatsuya Ikuta, Kouki Kawakami, Asuka Inoue, Katsumi Maenaka, Jeremy R H Tame, Weontae Lee, Sam-Yong Park

  PLoS biology 20 8 e3001714  2022年08月 

  Galanin is a neuropeptide expressed in the central and peripheral nervous systems, where it regulates various processes including neuroendocrine release, cognition, and nerve regeneration. Three G-protein coupled receptors (GPCRs) for galanin have been discovered, which is the focus of efforts to treat diseases including Alzheimer's disease, anxiety, and addiction. To understand the basis of the ligand preferences of the receptors and to assist structure-based drug design, we used cryo-electron microscopy (cryo-EM) to solve the molecular structure of GALR2 bound to galanin and a cognate heterotrimeric G-protein, providing a molecular view of the neuropeptide binding site. Mutant proteins were assayed to help reveal the basis of ligand specificity, and structural comparison between the activated GALR2 and inactive hβ2AR was used to relate galanin binding to the movements of transmembrane (TM) helices and the G-protein interface.
• Intraocular human cytomegaloviruses of ocular diseases are distinct from those of viremia and are capable of escaping from innate and adaptive immunity by exploiting HLA-E-mediated peripheral and central tolerance.

  Mariko Shirane, Nobuyo Yawata, Daisuke Motooka, Kensuke Shibata, Seik-Soon Khor, Yosuke Omae, Toshikatsu Kaburaki, Ryoji Yanai, Hisashi Mashimo, Satoshi Yamana, Takako Ito, Akira Hayashida, Yasuo Mori, Akihiko Numata, Yusuke Murakami, Kohta Fujiwara, Nobuyuki Ohguro, Mayumi Hosogai, Masato Akiyama, Eiichi Hasegawa, Michael Paley, Atsunobu Takeda, Katsumi Maenaka, Koichi Akashi, Wayne M Yokoyama, Katsushi Tokunaga, Makoto Yawata, Koh-Hei Sonoda

  Frontiers in immunology 13 1008220 - 1008220 2022年07月 

  Human cytomegalovirus (HCMV) infections develop into CMV diseases that result in various forms of manifestations in local organs. CMV-retinitis is a form of CMV disease that develops in immunocompromised hosts with CMV-viremia after viruses in the peripheral circulation have entered the eye. In the HCMV genome, extensive diversification of the UL40 gene has produced peptide sequences that modulate NK cell effector functions when loaded onto HLA-E and are subsequently recognized by the NKG2A and NKG2C receptors. Notably, some HCMV strains carry UL40 genes that encode peptide sequences identical to the signal peptide sequences of specific HLA-A and HLA-C allotypes, which enables these CMV strains to escape HLA-E-restricted CD8⁺T cell responses. Variations in UL40 sequences have been studied mainly in the peripheral blood of CMV-viremia cases. In this study, we sought to investigate how ocular CMV disease develops from CMV infections. CMV gene sequences were compared between the intraocular fluids and peripheral blood of 77 clinical cases. UL40 signal peptide sequences were more diverse, and multiple sequences were typically present in CMV-viremia blood compared to intraocular fluid. Significantly stronger NK cell suppression was induced by UL40-derived peptides from intraocular HCMV compared to those identified only in peripheral blood. HCMV present in intraocular fluids were limited to those carrying a UL40 peptide sequence corresponding to the leader peptide sequence of the host's HLA class I, while UL40-derived peptides from HCMV found only in the peripheral blood were disparate from any HLA class I allotype. Overall, our analyses of CMV-retinitis inferred that specific HCMV strains with UL40 signal sequences matching the host's HLA signal peptide sequences were those that crossed the blood-ocular barrier to enter the intraocular space. UL40 peptide repertoires were the same in the intraocular fluids of all ocular CMV diseases, regardless of host immune status, implying that virus type is likely to be a common determinant in ocular CMV disease development. We thus propose a mechanism for ocular CMV disease development, in which particular HCMV types in the blood exploit peripheral and central HLA-E-mediated tolerance mechanisms and, thus, escape the antivirus responses of both innate and adaptive immunity.
• Identification of RPL15 60S Ribosomal Protein as a Novel Topotecan Target Protein That Correlates with DAMP Secretion and Antitumor Immune Activation.

  Shunsuke Yamada, Yuichi Kitai, Takashi Tadokoro, Runa Takahashi, Haruka Shoji, Taiga Maemoto, Marie Ishiura, Ryuta Muromoto, Jun-Ichi Kashiwakura, Ken J Ishii, Katsumi Maenaka, Taro Kawai, Tadashi Matsuda

  Journal of immunology (Baltimore, Md. : 1950) 209 1 171 - 179 2022年06月20日 

  Damage-associated molecular patterns (DAMPs) contribute to antitumor immunity during cancer chemotherapy. We previously demonstrated that topotecan (TPT), a topoisomerase I inhibitor, induces DAMP secretion from cancer cells, which activates STING-mediated antitumor immune responses. However, how TPT induces DAMP secretion in cancer cells is yet to be elucidated. Here, we identified RPL15, a 60S ribosomal protein, as a novel TPT target and showed that TPT inhibited preribosomal subunit formation via its binding to RPL15, resulting in the induction of DAMP-mediated antitumor immune activation independent of TOP1. TPT inhibits RPL15-RPL4 interactions and decreases RPL4 stability, which is recovered by CDK12 activity. RPL15 knockdown induced DAMP secretion and increased the CTL population but decreased the regulatory T cell population in a B16-F10 murine melanoma model, which sensitized B16-F10 tumors against PD-1 blockade. Our study identified a novel TPT target protein and showed that ribosomal stress is a trigger of DAMP secretion, which contributes to antitumor immunotherapy.
• Novel super-neutralizing antibody UT28K is capable of protecting against infection from a wide variety of SARS-CoV-2 variants.

  Tatsuhiko Ozawa, Hideki Tani, Yuki Anraku, Shunsuke Kita, Emiko Igarashi, Yumiko Saga, Noriko Inasaki, Hitoshi Kawasuji, Hiroshi Yamada, So-Ichiro Sasaki, Mayu Somekawa, Jiei Sasaki, Yoshihiro Hayakawa, Yoshihiro Yamamoto, Yoshitomo Morinaga, Nobuyuki Kurosawa, Masaharu Isobe, Hideo Fukuhara, Katsumi Maenaka, Takao Hashiguchi, Hiroyuki Kishi, Isao Kitajima, Shigeru Saito, Hideki Niimi

  mAbs 14 1 2072455 - 2072455 2022年06月 

  Many potent neutralizing SARS-CoV-2 antibodies have been developed and used for therapies. However, the effectiveness of many antibodies has been reduced against recently emerging SARS-CoV-2 variants, especially the Omicron variant. We identified a highly potent SARS-CoV-2 neutralizing antibody, UT28K, in COVID-19 convalescent individuals who recovered from a severe condition. UT28K showed efficacy in neutralizing SARS-CoV-2 in an in vitro assay and in vivo prophylactic treatment, and the reactivity to the Omicron strain was reduced. The structural analyses revealed that antibody UT28K Fab and SARS-CoV-2 RBD protein interactions were mainly chain-dominated antigen-antibody interactions. In addition, a mutation analysis suggested that the emergence of a UT28K neutralization-resistant SARS-CoV-2 variant was unlikely, as this variant would likely lose its competitive advantage over circulating SARS-CoV-2. Our data suggest that UT28K offers potent protection against SARS-CoV-2, including newly emerging variants.
• Low-Cost Cell-Surface-Mimic Analysis of Ligand Interactions of Biotinylated Immune Receptors Using Surface Plasmon Resonance.

  Kimiko Kuroki, Hideo Fukuhara, Takashi Tadokoro, Katsumi Maenaka

  Methods in molecular biology (Clifton, N.J.) 2421 21 - 35 2022年06月 

  On the immune cell surface, many immune receptors are expressed and modulate the inhibitory or activating signals to control the immune responses. Recently, some of these receptors have been categorized as immune checkpoint receptors and targeted for cancer immunity or autoimmune diseases. To analyze the weak and fast binding typical for immune receptor-ligand interactions, a real-time surface plasmon resonance (SPR) technique is useful. However, it sometimes becomes difficult to optimize the immobilization conditions and appropriate controls. Considering that receptor orientation is relevant for achieving function on the cell surface, it is important to immobilize ligand proteins using specific tags at the membrane proximal end to avoid steric hindrance and structural changes in specific binding regions. Here we introduce a sensor chip, Sensor Chip CAP (Cytiva), which enables reversible and orientation-controlled immobilization of biotinylated ligands, resulting in a significant cost-effective method. We further show preparation methods of several biotinylated immune receptor proteins for SPR analysis, which are also useful for structural and other functional analyses.
• Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection.

  Takayuki Chikata, Wayne Paes, Nozomi Kuse, Thomas Partridge, Hiroyuki Gatanaga, Yu Zhang, Kimiko Kuroki, Katsumi Maenaka, Nicola Ternette, Shinichi Oka, Persephone Borrow, Masafumi Takiguchi

  Journal of virology 96 10 e0043222  2022年05月25日 

  There is increasing evidence for the importance of human leukocyte antigen C (HLA-C)-restricted CD8+ T cells in HIV-1 control, but these responses are relatively poorly investigated. The number of HLA-C-restricted HIV-1 epitopes identified is much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 nonprotective alleles. We identified 25 8- to 11-mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+/14:03+ cells. Analysis of T cell responses to these peptides identified novel 6 T cell epitopes targeted in HIV-1-infected HLA-C*14:02+/14:03+ subjects. Analyses using HLA stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell surface stabilization of HLA-C*14:02 than HLA-C*14:03. T cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals, with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ ones, the present study indicates that these HLA-C*14 subtypes differentially impact HIV-1 control by T cells and NK cells. IMPORTANCE Some human leukocyte antigen (HLA) class I alleles are associated with good clinical outcomes in HIV-1 infection and are called protective HLA alleles. Identification of T cell epitopes restricted by protective HLA alleles can give important insight into virus-immune system interactions and inform design of immune-based prophylactic/therapeutic strategies. Although epitopes restricted by many protective HLA-A/B alleles have been identified, protective HLA-C alleles are relatively understudied. Here, we identified 6 novel T cell epitopes presented by both HLA-C*14:02 (no association with protection) and HLA-C*14:03 (protective) using a mass spectrometry-based immunopeptidome profiling approach. We found that these peptides bound to and stabilized HLA-C*14:02 better than HLA-C*14:03 and observed differences in induction/expansion of epitope-specific T cell responses in HIV-infected HLA-C*14:02+ versus HLA-C*14:03+ individuals. These results enhance understanding of how the microstructural difference at position 21 between these HLA-C*14 subtypes may influence cellular immune responses involved in viral control in HIV-1 infection.
• Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity.

  Takumi Maruhashi, Daisuke Sugiura, Il-Mi Okazaki, Kenji Shimizu, Takeo K Maeda, Jun Ikubo, Harunori Yoshikawa, Katsumi Maenaka, Naozumi Ishimaru, Hidetaka Kosako, Tatsuya Takemoto, Taku Okazaki

  Immunity 55 5 912 - 924 2022年04月05日 

  Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity in vitro and in vivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced T cell suppression in vitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity in vitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.
• Application of Acoustic Ejection MS System to High-Throughput Screening for SARS-CoV-2 3CL Protease Inhibitors.

  Tsukasa Hasegawa, Riyo M Imamura, Tateki Suzuki, Takao Hashiguchi, Takao Nomura, Satoko Otsuguro, Katsumi Maenaka, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Akihiko Sato, Takayoshi Okabe, Tetsuo Nagano, Hirotatsu Kojima

  Chemical & pharmaceutical bulletin 70 3 199 - 201 2022年03月01日 

  MS is a powerful methodology for chemical screening to directly quantify substrates and products of enzymes, but its low throughput has been an issue. Recently, an acoustic liquid-handling apparatus (Echo®) used for rapid nano-dispensing has been coupled to a high-sensitivity mass spectrometer to create the Echo® MS system, and we applied this system to screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease inhibitors. Primary screening of 32033 chemical samples was completed in 12 h. Among the hits showing selective, dose-dependent 3CL-inhibitory activity, 8 compounds showed antiviral activity in cell-based assay.
• 5-Ethynylimidazole-4-carboxamide(EICA)ヌクレオチドプロドラッグの合成と抗デングウイルス活性

  日野谷直人, 中村元紀, 田良島典子, 大場靖子, 澤洋文, 松田彰, 前仲勝実, 南川典昭

  日本薬学会年会要旨集(Web) 70 3 220 - 225 2022年03月01日 

  We previously showed that 5-ethynyl-(1-β-D-ribofuranosyl)imidazole-4-carboxamide (1; EICAR) is a potent anti-dengue virus (DENV) compound but is cytotoxic to some cell lines, while its 4-thio derivative, 5-ethynyl-(4-thio-1-β-D-ribofuranosyl)imidazole-4-carboxamide (2; 4'-thioEICAR), has less cytotoxicity but also less anti-DENV activity. Based on the hypothesis that the lower anti-DENV activity of 2 is due to reduced susceptibility to phosphorylation by cellular kinase(s), we investigated whether a monophosphate prodrug of 2 can improve its activity. Here, we first prepared two types of prodrug of 1, which revealed that the S-acyl-2-thioethyl (SATE) prodrug had stronger anti-DENV activity than the aryloxyphosphoramidate (so-called ProTide) prodrug. Based on these findings, we next prepared the SATE prodrug of 4'-thioEICAR 18. As expected, the resulting 18 showed potent anti-DENV activity, which was comparable to that of 1; however, its cytotoxicity was also increased relative to 2. Our findings suggest that prodrugs of 4'-thioribonucleoside derivatives such as EICAR (1) represent an effective approach to developing potent biologically active compounds; however, the balance between antiviral activity and cytotoxicity remains to be addressed.
• Correction: Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity.

  Takanori Matsumaru, Risa Ikeno, Yusuke Shuchi, Toshiki Iwamatsu, Takashi Tadokoro, Sho Yamasaki, Yukari Fujimoto, Atsushi Furukawa, Katsumi Maenaka

  Chemical communications (Cambridge, England) 58 15 2580 - 2580 2022年02月17日 

  Correction for 'Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity' by Takanori Matsumaru et al., Chem. Commun., 2019, 55, 711-714, DOI: 10.1039/C8CC07322H.
• Characterization of Single-Chain Fv Fragments of Neutralizing Antibodies to Rabies Virus Glycoprotein.

  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.
• 5-Hydroxymethyltubercidin exhibits potent antiviral activity against flaviviruses and coronaviruses, including SARS-CoV-2.

  Kentaro Uemura, Haruaki Nobori, Akihiko Sato, Takao Sanaki, Shinsuke Toba, Michihito Sasaki, Akiho Murai, Noriko Saito-Tarashima, Noriaki Minakawa, Yasuko Orba, Hiroaki Kariwa, William W Hall, Hirofumi Sawa, Akira Matsuda, Katsumi Maenaka

  iScience 24 10 103120 - 103120 2021年10月22日 

  Newly emerging or re-emerging viral infections continue to cause significant morbidity and mortality every year worldwide, resulting in serious effects on both health and the global economy. Despite significant drug discovery research against dengue viruses (DENVs) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), no fully effective and specific drugs directed against these viruses have been discovered. Here, we examined the anti-DENV activity of tubercidin derivatives from a compound library from Hokkaido University and demonstrated that 5-hydroxymethyltubercidin (HMTU, HUP1108) possessed both potent anti-flavivirus and anti-coronavirus activities at submicromolar levels without significant cytotoxicity. Furthermore, HMTU inhibited viral RNA replication and specifically inhibited replication at the late stages of the SARS-CoV-2 infection process. Finally, we demonstrated that HMTU 5'-triphosphate inhibited RNA extension catalyzed by the viral RNA-dependent RNA polymerase. Our findings suggest that HMTU has the potential of serving as a lead compound for the development of a broad spectrum of antiviral agents, including SARS-CoV-2.
• A SARS-CoV-2 Antibody Broadly Neutralizes SARS-related Coronaviruses and Variants by Coordinated Recognition of a Virus Vulnerable Site

  Taishi Onodera, Shunsuke Kita, Yu Adachi, Saya Moriyama, Akihiko Sato, Takao Nomura, Shuhei Sakakibara, Takeshi Inoue, Takashi Tadokoro, Yuki Anraku, Kohei Yumoto, Cong Tian, Hideo Fukuhara, Michihito Sasaki, Yasuko Orba, Nozomi Shiwa, Naoko Iwata, Noriyo Nagata, Tateki Suzuki, Jiei Sasaki, Tsuyoshi Sekizuka, Keisuke Tonouchi, Lin Sun, Shuetsu Fukushi, Hiroyuki Satofuka, Yasuhiro Kazuki, Mitsuo Oshimura, Tomohiro Kurosaki, Makoto Kuroda, Yoshiharu Matsuura, Tadaki Suzuki, Hirofumi Sawa, Takao Hashiguchi, Katsumi Maenaka, Yoshimasa Takahashi

  Immunity 54 10 2385 - 2398.e10 2021年08月 

  Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.
• Potential anti-COVID-19 agents, cepharanthine and nelfinavir, and their usage for combination treatment.

  Hirofumi Ohashi, Koichi Watashi, Wakana Saso, Kaho Shionoya, Shoya Iwanami, Takatsugu Hirokawa, Tsuyoshi Shirai, Shigehiko Kanaya, Yusuke Ito, Kwang Su Kim, Takao Nomura, Tateki Suzuki, Kazane Nishioka, Shuji Ando, Keisuke Ejima, Yoshiki Koizumi, Tomohiro Tanaka, Shin Aoki, Kouji Kuramochi, Tadaki Suzuki, Takao Hashiguchi, Katsumi Maenaka, Tetsuro Matano, Masamichi Muramatsu, Masayuki Saijo, Kazuyuki Aihara, Shingo Iwami, Makoto Takeda, Jane A McKeating, Takaji Wakita

  iScience 24 4 102367 - 102367 2021年04月23日 

  Antiviral treatments targeting the coronavirus disease 2019 are urgently required. We screened a panel of already approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new agents having higher antiviral potentials than the drug candidates such as remdesivir and chroloquine in VeroE6/TMPRSS2 cells: the anti-inflammatory drug cepharanthine and human immunodeficiency virus protease inhibitor nelfinavir. Cepharanthine inhibited SARS-CoV-2 entry through the blocking of viral binding to target cells, while nelfinavir suppressed viral replication partly by protease inhibition. Consistent with their different modes of action, synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation was highlighted. Mathematical modeling in vitro antiviral activity coupled with the calculated total drug concentrations in the lung predicts that nelfinavir will shorten the period until viral clearance by 4.9 days and the combining cepharanthine/nelfinavir enhanced their predicted efficacy. These results warrant further evaluation of the potential anti-SARS-CoV-2 activity of cepharanthine and nelfinavir.
• Nickel‐Catalyzed Acyl Group Transfer of o‐ Alkynylphenol Esters Accompanied by C−O Bond Fission for Synthesis of Benzo[ b ]furan

  Ryohei Doi, Koji Shimizu, Yuma Ikemoto, Masashi Uchiyama, Mikiko Koshiba, Atsushi Furukawa, Katsumi Maenaka, Satoshi Watanabe, Yoshihiro Sato

  ChemCatChem 13 8 2086 - 2092 2021年04月21日
• Structure, solubility, and permeability relationships in a diverse middle molecule library.

  Hiroyuki Miyachi, Kayoko Kanamitsu, Mayumi Ishii, Eri Watanabe, Akira Katsuyama, Satoko Otsuguro, Fumika Yakushiji, Mizuki Watanabe, Kouhei Matsui, Yukina Sato, Satoshi Shuto, Takashi Tadokoro, Shunsuke Kita, Takanori Matsumaru, Akira Matsuda, Tomoyasu Hirose, Masato Iwatsuki, Yasuteru Shigeta, Tetsuo Nagano, Hirotatsu Kojima, Satoshi Ichikawa, Toshiaki Sunazuka, Katsumi Maenaka

  Bioorganic & medicinal chemistry letters 37 127847 - 127847 2021年04月01日 

  To develop methodology to predict the potential druggability of middle molecules, we examined the structure, solubility, and permeability relationships of a diverse library (HKDL ver.1) consisting of 510 molecules (359 natural product derivatives, 76 non-natural products, 46 natural products, and 29 non-natural product derivatives). The library included peptides, depsipeptides, macrolides, and lignans, and 476 of the 510 compounds had a molecular weight in the range of 500-2000 Da. The solubility and passive diffusion velocity of the middle molecules were assessed using the parallel artificial membrane permeability assay (PAMPA). Quantitative values of solubility of 471 molecules and passive diffusion velocity of 287 molecules were obtained, and their correlations with the structural features of the molecules were examined. Based on the results, we propose a method to predict the passive diffusion characteristics of middle molecules from their three-dimensional structural features.
• The physiological TMPRSS2 inhibitor HAI-2 alleviates SARS-CoV-2 infection.

  Yuriko Tomita, Shutoku Matsuyama, Hideo Fukuhara, Katsumi Maenaka, Hiroaki Kataoka, Takao Hashiguchi, Makoto Takeda

  Journal of virology 95 12 2021年03月31日 

  The largest disease pandemic in modern human history caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is still ongoing.….
• Canonical versus non-canonical transsynaptic signaling of neuroligin 3 tunes development of sociality in mice.

  Tomoyuki Yoshida, Atsushi Yamagata, Ayako Imai, Juhyon Kim, Hironori Izumi, Shogo Nakashima, Tomoko Shiroshima, Asami Maeda, Shiho Iwasawa-Okamoto, Kenji Azechi, Fumina Osaka, Takashi Saitoh, Katsumi Maenaka, Takashi Shimada, Yuko Fukata, Masaki Fukata, Jumpei Matsumoto, Hisao Nishijo, Keizo Takao, Shinji Tanaka, Shigeo Okabe, Katsuhiko Tabuchi, Takeshi Uemura, Masayoshi Mishina, Hisashi Mori, Shuya Fukai

  Nature communications 12 1 1848 - 1848 2021年03月23日 [査読無し]
   

  Neuroligin 3 (NLGN3) and neurexins (NRXNs) constitute a canonical transsynaptic cell-adhesion pair, which has been implicated in autism. In autism spectrum disorder (ASD) development of sociality can be impaired. However, the molecular mechanism underlying NLGN3-mediated social development is unclear. Here, we identify non-canonical interactions between NLGN3 and protein tyrosine phosphatase δ (PTPδ) splice variants, competing with NRXN binding. NLGN3-PTPδ complex structure revealed a splicing-dependent interaction mode and competition mechanism between PTPδ and NRXNs. Mice carrying a NLGN3 mutation that selectively impairs NLGN3-NRXN interaction show increased sociability, whereas mice where the NLGN3-PTPδ interaction is impaired exhibit impaired social behavior and enhanced motor learning, with imbalance in excitatory/inhibitory synaptic protein expressions, as reported in the Nlgn3 R451C autism model. At neuronal level, the autism-related Nlgn3 R451C mutation causes selective impairment in the non-canonical pathway. Our findings suggest that canonical and non-canonical NLGN3 pathways compete and regulate the development of sociality.
• MRC5 cells engineered to express ACE2 serve as a model system for the discovery of antivirals targeting SARS-CoV-2.

  Kentaro Uemura, Michihito Sasaki, Takao Sanaki, Shinsuke Toba, Yoshimasa Takahashi, Yasuko Orba, William W Hall, Katsumi Maenaka, Hirofumi Sawa, Akihiko Sato

  Scientific reports 11 1 5376 - 5376 2021年03月08日 

  Although the spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has resulted in a worldwide pandemic, there are currently no virus-specific drugs that are fully effective against SARS-CoV-2. Only a limited number of human-derived cells are capable of supporting SARS-CoV-2 replication and the infectivity of SARS-CoV-2 in these cells remains poor. In contrast, monkey-derived Vero cells are highly susceptibility to infection with SARS-CoV-2, although they are not suitable for the study of antiviral effects by small molecules due to their limited capacity to metabolize drugs compared to human-derived cells. In this study, our goal was to generate a virus-susceptible human cell line that would be useful for the identification and testing of candidate drugs. Towards this end, we stably transfected human lung-derived MRC5 cells with a lentiviral vector encoding angiotensin-converting enzyme 2 (ACE2), the cellular receptor for SARS-CoV-2. Our results revealed that SARS-CoV-2 replicates efficiently in MRC5/ACE2 cells. Furthermore, viral RNA replication and progeny virus production were significantly reduced in response to administration of the replication inhibitor, remdesivir, in MRC5/ACE2 cells compared with Vero cells. We conclude that the MRC5/ACE2 cells will be important in developing specific anti-viral therapeutics and will assist in vaccine development to combat SARS-CoV-2 infections.
• 全身性エリテマトーデスモデルマウスにおけるHLA-G2蛋白質の発症抑制効果の検証

  渡邊 紘士, 黒木 喜美子, 山田 千聖, 佐分利 由香里, 高橋 愛実, 前田 直良, 前仲 勝実

  日本薬学会年会要旨集 141年会 27V08 - am09S (公社)日本薬学会 2021年03月
• 免疫抑制分子HLA-G2特異的新規モノクローナル抗体の作製

  赤岩 愛記, 黒木 喜美子, 引地 和馬, 古川 敦, 前田 直良, 前仲 勝実

  日本薬学会年会要旨集 141年会 29P01 - 105S (公社)日本薬学会 2021年03月
• Enzootic nasal tumor virus type 2 envelope of goats acts as a retroviral oncogene in cell transformation.

  Naoyoshi Maeda, Yasuo Inoshima, Marcelo De Las Heras, Katsumi Maenaka

  Virus genes 57 1 50 - 59 2021年02月 

  Enzootic nasal tumor virus type 1 (ENTV-1) (ovine nasal tumor virus) and ENTV-2 (caprine nasal tumor virus) are known to be causative agents of enzootic nasal adenocarcinoma (ENA) in sheep and goats, respectively. Although the nucleotide and amino acid sequences of ENTV-1 and ENTV-2 are quite similar, they are recognized as phylogenetically distinct viruses. The envelope protein of ENTV-1 functions as an oncoprotein in the in vitro transformation of epithelial cells and fibroblasts. Thus, it is the primary determinant of in vivo tumorigenesis in ENA. As per our knowledge, no previous studies have reported in detail the role of ENTV-2 in ENA tumorigenesis. Here, in order to investigate the molecular mechanism of caprine ENA oncogenesis by ENTV-2, we have attempted to identify the transforming potential of ENTV-2 envelope, and investigated the activation of cell signaling pathways in oncogenic transformation. Our findings confirmed that ENTV-2 envelope was capable of inducing oncogenic transformation of rat cell lines in vitro. Further, we found that MAPK, Akt, and p38 were constitutively activated in ENTV-2 envelope-transformed clone cells. In addition, inhibitor experiments revealed that MEK-MAPK and PI3K-Akt signaling pathways are involved in the ENTV-2 envelope-induced cell transformation. These data indicate that ENTV-2 envelope could induce oncogenic transformation by signaling pathways that are also utilized by ENTV-1 envelope.
• SARS-CoV-2 variants with mutations at the S1/S2 cleavage site are generated in vitro during propagation in TMPRSS2-deficient cells.

  Michihito Sasaki, Kentaro Uemura, Akihiko Sato, Shinsuke Toba, Takao Sanaki, Katsumi Maenaka, William W Hall, Yasuko Orba, Hirofumi Sawa

  PLoS pathogens 17 1 e1009233  2021年01月 

  The spike (S) protein of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) binds to a host cell receptor which facilitates viral entry. A polybasic motif detected at the cleavage site of the S protein has been shown to broaden the cell tropism and transmissibility of the virus. Here we examine the properties of SARS-CoV-2 variants with mutations at the S protein cleavage site that undergo inefficient proteolytic cleavage. Virus variants with S gene mutations generated smaller plaques and exhibited a more limited range of cell tropism compared to the wild-type strain. These alterations were shown to result from their inability to utilize the entry pathway involving direct fusion mediated by the host type II transmembrane serine protease, TMPRSS2. Notably, viruses with S gene mutations emerged rapidly and became the dominant SARS-CoV-2 variants in TMPRSS2-deficient cells including Vero cells. Our study demonstrated that the S protein polybasic cleavage motif is a critical factor underlying SARS-CoV-2 entry and cell tropism. As such, researchers should be alert to the possibility of de novo S gene mutations emerging in tissue-culture propagated virus strains.
• Antitumor Effect of Sugar-Modified Cytosine Nucleosides on Growth of Adult T-Cell Leukemia Cells in Mice.

  Naoyoshi Maeda, Akira Matsuda, Satoko Otsuguro, Masahiko Takahashi, Masahiro Fujii, Katsumi Maenaka

  Vaccines 8 4 2020年11月05日 

  Adult T-cell leukemia (ATL) is a CD4+ T-cell neoplasm caused by human T-cell leukemia virus type I. As the prognosis for patients with ATL remains extremely poor due to resistance to conventional chemotherapy regimens, introduction of novel therapeutic agents is needed. Previous studies have reported that nucleosides 2'-deoxy-2'-methylidenecytidine (DMDC) and its derivative 2'-deoxy-2'-methylidene-5-fluorocytidine (FDMDC) exhibit antitumor activities in T-cell acute lymphoblastic leukemia (T-ALL) and solid tumor cell lines. Another nucleoside, 1-(2-azido-2-deoxy-β-D-arabinofuranosyl)cytosine (cytarazid), is considered a therapeutic drug with antitumor activity in human solid tumors. In this study, we investigated the effects of these nucleosides on cell growth in vitro and in vivo using relevant leukemia cell lines and NOD/Shi-scid, IL-2Rgnull (NOG) mice, respectively. The nucleosides demonstrated significant cytotoxic effects in ATL and T-ALL cell lines. Intraperitoneal administration of FDMDC and DMDC into tumor-bearing NOG mice resulted in significant suppression of tumor growth without lethal side effects. Our findings support a therapeutic application of these nucleosides against tumor progression by targeting DNA polymerase-dependent DNA synthesis in patients with ATL.
• Structural comparison of the C-terminal domain of functionally divergent lyssavirus P proteins.

  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.
• The Measles Virus V Protein Binding Site to STAT2 Overlaps That of IRF9.

  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.
• Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.

  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.
• Molecular mechanism of the recognition of bacterially cleaved immunoglobulin by the immune regulatory receptor LILRA2.

  Rika Yamazaki, Atsushi Furukawa, Kouyuki Hirayasu, Kohei Yumoto, Hideo Fukuhara, Hisashi Arase, Katsumi Maenaka

  The Journal of biological chemistry 295 28 9531 - 9541 2020年07月10日 [査読有り][通常論文]
   

  Human leukocyte immunoglobulin-like receptors (LILRs) typically regulate immune activation by binding to the human leukocyte antigen class I molecules. LILRA2, a member of the LILR family, was recently reported to bind to other unique ligands, the bacterially degraded Igs (N-truncated Igs), for the activation of immune cells. Therefore, LILRA2 is currently attracting significant attention as a novel innate immune receptor. However, the detailed recognition mechanisms required for this interaction remain unclear. In this study, using several biophysical techniques, we uncovered the molecular mechanism of N-truncated Ig recognition by LILRA2. Surface plasmon resonance analysis disclosed that LILRA2 specifically binds to N-truncated Ig with weak affinity (Kd = 4.8 μm) and fast kinetics. However, immobilized LILRA2 exhibited a significantly enhanced interaction with N-truncated Ig due to avidity effects. This suggests that cell surface-bound LILRA2 rapidly monitors and identifies bi- or multivalent abnormal N-truncated Igs through specific cross-linking to induce immune activation. Van't Hoff analysis revealed that this interaction is enthalpy-driven, with a small entropy loss, and results from differential scanning calorimetry indicated the instability of the putative LILRA2-binding site, the Fab region of the N-truncated Ig. Atomic force microscopy revealed that N truncation does not cause significant structural changes in Ig. Furthermore, mutagenesis analysis identified the hydrophobic region of LILRA2 domain 2 as the N-truncated Ig-binding site, representing a novel ligand-binding site for the LILR family. These results provide detailed insights into the molecular regulation of LILR-mediated immune responses targeting ligands that have been modified by bacteria.
• Structural characteristics of measles virus entry.

  Hideo Fukuhara, Mwila Hilton Mwaba, Katsumi Maenaka

  Current opinion in virology 41 52 - 58 2020年04月 

  Measles virus, a member of the genus Morbillivirus, is highly contagious and still shows considerable mortality with over 100000 deaths annually, although efficient attenuated vaccines exist. Recent studies of measles virus haemagglutinin (MeV-H) and its receptor, including crystallographic and electron microscopic structural analyses combined with functional assays, have revealed how the MeV-H protein recognizes its cognate receptors, SLAM and Nectin-4, and how the glycan shield ensures effective vaccination. In addition, the crystal structure of the MeV-F protein indicated its similarity to those of other paramyxoviruses. Taking into account these data, several models of viral entry/membrane fusion of measles viruses and related paramyxoviruses have been proposed. Furthermore, anti-MeV-F inhibitors targeted to specific regions to inhibit MeV-F protein activation were reported, with potency for preventing MeV infection. The inhibitors targeted for entry events may potentially be applied to treatment of MeV-derived diseases, although escape mutations and drug profiles should be considered.
• Biophysical research in Hokkaido University, Japan.

  Tomoyasu Aizawa, Makoto Demura, Kazutoshi Gohara, Hisashi Haga, Koichiro Ishimori, Masataka Kinjo, Tamiki Komatsuzaki, Katsumi Maenaka, Min Yao

  Biophysical reviews 12 2 233 - 236 2020年04月 [査読有り][通常論文]
  
• Therapeutic effects of soluble human leukocyte antigen G2 isoform in lupus-prone MRL/lpr mice.

  Hiroshi Watanabe, Kimiko Kuroki, Chisato Yamada, Yukari Saburi, Naoyoshi Maeda, Katsumi Maenaka

  Human immunology 81 4 186 - 190 2020年04月 [査読有り][通常論文]
   

  Human leukocyte antigen (HLA)-G, a non-classical HLA class I molecule, has one of the splicing isoforms, HLA-G2, which lacks one domain (α2) and forms a non-covalent homodimer. HLA-G2 is expressed on placental cells, regulatory T cells, tumor cells, and virus-infected cells, and is involved in immunosuppression. The major isoform of HLA-G, HLA-G1, binds to leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2, on the contrary, HLA-G2 binds to only LILRB2. We previously reported that HLA-G2 bound LILRB2 more strongly than HLA-G1 and also to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs. Furthermore, HLA-G2 showed immunosuppressive effects in both collagen-induced arthritis (CIA) and atopic dermatitis-like model mice. In this study, we examine in vivo effects of HLA-G2 in systemic lupus erythematosus (SLE) model mice. HLA-G2 showed the suppression of the typical SLE symptoms such as serum anti-dsDNA antibody level and urinary albumin index. Furthermore, HLA-G2 tended to downregulate B-lymphocyte stimulator (BLyS) production. This is the first observation of the immunosuppressive effects of HLA-G2 isoform in SLE model mice, suggesting that HLA-G2 could be a useful therapeutic agent for SLE.
• Cysteine-Rich Secretory Proteins (CRISPs) From Venomous Snakes: An Overview of the Functional Diversity in A Large and Underappreciated Superfamily.

  Takashi Tadokoro, Cassandra M Modahl, Katsumi Maenaka, Narumi Aoki-Shioi

  Toxins 12 3 2020年03月12日 [査読有り][通常論文]
   

  The CAP protein superfamily (Cysteine-rich secretory proteins (CRISPs), Antigen 5 (Ag5), and Pathogenesis-related 1 (PR-1) proteins) is widely distributed, but for toxinologists, snake venom CRISPs are the most familiar members. Although CRISPs are found in the majority of venoms, very few of these proteins have been functionally characterized, but those that have been exhibit diverse activities. Snake venom CRISPs (svCRISPs) inhibit ion channels and the growth of new blood vessels (angiogenesis). They also increase vascular permeability and promote inflammatory responses (leukocyte and neutrophil infiltration). Interestingly, CRISPs in lamprey buccal gland secretions also manifest some of these activities, suggesting an evolutionarily conserved function. As we strive to better understand the functions that CRISPs serve in venoms, it is worth considering the broad range of CRISP physiological activities throughout the animal kingdom. In this review, we summarize those activities, known crystal structures and sequence alignments, and we discuss predicted functional sites. CRISPs may not be lethal or major components of venoms, but given their almost ubiquitous occurrence in venoms and the accelerated evolution of svCRISP genes, these venom proteins are likely to have functions worth investigating.
• The mitochondrial inner membrane protein LETM1 modulates cristae organization through its LETM domain.

  Seiko Nakamura, Aiko Matsui, Shiori Akabane, Yasushi Tamura, Azumi Hatano, Yuriko Miyano, Hiroshi Omote, Mizuho Kajikawa, Katsumi Maenaka, Yoshinori Moriyama, Toshiya Endo, Toshihiko Oka

  Communications biology 3 1 99 - 99 2020年03月05日 [査読有り][通常論文]
   

  LETM1 is a mitochondrial inner membrane protein that is required for maintaining the mitochondrial morphology and cristae structures, and regulates mitochondrial ion homeostasis. Here we report a role of LETM1 in the organization of cristae structures. We identified four amino acid residues of human LETM1 that are crucial for complementation of the growth deficiency caused by gene deletion of a yeast LETM1 orthologue. Substituting amino acid residues with alanine disrupts the correct assembly of a protein complex containing LETM1 and prevents changes in the mitochondrial morphology induced by exogenous LETM1 expression. Moreover, the LETM1 protein changes the shapes of the membranes of in vitro-reconstituted proteoliposomes, leading to the formation of invaginated membrane structures on artificial liposomes. LETM1 mutant proteins with alanine substitutions fail to facilitate the formation of invaginated membrane structures, suggesting that LETM1 plays a fundamental role in the organization of mitochondrial membrane morphology.
• Structure of HIV-2 Nef Reveals Features Distinct from HIV-1 Involved in Immune Regulation.

  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.
• Measles Virus Hemagglutinin Protein Establishes a Specific Interaction With the Extreme N-Terminal Region of Human Signaling Lymphocytic Activation Molecule to Enhance Infection.

  Fumio Seki, Yuta Yamamoto, Hideo Fukuhara, Kazue Ohishi, Tadashi Maruyama, Katsumi Maenaka, Hiroaki Tokiwa, Makoto Takeda

  Frontiers in microbiology 11 1830 - 1830 2020年 

  Measles virus (MV) is a human pathogen that is classified in the genus Morbillivirus in the family Paramyxoviridae together with several non-human animal morbilliviruses. They cause severe systemic infections by using signaling lymphocytic activation molecule (SLAM) and poliovirus receptor-like 4 expressed on immune and epithelial cells, respectively, as receptors. The viral hemagglutinin (H) protein is responsible for the receptor-binding. Previously determined structures of MV-H and SLAM complexes revealed a major binding interface between the SLAM V domain and MV-H with four binding components (sites 1-4) in the interface. We studied the MV-H and human SLAM (hSLAM) complex structure in further detail by in silico analyses and determined missing regions or residues in the previously determined complex structures. These analyses showed that, in addition to sites 1-4, MV-H establishes a unique interaction with the extreme N-terminal region (ExNTR) of hSLAM. The first principles calculation-based fragment molecular orbital computation method revealed that methionine at position 29 (hSLAM-Met29) is the key residue for the interaction. hSLAM-Met29 was predicted to establish a CH-π interaction with phenylalanine at position 549 of MV-H (MVH-Phe549). A cell-cell fusion assay showed that the hSLAM-Met29 and MVH-Phe549 interaction is important for hSLAM-dependent MV membrane fusion. Furthermore, Jurkat cell lines expressing hSLAM with or without Met29 and recombinant MV possessing the H protein with or without Phe549 showed that the hSLAM-Met29 and MVH-Phe549 interaction enhanced hSLAM-dependent MV infection by ~10-fold. We speculate that in the evolutionary history of morbilliviruses, this interaction may have contributed to MV adaptation to humans because this interaction is unique for MV and only MV uses hSLAM efficiently among morbilliviruses.
• Biophysical characterization and single-chain Fv construction of a neutralizing antibody to measles virus.

  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.
• Relation of Colloidal and Conformational Stabilities to Aggregate Formation in a Monoclonal Antibody.

  Hiroaki Oyama, Hiroki Koga, Takashi Tadokoro, Katsumi Maenaka, Akira Shiota, Masami Yokoyama, Masanori Noda, Tetsuo Torisu, Susumu Uchiyama

  Journal of pharmaceutical sciences 109 1 308 - 315 2020年01月 [査読有り][通常論文]
   

  Aggregation of therapeutic monoclonal antibodies has a potential risk of immunogenicity, requiring minimization of aggregate formation. We have developed a fitting formula for antibody aggregation at 40°C based on physicochemical parameters, including colloidal and conformational stabilities. An IgG1 monoclonal antibody, MAb-T, was formulated in 24 combinations of different buffer types and pH with or without sodium chloride. The fitting formula for monomer loss was successfully established by nonlinear regression analysis of the results from accelerated stability testing. Calculated monomer fraction values by the fitting formula were strongly correlated with experimental values (R2 = 0.92). The model includes secondary virial coefficient, B22, as the representative parameter of colloidal stability, and aggregation temperature, Tagg, representing conformational stability. Then, we examined charge state, conformational flexibility, and thermal unfolding profile of MAb-T to clarify the molecular basis for the different aggregation propensities in sodium acetate buffer and in sodium citrate buffer at the same pH and buffer concentration. We concluded that the accumulation of citrate anions on the surface of MAb-T is the primary source of the less colloidal and conformational stabilities, resulting in the higher aggregation propensity in sodium citrate buffer.
• Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

  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.
• Repurposing existing drugs: identification of irreversible IMPDH inhibitors by high-throughput screening.

  Albertus Eka Yudistira Sarwono, Shinya Mitsuhashi, Mohammad Hazzaz Bin Kabir, Kengo Shigetomi, Tadashi Okada, Fumina Ohsaka, Satoko Otsuguro, Katsumi Maenaka, Makoto Igarashi, Kentaro Kato, Makoto Ubukata

  Journal of enzyme inhibition and medicinal chemistry 34 1 171 - 178 2019年12月 [査読有り][通常論文]
   

  Inosine 5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for the production of guanine nucleotides. Disruption of IMPDH activity has been explored as a therapeutic strategy for numerous purposes, such as for anticancer, immunosuppression, antiviral, and antimicrobial therapy. In the present study, we established a luciferase-based high-throughput screening system to identify IMPDH inhibitors from our chemical library of known bioactive small molecules. The screening of 1400 compounds resulted in the discovery of three irreversible inhibitors: disulfiram, bronopol, and ebselen. Each compound has a distinct chemical moiety that differs from other reported IMPDH inhibitors. Further evaluation revealed that these compounds are potent inhibitors of IMPDHs with kon values of 0.7 × 104 to 9.3 × 104 M-1·s-1. Both disulfiram and bronopol exerted similar degree of inhibition to protozoan and mammalian IMPDHs. Ebselen showed an intriguing difference in mode of inhibition for different IMPDHs, with reversible and irreversible inhibition to each Cryptosporidium parvum IMPDH and human IMPDH type II, respectively. In the preliminary efficacy experiment against cryptosporidiosis in severe combined immunodeficiency (SCID) mouse, a decrease in the number of oocyst shed was observed upon the oral administration of disulfiram and bronopol, providing an early clinical proof-of-concept for further utilization of these compounds as IMPDH inhibitors.
• Evaluation of the Reactivity and Receptor Competition of HLA-G Isoforms toward Available Antibodies: Implications of Structural Characteristics of HLA-G Isoforms.

  Atsushi Furukawa, Manami Meguro, Rika Yamazaki, Hiroshi Watanabe, Ami Takahashi, Kimiko Kuroki, Katsumi Maenaka

  International journal of molecular sciences 20 23 2019年11月26日 [査読有り][通常論文]
   

  The human leucocyte antigen (HLA)-G, which consists of seven splice variants, is a tolerogenic immune checkpoint molecule. It plays an important role in the protection of the fetus from the maternal immune response by binding to inhibitory receptors, including leukocyte Ig-like receptors (LILRs). Recent studies have also revealed that HLA-G is involved in the progression of cancer cells and the protection from autoimmune diseases. In contrast to its well characterized isoform, HLA-G1, the binding activities of other major HLA-G isoforms, such as HLA-G2, toward available anti-HLA-G antibodies are only partially understood. Here, we investigate the binding specificities of anti-HLA-G antibodies by using surface plasmon resonance. MEM-G9 and G233 showed strong affinities to HLA-G1, with a nM range for their dissociation constants, but did not show affinities to HLA-G2. The disulfide-linker HLA-G1 dimer further exhibited significant avidity effects. On the other hand, 4H84 and MEM-G1, which can be used for the Western blotting of HLA-G isoforms, can bind to native HLA-G2, while MEM-G9 and G233 cannot. These results reveal that HLA-G2 has a partially intrinsically disordered structure. Furthermore, MEM-G1, but not 4H84, competes with the LILRB2 binding of HLA-G2. These results provide novel insight into the functional characterization of HLA-G isoforms and their detection systems.
• Novel Notch signaling inhibitor NSI‑1 suppresses nuclear translocation of the Notch intracellular domain.

  Shiraishi T, Sakaitani M, Otsuguro S, Maenaka K, Suzuki T, Nakaya T

  International journal of molecular medicine 44 4 1574 - 1584 2019年10月 [査読有り][通常論文]
   

  The Notch receptor serves a fundamental role in the regulation of cell fate determination through intracellular signal transmission. Mutation of the Notch receptor results in abnormal active signaling, leading to the development of diseases involving abnormal cell growth, including malignant tumors. Therefore, the Notch signaling pathway is a useful pharmacological target for the treatment of cancer. In the present study, a compound screening system was designed to identify inhibitors of the Notch signaling targeting Notch intracellular domain (NICD). A total of 9,600 compounds were analyzed using the Michigan Cancer Foundation‑7 (MCF7) human breast adenocarcinoma cell line and the SH‑SY5Y human neuroblastoma cell line with the reporter assay system using an artificial protein encoding a partial Notch carboxyl‑terminal fragment fused to the Gal4 DNA‑binding domain. The molecular mechanism underlying the inhibition of Notch signaling by a hit compound was further validated using biochemical and cell biological approaches. Using the screening system, a potential candidate, Notch signaling inhibitor‑1 (NSI‑1), was isolated which showed 50% inhibition at 6.1 µM in an exogenous Notch signaling system. In addition, NSI‑1 suppressed the nuclear translocation of NICD and endogenous gene expression of hairy and enhancer of split‑1, indicating that NSI‑1 specifically targets Notch. Notably, NSI‑1 suppressed the cell viability of MCF7 cells and another human breast adenocarcinoma cell line, MDA‑MB‑231 exhibiting constitutive and high Notch signaling activity, whereas no significant effect was observed in the SH‑SY5Y cells bearing a lower Notch signaling activity. NSI‑1 significantly suppressed the viability of SH‑SY5Y cells expressing exogenous human Notch1. These results indicate that NSI‑1 is a novel Notch signaling inhibitor and suggest its potential as a useful drug for the treatment of diseases induced by constitutively active Notch signaling.
• Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species.

  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.
• Identification of IgG1 Aggregation Initiation Region by Hydrogen Deuterium Mass Spectrometry.

  Masanori Noda, Kentaro Ishii, Mika Yamauchi, Hiroaki Oyama, Takashi Tadokoro, Katsumi Maenaka, Tetsuo Torisu, Susumu Uchiyama

  Journal of pharmaceutical sciences 108 7 2323 - 2333 2019年07月 [査読有り][通常論文]
   

  Antibody aggregates are a potential risk for immunogenicity; therefore, rational approaches to improve associated aggregation properties need to be developed. Here, we report the amino acid region responsible for aggregation initiation. Two types of therapeutic IgG1 antibody monomer samples were prepared: IgG1 mAb40-3M stored at 40°C for 3 months, which existed in monodisperse state, and the monomer mAb65-5m, which was dissociated from small soluble aggregates by heating at 65°C for 5 min. Hydrogen deuterium exchange mass spectrometry of mAb40-3M identified 2 sites in the Fc region (site 1, F239-M256; site 2, S428-G450) with increased exchange rates. Site 1 includes a region reported as being susceptible to structural change induced by stress. Exposure of site 1 was undetected after 2 months of storage at 40°C but was subsequently detectable after 3 months. As site 2 is spatially close to site 1, the structural change of site 1 could propagate site 2. Besides these 2 regions, hydrogen deuterium exchange mass spectrometry of mAb65-5m identified an exposure of I257-W281 in Fc (site 3), within which a peptide sequence with high aggregation tendency was discovered. We thus concluded that exposure of site 3 is a trigger for the association of a partially denatured antibody.
• HLA-G dimer targets Granzyme B pathway to prolong human renal allograft survival.

  Ashwin Ajith, Vera Portik-Dobos, Anh Thu Nguyen-Lefebvre, Christine Callaway, Daniel D Horuzsko, Rajan Kapoor, Carlos Zayas, Katsumi Maenaka, Laura L Mulloy, Anatolij Horuzsko

  FASEB journal : official publication of the Federation of American Societies for Experimental Biology 33 4 5220 - 5236 2019年04月 [査読有り][通常論文]
   

  Human leukocyte antigen G (HLA-G), a nonclassic HLA class Ib molecule involved in the maintenance of maternal tolerance to semiallogeneic fetal tissues during pregnancy, has emerged as a potential therapeutic target to control allograft rejection. We demonstrate here that the level of soluble HLA-G dimer was higher in a group of 90 patients with a functioning renal allograft compared with 40 patients who rejected (RJ) their transplants. The HLA-G dimer level was not affected by demographic status. One of the potential mechanisms in tissue-organ allograft rejection involves the induction of granzymes and perforin, which are the main effector molecules expressed by CD8+ cytotoxic T lymphocytes and function to destroy allogeneic transplants. Using genomics and molecular and cellular analyses of cells from T-cell-mediated RJ and nonrejected kidney transplant patients, cells from leukocyte Ig-like receptor B1 (LILRB1) transgenic mice, humanized mice, and genetically engineered HLA-G dimer, we demonstrated a novel mechanism by which HLA-G dimer inhibits activation and cytotoxic capabilities of human CD8+ T cells. This mechanism implicated the down-regulation of Granzyme B expression and the essential involvement of LILRB1. Thus, HLA-G dimer has the potential to be a specific and effective therapy for prevention of allograft rejection and prolongation of graft survival.-Ajith, A., Portik-Dobos, V., Nguyen-Lefebvre, A. T., Callaway, C., Horuzsko, D. D., Kapoor, R., Zayas, C., Maenaka, K., Mulloy, L. L., Horuzsko, A. HLA-G dimer targets Granzyme B pathway to prolong human renal allograft survival.
• Crystal structure of the complex between venom toxin and serum inhibitor from Viperidae snake.

  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.
• Synthesis of glycerolipids containing simple linear acyl chains or aromatic rings and evaluation of their Mincle signaling activity.

  Takanori Matsumaru, Risa Ikeno, Yusuke Shuchi, Toshiki Iwamatsu, Takashi Tadokoro, Sho Yamasaki, Yukari Fujimoto, Atsushi Furukawa, Katsumi Maenaka

  Chemical communications (Cambridge, England) 55 5 711 - 714 2019年01月10日 [査読有り][通常論文]
   

  Mincle, expressed in activated phagocytes, recognizes the lipid ligand to activate the innate immune system. We have synthesized glycerol derivatives possessing simple alkyl chains or aromatic rings and elucidated their structure-activity relationships using a Mincle-mediated signaling assay. The activity depends on the length of the simple acyl chains of the glycerol derivatives.
• Suramin, screened from an approved drug library, inhibits HuR functions and attenuates malignant phenotype of oral cancer cells.

