前仲 勝実 (マエナカ カツミ)

薬学研究院 創薬科学部門 生体機能科学分野教授
人獣共通感染症国際共同研究所教授
産学・地域協働推進機構教授
総合イノベーション創発機構ワクチン研究開発拠点教授
One Healthリサーチセンター教授

研究者基本情報

■ 学位
  • 博士(工学), 東京大学
■ URL
researchmap URLホームページURL■ ID 各種
J-Global ID■ 研究キーワード・分野
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
■ 担当教育組織

経歴

■ 経歴
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, 日本国
委員歴
  • 2018年06月 - 現在
    日本生物物理学会, 代議員, 学協会
  • 2017年05月 - 現在
    日本ケミカルバイオロジー学会, 幹事, 学協会
  • 2017年04月 - 現在
    日本薬学会, 代議員, 学協会
  • 2015年08月 - 現在
    International Structural Genomics Organization (ISGO), Advisory Board Member, 学協会
  • 2014年06月 - 現在
    日本生化学会, 理事(2025.11~2027.10)、評議員, 学協会
  • 2011年04月 - 現在
    日本蛋白質科学会, 会長(2024~2026)、理事、執行役員, 学協会
  • 2024年 - 2026年
    Protein Society, Executive council member, 学協会
  • 2014年06月 - 2020年06月
    Journal of Biochemistry, Editor, Associate Editor, 学協会
  • 2015年03月 - 2017年03月
    日本生物物理学会北海道支部, 支部長, 学協会
  • 2002年06月 - 2003年06月
    Japanese Society for Biophysics, Associate editor, 学協会
  • 2002年 - 2003年
    日本生物物理学会, 編集委員, 学協会
学内役職歴
  • 教育研究評議会評議員, 2013年4月1日 - 2015年3月31日
  • 大学院生命科学院副学院長, 2013年4月1日 - 2014年3月31日
  • 大学院生命科学院副学院長, 2014年4月1日 - 2015年3月31日
  • 大学院薬学研究院副研究院長, 2013年4月1日 - 2015年3月31日
  • 薬学部附属薬用植物園長, 2014年4月1日 - 2016年3月31日
  • 薬学部附属薬用植物園長, 2016年4月1日 - 2018年3月31日
  • 薬学部附属薬用植物園長, 2018年4月1日 - 2020年3月31日
  • 薬学部附属薬用植物園長, 2020年4月1日 - 2022年3月31日

研究活動情報

■ 受賞
  • 2015年06月, 北海道大学, 北海道大学総長賞研究奨励賞
    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
    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
    前仲 勝実
■ 論文
  • Molecular basis of fibrinogen recognition by the innate immune receptor LILRA2
    Jiaqi Wang; Atsushi Furukawa; Liuan Chen; Ryo Suzuki; Hisashi Arase; Kouyuki Hirayasu; Katsumi Maenaka
    Journal of Biological Chemistry, 2026年04月
    研究論文(学術雑誌)
  • Exploring potential targets of Chlamydia trachomatis required for cellular adaptation through screening approved-drug libraries
    Ruiyu Li; Saicheng Zhang; Satoko Otsuguro; Akira Matsuda; Sora Kuroiwa; Tsubasa Tatsumiya; Jeewan Thapa; Torahiko Okubo; Katsumi Maenaka; Yuichi Takizawa; Hideaki Higashi; Hiroyuki Yamaguchi
    Next Research, 2026年03月
    研究論文(学術雑誌)
  • Structural basis of lenacapavir-induced HIV-1 capsid disruption during virion maturation
    Hiroki Tanaka; Reina Morita; Tomomasa Oka; Shunsuke Kita; Mina Sasaki; Katsumi Maenaka; Shinichi Machida
    2026年02月16日
  • Exploratory transcriptomics and in vivo analyses of suramin in tongue squamous cell carcinoma
    Wataru Kakuguchi; Masahiro Morimoto; Kenta Takahashi; Nako Maishi; Kyoko Hida; Takao Nomura; Satoko Otsuguro; Katsumi Maenaka; Yoichi Ohiro
    Biomedical Reports, 24, 3, 1, 10, Spandidos Publications, 2026年01月23日
    研究論文(学術雑誌)
  • The effect of guide RNA thermal denaturation on the quality of Cas9 ribonucleoprotein-loaded lipid nanoparticle formulations
    Rina Shimizu; Yuji Kashiwakura; Morisada Hayakawa; Shunsuke Kita; Mina Sato; Masatoshi Maeki; Manabu Tokeshi; Katsumi Maenaka; Tsukasa Ohmori; Yuma Yamada; Hideyoshi Harashima; Yusuke Sato
    RSC Pharmaceutics, 2026年
    研究論文(学術雑誌)
  • Enhanced Sampling Reveals Metastable Conformations Driving K417N-Mediated Class I Antibody Escape.