  Wataru Kakuguchi, Takao Nomura, Tetsuya Kitamura, Satoko Otsuguro, Kazuhiro Matsushita, Masahiro Sakaitani, Katsumi Maenaka, Kanchu Tei

  Cancer medicine 7 12 6269 - 6280 2018年12月 [査読有り][通常論文]
   

  AU-rich elements (ARE) exist in the 3'-untranslated regions of the mRNA transcribed from cell growth-related genes such as proto-oncogenes, cyclin-related genes, and growth factors. HuR binds and stabilizes ARE-mRNA. HuR is expressed abundantly in cancer cells and related malignant phenotypes. HuR knockdown attenuates the malignant phenotype of oral cancer cells. In this study, we screened 1570 compounds in the approved drug library by differential scanning fluorimetry (DSF) to discover a HuR-targeted compound. Firstly, 55 compounds were selected by DSF. Then, 8 compounds that showed a shift in the melting temperature value in a concentration-dependent manner were selected by DSF. Of them, suramin, an anti-trypanosomal drug, binds to HuR, exhibiting fast-on and fast-off kinetic behavior on surface plasmon resonance (SPR). We confirmed that suramin significantly decreased mRNA and protein expression of cyclin A2 and cyclin B1. The cyclin A2 and cyclin B1 mRNAs were destabilized by suramin. Furthermore, the motile and invasive activities of a tongue carcinoma cell line treated with suramin were markedly lower than those of control cells. The above findings suggest that suramin binds to HuR and inhibits its function. We also showed that the anticancer effects of suramin were caused by the inhibition of HuR function, indicating its potential as a novel therapeutic agent in the treatment of oral cancer. Our results suggest that suramin, via its different mechanism, may effectively suppress progressive oral cancer that cannot be controlled using other anticancer agents.
• Structure of MHC class I-like MILL2 reveals heparan-sulfate binding and interdomain flexibility.

  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.
• Structural insights into modulation and selectivity of transsynaptic neurexin-LRRTM interaction.

  Atsushi Yamagata, Sakurako Goto-Ito, Yusuke Sato, Tomoko Shiroshima, Asami Maeda, Masahiko Watanabe, Takashi Saitoh, Katsumi Maenaka, Tohru Terada, Tomoyuki Yoshida, Takeshi Uemura, Shuya Fukai

  Nature communications 9 1 3964 - 3964 2018年09月27日 [査読有り][通常論文]
   

  Leucine-rich repeat transmembrane neuronal proteins (LRRTMs) function as postsynaptic organizers that induce excitatory synapses. Neurexins (Nrxns) and heparan sulfate proteoglycans have been identified as presynaptic ligands for LRRTMs. Specifically, LRRTM1 and LRRTM2 bind to the Nrxn splice variant lacking an insert at the splice site 4 (S4). Here, we report the crystal structure of the Nrxn1β-LRRTM2 complex at 3.4 Å resolution. The Nrxn1β-LRRTM2 interface involves Ca2+-mediated interactions and overlaps with the Nrxn-neuroligin interface. Together with structure-based mutational analyses at the molecular and cellular levels, the present structural analysis unveils the mechanism of selective binding between Nrxn and LRRTM1/2 and its modulation by the S4 insertion of Nrxn.
• A Sialylated Voltage-Dependent Ca2+ Channel Binds Hemagglutinin and Mediates Influenza A Virus Entry into Mammalian Cells.

  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.
• Ribavirin-related compounds exert in vitro inhibitory effects toward rabies virus.

  Anindita PD, Sasaki M, Okada K, Ito N, Sugiyama M, Saito-Tarashima N, Minakawa N, Shuto S, Otsuguro S, Ichikawa S, Matsuda A, Maenaka K, Orba Y, Sawa H

  Antiviral research 154 1 - 9 2018年03月 [査読有り][通常論文]
   

  Rabies remains an invariably fatal neurological disease despite the availability of a preventive vaccination and post-exposure prophylaxis that must be immediately administered to the exposed individual before symptom onset. There is no effective medication for treatment during the symptomatic phase. Ribavirin, a guanine nucleoside analog, is a potent inhibitor of rabies virus (RABV) replication in vitro but lacks clinical efficacy. Therefore, we attempted to identify potential ribavirin analogs with comparable or superior anti-RABV activity. Antiviral activity and cytotoxicity of the compounds were initially examined in human neuroblastoma cells. Among the tested compounds, two exhibited a 5- to 27-fold higher anti-RABV activity than ribavirin. Examination of the anti-RABV mechanisms of action of the compounds using time-of-addition and minigenome assays revealed that they inhibited viral genome replication and transcription. Addition of exogenous guanosine to RABV-infected cells diminished the antiviral activity of the compounds, suggesting that they are involved in guanosine triphosphate (GTP) pool depletion by inhibiting inosine monophosphate dehydrogenase (IMPDH). Taken together, our findings underline the potency of nucleoside analogs as a class of antiviral compounds for the development of novel agents against RABV.
• Heparan sulfate proteoglycans serve as an attachment factor for rabies virus entry and infection.

  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02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.
• Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor.

  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.
• X-ray crystal structure of Escherichia coli HspQ, a protein involved in the retardation of replication initiation

  Yoshito Abe, Seijiro Shioi, Shunsuke Kita, Hikaru Nakata, Katsumi Maenaka, Daisuke Kohda, Tsutomu Katayama, Tadashi Ueda

  FEBS LETTERS 591 22 3805 - 3816 2017年11月 [査読有り][通常論文]
   

  The heat shock protein HspQ (YccV) of Escherichia coli has been proposed to participate in the retardation of replication initiation in cells with the dnaA508 allele. In this study, we have determined the 2.5-angstrom- resolution X-ray structure of the trimer of HspQ, which is also the first structure of a member of the YccV superfamily. The acidic character of the HspQ trimer suggests an interaction surface with basic proteins. From these results, we discuss the cellular function of HspQ, including its relationship with the DnaA508 protein.
• X-ray crystal structure of Escherichia coli HspQ, a protein involved in the retardation of replication initiation.

  Abe Y, Shioi S, Kita S, Nakata H, Maenaka K, Kohda D, Katayama T, Ueda T

  FEBS letters 591 22 3805 - 3816 2017年11月01日 [査読有り][通常論文]
   

  The heat shock protein HspQ (YccV) of Escherichia coli has been proposed to participate in the retardation of replication initiation in cells with the dnaA508 allele. In this study, we have determined the 2.5-Å-resolution X-ray structure of the trimer of HspQ, which is also the first structure of a member of the YccV superfamily. The acidic character of the HspQ trimer suggests an interaction surface with basic proteins. From these results, we discuss the cellular function of HspQ, including its relationship with the DnaA508 protein.
• Production of Single-Chain Fv Antibodies Specific for GA-Pyridine, an Advanced Glycation End-Product (AGE), with Reduced Inter-Domain Motion

  Natsuki Fukuda, Kentaro Noi, Lidong Weng, Yoshihiro Kobashigawa, Hiromi Miyazaki, Yukari Wakeyama, Michiyo Takaki, Yusuke Nakahara, Yuka Tatsuno, Makiyo Uchida-Kamekura, Yoshiaki Suwa, Takashi Sato, Naoki Ichikawa-Tomikawa, Motoyoshi Nomizu, Yukio Fujiwara, Fumina Ohsaka, Takashi Saito, Katsumi Maenaka, Hiroyuki Kumeta, Shoko Shinya, Chojiro Kojima, Teru Ogura, Hiroshi Morioka

  MOLECULES 22 10 2017年10月 [査読有り][通常論文]
   

  Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 degrees C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open-close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs.
• The Roles of Matricellular Proteins in Oncogenic Virus-Induced Cancers and Their Potential Utilities as Therapeutic Targets

  Naoyoshi Maeda, Katsumi Maenaka

  INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 18 10 2017年10月 [査読有り][通常論文]
   

  Matricellular proteins differ from other classical extracellular matrix proteins; for instance, they are transiently expressed as soluble proteins rather than being constitutively expressed in pathological conditions, such as acute viral infections. Accumulating studies have revealed that matricellular proteins, including osteopontin and tenascin-C, both of which interact with integrin heterodimers, are involved in inflammatory diseases, autoimmune disorders, and cancers. The concentrations of these matricellular proteins are elevated in the plasma of patients with certain types of cancers, indicating that they play important roles in oncogenesis. Chronic viral infections are associated with certain cancers, which are distinct from non-viral cancers. Viral oncogenes play critical roles in the development and progression of such cancers. It is vital to investigate the mechanisms of tumorigenesis and, particularly, the mechanism by which viral proteins induce tumor progression. Viral proteins have been shown to influence not only the viral-infected cancer cells, but also the stromal cells and matricellular proteins that constitute the extracellular matrix that surrounds tumor tissues. In this review, we summarize the recent progress on the involvement of matricellular proteins in oncogenic virus-induced cancers to elucidate the mechanism of oncogenesis and consider the possible role of matricellular proteins as therapeutic targets in virus-induced cancers.
• Therapeutic application of human leukocyte antigen-G1 improves atopic dermatitis-like skin lesions in mice

  Naoyoshi Maeda, Chisato Yamada, Ami Takahashi, Kimiko Kuroki, Katsumi Maenaka

  INTERNATIONAL IMMUNOPHARMACOLOGY 50 202 - 207 2017年09月 [査読有り][通常論文]
   

  Human leukocyte antigen (HLA)-G is an immune checkpoint molecule that plays critical roles in immune response and in triggering inhibitory signaling to immune cells such as T cells, natural killer cells, and antigen presenting cells. Thus, the application of HLA-G can be considered for treating immune response-related inflammatory disorders. We have previously reported that treatment with HLA-Gl and HLA-G2 ameliorates the joint swelling associated with collagen-induced arthritis of DBA/1 mice, an animal model for rheumatoid arthritis. In this study, we further investigated the effects of HLA-G1 on atopic dermatitis (AD), the most common inflammatory skin disorder. AD-like lesions were induced with the extract of the house dust mite Dermatophagoides farinae in NC/Nga mice. Continuous administration of HLA-G1 ameliorated the AD-like skin lesions in the mice. Furthermore, production of immunoglobulin E, interleukin (IL)-13, and IL-17A was significantly reduced in HLA-Gl-treated mice, suggesting a Th2/Th17-mediated immune-inhibitory function of HLA-G1 in vivo. Our studies shed light on novel therapeutic strategies with recombinant HLA-G proteins for immune reaction-mediated chronic inflammatory disorders.
• Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin.

  Tadato Ban, Takaya Ishihara, Hiroto Kohno, Shotaro Saita, Ayaka Ichimura, Katsumi Maenaka, Toshihiko Oka, Katsuyoshi Mihara, Naotada Ishihara

  Nature cell biology 19 7 856 - 863 2017年07月 [査読有り][通常論文]
   

  Mitochondria are highly dynamic organelles that undergo frequent fusion and fission. Optic atrophy 1 (OPA1) is an essential GTPase protein for both mitochondrial inner membrane (IM) fusion and cristae morphology. Under mitochondria-stress conditions, membrane-anchored L-OPA1 is proteolytically cleaved to form peripheral S-OPA1, leading to the selection of damaged mitochondria for mitophagy. However, molecular details of the selective mitochondrial fusion are less well understood. Here, we showed that L-OPA1 and cardiolipin (CL) cooperate in heterotypic mitochondrial IM fusion. We reconstituted an in vitro membrane fusion reaction using purified human L-OPA1 protein expressed in silkworm, and found that L-OPA1 on one side of the membrane and CL on the other side are sufficient for fusion. GTP-independent membrane tethering through L-OPA1 and CL primes the subsequent GTP-hydrolysis-dependent fusion, which can be modulated by the presence of S-OPA1. These results unveil the most minimal intracellular membrane fusion machinery. In contrast, independent of CL, a homotypic trans-OPA1 interaction mediates membrane tethering, thereby supporting the cristae structure. Thus, multiple OPA1 functions are modulated by local CL conditions for regulation of mitochondrial morphology and quality control.
• Cutting Edge: Class II-like Structural Features and Strong Receptor Binding of the Nonclassical HLA-G2 Isoform Homodimer

  Kimiko Kuroki, Kazuhiro Mio, Ami Takahashi, Haruki Matsubara, Yoshiyuki Kasai, Sachie Manaka, Masahide Kikkawa, Daizo Hamada, Chikara Sato, Katsumi Maenaka

  JOURNAL OF IMMUNOLOGY 198 9 3399 - 3403 2017年05月 [査読有り][通常論文]
   

  HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and (beta 2-microglobulin (beta 2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the alpha 2 domain-deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown. In this study, we unexpectedly found that HLA-G2 naturally forms a beta 2m-free and nondisulfide-linked homodimer, which is in contrast to the disulfide-bonded beta 2m-associated HLA-G1 homodimer. Furthermore, single-particle analysis, using electron microscopy, revealed that the overall structure and domain organization of the HLA-G2 homodimer resemble those of the HLA class II heterodimer. The HLA-G2 homodimer binds to leukocyte Ig-like receptor B2 with slow dissociation and a significant avidity effect. These findings provide novel insights into leukocyte Ig-like receptor B2-mediated immune regulation by the HLA-G2 isoform, as well as the gene evolution of HLA classes.
• Divergent synthesis of kinase inhibitor derivatives, leading to discovery of selective Gck inhibitors

  Takanori Matsumaru, Makoto Inai, Kana Ishigami, Toshiki Iwamatsu, Hiroshi Maita, Satoko Otsuguro, Takao Nomura, Akira Matsuda, Satoshi Ichikawa, Masahiro Sakaitani, Satoshi Shuto, Katsumi Maenaka, Toshiyuki Kan

  BIOORGANIC & MEDICINAL CHEMISTRY LETTERS 27 10 2144 - 2147 2017年05月 [査読有り][通常論文]
   

  We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors. (C) 2017 Elsevier Ltd. All rights reserved.
• A trimeric structural fusion of an antagonistic tumor necrosis factor-α mutant enhances molecular stability and enables facile modification.

  Inoue M, Ando D, Kamada H, Taki S, Niiyama M, Mukai Y, Tadokoro T, Maenaka K, Nakayama T, Kado Y, Inoue T, Tsutsumi Y, Tsunoda SI

  The Journal of biological chemistry 292 16 6438 - 6451 2017年04月 [査読有り][通常論文]
   

  Tumor necrosis factor-alpha (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo. Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.
• Isolated Polar Amino Acid Residues Modulate Lipid Binding in the Large Hydrophobic Cavity of CD1d

  Shinsuke Inuki, Toshihiko Aiba, Natsumi Hirata, Osamu Ichihara, Daisuke Yoshidome, Shunsuke Kita, Katsumi Maenaka, Koichi Fukase, Yukari Fujimoto

  ACS CHEMICAL BIOLOGY 11 11 3132 - 3139 2016年11月 [査読有り][通常論文]
   

  The CD1d protein is a nonpolymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self- and foreign-lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A' pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipid protein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A' pocket could be used for the modulation of the lipid binding. A series of the ligands, alpha-galactosyl ceramide (alpha-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.
• Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection

  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.
• Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability (vol 8, 216, 2016)

  Maino Tahara, Jean-Philippe Buerckert, Kazuhiko Kanou, Katsumi Maenaka, Claude P. Muller, Makoto Takeda

  VIRUSES-BASEL 8 11 2016年11月 [査読有り][通常論文]
   

  The authors wish to make the following change to their paper [1].[...].
• HIV-1 Control by NK Cells via Reduced Interaction between KIR2DL2 and HLA-C^∗12:02/C^∗14:03.

  Lin Z, Kuroki K, Kuse N, Sun X, Akahoshi T, Qi Y, Chikata T, Naruto T, Koyanagi M, Murakoshi H, Gatanaga H, Oka S, Carrington M, Maenaka K, Takiguchi M

  Cell reports 17 9 2210 - 2220 2016年11月 [査読有り][通常論文]
   

  Natural killer (NK) cells control viral infection in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands. We investigated 504 anti-retroviral (ART)-free Japanese patients chronically infected with HIV-1 and identified two KIR/HLA combinations, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03, that impact suppression of HIV-1 replication. KIR2DL2(+) NK cells suppressed viral replication in HLA-C*14:03(+) or HLA-C*12:02(+) cells to a significantly greater extent than did KIR2DL2(-) NK cells in vitro. Functional analysis showed that the binding between HIV-1-derived peptide and HLA-C*14:03 or HLA-C*12:02 influenced KIR2DL2(+) NK cell activity through reduced expression of the peptide-HLA (pHLA) complex on the cell surface (i.e., reduced KIR2DL2 ligand expression), rather than through reduced binding affinity of KIR2DL2 to the respective pHLA complexes. Thus, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03 compound genotypes have protective effects on control of HIV-1 through a mechanism involving KIR2DL2-mediated NK cell recognition of virusinfected cells, providing additional understanding of NK cells in HIV-1 infection.
• The immunosuppressive effect of domain-deleted dimer of HLA-G2 isoform in collagen-induced arthritis mice

  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.
• Establishment of the BacMam system using silkworm baculovirus

  Atsutoshi Imai, Takashi Tadokoro, Shunsuke Kita, Masataka Horiuchi, Hideo Fukuhara, Katsumi Maenaka

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 478 2 580 - 585 2016年09月 [査読有り][通常論文]
   

  The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy. (C) 2016 Elsevier Inc. All rights reserved.
• Chemical Screening Identifies EUrd as a Novel Inhibitor Against Temozolomide-Resistant Glioblastoma-Initiating Cells

  Yoshihiro Tsukamoto, Naoki Ohtsu, Smile Echizenya, Satoko Otsuguro, Ryosuke Ogura, Manabu Natsumeda, Mizuho Isogawa, Hiroshi Aoki, Satoshi Ichikawa, Masahiro Sakaitani, Akira Matsuda, Katsumi Maenaka, Yukihiko Fujii, Toru Kondo

  STEM CELLS 34 8 2016 - 2025 2016年08月 [査読有り][通常論文]
   

  Glioblastoma (GBM), one of the most malignant human cancers, frequently recurs despite multi-modal treatment with surgery and chemo/radiotherapies. GBM-initiating cells (GICs) are the likely cell-of-origin in recurrences, as they proliferate indefinitely, form tumors in vivo, and are resistant to chemo/radiotherapies. It is therefore crucial to find chemicals that specifically kill GICs. We established temozolomide (the standard medicine for GBM)-resistant GICs (GICRs) and used the cells for chemical screening. Here, we identified 1-(3-C-ethynyl-beta-D-ribopentofuranosyl) uracil (EUrd) as a selective drug for targeting GICRs. EUrd induced the death in GICRs more effectively than their parental GICs, while it was less toxic to normal neural stem cells. We demonstrate that the cytotoxic effect of EUrd on GICRs partly depended on the increased expression of uridine-cytidine kinase-like 1 (UCKL1) and the decreased one of 5'-nucleotidase cytosolic III (NT5C3), which regulate uridine-monophosphate synthesis positively and negatively respectively. Together, these findings suggest that EUrd can be used as a new therapeutic drug for GBM with the expression of surrogate markers UCKL1 and NT5C3.
• Synthesis and Th1-immunostimulatory activity of α-galactosylceramide analogues bearing a halogen-containing or selenium-containing acyl chain.

  Hossain MI, Hanashima S, Nomura T, Lethu S, Tsuchikawa H, Murata M, Kusaka H, Kita S, Maenaka K

  Bioorganic & medicinal chemistry 24 16 3687 - 3695 2016年08月 [査読有り][通常論文]
   

  A novel series of CD1d ligand alpha-galactosylceramides (alpha-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of alpha-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes. (C) 2016 Elsevier Ltd. All rights reserved.
• Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

  Maino Tahara, Jean-Philippe Burckert, Kazuhiko Kanou, Katsumi Maenaka, Claude P. Muller, Makoto Takeda

  VIRUSES-BASEL 8 8 2016年08月 [査読有り][通常論文]
   

  Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.
• Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes

  Yuichiro Fujieda, Olga Amengual, Masaki Matsumoto, Kimiko Kuroki, Hidehisa Takahashi, Michihito Kono, Takashi Kurita, Kotaro Otomo, Masaru Kato, Kenji Oku, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Katsumi Maenaka, Shigetsugu Hatakeyama, Keiichi I. Nakayama, Tatsuya Atsumi

  RHEUMATOLOGY 55 6 1117 - 1126 2016年06月 [査読有り][通常論文]
   

  Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression. Methods. RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR). Results. RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT. Conclusion. We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.
• Structures and Functions of MHC-like Proteins

  Shunsuke Kita, Katsumi Maenaka

  Encyclopedia of Immunobiology 2 271 - 278 2016年04月27日 [査読有り][通常論文]
   

  Classical MHC molecules utilize their α1-α2 domains to display endogenous (self and nonself) peptides for antigen presentation to immune cells. However, over the past few decades many investigations have revealed a wide range of structural and functional variations of MHC superfamily members. Here we focus on the MHC-like proteins harboring distinct functions from those of the classical MHCs, such as Fc binding to transport IgG (FcRn), fatty acid homeostasis (ZAG), Fe ion metabolism (HFE), other immune responses (T proteins, M proteins, and ULBPs), and immune evasion of viral infection (UL18, UL142, m138, m144, m145, m152, m155, m157, etc.). These MHC-like proteins have basically lost the ability to present peptides, due to modulations of the typical antigen-binding groove. Instead, the surfaces have become receptor recognition sites. We further highlight the various tactics employed to elegantly control the physiological functions by using expanded and diversified protein surfaces, such as those generated by domain deletion and heavy sugar modification.
• Binding affinity and biological activity evaluation of novel C-type lectin Mincle ligands

  Matsumaru Takanori, Furukawa Atsushi, Ikeno Risa, Shuchi Yusuke, Maenaka Katsumi

  ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY 251 2016年03月13日 [査読有り][通常論文]
  
• Antibody-mediated molecular-targeted therapy for adult T-cell leukemia: recent progress and future challenges in the treatment of cancers.

  Maeda N, Matsuda A, Maenaka K

  Cancer Cell & Microenvironment 3 2 e1201  2016年03月 [査読有り][通常論文]
  
• Structural aspects of C-type lectin receptors

  Atsushi Furukawa, Shunsuke Kita, Takashi Tadokoro, Hideo Fukuhara, Katsumi Maenaka

  C-Type Lectin Receptors in Immunity 179 - 190 2016年01月01日 [査読有り][通常論文]
   

  Numerous structural analyses (X-ray crystallography and NMR) of C-type lectin receptors (CLRs) have been performed, because CLRs are not only attractive as important molecules in immunity and infectious diseases but also as drug targets. In CLRs, high amino acid sequence similarity exists in the extracellular carbohydrate recognition domains (CRDs), which are responsible for ligand binding. However, recent functional analyses of CLRs implied that these molecules recognize a wide variety of ligands in addition to saccharides, including glycopeptides, glycolipids, and proteins. In this chapter, we focus on structural studies of CLRs. We first summarize the structural features conserved among the CRDs and then describe how each C-type lectin receptor elegantly achieves its distinct ligand specificity, by illustrating the structural aspects of several representative CLRs.
• Osteopontin-integrin interaction as a novel molecular target for antibody-mediated immunotherapy in adult T-cell leukemia

  Naoyoshi Maeda, Takashi Ohashi, Haorile Chagan-Yasutan, Toshio Hattori, Yayoi Takahashi, Hideo Harigae, Hiroo Hasegawa, Yasuaki Yamada, Masahiro Fujii, Katsumi Maenaka, Toshimitsu Uede

  RETROVIROLOGY 12 99 - 99 2015年11月 [査読有り][通常論文]
   

  Background: Adult T-cell leukemia (ATL) is a CD4(+) T-cell neoplasm with a poor prognosis. A previous study has shown that there is a strong correlation between the secreted matricellular protein osteopontin (OPN) level and disease severity in ATL patients. Here, we investigated the role of OPN in ATL pathogenesis and the possible application of anti-OPN monoclonal antibody (mAb) for ATL immunotherapy in NOD/Shi-scid, IL-2Rg(null) (NOG) mice. Results: Subcutaneous inoculation of ATL cell lines into NOG mice increased the plasma level of OPN, which significantly correlated with metastasis of the inoculated cells and survival time. Administration of an SVVYGLR motifrecognizing anti-OPN mAb resulted in inhibition not only of tumor growth but also of tumor invasion and metastasis. The number of fibroblast activating protein-positive fibroblasts was also reduced by this mAb. We then co-inoculated mouse embryonic fibroblasts (MEFs) isolated from wild-type (WT) or OPN knockout mice together with ATL-derived TL-OmI cells into the NOG mice. The mice co-inoculated with WT MEFs displayed a significant decrease in survival relative to those injected with TL-OmI cells alone and the absence of OPN in MEFs markedly improved the survival rate of TL-OmI-inoculated mice. In addition, tumor volume and metastasis were also reduced in the absence of OPN. Conclusion: We showed that the xenograft NOG mice model can be a useful system for assessment of the physiological role of OPN in ATL pathogenesis. Using this xenograft model, we found that fibroblast-derived OPN was involved in tumor growth and metastasis, and that this tumor growth and metastasis was significantly suppressed by administration of the anti-OPN mAbs. Our findings will lead to a novel mAb-mediated immunotherapeutic strategy targeting against the interaction of OPN with integrins on the tumor of ATL patients.
• Solution structure of an avirulence protein, AVR-Pia, from Magnaporthe oryzae

  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]
  
• The cell competition-based high-throughput screening identifies small compounds that promote the elimination of RasV12-transformed cells from epithelia

  Hajime Yamauchi, Takanori Matsumaru, Tomoko Morita, Susumu Ishikawa, Katsumi Maenaka, Ichigaku Takigawa, Kentaro Semba, Shunsuke Kon, Yasuyuki Fujita

  SCIENTIFIC REPORTS 5 15336 - 15336 2015年10月 [査読有り][通常論文]
   

  Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.
• Effect of hydrophobic scaffold on the cellular uptake and gene transfection activities of DNA-encapsulating liposomal nanoparticles via intracerebroventricular administration

  Hidetaka Akita, Taichi Nakatani, Kimiko Kuroki, Katsumi Maenaka, Kota Tange, Yuta Nakai, Hideyoshi Harashima

  INTERNATIONAL JOURNAL OF PHARMACEUTICS 490 1-2 142 - 145 2015年07月 [査読有り][通常論文]
   

  Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold. (C) 2015 Elsevier B.V. All rights reserved.
• Screening for FtsZ Dimerization Inhibitors Using Fluorescence Cross-Correlation Spectroscopy and Surface Resonance Plasmon Analysis

  Shintaro Mikuni, Kota Kodama, Akira Sasaki, Naoki Kohira, Hideki Maki, Masaharu Munetomo, Katsumi Maenaka, Masataka Kinjo

  PLOS ONE 10 7 e0130933  2015年07月 [査読有り][通常論文]
   

  FtsZ is an attractive target for antibiotic research because it is an essential bacterial cell division protein that polymerizes in a GTP-dependent manner. To find the seed chemical structure, we established a high-throughput, quantitative screening method combining fluorescence cross-correlation spectroscopy (FCCS) and surface plasmon resonance (SPR). As a new concept for the application of FCCS to polymerization-prone protein, Staphylococcus aureus FtsZ was fragmented into the N-terminal and C-terminal, which were fused with GFP and mCherry (red fluorescent protein), respectively. By this fragmentation, the GTP-dependent head-to-tail dimerization of each fluorescent labeled fragment of FtsZ could be observed, and the inhibitory processes of chemicals could be monitored by FCCS. In the first round of screening by FCCS, 28 candidates were quantitatively and statistically selected from 495 chemicals determined by in silico screening. Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening. Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR. Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).
• Crystal structure of extracellular domain of human lectin-like transcript 1 (LLT1), the ligand for natural killer receptor-P1A

  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.
• Heat Shock Protein 70 Regulates Degradation of the Mumps Virus Phosphoprotein via the Ubiquitin-Proteasome Pathway

  Hiroshi Katoh, Toru Kubota, Shunsuke Kita, Yuichiro Nakatsu, Natsuko Aoki, Yoshio Mori, Katsumi Maenaka, Makoto Takeda, Minoru Kidokoro

  JOURNAL OF VIROLOGY 89 6 3188 - 3199 2015年03月 [査読有り][通常論文]
   

  Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection.
• New Binding Face of C-type Lectin-like Domains

  Hideo Fukuhara, Atsushi Furukawa, Katsumi Maenaka

  STRUCTURE 22 12 1694 - 1696 2014年12月 [査読有り][通常論文]
   

  C-type lectin-like receptor 2 (CLEC-2) is a member of the C-type lectin (like) receptor (CLR) family that uses a Ca2+ binding domain to bind specific glycans. However, in this issue of Structure, Nagae and colleagues report on how the structures of CLEC-2 in complex with a glycopeptide podoplanin and a snake venom protein, rhodocytin, show a different mode of binding.
• The structural basis for receptor recognition of human interleukin-18

  Naotaka Tsutsumi, Takeshi Kimura, Kyohei Arita, Mariko Ariyoshi, Hidenori Ohnishi, Takahiro Yamamoto, Xiaobing Zuo, Katsumi Maenaka, Enoch Y. Park, Naomi Kondo, Masahiro Shirakawa, Hidehito Tochio, Zenichiro Kato

  NATURE COMMUNICATIONS 5 5340 - 5340 2014年12月 [査読有り][通常論文]
   

  Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor alpha (R alpha) and beta (R beta) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors' recognition mode for IL-18 is similar to IL-1 beta; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.
• The N-terminal Arg Residue Is Essential for Autocatalytic Activation of a Lipopolysaccharide-responsive Protease Zymogen

  Yuki Kobayashi, Takafumi Shiga, Toshio Shibata, Miyuki Sako, Katsumi Maenaka, Takumi Koshiba, Hikaru Mizumura, Toshio Oda, Shun-ichiro Kawabata

  JOURNAL OF BIOLOGICAL CHEMISTRY 289 37 25987 - 25995 2014年09月 [査読有り][通常論文]
   

  Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.
• Efficient and cost effective production of active-form human PKB using silkworm larvae

  Ryoko Maesaki, Ryosuke Satoh, Masato Taoka, Teppei Kanaba, Tsunaki Asano, Chiharu Fujita, Toshinobu Fujiwara, Yutaka Ito, Toshiaki Isobe, Toshio Hakoshima, Katsumi Maenaka, Masaki Mishima

  SCIENTIFIC REPORTS 4 6016 - 6016 2014年08月 [査読有り][通常論文]
   

  Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKB alpha using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKB alpha protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKB alpha/20 larvae was recorded. Kinase assays showed that the recombinant PKB alpha possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKB alpha with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
• Structural basis for simultaneous recognition of an O-glycan and its attached peptide of mucin family by immune receptor PILRα.

  Kuroki K, Wang J, Ose T, Yamaguchi M, Tabata S, Maita N, Nakamura S, Kajikawa M, Kogure A, Satoh T, Arase H, Maenaka K

  Proceedings of the National Academy of Sciences of the United States of America 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.
• The Host Protease TMPRSS2 Plays a Major Role in In Vivo Replication of Emerging H7N9 and Seasonal Influenza Viruses

  Kouji Sakai, Yasushi Ami, Maino Tahara, Toru Kubota, Masaki Anraku, Masako Abe, Noriko Nakajima, Tsuyoshi Sekizuka, Kazuya Shirato, Yuriko Suzaki, Akira Ainai, Yuichiro Nakatsu, Kazuhiko Kanou, Kazuya Nakamura, Tadaki Suzuki, Katsuhiro Komase, Eri Nobusawa, Katsumi Maenaka, Makoto Kuroda, Hideki Hasegawa, Yoshihiro Kawaoka, Masato Tashiro, Makoto Takeda

  JOURNAL OF VIROLOGY 88 10 5608 - 5616 2014年05月 [査読有り][通常論文]
   

  Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2(+/+) wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with >= 1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo.
• Heterologous production, purification, and immunodetection of Magnaporthe oryzae avirulence protein AVR-Pia

  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.
• NKG2D triggers cytotoxicity in murine epidermal γδ T cells via PI3K-dependent, Syk/ZAP70-independent signaling pathway.

  Ibusuki A, Kawai K, Yoshida S, Uchida Y, Nitahara-Takeuchi A, Kuroki K, Kajikawa M, Ose T, Maenaka K, Kasahara M, Kanekura T

  The Journal of investigative dermatology 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.
• Allosteric Regulation of Epoxide Opening Cascades by a Pair of Epoxide Hydrolases in Monensin Biosynthesis

  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.
• 1P005 Lectin-like transcript 1 (LLT1)の構造解析とLLT1-CD161 複合体のモデル作製(蛋白質 : 構造,ポスター,第52回日本生物物理学会年会(2014年度))

  Shunsuke Kita, Haruki Matsubara, Jun Kamishikiryo, Yuki Okabe, Hideo Fukuhara, Kimiko Kuroki, Katsumi Maenaka

  生物物理 54 1 S141  一般社団法人 日本生物物理学会 2014年
• Crystal Structure of the Lamprey Variable Lymphocyte Receptor C Reveals an Unusual Feature in Its N-Terminal Capping Module

  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.
• TMPRSS2 Is an Activating Protease for Respiratory Parainfluenza Viruses

  Masako Abe, Maino Tahara, Kouji Sakai, Hiromi Yamaguchi, Kazuhiko Kanou, Kazuya Shirato, Miyuki Kawase, Masahiro Noda, Hirokazu Kimura, Shutoku Matsuyama, Hideo Fukuhara, Katsumi Mizuta, Katsumi Maenaka, Yasushi Ami, Mariko Esumi, Atsushi Kato, Makoto Takeda

  JOURNAL OF VIROLOGY 87 21 11930 - 11935 2013年11月 [査読有り][通常論文]
   

  Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.
• Structural and functional mosaic nature of MHC class I molecules in their peptide-free form

  Eiji Kurimoto, Kimiko Kuroki, Yoshiki Yamaguchi, Maho Yagi-Utsumi, Takahiro Igaki, Takeshi Iguchi, Katsumi Maenaka, Koichi Kato

  Molecular Immunology 55 3-4 393 - 399 2013年10月 [査読有り][通常論文]
   

  Despite well-organized peptide-loading mechanisms within the endoplasmic reticulum, major histocompatibility complex class I (MHC-I) molecules can be displayed on cell surfaces in peptide-free forms. Although these empty MHC-I (eMHC-I) molecules are presumably involved in physiological and pathological processes, little is known about their structures and functions due to their instability. Using bacterially expressed HLA-Cw*07:02 heavy chain and β2 microglobulin molecules, we successfully established an in vitro refolding method to prepare eMHC-I molecules in sufficient quantities for detailed structural analyses. NMR spectroscopy in conjunction with subunit-specific 15N-labeling techniques revealed that the peptide-binding domains and the adjacent regions were unstructured in the peptide-free form, while the remaining regions maintained their structural integrity. Consistent with our spectroscopic data, the eMHC-I complex could interact with leukocyte Ig-like receptor B1, but not with killer cell Ig-like receptor 2DL3. Thus, eMHC-I molecules have a mosaic nature in terms of their three-dimensional structure and binding to immunologically relevant molecules. © 2013 Elsevier Ltd.
• Structural analysis for glycolipid recognition by the C-type lectins Mincle and MCL

  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.
• Functional Characterization of a Juvenile Hormone Esterase Related Gene in the Moth Sesamia nonagrioides through RNA Interference

  Dimitrios Kontogiannatos, Luc Swevers, Katsumi Maenaka, Enoch Y. Park, Kostas Iatrou, Anna Kourti

  PLOS ONE 8 9 e73834  2013年09月 [査読有り][通常論文]
   

  Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.
• Entry Mechanism of Morbillivirus Family

  Hideo Fukuhara, Surui Chen, Shin Takeda, Katsumi Maenaka

  YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 133 5 549 - 559 2013年05月 [査読有り][通常論文]
   

  The genus Morbillivirus includes measles virus, canine distemper virus and rinderpest virus. These are highly contagious and exhibit high mortality. These viruses have the attachment glycoprotein, hemagglutinin (H), at the virus surface, which bind to signaling lymphocyte activation molecule (SLAM) and Nectin 4 as receptors for the entry. However, the molecular mechanism for this entry has been limitedly understood. Here we summarize the current topics, (1) newly identified receptor, Nectin 4, (2) crystal structures of H-receptor complexes and (3) detail biochemical studies of the H-F communication for the entry. These provide insight on the mechanism of morbillivirus entry event and furthermore drug developments.
• The long-term immunosuppressive effects of disulfide-linked HLA-G dimer in mice with collagen-induced arthritis

  Kimiko Kuroki, Kaoru Hirose, Yuki Okabe, Yuko Fukunaga, Ami Takahashi, Mitsunori Shiroishi, Mizuho Kajikawa, Shigekazu Tabata, Seiko Nakamura, Toshiyuki Takai, Satoru Koyanagi, Shigehiro Ohdo, Katsumi Maenaka

  HUMAN IMMUNOLOGY 74 4 433 - 438 2013年04月 [査読有り][通常論文]
   

  HLA-G, a natural immunosuppressant present in the human placenta during pregnancy, prevents fetal destruction by the maternal immune system. The immunosuppressive effect of HLA-G is mediated by the immune cell inhibitory receptors, LILRB1 and LILRB2. HLA-G forms disulfide-linked dimers by natural oxidation, and the dimer associates with LILRB1/B2 much more strongly than the monomer. Furthermore, the dimer formation remarkably enhanced the LILRB-mediated signaling. In this report, we studied the in vivo immunosuppressive effect of the HLA-G dimer, using the collagen-induced arthritis model mouse. Mice were treated with the HLA-G monomer or dimer intracutaneously at the left foot joint, once or for 5 days, and the clinical severity was evaluated daily in a double-blind study. The HLA-G monomer and dimer both produced excellent anti-inflammatory effects with a single, local administration. Notably, as compared to the monomer, the dimer exhibited significant immunosuppressive effects at lower concentrations, which persisted for about two months. In accordance with this result, a binding study revealed that the HLA-G dimer binds PIR-B, the mouse homolog of the LILRBs, with higher affinity and avidity than the monomer. The HLA-G dimer is expected to be quite useful as an anti-rheumatoid arthritis agent, in small amounts with minimal side effects. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
• The Receptor-Binding Site of the Measles Virus Hemagglutinin Protein Itself Constitutes a Conserved Neutralizing Epitope

  Maino Tahara, Shinji Ohno, Kouji Sakai, Yuri Ito, Hideo Fukuhara, Katsuhiro Komase, Melinda A. Brindley, Paul A. Rota, Richard K. Plemper, Katsumi Maenaka, Makoto Takeda

  JOURNAL OF VIROLOGY 87 6 3583 - 3586 2013年03月 [査読有り][通常論文]
   

  Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.
• Distinct HIV-1 Escape Patterns Selected by Cytotoxic T Cells with Identical Epitope Specificity

  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.
• Functional and Structural Characterization of Neutralizing Epitopes of Measles Virus Hemagglutinin Protein

  Maino Tahara, Yuri Ito, Melinda A. Brindley, Xuemin Ma, Jilan He, Songtao Xu, Hideo Fukuhara, Kouji Sakai, Katsuhiro Komase, Paul A. Rota, Richard K. Plemper, Katsumi Maenaka, Makoto Takeda

  JOURNAL OF VIROLOGY 87 1 666 - 675 2013年01月 [査読有り][通常論文]
   

  Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.
• Canine distemper virus with the intact C protein has the potential to replicate in human epithelial cells by using human nectin4 as a receptor

  Noriyuki Otsuki, Tsuyoshi Sekizuka, Fumio Seki, Kouji Sakai, Toru Kubota, Yuichiro Nakatsu, Surui Chen, Hideo Fukuhara, Katsumi Maenaka, Ryoji Yamaguchi, Makoto Kuroda, Makoto Takeda

  VIROLOGY 435 2 485 - 492 2013年01月 [査読有り][通常論文]
   

  Recent outbreaks in monkeys have proven that canine distemper virus (CDV) causes diseases in a wide range of mammals. CDV uses SLAM and nectin4 as receptors to replicate in susceptible animals. Here, we show that human nectin4, but not human SLAM, is fully functional as a CDV receptor. The CDV Ac96I strain hardly replicated in nectin4-expressing human epithelial NCI-H358 cells, but readily adapted to grow in them. Unsurprisingly, no amino acid change in the H protein was required for the adaptation. The original Ac96I strain possessed a truncated C protein, and a subpopulation possessing the intact C protein was selected after growth in NCI-H358 cells. Other CDV strains possessing the intact C protein showed significantly higher growth abilities in NCI-H358 cells than the Ac96I strain with the truncated C protein. These findings suggest that the C protein is functional in human epithelial cells and critical for CDV replication in them. (C) 2012 Elsevier Inc. All rights reserved.
• 3P006 HLA-G2/G6アイソフォームの単粒子構造解析(01A.蛋白質:構造,ポスター,日本生物物理学会年会第51回(2013年度))

  Mio Kazuhiro, Kuroki Kimiko, Matsubara Haruki, Kasai Yoshiyuki, Sato Chikara, Maenaka Katsumi

  生物物理 53 1 S212  一般社団法人 日本生物物理学会 2013年
• Fabrication of growth factor- and extracellular matrix-loaded, gelatin-based scaffolds and their biocompatibility with Schwann cells and dorsal root ganglia

  Rodolfo E. Gamez Sazo, Katsumi Maenaka, Weiyong Gu, Patrick M. Wood, Mary Bartlett Bunge

  BIOMATERIALS 33 33 8529 - 8539 2012年11月 [査読有り][通常論文]
   

  One of the most exciting new avenues of research to repair the injured spinal cord is to combine cells for implantation with scaffolds that protect the cells and release growth factors to improve their survival and promote host axonal regeneration. To realize this goal, we fabricated biodegradable, photocurable gelatin tubes and membranes for exploratory in vitro studies. Detailed methods are described for their fabrication with a high gelatin concentration. Gelatin membranes fabricated in the same way as tubes and photo-co-immobilized with rhBDNF or rhNT-3, with or without Schwann cells (SCs), showed an initial burst of neurotrophin release within 24 h, with release diminishing progressively for 21 days thereafter. SCs attained their typical bipolar conformation on membranes without neurotrophins but adhesion, alignment and proliferation were improved with neurotrophins, particularly rhBDNF. When dorsal root ganglion explants were cultured on membranes containing laminin and fibronectin plus both neurotrophins, neurite outgrowth was lengthier compared to combining one neurotrophin with laminin and fibronectin. Thus, these gelatin membranes allow SC survival and effectively release growth factors and harbor extracellular matrix components to improve cell survival and neurite growth. These scaffolds, based on the combination of cross-linked gelatin technology and incorporation of neurotrophins and extracellular matrix components, are promising candidates for spinal cord repair. (C) 2012 Elsevier Ltd. All rights reserved.
• Inhibiting HLA-B27 homodimer-driven immune cell inflammation in spondylarthritis

  Sravan K. Payeli, Simon Kollnberger, Osiris Marroquin Belaunzaran, Markus Thiel, Kirsty McHugh, Joanna Giles, Jacqueline Shaw, Sascha Kleber, Anna Ridley, Isabel Wong-Baeza, Sarah Keidel, Kimiko Kuroki, Katsumi Maenaka, Andreas Wadle, Christoph Renner, Paul Bowness

  ARTHRITIS AND RHEUMATISM 64 10 3139 - 3149 2012年10月 [査読有り][通常論文]
   

  Objective Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLAB27 normally forms complexes with beta 2-microglobulin (beta 2m) and peptide to form heterotrimers. However, an unusual characteristic of HLAB27 is its ability to form beta 2m-free heavy chain homodimers (HLAB272), which, unlike classic HLAB27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLAB272 to KIR-3DL2positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLAB272 in order to confirm its expression in SpA and to inhibit its proinflammatory properties. Methods We generated monoclonal antibodies by screening a human phage display library positively against B272 and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B272-expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2B272 interactions was tested using cell lines and PBMCs from patients with SpA. Results Monoclonal antibody HD6 specifically recognized recombinant HLAB272 by ELISA and by SPR assay. HD6 bound to cell lines expressing B272. FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLAB272 to KIR-3DL2 and the survival and proliferation of KIR-3DL2positive NK cells. Finally, HD6 inhibited production of the proinflammatory diseaseassociated cytokine interleukin-17 by PBMCs from patients with AS. Conclusion These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.
• Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain

  Jae Man Lee, Naoya Kawakami, Hiroaki Mon, Hitoshi Mitsunobu, Kazuhiro Iiyama, Satoshi Ninaki, Katsumi Maenaka, Enoch Y. Park, Takahiro Kusakabe

  BIOTECHNOLOGY LETTERS 34 10 1773 - 1779 2012年10月 [査読有り][通常論文]
   

  Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90 % on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.
• Ribophorin II Is Involved in the Tissue Factor Expression Mediated by Phosphatidylserine-Dependent Antiprothrombin Antibody On Monocytes.

  Yuichiro Fujieda, Olga Amengual, Yusaku Kanetsuka, Toshio Odani, Kotaro Otomo, Kenji Oku, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Kimiko Kuroki, Katsumi Maenaka, Masaki Matsumoto, Shigetsugu Hatakeyama, Tatsuya Atsumi

  ARTHRITIS AND RHEUMATISM 64 10 S741 - S741 2012年10月 [査読有り][通常論文]
  
• Simultaneous inhibition of Src and Aurora kinases by SU6656 induces therapeutic synergy in human synovial sarcoma growth, invasion and angiogenesis in vivo

  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.
• Nectin4 Is an Epithelial Cell Receptor for Canine Distemper Virus and Involved in Neurovirulence

  Watanyoo Pratakpiriya, Fumio Seki, Noriyuki Otsuki, Kouji Sakai, Hideo Fukuhara, Hiromu Katamoto, Takuya Hirai, Katsumi Maenaka, Somporn Techangamsuwan, Nguyen Thi Lan, Makoto Takeda, Ryoji Yamaguchi

  JOURNAL OF VIROLOGY 86 18 10207 - 10210 2012年09月 [査読有り][通常論文]
   

  Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.
• HLA-B27 Homodimers and Free H Chains Are Stronger Ligands for Leukocyte Ig-like Receptor B_2 than Classical HLA Class I.

  前仲 勝実

  J Immunol 188 12 6184 - 6193 2012年06月 [査読有り][通常論文]
   

  Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with beta(2)-microglobulin (beta(2)m) and peptide and (beta 2m free) free H chain (FHC) forms including B27 dimers (termed B27(2)) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR) B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a K-D of 5.3 +/- 1.5 mu M but did not bind B27 FHC. LILRB2 bound to B27(2) and B27 FHC and B27 heterotrimers with K(D)s of 2.5, 2.6, and 22 +/- 6 mu M, respectively. Domain exchange experiments showed that B27(2) bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with K(D)s of 15.0 +/- 0.8 and 16.0 +/- 2.0 mu M, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis. The Journal of Immunology, 2012, 188: 6184-6193.
• InvolvementofanNKG2DLigandH60c in Epidermal Dendritic T Cell-Mediated Wound Repair

  前仲 勝実

  J Immunol 188 8 3972 - 3979 2012年04月15日 [査読有り][通常論文]
   

  Dendritic epidermal T cells (DETCs) found in mouse skin are NKG2D-positive γδ T cells involved in immune surveillance and wound repair. It is assumed that the interaction of an NKG2D receptor on DETCs and an MHC class I-like NKG2D ligand on keratinocytes activates DETCs, which then secrete cytokines promoting wound repair. However, direct evidence that DETC activation through NKG2D signaling promotes wound repair is not available. In the present study, we generated mAbs for an NKG2D ligand H60c previously suggested to be expressed specifically on skin keratinocytes. Local administration of H60c-specific mAb inhibited activation of DETCs and significantly delayed wound repair. Likewise, administration of NKG2D-specific mAb impaired wound repair to a similar extent. The delay in wound closure resulting from the blockade of the NKG2D pathway was comparable to that observed in γδ T cell-deficient mice. These results indicate that H60c/NKG2D interactions play a critical role in wound repair. Reassessment of binding affinities showed that H60c monomers bind to NKG2D with affinity (K d = 26 ± 3.2 nM) comparable to those of other high-affinity NKG2D ligands. H60c is transcribed not only in skin but also in tissues such as tongue and female reproductive tract known to contain epithelium-resident γδ T cells expressing invariant TCRs, suggesting a more general role for H60c in the maintenance of epithelial integrity. © 2012 by The American Association of Immunologists, Inc.
• Blockade of interaction between NKG2D and an NKG2D ligand H60c delays wound repair

  Yoshida S, Mohamed RH, Kajikawa M, Koizumi J, Tanaka M, Fugo K, Otsuka N, Maenaka K, Yagita H, Chiba H, Kasahara M

  Journal of Immunology 188 8 3972 - 3979 2012年04月 [査読有り][通常論文]
  
• Crystallization Strategy for the Glycoprotein-Receptor Complex Between Measles Virus Hemagglutinin and Its Cellular Receptor SLAM

  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.
• [Structural basis for measles virus-receptor recognition and its functional implications for viral entry and vaccination].

  Maenaka K, Hashiguchi T, Yanagi Y

  Nihon rinsho. Japanese journal of clinical medicine 70 4 695 - 703 2012年04月 [査読有り][通常論文]
   

  Measles is one of the most contagious and devastating viral diseases. However, highly effective live vaccines have been successfully used for more than five decades. Recent structural studies of measles virus hemagglutinin and its complexes with receptors (the signaling lymphocyte activation molecule (SLAM, CD150) and CD46) have provided many insights into measles virus entry mechanism. Furthermore, the sugar shields that cover the large surface areas of the hemagglutinin have implications for effective measles virus vaccination. These studies should help strengthen current global efforts to control and eliminate measles worldwide.
• Molecular recognition of paired receptors in the immune system

  Kimiko Kuroki, Atsushi Furukawa, Katsumi Maenaka

  FRONTIERS IN MICROBIOLOGY 3 429 - 429 2012年 [査読有り][通常論文]
   

  Cell surface receptors are responsible for regulating cellular function on the front line, the cell membrane. Interestingly, accumulating evidence clearly reveals that the members of cell surface receptor families have very similar extracellular ligand-binding regions but opposite signaling systems, either inhibitory or stimulatory. These receptors are designated as paired receptors. Paired receptors often recognize not only physiological ligands but also non-self ligands, such as viral and bacterial products, to fight infections. In this review, we introduce several representative examples of paired receptors, focusing on two major structural superfamilies, the immunoglobulin-like and the C-type lectin-like receptors, and explain how these receptors distinguish self and non-self ligands to maintain homeostasis in the immune system. We further discuss the evolutionary aspects of these receptors as well as the potential drug targets for regulating diseases.
• Molecular Basis for Herpesvirus Entry Mediator Recognition by the Human Immune Inhibitory Receptor CD160 and Its Relationship to the Cosignaling Molecules BTLA and LIGHT

  Rieko Kojima, Mizuho Kajikawa, Mitsunori Shiroishi, Kimiko Kuroki, Katsumi Maenaka

  JOURNAL OF MOLECULAR BIOLOGY 413 4 762 - 772 2011年11月 [査読有り][通常論文]
   

  CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4(+) T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function. (C) 2011 Elsevier Ltd. All rights reserved.
• Construction, Expression, and Characterization of a Single-Chain Variable Fragment Antibody Against 2,4-Dichlorophenoxyacetic Acid in the Hemolymph of Silkworm Larvae

  Seiichi Sakamoto, Benyakan Pongkitwitoon, Seiko Nakamura, Kaori Sasaki-Tabata, Yusuke Tanizaki, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto

  APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY 164 6 715 - 728 2011年07月 [査読有り][通常論文]
   

  A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 mu g per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
• Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

  Jun Kamishikiryo, Hideo Fukuhara, Yuki Okabe, Kimiko Kuroki, Katsumi Maenaka

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 27 23823 - 23830 2011年07月 [査読有り][通常論文]
   

  Human Th17 cells express high levels of CD161, a member of the killer cell lectin-like receptor (KLR) family (also referred to as NK receptor-P1A (NKRP1A) or KLRB1), as a representative marker. CD161 is also expressed on natural killer (NK) cells and NKT cells. Lectin-like transcript 1 (LLT1), another KLR family member, was recently identified as a ligand for CD161. This interaction may play pivotal roles in the immunomodulatory functions of Th17 cells as well as those of NK and NKT cells. However, the molecular basis for the interaction is poorly understood. Here we show that the extracellular domain of CD161 bound directly to LLT1 with a K(d) of 48 mu M and with the fast kinetics typical of cell-cell recognition receptors. Mutagenesis revealed that the similar membrane-distal beta-sheet and loop regions of both CD161 and LLT1 were utilized for the binding, and notably, these regions correspond to the ligand-binding sites for major histocompatibility complex (MHC)-recognizing KLRs. Furthermore, we found a pair of detrimental mutations for both molecules that restored the binding. These results reveal a new template model for the recognition mode between the KLR family members and provide insights into the molecular mechanism underlying Th17/NK/NKT-mediated immune responses.
• Differential but Competitive Binding of Nogo Protein and Class I Major Histocompatibility Complex (MHCI) to the PIR-B Ectodomain Provides an Inhibition of Cells

  Haruka Matsushita, Shota Endo, Eiji Kobayashi, Yuzuru Sakamoto, Keisuke Kobayashi, Kohji Kitaguchi, Kimiko Kuroki, Arvid Soderhall, Katsumi Maenaka, Akira Nakamura, Stephen M. Strittmatter, Toshiyuki Takai

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 29 25739 - 25747 2011年07月 [査読有り][通常論文]
   

  Binding of class I MHC molecules (MHCI) to an inhibitory receptor, PIR-B, expressed on B cells and myeloid cells provides constitutive cellular inhibition, thus ensuring peripheral tolerance. Recent unexpected findings pointed to a novel inhibitory role of PIR-B in neuritere generation through binding to three axonal outgrowth inhibitors of myelin, including Nogo. Thus, it becomes interesting to determine whether the actions of the inhibitory myelin proteins and MHCI could coexist independently or be mutually exclusive as to the PIR-B-mediated immune and neural cell inhibition. Here, we present data supporting the competition of Nogo-and MHCI-mediated inhibition where they coexist. Kinetic analyses of Nogo and MHCI binding to the whole or a part of the recombinant PIR-B ectodomain revealed that PIR-B binds with higher affinity to Nogo than MHCI and that the MHCI binding only occurred with the N-terminal domains of PIR-B, whereas Nogo binding occurred with either the N- or C-terminal ectodomains. Importantly, kinetic tests indicated that the binding to PIR-B of Nogo and MHCI was competitive. Both endogenous and exogenous Nogo intensified the PIR-B-mediated suppression of interleukin-6 release from lipopolysaccharide-stimulated wildtype, but not PIR-B-deficient, cultured mast cells, indicating that PIR-B mediates Nogo-induced inhibition. Thus, we propose a novel mechanism by which PIR-B-mediated regulation is achieved differentially but competitively via MHCI and Nogo in cells of the immune system.
• Structure of the measles virus hemagglutinin bound to its cellular receptor SLAM

  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.
• A fluorescent single domain antibody against plumbagin expressed in silkworm larvae for fluorescence-linked immunosorbent assay (FLISA)

  Seiichi Sakamoto, Benyakan Pongkitwitoon, Kaori Sasaki-Tabata, Waraporn Putalun, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto

  ANALYST 136 10 2056 - 2063 2011年 [査読有り][通常論文]
   

  A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 mu g mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.
• Analysis of receptor-ligand interactions by surface plasmon resonance.

  Kuroki K, Maenaka K

  Methods in molecular biology (Clifton, N.J.) 748 83 - 106 2011年 [査読有り][通常論文]
   

  Many immunological responses are often regulated by cell surface receptors in cell-cell recognition events. Such immune receptors on the cell surface typically exhibit low-affinity and fast-kinetic ligand interactions (e.g., K (d) in the μM range, k (off)  =  10(-2) to 20 s(-1)). Real-time surface plasmon resonance (SPR) detection systems are generally useful for determining these binding parameters. However, several technical points should be considered because the determination of low-affinity binding and fast kinetics is often rather difficult. Here, we introduce a general procedure for SPR experiments and, moreover, show typical examples for ligand binding of immune cell surface receptors, including experimentally useful tips. We also show how to determine the thermodynamic characteristics using the nonlinear van't Hoff and Arrhenius analyses. These affinity, kinetic, and thermodynamic parameters of immune-receptor binding are important for understanding immunological events as well as developing drugs and vaccines.
• Leukocyte Immunoglobulin-Like Receptor (LILR)ファミリーの分子認識—Structures and molecular recognition for Leukocyte Immunoglobulin-Like Receptor family

  黒木 喜美子, 前仲 勝実

  生化学 / 日本生化学会 編 83 8 715 - 726 東京 : 日本生化学会 2011年 [査読有り][通常論文]
  
• Measles virus hemagglutinin: structural insights into cell entry and measles vaccine

  Takao Hashiguchi, Katsumi Maenaka, Yusuke Yanagi

  FRONTIERS IN MICROBIOLOGY 2 247 - 247 2011年 [査読有り][通常論文]
   

  Measles is one of the most contagious viral diseases, and remains a major cause of childhood morbidity and mortality worldwide. The measles virus (MV), a member of the family Paramyxoviridae, enters cells through a cellular receptor, the signaling lymphocyte activation molecule (SLAM), CD46 or nectin-4. Entry is mediated by two MV envelope glycoproteins, the hemagglutinin (H) and the fusion (F) protein. The H protein mediates receptor attachment, while the F protein causes membrane fusion. The interaction between the H and F proteins is essential to initiate the cell entry process. Recently determined crystal structures of the MV-H protein unbound and bound to SLAM or CD46 have provided insights into paramyxovirus entry and the effectiveness of measles vaccine.
• Efficient silkworm expression of single-chain variable fragment antibody against ginsenoside Re using Bombyx mori nucleopolyhedrovirus bacmid DNA system and its application in enzyme-linked immunosorbent assay for quality control of total ginsenosides

  Seiichi Sakamoto, Benyakan Pongkitwitoon, Seiko Nakamura, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto

  JOURNAL OF BIOCHEMISTRY 148 3 335 - 340 2010年09月 [査読有り][通常論文]
   

  A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 mu g/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.
• Long-term control of HIV-1 in hemophiliacs carryin slow-progressing allele HLA-B*5101

  前仲 勝実

  J Virol 84 14 7151 - 7160 2010年07月 [査読有り][通常論文]
   

  HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101(+) hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101(+) hemophiliacs showed that the frequency of Pol283-8-specific CD8(+) T cells was inversely correlated with the viral load, whereas the frequencies of CD8(+) T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101(+) hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101(+) hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8(+) T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants.
• Dictyostelium Differentiation-Inducing Factor-1 Binds to Mitochondrial Malate Dehydrogenase and Inhibits Its Activity

  Tomoko Matsuda, Fumi Takahashi-Yanaga, Tatsuya Yoshihara, Katsumi Maenaka, Yutaka Watanabe, Yoshikazu Miwa, Sachio Morimoto, Yuzuru Kubohara, Masato Hirata, Toshiyuki Sasaguri

  JOURNAL OF PHARMACOLOGICAL SCIENCES 112 3 320 - 326 2010年03月 [査読有り][通常論文]
   

  We have reported that the differentiation-inducing factors (DIFs) DIF-1 and DIF-3, morphogens secreted from Dictyostelium discoideum, inhibit proliferation of several cancer cells via suppression of the Wnt/beta-catenin signaling pathway. However, the target molecules of DIFs involved in the anti-proliferative effects are still unknown. In the present study, DIF-1 tethered resins were synthesized to explore the target molecules of DIFs, and mitochondrial malate dehydrogenase (mMDH) was identified as one of the target molecules. In the in vitro assay, DIF-1 and other analogs including 2-MIDIF-1, DIF-3, and 6-MIDIF-3 were found to be capable of binding to mMDH but not to cytoplasmic MDH. However, only DIF-1 and 2-MIDIF-1 inhibited the enzymatic activity of mMDH. The effects of DIF analogs on ATP content and cell proliferation were then analyzed using HeLa cells. DIF-1 and 2-MIDIF-1 were found to lower the ATP content and both chemicals inhibited HeLa cell proliferation, suggesting that inhibition of mMDH activity affected cell energy production, probably leading to the inhibition of proliferation. These results suggest that the inhibition of mMDH activity by DIF-1 and 2-MIDIF-1 could be one of the mechanisms to induce anti-proliferative effects, independent of the inhibition of the Wnt/beta-catenin signaling pathway.
• Silkworm expression system as a platform technology in life science

  Tatsuya Kato, Mizuho Kajikawa, Katsumi Maenaka, Enoch Y. Park

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 85 3 459 - 470 2010年01月 [査読有り][通常論文]
   

  Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3-6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the "Silkroad," we expect that the "Bioroad" from Asia to Europe will be established by the silkworm expression system.
• Importance of the Hydrogen Bonding Network Including Asp52 for Catalysis, as Revealed by Asn59 Mutant Hen Egg-white Lysozymes

  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.
• Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).

  大木 出, 前仲 勝実

  J.Biol.Chem. 284 284 40 27327 - 27335 2009年10月 [査読有り][通常論文]
   

  The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca(2+)-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K(d) similar to 7-12 mu M), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1(+)NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.
• Silkworm expression and sugar profiling of human immune cell surface receptor, KIR2DL1.

  Kaori Sasaki, Mizuho Kajikawa, Kimiko Kuroki, Tomoko Motohashi, Tsukasa Shimojima, Enoch Y Park, Sachiko Kondo, Hirokazu Yagi, Koichi Kato, Katsumi Maenaka

  Biochemical and biophysical research communications 387 3 575 - 80 2009年09月25日 [査読有り][通常論文]
   

  Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni(2+) affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc and Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.
• HLA-G Molecule

  Jun Kamishikiryo, Katsumi Maenaka

  CURRENT PHARMACEUTICAL DESIGN 15 28 3318 - 3324 2009年09月 [査読有り][通常論文]
   

  Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule, which was first discovered in 1987 by Geraghty and colleagues [1]. While classical HLA class I molecules are expressed on all nucleated cells, the expression of the HLA-G molecule is highly tissue-restricted, such as to placental trophoblast cells. HLA-G binds inhibitory receptors such as leukocyte immunoglobulin-like receptors B1 (LILRB1/ILT2/CD85j) and LILRB2 (ILT4/CD85d), which are widely expressed on immune cells, to suppress a broad range of immune responses [2-4]. Thus, the expression of HLA-G in placenta protects the fetus from the maternal immune system. On the other hand, emerging studies have shown the relevance of the HLA-G molecule in pathologic conditions, such as transplantation rejection, autoimmunity, and cancer. HLA-G has other unique characteristics, in contrast with classical HLA molecules, including the existence of various forms of HLA-G: several splice variants, subunit-deficient conformations, homodimers, and their combinations have been found [5]. In this review, we highlight the molecular basis for the tolerogenic ability of the HLA-G molecule, especially by LILR recognition of various forms of HLA-G. We also discuss the potential clinical applications of HLA-G molecules.
• Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

  Mizuho Kajikawa, Kaori Sasaki, Yoshitaro Wakimoto, Masaru Toyooka, Tomoko Motohashi, Tsukasa Shimojima, Shigeki Takeda, Enoch Y. Park, Katsumi Maenaka

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 385 3 375 - 379 2009年07月 [査読有り][通常論文]
   

  Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS Using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha Subunit (G(i)alpha) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [(35)S]GTP gamma S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production. (C) 2009 Elsevier Inc. All rights reserved.
• Crystallization and Preliminary X-Ray Analysis of the Low-Affinity Complex Between Human Leukocyte Antigen-G (HLA-G) and Leukocyte Ig-Like Receptor B2 (LILRB2)

  Mitsunori Shiroishi, Katsumi Maenaka

  PROTEIN AND PEPTIDE LETTERS 16 4 447 - 449 2009年04月 [査読有り][通常論文]
   

  Human leukocyte antigen-G (HLA-G) is a nonclassical MHC class I (MHCI) molecule that is expressed mainly on placenta trophoblast cells. Leukocyte Ig-like receptor B2 (LILRB2) is a human inhibitory immune receptor that recognizes HLA-G with a higher affinity than any other MHCI although this interaction is only in the mu M range. The interaction between HLA-G and LILRB2 seems to play a dominant role in the escape of the fetus from the maternal immune response. Here we report the crystallization and x-ray analysis of the LILRB2/HLA-G complex. The extracellular domains of HLA-G and LILRB2 were expressed in Escherichia coli, refolded and purified. The initial crystallization trials using novel PEG-based screening sets provided crystals of the LILRB2/HLA-G complex with 40-50% PEG400 as the precipitant. These crystals belong to space group P3(1)21 (a=b=81.4 angstrom, c=186.7 angstrom, gamma=120 degrees). Dehydration of the crystals by soaking them in a solution containing a higher concentration of PEG400 dramatically improved the resolution and also the mosaicity.
• Reciprocal recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope by CD8(+) T cells and NK cells

  Hathairat Thananchai, Tariro Makadzange, Katsumi Maenaka, Kimiko Kuroki, Yanchun Peng, Chris Conlon, Sarah Rowland-Jones, Tao Dong

  AIDS 23 2 189 - 193 2009年01月 [査読有り][通常論文]
   

  Objectives: The HIV-1 Nef protein selectively downregulates human leukocyte antigen (HLA)-A and HLA-B but not HLA-C molecules on the Surface of infected cells. This allows HIV-infected cells to evade recognition by most cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We investigated the recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope SFNCGGEFF (SF9) and its variant SFNCGGEFL (SL9) by T cells and NK receptors. Design and method: Recognition of HIV-1 gp120 peptides (SF9 and SL9) by T-cell clones was measured by staining with HLA-Cw4-peptide tetrameric complexes and cytolytic assays using target cell pulsed with either peptides. KIR2DL1 binding to these two peptides was measured using surface plasmon resonance and tetramer staining of an NK cell line. Result: CTLs could recognize SF9 better than the variant SL9, as shown by both tetramer staining and cytolytic assays. Intriguingly, an HLA-Cw4 tetramer folded with the 'escape' variant SL9 could bind to KIR2DL1 on NK cell lines with higher affinity than HLA-Cw4-SF9. The binding of KIR2DL1 to its ligand results in inhibition of NK cell function. Our results indicate that the HIV-1 gp120 variant peptide SL9 could potentially escape both from NK cell and CTL recognition by increasing its affinity for KIR2DL1 binding. Conclusion: These data suggest that HIV-1 can acquire mutations that are capable of escaping from both CTL and NK cell recognition, a phenomenon we have termed 'double escape'. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins
• Enzyme-mediated protein refolding

  Jun Okada, Tatsuo Maruyama, Konomi Motomura, Kimiko Kuroki, Katsumi Maenaka, Masafumi Sakono, Masahiro Goto

  CHEMICAL COMMUNICATIONS 46 7197 - 7199 2009年 [査読有り][通常論文]
   

  We employed a urease-catalyzed reaction to gradually remove a high concentration of a chaotropic agent (urea) from a denatured protein solution and demonstrated that efficient protein refolding can be achieved by the urease-catalyzed reaction, without large-volume dilution.
• Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B-Mori nucleopolyhedrovirus

  Naoya Kawakami, Jae Man Lee, Hiroaki Mon, Yuji Kubo, Yutaka Banno, Yutaka Kawaguchi, Katsumi Maenaka, Enoch Y. Park, Katsumi Koga, Takahiro Kusakabe

  MOLECULAR BIOTECHNOLOGY 40 2 180 - 185 2008年10月 [査読有り][通常論文]
   

  The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a "factory" for large-scale expression using the BmNPV bacmid system.
• Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

  Narumi Aoki, Akie Sakiyama, Kimiko Kuroki, Katsumi Maenaka, Daisuke Kohda, Masanobu Deshimaru, Shigeyuki Terada

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1784 4 621 - 628 2008年04月 [査読有り][通常論文]
   

  Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habit serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with scrotriflin. It was bound to triflin and scrotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood. (0 2007 Elsevier B.V. All rights reserved.
• Association of LILRA2 (ILT1, LIR7) splice site polymorphism with systemic lupus erythematosus and microscopic polyangiitis

  K. Mamegano, K. Kuroki, R. Miyashita, M. Kusaoi, S. Kobayashi, K. Matsuta, K. Maenaka, M. Colonna, S. Ozaki, H. Hashimoto, Y. Takasaki, K. Tokunaga, N. Tsuchiya

  GENES AND IMMUNITY 9 3 214 - 223 2008年04月 [査読有り][通常論文]
   

  Leukocyte immunoglobulin-like receptors (LILRs) are inhibitory, stimulatory or soluble receptors encoded within the leukocyte receptor complex. Some LILRs are extensively polymorphic, and exhibit evidence for balancing selection and association with disease susceptibility. LILRA2 (LIR7/ILT1) is an activating receptor highly expressed in inflammatory tissues, and is involved in granulocyte and macrophage activation. In this study, we examined the association of LILRA2 and adjacently located LILRA1 with systemic lupus erythematosus (SLE), rheumatoid arthritis ( RA) and microscopic polyangiitis (MPA). Polymorphism screening detected a LILRA2 SNP ( rs2241524 G > A) that disrupts splice acceptor site of intron 6. Case-control association studies on 273 Japanese SLE, 296 RA, 50 MPA and 284 healthy individuals revealed increase of genotype A/A in SLE (12.1%, odds ratio ( OR) 1.82, 95% confidence interval (CI) 1.02-3.24, P=0.041) and in MPA (16.0%, OR 2.52, 95% CI 1.07-5.96, P=0.049) compared with healthy individuals (7.0%). The risk allele caused an activation of a cryptic splice acceptor site that would lead to a novel LILRA2 isoform lacking three amino acids in the linker region (Delta 419-421). Flow cytometry indicated that this isoform was expressed on the surface of monocytes. These findings suggested that LILRA2 Delta 419-421 isoform encoded by the splice site SNP may play a role in SLE and MPA.
• Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum.

  Aoki N, Sakiyama A, Kuroki K, Maenaka K, Kohda D, Deshimaru M, Terada S

  Biochimica et biophysica acta 1784 4 621 - 628 2008年04月 [査読有り][通常論文]
   

  Habu (Trimeresurus flavoviridis) serum contains 3 small serum proteins (SSP-1, SSP-2, and SSP-3) with molecular masses of 6.5 to 10 kDa. Gel filtration analysis showed that all the SSPs exist in high molecular mass forms of approximately 60 kDa in the serum. Ultrafiltration of Habu serum showed that SSPs dissociated from the complex below a pH of 4. An SSP-binding protein was purified from Habu serum by gel filtration, ion exchange, and reverse-phase HPLC. N-terminal sequencing yielded a 39-amino acid sequence, similar to the N-terminal region of triflin, which is a snake venom-derived Ca2+ channel blocker that suppresses smooth muscle contraction. The amino acid sequence of this protein, termed serotriflin, was established by peptide analysis and cDNA cloning. Serotriflin is a glycosylated protein and consists of 221 amino acids. Among the 3 SSPs, only SSP-2 formed a noncovalent complex with serotriflin. It was bound to triflin and serotriflin with high affinity, as evidenced by surface plasmon resonance. SSP-2 is considered to be a protein that prevents self injury by accidental leaking of venom into the blood.
• Biophysical characterization of O-glycosylated CD99 recognition by paired Ig-like type 2 receptors (PILR)

  前仲 勝実

  J. Biol. Chem. 283 283 14 8893 - 8901 2008年04月 [査読有り][通常論文]
   

  Paired Ig-like type 2 receptors ( PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory ( PILR alpha) and activating ( PILR beta) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance ( SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILR alpha can bind to CD99 with low affinity ( K-d similar to 2.2 mu M), but activating PILR beta binds with similar to 40 times lower affinity ( K-d similar to 85 mu M). In addition to our previous mutagenesis study ( Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. ( 2008) J. Immunol. 180, 1686 - 1693), the SPR analysis showed that PILR alpha can bind to each Ala mutant of the two CD99 O-glycosylated sites ( Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILR alpha binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILR beta can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILR alpha and PILR beta. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILR alpha-CD99 interaction is enthalpically driven with a large entropy loss (-T Delta S = 8.9 kcal.mol(-1)), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.
• Dynamic interaction of the measles virus hemagglutinin with its receptor signaling lymphocytic activation molecule (SLAM, CD150)

  Chanakha K. Navaratnarajah, Sompong Vongpunsawad, Numan Oezguen, Thilo Stehle, Werner Braun, Takao Hashiguchi, Katsumi Maenaka, Yusuke Yanagi, Roberto Cattaneo

  JOURNAL OF BIOLOGICAL CHEMISTRY 283 17 11763 - 11771 2008年04月 [査読有り][通常論文]
   

  The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.
• Homogeneous sugar modification improves crystallization of measles virus hemagglutinin

  Takao Hashiguchi, Mizuho Kajikawa, Maita Nobuo, Makoto Takeda, Kimiko Kuroki, Kaori Sasaki, Daisuke Kohda, Yusuke Yanagi, Katsumi Maenaka

  JOURNAL OF VIROLOGICAL METHODS 149 1 171 - 174 2008年04月 [査読有り][通常論文]
   

  Measles virus (MV) enters cells by binding to the signaling lymphocyte activation molecule (also called CD150) on the cell surface, and thus shows the lymphotropism and immunosuppressive effects. The head domain (residues Asp(149) to Arg(617)) of the MV hemagglutinin (MV-H), the attachment protein, was produced using a transient expression system in HEK293T cells. The purified MV-H protein was heterogeneous because of a variety of complex-sugar modifications. The complex-sugar-type MV-H was crystallized successfully, and the crystals belonged to the space group P41212 with the unit cell dimension of a = b = 134 angstrom, c = 100 angstrom, but diffracted only to 3.0 A resolution. MV-H was also expressed in HEK293SGnTI(-) cells lacking the N-acetylglucosaminyltransferase I activity, which render N-linked glycans of the proteins restricted and homogeneous, producing the oligomannose, Man(5)GlcNAc(2). The native and selenomethionyl derivative proteins of the oligomannose-type MV-H were crystallized, and the native crystals well diffracted to 2.6 angstrom resolution. Thus, homogeneous sugar modification may be useful for improved crystallization of heavily sugar-modified viral envelope proteins. (C) 2008 Elsevier B.V. All rights reserved.
• CENP-O class proteins form a stable complex and are required for proper kinetochore function

  Tetsuya Hori, Masahiro Okada, Katsumi Maenaka, Tatsuo Fukagawa

  MOLECULAR BIOLOGY OF THE CELL 19 3 843 - 854 2008年03月 [査読有り][通常論文]
   

  We previously identified a multisubunit complex (CENP-H/I complex) in kinetochores from human and chicken cells. We showed that the CENP-H/I complex is divided into three functional classes. In the present study, we investigated CENP-O class proteins, which include CENP-O, -P, -Q, -R, and -50 (U). We created chicken DT40 cell knockouts of each of these proteins, and we found that all knockout lines were viable, but that they showed slow proliferation and mitotic defects. Kinetochore localization of CENP-O, -P, -Q, and -50 was interdependent, but kinetochore localization of these proteins was observed in CENP-R -deficient cells. A coexpression assay in bacteria showed that CENP-O, -P, -Q, and -50 proteins form a stable complex that can associate with CENP-R. Phenotype analysis of knockout cells showed that all proteins except for CENP-R were required for recovery from spindle damage, and phosphorylation of CENP-50 was essential for recovery from spindle damage. We also found that treatment with the proteasome inhibitor MG132 partially rescued the severe mitotic phenotype observed in response to release from nocodazole block in CENP-50-deficient cells. This suggests that CENP-O class proteins are involved in the prevention of premature sister chromatid separation during recovery from spindle damage.
• Two novel NKG2D ligands of the mouse H60 family with differential expression patterns and binding affinities to NKG2D

  Akio Takada, Shigeru Yoshida, Mizuho Kajikawa, Yukiko Miyatake, Utano Tomaru, Masaharu Sakai, Hitoshi Chiba, Katsumi Maenaka, Daisuke Kohda, Kazunori Fugo, Masanori Kasahara

  JOURNAL OF IMMUNOLOGY 180 3 1678 - 1685 2008年02月 [査読有り][通常論文]
   

  H60, originally described as a dominant minor histocompatibility Ag, is an MHC class I-like molecule that serves as a ligand for the NKG2D receptor. In the present study, we identified two novel mouse chromosome 10-encoded NKG2D ligands structurally resembling H60. These ligands, which we named H60b and H60c, encode MHC class I-like molecules with two extracellular domains. Whereas H60b has a transmembrane region, H60c is a GPI-anchored protein. Recombinant soluble H60b and H60c proteins bound to NKG2D with affinities typical of cell-cell recognition receptors (K(d) = 310 nM for H60b and K(d) = 8.7 mu M for H60c). Furthermore, expression of H60b or H60c rendered Ba/F3 cells susceptible to lysis by NK cells, thereby establishing H60b and H60c as functional ligands for NKG2D. H60b and H60c transcripts were detected only at low levels in tissues of healthy adult mice. Whereas H60b transcripts were detectable in various tissues, H60c transcripts were detected mainly in the skin. Infection of mouse embryonic fibroblasts with murine cytomegalovirus induced expression of H60b, but not H60c or the previously known H60 gene, indicating that transcriptional activation of the three types of H60 genes is differentially regulated. The present study adds two new members to the current list of NKG2D ligands.
• 3P-015 MHC分子を構成するβ2ミクログロブリンのNMR構造解析(蛋白質・物性(3),第46回日本生物物理学会年会)

  Kurimoto Eiji, Yamaguchi Yoshiki, Kuroki Kimiko, Maenaka Katsumi, Kohda Daisuke, Kato Koichi

  生物物理 48 S130  一般社団法人 日本生物物理学会 2008年
• Structure-guided identification of a new catalytic motif of oligosaccharyltransferase.

  前仲 勝実, 神田 大輔

  EMBO J 27 27 1 234 - 243 2008年01月 [査読有り][通常論文]
   

  Asn-glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn-X-Thr/ Ser-motif-dependent manner. We also determined the 2.7-angstrom resolution crystal structure of the C-terminal soluble domain of Pyrococcus STT3. The structure-based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well-conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipid-linked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide-asparagine bond.
• Expression, crystallization and preliminary X-ray diffraction analysis of human paired Ig-like type 2 receptor alpha (PILR alpha)

  Shigekazu Tabata, Kimiko Kuroki, Nobuo Maita, Jing Wang, Ikuo Shiratori, Hisashi Arase, Daisuke Kohda, Katsumi Maenaka

  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 64 Pt 1 44 - 46 2008年01月 [査読有り][通常論文]
   

  Human paired immunoglobulin-like (Ig-like) type 2 receptor alpha (PILR alpha) is a type I membrane protein that is mainly expressed in immune-related cells such as monocytes, granulocytes and dendritic cells. PILR alpha can suppress the functions of such immune cells because it has the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region, which recruits the phosphatase Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-2) to inhibit phophorylations induced by activation signals. The extracellular region of human PILR alpha comprises one immunoglobulin superfamily V-set domain and a stalk region. The V-set domain (residues 13-131) of human PILR alpha was overexpressed in Escherichia coli as inclusion bodies, refolded by rapid dilution and purified. The PILR alpha protein was successfully crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.3 angstrom resolution at SPring-8 BL41XU; they belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 angstrom, and contain one molecule per asymmetric unit.
• Crystal structure of measles virus hemagglutinin provides insight into effective vaccines

  Takao Hashiguchi, Mizuho Kajikawa, Nobuo Maita, Makoto Takeda, Kimiko Kuroki, Kaori Sasaki, Daisuke Kohda, Yusuke Yanagi, Katsumi Maenaka

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 49 19535 - 19540 2007年12月 [査読有り][通常論文]
   

  Measles still remains a major cause of childhood morbidity and mortality worldwide. Measles virus (MV) vaccines are highly successful, but the mechanism underlying their efficacy has been unclear. Here we report the crystal structure of the MV attachment protein, hemagglutinin, responsible for MV entry. The receptor-binding head domain exhibits a cubic-shaped beta-propeller structure and forms a homodimer. N-linked sugars appear to mask the broad regions and cause the two molecules forming the dimer to tilt oppositely toward the horizontal plane. Accordingly, residues of the putative receptor-binding site, highly conserved among MV strains, are strategically positioned in the unshielded area of the protein. These conserved residues also serve as epitopes for neutralizing antibodies, ensuring the serological monotype, a basis for effective MV vaccines. Our findings suggest that sugar moieties in the MV hemagglutinin critically modulate virus-receptor interaction as well as antiviral antibody responses, differently from sugars of the HIV gp120, which allow for immune evasion.
• New oligosaccharyltransferase assay method

  神田 大輔, 前仲 勝実

  Glycobiology 17 17 11 1175 - 1182 2007年11月 [査読有り][通常論文]
   

  We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.
• Tom20 recognizes mitochondrial presequences through dynamic equilibrium amongmultiple bound states

  斉藤 貴士, 前仲 勝実, 神田 大輔

  EMBO J. 26 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.
• Purification,crystallization and preliminary X-ray diffraction studies of the soluble domain of the oligosaccharyltransferase STT3 subunit from the thermophilic archaeon Pyrococcus furiosus

  前仲 勝実, 神田 大輔

  Acta Crystallogr Sect F Struct Biol Cryst Commun 63 63 Pt 9 798 - 801 2007年09月 [査読有り][通常論文]
   

  Oligosaccharyltransferase catalyzes the transfer of preassembled oligosaccharides onto asparagine residues in nascent polypeptide chains. The STT3 subunit is thought to bear the catalytic site. The C-terminal domain of the STT3 protein of Pyrococcus furiosus was expressed in Escherichia coli cells. STT3 protein prepared from two different sources, the soluble fraction and the inclusion bodies, produced crystals that diffracted to 2.7 angstrom. During crystallization screening, cocrystals of P. furiosus STT3 with an E. coli 50S ribosomal protein, L7/ L12, were accidentally obtained. This cross-species interaction is not biologically relevant, but may be used to design a built-in polypeptide substrate for the STT3 crystals.
• Immune modulation of HLA-G dimer in maternal-fetal interface

  Kirniko Kuroki, Katsurni Maenaka

  EUROPEAN JOURNAL OF IMMUNOLOGY 37 7 1727 - 1729 2007年07月 [査読有り][通常論文]
   

  HLA-G is a non-classical human MHC class I molecule, which has several characteristics distinct from classical MHC, such as low polymorphism and restricted tissue distribution. HLA-G is expressed on placenta, thymus and some tumors. At the maternal-fetal interface, trophoblasts do not express major classical MHC class I molecules (MHCI), HLA-A and -B, to prevent normal T cell responses. Instead, HLA-G is expressed and can suppress a wide range of immune responses by binding to inhibitory immune cell surface receptors, such as leukocyte Ig-like receptor (LILR) B1 and LILRB2. HLA-G exists in various forms, including beta 2m-associated or -free disulfide-linked dimers that can be expressed either at the cell surface or in soluble form. However, until recently the physiological role of these different molecular forms has been unclear. In this issue of the European Journal of Immunology, one article demonstrates that the disulfide-linked homodimer of beta 2m-associated HLA-G is the major fraction expressed by trophoblast cells. The HLA-G dimer modulates the function of LILRB1-expressing antigen-presenting cells by principally binding to LILRB1. On the other hand, another recent report showed that beta 2m-free disulfide-linked HLA-G dimers are produced by villous cytotrophoblast cells. Taken together, these results provide strong evidence in support of the hypothesis that HLA-G dimers play a role in immune suppression at the maternal-fetal interface. Further in-depth investigation will help to clarify the precise mechanism of HLA-G receptor recognition and signaling in vivo and the role of these interactions in successful reproduction.
• Structural basis of the 3'-end recognition of a leading strand in stalled replication forks by PriA

  前仲 勝実, 神田 大輔

  EMBO J 28 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.
• Structural basis for dynamic interdomain movement and RNA recognition of the selenocysteine-specific elongation factor SelB

  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.
• Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy

  Kimiko Kuroki, Sayoko Kobayashi, Mitsunori Shiroishi, Mizuho Kajikawa, Naoaki Okamoto, Daisuke Kohda, Katsumi Maenaka

  JOURNAL OF IMMUNOLOGICAL METHODS 320 1-2 172 - 176 2007年03月 [査読有り][通常論文]
   

  Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein-protein interactions have been performed, the very weak interactions generally observed in cell-cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K-d greater than mu M range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 mu M (HLA-Cw4) and 5.7 mu M (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell-cell recognition receptors involved in immunologically relevant events. (c) 2006 Elsevier B.V. All rights reserved.
• Cutting edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B

  Hathairat Thananchai, Geraldine Gillespie, Maureen P. Martin, Arman Bashirova, Nobuyo Yawata, Makoto Yawata, Philippa Easterbrook, Daniel W. McVicar, Katsumi Maenaka, Peter Parham, Mary Carrington, Tao Dong, Sarah Rowland-Jones

  JOURNAL OF IMMUNOLOGY 178 1 33 - 37 2007年01月 [査読有り][通常論文]
   

  Although it is clear that KIR3DL1 recognizes Bw4(+) HLA-B, the role of Bw4(+) HLA-A allotypes as KIR3DL1 ligands is controversial We therefore examined the hinding of tetrameric HLA-A and -B complexes, including HLA*2402, a common Bw4(+) HLA-A allotype, to KIR3DLI*001, *005, *007, and *1502 allotypes. Only Bw4(+) tetramers hound K7R3DL1. Three of four HLA-A*2402 tetramers bound one or more KIR3DL1 allotypes and all four KIR3DL1 allotypes bound to one or more HLA-A*2402 tetramers, but with different binding specificities. Only KTR3DL1*005 bound both HLA-A*2402 and HLA-B*5703 tetramers. HLA-A*2402-expressing target cells were resistant to lysis by NK cells expressing KIR3DL1*001 or *005. This study shows that HLA-A*2402 is a ligand for KIR3DL1 and demonstrates how the binding of KIR3DL1 to Bw4(+) ligands depends upon the bound peptide as well as HLA and KIR3DL1 polymorphism.
• Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d)

  Mitsunori Shiroishi, Kimiko Kuroki, Linda Rasubala, Kouhei Tsumoto, Izumi Kumagai, Eiji Kurimoto, Koichi Kato, Daisuke Kohda, Katsumi Maenaka

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 103 44 16412 - 16417 2006年10月 [査読有り][通常論文]
   

  HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte lg-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including beta(2)-microglobulin (beta(2)m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the beta(2)m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the beta(2)m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-A resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCI recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G alpha 3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/beta(2)m) and free forms of beta(2)m. Binding studies using beta(2)m-free MHCIs revealed differential beta(2)m-dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCIs, which may in turn regulate immune suppression.
• MHC class I-like MILL molecules are beta(2)-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression

  Mizuho Kajikawa, Tomohisa Baba, Utano Tomaru, Yutaka Watanabe, Satoru Koganei, Sachiyo Tsuji-Kawahara, Naoki Matsumoto, Kazuo Yamamoto, Masaaki Miyazawa, Katsumi Maenaka, Akihiro Shizu, Masanori Kasahara

  JOURNAL OF IMMUNOLOGY 177 5 3108 - 3115 2006年09月 [査読有り][通常論文]
   

  MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta(2)-microglobulin (beta(2)m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta(2)m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.
• Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11)

  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.
• Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system

  Yungen Miao, Yuansong Zhang, Koichi Nakagaki, Tianfu Zhao, Aichun Zhao, Yan Meng, Masao Nakagaki, Enoch Y. Park, Katsumi Maenaka

  APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 71 2 192 - 199 2006年06月 [査読有り][通常論文]
   

  Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.
• Preparation and crystallization of the disulfide-linked HLA-G dimer

  M Shiroishi, D Kohda, K Maenaka

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1764 5 985 - 988 2006年05月 [査読有り][通常論文]
   

  HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dinier both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-angstrom data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines. (c) 2005 Elsevier B.V. All rights reserved.
• Preparation and crystallization of the disulfide-linked HLA-G dimer.

  Shiroishi M, Kohda D, Maenaka K

  Biochimica et biophysica acta 1764 5 985 - 988 2006年05月 [査読有り][通常論文]
   

  HLA-G is a non-classical MHC class I, which binds to inhibitory receptors, such as Leukocyte Ig-like receptors, to induce a wide range of tolerogenic immunological effects. HLA-G can be expressed as a disulfide-liked dimer both in solution and at the cell surface. However, the three-dimensional structure of the HLA-G dimer is unknown. Here, we report the crystallization of the disulfide-linked dimer form of HLA-G by adding dithiothreitol (DTT), enabling a 3.2-A data set to be collected. We also show that DTT promotes disulfide bond exchange of refolded HLA-G, whose free cysteine was protected, thus facilitating its dimerization. This technique could also be applied for disulfide-mediated dimer/multimer formation of refolded proteins harbouring free cysteines.
• Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer

  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.
• 2P031 Crystallization of the catalytic subunit of archaeal oligosaccharyltransferase(29. Protein structure and dynamics (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Igura Mayumi, Maita Nobuo, Kamishikiryou Jun, Maenaka Katsumi, Kohda Daisuke

  生物物理 46 2 S303  一般社団法人 日本生物物理学会 2006年
• 1P139 BmNPV expression system; Application to human natural killer cell receptors(4. Protein engineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Kajikawa Mizuho, Sasaki Kaori, Kuroki Kimiko, Motohashi Tomoko, Shimojima Tsukasa, Park Enoch Y., Kohda Daisuke, Maenaka Katsumi

  生物物理 46 2 S181  一般社団法人 日本生物物理学会 2006年
• 1P064 Comprehensive analysis of the MHCI binding of leukocyte immunoglobulin-like receptors using surface plasmon resonance technique(2. Protein function (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Kuroki Kimiko, Shiroishi Mitsunori, Fukunaga Yuko, Kohda Daisuke, Maenaka Katsumi

  生物物理 46 2 S162  一般社団法人 日本生物物理学会 2006年
• 1P076 Molecular basis for recognition of E-cadherin by killer cell-lectin like receptor, KLRG1(2. Protein function (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Nakamura Seiko, Kuroki Kimiko, Ohki Izuru, Sasaki Kaori, Maruyama Takuma, Ito Masayuki, Ikura Mitsuhiko, Yamamoto Kazuo, Matsumoto Naoki, Kohda Daisuke, Maenaka Katsumi

  生物物理 46 2 S165  一般社団法人 日本生物物理学会 2006年
• 1P088 N-linked protein glycosylation in hyperthermophilic archaeon Pyrococcus furiosus(2. Protein function (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Kamishikiryou Jun, Igura Mayumi, Maenaka Katsumi, Kohda Daisuke

  生物物理 46 2 S168  一般社団法人 日本生物物理学会 2006年
• 1P027 Structual and biochemical studies of MHC class I recognition by human Leukocyte Ig-like receptors(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

  Kuroki Kimiko, Shiroishi Mitsunori, Kajikawa Mizuho, Rasubala Linda, Kohda Daisuke, Maenaka Katsumi

  生物物理 46 2 S153  一般社団法人 日本生物物理学会 2006年
• Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli.

  Sasaki K, Ose T, Tanaka T, Mizukoshi T, Ishigaki T, Maenaka K, Masai H, Kohda D

  Biochimica et biophysica acta 1764 1 157 - 160 2006年01月 [査読有り][通常論文]
   

  PriA, a DEXH-type DNA helicase, binds specifically to the 3' end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3' terminus of DNA.
• Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like receptor B1 (LILRB1/ILT2/CD85j)

  M Shiroishi, K Kuroki, K Tsumoto, A Yokota, T Sasaki, K Amano, T Shimojima, Y Shirakihara, L Rasubala, PA van der Merwe, Kumagai, I, D Kohda, K Maenaka

  JOURNAL OF MOLECULAR BIOLOGY 355 2 237 - 248 2006年01月 [査読有り][通常論文]
   

  The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHO-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCl recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses showed that LILRB1-MHCI interactions are entropically driven (-T Delta S = -9.4 similar to-6.6 kcal mol(-1)) with low heat capacity changes (Delta C-p= -0.22 similar to-0.10 kcal mol(-1) K-1). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1-MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from '' Induced-fit '' binding, which is associated with large reductions in conformational flexibility and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells. (c) 2005 Elsevier Ltd. All rights reserved.
• Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis

  K Kuroki, N Tsuchiya, M Shiroish, L Rasubala, Y Yamashita, K Matsuta, T Fukazawa, M Kusaoi, Y Murakami, M Takiguchi, T Juji, H Hashimoto, D Kohda, K Maenaka, K Tokunaga

  HUMAN MOLECULAR GENETICS 14 16 2469 - 2480 2005年08月 [査読有り][通常論文]
   

  Leukocyte immunoglobulin-like receptor subfamily B member I (LILRB1/LIR1/ILT2) is an inhibitory receptor broadly expressed on leukocytes and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated to be a susceptibility region to systemic lupus erythematosus (SLE). In this study, we screened for polymorphisms of LILRB1 and examined their association with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four non-synonymous substitutions within the ligand-binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a non-synonymous substitution of the cytoplasmic region were identified. CY01 and 02 did not co-segregate with PE01-03. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 diplotype was significantly associated with RA (odds ratio 2.05, P = 0.019 and Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1 was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.
• Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR

  K Maenaka, K Fukushi, H Aramaki, Y Shirakihara

  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 Pt 8 796 - 798 2005年08月 [査読有り][通常論文]
   

  The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer D-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12-14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P(i), 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl2 pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2(1)2(1)2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 A. Native and selenomethionyl derivative data sets were collected to 3 A resolution at SPring-8 and the Photon Factory.
• Crystallization and preliminary X-ray analysis of mitochondrial presequence receptor Tom20 in complexes with a presequence from aldehyde dehydrogenase

  前仲 勝実, 神田 大輔

  Acta Crystallogr Sect F Struct Biol Cryst Commun 61 61 Pt 5 514 - 517 2005年05月 [査読有り][通常論文]
   

  Most mitochondrial proteins are synthesized in the cytosol and must be imported into the mitochondria. Many mitochondrial precursor proteins have an extra leader sequence at their N-terminus called a presequence. Presequences are recognized by the Tom20 receptor protein. Based on the previously determined NMR structure of rat Tom20, a fragment corresponding to the core structure was generated. A cysteine residue was added at the C-terminus of the rat aldehyde dehydrogenase presequence to fix the presequence peptide onto the Tom20 fragment via an intermolecular disulfide bond. Two crystal forms of the complex were successfully obtained with different designs of the linker sequence which diffracted to 2.1 and 1.9 A. Crystal dehydration and subsequent annealing was essential to obtain good diffraction data for the 2.1 A crystal form.
• Crystallization and preliminary X-ray analysis of the mRNA-binding domain of elongation factor SelB in complex with RNA

  L Rasubala, D Fourmy, T Ose, D Kohda, K Maenaka, S Yoshizawa

  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 61 Pt 3 296 - 298 2005年03月 [査読有り][通常論文]
   

  In bacteria, the selenocysteine-specific elongation factor SelB is necessary for incorporation of selenocysteine, the 21st amino acid, into proteins by the ribosome. SelB binds to an mRNA hairpin formed by the selenocysteine-insertion sequence (SECIS) and delivers selenocysteyl-tRNA (Sec-tRNASec) at the ribosomal A site. The minimum fragment (residues 512-634) of Moorella thermoacetica SelB (SelB-M) required for mRNA binding has been overexpressed and purified. The complex of SelB-M with 23 nucleotides of the SECIS mRNA hairpin was crystallized at 293 K using the hanging-drop vapour-diffusion or oil-batch methods. The crystals diffract to 2.3 A resolution using SPring-8 BL41XU and belong to the space group P2(1)2(1)2, with unit-cell parameters a = 81.69, b = 169.58, c = 71.69 A.
• Structural basis for mRNA recognition by elongation factor SelB

  Satoko Yoshizawa, Linda Rasubala, Toyoyuki Ose, Daisuke Kohda, Dominique Fourmy, Katsumi Maenaka

  Nature Structural and Molecular Biology 12 2 198 - 203 2005年02月20日 [査読有り][通常論文]
   

  In bacteria, incorporation of selenocysteine, the 21st 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 Å. 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. © 2005 Nature Publishing Group.
• Structural basis for mRNA recognition by elongation factor SelB

  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.
• Genetic variability of the major histocompatibility complex class I homologue encoded by human cytomegalovirus leads to differential binding to the inhibitory receptor ILT2

  M Vales-Gomez, M Shiroishi, K Maenaka, HT Reyburn

  JOURNAL OF VIROLOGY 79 4 2251 - 2260 2005年02月 [査読有り][通常論文]
   

  Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with beta2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted (alpha1, alpha2, and alpha3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.
• Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system

  T Motohashi, T Shimojima, T Fukagawa, K Maenaka, EY Park

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 326 3 564 - 569 2005年01月 [査読有り][通常論文]
   

  Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. (C) 2004 Elsevier Inc. All rights reserved.
• Crystal structure of a biologically functional form of PriB from Escherichia coli reveals a potential single-stranded DNA-binding site

  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.
• VHL-box and SOCS-box domains determine binding specificity for Cul2-Rbx1 and Cul5-Rbx2 modules of ubiquitin ligases

  T Kamura, K Maenaka, S Kotoshiba, M Matsumoto, D Kohda, RC Conaway, JW Conaway, KI Nakayama

  GENES & DEVELOPMENT 18 24 3055 - 3065 2004年12月 [査読有り][通常論文]
   

  The ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) complex is a member of a family of ubiquitin ligases that share a Cullin-Rbx module. SOCS-box proteins recruit substrates to the ECS complex and are linked to Cullin-Rbx via Elongin B/C. VHL has been implicated as a SOCS-box protein, but lacks a C-terminal sequence (downstream of the BC box) of the SOCS box. We now show that VHL specifically interacts with endogenous Cul2-Rbx1 in mammalian cells, whereas SOCS-box proteins associate with Cul5-Rbx2. We also identify LRR-1 and FEM1B as proteins that share a region of homology with VHL (the VHL box, including the BC box and downstream residues) and associate with Cul2-Rbx1. ECS complexes can thus be classified into two distinct protein assemblies, that is, those that contain a subunit with a VHL box (composed of the BC box and a downstream Cul2 box) that interacts with Cul2-Rbx1, and those that contain a subunit with a SOCS box (BC box and downstream Cul5 box) that interacts with Cul5-Rbx2. Domain-swapping analyses showed that the specificity of interaction of VHL-box and SOCS-box proteins with Cullin-Rbx modules is determined by the Cul2 and Cul5 boxes, respectively. Finally, RNAi-mediated knockdown of the Cul2-Rbx1 inhibited the VHL-mediated degradation of HIF-2alpha, whereas knockdown of Cul5-Rbx2 did not affect it. These data suggest that the functions of the Cul2-Rbx1 and Cul5-Rbx2 modules are distinct.
• The inhibitory NK cell receptor CD94/NKG2A and the activating receptor CD94/NKG2C bind the top of HLA-E through mostly shared but partly distinct sets of HLA-E residues

  H Wada, N Matsumoto, K Maenaka, K Suzuki, K Yamamoto

  EUROPEAN JOURNAL OF IMMUNOLOGY 34 1 81 - 90 2004年01月 [査読有り][通常論文]
   

  The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2d). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A or CD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/ NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/ NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.
• Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G

  M Shiroishi, K Tsumoto, K Amano, Y Shirakihara, M Colonna, VM Braud, DSJ Allan, A Makadzange, S Rowland-Jones, B Willcox, EY Jones, PA van der Merwe, Kumagai, I, K Maenaka

  PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 100 15 8856 - 8861 2003年07月 [査読有り][通常論文]
   

  Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (K-d = 2-45 muM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8(+) T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.
• Structure-guided design of sialic acid-based Siglec inhibitors and crystallographic analysis in complex with sialoadhesin

  NR Zaccai, K Maenaka, T Maenaka, PR Crocker, R Brossmer, S Kelm, EY Jones

  STRUCTURE 11 5 557 - 567 2003年05月 [査読有り][通常論文]
   

  The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC50 values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyi-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.
• Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs

  Y Uemura, S Senju, K Maenaka, LK Iwai, S Fujii, H Tabata, H Tsukamoto, S Hirata, YZ Chen, Y Nishimura

  JOURNAL OF IMMUNOLOGY 170 2 947 - 960 2003年01月 [査読有り][通常論文]
   

  Accumulating evidence indicates that recognition by TCRs is far more degenerate than formerly presumed. Cross-recognition of microbial Ags by autoreactive T cells is implicated in the development of autoimmunity, and elucidating the recognition nature of TCRs has great significance for revelation of the disease process. A major drawback of currently used means, including positional scanning synthetic combinatorial peptide libraries, to analyze diversity of epitopes recognized by certain TCRs is that the systematic detection of cross-recognized epitopes considering the combinatorial effect of amino acids within the epitope is difficult. We devised a novel method to resolve this issue and used it to analyze cross-recognition profiles of two glutamic acid decarboxylase 65-autoreactive CD4(+) T cell clones, established from type I diabetes patients. We generated a DNA-based randomized epitope library based on the original glutamic acid decarboxylase epitope using class II-associated invariant chain peptide-substituted invariant chains. The epitope library was composed of seven sublibraries, in which three successive residues within the epitope were randomized simultaneously. Analysis of agonistic epitopes indicates that recognition by both TCRs was significantly affected by combinations of amino acids in the antigenic peptide, although the degree of combinatorial effect differed between the two TCRs. Protein database searching based on the TCR recognition profile proved successful in identifying several microbial and self-protein-derived mimicry epitopes. Some of the identified mimicry epitopes were actually produced from recombinant microbial proteins by APCs to stimulate T cell clones. Our data demonstrate the importance of the combinatorial nature of amino acid residues of epitopes in molecular mimicry.
• Specificity, degeneracy, and molecular mimicry in antigen recognition by HLA-Class II restricted T cell receptors: Implications for clinical medicine

  Yasushi Uemura, Satoru Senju, Shinji Fujii, Leo Kei Iwai, Katsumi Maenaka, Hiroki Tabata, Takayuki Kanai, Yu-Zhen Chen, Yasuharu Nishimura

  Modern Rheumatology 13 3 205 - 214 2003年 [査読有り][通常論文]
   

  In humans, increased susceptibility to specific autoimmune diseases is closely associated with specific HLA-class II alleles. CD4+ T cells that recognize short self-peptides in the context of HLA-class II molecules via their T cell receptor (TCR) are considered to mediate the central role of pathogenesis in autoimmunity. Although both self-and nonself-peptides are presented on HLA-class II molecules under physiological conditions, several mechanisms exist to avoid the T cell response to the self-peptide/HLA-class II complex. One of the mechanisms that account for the breakdown in immune tolerance is cross-recognition by TCR between a pathogen-derived antigen and a host antigen (molecular mimicry theory). Epidemiological studies have indicated that a number of autoimmune diseases are developed or exacerbated after infections. Therefore, elucidating the recognition nature of HLA-class II restricted TCR in detail is necessary in order to understand disease processes. A large body of evidence indicates that T cell recognition is highly degenerate, and many different peptides can activate an individual T cell. Degeneracy of TCR recognition also can appear in various physiological outcomes, ranging from full activation to strong antagonism. Here, we review the clinical implications of our findings on T cell recognition, as well as a new direction of future applications for analyses in molecular mimicry. We also describe the latest developments in methods of mapping TCR epitopes for CD4+ T cells using a peptide epitope expression library generated in the class II-associated invariant chain peptide substituted invariant chain gene format.
• Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the transcriptional activator protein PhoB from Escherichia coli

  K Shindoh, K Maenaka, T Akiba, H Okamura, Y Nishimura, K Makino, Y Shirakihara

  ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 58 Pt 10 Pt 2 1862 - 1864 2002年10月 [査読有り][通常論文]
   

  PhoB is a transcriptional factor that activates more than 30 genes of the pho regulon in response to phosphate starvation. Crystals of its C-terminal domain (PhoBC) were obtained in two forms. The first crystal form, obtained from phosphate solution, belongs to space group P2(1), with unit-cell parameters a = 30.7, b = 105.9, c = 30.9 Angstrom, beta = 110.3degrees. The second form, crystallized from PEG solution, belongs to the same space group, but has a smaller unit cell (a = 30.6, b = 37.5, c = 44.4 Angstrom, beta = 109.4degrees). Crystals of selenomethionyl-derivatized PhoBC were obtained using the conditions for the second crystal form. Diffraction data from wild-type PhoBC (2.0 Angstrom resolution) and MAD data sets from selenomethionyl-derivative PhoBC (3.0 Angstrom resolution) have been collected at 100 K with a synchrotron-radiation source. MAD data analysis is in progress.
• Molecular recognition by Ig-like receptors, KIRs and Fc gamma Rs

  K Maenaka, PA van der Merwe, DI Stuart, P Sondermann, EY Jones

  ACTIVATING AND INHIBITORY IMMUNOGLOBULIN-LIKE RECEPTORS 45 - 54 2001年 [査読有り][通常論文]
   