    Xu Pan; Takashi Tadokoro; Taishi Onodera; Shunsuke Kita; Yoshimasa Takahashi; Katsumi Maenaka; Hisham M Dokainish
    Journal of chemical information and modeling, 65, 21, 11925, 11936, 2025年11月10日, [国際誌]
    英語, 研究論文(学術雑誌), The continuous emergence of SARS-CoV-2 variants compromises the effectiveness of neutralizing antibodies (nAbs), contributing to immune evasion. Several single-point mutations in the receptor-binding domain (RBD) have been shown to drastically affect the recognition of Class I antibodies. Understanding the molecular mechanisms behind such single-point mutations remains challenging, especially as weak binding affinity prevents the characterization of complex structures through conventional experimental methods. Here, we employ enhanced sampling molecular dynamics (MD) simulations, the generalized replica exchange with solute tempering (gREST), to investigate the structural and dynamic impacts of the K417N mutation on NT-193 (Class I) binding to RBD. Although stable conformations of wild-type and mutants show similar binding interactions, the mutation profoundly reshapes the transient metastable states. At the key interface between the heavy and light chains, the substitution of lysine with asparagine disrupts key interactions with the heavy chain in metastable states, facilitating antibody dissociation. Consistent with simulation results, surface plasmon resonance (SPR) experiments confirm that K417N significantly increases the dissociation rate, reducing the overall binding affinity to RBD. These findings illustrate the importance of capturing metastable conformations to fully understand mutation-driven immune evasion mechanisms. Moreover, this study demonstrates the power of applying gREST to unravel essential insights into transient and weakly interacting antibody-antigen complexes, facilitating the rational design and optimization of next-generation antibody therapeutics capable of overcoming viral immune escape.
  • Molecular basis for selection and inhibition of HIV-1 escape virus by T cells and KIR2DL2+NK cells.
    Takayuki Chikata; Kimiko Kuroki; Nozomi Kuse; Anna E Kliszczak; Wayne Paes; Nanami Tomioka; Robert Parker; Aure Aflalo; Tomohiro Akahoshi; Yu Zhang; Ryoya Yamashita; Ryuma Sakata; Hiroki Kusaka; Yosuke Watanabe; Annalisa Nicastri; Haruki Matsubara; Toyoyuki Ose; Shunsuke Kita; Shinichi Oka; Hiroyuki Gatanaga; Zhansong Lin; Nicola Ternette; Persephone Borrow; Katsumi Maenaka; Masafumi Takiguchi
    Nature communications, 16, 1, 9796, 9796, 2025年11月06日, [国際誌]
    英語, 研究論文(学術雑誌), NK cells and CD8+ T cells both contribute to HIV-1 control. These cells not only suppress HIV-1 replication, but also select HIV-1 escape mutant viruses. Most viruses bearing T cell escape mutations are expected to remain susceptible to NK cell suppression, but their inhibition by NK cells is unclear. We investigated the role of HIV-1-specific CD8+ T cells and NK cells recognizing superimposed Pol peptides in selection and control of HIV-1 mutant virus. KIR2DL2+NK cells have an enhanced ability to recognize HIV-1-infected cells after selection of Pol mutant virus by PolIY11-specific HLA-C*12:02-restricted T cells. Mass spectrometry-based immunopeptidome profiling of HIV-1-infected cells and analysis of crystal structures of TCR- and KIR2DL2-HLA-C*12:02-peptide complexes demonstrate the molecular basis for selection and recognition of the escape mutant epitope by TCR and KIR2DL2. The present study elucidates the mechanism for selection and inhibition of an HIV-1 escape virus by T cells and NK cells.