  Structural studies by us and other groups have shown that Killer cell Ig-like receptors (KIRs) and Fc gamma receptors (Fc gammaR) have a similar, unique topology (intermediate between I set and C2 set). In order to gain further insight into molecular recognition by these receptors, we have used surface plasmon resonance (SPR) to analyze the kinetic and thermodynamic properties of their interactions with their natural ligands. A repertoire of KIRs with two or three tandem Ig domains in their extracellular regions is expressed on human natural killer (NK) cells. These KIRs activate or inhibit NK cell cytotoxicity following recognition of MHC class I molecules on target cells. Different two-domain KIRs (KIR2Ds) recognise distinct subsets of HLA-C alleles. SPR analysis showed that, like other cell-cell recognition molecules interactions, the KIR2DL3 binds peptide-HLA-Cw7 with a low affinity (Kd similar to 10(-5)M), fast kinetics, and favourable entropic changes. In contrast, recent studies have shown that TCR/peptide-MHC interactions are characterised by slow kinetics and highly unfavourable entropic changes. Thus, although the TCR and KIRs both show allele- and peptide-specific MHC recognition, they bind with very different thermodynamic and kinetic properties. Fc gamma receptors (Fc gammaR) are expressed on immunologically active cells, bind the Fc portion of IgG and contribute to phagocytosis, cytotoxicity and the clearance of immune complexes. SPR analysis showed that the human low-affinity Fc gamma Rs (Fc gamma RIIa, Fc gamma RIIb and Fc gamma RIII) bind Fc with fast kinetics and a low affinity (Kd similar to 10(-6)M), as observed with other cell-cell recognition interactions, including KIR/HLA interactions. Interestingly, whereas the Fc gamma RIIa/Fc and Fc gamma RIIb/Fc interactions exhibited favourable entropic changes, comparable to the KIR/HLA interaction, the Fc gamma RIII/Fc interaction was characterized by large unfavourable entropic changes.
• Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I

  K Maenaka, T Juji, DI Stuart, EY Jones

  STRUCTURE WITH FOLDING & DESIGN 7 4 391 - 398 1999年04月 [査読有り][通常論文]
   

  Background: T cells and natural killer (NK) cells perform complementary tales in the cellular immune system. T cells identify infected cells directly through recognition of antigenic peptides that are displayed at the target cell surface by the classical major histocompatibility complex (MHC) class I molecules. NK cells monitor the target cell surface for malfunction of this display system, lysing potentially infected cells that might otherwise evade recognition by the T cells. Human killer cell inhibitory receptors (KIRs) control this process by either inhibiting or activating the cytotoxic activity of NK cells via specific binding to MHC class I molecules on the target cell. Results: We report the crystal structure of the extracellular region of the human p58 KIR (KIR2DL3), which is specific for the human MHC class I molecule HLA-Cw3 and related alleles. The structure shows the predicted topology of two tandem immunoglobulin-like domains, but comparison with the previously reported structure of the related receptor KIR2DL1 reveals an unexpected change of 23 degrees in the relative orientation of these domains. Conclusions: The altered orientation of the immunoglobulin-like domains maintains an unusually acute interdomain elbow angle, which therefore appears to be a distinctive feature of the KIRs. The putative MHC Glass I binding site is located on the outer surface of the elbow, spanning both domains. The unexpected observation that this binding site can be modulated by differences in the relative domain orientations has implications for the general mechanism of KIR-MHC class I complex formation.
• Redesigning of oligosaccharide binding sites of hen egg-white lysozyme and a homologous protein, α-lactalbumin

  I. Kumagai, K. Maenaka, H. Uchiyama, K. Watanabe, K. Miura

  Protein Engineering, Design and Selection 6 99  1993年 [査読有り][通常論文]
  

MISC

• 一本鎖抗体のVL領域に存在する2つのcis-Proを標的とした抗体工学研究

  楊一帆, 楊一帆, 岡崎匡, 田所高志, 喜多俊介, 佐藤卓史, 小橋川敬博, 前仲勝実, 森岡弘志 日本蛋白質科学会年会プログラム・要旨集 23rd (CD-ROM) 2023年06月
• 新型コロナウイルスに対するscFv化抗体の機能評価

  染谷太陽, 谷秀顕, 安楽佑樹, 田聡, 福原秀雄, 福原秀雄, 野村尚生, 田所高志, 小野寺大志, 安達悠, 森山彩野, 湯本航平, 鈴木干城, 佐々木慈英, 橋口隆生, 高橋宜聖, 喜多俊介, 前仲勝実, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 23rd (CD-ROM) 2023年06月
• N460K変異を有するSARS-CoV-2BA.2.75スパイクタンパク質のACE2親和性に関する構造的洞察

  安楽佑樹, 喜多俊介, 福原秀雄, 福原秀雄, 佐藤佳, 橋口隆生, 前仲勝実, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 23rd (CD-ROM) 2023年06月
• クライオ電子顕微鏡構造解析と高速AFM観察から見えてきたABCトランスポーター(P-gp)の構造ダイナミクス

  濱口紀江, 濱口紀江, 安達成彦, 野中雄仁, 喜多俊介, 今野翔, 金岡優依, 守屋俊夫, 川崎政人, 安田賢司, 安西尚彦, 林良雄, 前仲勝実, 千田俊哉, 小笠原諭, 内橋貴之, 村田武士 日本蛋白質科学会年会プログラム・要旨集 23rd (CD-ROM) 2023年06月
• アダプター蛋白質介在に伴う,乳がん特異的キナーゼの活性亢進評価

  武井梓穂, 宇賀神魁, 中迫純希, 松尾友樹, 神田諒, 前仲勝実, 松田正, 姚閔, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 23rd (CD-ROM) 2023年
• SARS CoV-2スパイク蛋白質と各種中和抗体の複合体構造解析より明らかとなった中和活性メカニズム

  山本旭麻, 東浦彰史, 下岡清美, 河野洋平, 喜多俊介, 安楽佑樹, 橋口隆生, 前仲勝実, 保田朋波流, 坂口剛正 日本ウイルス学会学術集会プログラム・予稿集(Web) 70th 2023年
• クライオ電子顕微鏡構造解析によるSARS-CoV-2XBB.1.5スパイク蛋白質のACE2受容体認識機構とXBB.1との構造比較

  矢島久乃, 安楽佑樹, 喜多俊介, 佐々木慈英, 木村(寺角)香菜子, 前仲勝実, 佐藤佳, 福原崇介, 橋口隆生 日本ウイルス学会学術集会プログラム・予稿集(Web) 70th 2023年
• SARS-CoV-2XBB.1スパイクタンパク質およびACE2複合体の立体構造解析

  安楽佑樹, 喜多俊介, 矢島久乃, 佐藤佳, 橋口隆生, 前仲勝実 日本ウイルス学会学術集会プログラム・予稿集(Web) 70th 2023年
• NIV抗体とSARS-CoV-2スパイクタンパク質複合体構造に基づく,中和活性と抗原認識機構の関係性の解析

  染谷太陽, 安楽佑樹, 森山彩野, 橋口隆生, 高橋宜聖, 喜多俊介, 前仲勝実 日本ウイルス学会学術集会プログラム・予稿集(Web) 70th 2023年
• X-206はSARS-CoV-2各種薬剤耐性株およびVOC変異株に対して広域かつ強力な阻害作用を示す

  佐々木慈英, 岡部伊織, 佐藤彰彦, 佐藤彰彦, 佐藤彰彦, 児玉耕太, 乙黒聡子, 佐々木道仁, 大場靖子, 澤洋文, 澤洋文, 前仲勝実, 柳雄介, 橋口隆生 日本ウイルス学会学術集会プログラム・予稿集(Web) 70th 2023年
• SARS-CoV-2中和抗体の一本鎖Fv断片を用いた構造解析

  田所高志, 田所高志, 喜多俊介, 染谷太陽, 安楽佑樹, 福原秀雄, 福原秀雄, 野村尚生, 小野寺大志, 森山彩野, 橋口隆生, 高橋宜聖, 前仲勝実, 前仲勝実 日本結晶学会年会講演要旨集 2023 2023年
• SARS-CoV-2スパイク蛋白質と広域中和抗体の複合体構造解析

  山本旭麻, 東浦彰史, 下岡清美, 河野洋平, 喜多俊介, 安楽佑樹, 野間井智, 橋口隆生, 前仲勝実, 保田朋波流, 坂口剛正 日本結晶学会年会講演要旨集 2023 2023年
• 腫瘍免疫活性化を目指したHLA-G抗体スクリーニング

  下柿元咲瑛, 黒木喜美子, 引地和馬, 赤岩愛記, 古川敦, 古川敦, 前田直良, 前仲勝実 日本薬学会年会要旨集(Web) 143rd 2023年
• 電子不足Cp^EIr(III)錯体の合成及びエーテルを配向基として用いたC-H官能基化反応への応用

  冨田永希, 小島正寛, 永島佑貴, 田中健, 杉山晴紀, 瀬川泰知, 古川敦, 前仲勝実, 前仲勝実, 前田理, 前田理, 吉野達彦, 吉野達彦, 松永茂樹, 松永茂樹 日本薬学会年会要旨集(Web) 143rd 2023年
• タバコ煙ガス相はJ774マクロファージにおいてPKCβを介してフェロトーシスを誘導する

  東 恒仁, 半田 悠, 眞井 洋輔, 前仲 勝実, 田所 高志 日本薬理学会年会要旨集 97 3-B-P-069 2023年 [査読無し]
   

  Cigarette smoking is a risk factor for various types of diseases including atherosclerosis, hypertension, chronic obstructive pulmonary disease, and respiratory infection. The respiratory infection caused by cigarette smoking is due to immune cell dysfunction by cigarette smoke, although its molecular mechanism remains to be clarified. The cigarette smoke can be divided into two phases: tar (particle) phase and gas phase. We have previously reported that gas phase extract of cigarette smoke (CSE) induces cell death. In this study, we have examined the effects of CSE on J774 macrophages. CSE and unsaturated carbonyl compounds, cytotoxic factors in the CSE, induced cell death in J774 macrophages. Ferrostatin-1 and liproxstatin-1, ferroptosis inhibitors, suppressed cell death caused by CSE and unsaturated carbonyl compounds. A broad-range protein kinase C (PKC) inhibitor Gö6983 suppressed CSE- and unsaturated carbonyl compounds-induced cell death. To identify PKC isoforms involved in the process, we have examined isoform-specific inhibitors. Enzastaurin, a PKCβ-specific inhibitor, suppressed the cell death. Enzastaurin also suppressed RSL3-induced ferroptosis. These results suggest that CSE and unsaturated carbonyl compounds induce PKCβ-dependent ferroptosis in J774 macrophages.
• サイトメガロウイルス(CMV)眼感染症眼内液とCMV血症末梢血のCMV UL40多型の違いと眼内への感染進展における意義

  白根 茉利子, 八幡 信代, 元岡 大祐, 柴田 健輔, Khor Seik-Soon, 大前 陽輔, 柳井 亮二, 眞下 永, 蕪城 俊克, 森 康雄, 沼田 晃彦, 秋山 雅人, 長谷川 英一, 武田 篤信, 大黒 伸行, 前仲 勝実, 赤司 浩一, 徳永 勝士, 八幡 真人, 園田 康平 日本臨床免疫学会総会プログラム・抄録集 50回 76 -76 2022年10月
• SARS-CoV-2スパイク蛋白質と中和抗体NT-193複合体のX線結晶構造解析

  喜多俊介, 小野寺大志, 安達悠, 森山彩野, 野村尚生, 田所高志, 安楽佑樹, 湯本航平, 田聡, 福原秀雄, 鈴木干城, 橋口隆生, 高橋宜聖, 前仲勝実 量子ビームサイエンスフェスタ(Web) 2021 2022年10月
• 汎SARS-CoV-2抗体の中和機構とBA.4/5変異株によるACE2受容体認識機構の構造基盤

  鈴木干城, 安楽佑樹, 木村香菜子, 喜多俊介, 佐々木慈英, 小澤龍彦, 仁井見英樹, 佐藤佳, 前仲勝実, 橋口隆生 日本ウイルス学会学術集会プログラム・予稿集(Web) 69th 2022年09月
• VCP化合物による脊髄損傷後血液脊髄関門機能保護作用

  鈴木 裕貴, 角家 健, 五月女 慧人, 遠藤 健, 船木 智, 周東 智, 前仲 勝実, 岩崎 倫政 日本整形外科学会雑誌 96 (8) S1631 -S1631 2022年09月
• 次硝酸ビスマスの急性期脊髄損傷に対する神経保護効果とその機序の解明

  五月女 慧人, 角家 健, 鈴木 裕貴, 遠藤 健, 中川 慎介, 前仲 勝実, 岩崎 倫政 日本整形外科学会雑誌 96 (8) S1691 -S1691 2022年09月
• 蛋白質科学における計画的セレンディピティ—Planned serendipity in protein science—特集 計画的セレンディピティが医学・創薬を革新する!

  前仲 勝実 医学のあゆみ 282 (9) 801 -804 2022年08月27日 

  本稿では、筆者が取り組んできた免疫と感染症に関わる表面抗原/受容体の蛋白質科学研究から計画的セレンディピティを考えたい。はじめに麻疹ウイルスの表面にある受容体結合蛋白質を取り上げ、その受容体との結合・構造について蛋白質科学解析を行った結果から、その免疫応答・制御を考察した例を説明する。次に、免疫チェックポイント受容体LILRとリガンドHLA-Gとの分子認識に関する解析から、想定外の新規HLA-G分子形態によるavidity効果を介した免疫制御を見出したことを説明する。最後に、新型コロナウイルス(SARS-CoV-2)に対する中和抗体の認識機構についての蛋白質レベルでの解析から見出された変異ウイルスに対する活性とavidity効果について紹介したい。(著者抄録)
• 電子不足Cp^EIr(III)錯体の合成及びエーテルを配向基として用いたC-H官能基化反応への応用

  冨田永希, 小島正寛, 永島佑貴, 田中健, 杉山晴紀, 杉山晴紀, 瀬川泰知, 瀬川泰知, 古川敦, 前仲勝実, 前仲勝実, 前田理, 前田理, 前田理, 吉野達彦, 吉野達彦, 松永茂樹, 松永茂樹 次世代を担う有機化学シンポジウム講演要旨集 20th 2022年06月
• 北大創薬センターのCOVID-19治療薬開発の現状と展望

  前仲勝実, 前仲勝実 日本分析化学会有機微量分析研究懇談会・計測自動制御学会力学量計測部会合同シンポジウム講演要旨集 89th-119th 2022年06月
• 平滑筋弛緩薬パパベリンの新規効能:血液脊髄関門保護を介した脊髄損傷に対する神経保護効果

  鈴木裕貴, 角家健, 五月女慧人, 遠藤健, 浅野毅, 前仲勝実, 中川慎介, 岩崎倫政 日本整形外科学会雑誌 96 (2) 2022年06月
• MHC class I様分子CD1dと糖脂質複合体のX線結晶構造解析

  喜多俊介, 秋田穂, 日下裕規, TIAN Cong, 田所高志, 井貫晋輔, 新山真由美, 杉山成, 村田道雄, 藤本ゆかり, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 22nd (Web) 2022年06月
• SARS-CoV-2感染と中和抗体開発

  安楽佑樹, 喜多俊介, 福原秀雄, 前仲勝実 月刊臨床免疫・アレルギー科 77 (5) 604 -609 2022年05月
• 免疫抑制性タンパク質HLA-G2の新規受容体探索と結合親和性評価

  渡邊 紘士, 黒木 喜美子, 前仲 勝実 日本薬学会年会要旨集 142年会 26L -pm03S 2022年03月
• 耐熱性放線菌JA74株が生産する熱ショック代謝物maniwamycin類の全合成及び生物活性評価

  船山 佳世, 齋藤 駿, 加藤 航, 新藤 一敏, 川本 芽子, 松原 輝彦, 佐藤 智典, 乙黒 聡子, 前仲 勝実, 井本 正哉, 荒井 緑 日本薬学会年会要旨集 142年会 27T -am06S 2022年03月
• 5-Ethynylimidazole-4-carboxamide(EICA)ヌクレオチドプロドラッグの合成と抗デングウイルス活性

  日野谷 直人, 中村 元紀, 田良島 典子, 大場 靖子, 澤 洋文, 松田 彰, 前仲 勝実, 南川 典昭 日本薬学会年会要旨集 142年会 28S -pm03S 2022年03月
• 免疫系受容体の弱く速い結合を介したリガンド認識機構—Ligand recognition mechanisms by immune system receptors via weak and fast binding—特集 物質共生の観点からDDSを考える

  渡邊 紘士, 黒木 喜美子, 前仲 勝実 Drug delivery system : DDS : official journal of the Japan Society of Drug Delivery System / 日本DDS学会 編 37 (2) 112 -121 2022年03月 [査読有り]
   

  免疫系受容体は、自己・非自己を見分け、免疫活性あるいは抑制シグナルの起点となることで生体内の恒常性を維持するという重要な役割をもつ。しかし、この免疫系受容体とリガンドとの結合は、抗体等の可溶性タンパク質と異なり、弱い相互作用であり、解離速度が速いことから、正確な結合の評価に困難が伴う。本稿では、この免疫系受容体のリガンド認識における弱く速い相互作用について、物理化学的および構造生物学的手法を用いて明らかとなってきた速度論的および熱力学的特徴とその立体構造との関連について概説する。(著者抄録)
• 独自の設計による高機能化抗体の調製と相互作用解析

  田所高志, 田所高志, 大村玲央, 冨田麻美, 坪井晴美, 中村光太, 前仲勝実 バイオメディカル分析科学シンポジウム講演要旨集 34th 2022年
• 世界初IDP酵素の検証

  大和田ゆうき, 澤田光平, 荒井彩花, 田所高志, 南篤志, 前仲勝実, 久米田博之, 姚閔, 及川英秋, 尾瀬農之, 尾瀬農之 日本結晶学会年会講演要旨集 2022 2022年
• 強力な中和抗体NT-108によるSARS-CoV-2スパイクタンパク質認識の構造的基盤

  安楽佑樹, 小野寺大志, 喜多俊介, 安達悠, 森山彩野, 佐藤彰彦, 佐藤彰彦, 野村尚生, 田所高志, 田所高志, 湯本航平, 湯本航平, 伊東詩織, 田聡, 福原秀雄, 福原秀雄, 佐々木道仁, 大場靖子, 志和希, 岩田奈織子, 永田典代, 鈴木干城, 佐々木慈英, 関塚剛史, 登内奎介, 福士秀悦, 里深博幸, 香月康宏, 孫琳, 押村光雄, 黒田誠, 鈴木忠樹, 澤洋文, 橋口隆生, 高橋宜聖, 前仲勝実 日本ウイルス学会学術集会プログラム・予稿集(Web) 69th 2022年
• 物質共生の観点からDDSを考える 免疫系受容体の弱く速い結合を介したリガンド認識機構

  渡邊紘士, 黒木喜美子, 前仲勝実 Drug Delivery System 37 (2) 112 -121 2022年 [査読有り]
   

  免疫系受容体は、自己・非自己を見分け、免疫活性あるいは抑制シグナルの起点となることで生体内の恒常性を維持するという重要な役割をもつ。しかし、この免疫系受容体とリガンドとの結合は、抗体等の可溶性タンパク質と異なり、弱い相互作用であり、解離速度が速いことから、正確な結合の評価に困難が伴う。本稿では、この免疫系受容体のリガンド認識における弱く速い相互作用について、物理化学的および構造生物学的手法を用いて明らかとなってきた速度論的および熱力学的特徴とその立体構造との関連について概説する。
• Ca²⁺によるERp57-CNX複合体の構造機能調節メカニズムの解明

  金村進吾, 谷川雄哉, 伊藤大, LIN Yuxi, 松崎元紀, 黒木喜美子, 山口宏, 前仲勝実, LEE Young-Ho, 稲葉謙次, 奥村正樹 日本分子生物学会年会プログラム・要旨集(Web) 44th 2021年12月
• 北海道大学創薬拠点におけるクライオ電子顕微鏡の運用について

  前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 44th 2021年12月
• 脂質抗原提示分子CD1dの熱安定性解析

  秋田穂, 日下裕規, TIAN Cong, 田所高志, 井貫晋輔, 新山真由美, 杉山成, 村田道雄, 藤本ゆかり, 喜多俊介, 前仲勝実, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 44th 2021年12月
• カルシウムイオンによるERp57-CNX複合体の構造機能調節

  谷川 雄哉, 金村 進吾, 伊藤 大, 林 雨曦, 松崎 元紀, 黒木 喜美子, 山口 宏, 前仲 勝実, 李 映昊, 稲葉 謙次, 奥村 正樹 日本生化学会大会プログラム・講演要旨集 94回 [3T14m -245)] 2021年11月
• カイコ由来の内因性リガンドとの複合体における脂質抗原提示分子CD1bのX線結晶構造解析【JST・京大機械翻訳】

  TIAN Cong, KUSAKA Hiroki, TADOKORO Takashi, KITA Shunsuke, MAENAKA Katsumi 日本結晶学会年会講演要旨集 2021 2021年11月
• MHC classI様分子CD1dとアミド型α-GarCer複合体のX線結晶構造解析

  秋田穂, 日下裕規, 田聡, 田所高志, 井貫晋輔, 新山真由美, 杉山成, 村田道雄, 藤本ゆかり, 喜多俊介, 前仲勝実 日本結晶学会年会講演要旨集 2021 2021年11月
• MicroED法を用いたNi錯体の構造決定

  古川敦, 土井良平, 土井良平, 池本優真, 内山雅史, 小芝未希子, 前仲勝実, 佐藤美洋 日本結晶学会年会講演要旨集 2021 2021年11月
• 北大創薬センターの新型コロナウイルス治療薬開発の現状と展望

  前仲勝実 日本薬学会九州支部大会講演要旨集 38th (CD-ROM) 2021年11月
• カルシウムイオンによるERp57-CNX複合体の構造機能調節

  谷川 雄哉, 金村 進吾, 伊藤 大, 林 雨曦, 松崎 元紀, 黒木 喜美子, 山口 宏, 前仲 勝実, 李 映昊, 稲葉 謙次, 奥村 正樹 日本生化学会大会プログラム・講演要旨集 94回 [3T14m -245)] 2021年11月
• 新型コロナウイルスspike蛋白質と中和抗体NT-193複合体のX線結晶構造解析

  喜多俊介, 小野寺大志, 安達悠, 森山彩野, 野村尚生, 田所高志, 安楽佑樹, 湯本航平, 田聡, 福原秀雄, 鈴木干城, 佐々木慈英, 福士秀悦, 里深博幸, 香月康宏, 押村光雄, 橋口隆生, 高橋宜聖, 前仲勝実 日本結晶学会年会講演要旨集 2021 2021年09月
• 毒ヘビ由来Ca²⁺チャネル阻害型神経毒と毒ヘビ血清蛋白質の複合体構造

  田所高志, 岡部由紀, 松原永季, 喜多俊介, 尾瀬農之, 黒木喜美子, 前仲勝実, 寺田成之, 塩井(青木)成留実 日本結晶学会年会講演要旨集 2021 2021年08月
• 構造解析から創薬へ タンパク質立体構造に基づいた抗ウイルス薬の創製戦略

  安楽佑樹, 喜多俊介, 前仲勝実 医学のあゆみ 278 (6) 532 -538 2021年08月 

  Structure-based drug design(SBDD)は強力な標的阻害活性を示すリード化合物を、より迅速かつ費用対効果の高い方法で発見することを目的とした新規治療薬創製のアプローチのひとつである。X線結晶構造解析や核磁気共鳴法(NMR)、クライオ電子顕微鏡解析といった手法で得られたタンパク質の立体構造は薬剤が結合可能なポケットを可視化し、薬剤開発への重要な知見を与える。SBDDによって開発された薬剤の代表例として、ヒト免疫不全ウイルス(HIV)に対抗するプロテアーゼ阻害薬があげられる。後天性免疫不全症候群(AIDS)関連死亡者数を大きく減少させることに成功したプロテアーゼ阻害薬は、以後の創薬戦略に大きな影響を与えた。2019年末に出現したSARSコロナウイルス2(SARS-CoV-2)では驚異的な速さで創薬標的分子の立体構造が決定され、抗ウイルス薬の開発が進められている。本稿では抗HIV薬の開発を例にあげてSBDDについて概説し、さらにSARS-CoV-2に対する取り組みについて触れていきたい。(著者抄録)
• 蛋白質科学を基盤としたアカデミア創薬の現状と展望:北大創薬センターの活動

  前仲勝実, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 21st 2021年06月
• カルシウムによるERp57-CNXの構造機能調節

  谷川雄哉, 金村進吾, 伊藤大, LIN Yuxi, 松崎元紀, 黒木喜美子, 山口宏, LEE Young-Ho, 前仲勝実, 稲葉謙次, 奥村正樹, 奥村正樹 日本蛋白質科学会年会プログラム・要旨集 21st 2021年06月
• サポニン蛋白質に仲介されるCD1bへの脂質負荷の機能解析

  TIAN Cong, KUSAKA Hiroki, TADOKORO Takashi, KITA Shunsuke, MAENAKA Katsumi 日本蛋白質科学会年会プログラム・要旨集 21st 2021年06月
• 脂質抗原提示分子CD1bのX線結晶構造解析

  TIAN Cong, KUSAKA Hiroki, TADOKORO Takashi, KITA Shunsuke, MAENAKA Katsumi 量子ビームサイエンスフェスタ(Web) 2020 2021年03月
• 【ウイルス感染症に取り組む生物物理学】ウイルス生物物理学 創薬モダリティへの貢献

  前仲 勝実, 福原 秀雄, 橋口 隆生, Caaveiro Jose M.M., 長門石 曉, 黒田 大祐, 津本 浩平 生物物理 61 (2) 082 -089 2021年03月 [査読無し]
   

  Recent virological researches using biophysical methods, termed as virus biophysics or virophysics, largely contribute to the development of anti-viral drugs and vaccines. In this review, examples of structural and physicochemical analyses for representative viruses to develop drug modalities such as small compounds, antibodies, and vaccines are explained, and future direction of biophysical research for virus research is also discussed.
• 免疫活性化受容体LILRA2のANGPTL6認識機構の解明(The understanding of binding mechanism of LILRA2 and ANGPTL6)

  王 嘉き, 古川 敦, 山崎 莉佳, 平安 恒幸, 門松 毅, 尾池 雄一, 荒瀬 尚, 前仲 勝実 日本薬学会年会要旨集 141年会 28P01 -105S 2021年03月
• ウイルス生物物理学:創薬モダリティへの貢献

  前仲勝実, 福原秀雄, 橋口隆生, CAAVEIRO Jose M. M., 長門石曉, 黒田大祐, 津本浩平, 津本浩平 生物物理(Web) 61 (2) 2021年03月
• 狂犬病ウイルスP蛋白質による宿主の免疫経路阻害機構の解明

  南未来, 杉山葵, 野間井智, JIANG Xinxin, 前仲勝実, YAO Min, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 21st 2021年
• 病原性理解のためのLyssavirus P蛋白質C末端ドメイン構造解析

  杉山葵, 野間井智, JIANG Xinxin, 南未来, 前仲勝実, 伊藤直人, GOOLEY Paul R., MOSELEY Gregory W., YAO Min, 尾瀬農之, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 21st 2021年
• 麻疹ウイルスVタンパク質によるType I IFN経路阻害機構の解明

  木本円花, 永野悠馬, 杉山葵, 姚閔, 田所高志, 前仲勝実, 尾瀬農之 日本結晶学会年会講演要旨集 2021 2021年
• 平滑筋弛緩薬パパベリンの新規効能:血液脊髄関門保護を介した脊髄損傷に対する神経保護効果

  鈴木裕貴, 角家健, 五月女慧人, 遠藤健, 浅野毅, 岩崎倫政, 中川慎介, 前仲勝実 北海道整形災害外科学会 139th (2) S73 -S73 2021年
• HLA-G-LILRB 複合体構造を基にした機能解析

  黒木, 喜美子, 松原, 永季, 神田, 諒, 宮下, 尚之, 白石, 充典, 福永, 裕子, 上敷領, 淳, 福永, 淳, 福原, 秀雄, 廣瀬, 薫, Hunt, Joan S, 杉田, 有治, 喜多, 俊介, 尾瀬, 農之, 前仲, 勝実 福山大学薬学部研究年報 = Annual report of the Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University 38 (38) 35 -36 2020年12月25日
• COVID-19対策の創薬開発へ向けた北大創薬科学研究教育センターの取り組み

  前仲勝実 CBI学会大会 2020 (CD-ROM) 2020年10月
• 北大創薬センターの医薬品モダリティと臨床に向けたアカデミア創薬の取り組み

  前仲 勝実 日本薬理学会年会要旨集 93 2-S19-4 2020年03月 [査読無し]
   

  Hokkaido University Institute of Pharmaceutical Sciences established an affiliated drug discovery science research and education center, Center for Research and Education on Drug Discovery of Hokkaido University (CRED), in 2011 to promote academia drug discovery research. We have set up over the last 8 years the ‘state of the art' instruments necessary for chemical screening, and provided the expertise for drug discovery to novice users, supported by the projects, such as "Basis for Supporting Innovative Drug Discovery and Life Science Research". We also organize a "Platform for medical care and drug discovery" network with related academic departments and universities, Hokkaido University hospital and pharmaceutical companies mainly in Hokkaido Area. We are proceeding drug discovery modality research using a seamless "from drug seeds to preclinical" development system with a cryo-electron microscope: (1) unique Hokkaido University chemical library (peptides, nucleic acids and natural compounds) and semi-automatic preparation system, (2) preparation technology of difficult-to-express proteins, (3) complete physicochemical measurement technology, (4) Integrated structural analysis technology with cryo EM. In this lecture, I would like to look back over the past eight years and introduce the current efforts of CRED with some specific examples including the cooperation with the Development Unit of Drug Discovery Initiative of the University of Tokyo to proceed ADME evaluation and derivative synthesis.
• アカデミア創薬に向けたクライオ電子顕微鏡の利用-北大創薬センターの試み

  前仲勝実 日本薬学会年会要旨集(CD-ROM) 140th (Web) LS08 -LS08 2020年03月
• 血液脊髄関門機能保護を介した脊髄損傷治療薬の開発

  鈴木 裕貴, 角家 健, 遠藤 健, 袁 儒非, 浅野 毅, 前仲 勝実, 中川 慎介, 岩崎 倫政 Journal of Spine Research 11 (3) 589 -589 2020年03月
• 〈Basic Science〉抑制化受容体と免疫応答 抑制化受容体の構造

  渡邊紘士, 黒木喜美子, 前仲勝実 炎症と免疫 29 (1) 2 -7 2020年02月 

  近年、抑制化受容体は免疫チェックポイント阻害薬の標的として注目され、抗体医薬品を中心に創薬開発・臨床試験が精力的に実施されている。一方で、抑制化受容体のなかには、相同性の高いファミリーを形成する受容体や、一つの受容体が非自己リガンドを含む多様な分子種を認識するものも存在する。これらの受容体に対する創薬開発においては、リガンド認識機構を構造的に理解したうえで、目的に応じた阻害薬をデザインすることが必須となる。(著者抄録)
• リッサウイルスP蛋白質C末端ドメインの構造比較

  杉山葵, 野間井智, 蒋欣欣, 南未来, 前仲勝実, 伊藤直人, GOOLEY Paul R., MOSELEY Gregory W., 姚閔, 尾瀬農之, 尾瀬農之 日本結晶学会年会講演要旨集 2020 (CD-ROM) 2020年
• 血液脊髄関門機能保護を介した脊髄損傷治療薬の開発

  鈴木裕貴, 角家健, 遠藤健, 袁儒非, 浅野毅, 前仲勝実, 中川慎介, 岩崎倫政 Journal of Spine Research (Web) 11 (3) 2020年
• DuvenhageウイルスP蛋白質C末端ドメインの構造解析

  杉山葵, 蒋欣欣, 前仲勝実, 姚閔, 尾瀬農之 量子ビームサイエンスフェスタ(Web) 2019 2020年
• MHCバイオロジー 小胞体におけるMHCの品質管理

  金村進吾, 金村進吾, 松崎元紀, 前仲勝実, 稲葉謙次, 奥村正樹 月刊臨床免疫・アレルギー科 74 (5) 419 -426 2020年
• LILRA2のリガンド認識機構の解明

  古川敦, 山崎莉佳, WANG Jiaqi, 平安恒幸, 湯本航平, 福原秀雄, 荒瀬尚, 前仲勝実 日本生化学会大会(Web) 93rd [2Z13 -659)] 2020年
• アカデミア創薬における小胞体ストレス応答性酵素を標的とした抗癌剤スクリーニング

  野村尚生, 松丸尊紀, 春山知樹, 前田直良, 奥村正樹, 金村進吾, 稻葉謙次, 田村保明, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 42nd 2019年12月
• CD1dとアミド基導入抗原複合体のX線結晶構造解析

  喜多 俊介, 日下 裕規, 井貫 晋輔, Md. Imran Hossain, 花島 慎弥, 田所 高志, 新山 真由美, 杉山 成, 相羽 俊彦, 尾瀬 農之, 黒木 喜美子, 深瀬 浩一, 村田 道雄, 藤本 ゆかり, 前仲 勝実 日本結晶学会 講演要旨集 2019年11月 [査読無し][通常論文]
  
• 免疫系受容体によるウイルス感染制御の構造基盤

  前仲 勝実 日本小児感染症学会総会・学術集会プログラム・抄録集 51回 89 -89 2019年10月
• 免疫系受容体によるウイルス感染制御の構造基盤

  前仲 勝実 日本小児感染症学会総会・学術集会プログラム・抄録集 51回 89 -89 2019年10月 [査読無し][通常論文]
  
• タンパク質の運命を制御する生体システムと疾病のフロンティア 小胞体ストレス応答性酸化還元調節酵素を標的とした創薬スクリーニング

  野村 尚生, 松丸 尊紀, 春山 知樹, 前田 直良, 奥村 正樹, 金村 進吾, 稲葉 謙次, 田村 保明, 前仲 勝実 日本生化学会大会プログラム・講演要旨集 92回 [2S03m -05] 2019年09月
• 抗体医薬のための人工的三量体一本鎖Fvフラグメントの開発(Development of the engineered trimeric single-chain Fv fragment of the therapeutic antibody)

  Tadokoro Takashi, Nakamura Kota, Tsuboi Harumi, Maenaka Katsumi 生物物理 59 (Suppl.1-2) S391 -S391 2019年08月 [査読無し][通常論文]
  
• C型レクチン受容体Mincleリガンドとしての複合脂質:合成と機能解析

  松丸尊紀, 齋藤良太, 古川敦, 山崎晶, 前仲勝実, 藤本ゆかり 日本化学会春季年会講演予稿集(CD-ROM) 99th ROMBUNNO.2F6‐08 2019年03月 [査読無し][通常論文]
  
• SPECIFICITY OF ENDOGENOUS INHIBITOR FOR THE SNAKE NEUROTOXIN TRIFLIN: INTERACTION AND STRUCTURE ANALYSIS

  Narumi Shioi, Takashi Tadokoro, Yaopeng Hu, Lin Hai Kurahara, Keizo Hiraishi, Katsumi Maenaka, Isao Kuraoka, Shigeyuki Terada TOXICON 158 S34 -S34 2019年02月
• 脂質抗原提示分子CD1dの結晶構造解析

  喜多俊介, 日下裕規, HOSSAIN Md. Imran, 花島慎弥, 井貫晋輔, 田所高志, 新山真由美, 杉山成, 相羽俊彦, 相羽俊彦, 尾瀬農之, 黒木喜美子, 深瀬浩一, 藤本ゆかり, 村田道雄, 前仲勝実 量子ビームサイエンスフェスタ(Web) 2018 2019年
• イヌジステンパーウイルス受容体結合タンパク質の構造と膜融合の活性化機構

  福原秀雄, 酒匂幸, 河村美尋, 梶川瑞穂, 橋口隆生, 竹田誠, PHILIPPE Plattet, 尾瀬農之, 前仲勝実 日本ウイルス学会学術集会プログラム・予稿集(Web) 67th 2019年
• ヒトSLAMのアミノ最末端領域は麻疹ウイルスの受容体として機能するために重要である

  關文緒, 福原秀雄, 山本雄大, SUNDARAM Arulmozhiraja, 大石和恵, 丸山正, 常盤広明, 前仲勝実, 竹田誠 日本ウイルス学会学術集会プログラム・予稿集(Web) 67th 2019年
• 小胞体ストレス応答性酸化還元調節酵素を標的とした創薬スクリーニング

  野村尚生, 松丸尊紀, 春山知樹, 前田直良, 奥村正樹, 金村進吾, 稲葉謙次, 田村保明, 前仲勝実 日本生化学会大会(Web) 92nd [2S03m -05] 2019年 [査読無し][通常論文]
  
• 麻疹中和抗体のフラグメント化と物理化学的特性の解析

  田所高志, LUBNA Jahan Mst, 伊藤由梨, 田原舞乃, 橋口隆生, 竹田誠, 福原秀雄, 前仲勝実 日本細胞生物学会大会(Web) 71st ROMBUNNO.2P‐209 (WEB ONLY) 2019年 [査読無し][通常論文]
  
• 狂犬病ウイルスP蛋白質によるJAK-STATシグナル阻害機構の解明

  杉山葵, 蒋欣欣, 永野悠馬, 野間井智, 若原拓也, 前仲勝実, 姚閔, MOSLEY Gregory, 尾瀬農之 日本細胞生物学会大会(Web) 71st ROMBUNNO.2P‐005 (WEB ONLY) 2019年 [査読無し][通常論文]
  
• 免疫受容体LILRA2のリガンド認識機構の解明

  山崎莉佳, 古川敦, 平安恒幸, 平安恒幸, 門松毅, 尾池雄一, 前仲勝実 日本細胞生物学会大会(Web) 71st ROMBUNNO.2P‐127 (WEB ONLY) 2019年 [査読無し][通常論文]
  
• モリビリウイルス属の細胞侵入機構と創薬への取り組み

  前仲勝実, 前仲勝実, 前仲勝実 生体分子科学討論会講演要旨集 46th 14 2019年 [査読無し][通常論文]
  
• IDPを使用した新規ペア型酵素の戦略

  澤田光平, 田所高志, 大和田ゆうき, 南篤志, 久米田博之, 斎尾智英, 姚閔, 及川英秋, 前仲勝実, 尾瀬農之, 尾瀬農之 日本結晶学会年会講演要旨集 2018 32 2018年11月01日 [査読無し][通常論文]
  
• [北大は新入生を歓迎する] ： 先生からのメッセージ

  北村, 清彦, 金沢, 英之, 岡田, 信弘, 鈴木, 恵二, 藤原, 正智, 岩﨑, 克則, 前仲, 勝実, 志村, 華子, 高見, 敏子, 渡辺, 将人, 東藤, 正浩 リテラポプリ 44 11 -14 2018年10月05日
• NMRを用いた自然免疫受容体Mincleによる糖脂質認識機構の解析

  古川敦, 須知祐介, 久米田博之, 松丸尊紀, 齊藤貴士, 前仲勝実 Abstracts. Annual Meeting of the NMR Society of Japan 57th 178‐179 2018年10月 [査読無し][通常論文]
  
• 高精度分子シミュレーションに基づく中分子化合物の脂質二重膜透過解析

  清水奏, 清水奏, ARULMOZHIRAJA Sundaram, ARULMOZHIRAJA Sundaram, 山本雄大, 山本雄大, 前仲勝実, 前仲勝実, 常盤広明, 常盤広明 日本薬学会関東支部大会講演要旨集 62nd 97 2018年09月10日 [査読無し][通常論文]
  
• ヒト単純ヘルペスウイルス由来糖ペプチドと免疫受容体PILRα相互作用解析

  野村尚生, 柿田浩輔, 古川敦, 穴田仁洋, 橋下俊一, 松永茂樹, 齊藤貴士, 前仲勝実, 前仲勝実 Abstracts. Annual Meeting of the NMR Society of Japan 57th 198‐199 2018年09月03日 [査読無し][通常論文]
  
• エポキシド加水分解機能を持つタンパク質の分子進化的考察

  飯淵 友直, 尾瀬 農之, 前仲 勝実, 及川 英秋, 園山 正史, 向井 有理 日本生化学会大会プログラム・講演要旨集 91回 [2P -110] 2018年09月 [査読無し][通常論文]
  
• カイコ脂質輸送蛋白質の機能解析

  喜多 俊介, 日下 裕規, 前仲 勝実 日本生化学会大会プログラム・講演要旨集 91回 [3P -070] 2018年09月 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dによる抗原認識機構の解析

  日下 裕規, 喜多 俊介, 大野 祐介, 木原 章雄, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 日本生化学会大会プログラム・講演要旨集 91回 [3P -090] 2018年09月 [査読無し][通常論文]
  
• エポキシド加水分解機能を持つタンパク質の分子進化的考察

  飯淵 友直, 尾瀬 農之, 前仲 勝実, 及川 英秋, 園山 正史, 向井 有理 日本生化学会大会プログラム・講演要旨集 91回 [2P -110] 2018年09月 [査読無し][通常論文]
  
• 狂犬病ウイルスP蛋白質によるJAK‐STATシグナル阻害機構の解明

  杉山葵, JIANG Xinxin, 永野悠馬, 野間井智, 若原拓也, 前仲勝実, YAO Min, MOSLEY Gregory, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 18th 48 2018年05月23日 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dによる抗原認識における種差

  日下裕規, 喜多俊介, HOSSAIN Imran, 花島慎弥, 井貫晋輔, 新山真由美, 杉山成, 相羽俊彦, 尾瀬農之, 黒木喜美子, 深瀬浩一, 藤本ゆかり, 村田道雄, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 18th 84 2018年05月23日 [査読無し][通常論文]
  
• 慢性炎症関連因子ANGPTL2の免疫細胞受容体LILRB2結合部位の同定

  荒牧峻彦, 黒木喜美子, 門松毅, 尾池雄一, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 18th 146 2018年05月23日 [査読無し][通常論文]
  
• 毒ヘビ由来Ca2+チャネル阻害型神経毒と毒ヘビ血清蛋白質の複合体構造

  田所高志, 塩井(青木, 成留実, 岡部由紀, 松原永季, 喜多俊介, 尾瀬農之, 黒木喜美子, 前仲勝実, 寺田成之 日本蛋白質科学会年会プログラム・要旨集 18th (10) 90 2018年05月23日 [査読無し][通常論文]
   

  ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.
• NMRを用いた自然免疫受容体Mincleによる糖脂質認識機構の解析

  古川敦, 須知佑介, 久米田博之, 松丸尊紀, 齊藤貴士, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 18th 120 2018年05月23日 [査読無し][通常論文]
  
• ヒトの自然免疫系を阻害する麻疹ウイルスV蛋白質の機能解析

  永野悠馬, 若原拓也, 秦玉瑩, 柳雄介, 前仲勝実, 尾瀬農之, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 18th 64 2018年05月23日 [査読無し][通常論文]
  
• 免疫系細胞表面受容体群の多様な分子認識

  前仲勝実 日本比較免疫学会学術集会講演要旨 30th 2018年05月
• 新規Notch signaling阻害剤NSI-1の開発

  白石 昂也, 堺谷 政弘, 乙黒 聡子, 前仲 勝実, 中矢 正, 鈴木 利治 日本薬学会年会要旨集 138年会 (3) 128 -128 2018年03月 [査読無し][通常論文]
  
• イヌジステンパーウイルスの株間の感染性の違いに着目したウイルスタンパク質と受容体の相互作用解析

  松尾 直也, 關 文緒, 中野 祥吾, 伊藤 創平, 前仲 勝実, 竹田 誠, 常盤 広明 日本薬学会年会要旨集 138年会 (3) 185 -185 2018年03月 [査読無し][通常論文]
  
• HSV-1感染における糖ペプチドと免疫受容体PILRα相互作用解析

  野村 尚生, 柿田 浩輔, 古川 敦, 穴田 仁洋, 橋下 俊一, 松永 茂樹, 齊藤 貴士, 前仲 勝実 日本薬学会年会要旨集 138年会 (2) 245 -245 2018年03月 [査読無し][通常論文]
  
• 細菌に分解された抗体の免疫活性化レセプターLILRA2による認識機構の解明

  山崎 莉佳, 古川 敦, 平安 恒幸, 荒瀬 尚, 前仲 勝実 日本薬学会年会要旨集 138年会 (2) 247 -247 2018年03月 [査読無し][通常論文]
  
• PILRαによる糖ペプチド認識機構の解明

  古川 敦, 柿田 浩輔, 荒瀬 尚, 穴田 仁洋, 尾瀬 農之, 橋本 俊一, 前仲 勝実 日本薬学会年会要旨集 138年会 (2) 261 -261 2018年03月 [査読無し][通常論文]
  
• イヌジステンパーウイルスの株間の感染性の違いに着目したウイルスタンパク質と受容体の相互作用解析

  松尾直也, 關文緒, 中野祥吾, 伊藤創平, 前仲勝実, 竹田誠, 常盤広明, 常盤広明 日本薬学会年会要旨集(CD-ROM) 138th (3) ROMBUNNO.26PA‐pm161S -185 2018年03月 [査読無し][通常論文]
  
• HSV‐1感染における糖ペプチドと免疫受容体PILRα相互作用解析

  野村尚生, 柿田浩輔, 古川敦, 穴田仁洋, 橋下俊一, 松永茂樹, 齊藤貴士, 前仲勝実, 前仲勝実 日本薬学会年会要旨集(CD-ROM) 138th (2) ROMBUNNO.28X‐am02 -245 2018年03月 [査読無し][通常論文]
  
• 細菌に分解された抗体の免疫活性化レセプターLILRA2による認識機構の解明

  山崎莉佳, 古川敦, 平安恒幸, 荒瀬尚, 前仲勝実 日本薬学会年会要旨集(CD-ROM) 138th (2) ROMBUNNO.28X‐pm02S -247 2018年03月 [査読無し][通常論文]
  
• PILRαによる糖ペプチド認識機構の解明

  古川敦, 柿田浩輔, 荒瀬尚, 穴田仁洋, 尾瀬農之, 橋本俊一, 前仲勝実 日本薬学会年会要旨集(CD-ROM) 138th (2) ROMBUNNO.27PA‐am327 -261 2018年03月 [査読無し][通常論文]
  
• B型肝炎治療 2018 核酸アナログ製剤によるIFN‐λ誘導とそれを標的としたB型肝炎治療薬の探索

  村田一素, 前仲勝実, 溝上雅史 Mebio 35 (1) 80‐87 -87 2018年01月10日 [査読無し][通常論文]
   

  ●核酸アナログ製剤はB型肝炎ウイルスの逆転写酵素を阻害するが、アデホビル、テノホビルには、インターフェロン(interferon;IFN)-λ3を誘導するという追加効果がある。●誘導されたIFN-λ3は、HBs抗原産生を抑制し、ISG(IFN-stimulated genes;IFN誘導遺伝子)を誘導する。●アデホビル、テノホビルの単独療法によるHBs抗原もしくはdrug-offはまれであるので、さらに強力にIFN-λ3を誘導する薬剤などIFN-λ3を標的とした新規治療法の開発が期待される。(著者抄録)
• 狂犬病ウイルスP蛋白質変異体の結晶および機能解析

  野間井智, 蒋欣欣, 永野悠馬, 杉山葵, 姚閔, GOOLEY Paul, MOSELEY Gregory, 前仲勝実, 尾瀬農之, 尾瀬農之 量子ビームサイエンスフェスタ(Web) 2017 2018年
• 39. ポリエーテル生合成経路中に存在する新型ペア型酵素の戦略を解明する

  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 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，
• 【B型肝炎治療2018】 核酸アナログ製剤によるIFN-λ誘導とそれを標的としたB型肝炎治療薬の探索

  村田 一素, 前仲 勝実, 溝上 雅史 Mebio 35 (1) 80 -87 2018年01月 [査読無し][通常論文]
   

  ●核酸アナログ製剤はB型肝炎ウイルスの逆転写酵素を阻害するが、アデホビル、テノホビルには、インターフェロン(interferon;IFN)-λ3を誘導するという追加効果がある。●誘導されたIFN-λ3は、HBs抗原産生を抑制し、ISG(IFN-stimulated genes;IFN誘導遺伝子)を誘導する。●アデホビル、テノホビルの単独療法によるHBs抗原もしくはdrug-offはまれであるので、さらに強力にIFN-λ3を誘導する薬剤などIFN-λ3を標的とした新規治療法の開発が期待される。(著者抄録)
• カイコ脂質輸送蛋白質の機能解析

  喜多俊介, 日下裕規, 前仲勝実 日本生化学会大会(Web) 91st [3P -070] 2018年 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dによる抗原認識機構の解析

  日下裕規, 喜多俊介, 大野祐介, 木原章雄, 尾瀬農之, 黒木喜美子, 前仲勝実 日本生化学会大会(Web) 91st [3P -090] 2018年 [査読無し][通常論文]
  
• 新規Notch signaling阻害剤NSI-1の開発

  白石昂也, 堺谷政弘, 乙黒聡子, 前仲勝実, 前仲勝実, 中矢正, 鈴木利治 日本薬学会年会要旨集(CD-ROM) 138th (3) ROMBUNNO.26PA‐am139S -128 2018年 [査読無し][通常論文]
  
• ヌクレアーゼ抵抗性4’修飾型ヌクレオシドのアンチセンスgapmerへの導入

  平塚隆寛, 田所高志, 前仲勝実, 松田彰 日本薬学会年会要旨集(CD-ROM) 138th ROMBUNNO.26M‐pm06S 2018年 [査読無し][通常論文]
  
• 細菌によって分解された抗体と免疫活性化受容体LILRA2との特異的相互作用機構の解明

  山崎莉佳, 古川敦, 平安恒幸, 荒瀬尚, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 18th 64 2018年 [査読無し][通常論文]
  
• 免疫制御分子PILRαと免疫細胞分化マーカーCD45の相互作用解析

  石塚幹広, 古川敦, 荒瀬尚, 前仲勝実 日本生化学会大会(Web) 90th ROMBUNNO.1P‐0031 (WEB ONLY) -0031] 2017年12月 [査読無し][通常論文]
  
• HLA-Cw12拘束性HIV-1ペプチドに対する宿主免疫受容体認識機構

  山下 諒也, 黒木 喜美子, 渡邊 洋介, 阪田 竜馬, 村越 勇人, 赤星 智寛, 滝口 雅文, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [1P -0139] 2017年12月 [査読無し][通常論文]
  
• 免疫補助制御分子CD160とリガンドHVEMの分子認識機構に向けた研究

  岩森 美樹, 黒木 喜美子, 齊藤 貴士, 阿部 千紘, 小島 理恵子, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [3P -0103] 2017年12月 [査読無し][通常論文]
  
• ヒト免疫受容体NKRP1AによるリガンドLLT1の分子認識機構

  田所 高志, 喜多 俊介, 松原 永季, 笠井 宣征, 玉置 貴晴, 岡部 由紀, 日下 裕規, 石山 夢美, 福原 秀雄, 上敷領 淳, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [2P -0185] 2017年12月 [査読無し][通常論文]
  
• PEG化タンパク質精製法の確立に向けた新規構造化PEG分子の設計と機能解析

  山田 千聖, Wawro Adam, 黒木 喜美子, 高橋 愛実, 村岡 貴博, 金原 数, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [3P -0167] 2017年12月 [査読無し][通常論文]
  
• HIV-2 Nefタンパク質のX線結晶構造

  平尾 憲吾, 黒木 喜美子, Andrews Sophie, 尾瀬 農之, Rowland-Jones Sarah, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [2P -0091] 2017年12月 [査読無し][通常論文]
  
• 単純ヘルペスウイルス由来糖蛋白質がヒト受容体を認識するメカニズム

  尾瀬農之, 石塚幹広, 古川敦, 黒木喜美子, 前仲勝実 日本結晶学会年会講演要旨集 2017 47 2017年11月23日 [査読無し][通常論文]
  
• 糖脂質α-GalCerを基盤とした脂質改変型CD1dリガンドの創製研究

  井貫 晋輔, 相羽 俊彦, 平田 菜摘, 柏原 瑛美, 喜多 俊介, 前仲 勝実, 深瀬 浩一, 藤本 ゆかり エンドトキシン・自然免疫研究 20 64 -67 2017年10月 [査読無し][通常論文]
  
• 狂犬病ウイルスの細胞吸着に関与する宿主因子の解析

  佐々木道仁, アニンディタ パウリナ, 伊藤直人, 杉山誠, 福原秀雄, 尾瀬農之, 尾瀬農之, 前仲勝実, 澤洋文, 澤洋文 日本獣医学会学術集会講演要旨集 160th 400 -400 2017年08月30日 [査読無し][通常論文]
  
• Examination of antiviral activity of 5-ethynyl-1-ribofuranosylimidazole-4-carboxamide(EICAR) against rabies virus in vitro(和訳中)

  Anindita Paulina Duhita, 佐々木 道仁, 伊藤 直人, 杉山 誠, 南川 典昭, 周東 智, 乙黒 聡子, 市川 聡, 松田 彰, 前仲 勝実, 大場 靖子, 澤 洋文 日本獣医学会学術集会講演要旨集 160回 390 -390 2017年08月 [査読無し][通常論文]
  