  • Thermoresponsive hydrogelation of N-acetyl chitohexaose: Gelation mechanism and application in controlled protein release.
    Hiroyuki Kono; Yuya Nagaoka; Ayumu Izutsu; Shunsuke Kita; Katsumi Maenaka; Shunsuke Nishio; Takeshi Hattori; Yuka Yoshikawa; Kousuke Michishita; Taichi Usui; Takuya Isono; Makoto Ogata
    Carbohydrate polymers, 366, 123897, 123897, 2025年10月15日, [国際誌]
    英語, 研究論文(学術雑誌), Chitin is a polysaccharide composed of N-acetylglucosamine units that form highly crystalline structures in biological systems; however, the precise mechanism underlying its self-assembly in aqueous environments remains unclear. In this study, we demonstrated that N-acetyl chitohexaose (GN6), a chitin oligosaccharide with a degree of polymerization of six, uniquely undergoes thermally induced self-assembly into crystalline nanofibers and forms reversible hydrogels in water. Among chitin oligosaccharides ranging from monomer to hexamer, only GN6 exhibited gelation upon heating, which is attributed to its optimal molecular length and balanced hydrophilic-hydrophobic characteristics. Comprehensive structural analyses using solid-state nuclear magnetic resonance spectroscopy, small- and wide-angle X-ray scattering, transmission electron microscopy, atomic force microscopy, and microcrystal electron diffraction revealed that GN6 molecules align along the crystallographic β-axis and assemble into nanofibers with highly ordered α-chitin-type molecular packing. This directional organization is driven by cooperative hydrogen bonding and hydrophobic interactions. Furthermore, the hydrogels exhibited a sustained release of encapsulated proteins, including insulin, albumin, and immunoglobulin G, for more than four weeks under physiological conditions, without using chemical crosslinking agents. These findings highlight the potential of this oligosaccharide as a biocompatible, drug delivery material and as a minimalistic model system for studying polysaccharide self-assembly in aqueous environments.
  • Examining the Impact of Storage Conditions on the Stability of a Liquid Formulation of mRNA-Loaded Lipid Nanoparticles.
    Mina Sato; Eleni Samaridou; Moritz Beck-Broichsitter; Masatoshi Maeki; Shunsuke Kita; Manabu Tokeshi; Katsumi Maenaka; Hideyoshi Harashima; Yusuke Sato
    Pharmaceutics, 17, 9, 2025年09月14日, [国際誌]
    英語, 研究論文(学術雑誌), Background/Objectives: This study investigated the effect of storage conditions on mRNA-LNPs in situ via identification of the formulation traits necessary for improving storage stability. Methods: We synthesized an ionizable lipid, namely TOT-28, which has a hydrolysis-susceptible ester bond in its hydrophilic head group that allows it to act as an indicator of the hydrophilic environment within the mRNA-LNPs. LNPs were stored either at 4 or 25 °C for up to 8 weeks to investigate the effect of pH and temperature on ester hydrolysis, internal mRNA integrity, physicochemical properties of the LNPs, and mRNA gene expression. Results: The results indicate that, at 25 °C, a lower buffer pH increases ester hydrolysis, whereas an opposite trend slightly occurs in ester hydrolysis with storage at 4 °C. We also found that TOT-28-based LNPs were less hydrated and microviscosity was higher at 4 °C compared with storage temperature at 25 °C. Therefore, TOT-28-based LNPs seem less sensitive to external buffer solutions because of a higher-order structure when stored at lower temperatures. In addition, we found that LNPs with different ionizable lipid structures exhibit distinct responses to pH changes at specific storage temperatures. Conclusions: Our findings provide novel insights into the appropriate conditions for long-term storage of the mRNA-LNPs as a liquid formulation.
  • Understanding the structure of measles virus and its implications for novel drug discovery.
    Liuan Chen; Shunsuke Kita; Hideo Fukuhara; Katsumi Maenaka
    Expert opinion on drug discovery, 20, 9, 1131, 1140, 2025年09月, [国際誌]
    英語, 研究論文(学術雑誌), INTRODUCTION: Despite having a stably effectively vaccine for decades, the Measles virus (MV) still causes periodic outbreaks given its highly contagious nature and a consistent decline in immunization coverage, which was further exacerbated during the COVID-19 pandemic, leading to reduced immunization rates. Equally concerning, there are also no approved treatments for measles. AREAS COVERED: Herein, the authors explore the current challenges of MV therapy discovery. Firstly, the article will provide an overview of the potential drug-targeted steps in the MV infection process, followed by discussion on the characteristics of existing drugs as well as the feasibility of structure-based drug discovery. Finally, the authors highlight the current progress in the field and the future opportunities for antiviral development. This article is based on a literature review including original publications, standard sources, the Protein Data Bank and clinical trials. EXPERT OPINION: First and foremost, a comprehensive structural analysis of neutralizing antibodies and RdRp inhibitors is required for efficient antiviral development. Moreover, the therapeutic prospects and current limitations for acute MV and subacute sclerosing panencephalitis (SSPE) treatments should be considered. Due to various factors including mutations, the development of broad-spectrum antivirals may minimize many of the existing barriers.
  • An FDA-approved drug library screening identifies proteasome inhibitors as selective cytotoxic agents for angiosarcoma cells.
    Che-Yuan Hsu; Teruki Yanagi; Kodai Miyamoto; Satoko Otsuguro; Katsumi Maenaka; Hiroshi Nishihara; Hideki Nakamura; Kenzo Takahashi; Hideyuki Ujiie
    British journal of cancer, 133, 1, 3, 13, 2025年07月, [国際誌]
    英語, 研究論文(学術雑誌), Cutaneous angiosarcoma (CAS) is a life-threatening neoplasm with a 5-year survival rate of under 40% in advanced cases. As available treatments for CAS are limited, novel therapeutics must be explored. To identify potential therapeutic candidates, we conducted a drug screening analysis using an angiosarcoma cell line, HAMON. Cancer-related gene analysis revealed alterations in FGFR4, MYCN, CDKN2A, NF1, TP53, KDM6A, ATRX, MSH6, ATM, and NOTCH1 in HAMON cells. Screening of 4681 FDA-approved drugs identified four candidate compounds, with the proteasome inhibitor bortezomib selected for further study. ATP and MTT assays revealed bortezomib to be the most effective candidate against HAMON cells. Clonogenic assays revealed fewer HAMON cell colonies in the range of 1-10 nM bortezomib. DNA-content fluorescence-activated cell sorting analysis revealed a notable increase in the sub-G0/G1 phases, suggesting cell death without cell cycle arrest. Annexin V-propidium iodide staining revealed a significant increase in the percentage of early and late apoptotic cells in the bortezomib group. Mechanistically, bortezomib induced activation of NF-κB and endoplasmic reticulum stress signaling. The administration of bortezomib to immunocompromised mice implanted with HAMON cells induced apoptosis of tumor cells. This study identified the proteasome inhibitor bortezomib as a potential candidate for angiosarcoma in vitro and in vivo.
  • Structural basis of the hepatitis B virus X protein in complex with DDB1.