• In vitroでの狂犬病ウイルスに対する5-エチニル-1-リボフラノシルイミダゾール-4-カルボキサミド(EICAR)の抗ウイルス活性に関する検討(Examination of antiviral activity of 5-ethynyl-1-ribofuranosylimidazole-4-carboxamide(EICAR) against rabies virus in vitro)

  Anindita Paulina Duhita, 佐々木 道仁, 伊藤 直人, 杉山 誠, 南川 典昭, 周東 智, 乙黒 聡子, 市川 聡, 松田 彰, 前仲 勝実, 大場 靖子, 澤 洋文 日本獣医学会学術集会講演要旨集 160回 390 -390 2017年08月 [査読無し][通常論文]
  
• 狂犬病ウイルスの細胞吸着に関与する宿主因子の解析

  佐々木 道仁, アニンディ・タパウリナ, 伊藤 直人, 杉山 誠, 福原 秀雄, 尾瀬 農之, 前仲 勝実, 澤 洋文 日本獣医学会学術集会講演要旨集 160回 400 -400 2017年08月 [査読無し][通常論文]
  
• ハブ血清蛋白質SSPの毒蛇CRISPsの阻害剤としての応用

  塩井成留実, 田所高志, 胡耀鵬, 倉原琳, 平石敬三, 前仲勝実 トキシンシンポジウム予稿集 64th 76‐79 2017年07月01日 [査読無し][通常論文]
  
• WetとDryのコミュニケーションによるアカデミア創薬

  児玉 耕太, 斉尾 智英, 前仲 勝実, 金城 政孝 バイオサイエンスとインダストリー 75 (4) 323 -325 2017年07月 [査読無し][通常論文]
  
• 麻疹ウイルスV蛋白質C末端領域の構造的特徴

  永野悠馬, 若原拓也, 蒋欣欣, 柳雄介, 前仲勝実, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 17th 48 2017年05月22日 [査読無し][通常論文]
  
• 毒ヘビ神経毒とその内在性阻害蛋白質複合体の立体構造解析

  塩井(青木, 成留実, 田所高志, 胡耀鵬, 前仲勝実, 寺田成之 日本蛋白質科学会年会プログラム・要旨集 17th 130 2017年05月22日 [査読無し][通常論文]
  
• 分泌タンパク質ANGPTL2を介したシグナル伝達を阻害可能な低分子化合物探索

  荒牧峻彦, 黒木喜美子, 門松毅, 尾池雄一, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 17th 147 2017年05月22日 [査読無し][通常論文]
  
• 細菌の抗体分解による免疫レセプター活性化の分子基盤

  山崎莉佳, 古川敦, 平安恒幸, 黒木喜美子, 荒瀬尚, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 17th 64 2017年05月22日 [査読無し][通常論文]
  
• トラスツズマブ一本鎖Fv抗体断片の構築および高機能化

  田所高志, 中村光太, 坪井晴美, 前田龍, 尾瀬農之, 松田彰, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 17th 148 2017年05月22日 [査読無し][通常論文]
  
• CD1dの一本鎖化による安定化と結晶構造解析

  日下裕規, 喜多俊介, HOSSAIN Imuran Md, 花島慎弥, 井貫晋輔, 新山真由美, 杉山成, 尾瀬農之, 黒木喜美子, 藤本ゆかり, 村田道雄, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 17th 120 2017年05月22日 [査読無し][通常論文]
  
• インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定

  藤岡容一朗, 西出真也, 尾瀬農之, 加藤いづみ, 福原秀雄, 藤岡真理, 堀内浩水, 佐藤絢, NEPAL Prabha, 柏木彩花, WANG Jing, 堀口美香, PAUDEL Sarad, 南保明日香, 宮崎忠昭, 前仲勝実, 前仲勝実, 大場雄介 日本細胞生物学会大会(Web) 69th ROMBUNNO.T8‐04(P2‐028) (WEB ONLY) -61 2017年05月 [査読無し][通常論文]
  
• インフルエンザウイルス細胞侵入において鍵となる宿主タンパク質の同定

  藤岡 容一朗, 西出 真也, 尾瀬 農之, 加藤 いづみ, 福原 秀雄, 藤岡 真理, 堀内 浩水, 佐藤 絢, Nepal Prabha, 柏木 彩花, Wang Jing, 堀口 美香, Paudel Sarad, 南保 明日香, 宮崎 忠昭, 前仲 勝実, 大場 雄介 日本細胞生物学会大会講演要旨集 69回 61 -61 2017年05月 [査読無し][通常論文]
  
• IFN‐lambdaを標的とした新規B型肝炎治療薬の探索

  村田一素, 村田一素, 前仲勝実, 溝上雅史 肝臓 58 (Supplement 1) A45 2017年04月20日 [査読無し][通常論文]
  
• B型肝炎研究の新展開 IFN-lambdaを標的とした新規B型肝炎治療薬の探索

  村田 一素, 前仲 勝実, 溝上 雅史 肝臓 58 (Suppl.1) A45 -A45 2017年04月 [査読無し][通常論文]
  
• 脂質結合タンパク質の構造生物学 最近の展開 自然免疫受容体Mincleの糖脂質認識機構

  古川 敦, 須知 佑介, 池野 里紗, 松丸 尊紀, 上敷領 淳, 齊藤 貴士, 尾瀬 農之, 山崎 晶, 前仲 勝実 日本薬学会年会要旨集 137年会 (1) 136 -136 2017年03月 [査読無し][通常論文]
  
• 脂質結合タンパク質の構造生物学 最近の展開 脂質抗原受容体CD1dの脂質認識部位に存在する親水性アミノ酸残基の機能解析とその制御

  井貫 晋輔, 相羽 俊彦, 平田 菜摘, 相原 瑛美, 市原 収, 吉留 大輔, 喜多 俊介, 前仲 勝実, 深瀬 浩一, 藤本 ゆかり 日本薬学会年会要旨集 137年会 (1) 137 -137 2017年03月 [査読無し][通常論文]
  
• 脂質結合タンパク質の構造生物学 最近の展開 脂質-タンパク質相互作用の解明を目指した重原子化CD1dリガンドの合成と生理活性の評価

  花島 慎弥, Hossain Imran, 土川 博史, 村田 道雄, 日下 裕規, 喜多 俊介, 前仲 勝実 日本薬学会年会要旨集 137年会 (1) 137 -137 2017年03月 [査読無し][通常論文]
  
• 次世代のアカデミア創薬を担う若手の力 抗がん剤を目指した若手研究者連携による創薬スクリーニング

  野村 尚生, 松丸 尊紀, 奥村 正樹, 稲葉 謙次, 田村 保明, 前仲 勝実 日本薬学会年会要旨集 137年会 (1) 142 -142 2017年03月 [査読無し][通常論文]
  
• 脂質結合タンパク質の構造生物学 最近の展開 脂質-タンパク質相互作用の解明を目指した重原子化CD1dリガンドの合成と生理活性の評価

  花島 慎弥, Hossain Imran, 土川 博史, 村田 道雄, 日下 裕規, 喜多 俊介, 前仲 勝実 日本薬学会年会要旨集 137年会 (1) 137 -137 2017年03月 [査読無し][通常論文]
  
• 脂質結合タンパク質の構造生物学 最近の展開 脂質抗原受容体CD1dの脂質認識部位に存在する親水性アミノ酸残基の機能解析とその制御

  井貫 晋輔, 相羽 俊彦, 平田 菜摘, 相原 瑛美, 市原 収, 吉留 大輔, 喜多 俊介, 前仲 勝実, 深瀬 浩一, 藤本 ゆかり 日本薬学会年会要旨集 137年会 (1) 137 -137 2017年03月 [査読無し][通常論文]
  
• Breast tumor kinase活性化機構の解明

  松尾友樹, 神田諒, 西條慎也, 清水伸隆, 前仲勝実, 尾瀬農之 量子ビームサイエンスフェスタ(Web) 2016 2017年
• ヒストンメチル化酵素複合体構成因子EZH2-EED相互作用阻害剤の創製研究

  塩田勇介, 薬師寺文華, 加藤いづみ, 岡田ゆかり, 堀内正隆, 児玉耕太, 松田彰, 前仲勝実, 市川聡 メディシナルケミストリーシンポジウム講演要旨集 35th 2017年
• 自然免疫受容体Mincleの糖脂質認識機構

  古川敦, 須知佑介, 池野里紗, 松丸尊紀, 上敷領淳, 齊藤貴士, 尾瀬農之, 山崎晶, 前仲勝実 日本薬学会年会要旨集(CD-ROM) 137th ROMBUNNO.S12‐1 2017年 [査読無し][通常論文]
  
• 抗がん剤を目指した若手研究者連携による創薬スクリーニング

  野村尚生, 松丸尊紀, 奥村正樹, 稲葉謙次, 田村保明, 前仲勝実 日本薬学会年会要旨集(CD-ROM) 137th ROMBUNNO.S13‐4 2017年 [査読無し][通常論文]
  
• 免疫受容体Mincleによる糖脂質認識機構のNMR解析

  齊藤貴士, 須知佑介, 古川敦, 前仲勝実 Abstracts. Annual Meeting of the NMR Society of Japan 55th 146‐147 2016年11月16日 [査読無し][通常論文]
  
• 緑膿菌のレクチンLecA・LecB阻害剤リード化合物の創製研究

  大形悠梨子, 高木朋之, 阿部裕希, 加藤いづみ, 前仲勝実, 市川聡, 堺谷政弘 メディシナルケミストリーシンポジウム講演要旨集 34th 161 2016年11月11日 [査読無し][通常論文]
  
• 糖脂質リガンドにおける脂質部位の構造展開を基盤とした新規CD1dリガンドの創製研究

  井貫晋輔, 相羽俊彦, 相羽俊彦, 平田菜摘, 柏原瑛美, 市原収, 吉留大輔, 喜多俊介, 前仲勝実, 深瀬浩一, 藤本ゆかり メディシナルケミストリーシンポジウム講演要旨集 34th 139 2016年11月11日 [査読無し][通常論文]
  
• 悪性脳腫瘍治療を指向した新規ベンゾオキサゾール誘導体の合成と活性評価

  林健人, 阿部祐希, 前田直良, 前仲勝実, 市川聡, 堺谷政弘 メディシナルケミストリーシンポジウム講演要旨集 34th 103 2016年11月11日 [査読無し][通常論文]
  
• HLAの立体構造と免疫制御受容体の分子認識機構

  前仲勝実, 前仲勝実, 喜多俊介, 黒木喜美子 MHC (Web) 23 (2 Suppl) 63 -63 2016年10月15日 [査読無し][通常論文]
  
• HLAの立体構造と免疫制御受容体の分子認識機構

  前仲 勝実, 喜多 俊介, 黒木 喜美子 MHC: Major Histocompatibility Complex 23 (2Suppl.) 63 -63 2016年10月 [査読無し][通常論文]
  
• HLAの立体構造と免疫制御受容体の分子認識機構

  前仲 勝実, 喜多 俊介, 黒木 喜美子 MHC: Major Histocompatibility Complex 23 (2Suppl.) 63 -63 2016年10月 [査読無し][通常論文]
  
• HLAの立体構造と免疫制御受容体の分子認識機構

  黒木 喜美子, 喜多 俊介, 前仲 勝実 MHC: Major Histocompatibility Complex 23 (2) 80 -95 2016年09月 [査読無し][通常論文]
   

  HLAは非常に遺伝子多型性が高く,多数の遺伝子ファミリーを形成することによって多重性も獲得し,自己・非自己認識を担っている糖タンパク質である。通常,幅広い抗原由来のペプチドをT細胞へ提示するが,さらに,様々な免疫制御受容体との相互作用を介して免疫応答を多面的に調節し,個体の恒常性を維持していることが明らかになってきた。このようなHLAが持つ多面的機能の理解には,X線結晶構造解析による立体構造の決定や物理化学的な相互作用解析が大きな貢献を果たしてきた。本稿では,HLAの分子構造から特にHLAクラスIと受容体群との分子認識機構に着目し,どのようにHLAが免疫反応を制御しているかを概説するとともに,疾患との関連を考察する。(著者抄録)
• 脂質認識部位を制御する新規リガンドを用いたCD1dの機能解析

  平田菜摘, 相羽俊彦, 相羽俊彦, 内梨洋介, 市原収, 吉留大輔, 喜多俊介, 前仲勝実, 深瀬浩一, 井貫晋輔, 藤本ゆかり 日本糖質学会年会要旨集 35th 115 2016年08月01日 [査読無し][通常論文]
  
• アンジオポエチン様因子Angptl2の結晶構造解析および免疫受容体LILRB2との相互作用解析

  荒牧峻彦, 黒木喜美子, 門松毅, 寺田和豊, 尾池雄一, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 104 2016年05月19日 [査読無し][通常論文]
  
• I型IFNシグナルを回避する麻疹ウイルスVタンパク質の分子基盤解析

  永野悠馬, 若原拓也, 野間井智, 市川聡, 柳雄介, 前仲勝実, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 16th 85 2016年05月19日 [査読無し][通常論文]
  
• 可溶性HLA‐Gの製剤化に向けて

  田所高志, 可野巧, 市川聡, 松田彰, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 155 2016年05月19日 [査読無し][通常論文]
  
• 生体内AGE化タンパク質検出系の構築に向けた一本鎖抗体(scFv)の創製と評価

  福田夏希, 宮崎広海, 分山結加里, 中原悠介, 佐藤卓史, 小橋川敬博, 逢坂文那, 斉藤貴士, 前仲勝実, 野井健太郎, 小椋光, 中村照也, 山縣ゆり子, 森岡弘志 日本蛋白質科学会年会プログラム・要旨集 16th 118 2016年05月19日 [査読無し][通常論文]
  
• カイコバキュロウイルスを用いた新規Bacmamの開発

  今井徳俊, 田所高志, 福原秀雄, 堀内正隆, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 78 2016年05月19日 [査読無し][通常論文]
  
• 免疫疾患患者血清中のHLA‐Gの定量に向けた抗HLA‐G抗体の特性評価

  目黒愛実, 古川敦, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 81 2016年05月19日 [査読無し][通常論文]
  
• NMRを用いた自然免疫受容体Mincleの糖脂質認識機構の解析

  須知佑介, 古川敦, 齊藤貴士, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 50 2016年05月19日 [査読無し][通常論文]
  
• カイコバキュロウイルス発現系による急性骨髄性白血病に特異的なFLT3/ITDタンパク質の効率的な生産

  開俊樹, 菊池次郎, 喜多俊介, 前仲勝実, 古川雄祐, 柴山修哉 日本農芸化学会大会講演要旨集(Web) 2016 2C004 (WEB ONLY) 2016年03月05日 [査読無し][通常論文]
  
• 抗ウイルス感染症研究のフロンティア ウイルス侵入過程の重要性 モルビリウイルス属の細胞侵入機構の構造基盤と阻害剤開発

  前仲 勝実 日本薬学会年会要旨集 136年会 (1) 221 -221 2016年03月 [査読無し][通常論文]
  
• ガレクチン‐3阻害剤の創薬研究

  渡邊友章, 大形悠梨子, 阿部祐希, 手塚洋平, 前田直良, 前仲勝実, 堺谷政弘 日本薬学会年会要旨集(CD-ROM) 136th (2) ROMBUNNO.29T‐PM10S -127 2016年03月 [査読無し][通常論文]
  
• 小胞体ストレスタンパク質を標的とした抗癌剤スクリーニング

  野村尚生, 鈴木華央, 松丸尊紀, 奥村正樹, 稲葉謙次, 田村保明, 前仲勝実 日本薬学会年会要旨集(CD-ROM) 136th (2) ROMBUNNO.28S‐PM06 -66 2016年03月 [査読無し][通常論文]
  
• 単純ヘルペスウイルス感染阻害活性を示す糖ペプチドの合成研究

  柿田浩輔, 古川敦, 穴田仁洋, 南部寿則, 前仲勝実, 松永茂樹, 橋本俊一 日本薬学会年会要旨集(CD-ROM) 136th (2) ROMBUNNO.28AB‐PM065 -194 2016年03月 [査読無し][通常論文]
  
• CD1dの脂質認識部位への高親和性を指向した新規リガンドの創製研究

  井貫 晋輔, 相羽 俊彦, 平田 菜摘, 市原 収, 吉留 大輔, 喜多 俊介, 前仲 勝実, 深瀬 浩一, 藤本 ゆかり 天然有機化合物討論会講演要旨集 58 Oral28 2016年 

  背景・目的 脂質抗原受容体CD1dは樹状細胞等に存在しT細胞の分化、活性化を制御することが知られている。CD1dは糖脂質リガンドと結合すると、ナチュラルキラーT（NKT）細胞上のT細胞抗原受容体（TCR）に認識され、様々なサイトカイン（INF-g、IL-4、IL-10など）を誘導する¹⁾。これらのサイトカインは免疫応答のバランス制御に関わっており、制御を可能とするリガンドの創製は重要な研究課題である。既知のCD1dリガンドとしては海洋天然物を基盤に開発された糖脂質a-GalCer (KRN7000) が知られている（Fig.1）²⁾。これまでのa-GalCerの構造活性相関研究によりCD1dに対するリガンド結合様式の解明が精力的に進められてきたが³⁾、CD1dの疎水性ポケットにおける脂質リガンド認識機構の詳細な解析はあまり行われていない。このような背景のもと、我々はa-GalCerの長鎖アルキル基の構造展開を基に、CD1dの脂質結合部位におけるリガンド認識機構の解明とその制御を目指し、本研究に着手した。 化合物デザイン・合成 糖脂質やリン脂質など生体関連脂質分子は、主に糖やリン酸基等の親水性ヘッドグループと長鎖アルキル基等の疎水性領域から構成されている。長鎖アルキル基等は、脂質認識タンパク質中の非極性アミノ酸から構成される疎水性ポケットによって疎水性相互作用を介して認識される。しかしながら、いくつかの脂質認識タンパク質において、これらの疎水性ポケット中の、非常に限定的な領域に、極性アミノ酸を見出すことができる。疎水性領域における水素結合はタンパク質表面などの親水性領域における水素結合と比較し、より安定な結合を形成することが報告されているが⁴⁾、脂質認識において疎水性ポケット中の親水性アミノ酸残基に着目して、水素結合形成を狙う試みは限られている。我々は、これらの親水性残基との水素結合形成による活性制御を指向したリガンド構造のデザインを行った。報告されているmCD1d—a-GalCer複合体のX線結晶構造（PDB: 3G08）⁵⁾を精査した結果、a-GalCerの長鎖アルキル基との相互作用領域である疎水性ポケット（A’ pocket）中に、水素結合可能な親水性領域（Ser28およびCys12, His38周辺部）を見出した（Fig.1）。これまでにこれらの親水性領域との相互作用を意図したリガンドとして、脂質末端に水酸基を含むa-GalCer誘導体が報告されているが、詳細な構造活性相関研究は行われていない⁶⁾ 。今回、我々は親水性領域に対して水素結合等を介して相互作用可能なアミド基をa-GalCerの長鎖アルキル基に導入することを計画した（Fig.2）。まずCD1d のSer28との相互作用が予想される部位近傍にアミド基を導入した5数種のリガンド(1a-e)を合成した。またCys12, His38との相互作用を意図したリガンド（2a、2b）も併せて合成した。 生物活性評価 最初にmCD1dタンパク質とNKTハイブリドーマ（2E10）⁷⁾を用いたサイトカイン誘導能評価を行った（APC-free assay）⁸⁾。すなわち、CD1dをプレートに固定化し、各リガンドを加えた後、NKTハイブリドーマを加え、誘導されるサイトカイン（IL-2）の量を定量した（Fig.3）。1a、1bはa-GalCerと比較し、 (View PDFfor the rest of the abstract.)
• モルビリウイルス属の細胞侵入機構の構造基盤と阻害剤開発

  前仲勝実 日本薬学会年会要旨集(CD-ROM) 136th ROMBUNNO.S40‐4 2016年 [査読無し][通常論文]
  
• 麻疹ウイルスHタンパクの5つの主要な抗原エピトープ

  竹田誠, 田原舞乃, 前仲勝実 日本小児感染症学会総会・学術集会プログラム・抄録集 48th 316 2016年 [査読無し][通常論文]
  
• ヘパラン硫酸を介した狂犬病ウイルスの細胞吸着機構の解析

  佐々木道仁, アニンディタ パウリナ, 伊藤直人, 杉山誠, 福原秀雄, 尾瀬農之, 前仲勝実, 大場靖子, 澤洋文, 澤洋文 日本分子生物学会年会プログラム・要旨集(Web) 39th ROMBUNNO.2P‐0314 (WEB ONLY) 2016年 [査読無し][通常論文]
  
• HLA‐Cw12拘束性HIV‐1由来ペプチドの探索

  山下諒也, 黒木喜美子, 渡邊洋介, 小柳円, 滝口雅文, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 39th ROMBUNNO.1P‐0042 (WEB ONLY) 2016年 [査読無し][通常論文]
  
• 狂犬病ウイルスGタンパク質特異的抗体の中和機構の解明

  有坂知朗, 荒牧峻彦, 青木亨丞, 伊藤直人, 杉山誠, 尾瀬農之, 福原秀雄, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 39th ROMBUNNO.1P‐0598 (WEB ONLY) 2016年 [査読無し][通常論文]
  
• 糖鎖を作る・読む・壊す分子システム ヒト免疫系受容体PILRの糖ペプチド認識機構

  前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [1S2 -5] 2015年12月 [査読無し][通常論文]
  
• PEG化タンパク質の精製に向けた新規PEG化試薬の設計

  山田 千聖, Wawro Adam, 黒木 喜美子, 高橋 愛実, 村岡 貴博, 金原 数, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [2P0459] -[2P0459] 2015年12月 [査読無し][通常論文]
  
• 構造分子生物学・生化学の進展 糖脂質認識C型レクチン受容体Mincleの構造解析と新規アジュバント探索

  古川 敦, 上敷領 淳, 須知 裕介, 池野 里紗, 松丸 尊紀, 児玉 耕太, 尾瀬 農之, 山崎 晶, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [3W7 -6] 2015年12月 [査読無し][通常論文]
  
• (プロ)レニン受容体の調製と機能評価系の構築

  石山 夢美, 喜多 俊介, 田所 高志, 笠井 宣征, 北辻 千展, Krayukhina Elena, 内山 進, 神田 敦宏, 石田 晋, 黒木 喜美子, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [2P1235] -[2P1235] 2015年12月 [査読無し][通常論文]
  
• Lectin-like transcript 1(LLT1)C型レクチン様ドメインの結晶構造解析

  喜多 俊介, 田所 高志, 松原 永季, 笠井 宣征, 玉置 貴晴, 岡部 由紀, 日下 裕規, 石山 夢美, 福原 秀雄, 上敷領 淳, Krayukhina Elena, 内山 進, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [3T17p -02(3P0328)] 2015年12月 [査読無し][通常論文]
  
• カイコバキュロウイルスを用いた新規Bacmamの開発

  今井徳俊, 田所高志, 堀内正隆, 福原秀雄, 前仲勝実 日本生化学会大会(Web) 88th 3P0912 (WEB ONLY) -[3P0912] 2015年12月 [査読無し][通常論文]
  
• Crystal structure of human CD1d-β2m produced by silkworm-baculovirus expression system

  Kita S, Kusaka H, Yoshida K, Kasai Y, Niiyama M, Hanashima S, Sugiyama S, Murata M, Kuroki K, Maenaka K CD1-MR1-2015 2015年11月 [査読無し][通常論文]
  
• THE IMMUNOSUPPRESSIVE EFFECT OF HLA-G2/G6 HOMODIMER IN COLLAGEN-INDUCED ARTHRITIS MICE

  Ami Takahashi, Kimiko Kuroki, Katsumi Maenaka TISSUE ANTIGENS 86 (2) 78 -78 2015年08月 [査読無し][通常論文]
  
• MECHANISMS REPROGRAMMING B CELLS BY HLA-G AND THEIR SIGNATURE IN KIDNEY TRANSPLANT PATIENTS

  Maureen Ezeakile, Vera Portik-Dobos, Daniel D. Horuzsko, Rajan Kapoor, Katsumi Maenaka, Carlos F. Zayas, Laura L. Mulloy, Anatolij Horuzsko TISSUE ANTIGENS 86 (2) 90 -91 2015年08月 [査読無し][通常論文]
  
• 【構造生物学が推進する医薬基盤とその応用】抑制型免疫受容体PILRαによるヘルペスウイルス1型由来O型糖鎖およびペプチド配列の同時認識と創薬

  尾瀬 農之, 古川 敦, 黒木 喜美子, 前仲 勝実 医学のあゆみ 254 (8) 559 -565 2015年08月 [査読無し][通常論文]
   

  ウイルスは、体内・細胞侵入から放出に至るまでその生活環のすべてにおいて宿主の生命活動を巧妙に利用している。宿主因子とウイルス分子の相互作用を原子レベルで解明できれば、宿主とウイルスが攻防を繰り広げている進化の過程を目の当たりにし、感染防御に欠かせない宿主の生命現象を深く理解することができる。本研究では、単純ヘルペスウイルスI型(HSV-I)がヒト細胞に侵入する際に免疫系受容体paired Ig-like type 2レセプターα(PILRα)を利用する仕掛けを、構造生物学的手法により明らかにした。PILRαはHSV-1の細胞融合タンパク質gBの特定領域を認識するが、この認識は、修飾されたO型糖鎖と糖鎖が結合しているポリペプチド鎖を同時に認識する新奇なものであった。O型糖鎖とペプチドの両者がgBとPILRαの結合に必要なことを、表面プラズモン法により確認し、実際のウイルス感染も阻害されることが明らかとなった。この発見を利用し、HSV-I感染予防などの創薬に向けてスクリーニングや化合物の設計・合成を行っている。(著者抄録)
• 単純ヘルペスウイルス感染阻害活性を有する糖ペプチドの設計と合成

  柿田浩輔, 羽鳥菜々生, 古川敦, 山田友樹, 穴田仁洋, 南部寿則, 前仲勝実, 橋本俊一 日本糖質学会年会要旨集 34th 252 2015年07月01日 [査読無し][通常論文]
  
• 可溶性HLA‐G分子の効率的生産に向けてのアプローチ

  可野巧, 田所高志, 市川聡, 松田彰, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 115 2015年05月26日 [査読無し][通常論文]
  
• ポリエーテル化合物生合成酵素の結晶構造解析

  尾瀬農之, 笈川あずさ, 野間井智, 澤田光平, 道仙卓也, 南篤志, 及川英秋, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 67 2015年05月26日 [査読無し][通常論文]
  
• マウス由来TMPRSS2の相互作用解析に向けたタンパク質発現系の構築

  宮本優介, 福原秀雄, 竹田誠, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 115 2015年05月26日 [査読無し][通常論文]
  
• 毒ヘビがもつ自己の毒に対する内在性阻害タンパク質Small Serum Proteinの機能と構造解析

  塩井(青木, 成留実, 塩井誠次郎, 田所高志, 黒木喜美子, 前仲勝実, 寺田成之 日本蛋白質科学会年会プログラム・要旨集 15th 78 2015年05月26日 [査読無し][通常論文]
  
• ヒト免疫受容体NKRP1AによるリガンドLLT1の分子認識機構の解明

  田所高志, 喜多俊介, 松原永季, 笠井宣征, 玉置貴晴, 岡部由紀, 日下裕規, 石山夢美, 福原秀雄, 上敷領淳, 尾瀬豊之, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 145 2015年05月26日 [査読無し][通常論文]
  
• カイコバキュロウイルス発現系を用いた麻疹ウイルスHタンパク質およびヒト免疫受容体SLAMの調製と相互作用解析

  今井徳俊, 田所高志, 吉田康貴, 喜多俊介, 黒木喜美子, 橋口隆生, 柳雄介, 竹田誠, 福原秀雄, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 114 2015年05月26日 [査読無し][通常論文]
  
• 単純ヘルペスウイルス侵入阻害糖ペプチドの最適化を目指した熱力学的相互作用解析およびX線結晶構造解析

  山田友樹, 古川敦, 柿田浩輔, 坂本二郎, 前田直良, 逢坂文那, 斉藤貴士, 黒木喜美子, 荒瀬尚, 穴田仁洋, 尾瀬農之, 橋本俊一, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 90 2015年05月26日 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dの結晶構造解析

  日下裕規, 喜多俊介, 吉田康貴, 笠井宜征, 新山真由美, 杉山成, 村田道雄, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 89 2015年05月26日 [査読無し][通常論文]
  
• JAK‐STAT経路を阻害するウイルスタンパク質の機能・構造解析

  永野悠馬, 野間井智, 前仲勝実, 柳雄介, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 15th 88 2015年05月26日 [査読無し][通常論文]
  
• カイコ幼虫を用いた活性型PKBの発現と精製

  前崎綾子, 佐藤亮介, 田岡万悟, 金場哲平, 朝野維起, 藤田千春, 藤原俊伸, 伊藤隆, 礒辺俊明, 箱嶋敏雄, 前仲勝実, 三島正規 日本蛋白質科学会年会プログラム・要旨集 15th 123 2015年05月26日 [査読無し][通常論文]
  
• アンジオポエチン様因子Angptl2の発現系構築と免疫受容体LILRB2との相互作用解析

  荒牧峻彦, 黒木喜美子, 門松毅, 寺田和豊, 尾池雄一, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 138 2015年05月26日 [査読無し][通常論文]
  
• ハーセプチン抗体Fv断片の相互作用解析および熱安定性解析

  中村光太, 田所高志, 前田龍, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 114 2015年05月26日 [査読無し][通常論文]
  
• 脂質活性構造研究の最前線 免疫系受容体MincleとMCLの糖脂質認識の分子基盤

  前仲 勝実 日本薬学会年会要旨集 135年会 (1) 93 -93 2015年03月 [査読無し][通常論文]
  
• 動き出した創薬オープンイノベーションネットワーク 北海道大学スクリーニング拠点の現状と展望

  前仲 勝実 日本薬学会年会要旨集 135年会 (1) 155 -155 2015年03月 [査読無し][通常論文]
  
• 単純ヘルペスウイルスがペア型レセプターPILRαを利用するメカニズム

  尾瀬農之, 黒木喜美子, 山口宗親, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実 物構研サイエンスフェスタ要旨集 3rd 79 2015年 [査読無し][通常論文]
  
• ゲノム網羅的解析によるB型肝炎ウイルス感染の病態関連遺伝子の同定と新規診断法の開発 HLA等機能解析

  前仲勝実 ゲノム網羅的解析によるB型肝炎ウイルス感染の病態関連遺伝子の同定と新規診断法の開発に関する研究 平成26年度 委託業務成果報告書 59 -60 2015年 [査読無し][通常論文]
  
• 北海道大学スクリーニング拠点の現状と展望

  前仲勝実 日本薬学会年会要旨集(CD-ROM) 135th ROMBUNNO.S20-2 2015年 [査読無し][通常論文]
  
• 免疫系受容体MincleとMCLの糖脂質認識の分子基盤

  前仲勝実 日本薬学会年会要旨集(CD-ROM) 135th ROMBUNNO.S02-3 2015年 [査読無し][通常論文]
  
• 高収率な一本鎖抗体のデザインに向けた検討

  山下駿, 山上紗矢佳, 逢坂文那, 齊藤貴士, 片岡千和, 澤田石一之, 前仲勝実, 小橋川敬博, 森岡弘志 日本薬学会九州支部大会講演要旨集 31st 90 2014年11月21日 [査読無し][通常論文]
  
• クロマチンリモデリング関連因子間の相互作用解析

  谷口岳史, 辻美保子, 森田和美, 阿川大貴, 諏訪喜昭, 小橋川敬博, 池鯉鮒麻美, 中村照也, 逢坂文那, 齊藤貴士, 前仲勝実, 菅野新一郎, 安井明, 山縣ゆり子, 森岡弘志 日本薬学会九州支部大会講演要旨集 31st 43 2014年11月21日 [査読無し][通常論文]
  
• 麻疹ウイルスHタンパク質を標的とした侵入阻害剤の開発

  東端将哲, 福原秀雄, 逢坂文那, 橋口隆生, 柳雄介, 竹田誠, 児玉耕太, 齊藤貴士, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 62nd 204 2014年10月31日 [査読無し][通常論文]
  
• 犬ジステンパーウイルスエンベロープタンパク質HおよびFの相互作用解析と電子顕微鏡による構造解析

  依田芽生, 福原秀雄, 武田森, 三尾和弘, PLATTET Philippe, 竹田誠, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 62nd 218 2014年10月31日 [査読無し][通常論文]
  
• ムンプスウイルス感染におけるHeat Shock Protein70の役割

  加藤大志, 喜多俊介, 久保田耐, 中津祐一郎, 前仲勝実, 木所稔, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 62nd 220 2014年10月31日 [査読無し][通常論文]
  
• イヌジステンパーウイルスH蛋白質におけるP541S変異が受容体SLAMとの相互作用に及ぼす影響

  吉田康貴, 酒井宏治, 喜多俊介, 福原秀雄, 柳雄介, 竹田誠, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 62nd 219 2014年10月31日 [査読無し][通常論文]
  
• 単純ヘルペスウイルス1型感染阻害物質の探索

  前田直良, 古川敦, 坂本二郎, 黒木喜美子, 尾瀬農之, 柿田浩輔, 穴田仁洋, 橋本俊一, 有井潤, 加藤哲久, 川口寧, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 62nd 273 2014年10月31日 [査読無し][通常論文]
  
• II型膜貫通型セリンプロテアーゼTMPRSS2は,HA開裂部位にmono‐basicなアミノ酸配列をもつA型インフルエンザウイルスに対する肺内必須活性化酵素である

  酒井宏治, 網康至, 田原舞乃, 久保田耐, 安楽正輝, 中島典子, 高下恵美, 関塚剛史, 駒瀬勝啓, 信澤枝里, 小田切孝人, 前仲勝実, 黒田誠, 長谷川秀樹, 河岡義裕, 田代眞人, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 62nd 155 2014年10月31日 [査読無し][通常論文]
  
• 構造細胞生物学の展開 医薬イノベーションの起爆剤としての構造 糖脂質・糖ペプチドを認識する免疫系受容体の構造生物学的研究

  前仲 勝実 日本生化学会大会プログラム・講演要旨集 87回 [2S06p -4] 2014年10月 [査読無し][通常論文]
  
• マラリア原虫におけるアピコプラストTic22とトランジット配列の複合体形成

  逢坂文那, 山田友樹, 松丸尊紀, 前仲勝実, 齊藤貴士 日本生化学会大会(Web) 87th 4P-234 (WEB ONLY) -234] 2014年10月 [査読無し][通常論文]
  
• 自己免疫疾患に関わるHLAクラスI分子による免疫制御の分子基盤

  前仲 勝実, 黒木 喜美子 日本脊椎関節炎学会誌 6 (1) 33 -38 2014年10月 [査読無し][通常論文]
   

  ヒト白血球抗原(HLA)-B27は、多集団において、リウマチ性疾患の一つである強直性脊椎炎(AS)患者のほぼ90%が陽性であること、HLA-B27遺伝子導入ラット・マウスがAS様症状を示すことから、AS病因遺伝子として強く示唆されてきた。HLA-B27ホモ二量体と類似したホモ二量体を形成するHLA-Gの分子形態とも比較しながら、その分子構造基盤につき考察し、AS発症の機序について述べた。
• 自己免疫疾患に関わるHLAクラスI分子による免疫制御の分子基盤

  前仲 勝実, 黒木 喜美子 日本脊椎関節炎学会誌 6 (1) 33 -38 2014年10月 [査読無し][通常論文]
   

  ヒト白血球抗原(HLA)-B27は、多集団において、リウマチ性疾患の一つである強直性脊椎炎(AS)患者のほぼ90%が陽性であること、HLA-B27遺伝子導入ラット・マウスがAS様症状を示すことから、AS病因遺伝子として強く示唆されてきた。HLA-B27ホモ二量体と類似したホモ二量体を形成するHLA-Gの分子形態とも比較しながら、その分子構造基盤につき考察し、AS発症の機序について述べた。
• Application of the silkworm expression system for the structural biology: a case study of human CD1d-β2m

  Kita S, Kusaka H, Yoshida K, Kasai Y, Niiyama M, Sugiyama S, Murata M, Kuroki K, Maenaka K ICCBM15 2014年09月 [査読無し][通常論文]
  
• 抗ヘルペスウイルス活性をもつシアル酸含有糖ペプチドおよび類縁体の合成

  柿田浩輔, 羽鳥菜々生, 坂本二郎, 古川敦, 穴田仁洋, 南部寿則, 前仲勝実, 橋本俊一 日本糖質学会年会要旨集 33rd 140 2014年07月23日 [査読無し][通常論文]
  
• Structural Analysis for the Interaction of sialic T antigen glycopeptide of HSV-1 with Entry Receptor PILR alpha

  Takao Nomura, Jiro Sakamoto, Fumina Oosaka, Kosuke Kakita, Atsushi Furukawa, Masahiro Anada, Shunichi Hashimoto, Kimiko Kuroki, Toyoyuki Ose, Hisashi Arase, Takashi Saitoh, Katsumi Maenaka PROTEIN SCIENCE 23 230 -230 2014年07月 [査読無し][通常論文]
  
• 熱力学的解析から見えてきた一本鎖抗体の効率的生産へ向けてのアプローチ

  山下駿, 山上紗矢佳, 逢坂文那, 斉藤貴士, 片岡千和, 澤田石一之, 前仲勝実, 小橋川敬博, 森岡弘志 日本蛋白質科学会年会プログラム・要旨集 14th 112 2014年05月26日 [査読無し][通常論文]
  
• 表面蛋白質の発現ストラテジー

  前仲勝実 日本蛋白質科学会年会プログラム・要旨集 14th 39 2014年05月26日 [査読無し][通常論文]
  
• C型レクチン様受容体MincleとMCLの糖脂質認識機構

  古川敦, 上敷領淳, 森大輝, 豊永憲司, 岡部由紀, 藤司亜也, 神田諒, 三宅靖延, 尾瀬農之, 山崎晶, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 14th 121 2014年05月26日 [査読無し][通常論文]
  
• 「構造生物学と免疫」MHCクラスI受容体の構造生物学

  黒木喜美子, 前仲勝実 JSI Newsl 22 (2) 22 2014年04月09日 [査読無し][通常論文]
  
• 創薬研究の基盤となる相互作用解析 一本鎖抗体の実用化に向けた構造機能解析

  森岡 弘志, 小橋川 敬博, 諏訪 喜昭, 山下 駿, 山本 珠実, 福田 夏希, 二階堂 里那, 中原 悠介, 龍野 友花, 斉藤 貴士, 逢坂 文那, 手塚 洋平, 前仲 勝実, 澤田石 一之, 片岡 千和 日本薬学会年会要旨集 134年会 (1) 242 -242 2014年03月 [査読無し][通常論文]
  
• 含リン脱離基法を機軸とする抗ヘルペスウイルス活性をもつ糖ペプチドの立体選択的合成

  柿田浩輔, 羽鳥菜々生, 穴田仁洋, 南部寿則, 前仲勝実, 橋本俊一 日本薬学会年会要旨集(CD-ROM) 134th (2) ROMBUNNO.29PMS-079 -247 2014年03月 [査読無し][通常論文]
  
• アカデミア創薬実現に必要な10の視点 5.合理的創薬と構造解析

  齊藤貴士, 堺谷政弘, 前仲勝実 実験医学 32 (2) 222 -229 2014年02月01日 [査読無し][通常論文]
  
• 【研究成果を薬につなげるアカデミア創薬の戦略と実例】 (第1章)アカデミア創薬実現に必要な10の視点 合理的創薬と構造解析

  齊藤 貴士, 堺谷 政弘, 前仲 勝実 実験医学 32 (2) 222 -229 2014年02月 [査読無し][通常論文]
   

  創薬のターゲットとなる疾患関連タンパク質の立体構造情報を利用した合理的な薬剤設計は、近年の創薬では必須の技術と言える。本稿では、合理的創薬を行うためのタンパク質の立体構造の決定法と、その立体構造情報を利用したインシリコスクリーニング、SBDD(structure based drug design)、FBDD(fragment based drug discovery)について、実例を示しながら解説する。(著者抄録)
• 一本鎖抗体の実用化に向けた構造機能解析

  森岡弘志, 小橋川敬博, 諏訪喜昭, 山下駿, 山本珠実, 福田夏希, 二階堂里那, 中原悠介, 龍野友花, 斉藤貴士, 逢坂文那, 手塚洋平, 前仲勝実, 澤田石一之, 片岡千和 日本薬学会年会要旨集(CD-ROM) 134th ROMBUNNO.S50-5 2014年 [査読無し][通常論文]
  
• 単純ヘルペスウイルスがペア型レセプターPILRαを利用する分子基盤

  尾瀬農之, 黒木喜美子, 山口宗親, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実 日本結晶学会年会講演要旨集 2014 110 2014年 [査読無し][通常論文]
  
• 糖脂質・糖ペプチドを認識する免疫系受容体の構造生物学的研究

  前仲勝実 日本生化学会大会(Web) 87th 2S06P-4 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• NMRによるヒトCD160細胞外ドメインの立体構造解析

  阿部千紘, 黒木喜美子, 小島理恵子, 齊藤貴士, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0723 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• 一本鎖抗体の高収率化クローンの溶液挙動および耐熱性の解析

  山下駿, 山上紗矢佳, 逢坂文那, 齊藤貴士, 片岡千和, 澤田石一之, 前仲勝実, 小橋川敬博, 森岡弘志 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0936 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• クロマチンリモデリング複合体形成の構造生物学的研究

  谷口岳史, 辻美保子, 森田和美, 阿川大貴, 諏訪喜昭, 小橋川敬博, 池鯉鮒麻美, 中村照也, 逢坂文那, 齊藤貴士, 前仲勝実, 菅野新一郎, 安井明, 山縣ゆり子, 森岡弘志 日本分子生物学会年会プログラム・要旨集(Web) 37th 3P-0138 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• HEK293細胞を用いたHLA‐Cw12拘束性HIV‐1由来免疫制御ペプチドの探索

  渡邊洋介, 黒木喜美子, 小柳円, 滝口雅文, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0721 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• 合理的HIVワクチン設計のためのgp120エピトープ探索系の確立に向けて

  阪田竜馬, 黒木喜美子, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0722 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• 2SBP-02 C 型レクチン受容体Mincle の糖脂質認識機構(シグナル伝達機構における構造細胞生物学的新展開,新学術領域研究「細胞シグナリング複合体によるシグナル検知・伝達・応答の構造的基礎」共催,シンポジウム,第52回日本生物物理学会年会(2014年度))

  古川 敦, 上敷領 淳, 尾瀬 農之, 山崎 晶, 前仲 勝実 生物物理 54 (1) S131 2014年 [査読無し][通常論文]
  
• 構造生命科学-構造生物学を超えて-

  前仲 勝実, 寺本 央 生物物理 53 (6) 334 -335 2013年11月25日 [査読無し][通常論文]
  
• マウスリゾチームにおける部位特異的なアスパラギン残基のラセミ化機構の解析

  藤田玲奈, 斎藤貴士, 前仲勝実, 林いづみ, 白石充典, 阿部義人, 植田正 日本薬学会九州支部大会講演要旨集 30th 159 2013年11月20日 [査読無し][通常論文]
  
• ウイルスの膜融合に関与するTMPRSS2タンパク質の調製と結晶化

  佐藤亮, 福原秀雄, 竹田誠, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 61st 419 2013年10月29日 [査読無し][通常論文]
  
• 単純ヘルペスウイルス1型(HSV‐1)のglycoprotein B(gB)とmyelin associated glycoprotein(MAG)との相互作用解明

  青木亨丞, 福原秀雄, 黒木喜美子, 武田森, 末永忠広, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 61st 329 2013年10月29日 [査読無し][通常論文]
  
• 受容体結合領域を含む複数の主要エピトープを変化させた麻疹ウイルスの解析

  田原舞乃, 酒井宏治, 駒瀬勝啓, 前仲勝実, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 61st 225 2013年10月29日 [査読無し][通常論文]
  
• Nectin4を介するモルビリウイルスの細胞侵入機構の解明

  陳甦ルイ, 福原秀雄, 伊藤由梨, 酒匂幸, 橋口隆生, 梶川瑞穂, 柳雄介, 竹田誠, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 61st 315 2013年10月29日 [査読無し][通常論文]
  
• 1回膜貫通型 受容体の遺伝子:KIR,LILR,PILR 免疫系細胞表面のペア型受容体

  黒木喜美子, 前仲勝実 生体の科学 64 (5) 490 -491 2013年10月15日 [査読無し][通常論文]
  
• monensin生合成酵素の結晶構造解析

  道仙卓也, 南篤志, 笈川あずさ, 佐藤恭平, 前仲勝実, 及川英秋, 尾瀬農之 日本結晶学会年会講演要旨集 2013 75 2013年10月12日 [査読無し][通常論文]
  
• 【細胞表面受容体】構造的特徴:1回膜貫通型 受容体の遺伝子:KIR,LILR,PILR 免疫系細胞表面のペア型受容体

  黒木 喜美子, 前仲 勝実 生体の科学 64 (5) 490 -491 2013年10月 [査読無し][通常論文]
  
• Nectin4は上皮に局在する犬ジステンパーウイルス特異的受容体である

  PRATAKPIRIYA Watanyoo, 關文緒, 大槻紀之, 酒井宏治, 福原秀雄, 片本宏, 平井卓哉, TECHANGAMSUWAN Somporn, LAN Nguyen, 前仲勝実, 竹田誠, 山口良二 日本獣医学会学術集会講演要旨集 156th 215 -215 2013年08月30日 [査読無し][通常論文]
  
• MurDの大量発現系の構築と結晶化

  笈川あずさ, 北辻千展, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 13th 123 2013年05月31日 [査読無し][通常論文]
  
• C型レクチン様受容体Mindeの糖脂質認識機構

  古川敦, 上敷領淳, 岡部由紀, 神田涼, 豊永憲司, 森大輝, 三宅靖延, 山崎晶, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 13th 115 2013年05月31日 [査読無し][通常論文]
  
• モネンシン生合成酵素の結晶構造解析

  尾瀬農之, 笈川あずさ, 南篤志, 佐藤恭平, 及川英秋, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 13th 122 2013年05月31日 [査読無し][通常論文]
  
• 樹状細胞免疫受容体DCIRの発現・精製およびX線結晶構造解析

  福原秀雄, 松原永季, 神田諒, 矢部力朗, 海部知則, 尾瀬農之, 岩倉洋一郎, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 13th 119 2013年05月31日 [査読無し][通常論文]
  
• バキュロウイルス‐カイコ発現系を用いて産生したヒトチロシナーゼの性質

  梅田彩奈, 佐藤正幸, 忠平和子, 日下部宜宏, PARK Enock Y, 前仲勝実, 蜷木理, 殿塚隆史, 西河淳 日本農芸化学会大会講演要旨集(Web) 2013 2C16A15 (WEB ONLY) 2013年03月05日 [査読無し][通常論文]
  
• 創薬を指向した構造生物学の最前線 パラミクソウイルス侵入機構と阻害剤開発

  東端 将哲, 福原 秀雄, 齊藤 貴士, 陳 甦へい, 逢坂 文那, 児玉 耕太, 尾瀬 農之, 前仲 勝実 日本薬学会年会要旨集 133年会 (1) 100 -100 2013年03月 [査読無し][通常論文]
  
• 創薬を指向した構造生物学の最前線 パラミクソウイルス侵入機構と阻害剤開発

  東端 将哲, 福原 秀雄, 齊藤 貴士, 陳 甦へい, 逢坂 文那, 児玉 耕太, 尾瀬 農之, 前仲 勝実 日本薬学会年会要旨集 133年会 (1) 100 -100 2013年03月 [査読無し][通常論文]
  
• 細胞外マトリックス分子/ケモカインを標的とした成人T細胞白血病制御法の開発

  前田直良, 上出利光, 服部俊夫, 前仲勝実, 大橋貴 金沢大学がん進展制御研究所がんの転移・薬剤耐性に関わる先導的研究拠点 2012 183 -185 2013年 [査読無し][通常論文]
  
• 重症呼吸器ウイルス感染症のサーベイランス・病態解明及び制御に関する研究 呼吸器感染ヒトパラミクソウイルス全般の病態解明・制御に関する研究 呼吸器感染症ウイルス増殖におけるTMPRSS2の役割ならびに膜融合タンパクP3位保存グルタミンの重要性について

  竹田誠, 安部昌子, 田原舞乃, 酒井宏治, 白戸憲也, 松山州徳, 加納和彦, 野田雅博, 木村博一, 網康至, 加藤篤, 水田克巳, 前仲勝実, 福原秀雄, 江角眞理子 重症呼吸器ウイルス感染症のサーベイランス・病態解明及び制御に関する研究 平成24年度 総括・分担研究報告書 139 -148 2013年 [査読無し][通常論文]
  
• 新型インフルエンザ等新興・再興感染症研究事業 早期麻疹排除及び排除状態の維持に関する研究 麻疹ウイルスの抗原エピトープの構造解析と効果的なワクチン維持のための研究

  前仲勝実 早期麻疹排除及び排除状態の維持に関する研究 平成22-24年度 総合研究報告書 平成24年度 総括・分担研究報告書 173 -176 2013年 [査読無し][通常論文]
  
• 免疫系受容体LILRファミリーの構造と創薬への試み

  前仲勝実 千里ライフサイエンスセミナー講演要旨集 2013 16 -17 2013年 [査読無し][通常論文]
  
• HIV由来ペプチドライブラリーを用いたHLA‐Cw12拘束性免疫制御ペプチドの同定

  渡邊洋介, 黒木喜美子, 小柳円, 滝口雅文, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 36th 3P-0758 (WEB ONLY) 2013年 [査読無し][通常論文]
  
• 結晶構造解析に向けた,乳がん特異的キナーゼBrkとアダプタータンパク質STAP‐2の機能解析

  神田諒, 関根勇一, 前仲勝実, 松田正, 尾瀬農之 日本分子生物学会年会プログラム・要旨集(Web) 36th 2P-0302 (WEB ONLY) 2013年 [査読無し][通常論文]
  
• モルビリウイルス属の細胞侵入機構

  福原秀雄, CHEN Surui, 武田森, 前仲勝実 薬学雑誌 133 (5) 549 -559 2013年 [査読無し][通常論文]
   

  The genus Morbillivirus includes measles virus, canine distemper virus and rinderpest virus. These are highly contagious and exhibit high mortality. These viruses have the attachment glycoprotein, hemagglutinin (H), at the virus surface, which bind to signaling lymphocyte activation molecule (SLAM) and Nectin 4 as receptors for the entry. However, the molecular mechanism for this entry has been limitedly understood. Here we summarize the current topics, (1) newly identified receptor, Nectin 4, (2) crystal structures of H-receptor complexes and (3) detail biochemical studies of the H-F communication for the entry. These provide insight on the mechanism of morbillivirus entry event and furthermore drug developments. © 2013 The Pharmaceutical Society of Japan.
• 創薬に向けた構造生物学

  前仲 勝実, 加藤 晃一 YAKUGAKU ZASSHI 133 (5) 507 -507 2013年 [査読無し][通常論文]
  
• 創薬に向けた構造生物学 モルビリウイルス属の細胞侵入機構

  福原 秀雄, 陳 甦へい, 武田 森, 前仲 勝実 薬学雑誌 133 (5) 549 -559 2013年 [査読無し][通常論文]
   

  モルビリウイルス属は一本鎖マイナス鎖RNAをゲノムとするエンベロープウイルスである。そのウイルス表面には、細胞接着に係わる受容体結合タンパク質と細胞融合を担う融合タンパク質が存在し、基本的にはこの二つのタンパク質が協調して働くことにより、宿主細胞への侵入が起こる。受容体情報伝達リンパ球活性化分子、受容体Nectin4、モルビリウイルスの侵入と体内伝搬、モルビリウイルスの細胞侵入機構、について述べた。
• カニクイザルで致死的感染症を起こしたジステンパーウイルスのサルレセプターの効率的な利用:ジステンパーウイルスはヒトへの脅威となり得るのか?

  酒井宏治, 關文緒, 網康至, 田原舞乃, 中津祐一郎, 大槻紀之, 福原秀雄, 福士秀悦, 吉河智城, 西條政幸, 森川茂, 前仲勝実, 山口良二, 駒瀬勝啓, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 60th 282 2012年10月31日 [査読無し][通常論文]
  
• 野外イヌジステンパーウイルスはヒトNectin4を受容体として利用できる

  大槻紀之, 関塚剛史, 關文緒, 酒井宏治, 久保田耐, 福原秀雄, 前仲勝実, 山口良二, 黒田誠, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 60th 381 2012年10月31日 [査読無し][通常論文]
  
• 麻疹ウイルス単一血溝型決定の分子基盤

  田原舞乃, BRINDLEY Melinda A, 福原秀雄, 酒井宏治, 大野真治, 駒瀬勝啓, ROTA Paul A, PLEMPER Richard K, 前仲勝実, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 60th 282 2012年10月31日 [査読無し][通常論文]
  
• イヌジステンパーウイルスの上皮および中枢神経感染におけるイヌNectin4の利用

  關文緒, PRATAKPIRIYA Watanyoo, 大槻紀之, 酒井宏治, 福原秀雄, 前仲勝実, 山口良二, 竹田誠 日本ウイルス学会学術集会プログラム・抄録集 60th 381 2012年10月31日 [査読無し][通常論文]
  
• オステオポンチン-インテグリン相互作用を分子標的とした成人T細胞白血病に対する抗体免疫療法(Osteopontin-integrin interaction as a molecular target for antibody-mediated immunotherapy in adult T-cell leukemia)

  前田 直良, 大橋 貴, 浩 日勒, 服部 俊夫, 高橋 弥生, 張替 秀郎, 長谷川 寛雄, 藤井 雅寛, 前仲 勝実, 上出 利光 日本癌学会総会記事 71回 117 -117 2012年08月 [査読無し][通常論文]
  
• Structural Biologyとがん治療創薬 ヒトKiller cell Ig-like receptor群のNK細胞アロ反応性の構造基盤(Structural biology and cancer therapy Structural basis for natural killer cell alloreactivity of human killer cell Ig-like receptors)

  前仲 勝実 日本癌学会総会記事 71回 227 -228 2012年08月 [査読無し][通常論文]
  
• HIV由来変異ペプチドによる宿主免疫の二重逃避機構

  黒木 喜美子, 前仲 勝実 臨床免疫・アレルギー科 58 (2) 210 -216 2012年08月 [査読無し][通常論文]
  
• 麻疹ウイルス及び犬ジステンパーウイルスH蛋白質の構造学的研究

  尾瀬農之, 橋口隆生, 酒匂幸, 伊藤由梨, 福原秀雄, 黒木喜美子, 柳雄介, 竹田誠, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 12th 83 2012年05月31日 [査読無し][通常論文]
  
• 機能および構造解析に向けた細胞表面蛋白質の調製法

  福原秀雄, 橋口隆生, 黒木喜美子, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 12th 19 2012年05月31日 [査読無し][通常論文]
  
• ヤツメウナギ由来可変性リンパ球受容体VLRCの結晶構造

  神田諒, 須藤洋一, 笠松純, 笠原正典, 前仲勝実, 尾瀬農之 日本蛋白質科学会年会プログラム・要旨集 12th 117 2012年05月31日 [査読無し][通常論文]
  
• 【抗ウイルス薬-最新の動向-】 麻疹ウイルスと受容体との複合体の立体構造と今後の展望

  前仲 勝実, 橋口 隆生, 柳 雄介 日本臨床 70 (4) 695 -703 2012年04月 [査読無し][通常論文]
  
• 麻疹ウイルスと受容体との複合体の立体構造と今後の展望 (特集 抗ウイルス薬) -- (新規抗ウイルス薬の開発動向と展望)

  前仲 勝実, 橋口 隆生, 柳 雄介 日本臨床 70 (4) 695 -703 2012年04月 [査読無し][通常論文]
  
• 抗ウイルス薬 IV.特論 麻疹ウイルスと受容体との複合体の立体構造と今後の展望

  前仲勝実, 前仲勝実, 橋口隆生, 橋口隆生, 柳雄介 日本臨床 70 (4) 695-703 -703 2012年04月01日 [査読無し][通常論文]
  
• ロバスト設計最適化支援ツールを用いたin silicoスクリーニング

  児玉耕太, 山口政隆, 小川修一, 尾瀬農之, 前仲勝実 日本薬学会年会要旨集 132nd (4) 115 2012年03月05日 [査読無し][通常論文]
  
• ウイルス感染機構の構造基盤と創薬への展開

  前仲勝実, 前仲勝実 日本薬学会年会要旨集 132nd (1) 99 2012年03月05日 [査読無し][通常論文]
  
• 創薬に向けた構造生物学 ウイルス感染機構の構造基盤と創薬への展開

  前仲 勝実 日本薬学会年会要旨集 132年会 (1) 99 -99 2012年03月 [査読無し][通常論文]
  
• オステオポンチン‐インテグリン相互作用を標的とした新規成人T細胞白血病治療法の開発

  前田直良, 大橋貴, CHAGAN‐YASUTAN Haorile, 服部俊夫, 高橋弥生, 張替秀郎, 長谷川寛雄, 藤井雅寛, 前仲勝実, 上出利光 日本生体防御学会学術総会講演抄録集 23rd 66 2012年 [査読無し][通常論文]
  
• 早期麻疹排除及び排除状態の維持に関する研究 麻疹ウイルスの抗原エピトープの構造解析と効果的なワクチン維持のための研究

  前仲勝実 早期麻疹排除及び排除状態の維持に関する研究 平成23年度 総括・分担研究報告書 114 -117 2012年 [査読無し][通常論文]
  
• 樹状細胞免疫受容体DCIRの結晶化と構造解析

  福原秀雄, 松原永季, 神田諒, 矢部力朗, 海部知則, 上敷領淳, 尾瀬農之, 岩倉洋一郎, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 35th 1P-0056 (WEB ONLY) 2012年 [査読無し][通常論文]
  
• ヒト免疫制御受容体LILRB1とリガンドHLA‐Gの複合体の結晶構造解析

  黒木喜美子, 松原永季, 神田諒, 上敷領淳, 尾瀬農之, 福永裕子, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 35th 1P-0060 (WEB ONLY) 2012年 [査読無し][通常論文]
  
• 各臓器移植についての拒絶反応の解説・移植の新しい治療法の紹介(第5回) NKレセプターとHLAの最前線

  黒木 喜美子, 北辻 千展, 前仲 勝実 MHC: Major Histocompatibility Complex 18 (3) 215 -233 2011年12月 [査読無し][通常論文]
   

  ナチュラルキラー(NK)細胞上には多数のNKレセプターが存在し、自然免疫で中心的な役割を果たすNK細胞の傷害活性を制御している。ヒトNKレセプターは、構造上免疫グロブリン(Ig)様レセプターに属するkiller cell immunoglobulin(Ig)-like receptor群、leukocyte Ig-like receptor群およびC型レクチン様レセプターに属するNKG2/CD94群に分類され、いずれも活性型と抑制型が存在するペア型レセプターの一員である。抑制型NKレセプターの多くはMHCクラスIをリガンドとして認識し、正常な自己細胞に対してNK細胞が傷害しないように制御しているが、活性型NKレセプターのリガンドについては未だ不明な点も多い。本稿では特にMHCクラスIをリガンドとするNKレセプターに焦点を当て、その機能、構造、疾患との関連について最近の知見を概説する。(著者抄録)
• 各臓器移植についての拒絶反応の解説・移植の新しい治療法の紹介(第5回) NKレセプターとHLAの最前線

  黒木 喜美子, 北辻 千展, 前仲 勝実 MHC: Major Histocompatibility Complex 18 (3) 215 -233 2011年12月 [査読無し][通常論文]
   

  ナチュラルキラー(NK)細胞上には多数のNKレセプターが存在し、自然免疫で中心的な役割を果たすNK細胞の傷害活性を制御している。ヒトNKレセプターは、構造上免疫グロブリン(Ig)様レセプターに属するkiller cell immunoglobulin(Ig)-like receptor群、leukocyte Ig-like receptor群およびC型レクチン様レセプターに属するNKG2/CD94群に分類され、いずれも活性型と抑制型が存在するペア型レセプターの一員である。抑制型NKレセプターの多くはMHCクラスIをリガンドとして認識し、正常な自己細胞に対してNK細胞が傷害しないように制御しているが、活性型NKレセプターのリガンドについては未だ不明な点も多い。本稿では特にMHCクラスIをリガンドとするNKレセプターに焦点を当て、その機能、構造、疾患との関連について最近の知見を概説する。(著者抄録)
• gamma/deltaT細胞とNK細胞 ヒトCD161(NKRP1A/KLRB1)とリガンドLLT1の分子認識機構(Molecular basis for LLT1 recognition by human CD161 (NKRP1A/KLRB1))

  黒木 喜美子, 上敷領 淳, 福原 秀雄, 岡部 由紀, 前仲 勝実 日本免疫学会総会・学術集会記録 40 77 -77 2011年11月 [査読無し][通常論文]
  
• SrcおよびAuroraキナーゼ2重阻害による滑膜肉腫の相乗的in vivo抗腫瘍効果(Dual inhibition of Src and Aurora kinases abrogates tumor growth, invasion, and angiogenesis of synovial sarcoma in vivo)

  津田 真寿美, 新井 隆太, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介 日本癌学会総会記事 70回 274 -274 2011年09月 [査読無し][通常論文]
  
• 抗ジンセノシドRe小型化抗体の作製及び酵素免疫吸着測定法(ELISA)の開発

  坂元政一, PONGKITWITOON Benyakan, 田中宏幸, 森元聡, 前仲勝実, 柴田攻 コロイドおよび界面化学討論会講演要旨集 63rd 512 2011年08月22日 [査読無し][通常論文]
  
• Leukocyte Immunoglobulin-Like Receptor(LILR)ファミリーの分子認識

  黒木 喜美子, 前仲 勝実 生化学 83 (8) 715 -726 2011年08月 [査読無し][通常論文]
   

  Leukocyte Immunoglobulin(Ig)-Like Receptor(LILR)は、細胞外にIg様ドメインを二つまたは四つ持ち、免疫系の細胞に広く発現するペア型受容体ファミリーであり、細胞内ドメインの構造から活性型、抑制型、分泌型の3種類に分類される。1997年以降、現在までに11種類の機能的LILRタンパク質が同定されており、疾患と発現量または遺伝子多型との関連が多数報告されているにも関わらず、その機能・構造・リガンド特異性については未だ不明な点が多い。本稿では特にリガンドとして主要組織適合性複合体(MHC)クラスI分子を認識する抑制性受容体LILRB1とLILRB2に焦点を当て、LILRファミリーの立体構造およびリガンドとの分子認識機構について、筆者らの研究成果を中心に最近の知見を概説する。(著者抄録)
• BmNPVバクミドシステムを用いたカイコでの膜タンパク質の発現

  佐々木香織, 梶川瑞穂, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 11th 152 2011年05月27日 [査読無し][通常論文]
  
• 表面タンパク質の不安定な複合体の分子解析法

  前仲勝実 日本蛋白質科学会年会プログラム・要旨集 11th 34 2011年05月27日 [査読無し][通常論文]
  
• T細胞機能の誘導と抑制 HVEMとその受容体CD160によるT細胞の制御

  小島理恵子, 前仲勝実 月刊臨床免疫・アレルギー科 55 (5) 515 -520 2011年05月25日 [査読無し][通常論文]
  
• HVEMとその受容体CD160によるT細胞の制御 (特集 T細胞機能の誘導と抑制)

  小島 理恵子, 前仲 勝実 臨床免疫・アレルギー科 55 (5) 515 -520 2011年05月 [査読無し][通常論文]
  
• EPITOPE MAPPING OF A NOVEL B27-SPECIFIC ANTIBODY

  Joanna L. Giles, Kirsty McHugh, Katilin DiGleria, Jackie Shaw, Simon Kollnberger, Katsumi Maenaka, Osiris Marroquin, Christoph Renner, Paul Bowness RHEUMATOLOGY 50 140 -141 2011年04月 [査読無し][通常論文]
  
• 生命現象を担う生体分子に着目した創薬と臨床応用 多様な形態を有するHLA-G蛋白質とヒト免疫制御受容体との分子認識

  黒木 喜美子, 福永 裕子, 上敷領 淳, 白石 充典, 尾瀬 農之, 前仲 勝実 日本薬学会年会要旨集 131年会 (1) 188 -188 2011年03月 [査読無し][通常論文]
  
• 生命現象を担う生体分子に着目した創薬と臨床応用 多様な形態を有するHLA-G蛋白質とヒト免疫制御受容体との分子認識

  黒木 喜美子, 福永 裕子, 上敷領 淳, 白石 充典, 尾瀬 農之, 前仲 勝実 日本薬学会年会要旨集 131年会 (1) 188 -188 2011年03月 [査読無し][通常論文]
  
• SU6656はSrcとAuroraキナーゼの二重阻害によってヒト滑膜肉腫の進展を抑制する

  新井 隆太, 津田 真寿美, 渡部 琢哉, 尾瀬 農之, 小布施 力史, 前仲 勝実, 三浪 明男, 大場 雄介 日本分子生物学会年会プログラム・要旨集(Web) 34th WEB ONLY 2P-0690 2011年 [査読無し][通常論文]
  
• ヤツメウナギ由来可変性リンパ球受容体の結晶学的研究

  神田諒, 須藤洋一, 笠松純, 笠原正典, 前仲勝実, 尾瀬農之 日本結晶学会年会講演要旨集 2011 90 2011年 [査読無し][通常論文]
  
• 麻疹ウイルスH蛋白質と受容体の複合体構造

  尾瀬農之, 橋口隆生, 久保田万理恵, 真板宣夫, 柳雄介, 前仲勝実 日本結晶学会年会講演要旨集 2011 87 2011年 [査読無し][通常論文]
  
• 抑制型レセプターCD160によるHVEM認識の分子基盤

  小島理恵子, 梶川瑞穂, 白石充典, 黒木喜美子, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 34th 2T7PI-2 (WEB ONLY) 2011年 [査読無し][通常論文]
  
• CDV‐Hの結晶構造解析と機能解析

  伊藤由梨, 福原秀雄, 酒匂幸, 橋口隆生, 梶川瑞穂, 竹田誠, 柳雄介, 尾瀬農之, 前仲勝実 日本結晶学会年会講演要旨集 2011 90 2011年 [査読無し][通常論文]
  
• 免疫抑制分子HLA‐Gダイマーの局所投与による関節炎抑制効果の検討

  黒木喜美子, 岡部由紀, 廣瀬薫, 福永裕子, 小柳悟, 大戸茂弘, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 34th 2P-0444 (WEB ONLY) 2011年 [査読無し][通常論文]
  
• 早期麻疹排除及び排除状態の維持に関する研究 麻疹ウイルスの抗原エピトープの構造解析と効果的なワクチン維持のための研究

  前仲勝実 早期麻疹排除及び排除状態の維持に関する研究 平成22年度 総括・分担研究報告書 117 -120 2011年 [査読無し][通常論文]
  
• ヒト免疫制御受容体LILRB1とリガンドHLA‐Gの複合体の結晶構造解析

  松原永季, 黒木喜美子, 上敷領淳, 尾瀬農之, 前仲勝実 日本結晶学会年会講演要旨集 2011 95 2011年 [査読無し][通常論文]
  
• HIV由来変異ペプチドによるKIR2DL1を介した免疫逃避機構の分子基盤

  黒木喜美子, 上敷領淳, 尾瀬農之, THANANCHAI Hathairat, MAKADZANGE Tariro, ROWLAND‐JONES Sarah, DONG Tao, 前仲勝実 日本結晶学会年会講演要旨集 2011 94 2011年 [査読無し][通常論文]
  
• HIV由来ペプチドライブラリーを用いたHLA-Cw4拘束性免疫制御ペプチドの同定

  黒木 喜美子, 福永 裕子, 尾瀬 豊之, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 1P -1023 2010年12月 [査読無し][通常論文]
  
• 構造細胞生物学の新展開 モルビリウイルス属の細胞侵入機構と免疫制御の構造基盤

  前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 4W16 -6 2010年12月 [査読無し][通常論文]
  
• 麻疹ウイルスと受容体SLAMの相互作用と膜融合

  橋口隆生, 白銀勇太, 前仲勝実, 柳雄介 日本ウイルス学会学術集会プログラム・抄録集 58th 147 2010年10月15日 [査読無し][通常論文]
  
• HSV‐1のエンベロープ蛋白質gBペプチドおよびペア型レセプターPILRαの複合体結晶構造解析

  尾瀬農之, 山口宗親, WAN Jing, 黒木喜美子, 田畑栄一, 真板宣夫, 中村聖子, 梶川瑞穂, 白鳥行大, 佐藤毅史, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 58th 420 2010年10月15日 [査読無し][通常論文]
  
• モルビリウイルス属ワクチンの有効性の構造基盤

  前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 58th 155 2010年10月15日 [査読無し][通常論文]
  
• イヌジステンパーウイルスHタンパク質と受容体SLAMとの分子認識

  伊藤由梨, 福原秀雄, 酒匂幸, 橋口隆生, 梶川瑞穂, 竹田誠, 柳雄介, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 58th 247 2010年10月15日 [査読無し][通常論文]
  
• NKおよびクラスI認識受容体研究の最前線 クラスI認識受容体の構造とリガンド認識

  黒木 喜美子, 福永 裕子, 上敷領 淳, 白石 充典, 尾瀬 農之, 前仲 勝実 MHC: Major Histocompatibility Complex 17 (2) 136 -136 2010年08月 [査読無し][通常論文]
  
• NKおよびクラスI認識受容体研究の最前線 クラスI認識受容体の構造とリガンド認識

  黒木 喜美子, 福永 裕子, 上敷領 淳, 白石 充典, 尾瀬 農之, 前仲 勝実 MHC: Major Histocompatibility Complex 17 (2) 136 -136 2010年08月 [査読無し][通常論文]
  
• 新規CRISP蛋白質の発見と毒蛇血清蛋白質SSP‐2との相互作用領域の解析

  塩井(青木, 成留実, 清村康子, 黒木喜美子, 前仲勝実, 寺田成之 日本蛋白質科学会年会プログラム・要旨集 10th 73 2010年05月15日 [査読無し][通常論文]
  
• LILR1/HLA‐GとLILR82/HLA‐G複合体の結合機構の違い

  宮下尚之, 黒木喜美子, 前仲勝実, 杉田有治 日本蛋白質科学会年会プログラム・要旨集 10th 66 2010年05月15日 [査読無し][通常論文]
  
• 麻疹ウイルスによる細胞侵入機構の構造基盤

  橋口隆生, 尾瀬農之, 上敷領淳, 柳雄介, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 10th 32 2010年05月15日 [査読無し][通常論文]
  
• 活性化型受容体LILRA1‐MHC相互作用を介した免疫反応制御の分子基盤

  上敷領淳, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 10th 163 2010年05月15日 [査読無し][通常論文]
  
• 細胞表面において生体防御に関わる分子認識の構造生物学

  福原秀雄, 橋口隆生, 黒木喜美子, 白石充典, 佐々木香織, 梶川瑞穂, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 10th 32 2010年05月15日 [査読無し][通常論文]
  
• HLA-B27 FORMS HEAVY CHAIN HOMODIMERS IN VITRO AND IN VIVO THAT BIND TO CELLULAR RECEPTORS INCLUDING LILR AND KIR FAMILY MEMBERS

  Kirsty McHugh, Joanna Giles, Simon Kollnberger, Kimiko Kuroi, Katsumi Maenaka, Paul Bowness RHEUMATOLOGY 49 I50 -I50 2010年04月 [査読無し][通常論文]
  
• モルビリウイルス属の細胞侵入機構と免疫制御の構造基盤

  前仲勝実 生化学 ROMBUNNO.4W16-6 2010年 [査読無し][通常論文]
  
• HLA多型が寄与する自己免疫疾患の発症機序の解明 HLA‐B27の構造的分子特性の解析と免疫制御機構の解明

  前仲勝実 HLA多型が寄与する自己免疫疾患の発症機序の解明 平成21年度 総括・分担研究報告書 18 -21 2010年 [査読無し][通常論文]
  
• ペア型レセプターPILRα及びHSV‐1のエンベロープ蛋白質gB模倣ペプチド複合体結晶構造

  尾瀬農之, 山口宗親, 黒木喜美子, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実 日本結晶学会年会講演要旨集 2010 97 2010年 [査読無し][通常論文]
  
• 蚕バイオテクノロジー : タンパク質生産工場として(第61回大会シンポジウム報告)

  朴 龍洙, 前仲 勝実 生物工学会誌 : seibutsu-kogaku kaishi 87 (11) 539 -539 2009年11月25日 [査読無し][通常論文]
  
• T細胞補助シグナル CD160-HVEM相互作用を中心としたT細胞制御機構の分子基盤

  小島 理恵子, 梶川 瑞穂, 白石 充典, 黒木 喜美子, 前仲 勝実 日本免疫学会総会・学術集会記録 39 161 -161 2009年11月 [査読無し][通常論文]
  
• T細胞補助シグナル CD160-HVEM相互作用を中心としたT細胞制御機構の分子基盤

  小島 理恵子, 梶川 瑞穂, 白石 充典, 黒木 喜美子, 前仲 勝実 日本免疫学会総会・学術集会記録 39 161 -161 2009年11月 [査読無し][通常論文]
  
• イヌジステンパーウイルスHタンパク質と受容体SLAMとの分子認識

  酒匂幸, 橋口隆生, 竹田誠, 柳雄介, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 57th 276 2009年10月01日 [査読無し][通常論文]
  
• 麻疹ウイルスHタンパク質と受容体SLAMの複合体の結晶構造と麻疹ウイルスの細胞侵入機構

  橋口隆生, 尾瀬農之, 上敷領淳, 竹田誠, 前仲勝実, 柳雄介 日本ウイルス学会学術集会プログラム・抄録集 57th 149 2009年10月01日 [査読無し][通常論文]
  
• Paired Immunoglobulin(Ig)Like type 2 Receptor(PILR)αによるGlycoprotein B(gB)認識機構の解明

  山口宗親, 黒木喜美子, 田畑栄一, 真板宣夫, 梶川瑞穂, 尾瀬農之, 中村聖子, 王静, 佐藤毅, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 57th 149 2009年10月01日 [査読無し][通常論文]
  
• 真正細菌におけるセレノシステイン特異的翻訳伸長因子SelBとSECIS mRNAの相互作用基盤

  尾瀬農之, RASUBALA Linda, 神田大輔, SOLER Nicolas, 吉澤聡子, FOURMY Dominique, 前仲勝実 生化学 ROMBUNNO.2S8A-2 2009年09月25日 [査読無し][通常論文]
  
• 翻訳されうる21番目のアミノ酸、セレノシステインを含有するタンパク質研究のブレーク・スルー 真正細菌におけるセレノシステイン特異的翻訳伸長因子SelBとSECIS mRNAの相互作用基盤

  尾瀬 農之, Rasubala Linda, 神田 大輔, Soler Nicolas, 吉澤 聡子, Fourmy Dominique, 前仲 勝実 日本生化学会大会プログラム・講演要旨集 82回 2S8a -2 2009年09月 [査読無し][通常論文]
  
• BmNPVバクミドを用いたヒト膜タンパク質受容体のカイコ個体での生産

  梶川瑞穂, 佐々木香織, 黒木喜美子, 橋口隆生, 岡部由紀, 福原秀雄, 竹田誠, 柳雄介, 脇本義太郎, 豊岡勝, 武田茂樹, 本橋智子, 霜島司, PARK Enoch Y, 前仲勝実 日本生物工学会大会講演要旨集 61st 281 2009年08月25日 [査読無し][通常論文]
  
• 皮膚免疫に重要な役割を果たすと推定される新しいMHCクラスI様分子の解析

  吉田繁, 富居一範, 梶川瑞穂, 近藤瑞穂, 大塚紀幸, 前仲勝実, 笠原正典 MHC 16 (2) 178 -178 2009年08月25日 [査読無し][通常論文]
  
• 3S13p03 BmNPVバクミドを用いたヒト膜タンパク質受容体のカイコ個体での生産(蚕バイオテクノロジ一-タンパク質生産工場として-,シンポジウム)

  梶川 瑞穂, 佐々木 香織, 黒木 喜美子, 橋口 隆生, 岡部 由紀, 福原 秀雄, 竹田 誠, 柳 雄介, 脇本 義太郎, 豊岡 勝, 武田 茂樹, 本橋 智子, 霜島 司, 朴 龍洙, 前仲 勝実 日本生物工学会大会講演要旨集 21 (0) 281 2009年08月25日 [査読無し][通常論文]
  
• HIVペプチドの変異によるNK細胞受容体を介した免疫逃避の分子基盤

  前仲勝実 日本蛋白質科学会年会プログラム・要旨集 9th 45 2009年04月24日 [査読無し][通常論文]
  
• CD160‐HVEM相互作用を中心としたT細胞活性制御の分子基盤

  小島理恵子, 梶川瑞穂, 白石充典, 黒木喜美子, 前仲勝実 日本分子生物学会年会講演要旨集 32nd (Vol.1) 63 2009年 [査読無し][通常論文]
  
• HLA多型が寄与する自己免疫疾患の発症機序の解明 HLA‐B27の構造的分子特性の解析と免疫制御機構の解明

  前仲勝実 HLA多型が寄与する自己免疫疾患の発症機序の解明 平成20年度 総括・分担研究報告書 16 -19 2009年 [査読無し][通常論文]
  
• マウスMHCクラスIb分子MILL2の結晶構造解析

  梶川瑞穂, 尾瀬農之, 笠原正典, 前仲勝実 生化学 81回・31回 2T20-1 -1 2008年11月 [査読無し][通常論文]
  
• シグナル伝達タンパク質の分子認識研究の新展開 麻疹ウイルス受容体結合タンパク質の立体構造

  前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 81回・31回 3S16 -2 2008年11月 [査読無し][通常論文]
  
• Association of LILRA2 (ILT1, LIR7) splice site polymorphism with systemic lupus erythematosus and microscopic polyangiitis (vol 9, page 214, 2008)

  K. Mamegano, K. Kuroki, R. Miyashita, M. Kusaoi, S. Kobayashi, K. Matsuta, K. Maenaka, M. Colonna, S. Ozaki, H. Hashimoto, Y. Takasaki, K. Tokunaga, N. Tsuchiya GENES AND IMMUNITY 9 (7) 650 -650 2008年10月 [査読無し][通常論文]
  
• “21番目のアミノ酸”セレノシステインを取り込むための分子基盤

  尾瀬農之, RASUBALA Linda, 神田大輔, SOLER Nicolas, 吉澤聡子, FOURMY Dominique, 前仲勝実 補体シンポジウム講演集 45th 194 -194 2008年07月 [査読無し][通常論文]
  
• ウイルス認識の分子基盤 麻疹ウイルスHタンパク質のX線結晶構造解析

  橋口 隆生, 梶川 瑞穂, 真板 宣夫, 竹田 誠, 黒木 喜美子, 佐々木 香織, 柳 雄介, 前仲 勝実 補体シンポジウム講演集 45 61 -61 2008年07月 [査読無し][通常論文]
  
• ウイルス認識の分子基盤 麻疹ウイルスHタンパク質のX線結晶構造解析

  橋口 隆生, 梶川 瑞穂, 真板 宣夫, 竹田 誠, 黒木 喜美子, 佐々木 香織, 柳 雄介, 前仲 勝実 補体シンポジウム講演集 45 61 -61 2008年07月 [査読無し][通常論文]
  
• 麻疹ウイルスHタンパク質のX線結晶構造

  橋口 隆生, 前仲 勝実, 柳 雄介 ウイルス 58 (1) 1 -10 2008年06月22日 [査読無し][通常論文]
   

  この10数年ほどの間にX線結晶構造解析や核磁気共鳴などの手法を用いることにより，多くのウイルスで重要なタンパク質の立体構造が明らかにされてきた．その結果，ウイルスと宿主の間で起きている相互作用を視覚的かつ原子レベルで議論することができるようになり，ウイルス側と宿主側双方の戦略的なメカニズムについての理解が大いに深まった．本稿では，麻疹ウイルスレセプター結合タンパク質のX線結晶構造解析から見えてきたウイルスの細胞侵入戦略やワクチンが成功を収めてきた分子基盤，さらに新たな上皮細胞レセプターに関する最新の知見について紹介する．また，今回の結晶化に用いた，均一な糖鎖を産生するヒト培養細胞株（293SGnTI(-)細胞）のタンパク質発現系についても取り上げる．
• 複数の結合様式を介したミトコンドリアTom20によるプレ配列の認識機構

  齊藤貴士, 井倉真由美, 尾瀬農之, 前仲勝実, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 8th 19 2008年05月23日 [査読無し][通常論文]
  
• DNAのメチル化シグナルを見分ける分子メカニズム―メチル化及び非メチル化CpG結合ドメインの立体構造解析より―

  大木出, 真板宣夫, 前仲勝実, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 8th 81 2008年05月23日 [査読無し][通常論文]
  
• BmNPVバクミドとカイコ個体を用いたヒトGタンパク質共役型受容体の生産

  梶川瑞穂, 佐々木香織, 脇本義太郎, 豊岡勝, 武田茂樹, 本橋智子, 霜島司, PARK Enoch Y, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 8th 124 2008年05月23日 [査読無し][通常論文]
  
• Asn結合型糖鎖修飾を決定するオリゴ糖転移酵素・膜タンパク質の大腸菌による発現

  井倉真由美, 上敷領淳, NYIRENDA James, 前仲勝実, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 8th 81 2008年05月23日 [査読無し][通常論文]
  
• BmNPVバクミドシステムによるヒト免疫細胞表面レセプターの発現と糖鎖解析

  佐々木香織, 梶川瑞穂, 矢木宏和, 近藤幸子, 黒木喜美子, 本橋智子, 霜島司, PARK Enoch Y, 加藤晃一, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 8th 98 2008年05月23日 [査読無し][通常論文]
  
• 免疫学 HLA-G 副作用の少ない生物学的製剤の可能性

  黒木 喜美子, 前仲 勝実 医学のあゆみ 224 (11) 874 -875 2008年03月 [査読無し][通常論文]
  
• BmNPVバクミドシステムによるヒト免疫細胞表面レセプターの発現と糖鎖解析

  佐々木香織, 梶川瑞穂, 矢木宏和, 近藤幸子, 黒木喜美子, 本橋智子, 霜島司, PARK Enoch Y, 加藤晃一, 前仲勝実 生化学 4P-0214 2008年 [査読無し][通常論文]
  
• 麻疹ウイルス受容体結合タンパク質の立体構造

  前仲勝実 生化学 3S16-2 2008年 [査読無し][通常論文]
  
• 【ウイルス学・構造生物学】結晶構造が解き明かす麻疹ウイルスワクチン成功の理由

  橋口隆生, 柳雄介, 前仲勝実 メディカルバイオ 5 (1) 10 -11 2008年01月01日 [査読無し][通常論文]
  
• 構造基盤解析に向けたミトコンドリアTom20‐プレ配列ペプチド複合体の安定化

  齊藤貴士, 井倉真由美, 尾瀬農之, 帯田孝之, 小島理恵子, 前仲勝実, 神田大輔 Abstr Annu Meet NMR Soc Jpn 46th 164 -165 2007年09月11日 [査読無し][通常論文]
  
• アスパラギン結合型糖鎖修飾を決定するオリゴ糖転移酵素の構造生化学

  井倉真由美, 上敷領淳, 真板宣夫, 山田真希, 前仲勝実, 神田大輔 日本糖質学会年会要旨集 27th 65 2007年07月10日 [査読無し][通常論文]
  
• 超好熱性古細菌Pyrococcus furiosusのN型糖鎖生合成機構の解明

  上敷領淳, 井倉真由美, 山田真希, 前仲勝実, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 7th 63 2007年05月07日 [査読無し][通常論文]
  
• Asn結合型糖鎖修飾を決定するオリゴ糖転移酵素の構造生物学研究

  井倉真由美, 真板宣夫, 上敷領淳, 山田真希, 前仲勝実, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 7th 28 2007年05月07日 [査読無し][通常論文]
  
• PriAタンパク質によるDNA3′末端の塩基非選択的認識機構の解明

  佐々木香織, 尾瀬農之, 岡本直明, 田中卓, 前仲勝実, 正井久雄, 斉藤美保子, 白井剛, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 7th 50 2007年05月07日 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig‐Like type2 Receptor(PILR)の構造解析

  田畑栄一, 黒木喜美子, 梶川瑞穂, 白鳥行大, 荒瀬尚, 神田大輔, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 7th 79 2007年05月07日 [査読無し][通常論文]
  
• ミトコンドリアTom20によるプレ配列の動的認識機構:緩和解析からのアプローチ

  齊藤貴士, 井倉真由美, 尾瀬農之, 帯田孝之, 小島理恵子, 前仲勝実, 神田大輔 日本蛋白質科学会年会プログラム・要旨集 7th 38 2007年05月07日 [査読無し][通常論文]
  
• エントロピー駆動型の受容体-リガンド相互作用 免疫細胞表面受容体LILR(ILT/LIR/CD85)群の分子認識

  白石 充典, 黒木 喜美子, 神田 大輔, 前仲 勝実 生物物理 47 (2) 93 -99 2007年03月 [査読無し][通常論文]
   

  Protein-protein and protein-nucleic acid interactions are fundamental in self and non-self recognition in immune system. Leukocyte immunoglobulin-like receptor B1 (LILRB1) and LILRB2 are human inhibitory receptors which recognize major histocompatibility complex (MHC) class I molecules and take part in immune regulation. In this review, we demonstrated detailed thermodynamic and kinetic studies of interactions between LILRs and MHC class I molecules, and structural analyses by X-ray crystallography and NMR. Our data shows the binding mode involving some conformational adjustment without a v...
• クロマチン構造制御に関わる新規CpG配列結合ドメインのDNA認識の分子メカニズム

  大木出, 前仲勝実, 神田大輔 生化学 1P-0034 2007年 [査読無し][通常論文]
  
• Asn結合型糖鎖修飾を決定するオリゴ糖転移酵素の構造生物学研究

  井倉真由美, 真板宣夫, 上敷領淳, 山田真希, 前仲勝実, 神田大輔 生化学 4P-0096 2007年 [査読無し][通常論文]
  
• 難治性血管炎に関する調査研究 日本人顕微鏡的多発血管炎の疾患感受性遺伝子に関する研究

  土屋尚之, 宮下リサ, 豆ケ野剛一, 徳永勝士, 川崎綾, 黒木喜美子, 前仲勝実, COLONNA Marco, 小林茂人, 橋本博史, 尾崎承一 難治性血管炎に関する調査研究 平成18年度総括・分担研究報告書 43 -48 2007年 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig‐Like type2 Receptor(PILR)のCD99認識機構の分子基盤

  田畑栄一, 黒木喜美子, 梶川瑞穂, WANG Jing, 荒瀬尚, 神田大輔, 前仲勝実 生化学 1P-0043 2007年 [査読無し][通常論文]
  
• 停止DNA複製フォークにおけるPriA蛋白質のDNA認識の構造基盤

  佐々木香織, 田中卓, 正井久雄, 前仲勝実, 神田大輔 生化学 1P-0701 2007年 [査読無し][通常論文]
  
• クロマチン工学への応用に向けたキネトコアタンパク質複合体の再構成

  堀哲也, 真柳浩太, 前仲勝実, 保木裕子, 佐渡敬, 岡田聖裕, 深川竜郎 生化学 2P-0788 2007年 [査読無し][通常論文]
  
• ジスルフィド結合で拘束したTom20‐プレ配列複合体の動的振る舞い

  斉藤貴士, 伊倉真由美, 小島理恵子, 帯田孝之, 前仲勝実, 神田大輔 Abstr Annu Meet NMR Soc Jpn 45th 244 -245 2006年11月20日 [査読無し][通常論文]
  
• Molecular basis for LILR recognition of HLA-G and its dimer

  M. Shiroishi, K. Kuroki, T. Ose, L. Rasubala, I. Shiratori, H. Arase, D. Kohda, K. Maenaka TISSUE ANTIGENS 68 (4) CP6 -CP6 2006年10月 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig‐Like type2Receptor(PILR)の構造解析

  田畑栄一, 黒木喜美子, 白鳥行大, 荒瀬尚, 神田大輔, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 6th 96 2006年03月31日 [査読無し][通常論文]
  
• BmNPVバクミドシステムを用いたカイコ個体でのヒト免疫細胞表面レセプターKIRの大量発現

  佐々木香織, 梶川瑞穂, 黒木喜美子, 本橋智子, 霜島司, 朴龍洙, 神田大輔, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 6th 62 2006年03月31日 [査読無し][通常論文]
  
• 21番目のアミノ酸を取り込むための分子機構

  尾瀬 農之, Rasubala Linda, 吉澤 聡子, Fourmy Dominique, 神田 大輔, 前仲 勝実 生物物理 46 (2) 102 -105 2006年03月25日 [査読無し][通常論文]
  
• Crystallization and preliminary crystallographic analysis of the N-terminal domain of PriA from Escherichia coli

  K Sasaki, T Ose, T Tanaka, T Mizukoshi, T Ishigaki, K Maenaka, H Masai, D Kohda BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1764 (1) 157 -160 2006年01月 [査読無し][通常論文]
   

  PriA, a DEXH-type DNA helicase, binds specifically to the 3' end of DNA through its N-terminal domain, and is a candidate sensor protein that recognizes arrested DNA replication forks in bacteria. We crystallized an N-terminal fragment of PriA in the absence and the presence of oligonucleotides to elucidate the structural basis for the specific recognition of the 3' terminus of DNA. (c) 2005 Elsevier B.V. All rights reserved.
• 大腸菌プライモソーム形成に関わるタンパク質PriBの立体構造とDNAとの相互作用

  塩井誠次郎, 阿部義人, 竹縄太一, 前仲勝実, 神田大輔, 片山勉, 植田正 日本分子生物学会年会講演要旨集 28th 56 2005年11月25日 [査読無し][通常論文]
  
• ミトコンドリアプレ配列認識のためのTom20タンパク質の動的構造

  井倉真由美, 帯田孝之, 尾瀬農之, 遠藤斗志也, 前仲勝実, 神田大輔 日本分子生物学会年会講演要旨集 28th 602 2005年11月25日 [査読無し][通常論文]
  
• MHCクラスI様分子MILLの構造・機能解析

  梶川瑞穂, 笠原正典, 神田大輔, 前仲勝実 日本分子生物学会年会講演要旨集 28th 282 2005年11月25日 [査読無し][通常論文]
  
• NK細胞抑制型レセプターKLRG1(Killer Cell Lectin‐Like Receptor G1)によるKLRG1リガンド認識の構造基盤

  中村聖子, 黒木喜美子, 佐々木香織, 丸山拓馬, 伊藤昌之, 山本一夫, 松本直樹, 神田大輔, 前仲勝実 日本分子生物学会年会講演要旨集 28th 572 2005年11月25日 [査読無し][通常論文]
  
• カイコ個体を用いたタンパク質の大量発現用バクミドの開発及び応用

  PARK Enoch Y, 前仲勝実 日本分子生物学会年会講演要旨集 28th 48 2005年11月25日 [査読無し][通常論文]
  
• E.coli由来PriAのN末端ドメインによるDNA3′末端認識の構造的基盤

  佐々木香織, 尾瀬農之, 田中卓, 岡本直明, 前仲勝実, 正井久雄, 神田大輔 日本分子生物学会年会講演要旨集 28th 393 2005年11月25日 [査読無し][通常論文]
  
• Killer cell lectin-like receptor G1(KLRG1)が認識するKLRG1リガンド上領域の同定

  丸山 拓馬, 松本 直樹, 伊藤 昌之, 中村 聖子, 黒木 喜美子, 前仲 勝実, 山本 一夫 日本免疫学会総会・学術集会記録 35 97 -97 2005年11月 [査読無し][通常論文]
  
• 白血球抑制性受容体LILRB1とMHCクラスIとのFCSおよびNMRを用いた相互作用解析

  黒木 喜美子, 白石 充典, リンダ・ラスバラ, 小林 佐代子, 梶川 瑞穂, 田畑 栄一, 福永 裕子, 岡本 直明, 神田 大輔, 前仲 勝実 日本免疫学会総会・学術集会記録 35 99 -99 2005年11月 [査読無し][通常論文]
  
• HLA-GダイマーのLILR/LIR/ILTレセプターを介した強いシグナル伝達とその構造基盤

  前仲 勝実, 白石 充典, 黒木 喜美子, リンダ・ラズバラ, 白鳥 行大, 荒瀬 尚, 神田 大輔 日本免疫学会総会・学術集会記録 35 99 -99 2005年11月 [査読無し][通常論文]
  
• NK細胞抑制型レセプターKLRG1(Killer Cell Lectin-Like Recepter G1)によるKLRG1リガンド認識の構造基盤

  中村 聖子, 黒木 喜美子, 佐々木 香織, 丸山 拓馬, 伊藤 昌之, 山本 一夫, 松本 直樹, 神田 大輔, 前仲 勝実 日本免疫学会総会・学術集会記録 35 98 -98 2005年11月 [査読無し][通常論文]
  
• 白血球抑制性受容体LILRB1とMHCクラスIとのFCSおよびNMRを用いた相互作用解析

  黒木 喜美子, 白石 充典, リンダ・ラスバラ, 小林 佐代子, 梶川 瑞穂, 田畑 栄一, 福永 裕子, 岡本 直明, 神田 大輔, 前仲 勝実 日本免疫学会総会・学術集会記録 35 99 -99 2005年11月 [査読無し][通常論文]
  
• 関節リウマチ(RA)関連Leukocyte Immunoglobulin-like Receptor(LILR)B1ハプロタイプの構造・発現解析

  黒木 喜美子, 白石 充典, リンダ・ラスバラ, 土屋 尚之, 神田 大輔, 徳永 勝士, 前仲 勝実 日本リウマチ学会総会・学術集会・国際リウマチシンポジウムプログラム・抄録集 49回・14回 211 -211 2005年04月 [査読無し][通常論文]
  
• 大腸菌由来PriAとオリゴヌクレオチドとの複合体の結晶化

  佐々木香織, 尾瀬農之, 岡本直明, 田中卓, 正井久雄, 前仲勝実, 神田大輔 日本分子生物学会年会プログラム・講演要旨集 27th 618 2004年11月25日 [査読無し][通常論文]
  
• ラットTom20タンパク質とALDHプレ配列複合体のX線結晶構造解析

  井倉真由美, 帯田孝之, 尾瀬農之, 遠藤斗志也, 前仲勝実, 神田大輔 日本分子生物学会年会プログラム・講演要旨集 27th 732 2004年11月25日 [査読無し][通常論文]
  
• ヒト免疫細胞受容体ILT/LIR/CD85ファミリーのリガンド認識の分子的基盤

  白石充典, 黒木喜美子, 小島恵理子, 津本浩平, 熊谷泉, 神田大輔, 前仲勝実 日本分子生物学会年会プログラム・講演要旨集 27th 354 2004年11月25日 [査読無し][通常論文]
  
• ミトコンドリア・プレ配列受容体Tom20‐ALDHプレ配列複合体の結晶構造と緩和解析

  帯田孝之, 井倉真由美, 尾瀬農之, 前仲勝実, 遠藤斗志也, 神田大輔 NMR討論会講演要旨集 43rd 212 -213 2004年11月10日 [査読無し][通常論文]
  
• ヒト免疫細胞抑制型受容体ILT4のリガンド認識の分子的基盤

  白石充典, 津本浩平, 栗本英治, 加藤晃一, 熊谷泉, 神田大輔, 前仲勝実 日本免疫学会総会・学術集会記録 34 230 -230 2004年11月05日 [査読無し][通常論文]
  
• 関節リウマチ(RA)関連Leukocyte Immunoglobulin-like Receptor(LIR)1ハプロタイプの構造・発現解析

  黒木 喜美子, 土屋 尚之, 白石 充典, リンダ・ラズバラ, 山下 由美, 小池 隆夫, 神田 大輔, 徳永 勝士, 前仲 勝実 日本免疫学会総会・学術集会記録 34 162 -162 2004年11月 [査読無し][通常論文]
  
• Leukocyte Ig‐like receptor群の構造解析

  前仲勝実 医学のあゆみ 209 (13) 1033 -1034 2004年06月26日 [査読無し][通常論文]
  
• ヒト免疫細胞抑制型受容体ILT2,4の相互作用解析

  白石充典, 津本浩平, 熊谷泉, 白木原康雄, 前仲勝実 生化学 76 (3) 320 -320 2004年03月25日 [査読無し][通常論文]
  
• 第三のナイシン類縁体,ナイシンQ

  米山史紀, 深尾匡憲, 帯田孝之, 善藤威史, 前仲勝実, 中山二郎, 神田大輔, 園元謙二 日本農芸化学会大会講演要旨集 2004 162 2004年03月05日 [査読無し][通常論文]
  
• NK細胞表面レセプター群の分子認識

  前仲勝実 生化学 76 (2) 135 -140 2004年02月25日 [査読無し][通常論文]
   

  コレクション : 国立国会図書館デジタルコレクション > デジタル化資料 > 雑誌
• Molecular recognitions of natural killer cell receptors

  K Maenaka SEIKAGAKU 76 (2) 135 -140 2004年02月 [査読無し][通常論文]
  
• ヒト免疫細胞受容体ILT2,4の相互作用解析

  白石充典, ラスバラ リンダ, 津本浩平, 熊谷泉, 神田大輔, 前仲勝実 日本免疫学会総会・学術集会記録 33 318 -318 2003年11月05日 [査読無し][通常論文]
  
• NMRを用いたempty MHCクラスIタンパク質の動的構造解析

  栗本英治, 山口芳樹, 前仲勝実, 神田大輔, 加藤晃一 日本免疫学会総会・学術集会記録 33 324 -324 2003年11月05日 [査読無し][通常論文]
  
• Distinct associations of the leukocyte immunoglobulin-like receptor (LIR)1 and LIR6 polymorphisms with susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

  K Kuroki, N Tsuchiya, K Maenaka, L Rasubala, M Shiroishi, Y Yamashita, K Matsuta, T Fukazawa, D Kohda, T Koike, T Juji, H Hashimoto, K Tokunaga ARTHRITIS AND RHEUMATISM 48 (9) S197 -S197 2003年09月 [査読無し][通常論文]
  
• 抗原ペプチド非存在下におけるMHCクラスI蛋白質のNMRによる立体構造解析

  栗本 英治, 山口 芳樹, 井垣 尚啓, 前仲 勝実, 加藤 晃一 生物物理 43 (1) S41 -S41 2003年08月25日 [査読無し][通常論文]
  
• Nathan R. Zaccai, Katsumi Maenaka, Taeko Maenaka, Paul R. Crocker, Reinhard Brossmer, S■rge Kelm, & E. Yvonne Jones. Structure-guided design of sialic Acid-based siglec inhibitors and crystallographic analysis in complex with sialoadhesin

  NR Zaccai, K Maenaka, T Maenaka, PR Crocker, R Brossmer, S Kelm, EY Jones Structure 11 (5) 557 -567 2003年05月 [査読無し][通常論文]
   

  The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC50 values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyi-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.
• Structure-guided design of sialic acid-based Siglec inhibitors and crystallographic analysis in complex with sialoadhesin

  NR Zaccai, K Maenaka, T Maenaka, PR Crocker, R Brossmer, S Kelm, EY Jones STRUCTURE 11 (5) 557 -567 2003年05月 [査読無し][通常論文]
   

  The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC50 values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyi-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.
• Bacmidを用いたBmNPVバキュロウイルス外来遺伝子発現系の構築

  本橋智子, 霜島司, 前仲勝実, 朴龍しゅ 日本農芸化学会大会講演要旨集 2003 154 2003年03月05日 [査読無し][通常論文]
  
• Yasushi Uemura, Satoru Senju, Katsumi Maenaka, Leo Kei Iwai, Shinji Fujii, Hiroki Tabata, Hirotake Tsukamoto, Shinya Hirata, Yu-Zhen Chen & Yasuharu Nishimura. Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to i・・・

  J. Immunol 170 947 -960 2003年 [査読無し][通常論文]
   

  Yasushi Uemura, Satoru Senju, Katsumi Maenaka, Leo Kei Iwai, Shinji Fujii, Hiroki Tabata, Hirotake Tsukamoto, Shinya Hirata, Yu-Zhen Chen & Yasuharu Nishimura. Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs.
• Mitsunori Shiroishi, Kouhei Tsumoto, Kimie Amano, Yasuo Shirakihara, Marco Colonna, Veronique M. Braud, David S. J. Allan, Azure Makadzange, Sarah Rowland-Jones, Benjamin Willcox, E. Yvonne Jones, P. Anton van der Merwe, Izumi Kumagai, & Katsumi Ma・・・

  Proc. Natl. Acad. Sci. USA 100 8856 -61 2003年 [査読無し][通常論文]
   

  Mitsunori Shiroishi, Kouhei Tsumoto, Kimie Amano, Yasuo Shirakihara, Marco Colonna, Veronique M. Braud, David S. J. Allan, Azure Makadzange, Sarah Rowland-Jones, Benjamin Willcox, E. Yvonne Jones, P. Anton van der Merwe, Izumi Kumagai, & Katsumi Maenaka. Human inhibitory receptors ILT2 and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G
• Yasushi Uemura, Satoru Senju, Shinji Fujii, Leo Kei Iwai, Katsumi Maenaka, Hiroki Tabata, Takayuki Kanai, Yu-Zhen Chen and Yasuharu Nishimura. Specificity, degeneracy, and molecular mimicry in antigen recognition by HLA-Class Ⅱ restricted T cell recep・・・

  Yasushi Uemura, Satoru Senju, Shinji Fujii, Leo Kei Iwai, Katsumi Maenaka, Hiroki Tabata, Takayuki Kanai, Yu-Zhen Chen, Yasuharu Nishimura Modern Rheumatology 13 (3) 205 -214 2003年 [査読無し][通常論文]
   

  
  Yasushi Uemura, Satoru Senju, Shinji Fujii, Leo Kei Iwai, Katsumi Maenaka, Hiroki Tabata, Takayuki Kanai, Yu-Zhen Chen and Yasuharu Nishimura. Specificity, degeneracy, and molecular mimicry in antigen recognition by HLA-Class Ⅱ restricted T cell receptors: implications for clinical medicine.
• Haruka Wada, Naoki Matsumoto, Katsumi Maenaka, Kazuhiro Suzuki and Kazuo Yamamoto. Two HLA-E Mutants Segregate the Inhibitory NK Cell Receptor CD94/NKG2A and the Activating Receptor CD94/NKG2C, Both of Whichi Bind the Top of HLA-E

  Eur. J. Immunol In the press 2003年 [査読無し][通常論文]
  
• Yasushi Uemura, Satoru Senju, Katsumi Maenaka, Leo Kei Iwai, Shinji Fujii, Hiroki Tabata, Hirotake Tsukamoto, Shinya Hirata, Yu-Zhen Chen & Yasuharu Nishimura. Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to i・・・

  J. Immunol 170 947 -960 2003年 [査読無し][通常論文]
   

  Yasushi Uemura, Satoru Senju, Katsumi Maenaka, Leo Kei Iwai, Shinji Fujii, Hiroki Tabata, Hirotake Tsukamoto, Shinya Hirata, Yu-Zhen Chen & Yasuharu Nishimura. Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs.
• Mitsunori Shiroishi, Kouhei Tsumoto, Kimie Amano, Yasuo Shirakihara, Marco Colonna, Veronique M. Braud, David S. J. Allan, Azure Makadzange, Sarah Rowland-Jones, Benjamin Willcox, E. Yvonne Jones, P. Anton van der Merwe, Izumi Kumagai, & Katsumi Ma・・・

  Proc. Natl. Acad. Sci. USA 100 8856 -61 2003年 [査読無し][通常論文]
   

  Mitsunori Shiroishi, Kouhei Tsumoto, Kimie Amano, Yasuo Shirakihara, Marco Colonna, Veronique M. Braud, David S. J. Allan, Azure Makadzange, Sarah Rowland-Jones, Benjamin Willcox, E. Yvonne Jones, P. Anton van der Merwe, Izumi Kumagai, & Katsumi Maenaka. Human inhibitory receptors ILT2 and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G
• Specificity, degeneracy, and molecular mimicry in antigen recognition by HLA-Class II restricted T cell receptors: Implications for clinical medicine

  Yasushi Uemura, Satoru Senju, Shinji Fujii, Leo Kei Iwai, Katsumi Maenaka, Hiroki Tabata, Takayuki Kanai, Yu-Zhen Chen, Yasuharu Nishimura Modern Rheumatology 13 (3) 205 -214 2003年 [査読無し][通常論文]
   

  In humans, increased susceptibility to specific autoimmune diseases is closely associated with specific HLA-class II alleles. CD4+ T cells that recognize short self-peptides in the context of HLA-class II molecules via their T cell receptor (TCR) are considered to mediate the central role of pathogenesis in autoimmunity. Although both self-and nonself-peptides are presented on HLA-class II molecules under physiological conditions, several mechanisms exist to avoid the T cell response to the self-peptide/HLA-class II complex. One of the mechanisms that account for the breakdown in immune tolerance is cross-recognition by TCR between a pathogen-derived antigen and a host antigen (molecular mimicry theory). Epidemiological studies have indicated that a number of autoimmune diseases are developed or exacerbated after infections. Therefore, elucidating the recognition nature of HLA-class II restricted TCR in detail is necessary in order to understand disease processes. A large body of evidence indicates that T cell recognition is highly degenerate, and many different peptides can activate an individual T cell. Degeneracy of TCR recognition also can appear in various physiological outcomes, ranging from full activation to strong antagonism. Here, we review the clinical implications of our findings on T cell recognition, as well as a new direction of future applications for analyses in molecular mimicry. We also describe the latest developments in methods of mapping TCR epitopes for CD4+ T cells using a peptide epitope expression library generated in the class II-associated invariant chain peptide substituted invariant chain gene format.
• Haruka Wada, Naoki Matsumoto, Katsumi Maenaka, Kazuhiro Suzuki and Kazuo Yamamoto. Two HLA-E Mutants Segregate the Inhibitory NK Cell Receptor CD94/NKG2A and the Activating Receptor CD94/NKG2C, Both of Whichi Bind the Top of HLA-E

  Eur. J. Immunol In the press 2003年 [査読無し][通常論文]
  
• Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for GAD65 specific TCRs

  Uemura, Y, Senju, S, Maenaka, K, Iwai, L.K, Fujii, S, Tabata, H, Tsukamoto, H, Hirata, S, Chen, Y-Z, Nishimura, Y J. Immunol. 170 (947) 960 -960 2003年 [査読無し][通常論文]
  
• エピトープ発現ライブラリーを用いたGAD65自己反応性TCRの認識エピトープの多様性に関する解析

  植村靖史, 千住覚, 前仲勝実, 藤井慎嗣, 塚本博丈, CHEN Y‐C, 西村泰治 日本免疫学会総会・学術集会記録 32 278 -278 2002年10月31日 [査読無し][通常論文]
  
• Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the transcriptional activator protein PhoB from Escherichia coli

  K Shindoh, K Maenaka, T Akiba, H Okamura, Y Nishimura, K Makino, Y Shirakihara ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 58 1862 -1864 2002年10月 [査読無し][通常論文]
   

  PhoB is a transcriptional factor that activates more than 30 genes of the pho regulon in response to phosphate starvation. Crystals of its C-terminal domain (PhoBC) were obtained in two forms. The first crystal form, obtained from phosphate solution, belongs to space group P2(1), with unit-cell parameters a = 30.7, b = 105.9, c = 30.9 Angstrom, beta = 110.3degrees. The second form, crystallized from PEG solution, belongs to the same space group, but has a smaller unit cell (a = 30.6, b = 37.5, c = 44.4 Angstrom, beta = 109.4degrees). Crystals of selenomethionyl-derivatized PhoBC were obtained using the conditions for the second crystal form. Diffraction data from wild-type PhoBC (2.0 Angstrom resolution) and MAD data sets from selenomethionyl-derivative PhoBC (3.0 Angstrom resolution) have been collected at 100 K with a synchrotron-radiation source. MAD data analysis is in progress.
• Crystallization and preliminary X-ray diffraction studies on the DNA-binding domain of the transcriptional activator protein PhoB from Escherichia coli

  K Shindoh, K Maenaka, T Akiba, H Okamura, Y Nishimura, K Makino, Y Shirakihara ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 58 1862 -1864 2002年10月 [査読無し][通常論文]
   

  PhoB is a transcriptional factor that activates more than 30 genes of the pho regulon in response to phosphate starvation. Crystals of its C-terminal domain (PhoBC) were obtained in two forms. The first crystal form, obtained from phosphate solution, belongs to space group P2(1), with unit-cell parameters a = 30.7, b = 105.9, c = 30.9 Angstrom, beta = 110.3degrees. The second form, crystallized from PEG solution, belongs to the same space group, but has a smaller unit cell (a = 30.6, b = 37.5, c = 44.4 Angstrom, beta = 109.4degrees). Crystals of selenomethionyl-derivatized PhoBC were obtained using the conditions for the second crystal form. Diffraction data from wild-type PhoBC (2.0 Angstrom resolution) and MAD data sets from selenomethionyl-derivative PhoBC (3.0 Angstrom resolution) have been collected at 100 K with a synchrotron-radiation source. MAD data analysis is in progress.
• バキュロウィルスBmNPV外来遺伝子発現系における効率的ベクターの構築

  本橋智子, PARK E Y, 前仲勝実 日本生物工学会大会講演要旨集 2002 66 -66 2002年09月25日 [査読無し][通常論文]
  
• 461 バキュロウィルスBmNPV外来遺伝子発現系における効率的ベクターの構築(遺伝子工学,一般講演)

  本橋 智子, 朴 龍洙, 前仲 勝実 日本生物工学会大会講演要旨集 14 (0) 66 -66 2002年09月25日 [査読無し][通常論文]
  
• ヒト免疫細胞抑制型受容体ILT2,4の相互作用解析

  白石充典, 前仲勝実, 白木原康雄, JONES Y, VAN DER MERWER A, 津本浩平, 熊谷泉 生化学 74 (8) 957 -957 2002年08月25日 [査読無し][通常論文]
  
• ヒト白血球抗原(HLA)とNK細胞受容体の相互作用の熱量計を用いた解析

  郷田秀一郎, 津本浩平, 横田亜紀子, 白石充典, 前仲勝実, 熊谷泉 生化学 74 (8) 871 -871 2002年08月25日 [査読無し][通常論文]
  
• HLA typing in a Kenyan cohort identifies novel class I alleles that restrict cytotoxic T cell response to local HIV-1 clades.