    Hiroki Tanaka; Joao Diogo Dias; Basile Jay; Shunsuke Kita; Mina Sasaki; Hiroyuki Takeda; Naoki Kishimoto; Shunsuke Sasaki; Shogo Misumi; Masashi Mizokami; Christine Neuveut; Takashi Sumikama; Mikihiro Shibata; Katsumi Maenaka; Shinichi Machida
    Proceedings of the National Academy of Sciences of the United States of America, 122, 24, e2421325122, 2025年06月17日, [国際誌]
    英語, 研究論文(学術雑誌), A cure for chronic hepatitis B requires eliminating or permanently silencing covalently closed circular DNA (cccDNA). A pivotal target of this approach is the hepatitis B virus (HBV) X protein (HBx), which is a key factor that promotes transcription from cccDNA. However, the HBx structure remains unsolved. Here, we present the cryoelectron microscopy structure of HBx in complex with DDB1, which is an essential complex for cccDNA transcription. In this structure, hydrophobic interactions within HBx were identified, and mutational analysis highlighted their importance in the HBV life cycle. Our biochemical analysis revealed that the HBx-DDB1 complex directly interacts simultaneously with NSE3, which is a component of the SMC5/6 complex, and Spindlin1. Additionally, HBx-DDB1 complex dynamics were explored via high-speed atomic force microscopy. These findings provide comprehensive insights into the structure and function of HBx in HBV replication.
  • Evolutionary and structural basis of SLAMF1 utilization in morbilliviruses-Implications for host range and cross-species transmission.
    Ayumu Hyodo; Fumio Seki; Kento Fukuda; Kaede Tashiro; Yuki Kitai; Yukiko Akahori; Hideko Watabe; Hiroshi Katoh; Rikuto Osaki; Daisuke Takaya; Norihito Kawashita; Hideo Fukuhara; Satoshi Ikegame; Tomoki Yoshikawa; Park Eunsil; Shigeru Morikawa; Ryoji Yamaguchi; Benhur Lee; Katsumi Maenaka; Tsuyoshi Shirai; Kaori Fukuzawa; Shigenori Tanaka; Makoto Takeda
    PLoS pathogens, 21, 6, e1012990, 2025年06月, [国際誌]
    英語, 研究論文(学術雑誌), Morbilliviruses, including measles virus (MV), canine distemper virus (CDV), peste des petits ruminants virus, and cetacean morbillivirus pose a significant threat to humans and animals. While the host range of morbilliviruses is generally well-defined, cross-species transmission events with significant mortality have also been reported. Their entry into immune cells, the primary targets of morbilliviruses, relies on the signaling lymphocytic activation molecule (SLAM), also known as SLAMF1 or CD150. In this study, we hypothesize that the ability of morbilliviruses to utilize heterologous SLAM receptors stems from evolutionarily conserved structural determinants within the SLAM protein and that minimal genetic changes in the viral receptor-binding H protein can enable adaptation to novel hosts. To test this, we systematically assessed SLAM utilization and adaptation by diverse morbilliviruses. We found that most morbilliviruses efficiently utilize SLAM from multiple host species, including Myotis bat SLAM, but not human SLAM. Only MV could efficiently utilize human SLAM. Additionally, unlike other morbilliviruses, MV utilized Myotis bat SLAM inefficiently. As an example of morbillivirus adaptation to non-host animal SLAM, we conducted an MV adaptation experiment with Myotis bat SLAM. We demonstrated that MV readily adapted to utilize Myotis bat SLAM by acquiring a single N187Y mutation in its hemagglutinin protein. Notably, hypothetical ancestral SLAMs acted as universal receptors for all morbilliviruses. These results reinforced that morbillivirus receptor usage is primarily supported by evolutionarily conserved structural features of SLAM, highlighting a molecular basis that enables morbilliviruses to rapidly adapt to diverse animal SLAMs.
  • Regulatory mimicry of cyclin-dependent kinases by a conserved herpesvirus protein kinase.
    Naoto Koyanagi; Kowit Hengphasatporn; Akihisa Kato; Moeka Nobe; Kosuke Takeshima; Yuhei Maruzuru; Katsumi Maenaka; Yasuteru Shigeta; Yasushi Kawaguchi
    Proceedings of the National Academy of Sciences of the United States of America, 122, 16, e2500264122, 2025年04月22日, [国際誌]
    英語, 研究論文(学術雑誌), Herpesviruses encode conserved protein kinases (CHPKs) that target cellular cyclin-dependent kinase (CDK) phosphorylation sites; thus, they are termed viral CDK-like kinases. Tyrosine 15 in the GxGxxG motifs of CDK1 and CDK2, whose phosphorylation down-regulates their catalytic activities, is conserved in the corresponding motifs of CHPKs. We found that CHPK UL13, the corresponding Tyr-162 in herpes simplex virus 2 (HSV-2), was phosphorylated in HSV-2-infected cells. Mutational analyses of HSV-2 UL13 Tyr-162 suggested that phosphorylation of UL13 Tyr-162 reduced the phosphorylation of all UL13 substrates tested in HSV-2-infected cells. These findings suggested that HSV-2 UL13 mimicked the regulatory mechanism of CDKs and that this CHPK has regulatory and functional mimicry with CDKs. Furthermore, phosphorylation of HSV-2 UL13 Tyr-162 was suggested to be required for the downregulation of viral replication and pathogenicity, specifically in the brains of mice, and for efficient viral recurrence in guinea pigs. These findings highlight the dual impact of the regulatory mimicry of CDKs by CHPK on the fine-tuned regulation of lytic and latent HSV-2 infections in vivo.
  • ARNAX is an ideal adjuvant for COVID-19 vaccines to enhance antigen-specific CD4+ and CD8+ T-cell responses and neutralizing antibody induction.
    Tomomi Kawakita; Toshiki Sekiya; Yayoi Kameda; Naoki Nomura; Marumi Ohno; Chimuka Handabile; Akari Yamaya; Hideo Fukuhara; Yuki Anraku; Shunsuke Kita; Shinsuke Toba; Hirotake Tsukamoto; Tomohiro Sawa; Hiroyuki Oshiumi; Yasushi Itoh; Katsumi Maenaka; Akihiko Sato; Hirofumi Sawa; Yasuhiko Suzuki; Lorena E Brown; David C Jackson; Hiroshi Kida; Misako Matsumoto; Tsukasa Seya; Masashi Shingai
    Journal of virology, e0229024, 2025年04月15日, [国際誌]
    英語, 研究論文(学術雑誌), UNLABELLED: ARNAX is a synthetic nucleotide-based Toll-like receptor 3 (TLR3) ligand that specifically stimulates the TLR3/TIR domain-containing adaptor molecule 1 (TICAM-1) pathway without activating inflammatory responses. ARNAX activates cellular immunity via cross-presentation; hence, its practical application has been demonstrated in cancer immunotherapy. Given the importance of cellular immunity in virus infections, ARNAX is expected to be a more effective vaccine adjuvant for virus infections than alum, an adjuvant approved for human use that mainly enhances humoral immunity. In the present study, the trimeric recombinant spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was prepared as a vaccine antigen and formulated with ARNAX. When T-cell and neutralizing antibody responses were evaluated in immunized mice, antigen formulated with ARNAX generated significantly larger numbers of antigenspecific CD4+ and CD8+ T cells, as well as higher titers of neutralizing antibodies, compared to antigen alone or antigen formulated with alum. In experiments where immunized mice were challenged with a SARS-CoV-2 mouse-adapted virus derived from the ancestral strain, immunization with antigen formulated with ARNAX reduced virus titers in the lungs at 3 days post-infection to a much greater extent than did immunization with either antigen alone or that formulated with alum. These results show that ARNAX potently enhances the levels of both cellular and humoral immunity above those seen with alum, providing significantly greater viral clearing responses. Thus, ARNAX may act as a useful adjuvant for prophylactic vaccines, particularly for viral infectious diseases. IMPORTANCE: Cellular immunity is a critical immunological defense system against virus infections. However, aluminum salts, the most widely used adjuvant for vaccines for human use, do not promote strong cellular immunity. To prepare for the next pandemic of viral origin, the development of Th1-type adjuvants with low adverse reactions that induce cellular immunity is necessary. ARNAX is a TLR3 agonist consisting of DNA-RNA hybrid nucleic acid, which is expected to be an adjuvant that induces cellular immunity. The present study using a coronavirus disease 2019 mouse model demonstrated that ARNAX potently induces cellular immunity in addition to humoral immunity with minimal induction of inflammatory cytokines. Therefore, ARNAX has the potential to be used as a potent and welltolerated adjuvant for vaccines against pandemic viruses emerging in the future.