  AIDS in press (16) 1899 -1904 2002年 [査読無し][通常論文]
  
• HLA typing in a Kenyan cohort identifies novel class I alleles that restrict cytotoxic T cell response to local HIV-1 clades.

  AIDS in press 2002年 [査読無し][通常論文]
  
• 免疫グロブリン様レセプター群のリガンド認識に関する速度論的熱力学的解析

  前仲 勝実, 白石 充典, 津本 浩平, Anton van, der Merwe, David Stuart, Yvonne Jones, Peter Sondermann, 熊谷 泉, 白木原 康雄 日本免疫学会総会・学術集会記録 31 233 -233 2001年12月 [査読無し][通常論文]
  
• The human low affinity Fc gamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties

  K Maenaka, PA van der Merwe, DI Stuart, EY Jones, P Sondermann JOURNAL OF BIOLOGICAL CHEMISTRY 276 (48) 44898 -44904 2001年11月 [査読無し][通常論文]
   

  Fc gamma receptors (Fc gamma Rs) are expressed on all immunologically active cells. They bind the Fe portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity Fc gamma Rs (Fc gamma RIIa, Fc gamma RIIb, and Fc gamma gamma RIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K-D similar to0.6-2.5 muM) and fast kinetics, suggesting that Fc gammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fe receptors exhibit distinct thermodynamic properties. Whereas the binding of the Fc gamma RIIa and Fc gamma RIIb to Fe is driven by favorable entropic and enthalpic changes, the binding of Fc gamma RIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity Fc gamma Rs.
• The human low affinity Fc gamma receptors IIa, IIb, and III bind IgG with fast kinetics and distinct thermodynamic properties

  K Maenaka, PA van der Merwe, DI Stuart, EY Jones, P Sondermann JOURNAL OF BIOLOGICAL CHEMISTRY 276 (48) 44898 -44904 2001年11月 [査読無し][通常論文]
   

  Fc gamma receptors (Fc gamma Rs) are expressed on all immunologically active cells. They bind the Fe portion of IgG, thereby triggering a range of immunological functions. We have used surface plasmon resonance to analyze the kinetic and thermodynamic properties of the interactions between the ectodomains of human low affinity Fc gamma Rs (Fc gamma RIIa, Fc gamma RIIb, and Fc gamma gamma RIIb-NA2) and IgG1 or the Fc fragment of IgG1. All three receptors bind Fc or IgG with similarly low affinities (K-D similar to0.6-2.5 muM) and fast kinetics, suggesting that Fc gammaR-mediated recognition of aggregated IgG and IgG-coated particles or cells is mechanistically similar to cell-cell recognition. Interestingly, the Fe receptors exhibit distinct thermodynamic properties. Whereas the binding of the Fc gamma RIIa and Fc gamma RIIb to Fe is driven by favorable entropic and enthalpic changes, the binding of Fc gamma RIII is characterized by highly unfavorable entropic changes. Although the structural bases for these differences remain to be determined, they suggest that the molecular events coupled to the binding differ among the low affinity Fc gamma Rs.
• 免疫グロブリン様レセプター群のリガンド認識に関する速度論的熱力学的解析

  前仲勝実, 白石充典, 津本浩平, VAN DER MERWE A, STUART D, JONES Y, SONDERMANN P, 熊谷泉, 白木原康雄 日本免疫学会総会・学術集会記録 31 233 2001年10月31日 [査読無し][通常論文]
  
• MHCクラスI分子及びヒトNK細胞阻害受容体(KIR)の効率的な巻き戻しと相互作用の熱力学的解析

  郷田秀一郎, 横田亜紀子, 白石充典, 津本浩平, 前仲勝実, 熊谷泉 日本生物物理学会年会講演予稿集 39th S47 2001年10月 [査読無し][通常論文]
  
• 不溶性か粒から巻き戻した各種受容体細胞外ドメインの機能評価

  北村武, 郷田秀一郎, 津本浩平, 前仲勝実, 三沢悟, 熊谷泉 日本生物物理学会年会講演予稿集 39th S47 2001年10月 [査読無し][通常論文]
  
• Ig‐like receptor群の分子認識機構

  前仲勝実 MHC 8 (2) 104 2001年09月30日 [査読無し][通常論文]
  
• 1P059MHCクラスI分子及びヒトNK細胞阻害受容体(KIR)の効率的な巻き戻しと相互作用の熱力学的解析

  郷田 秀一郎, 横田 亜紀子, 白石 充典, 津本 浩平, 前仲 勝実, 熊谷 泉 生物物理 41 (1) S47 2001年09月10日 [査読無し][通常論文]
  
• 1P060不溶性顆粒から巻き戻した各種受容体細胞外ドメインの機能評価

  北村 武, 郷田 秀一郎, 津本 浩平, 前仲 勝実, 三沢 悟, 熊谷 泉 生物物理 41 (1) S47 2001年09月10日 [査読無し][通常論文]
  
• MHCと移植 Ig-like receptor群の分子認識機構

  前仲 勝実 MHC: Major Histocompatibility Complex 8 (2) 104 -104 2001年09月 [査読無し][通常論文]
  
• 不溶性か粒で調製した各種受容体細胞外ドメインの効率よい巻き戻し系の構築

  北村武, 郷田秀一郎, 津本浩平, 前仲勝実, 三沢悟, 熊谷泉 生化学 73 (8) 959 2001年08月25日 [査読無し][通常論文]
  
• 生命現象の理解と医療・創薬に向けて 8.生体防御 免疫系レセプターの分子認識

  前仲勝実 蛋白質 核酸 酵素 46 (11) 1805 -1811 2001年08月10日 [査読無し][通常論文]
  
• 【新世紀における蛋白質科学の進展】 生命現象の理解と医療・創薬に向けて 生体防御 免疫系レセプターの分子認識

  前仲 勝実 蛋白質・核酸・酵素 46 (11) 1805 -1811 2001年08月 [査読無し][通常論文]
   

  免疫系レセプターの機能制御を理解するには,細胞表面レセプターの分子認識を解析することが必須であるが,ようやく最近,いくつかのレセプターについて機能と構造の両面からの統合的な解析が報告されてきた.その例として,自然免疫にかかわるナチュラルキラー(NK)細胞に発現するkiller cell Ig-like receptor(KIR)の主要組織適合性抗原(MHC)クラスIに対する分子認識の著者等の研究を中心に,同じくMHCを認識し,適応免疫にかかわるレセプター(T細胞レセプター,CD8など)や他のレセプターと比較しながら,多様な免疫系レセプターの分子認識について概説した
• 免疫系レセプターの分子認識 (新世紀における蛋白質科学の進展) -- (第3部 生命現象の理解と医療・創薬に向けて)

  前仲 勝実 蛋白質核酸酵素 46 (11) 1427 -1428,1805〜1811 2001年08月 [査読無し][通常論文]
  
• Detection of autoantibodies to killer immunoglobulin-like receptors using recombinant fusion proteins for two killer immunoglobulin-like receptors in patients with systemic autoimmune diseases

  T Matsui, M Otsuka, K Maenaka, H Furukawa, T Yabe, K Yamamoto, K Nishioka, T Kato ARTHRITIS AND RHEUMATISM 44 (2) 384 -388 2001年02月 [査読無し][通常論文]
   

  Objective. To investigate the existence of autoantibodies to killer immunoglobulin-like receptors (KIRs), especially p58.1 (KIR2DLI) and p58.2 (KIR2DL3), in patients with systemic autoimmune diseases. Methods. Sera from 30 patients with systemic lupus erythematosus (SLE), 30 patients with rheumatoid arthritis (RA), 22 patients with Behcet's disease, and 20 healthy control subjects were tested for anti-p58.1 and anti-p58.2 antibodies by Western blot analysis using recombinant p58.1 and p58.2 proteins. Furthermore, clinical features and laboratory data were compared between the anti-p58.1/58.2 antibody-positive and -negative patients. Results. Anti-p58.1 antibodies were detected in 7 (23.3%) of the 30 patients with SLE, 9 (30%) of the 30 patients with RA, and 6 (27.3%) of the 22 patients with Behcet's disease. Anti-p58.2 antibodies were detected in the same 22 patients who were positive for the anti-p58.1 antibodies. None of the serum samples from the healthy donors were positive for antibodies to the recombinant p58.1 or p58.2 molecules. Compared with the anti-p58.1/58.2 antibody-negative patients, the anti-p58.1/58.2 antibody-positive patients had significantly elevated levels of serum IgG in all 3 diseases tested, an accelerated erythrocyte sedimentation rate in RA and SLE, and decreased white blood cell counts in RA. Conclusion. This report is the first to describe the presence of autoantibodies to KIR2DL (p58.1 and p58.2) in the sera of patients with systemic autoimmune diseases. Considering the correlation with several clinical features, these autoantibodies may be involved in the pathologic process of the autoimmune diseases.
• Detection of autoantibodies to killer immunoglobulin-like receptors using recombinant fusion proteins for two killer immunoglobulin-like receptors in patients with systemic autoimmune diseases

  T Matsui, M Otsuka, K Maenaka, H Furukawa, T Yabe, K Yamamoto, K Nishioka, T Kato ARTHRITIS AND RHEUMATISM 44 (2) 384 -388 2001年02月 [査読無し][通常論文]
   

  Objective. To investigate the existence of autoantibodies to killer immunoglobulin-like receptors (KIRs), especially p58.1 (KIR2DLI) and p58.2 (KIR2DL3), in patients with systemic autoimmune diseases. Methods. Sera from 30 patients with systemic lupus erythematosus (SLE), 30 patients with rheumatoid arthritis (RA), 22 patients with Behcet's disease, and 20 healthy control subjects were tested for anti-p58.1 and anti-p58.2 antibodies by Western blot analysis using recombinant p58.1 and p58.2 proteins. Furthermore, clinical features and laboratory data were compared between the anti-p58.1/58.2 antibody-positive and -negative patients. Results. Anti-p58.1 antibodies were detected in 7 (23.3%) of the 30 patients with SLE, 9 (30%) of the 30 patients with RA, and 6 (27.3%) of the 22 patients with Behcet's disease. Anti-p58.2 antibodies were detected in the same 22 patients who were positive for the anti-p58.1 antibodies. None of the serum samples from the healthy donors were positive for antibodies to the recombinant p58.1 or p58.2 molecules. Compared with the anti-p58.1/58.2 antibody-negative patients, the anti-p58.1/58.2 antibody-positive patients had significantly elevated levels of serum IgG in all 3 diseases tested, an accelerated erythrocyte sedimentation rate in RA and SLE, and decreased white blood cell counts in RA. Conclusion. This report is the first to describe the presence of autoantibodies to KIR2DL (p58.1 and p58.2) in the sera of patients with systemic autoimmune diseases. Considering the correlation with several clinical features, these autoantibodies may be involved in the pathologic process of the autoimmune diseases.
• 免疫系レセプターの分子認識

  蛋白質核酸酵素 増刊号「新世紀における蛋白質科学の進展」 46(11)、1805-1811 2001年 [査読無し][通常論文]
  
• ヒトMHCクラスI HLA-B*5101とHIV由来の免疫優性エピトープペプチドの複合体のX線結晶構造解析

  前仲 勝実, 前仲 太恵子, 冨山 宏子, 滝口 雅文, David Stuart, Yvonne Jones 日本免疫学会総会・学術集会記録 30 315 -315 2000年11月 [査読無し][通常論文]
  
• ヒトMHCクラスI HLA‐B5101とHIV由来の免疫優性エピトープペプチドの複合体のX線結晶構造解析

  前仲勝実, 前仲太恵子, 冨山宏子, 滝口雅文, STUART D, JONES Y 日本免疫学会総会・学術集会記録 30 315 2000年09月26日 [査読無し][通常論文]
  
• Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes

  K Maenaka, T Maenaka, H Tomiyama, M Takiguchi, DI Stuart, EY Jones JOURNAL OF IMMUNOLOGY 165 (6) 3260 -3267 2000年09月 [査読無し][通常論文]
   

  The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography, In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171, This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.
• Nonstandard peptide binding revealed by crystal structures of HLA-B*5101 complexed with HIV immunodominant epitopes

  K Maenaka, T Maenaka, H Tomiyama, M Takiguchi, DI Stuart, EY Jones JOURNAL OF IMMUNOLOGY 165 (6) 3260 -3267 2000年09月 [査読無し][通常論文]
   

  The crystal structures of the human MHC class I allele HLA-B*5101 in complex with 8-mer, TAFTIPSI, and 9-mer, LPPVVAKEI, immunodominant peptide epitopes from HIV-1 have been determined by x-ray crystallography, In both complexes, the hydrogen-bonding network in the N-terminal anchor (P1) pocket is rearranged as a result of the replacement of the standard tyrosine with histidine at position 171, This results in a nonstandard positioning of the peptide N terminus, which is recognized by B*5101-restricted T cell clones. Unexpectedly, the P5 peptide residues appear to act as anchors, drawing the peptides unusually deeply into the peptide-binding groove of B51. The unique characteristics of P1 and P5 are likely to be responsible for the zig-zag conformation of the 9-mer peptide and the slow assembly of B*5101. A comparison of the surface characteristics in the alpha1-helix C-terminal region for B51 and other MHC class I alleles highlights mainly electrostatic differences that may be important in determining the specificity of human killer cell Ig-like receptor binding.
• ヒトkiller cell Ig‐like receptorの分子認識機構

  前仲勝実, VAN DER MERWE A, 前仲太恵子, 白木原康雄, STUART D, JONES Y 生化学 72 (8) 1029 -1029 2000年08月25日 [査読無し][通常論文]
  
• 3H1145 camphor代謝系酵素群オペロンを制御するリプレッサーCamR蛋白質の結晶化

  前仲 勝実, 福司 功治, 荒牧 弘範, 白木原 康雄 生物物理 40 (1) S195 2000年08月05日 [査読無し][通常論文]
  
• ヒトkiller cell immunoglobulin(Ig)‐like receptorの構造と機能

  前仲勝実, VAN DER MERWE A, 十字猛夫, 前仲太恵子, 白木原康雄, STUART D, JONES Y タンパク質構造討論会講演要旨集 51st 325 2000年06月07日 [査読無し][通常論文]
  
• Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8 alpha alpha

  GF Gao, BE Willcox, Wyer, JR, JM Boulter, CA O'Callaghan, K Maenaka, DI Stuart, EY Jones, PA Van Der Merwe, JI Bell, BK Jakobsen JOURNAL OF BIOLOGICAL CHEMISTRY 275 (20) 15232 -15238 2000年05月 [査読無し][通常論文]
   

  The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR), Here we use surface plasmon resonance to study the binding of CD8 alpha alpha to class I MHC molecules. CD8 alpha alpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K-d) of 90-220 mu M, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8 alpha alpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8 alpha alpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (greater than or equal to 1 mM), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8 alpha alpha bound normally to the nonclassical MHC molecule HLA-G (K-d similar to 150 mu M), but only weakly to the natural killer cell receptor ligand HLA-E (K-d greater than or equal to 1 mM). Site-directed mutagenesis experiments revealed that variation in CD8 alpha alpha binding affinity can be explained by amino acid differences within the alpha 3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha 3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.
• Classical and nonclassical class I major histocompatibility complex molecules exhibit subtle conformational differences that affect binding to CD8 alpha alpha

  GF Gao, BE Willcox, Wyer, JR, JM Boulter, CA O'Callaghan, K Maenaka, DI Stuart, EY Jones, PA Van Der Merwe, JI Bell, BK Jakobsen JOURNAL OF BIOLOGICAL CHEMISTRY 275 (20) 15232 -15238 2000年05月 [査読無し][通常論文]
   

  The cell surface molecules CD4 and CD8 greatly enhance the sensitivity of T-cell antigen recognition, acting as "co-receptors" by binding to the same major histocompatibility complex (MHC) molecules as the T-cell receptor (TCR), Here we use surface plasmon resonance to study the binding of CD8 alpha alpha to class I MHC molecules. CD8 alpha alpha bound the classical MHC molecules HLA-A*0201, -A*1101, -B*3501, and -C*0702 with dissociation constants (K-d) of 90-220 mu M, a range of affinities distinctly lower than that of TCR/peptide-MHC interaction. We suggest such affinities apply to most CD8 alpha alpha/classical class I MHC interactions and may be optimal for T-cell recognition. In contrast, CD8 alpha alpha bound both HLA-A*6801 and B*4801 with a significantly lower affinity (greater than or equal to 1 mM), consistent with the finding that interactions with these alleles are unable to mediate cell-cell adhesion. Interestingly, CD8 alpha alpha bound normally to the nonclassical MHC molecule HLA-G (K-d similar to 150 mu M), but only weakly to the natural killer cell receptor ligand HLA-E (K-d greater than or equal to 1 mM). Site-directed mutagenesis experiments revealed that variation in CD8 alpha alpha binding affinity can be explained by amino acid differences within the alpha 3 domain. Taken together with crystallographic studies, these results indicate that subtle conformational changes in the solvent exposed alpha 3 domain loop (residues 223-229) can account for the differential ability of both classical and nonclassical class I MHC molecules to bind CD8.
• MHC superfamily structure and the immune system

  K Maenaka, EY Jones CURRENT OPINION IN STRUCTURAL BIOLOGY 9 (6) 745 -753 1999年12月 [査読無し][通常論文]
   

  During the past year, a plethora of structural information has provided detailed insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells. Likewise, there have been major advances in our knowledge of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), HLA-E, HFE, ZAG and MIC-A.
• MHC superfamily structure and the immune system

  K Maenaka, EY Jones CURRENT OPINION IN STRUCTURAL BIOLOGY 9 (6) 745 -753 1999年12月 [査読無し][通常論文]
   

  During the past year, a plethora of structural information has provided detailed insights into the interactions between classical MHC class I molecules and their cognate receptors on T cells. Likewise, there have been major advances in our knowledge of the structures and functions of five nonclassical MHC-like molecules: HLA-DM (murine H2-M), HLA-E, HFE, ZAG and MIC-A.
• Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties

  K Maenaka, T Juji, T Nakayama, Wyer, JR, GF Gao, T Maenaka, NR Zaccai, A Kikuchi, T Yabe, K Tokunaga, K Tadokoro, DI Stuart, EY Jones, PA van der Merwe JOURNAL OF BIOLOGICAL CHEMISTRY 274 (40) 28329 -28334 1999年10月 [査読無し][通常論文]
   

  Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K-d similar to 7 mu M at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., WS er, J. R., Ladbury, J. E., Bell, J. I., Jakobsen, B. K,, and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.
• Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties

  K Maenaka, T Juji, T Nakayama, Wyer, JR, GF Gao, T Maenaka, NR Zaccai, A Kikuchi, T Yabe, K Tokunaga, K Tadokoro, DI Stuart, EY Jones, PA van der Merwe JOURNAL OF BIOLOGICAL CHEMISTRY 274 (40) 28329 -28334 1999年10月 [査読無し][通常論文]
   

  Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K-d similar to 7 mu M at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., WS er, J. R., Ladbury, J. E., Bell, J. I., Jakobsen, B. K,, and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition.
• Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I

  Katsumi Maenaka, Takeo Juji, David I. Stuart, E. Yvonne Jones Structure 7 (4) 391 -398 1999年04月15日 [査読無し][通常論文]
   

  Background: T cells and natural killer (NK) cells perform complementary roles in the cellular immune system. T cells identify infected cells directly through recognition of antigenic peptides that are displayed at the target cell surface by the classical major histocompatibility complex (MHC) class I molecules. NK cells monitor the target cell surface for malfunction of this display system, lysing potentially infected cells that might otherwise evade recognition by the T cells. Human killer cell inhibitory receptors (KIRs) control this process by either inhibiting or activating the cytotoxic activity of NK cells via specific binding to MHC class I molecules on the target cell. Results: We report the crystal structure of the extracellular region of the human p58 KIR (KIR2DL3), which is specific for the human MHC class I molecule HLA-Cw3 and related alleles. The structure shows the predicted topology of two tandem immunoglobulin-like domains, but comparison with the previously reported structure of the related receptor KIR2DL1 reveals an unexpected change of 23°in the relative orientation of these domains. Conclusions: The altered orientation of the immunoglobulin-like domains maintains an unusually acute interdomain elbow angle, which therefore appears to be a distinctive feature of the KIRs. The putative MHC class I binding site is located on the outer surface of the elbow, spanning both domains. The unexpected observation that this binding site can be modulated by differences in the relative domain orientations has implications for the general mechanism of KIR-MHC class I complex formation.
• Crystal structure of the human p58 killer cell inhibitory receptor (KIR2DL3) specific for HLA-Cw3-related MHC class I

  Katsumi Maenaka, Takeo Juji, David I. Stuart, E. Yvonne Jones Structure 7 (4) 391 -398 1999年04月15日 [査読無し][通常論文]
   

  Background: T cells and natural killer (NK) cells perform complementary roles in the cellular immune system. T cells identify infected cells directly through recognition of antigenic peptides that are displayed at the target cell surface by the classical major histocompatibility complex (MHC) class I molecules. NK cells monitor the target cell surface for malfunction of this display system, lysing potentially infected cells that might otherwise evade recognition by the T cells. Human killer cell inhibitory receptors (KIRs) control this process by either inhibiting or activating the cytotoxic activity of NK cells via specific binding to MHC class I molecules on the target cell. Results: We report the crystal structure of the extracellular region of the human p58 KIR (KIR2DL3), which is specific for the human MHC class I molecule HLA-Cw3 and related alleles. The structure shows the predicted topology of two tandem immunoglobulin-like domains, but comparison with the previously reported structure of the related receptor KIR2DL1 reveals an unexpected change of 23°in the relative orientation of these domains. Conclusions: The altered orientation of the immunoglobulin-like domains maintains an unusually acute interdomain elbow angle, which therefore appears to be a distinctive feature of the KIRs. The putative MHC class I binding site is located on the outer surface of the elbow, spanning both domains. The unexpected observation that this binding site can be modulated by differences in the relative domain orientations has implications for the general mechanism of KIR-MHC class I complex formation.
• ヒトkiller cell Ig-like receptor(KIR)の構造と機能

  構造生物 5, 5-15 1999年 [査読無し][通常論文]
  
• Structural and functional effect of Trp-62 -> Gly and Asp-101 -> Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes

  K Maenaka, M Matsushima, G Kawai, A Kidera, K Watanabe, R Kuroki, Kumagai, I BIOCHEMICAL JOURNAL 333 71 -76 1998年07月 [査読無し][通常論文]
   

  In order to clarify the structural role of subsite B of hen eggwhite lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62 --> Gly and Asp 101 --> Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-D-glucosamine)(3) [(GlcNAc)(3)], using X-ray crystallography, The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc), molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)(2), the reducing end of which is an alpha-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a beta-anomer-retaining manner, and so the alpha-anomer product was converted from the beta-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.
• Structural and functional effect of Trp-62 -> Gly and Asp-101 -> Gly substitutions on substrate-binding modes of mutant hen egg-white lysozymes

  K Maenaka, M Matsushima, G Kawai, A Kidera, K Watanabe, R Kuroki, Kumagai, I BIOCHEMICAL JOURNAL 333 71 -76 1998年07月 [査読無し][通常論文]
   

  In order to clarify the structural role of subsite B of hen eggwhite lysozyme in hydrolytic activity towards a carbohydrate substrate, we analysed the structures of Trp-62 --> Gly and Asp 101 --> Gly mutant hen lysozymes, which have no side chain at positions 62 or 101, complexed with a substrate analogue, (N-acetyl-D-glucosamine)(3) [(GlcNAc)(3)], using X-ray crystallography, The overall protein structures in the mutant lysozyme complexes were almost identical to those in the wild type. In the crystals of all the mutant complexes, the (GlcNAc), molecule, which is an inhibitor of wild-type lysozyme, had no inhibitory effect, but was hydrolysed as a substrate. One of the products, (GlcNAc)(2), the reducing end of which is an alpha-anomer, was bound in an unproductive binding mode, protruding from the active-site cleft, and was able to act as an inhibitor. Hydrolysis of the synthetic substrate by the mutants occurred in a beta-anomer-retaining manner, and so the alpha-anomer product was converted from the beta-anomer product. Thus the interactions of Asp-101 and Trp-62 in subsite B are not essential for the catalytic mechanism, but co-operatively enhance the affinity of the substrate in the productive binding mode, other than the inhibitor in the unproductive mode.
• Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2)

  K Maenaka, T Juji, K Tadokoro, K Harlos, DI Stuart, EY Jones ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 54 433 -435 1998年05月 [査読無し][通常論文]
   

  Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR's specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average M-r= 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6(1)22 or P6(5)22) with lattice constants a = b = 95.3, c = 130.8 Angstrom. A native data set (3 Angstrom resolution) has been collected at the Photon Factory (lambda = 1.0 Angstrom).
• Crystallization and preliminary diffraction studies of the extracellular region of human p58 killer cell inhibitory receptor (KIR2)

  K Maenaka, T Juji, K Tadokoro, K Harlos, DI Stuart, EY Jones ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY 54 433 -435 1998年05月 [査読無し][通常論文]
   

  Molecules of the human killer cell inhibitory receptor (KIR) family, which belong to the immunoglobulin superfamily (IgSF), are expressed on the surface of natural killer (NK) cells and some subsets of T cells. These receptors function to mediate the inhibition or activation of cytotoxic activity by recognizing HLA class I molecules on the target cell. The extracellular region of a p58 KIR's specific for HLA-Cw1,3,7 (KIR2) has been overproduced in Escherichia coli and purified. The recombinant KIR2 has been crystallized in 9-10% poly(ethylene glycol) methyl ether (average M-r= 8000), 50mM HEPES, 8% ethylene glycol, 0.5% octyl-beta-glucoside, pH 7.5, at 294 K using the sitting-drop vapour-diffusion method. Preliminary X-ray diffraction studies reveal the space group to be hexagonal (P6(1)22 or P6(5)22) with lattice constants a = b = 95.3, c = 130.8 Angstrom. A native data set (3 Angstrom resolution) has been collected at the Photon Factory (lambda = 1.0 Angstrom).
• Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode

  K Maenaka, M Matsushima, G Kawai, K Watanabe, R Kuroki, Kumagai, I BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1384 (1) 23 -31 1998年04月 [査読無し][通常論文]
   

  Trp62 in hen egg-white lysozyme has general features observed in protein-carbohydrate interactions, a stacking interaction toward nonpolar surface of the substrate sugar residue B and a hydrogen bonding network with the residue C, Our previous report (I. Kumagai, K. Maenaka, F. Sunada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) showed that the substitution of Trp62 changed the substrate binding modes, especially, the Trp62His mutant exhibited the drastic change of the binding mode and preferred to a minor binding mode of the wild-type enzyme. In order to clarify the relationship between functional and structural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, (GlcNAc)(3), by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild-type. His62 shared almost the same plane as the indole ring of Trp62. of the wild-type. Although the (GlcNAc)(3) molecule which is an inhibitor against the wild-type lysozyme was cocrystallized, the Trp62His mutant did not put it in the sites A-B-C but hydrolyzed it as a substrate. One of the products, (GlcNAc)(3), whose reducing end is alpha-anomer, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anomer product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interactions between His63 and the sugar residues B and C. (C) 1998 Elsevier Science B.V.
• Structural analysis of mutant hen egg-white lysozyme preferring a minor binding mode

  K Maenaka, M Matsushima, G Kawai, K Watanabe, R Kuroki, Kumagai, I BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY 1384 (1) 23 -31 1998年04月 [査読無し][通常論文]
   

  Trp62 in hen egg-white lysozyme has general features observed in protein-carbohydrate interactions, a stacking interaction toward nonpolar surface of the substrate sugar residue B and a hydrogen bonding network with the residue C, Our previous report (I. Kumagai, K. Maenaka, F. Sunada, S. Takeda, K. Miura, Eur. J. Biochem. 212 (1993) 151-156.) showed that the substitution of Trp62 changed the substrate binding modes, especially, the Trp62His mutant exhibited the drastic change of the binding mode and preferred to a minor binding mode of the wild-type enzyme. In order to clarify the relationship between functional and structural changes of the Trp62His mutant, we analyzed the structure of the Trp62His mutant hen lysozyme complexed with the substrate analogue, (GlcNAc)(3), by X-ray crystallography. The overall protein structure in the mutant lysozyme complex was almost identical to that in the wild-type. His62 shared almost the same plane as the indole ring of Trp62. of the wild-type. Although the (GlcNAc)(3) molecule which is an inhibitor against the wild-type lysozyme was cocrystallized, the Trp62His mutant did not put it in the sites A-B-C but hydrolyzed it as a substrate. One of the products, (GlcNAc)(3), whose reducing end is alpha-anomer, was bound in another binding mode sticking out from the active-site cleft. The hydrolytic activity against the synthetic substrate showed that the mutant was a beta-anomer retaining enzyme, so the alpha-anomer product was converted from the beta-anomer product. Therefore, the Trp62His mutant showed the remarkable change of the substrate binding modes not by alteration of the catalytic system but possibly by subtle rearrangement of general features of protein-carbohydrate interactions between His63 and the sugar residues B and C. (C) 1998 Elsevier Science B.V.
• ファージ提示法による抗体工学

  熊谷 泉, 津本 浩平, 前仲 勝実 生物物理 213, 219-222 (5) 219 -222 1997年09月25日 [査読無し][通常論文]
  
• The human natural killer gene complex (NKC) is located on chromosome 12p13.1-p13.2

  Y Suto, T Yabe, K Maenaka, K Tokunaga, K Tadokoro, T Juji IMMUNOGENETICS 46 (2) 159 -162 1997年 [査読無し][通常論文]
  
• The human natural killer gene complex (NKC) is located on chromosome 12p13.1-p13.2

  Y Suto, T Yabe, K Maenaka, K Tokunaga, K Tadokoro, T Juji IMMUNOGENETICS 46 (2) 159 -162 1997年 [査読無し][通常論文]
  
• The 1.8-angstrom X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding

  S Sugiyama, Y Matsuo, K Maenaka, DG Vassylyev, M Matsushima, K Kashiwagi, K Igarashi, K Morikawa PROTEIN SCIENCE 5 (10) 1984 -1990 1996年10月 [査読無し][通常論文]
   

  The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 Angstrom resolution and revealed the detailed substrate-binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.
• The 1.8-angstrom X-ray structure of the Escherichia coli PotD protein complexed with spermidine and the mechanism of polyamine binding

  S Sugiyama, Y Matsuo, K Maenaka, DG Vassylyev, M Matsushima, K Kashiwagi, K Igarashi, K Morikawa PROTEIN SCIENCE 5 (10) 1984 -1990 1996年10月 [査読無し][通常論文]
   

  The PotD protein from Escherichia coli is one of the components of the polyamine transport system present in the periplasm. This component specifically binds either spermidine or putrescine. The crystal structure of the E. coli PotD protein complexed with spermidine was solved at 1.8 Angstrom resolution and revealed the detailed substrate-binding mechanism. The structure provided the detailed conformation of the bound spermidine. Furthermore, a water molecule was clearly identified in the binding site lying between the amino-terminal domain and carboxyl-terminal domain. Through this water molecule, the bound spermidine molecule forms two hydrogen bonds with Thr 35 and Ser 211. Another periplasmic component of polyamine transport, the PotF protein, exhibits 35% sequence identity with the PotD protein, and it binds only putrescine, not spermidine. To understand these different substrate specificities, model building of the PotF protein was performed on the basis of the PotD crystal structure. The hypothetical structure suggests that the side chain of Lys 349 in PotF inhibits spermidine binding because of the repulsive forces between its positive charge and spermidine. On the other hand, putrescine could be accommodated into the binding site without any steric hindrance because its molecular size is much smaller than that of spermidine, and the positively charged amino group is relatively distant from Lys 349.
• Development of a novel phage display system and its application to conversion of molecular recognition activities of proteins

  Kumagai, I, K Tsumoto, K Maenaka PROTEIN ENGINEERING 9 (9) 3 -3 1996年09月 [査読無し][通常論文]
  
• ヒトNKクラスIレセプターの大腸菌での発現とファージ表面への提示

  前仲 勝実, 西村 元子, 菊地 安希子, 中山 貴博, 斎藤 彰一, 屋部 登志雄, 熊谷 泉, 田所 憲治, 十字 猛夫 日本分子生物学会年会プログラム・講演要旨集 19 (0) 527 -527 1996年08月01日 [査読無し][通常論文]
  
• ファージペプチドライブラリーを用いたヒト赤血球GPA分子上の抗体認識エピトープの解析

  屋部 登志雄, 古田 大, 前仲 勝実, 津本 浩平, 田所 憲治, 内川 誠, 熊谷 泉, 十字 猛夫 日本分子生物学会年会プログラム・講演要旨集 19 (0) 720 -720 1996年08月01日 [査読無し][通常論文]
  
• 抗原のファージ提示による抗原抗体反応の解析

  津本 浩平, 前仲 勝実, 熊谷 泉 日本分子生物学会年会プログラム・講演要旨集 19 (0) 720 -720 1996年08月01日 [査読無し][通常論文]
  
• Chromosomal localization of the human natural killer cell class I receptor family genes to 19q13.4 by fluorescence in situ hybridization

  Y Suto, K Maenaka, T Yabe, M Hirai, K Tokunaga, K Tadokoro, T Juji GENOMICS 35 (1) 270 -272 1996年07月 [査読無し][通常論文]
  
• Chromosomal localization of the human natural killer cell class I receptor family genes to 19q13.4 by fluorescence in situ hybridization

  Y Suto, K Maenaka, T Yabe, M Hirai, K Tokunaga, K Tadokoro, T Juji GENOMICS 35 (1) 270 -272 1996年07月 [査読無し][通常論文]
  
• ヒトNKクラスIレセプターの大腸菌での発現とファージ表面への提示

  前仲勝実, 西村元子, 菊地安希子, 中山貴博, 斎藤彰一, 屋部登志雄, 熊谷泉, 田所憲治, 十字猛夫 日本分子生物学会年会プログラム・講演要旨集 19th (7) 527 -1039 1996年07月 [査読無し][通常論文]
  
• 抗原のファージ提示による抗原抗体反応の解析

  津本浩平, 前仲勝実, 熊谷泉 日本分子生物学会年会プログラム・講演要旨集 19th 720 1996年07月 [査読無し][通常論文]
  
• ファージペプチドライブラリーを用いたヒト赤血球GPA分子上の抗体認識エピトープの解析

  屋部登志雄, 古田大, 前仲勝実, 津本浩平, 田所憲治, 内川誠, 熊谷泉, 十字猛夫 日本分子生物学会年会プログラム・講演要旨集 19th 720 1996年07月 [査読無し][通常論文]
  
• 触媒機構に基づく酵素分子の進化工学

  前仲勝実, 熊谷泉 化学 51 (6) 398 -399 1996年06月 [査読無し][通常論文]
  
• 触媒機構に基づく酵素分子の進化工学 (1996年の化学-6-)

  前仲 勝実, 熊谷 泉 化学 51 (6) 398 -399 1996年06月 [査読無し][通常論文]
   

  資料形態 : テキストデータ プレーンテキスト
• 触媒機構に基づく酵素分子の進化工学

  化学6 398-399 1996年 [査読無し][通常論文]
  
• A stable phage-display system using a phagemid vector: Phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline, phosphatase, and anti-HEL monoclonal antibody, HyHEL10

  K Maenaka, M Furuta, K Tsumoto, K Watanabe, Y Ueda, Kumagai, I BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218 (3) 682 -687 1996年01月 [査読無し][通常論文]
   

  A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed. A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII Fusion protein in the opposite direction to that of the lac promoter of pTZ18U. Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E. coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL 10, could be stably and functionally displayed. Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII. Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions. (C) 1996 Academic Press, Inc.
• A stable phage-display system using a phagemid vector: Phage display of hen egg-white lysozyme (HEL), Escherichia coli alkaline, phosphatase, and anti-HEL monoclonal antibody, HyHEL10

  K Maenaka, M Furuta, K Tsumoto, K Watanabe, Y Ueda, Kumagai, I BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218 (3) 682 -687 1996年01月 [査読無し][通常論文]
   

  A stable expression system for displaying the pIII fusion protein on the surface of a filamentous phage was constructed. A phagemid pIII display vector, pLUCK, was constructed by inserting the gene encoding the pIII Fusion protein in the opposite direction to that of the lac promoter of pTZ18U. Using this phage display system, two enzymes, hen egg-white lysozyme (HEL) and E. coli alkaline phosphatase, and the single-chain Fv fragment of anti-HEL monoclonal antibody HyHEL 10, could be stably and functionally displayed. Northern and primer extension analyses showed that a small amount of the sense mRNA encoding pIII-fused HEL was transcribed from the minor phage promoter located in the region encoding the C-terminus of pIII. Repressed expression of the pIII fusion protein can lead to the display of a wide range of proteins on filamentous phages without the need for strict expression conditions. (C) 1996 Academic Press, Inc.
• プロテアーゼインヒビターのファージコート蛋白質への提示

  津本浩平, 前仲勝実, 田口精一, 百瀬春生, 熊谷泉 日本分子生物学会年会プログラム・講演要旨集 18th 524 1995年11月 [査読無し][通常論文]
  
• NK細胞受容体の繊維状ファージ表面への提示の試み

  前仲勝実, 西村元子, 斉藤彰一, 屋部登志雄, 田所憲治, 熊谷泉, 十字猛夫 日本免疫学会総会・学術集会記録 25 424 1995年10月 [査読無し][通常論文]
  
• 安定なファージ提示系の開発と抗体工学への応用

  熊谷泉, 前仲勝実, 津本浩平 日本免疫学会総会・学術集会記録 25 424 1995年10月 [査読無し][通常論文]
  
• B‐LCLを標的細胞として測定したNK細胞のアロ特異的細胞障害活性

  斉藤彰一, 西村元子, 前仲勝実, 屋部登志雄, 田所憲治, 十字猛夫 日本免疫学会総会・学術集会記録 25 286 1995年10月 [査読無し][通常論文]
  
• Development of the stable phagemid pIII display system of proteins on the surface of the filamentous phage

  K Maenaka, K Tsumoto, Y Ueda, M Furuta, K Watanabe, Kumagai, I PROTEIN ENGINEERING 8 (9) 43 -43 1995年09月 [査読無し][通常論文]
  
• DISSECTION OF PROTEIN-CARBOHYDRATE INTERACTIONS IN MUTANT HEN EGG-WHITE LYSOZYME COMPLEXES AND THEIR HYDROLYTIC ACTIVITY

  K MAENAKA, M MATSUSHIMA, H SONG, F SUNADA, K WATANABE, KUMAGAI, I JOURNAL OF MOLECULAR BIOLOGY 247 (2) 281 -293 1995年03月 [査読無し][通常論文]
   

  Trp62 in the binding subsite B of hen egg-white lysozyme shows general features often observed in protein-carbohydrate interactions including a stacking interaction and a hydrogen bonding network with water molecules. A previous report by our group showed that the perturbation of these interactions by substitution of Trp62 with tyrosine or phenylalanine affects the substrate binding modes and also enhances the hydrolytic activity In order to elucidate the relationship between structural and functional changes of these protein-carbohydrate interactions, the Trp62Tyr and Trp62Phe mutants complexed with the substrate analogue, (GlcNAc)(3), were analyzed at 1.8 Angstrom resolution by X-ray crystallography. The overall structures of the mutant enzymes are indistinguishable from that of the wild type enzyme. Although the wild-type enzyme binds (GlcNAc)(3) in only one binding mode (A-B-C), the Trp62Tyr mutant binds (GlcNAc)(3) in two binding modes (A-B-C, B-C-D) and the Trp62Phe mutant has an even weaker binding mode. The aromatic rings of Tyr62 and Phe62 maintain their interactions with the carbohydrate molecules, but make fewer stacking interactions with the GlcNAc in the B site than the wild-type enzyme does. The hydroxyl group of Tyr62 interacts weakly with a water molecule which mediates hydrogen bonding in the GlcNAc residues in the B and C sites. The C-6 hydroxyl group of the GlcNAc residue in the C site rotates around the C-5-C-6 bond to complete the hydrogen bond network in the Trp62Tyr mutant-(GlcNAc)(3) complex. On the other hand, this hydrogen bonding network does not form in the Trp62Phe mutant-(GlcNAc)(3). In addition to these structural studies, the kinetic parameters of the hydrolysis of 4-methylumbelliferyl N-acetyl-chitotriose, ((GlcNAc)(3)-MeU), have been determined in order to further characterize the enzymatic properties of these mutant lysozymes. This demonstrates that the modulation of the hydrogen bonding network, including the flexible part of the carbohydrate and water molecules and/or the slight reduction of stacking interaction in the B site, alters the binding mode toward the carbohydrate and induces an enhancement of the hydrolytic activity.
• DISSECTION OF PROTEIN-CARBOHYDRATE INTERACTIONS IN MUTANT HEN EGG-WHITE LYSOZYME COMPLEXES AND THEIR HYDROLYTIC ACTIVITY

  K MAENAKA, M MATSUSHIMA, H SONG, F SUNADA, K WATANABE, KUMAGAI, I JOURNAL OF MOLECULAR BIOLOGY 247 (2) 281 -293 1995年03月 [査読無し][通常論文]
   

  Trp62 in the binding subsite B of hen egg-white lysozyme shows general features often observed in protein-carbohydrate interactions including a stacking interaction and a hydrogen bonding network with water molecules. A previous report by our group showed that the perturbation of these interactions by substitution of Trp62 with tyrosine or phenylalanine affects the substrate binding modes and also enhances the hydrolytic activity In order to elucidate the relationship between structural and functional changes of these protein-carbohydrate interactions, the Trp62Tyr and Trp62Phe mutants complexed with the substrate analogue, (GlcNAc)(3), were analyzed at 1.8 Angstrom resolution by X-ray crystallography. The overall structures of the mutant enzymes are indistinguishable from that of the wild type enzyme. Although the wild-type enzyme binds (GlcNAc)(3) in only one binding mode (A-B-C), the Trp62Tyr mutant binds (GlcNAc)(3) in two binding modes (A-B-C, B-C-D) and the Trp62Phe mutant has an even weaker binding mode. The aromatic rings of Tyr62 and Phe62 maintain their interactions with the carbohydrate molecules, but make fewer stacking interactions with the GlcNAc in the B site than the wild-type enzyme does. The hydroxyl group of Tyr62 interacts weakly with a water molecule which mediates hydrogen bonding in the GlcNAc residues in the B and C sites. The C-6 hydroxyl group of the GlcNAc residue in the C site rotates around the C-5-C-6 bond to complete the hydrogen bond network in the Trp62Tyr mutant-(GlcNAc)(3) complex. On the other hand, this hydrogen bonding network does not form in the Trp62Phe mutant-(GlcNAc)(3). In addition to these structural studies, the kinetic parameters of the hydrolysis of 4-methylumbelliferyl N-acetyl-chitotriose, ((GlcNAc)(3)-MeU), have been determined in order to further characterize the enzymatic properties of these mutant lysozymes. This demonstrates that the modulation of the hydrogen bonding network, including the flexible part of the carbohydrate and water molecules and/or the slight reduction of stacking interaction in the B site, alters the binding mode toward the carbohydrate and induces an enhancement of the hydrolytic activity.
• Development of the stable phagemid pIII display system of proteins on the surface of the filamentous phage

  Maenaka K, Tsumoto K, Ueda Y, Furuta M, Watanabe K, Kumagai I Protein Eng. 9 965 1995年 [査読無し][通常論文]
  
• STRUCTURAL-CHANGES OF ACTIVE-SITE CLEFT AND DIFFERENT SACCHARIDE BINDING MODES IN HUMAN LYSOZYME CO-CRYSTALLIZED WITH HEXA-N-ACETYL-CHITOHEXAOSE AT PH 4.0

  HW SONG, K INAKA, K MAENAKA, M MATSUSHIMA JOURNAL OF MOLECULAR BIOLOGY 244 (5) 522 -540 1994年12月 [査読無し][通常論文]
   

  Human lysozyme was co-crystallized with hexa-N-acetyl-chitohexaose, (GlcNAc)(6), at pH 4.0 and 4.0 degrees C in a new orthorhombic form, where two protein molecules, MOL1 and MOL2, were contained in an asymmetric unit. The three-dimensional structure was refined to an R-factor of 17.0% at 1.6 Angstrom resolution. It was found that (GlcNAc)(6) had already been cleaved to (GlcNAc)(4) and (GlcNAc)(2). In MOL1, (GlcNAc)(4) was bound to the A, B, C and D subsites, and the binding sites of (GlcNAc)(2) were close to the E and F subsites proposed on the basis of model building by Phillips and his colleagues. In MOL2, only the (GlcNAc)(4) moiety could be found in the A, B, C and D subsites. Significant shifts of the backbone atoms were observed in the region of residues 102 to 120, Which composed one side of the wall of the active site cleft. Consequently, the active cleft, with respect to the saccharide binding sites A, B and C, is narrower in both protein molecules. The residues 109 to 111 in site D of MOL1 are moved toward saccharide residue D, whereas those of MOL2 are only slightly shifted. In spite of these facts, the saccharide residues in site MOL1 and MOL2 are moved inside of the cleft. The distribution of water molecules and the hydrogen bond network in site D differ between the structures of MOL1 and MOL2. These structural changes in the active site cleft may be responsible for accommodating the substrate and releasing the products of hydrolysis. These results suggest that the three-dimensional structures of MOL1 and MOL2 remain in intermediate states between a transition state and an enzyme/product complex state.
• STRUCTURAL-CHANGES OF ACTIVE-SITE CLEFT AND DIFFERENT SACCHARIDE BINDING MODES IN HUMAN LYSOZYME CO-CRYSTALLIZED WITH HEXA-N-ACETYL-CHITOHEXAOSE AT PH 4.0

  HW SONG, K INAKA, K MAENAKA, M MATSUSHIMA JOURNAL OF MOLECULAR BIOLOGY 244 (5) 522 -540 1994年12月 [査読無し][通常論文]
   