  • Structural analysis shows the mode of inhibition for Staphylococcus aureus lipase by antipsychotic penfluridol.
    Julia Kitadokoro; Takatsugu Hirokawa; Masayuki Kamo; Naoki Furubayashi; Yukiko Okuno; Takaaki Hikima; Masaki Yamamoto; Koji Inaka; Katsumi Maenaka; Shigeki Kamitani; Kengo Kitadokoro
    Scientific reports, 15, 1, 11876, 11876, 2025年04月14日, [国際誌]
    英語, 研究論文(学術雑誌), It is now well-established that Staphylococcus aureus can produce a range of toxin proteins, resulting in a spectrum of pathological conditions when it infects individuals with pre-existing medical conditions or immunocompromised. Among these, MRSA is one of the most prominent antimicrobial-resistant organisms and a significant cause of mortality in many patients. It has been demonstrated that Staphylococcus aureus lipase (SAL) is a vital factor in the proliferation of this bacterium. A combination of in silico screening and X-ray crystallography was employed to analyze inhibitors of SAL, and the results were highly significant. In silico screening identified a number of compounds, and the enzyme activity assay demonstrated that the antipsychotic drug penfluridol exhibited potent inhibitory activity against SAL. We have conducted co-crystallization of penfluridol and SAL on the ground and in space. The resulting co-crystals were subjected to data measurement using the synchrotron radiation facility at SPring-8, and the complex structure was determined. The crystal structure of the penfluridol-SAL complex was determined at 2.2 Å resolution, thereby providing the structural basis for developing new anti-infective agents that inhibit the growth of Staphylococcus aureus. These findings are anticipated to facilitate the development of compounds with potent inhibitory activity.
  • Structural and virological identification of neutralizing antibody footprint provides insights into therapeutic antibody design against SARS-CoV-2 variants.
    Yuki Anraku; Shunsuke Kita; Taishi Onodera; Akihiko Sato; Takashi Tadokoro; Shiori Ito; Yu Adachi; Ryutaro Kotaki; Tateki Suzuki; Jiei Sasaki; Nozomi Shiwa-Sudo; Naoko Iwata-Yoshikawa; Noriyo Nagata; Souta Kobayashi; Yasuhiro Kazuki; Mitsuo Oshimura; Takao Nomura; Michihito Sasaki; Yasuko Orba; Tadaki Suzuki; Hirofumi Sawa; Takao Hashiguchi; Hideo Fukuhara; Yoshimasa Takahashi; Katsumi Maenaka
    Communications biology, 8, 1, 483, 483, 2025年03月22日, [国際誌]
    英語, 研究論文(学術雑誌), Medical treatments using potent neutralizing SARS-CoV-2 antibodies have achieved remarkable improvements in clinical symptoms, changing the situation for the severity of COVID-19 patients. We previously reported an antibody, NT-108 with potent neutralizing activity. However, the structural and functional basis for the neutralizing activity of NT-108 has not yet been understood. Here, we demonstrated the therapeutic effects of NT-108 in a hamster model and its protective effects at low doses. Furthermore, we determined the cryo-EM structure of NT-108 in complex with SARS-CoV-2 spike. The single-chain Fv construction of NT-108 improved the cryo-EM maps because of the prevention of preferred orientations induced by Fab orientation. The footprints of NT-108 illuminated how escape mutations such as E484K evade from class 2 antibody recognition without ACE2 affinity attenuation. The functional and structural basis for the potent neutralizing activity of NT-108 provides insights into the rational design of therapeutic antibodies.