  Human lysozyme was co-crystallized with hexa-N-acetyl-chitohexaose, (GlcNAc)(6), at pH 4.0 and 4.0 degrees C in a new orthorhombic form, where two protein molecules, MOL1 and MOL2, were contained in an asymmetric unit. The three-dimensional structure was refined to an R-factor of 17.0% at 1.6 Angstrom resolution. It was found that (GlcNAc)(6) had already been cleaved to (GlcNAc)(4) and (GlcNAc)(2). In MOL1, (GlcNAc)(4) was bound to the A, B, C and D subsites, and the binding sites of (GlcNAc)(2) were close to the E and F subsites proposed on the basis of model building by Phillips and his colleagues. In MOL2, only the (GlcNAc)(4) moiety could be found in the A, B, C and D subsites. Significant shifts of the backbone atoms were observed in the region of residues 102 to 120, Which composed one side of the wall of the active site cleft. Consequently, the active cleft, with respect to the saccharide binding sites A, B and C, is narrower in both protein molecules. The residues 109 to 111 in site D of MOL1 are moved toward saccharide residue D, whereas those of MOL2 are only slightly shifted. In spite of these facts, the saccharide residues in site MOL1 and MOL2 are moved inside of the cleft. The distribution of water molecules and the hydrogen bond network in site D differ between the structures of MOL1 and MOL2. These structural changes in the active site cleft may be responsible for accommodating the substrate and releasing the products of hydrolysis. These results suggest that the three-dimensional structures of MOL1 and MOL2 remain in intermediate states between a transition state and an enzyme/product complex state.
• CONTRIBUTION TO ANTIBODY-ANTIGEN INTERACTION OF STRUCTURALLY PERTURBED ANTIGENIC RESIDUES UPON ANTIBODY-BINDING

  K TSUMOTO, Y UEDA, K MAENAKA, K WATANABE, K OGASAHARA, K YUTANI, KUMAGAI, I JOURNAL OF BIOLOGICAL CHEMISTRY 269 (46) 28777 -28782 1994年11月 [査読無し][通常論文]
   

  For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E. W., Sheriff, S., Cohen, G. Il., Smith-Gill, S. J., and Davies, D. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5938-5942), was investigated. Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry. The results can be summarized as follows. 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli. Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant. 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss. 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex. 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute 60 the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction.
• CONTRIBUTION TO ANTIBODY-ANTIGEN INTERACTION OF STRUCTURALLY PERTURBED ANTIGENIC RESIDUES UPON ANTIBODY-BINDING

  K TSUMOTO, Y UEDA, K MAENAKA, K WATANABE, K OGASAHARA, K YUTANI, KUMAGAI, I JOURNAL OF BIOLOGICAL CHEMISTRY 269 (46) 28777 -28782 1994年11月 [査読無し][通常論文]
   

  For elucidating the contribution of structurally perturbed antigenic residues upon antibody binding to antigen-antibody interaction, the interaction between hen egg white lysozyme (HEL) and HyHEL10 Fv fragment, which is one of several monoclonal antibodies against HEL and structurally well defined (Padlan, E.A., Silverton, E. W., Sheriff, S., Cohen, G. Il., Smith-Gill, S. J., and Davies, D. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 5938-5942), was investigated. Asp-101 and Trp-62 of HEL, whose conformations are perturbed by the binding of antibody HyHEL10 in this interaction, were replaced with Gly, and the resulting interactions were studied by assay of the inhibition of the lysozyme activity with the Fv fragment and by titration calorimetry. The results can be summarized as follows. 1) It was possible to prepare the fully functional Fv fragment of HyHEL10 using a secretory expression system in Escherichia coli. Its inhibition profile for HEL activity was almost indistinguishable from that of HyHEL10 IgG, and the contribution of enthalpy to driving the interaction was shown to be significant. 2) A thermodynamic study of the interaction between the D101G mutant HEL and the Fv fragment revealed that, although the negative enthalpy change was smaller than that for the wild type, the Gibbs energy was almost identical to that of the wild type, which resulted from the smaller entropy loss. 3) Study of the interaction between the W62G mutant HEL and this Fv fragment indicated that the rotation of the Trp-62 indole ring upon binding of the antibody made an enthalpic contribution to antibody-antigen interaction, although Trp-62 of HEL was proposed not to be the direct contact residue in the HyHEL10.HEL complex. 4) From these results, it was confirmed experimentally that structural perturbations of antigenic residues upon antibody binding of antigen would contribute 60 the gain of enthalpic energy, in spite of partial offset by entropic loss, and to driving the interaction.
• ニワトリ卵白リゾチームの繊維状ファージ表面への提示

  前仲勝実, 津本浩平, 上田能孝, 渡辺公綱, 熊谷泉 日本分子生物学会年会プログラム・講演要旨集 17th 394 1994年11月 [査読無し][通常論文]
  
• ニワトリ卵白リゾチームTrp62改変体の基質類似体との複合体のX線結晶解析

  前仲勝実, 松島正明, 宋海衛, 渡辺公綱, 熊谷泉 日本蛋白工学会年会プログラム・要旨集 6th 10 1994年05月 [査読無し][通常論文]
  
• FUNCTIONAL AND STRUCTURAL ROLE OF A TRYPTOPHAN GENERALLY OBSERVED IN PROTEIN-CARBOHYDRATE INTERACTION - TRP-62 OF HEN EGG-WHITE LYSOZYME

  K MAENAKA, G KAWAI, K WATANABE, F SUNADA, KUMAGAI, I JOURNAL OF BIOLOGICAL CHEMISTRY 269 (10) 7070 -7075 1994年03月 [査読無し][通常論文]
   

  In order to elucidate the role of the aromatic ring in recognition of the sugar ring, Trp-62 of hen egg white lysozyme, which is proposed on the basis of x-ray crystallography data to make contact with a sugar ring through van der Waals interaction, was replaced with aliphatic amino acids (Leu, Ile, Val, and Ala) and Gly by site-directed mutagenesis. In spite of the loss of the aromatic effect, these mutant lysozymes, except for the Trp-62 --> Gly mutant, showed higher bacteriolytic activity than the wild-type lysozyme. Furthermore, the Trp-62 --> Gly mutant still retained appreciable bacteriolytic activity. On the other hand, by these replacements, the enzymatic activities toward non-charged substrates were markedly reduced. Additionally, the side-chain structure of position 62 was found to be largely responsible for recognition of a saccharide ring in its active site cleft. NMR analysis of the Trp-62 --> Leu and Trp-62 -->, Gly mutants indicated that the structural effects of Trp-62 replacements were localized in the loop region around position 62 and the part of the beta-sheet containing the hydrogen bonding network important for enzymatic activity. Thus, we conclude that Trp-62 not only interacts with oligosaccharide through van der Waals contact, but also maintains the local structural conformation to produce the lysozyme-oligosaccharide interaction.
• FUNCTIONAL AND STRUCTURAL ROLE OF A TRYPTOPHAN GENERALLY OBSERVED IN PROTEIN-CARBOHYDRATE INTERACTION - TRP-62 OF HEN EGG-WHITE LYSOZYME

  K MAENAKA, G KAWAI, K WATANABE, F SUNADA, KUMAGAI, I JOURNAL OF BIOLOGICAL CHEMISTRY 269 (10) 7070 -7075 1994年03月 [査読無し][通常論文]
   

  In order to elucidate the role of the aromatic ring in recognition of the sugar ring, Trp-62 of hen egg white lysozyme, which is proposed on the basis of x-ray crystallography data to make contact with a sugar ring through van der Waals interaction, was replaced with aliphatic amino acids (Leu, Ile, Val, and Ala) and Gly by site-directed mutagenesis. In spite of the loss of the aromatic effect, these mutant lysozymes, except for the Trp-62 --> Gly mutant, showed higher bacteriolytic activity than the wild-type lysozyme. Furthermore, the Trp-62 --> Gly mutant still retained appreciable bacteriolytic activity. On the other hand, by these replacements, the enzymatic activities toward non-charged substrates were markedly reduced. Additionally, the side-chain structure of position 62 was found to be largely responsible for recognition of a saccharide ring in its active site cleft. NMR analysis of the Trp-62 --> Leu and Trp-62 -->, Gly mutants indicated that the structural effects of Trp-62 replacements were localized in the loop region around position 62 and the part of the beta-sheet containing the hydrogen bonding network important for enzymatic activity. Thus, we conclude that Trp-62 not only interacts with oligosaccharide through van der Waals contact, but also maintains the local structural conformation to produce the lysozyme-oligosaccharide interaction.
• 回折データ収集法の進展 : より速く,より美しく : a)実験室系(<特集>タンパク質結晶学の新時代 : 1.タンパク質結晶学方法論の現状と将来)

  松島 正明, 熊谷 泉, 前仲 勝実 生物物理 33 (6) 311 -313 1993年11月25日 [査読無し][通常論文]
   

  記事分類: 物理学--分子・物性--結晶
• 実験室レベルの高精度X線結晶構造解析-回折データ収集法の進展-より美しく-

  松島 正明, 熊谷 泉, 前仲 勝実 生物物理 33, 311-313 (6) 7 -9 1993年11月01日 [査読無し][通常論文]
  
• タンパク質工学に基づくc型リゾチームの機能解析

  前仲 勝実, 熊谷 泉 生物物理 33 (4) 219 -224 1993年07月25日 [査読無し][通常論文]
   

  Protein engineering studies of the recombinant c-type lysozymes have provided incisive probes into the roles of individual amino acid residues in the enzymes. The amino acid replacements in the active site cleft of the lysozymes by site-directed mutagenesis and functional analyses of these mutant enzymes were summerized and discussed.
• EFFECTS OF SUBSITE ALTERATIONS ON SUBSTRATE-BINDING MODE IN THE ACTIVE-SITE OF HEN EGG-WHITE LYSOZYME

  KUMAGAI, I, K MAENAKA, F SUNADA, S TAKEDA, K MIURA EUROPEAN JOURNAL OF BIOCHEMISTRY 212 (1) 151 -156 1993年02月 [査読無し][通常論文]
   

  The subsite structures in the active site of hen egg-white lysozyme were altered by site-directed mutagenesis. Replacement of Trp62, which is involved in apolar interaction with a sugar ring, and Asp101, which is hydrogen bonded to the same sugar ring in subsite B, led to a shift of the oligosaccharide-binding mode in the active-site cleft. Consequently, the double-mutant lysozyme (Trp62His, Asp101Gly) exhibited a drastic change of substrate-binding without any significant loss of enzymic activity. Conversion of Asn37, which is postulated to be involved in interaction with a sugar ring in subsite F, had a reverse effect on substrate binding. Nuclear magnetic resonance analysis of mutant lysozymes, in which Trp62 was replaced with Phe or His, suggested that these replacements not only altered the structure of the amino acid side chain at position 62 of the lysozyme, but also induced local structural changes around the residue at position 62.
• Effects of subsite alterations on substrate‐binding mode in the active site of hen egg‐white lysozyme

  Izumi KUMAGAI, Katsumi MAENAKA, Futoshi SUNADA, Shigeki TAKEDA, Kin‐ichiro MIURA European Journal of Biochemistry 212 (1) 151 -156 1993年 [査読無し][通常論文]
   

  The subsite structures in the active site of hen egg‐white lysozyme were altered by site‐directed mutagenesis. Replacement of Trp62, which is involved in apolar interaction with a sugar ring, and Asp101, which is hydrogen bonded to the same sugar ring in subsite B, led to a shift of the oligosaccharide‐binding mode in the active‐site cleft. Consequently, the double‐mutant lysozyme (Trp62His, Asp101Gly) exhibited a drastic change of substrate‐binding without any significant loss of enzymic activity. Conversion of Asn37, which is postulated to be involved in interaction with a sugar ring in subsite F, had a reverse effect on substrate binding. Nuclear magnetic resonance analysis of mutant lysozymes, in which Trp62 was replaced with Phe or His, suggested that these replacements not only altered the structure of the amino acid side chain at position 62 of the lysozyme, but also induced local structural changes around the residue at position 62. Copyright © 1993, Wiley Blackwell. All rights reserved
• REDESIGNING OF OLIGOSACCHARIDE BINDING-SITES OF HEN EGG-WHITE LYSOZYME AND A HOMOLOGOUS PROTEIN, ALPHA-LACTALBUMIN

  KUMAGAI, I, K MAENAKA, H UCHIYAMA, K WATANABE, K MIURA PROTEIN ENGINEERING 6 99 -99 1993年 [査読無し][通常論文]
  
• Interaction between a recombinant Fv fragment and its antigen protein: Effect of structural conversions of the antigenic epitope

  Tsumoto K, Ueda Y, Maenaka K, Ogawawara K, Yutani K, Watanabe K, Kumagai I Protein Eng. 7 1010 1993年 [査読無し][通常論文]
  
• ニワトリリゾチームの基質結合部位の構造改変 機能変換とエピトーブ構造の解析

  熊谷泉, 前仲勝実, 上田能孝, 津本浩平, 河合剛太, 渡辺公綱, 三浦謹一郎 タンパク質構造討論会講演要旨集 43rd 73 -76 1992年09月 [査読無し][通常論文]
  
• ニワトリリゾチームTrp62改変体のNMRによる構造解析

  前仲勝実, 砂田太, 河合剛太, 渡辺公綱, 三浦謹一郎, 熊谷泉 生体分子の構造と機能に関する討論会講演要旨集 19th 18 -19 1992年07月 [査読無し][通常論文]
  

書籍等出版物

• 薬系免疫学

  植田, 正, 前仲, 勝実 
  南江堂 2022年12月 (ISBN: 9784524404117) x, 295p
• 創薬研究のための相互作用解析パーフェクト : 低中分子・抗体創薬におけるスクリーニング戦略と実例、in silico解析、一歩進んだ分析技術まで

  津本, 浩平, 前仲, 勝実 
  羊土社 2021年12月 (ISBN: 9784758122566) 365, 16p
• 薬系免疫学(改訂第3版)

  植田 正, 前仲 勝実 (担当:共編者(共編著者))
  南江堂 2018年01月 (ISBN: 4524403515) 278

  
• Advanced methods in structural biology

  千田, 俊哉, 前仲, 勝実 
  Springer 2016年06月 (ISBN: 9784431560289) x, 340 p.
• Essentialタンパク質科学

  Williamson, Michael P., 津本, 浩平, 植田, 正, 前仲, 勝実 (担当:共訳)
  南江堂 2016年02月 (ISBN: 9784524268641) xvi, 451p

  
• 薬系免疫学

  植田, 正, 前仲, 勝実 (担当:共著)
  南江堂 2012年09月 (ISBN: 9784524402960) x, 258p

  
• 薬系免疫学

  植田, 正, 前仲, 勝実 (担当:共編者(共編著者))
  南江堂 2007年10月 (ISBN: 9784524402465) ix, 238p

  
• Molecular recognition by Ig-like receptors, KIRs and Fc-gammma-Rs. In Activating and Inhibitory Immunoglobulin-Like Receptors (Eds. M.D.Cooper, T.Takai and J.V.Ravetch)

  Spring-Verlag Tokyo,Inc. 2001年
• Molecular recognition by Ig-like receptors, KIRs and Fc-gammma-Rs. In Activating and Inhibitory Immunoglobulin-Like Receptors

  Eds. M.D.Cooper, T.Takai and J.V.Ravetch 2001年
• リゾチーム-酵素および抗原のモデルタンパク質として-

  シリーズ分子生物学6 構造生物学(三浦謹一郎編)

担当経験のある科目（授業）

応用生物統計学 創薬発見科学北海道大学
創薬科学特別講義Ⅱ（バイオキャンプ）北海道大学
創薬科学特別講義Ⅰ（ライフイノベーションセミナー）北海道大学
バイオ医薬学基礎特論北海道大学
先端創薬学特論北海道大学
医療薬学特論北海道大学
物理化学II北海道大学
物理化学I北海道大学

所属学協会

• 日本薬学会   日本ウイルス学会   日本結晶学会   日本ケミカルバイオロジー学会   日本分子生物学会   日本生化学会   日本蛋白質科学会   日本免疫学会   日本生物物理学会   The Molecular Biology Society of Japan   The Japanese Biochemical Society   The Japanese Protein Society   Japanese Society for Biophysics   

Works（作品等）

• CD85レセプター群の分子認識

  2000年 -2005年
• Molecular recognition of CD85 receptors

  2000年 -2005年
• Fcレセプター群の分子認識

  1998年 -2005年
• Melcular recongnition of Fc receptors

  1998年 -2005年

共同研究・競争的資金等の研究課題

• マテリアル・シンバイオシスのための生命物理化学

  日本学術振興会：科学研究費助成事業 学術変革領域研究(A)

  研究期間 : 2020年11月 -2025年03月 

  代表者 : 山吉 麻子, 前仲 勝実, 荏原 充宏, 望月 慎一, 長谷 耕二, 大場 雄介, 白石 貢一, 植畑 拓也, 森 健, 山本 剛史, 天野 麻穂

   

  本領域は、免疫反応などの生体応答を物理化学的視点から捉え直し、次世代の生体と物質の共生（マテリアル・シンバイオシス）を目指して新たな学術的変革を推進することを目的としている。本年度は初年度であるため、領域推進の骨格分を構築することに尽力した。 （1）採択後間もない令和３年１月から公募研究の募集が始まることに向け、領域推進に必要な人材の応募を促すべく、適宜、総括班会議を設けることで、公募要領内容を議論し決定した。マテリアル・シンバオシスに関する研究は黎明期にあり、未知の研究対象が多く残されている。そこで、物質と生体分子に関する新たな相互作用の解明に繋がるポテンシャルを持った野心的な課題を、公募班として積極的に取り入れることが可能な領域体制の構築について重点的に議論した。 （2）計画班の研究内容を総括班内で共有し、領域全体の研究進捗の把握と研究協力・交流を図った。今後の方向性を詳細に議論し、明確かつ具体的な戦略目標を設定した。計画研究者による運営会議によって、領域全体の活動状況を把握し、国内外における関連分野の学術状況も鑑みながら、研究ビジョンに沿った運営方針を策定した。研究開始初年度は、計画班内の研究リソースの共有と連携を一層深める活動に重点を置いて実行した。 （3）計画班、公募班の共同研究を推進するためのAFMイメージング支援、マテリアル合成支援などの技術支援に必要な機材等の整備を行った。 （4）本領域の内容を全国の研究者に幅広く知って頂くため、キックオフシンポジウムを開催した。また、科学研究の成果を社会へ還元するため、ホームページを開設し、国内外問わず広く成果を公開した。また、公開シンポジウムやニュースレターなどの企画策定を行った。
• 細胞表面蛋白質の弱い分子認識の定量化・構造解析

  日本学術振興会：科学研究費助成事業 学術変革領域研究(A)

  研究期間 : 2020年11月 -2025年03月 

  代表者 : 前仲 勝実, 望月 慎一
• 高機能ゲルによるがん幹細胞リプログラミングと治療薬開発基盤の創出

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2019年04月 -2024年03月 

  代表者 : 田中 伸哉, 津田 真寿美, 高阪 真路, 前仲 勝実, 黒川 孝幸

   

  （研究の骨格）本研究は、研究代表者が高機能ポリマーハイドロゲルを用いることで、極めて短時間にがん細胞のリプログラミングを誘導して、がん幹細胞を同定する方法を見出したことにはじまる。これは従来のがん幹細胞分離同定法とは異なり、がんの種類を問わず24時間以内にがん幹細胞を同定することができる画期的な方法であり、本研究では、この高機能ゲルのどのような物理学的因子ががん細胞の遺伝子発現変化を短時間で誘導するのかについて検討し、高機能ゲルを基盤としたがん幹細胞診断法を開発し、さらにがん幹細胞標的治療薬を大規模スクリーニングにより創出するものである。 （具体的な実績）申請書においてR2年度は２つの目標を設定した。1番目は「がん細胞リプログラミングを誘導する物理的因子の解明と臓器別人工がん幹細胞ニッシェの開発」である。研究代表者らは高機能ゲルの中でも、ダブルネットワークゲル（DNゲル）の構成成分であるpoly-2-acrylamido- 2-methylpropanesulfonic acid (PAMPS) ゲルに着目し、弾性率や荷電状態を変化させることで最も効率的にがん幹細胞性を誘導させ得る条件を明らかにした（論文作成中）。また、２つ目の目標である「高機能ゲルによるエピジェネティカルなゲノム制御機構（メカノメモリー）の探索」は、poly N-(carboxymethl)-N, N-dimethyl-2-(methacryloyloxy) ethanaminium (PCDME)ゲル上で培養したがん細胞を通常培養皿に移して一定時間培養すると幹細胞性が亢進したことから、PCDMEゲルによるがん細胞のリプログラミングの過程でエピゲノムの変化が誘導されることが明らかとなった。
• HLA分子を介した免疫系を撹乱する薬物の新規同定法の開発

  日本学術振興会：科学研究費助成事業 挑戦的研究(萌芽)

  研究期間 : 2018年06月 -2020年03月 

  代表者 : 前仲 勝実

   

  人類共通のHLA-Gを例として、代表者等が構築したHLAタンパク質を用いたスクリーニング法により実施した既存薬スクリーニングの結果を受けて、疾患との関連が予想できる結合候補薬剤に着目した。この結合候補薬剤についてin vitroで調製したHLA-Gとの複合体を用いた質量分析法による解析により、候補薬剤とHLA-Gタンパク質との結合を実証することに成功した。 他方、薬剤過敏症と関連するHLAと薬剤の結合や提示されるペプチドパターンの変化が分子レベルで明らかにされている抗HIV薬・アバカビルとHLA-B*57:01多型について、我々のスクリーニング法を適用したところ、両者の結合を示唆する成果を得た。
• 終末糖化産物の構造を識別する機能性一本鎖抗体を用いた高感度画像解析法の開発

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2016年04月 -2018年03月 

  代表者 : 森岡 弘志, 小橋川 敬博, 佐藤 卓史, 前仲 勝実, 丸山 徹, 福田 夏希

   

  終末糖化産物（AGE）を認識する一本鎖抗体（scFv）融合タンパク質を作製し、生体内に局在するAGEをin vivoイメージングする方法の開発研究を行った。ファージディスプレイ法によって、抗原結合性ならびに熱安定性の高いscFv抗体を作製した。次にscFvの血中滞留性を改善するために、Sortase Aを用いてヒト血清アルブミンとの融合分子を作製し、血中滞留性の向上が確認できた。scFv抗体にランタノイド蛍光ペプチド配列を結合させ、時間分解蛍光測定が可能なランタノイドイオンの導入を試みたが、蛍光は観測されなかった。現在、クリック反応を用いて蛍光プローブの導入を進めている。
• 毒ヘビ神経毒を標的とした咬傷治療薬創製の基盤研究

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2016年04月 -2018年03月 

  代表者 : 塩井 成留実, 前仲 勝実, 倉原 琳, 胡 耀鵬, 平石 敬三, 平野 亨

   

  本研究では、毒ヘビ血液由来毒素阻害タンパク質群の標的毒素の認識機構および阻害機構を明らかにすることを目的としている。主な成果は、以下に示す3つである。 （１）ハブ毒中のイオンチャネルブロッカーの標的イオンチャネルを同定し、毒ヘビ血清タンパク質がその毒素の生理活性を阻害することを明らかにした。（２）ファージディスプレイ法および化学合成法を用いて、阻害タンパク質の毒素結合領域のペプチド合成を行った。（３）数種のヘビ粗毒を用いて、ヘビ血液成分との結合毒素を同定した。
• ジステンパーウイルスの細胞侵入機構の分子基盤と感染防御

  日本学術振興会：科学研究費助成事業 基盤研究(A)

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 前仲 勝実, 福原 秀雄

   

  犬ジステンパーウイルス（CDV）の膜融合タンパク質（CDV-F）について、クライオ電子顕微鏡による観察を実施した。クライオ電子顕微鏡に特有の試料調製条件およびグリッド作製条件の検討を行なったところ、より高分解能で解析できる目処がついた。一方、同ウイルスの受容体結合タンパク質（CDV-H）については、ラットを用いて抗体の作製を進め、モノクローナル抗体を産生するハイブリドーマを複数樹立した。得られた抗体のサブタイプやCDV-Hへの結合を確認したほか、膜融合活性を指標とした解析から、各抗体により結合親和性や特異性が異なることがわかった。これらにより、CDVワクチンの有効性の分子基盤を解明した。
• 脂質センサー型ITAM受容体による抗酸菌特異的脂質の認識と結核病態の制御

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 原 博満, 山崎 晶, 松崎 吾郎, 清原 秀康, 飯笹 英一, 前仲 勝実, 宮崎 敦

   

  抗酸菌細胞壁の脂質成分は免疫を賦活化あるいは抑制する活性を有する。我々は、抗酸菌の免疫制御性脂質として知られるミコール酸（MA）およびフェノール糖脂質（PGL）を認識するDAP12会合型のITAM共役受容体を同定した。これらの受容体の欠損はこれら脂質に対するMφの応答をほぼ完全に消失させた。また、MA受容体の活性化は抗酸菌増殖に許容的なMφの動員を促すことが判った。MA受容体欠損マウスは、C型レクチンMincleを介した炎症応答が促進され、BCG菌の排除が促進されることが判った。従って、免疫制御性の抗酸菌脂質を認識するDAP12会合型受容体が、菌の免疫回避に関わる可能性が示唆された。
• ジステンパーウイルスの細胞侵入機構の分子基盤と感染防御

  文部科学省：科学研究費補助金（基盤A）

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 前仲 勝実
• 細胞シグナリング複合体によるシグナル検知・伝達・応答の構造的基礎

  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 : 2010年04月 -2016年03月 

  代表者 : 箱嶋 敏雄, 前仲 勝実, 山下 栄樹, 佐藤 主税, 深井 周也, 千田 俊哉, 稲垣 冬彦, 三島 正規, 月原 冨武, 甲斐莊 正恒, 吉田 賢右, 田中 啓二, 貝淵 弘三, 磯貝 彰

   

  平成22年度に発足した新学術領域研究「動物・植物細胞のシグナル検知と伝達の構造生物学」では、平成23 年と25 年には公募研究を加えて、種々の手法による構造生物学、タンパク質化学、生化学、細胞生物学の研究者の相互協力により、重要な細胞機能の制御に関わるシグナル伝達経路で形成される複合体（細胞シグナリング複合体）の三次元構造決定を通して、相互作用の特異性と分子機能の制御機構を原子分解能で解明してきた。本終了研究では、総括班が中心となって領域の研究成果をとりまとめるとともに、本領域の到達点を明確にすることで、それに立脚した更なる発展・展開の方向性を関連研究者と共有して、我が国の学術に寄与すると考えた。具体的には、成果を取りまとめた成果報告書の作成を行うとともに、領域の成果を外部に公表するシンポジウム等を学会との共催も視野に入れて開催した。 ①成果報告書の作成:本領域によって得られた研究成果、および学会・シンポジウムの開催記録、若手育成のための取り組み、更にはニュースレター（全53報）の抜粋をまとめた成果報告書を作成して、製本した（337ページ）。また、シンポジウムの内容をまとめたものを作成する。 ②事後評価の取りまとめの準備:領域の事後評価の準備をして、ヒヤリングを受けた。 ③シンポジウムの開催:本新学術領域研究において新たに得られた成果を広く社会に公表することを目的として、本領域の研究者が企画したシンポジウム「構造分子生物学・生化学の進展」（オーガナイザー：箱嶋敏雄・奈良先端大教授，前仲勝実・北大教授）をBMB2015（12月3日神戸ポートピアホテル）で開催して，今後の構造細胞生物学の展開について活発な議論がなされた。
• 生体防御に関わる細胞表面受容体のシグナル検知機構の解析

  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 : 2010年04月 -2015年03月 

  代表者 : 前仲 勝実

   

  生体防御の最前線にあたる細胞表面の受容体とガン細胞や病原体などの非自己抗原との相互認識を構造生物学的に原子レベルで可視化することは医学的に重要な免疫応答や感染経路の作用機序を理解するために必須である。本研究では、免疫疾患・感染症に重要なシグナル制御複合体として、T細胞の共抑制分子CD160/HVEMシグナル制御複合体、ヒトTh17細胞に発現するNKR-P1/CD161受容体とリガンドLLT1との複合体、麻疹ウイルス表面蛋白質侵入複合体、糖脂質認識免疫受容体MincleとMCL等の複雑な構成となる複合体の立体構造と物理化学的視点から特徴を抽出し、構造分子医学研究を推進した。
• HIV変異ペプチドによる免疫逃避機構の構造基盤

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2010年 -2012年 

  代表者 : 前仲 勝実

   

  ウイルス感染を制御する受容体は、主要組織適合性抗原(MHC)を認識するNK細胞受容体KIR群やLILR群である。本研究ではHIV変異ペプチドを提示したMHCとLILR群との複合体の低分解能結晶構造解析の結果、ペプチドの変異によるLILR結合活性への影響は低いように思われ、結合実験の結果はこれを支持した。他方、KIRについて、異なるペプチドの結合が変化する構造基盤が明らかとなってきた。
• 免疫系細胞表面レセプター群の分子認識

  科学研究費補助金

  研究期間 : 1997年 -2010年
• Molecular recongnition of cell surface receptors in immune system

  Grant-in-Aid for Scientific Research

  研究期間 : 1997年 -2010年
• 非古典的MHC細胞表面抗原Millの立体構造解析

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2008年 -2009年 

  代表者 : 前仲 勝実

   

  細胞表面抗原である主要組織適合性抗原(MHC)の多様なメンバーのうち、特殊な生物学的機能を有するものが非古典的MHCと呼ばれる一群である。最近、見出された新規の非古典的MHCクラスI様分子のMILLファミリー(MILL1およびMILL2)は以下の性質を有することがわかった:(1)ペプチド提示能を有さない、(2)GPIアンカー型分子である、(3)軽鎖(β2ミクログロブリン)に結合する、(4)MILL1は胸腺髄質上皮細胞に発現し、MILL2は免疫系以外のかなり多くの細胞に発現する。しかし、MILLの生理機能の解明は不十分であり、立体構造も決定されていない。そこで、私たちはMILLの立体構造を明らかにすることで、MILLの生理機能を解明することを目指している。昨年度から引き続き、大腸菌発現と巻き戻しにより調製したMILL2の細胞外ドメインの結晶を用いて2.2Åの高分解能データにより構造解析を行った。重原子同型置換法により位相決定後、モデル構築を行った結果、通常のMHCでは見られない大きなドメイン間の構造の柔軟性が観測された。しかし、興味深いことに、β2ミクログロブリンとの会合状態は通常のMHCで見られる配向を維持していた。これらの構造的特徴を踏まえて、MILL特異的レセプターの結合部位を予測した。さらに、MILLと結合することを確認できた細胞群を用いて、先の予測に基づく変異体解析を行った。
• NK細胞受容体を介したHIV由来ペプチドによる免疫制御と構造基盤

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2008年 -2009年 

  代表者 : 前仲 勝実

   

  ウイルス感染の主たる防御は細胞傷害性T細胞(CTL)やナチュラルキラー(NK)細胞である。これらの細胞が最終的に機能しなくなった時点から、重篤な疾患の発症が起きる。主要組織適合性抗原(MHC)を認識するヒトNK細胞受容体Killer cell Ig-like receptor(KIR)群はNK細胞だけでなく、CTLにも発現し、特に抑制型KIR群はこれらの細胞の不活性化に関与すると考えられている。昨年度に引き続き、HIV-1 gp120由来ペプチドの変異が感染者由来のCTLの反応を落とし、かつ抑制型KIR2DL1との結合増強によるNK細胞の不活性化を行う2重免疫逃避機構の分子基盤を明らかにすることを目指した。大腸菌発現と巻き戻しにより調製したKIR2DL1と、HIV由来ペプチドと会合したHLA-Cw4との複合体の結晶を用いて2.5Åの高分解能データを用いて、X線結晶構造解析による構造決定を行った。その結果、HIVペプチドのC末端側の変異アミノ酸部位にある側鎖がHLA-Cw4の狭い溝に収まっており、その変異はペプチド全体の配向に影響を及ぼす可能性が示唆された。おそらくペプチドの配置の変化によるKIR群との結合に影響が出たものと考えられる。他方、HIVゲノム情報に基づくペプチドライブラリーからのHLA-Cw4結合ペプチドを同定し、全てのペプチドについて表面プラズモン共鳴法を用いた相互作用解析によりKIR群との結合を測定した。
• 多様な細胞表面抗原を認識するペア型免疫系受容体群

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2007年 -2009年 

  代表者 : 前仲 勝実

   

  細胞表面抗原は細胞の状況に合わせた多様な分子形態が存在し、その構造的特徴をペア型受容体群が認識することにより免疫応答が調整されている。本課題では代表的なペア型受容体群であるPILRおよびKLRG1などについてリガンドとの相互作用解析、立体構造解析を行った。その結果、リガンドの形態として、PILRについては糖とペプチド部分が、KLRG1では2量体化が制御に関わることがわかり、これらの免疫応答の人為的制御に向けた基盤を明らかに出来た。
• ミトコンドリア輸入装置と前駆体タンパク質の間のソフトな相互作用に基づく分子認識

  文部科学省：科学研究費補助金(特定領域研究)

  研究期間 : 2003年 -2008年 

  代表者 : 神田 大輔, 前仲 勝実, 斉藤 貴士

   

  ミトコンドリア内部へ輸送されるタンパク質はN末端にプレ配列が付加されて生合成される.Tom20タンパク質はミトコンドリア外膜にあって,プレ配列を最初に認識する受容体である.Tom20とプレ配列の複合体を共有結合で安定化する技術を新規に考案して,結晶構造解析とNMR緩和時間解析を行った.複数の結合状態が存在するが,それぞれは認識としては不完全である.この複数の状態の間の速い動的平衡が,Tom20が多様なプレ配列を認識するメカニズムであることを提唱した.
• 細胞表面抗原HLA-B27ホモ2量体と免疫抑制レセプターの複合体の立体構造解析

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2006年 -2007年 

  代表者 : 前仲 勝実

   

  細胞表面は全ての外界との最初の接点である。ヒトを感染症やガンから守る免疫系においても細胞表面のレセプター群が異物(非自己)認識の最前線にあたる。そのため、免疫系の機能制御には細胞表面レセプター群のリガンド分子認識機構の理解が欠かせない。そこで、本申請では、難治リウマチ性自己免疫疾患である強直性脊椎炎(ankylosing spondylitis、以下ASと略す)の原因遺伝子である細胞表面抗原HLA-B27(主要組織適合性抗原(MHC)の一つ)によるAS発症の分子機構を明らかにする。具体的には、疾患の進行に伴い、細胞表面に発現する軽鎖(β2m)欠損HLA-B27ホモダイマーについて、免疫細胞表面抑制レセプターであるLeukocyte Ig-like receptor(LILR)群との分子認識を相互作用解析と立体構造解析により明らかにする。 昨年度に引き続き、本年度は、X線結晶構造解析を目指して、β2m欠損HLA-B27のホモダイマーを大腸菌での封入体発現と巻き戻しにより安定なサンプルの調製を目指して複数のコンストラクトを作成したが、蛋白質の分解を抑制することができなかった。他方、NMRによる相互作用解析に向けて、β2m欠損HLA-B27のホモダイマーに結合するLILRB2を^<15>Nラベル体で作成したところ、HSQCスペクトルで充分に分離したシグナルを得ることができた。現在、アミノ酸の主鎖の帰属の作業に取組んでいる。帰属ができれば、β2m欠損HLA-B27のホモダイマーとHLA-B27の通常型で認識様式の差異を明らかにすることができる。
• セレノシステイン特異的伸長因子SelBの結晶構造解析

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2005年 -2006年 

  代表者 : 前仲 勝実

   

  セレノシステインSecは"21番目のアミノ酸"と呼ばれ、遺伝子上に巧みにコードされた特殊なアミノ酸である。mRNA上の通常終止コドンであるUGAが引き続く特殊な2次構造をもつRNA配列(SECIS)が存在するとき、UGAコドンがSecの遺伝子コードに変身し、蛋白質中に取り込まれる。その際に特殊な伸長因子SelBが必要となる。SelBは通常の伸長因子EF-Tuと異なり、セレノシステイン特異的tRNAを結合するEF-Tuに相同性の高いN末端ドメインと、SECISRNAを認識する特別なC末端ドメインを持つ。これまでに我々は4つのwinged helix(WH)様構造を有するC末端ドメインのうちmRNA結合最小ドメイン(WH3-WH4,512-634)とRNAとの複合体の結晶構造解析に成功し、新規のRNA認識機構を明らかにした。本研究では、引き続きC末端ドメイン全長の動的な構造変化をX線結晶構造解析により明らかにすることを目指す。 本年度は、昨年度得られたM.thermoacetica SelB C末端ドメイン全長(SelB-C、377-634)とSECIS mRNAヘアピンとの複合体の結晶からSpring8にて得られた回折データを用いて、分子置換法により構造決定を行うことに成功した。その結果、これまでのSelB-SECIS RNA相互作用以外に、予想外のRNA結合様式が存在することがわかった。これは、SelB-Cの4つのWHドメインうちWH3-4とWH2の間がRNA結合に伴い、正電荷に偏った表面構造を作り出すことにより形成されることがわかった。また、この部分ではRNAのリン酸骨格のみが認識され、塩基特異性はないと考えられたことから、tRNAやrRNAの認識に重要な役割を果たす可能性を明らかにすることができた。同時にSelB全体の動的な構造変化とリボソームの間でのコミュニケーションを考察することができた。
• ペア型レセプターPILRの結晶構造解析

  日本学術振興会：科学研究費助成事業 特定領域研究

  研究期間 : 2005年 -2006年 

  代表者 : 前仲 勝実

   

  本年度は多様な免疫監視の制御に関わるペア型レセプターとして、CD99様分子を認識するPILR(Paired type2 Ig-like receptor)とMHCクラスI分子を認識するLILR(Leukocyte Ig-like receptor、別名ILT/LIR/CD85)について、リガンド分子認識機構の構造基盤を相互作用解析と立体構造解析(NMR、X線結晶構造解析)により明らかにすることに取り組んだ。昨年度おおよそ組み上げた大腸菌を用いた発現と巻き戻しにより、ヒト及びマウス由来のPILR群の、細胞外ドメイン全長と免疫グロブリンフォールドVsetドメインのみの2種類を調製した。また、マウス抑制型PILRαおよび活性型PILRβについて、リガンドPILR-Lとの結合実験を表面プラズモン共鳴により行い、特異的結合を示した(Tabata et al. PILRの発現及び機能解析についての論文投稿準備中)。他方、ヒトの抑制型PILRαのVsetについて結晶化に成功し、セレノメチオニン誘導体を用いた結晶から2Åの高分解能データのデータ収集に成功し、多波長異常分散(MAD)法による構造決定を現在進めている。他方、LILR受容体についてはLILRB2とHLA-Gとの複合体の結晶構造解析に成功し、上述の機能的な特徴の構造基盤を明らかにすることができた(Shiroishi et al., PNAS2006)。HLA-Gの特殊な2量体型に注目し、その機能および構造の特徴を明らかにした(Shiroishi et al., JBC2006a, BBA2006)。機能解析からavidity効果により、通常の単量体型よりも強く結合し、効率良く細胞内へのシグナル伝達を行えることがわかった。胎盤内での生理的意義が大きい可能性が高い。
• 構造解析に基づくPXドメインのシグナル変換機能の解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2004年 -2006年 

  代表者 : 神田 大輔, 前仲 勝実

   

  タンパク質ドメインの立体構造と機能を明らかにすることは,「1つのタンパク質ドメインについての構造機能相関研究が多数のタンパク質の構造に関する情報や機能に関する示唆を一挙にもたらす」という意味において,非常に稔り多い研究分野である.たとえば,1990年代始めのSH2ドメインやSH3ドメインの構造とそれに基づいた機能研究が良い例である,本研究では,我々が世界で初めて立体構造決定に成功したPXドメインを対象に,SH3ドメインとの相互作用を網羅的に検出することを目指した.2つのドメインをそれぞれにもつタンパク質はゲノム中に多数存在するので,そのインパクトは非常に大きい. PXとSH3ドメインが相互作用する可能性は,本研究が開始される3年前の時点では,種々の状況証拠から考えて,極めてありそうな事であった.しかし,研究成果のなかで詳しく述べられているように,本研究において実験的に検出することはできなかったこと,あるいは論文などの報告が他グループから出てこないことから考えて,現在ではかなり否定的な状況である.これはPXドメインの機能について,否定的ではあるものの一つの事実を確定できたと考える. そこで,本研究では最後の1年間を使って,PXの新たなターゲットとして,PXAドメインを選定した,PXAドメインはアミノ酸配列だけから定義されたドメインであり,構造のみならず機能についても不明である.本研究ではPXAドメインの発現系の確立と,PXとPXAの間の相互作用の可能性を示す実験結果を得ることに成功した.PXとPXAの間の相互作用の生物学的な意義として,ドメイン間の相互作用が動的に変化して,PXドメインのマスキングが解消される結果,タンパク質が膜へ移行するメカニズムが想定される.
• NK細胞活性化レセプターCD160の分子認識解明

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2004年 -2005年 

  代表者 : 前仲 勝実

   

  CD160(BY55)レセプターは末梢血NK細胞(および細胞傷害性T細胞の一部)に発現し、NK細胞を活性化する。NK細胞活性化レセプター群の多くはそのリガンドが同定されていないが、最近CD160はホモ多量体を形成してMHCクラスI(MHCI)を認識することが細胞レベルで明らかにされた珍しいレセプターである。そこで、本研究ではCD160のMHCIに対する分子認識機構を機能(速度論及び熱力学)および立体構造解析(X線構造解析)の両面から分子レベルで明らかにすることにより、NK細胞の活性化機構を理解することを目的とする。 平成16年度に大腸菌の封入体からの巻き戻し系を用いて作製したCD160分子では、単量体しかえられないなど、生理的活性を有する状態にあるかどうか、不明な点が多くあった。また、このCD160多量体をゲル濾過、イオン交換クロマトグラフィーによって単一な多量体分子を単離することは困難であり、大腸菌の分泌発現系を用いてもCD160分子を発現することができなかった。そこで、本年度は、CD160分子の単一な多量体の大量調製のために、本来CD160分子が発現しているヒト細胞である293細胞を用いた分泌発現系を構築した。シグナル配列を発現ペクター由来のものを用いて発現させたところ、3量体と2量体の混合物が得られ、シグナル配列のプロセッシングに問題があることが明らかとなった。そこで、天然のCD160由来のシグナル配列に戻したところ、単一の蛋白質としてCD160分子が得られた。
• SH3ドメインに高親和性・特異的ペプチドの立体構造に基づいたライブラリデザイン

  日本学術振興会：科学研究費助成事業 萌芽研究

  研究期間 : 2004年 -2005年 

  代表者 : 神田 大輔, 前仲 勝実

   

  SH3ドメインはプロリンリッチ配列(PXXP配列)に結合するドメインである。市販のファージライブラリPh.D.-C7Cファージディスプレイペプチドライブラリキット(BioLabs社)を用いて,p67^蛋白質のSH3ドメインに結合するペプチドを選択した.p67^蛋白質のSH3ドメインをGST融合タンパク質として大腸菌を用いて発現・精製した.Glutathione coated96穴HS plateプレートを用いてGST部分でプラスチックに結合させ,方向が制御された状態の固相化をおこなった.また,溶液中での選択を行うためGST Magnet Agarose Beadsを用いてプルダウンを行った.特異的に吸着したファージを溶出し、大腸菌に感染させ、プラークを得た。このパンニング操作を3回繰り返した。結果はファージELISAを使って確認した.ファージライブラリPh.D.-C7Cペプチドライブラリキットは,7残基のランダムライブラリであり,両端のシステインがSS結合を形成して環化している.PXXPを含む配列が繰り返し得られた.得られた4つのアミノ酸配列をペプチド合成し,SS結合を作らせて環化した.質量分析とDTNBによるSH基の定量で確認した.^<15>Nラベルしたp67^蛋白質のSH3ドメインを調製した.p67^SH3はシステイン残基をセリンに変えた変異体であるが,これはリガンドが結合していない状態ではアンフォールドしているが,リガンドが結合するとフォールドする性質をもっている.4種のペプチドのうち1つがSH3ドメインをフォールドさせることができた.このペプチドはSS結合でコンホメーションが制限されているので,通常のポリプロリンヘリックス2型のコンホメーションで結合しているとは考えにくい.NMR測定をおこなって構造情報を収集した.
• ミトコンドリア外膜透過装置の立体構造と通過中のポリペプチド鎖のコンホメーション

  文部科学省：科学研究費補助金(特定領域研究)

  研究期間 : 2003年 -2004年 

  代表者 : 神田 大輔, 前仲 勝実

   

  ミトコンドリア外膜に存在する膜透過装置においてTom40タンパク質がチャネルを形成している。このチャネルのポアを通過して前駆体タンパク質が輸送される。立体構造はβバレルからなるポーリン型であると予想されている.大腸菌を用いて酵母のTom40全長を発現すると,インクルージョンボディとなった.6M尿素を用いて可溶化し,陽イオン交換カラムクロマトグラフィーを用いて精製を行い、大腸菌1L培養当たり60〜70mgの変成状態のTom40タンパク質を得ることができた。膜タンパク質の立体構造解析においては使用する界面活性剤の選択が鍵となる。そこで、我々はCD、NMR、電子顕微鏡・負染色、ブルーネイティブ-PAGE、界面活性剤存在下でのゲルろ過などを指標として27種類の界面活性剤のスクリーニングを行い、Tom40の巻き戻しに有効であると思われるものを5種類選択した(DDM,OG,SML,Brij35,C_<12>E_6).ついで,Tom40の巻き戻し方法を,希釈法,ゲルろ過に直接インジェクションする方法,ヒスタグを用いてカラムに固定しながら変性剤を取り除く3つの方法について検討した.その結果、ゲルろ過カラムによる巻き戻し法が最も有効であった.ボイドボリュームよりあとの位置のピークから,電子顕微鏡・負染色観察で穴が1つあいたリング状の粒子が確認された.また,CDおよびNMRでβシート構造が含まれ...
• 免疫系レセプターのファージ提示系による高機能化と立体構造解析による機構解明

  文部科学省：科学研究費補助金(奨励研究(A), 若手研究(B))

  研究期間 : 2001年 -2002年 

  代表者 : 前仲 勝実

   

  <背景と目的>免疫系細胞表面に発現する様々なレセプターは細胞間の情報伝達に関与し、様々な疾患に直接結びつくことから、医学的に大変重要である。申請者らは幅広い免疫細胞に見出されてきた免疫レセプター抑制性モチーフ(ITIM)を持つ抑制性免疫レセプタースーパーファミリー(Inhibitory-receptor superfamily,以下IRSと省略する)に着目した。このファミリーの多くは免疫グロブリン(Ig)様ドメインを持ちながら、多様なリガンドを認識するため、レセプターの分子認識を解析するには格好の標的であると考えられる。そこで本研究は、IRSレセプターの高機能化を目指して、ファージ提示系を用いて膨大なライブラリーからリガンドに対する高親和性を示すレセプターを選択し、さらに高親和性のメカニズムをX線結晶構造解析により明らかにすることを目的とした。<検討結果と考察>本年度はIRSファミリーであるKIRについて、ファージ提示系を持ちいた2量体レセプターファージの作製を行ってきたが、現在まで大腸菌の発現系を利用して作製したリガンドMHCとの結合の確認に成功していない。2量体蛋白質を作成し、実際の蛋白質レベルで結合の上昇が見られるかどうか今後確認する予定である。他方、IRSファミリーのFcγRファミリーについては、FcγRファージの作製に取り組んでいるが、現在までFcとの結合は確認でき...
• 免疫レセプター群の蛋白質間分子認識解明を目指したX線結晶構造解析データの収集

  文部科学省：科学研究費補助金(特定領域研究(C))

  研究期間 : 2000年 -2000年 

  代表者 : 前仲 勝実

   

  <背景と目的>ゲノム解析の進展により人間の病理に関わる多くの細胞表面レセプター群が見出されてくると、現在全体の約1/3を占めている免疫グロブリン(Ig)様ドメインを持つレセプターを統合的に解析することが重要になってくる。本研究では最近免疫系細胞に幅広く見出されてきた免疫レセプター抑制性モチーフ(ITIM)を細胞内ドメインにもつヒト抑制性免疫レセプタースーパーファミリー(Inhibitory-receptor superfamily,以下IRSと省略する)の多くがIg様ドメインを持ち、また多様なリガンドを認識するため、統合的な蛋白質間分子認識データを収集する格好の標的であることに着目した。我々はIRSの中から、主要組織適合性抗原(MHC)を認識するKiller cell Ig-like receptor(KIR)やIg-like transcript(ILT)、更には抗体Fc部位を認識するFcγRについてリガンド分子認識機構の機能解析及びX線結晶構造解析を行うことを目的とした。<検討結果と考察>本年度は、KIRのMHCに対する分子認識に関して2つのIg様ドメインを細胞外にもつKIR2Dのこれまでの解析を踏まえ、更に3つのIg様ドメインを持つKIR3DのリガンドMHC、HLA-B51の結晶構造解析及び活性型KIR2Dの機能解析を行った。決定したHLA-B51の立体構造から、KIR...
• ファージディスプレイ系の改良
• 免疫系レセプターに対する阻害剤開発
• Improvement of Phage display system
• Development of inhibitors for immunoreceptors

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• WO2019-177053:全身性エリテマトーデス又は全身性エリテマトーデスに起因して生じる疾患の予防又は治療剤及び膠原病の予防又は治療剤  2019年09月19日

  前仲 勝実, 黒木 喜美子, 前田 直良, 山田 千聖, ▲高▼橋 愛実  国立大学法人北海道大学  202103009615801818
• WO2019-177054:腫瘍予防又は治療剤、PD-L1抑制剤、腫瘍予防又は治療剤のスクリーニング方法及びPD-L1抑制剤のスクリーニング方法  2019年09月19日

  前仲 勝実, 黒木 喜美子, ▲高▼橋 愛実  国立大学法人北海道大学  202103010157614670
• WO2019-124461:抗体断片多量体分子、医薬品、抗腫瘍剤、自己免疫疾患治療剤及び抗体断片多量体分子の作製方法  2019年06月27日

  前仲 勝実, 田所 高志  国立大学法人北海道大学  202103002931165546
• WO2019-054409:HLAタンパク質に相互作用する物質のスクリーニング方法及びスクリーニング用キット  2019年03月21日

  前仲 勝実, 田所 高志, 野村 尚生, 黒木 喜美子, 可野 巧  国立大学法人北海道大学  202003014201864539
• 特開2018-087173:悪性脳腫瘍治療薬  2018年06月07日

  堺谷 政弘, 林 健人, 前田 直良, 前仲 勝実, 市川 聡  国立大学法人北海道大学  201803004142926807
• WO2016-031936:KIR2DS1に対するモノクローナル抗体  2016年03月03日

  前仲 勝実, 黒木 喜美子, 米田 宏, 福原 秀雄  国立大学法人北海道大学  201703012187382811
• 特開2015-140322:関節リウマチまたはその関連疾患の予防または治療剤  2015年08月03日

  前仲 勝実, 黒木 喜美子, 高橋 愛実  国立大学法人北海道大学  201503017468526054
• 特許第5637981号:関節リウマチまたはその関連疾患の予防または治療剤  2014年10月31日

  前仲 勝実, 黒木 喜美子, 大戸 茂弘, 小柳 悟  国立大学法人北海道大学  201403053538317146
• WO2010-101192:関節リウマチまたはその関連疾患の予防または治療剤  2010年09月10日

  前仲 勝実, 黒木 喜美子, 大戸 茂弘, 小柳 悟  国立大学法人九州大学  201203086087695096
• WO2010-101192:関節リウマチまたはその関連疾患の予防または治療剤  2010年09月10日

  前仲 勝実, 黒木 喜美子, 大戸 茂弘, 小柳 悟  国立大学法人九州大学  201203086087695096
• 特開2009-131151:PILRαと結合するポリペプチド、およびそれをコードするポリヌクレオチド、並びにその利用  2009年06月18日

  荒瀬 尚, 白鳥 行大, 前仲 勝実  国立大学法人大阪大学  200903083001524532
• 特開2009-106163:核酸配列、ベクター、形質転換体、製造方法、及び、核酸配列プライマー  2009年05月21日

  前仲 勝実, 黒木 喜美子, 神田 大輔, 梶川 瑞穂, 白石 充典  国立大学法人九州大学  200903052598489217
• 特許第4288389号:BmNPVシャトルベクター  2009年04月10日

  朴 龍洙, 本橋 智子, 前仲 勝実, 霜島 司  財団法人浜松科学技術研究振興会  201103018687704436
• 特許第4288389号:BmNPVシャトルベクター  2009年04月10日

  朴 龍洙, 本橋 智子, 前仲 勝実, 霜島 司  財団法人浜松科学技術研究振興会  200903091167851216
• 特開2004-173507:BmNPVシャトルベクター  2004年06月24日

  朴 龍洙, 本橋 智子, 前仲 勝実, 霜島 司  財団法人浜松科学技術研究振興会  200903091167851216
• 特開平8-298984:ファージミドおよびその使用方法  1996年11月19日

  前仲 勝実, 熊谷 泉  熊谷 泉  200903029676271833

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  期間 : 2019年05月23日 - 2019年05月23日

  役割 : 講師