  • Structural analysis reveals how tetrameric tyrosine-phosphorylated STAT1 is targeted by the rabies virus P-protein
    Aoi Sugiyama; Miku Minami; Kaito Ugajin; Satomi Inaba-Inoue; Nana Yabuno; Yuichiro Takekawa; Sun Xiaomei; Shiho Takei; Mina Sasaki; Tomo Nomai; Xinxin Jiang; Shunsuke Kita; Katsumi Maenaka; Mika Hirose; Min Yao; Paul R. Gooley; Gregory W. Moseley; Yukihiko Sugita; Toyoyuki Ose
    Science Signaling, 18, 878, American Association for the Advancement of Science (AAAS), 2025年03月18日
    研究論文(学術雑誌), Signal transducer and activator of transcription (STAT) family members mediate signaling in the Janus kinase (JAK)–STAT pathway and are activated by phosphorylation at a conserved tyrosine residue, resulting in dimerization through reciprocal interactions between the phosphotyrosine and a Src homology 2 (SH2) domain. Tyrosine-phosphorylated STAT (pY-STAT) then translocates to the nucleus to induce the expression of genes encoding antiviral proteins. Although the active and functional forms of STATs are conventionally considered to be dimers, STATs can undergo higher-order oligomerization, which is implicated in regulating transcriptional activity. We present the cryo–electron microscopy (cryo-EM) structure of the tetrameric form of intact pY-STAT1 in complex with DNA, which indicates that interactions between the amino-terminal domains (NTDs) of STAT1 induce oligomerization. The tetrameric structure revealed a compact conformation with a previously uncharacterized binding interface: Two DNA-bound dimers are twofold symmetrically aligned to transform into a tandem DNA-binding model without NTD dimer separation. Moreover, biochemical analyses indicated that the rabies virus P-protein selectively targeted tetrameric pY-STAT1. Combined with data showing which regions contribute to the interaction between pY-STAT1 and the P-protein, we constructed a binding model explaining how P recognizes the pY-STAT1 tetramer. These data provide insight into how pathogenic viruses target signaling pathways that mediate the host immune response.
  • The strategy used by naïve anti-PEG antibodies to capture flexible and featureless PEG chains.
    Yiwei Liu; Takahiro Mori; Yusei Ito; Kimiko Kuroki; Seiichiro Hayashi; Daisuke Kohda; Taro Shimizu; Tatsuhiro Ishida; Steve R Roffler; Mika K Kaneko; Yukinari Kato; Takao Arimori; Takamasa Teramoto; Kazuhiro Takemura; Kenta Ishibashi; Yoshiki Katayama; Katsumi Maenaka; Yoshimitsu Kakuta; Akio Kitao; Takeshi Mori
    Journal of controlled release : official journal of the Controlled Release Society, 380, 396, 403, 2025年02月10日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), Polyethylene glycol (PEG) is widely used as a standard stealth polymer, although the induction of anti-PEG antibodies and consequent effects have drawn attention in recent years. To date, several anti-PEG antibodies induced by PEG-modified proteins via the T cell-dependent (TD) pathway, in which affinity maturation occurs, have been reported. In contrast, structures of the naïve anti-PEG antibodies before affinity maturation have not been described in the literature. Here, to understand the details of the naïve anti-PEG antibodies capturing PEG, we studied a naïve anti-PEG antibody induced by a PEG-modified liposome in the absence of affinity maturation via the T cell-independent (TI) pathway. The mutation levels, structures as well as in vitro and in silico binding properties of TI and TD anti-PEG antibodies were compared. The TI anti-PEG antibody showed no mutation and a low binding affinity toward PEG, meanwhile, it allowed PEG chain sliding and weak interaction with the terminal group. Furthermore, the naïve anti-PEG antibodies may obtain high affinities by forming tunnel structures via minimal mutations. This research provides new insights into polymer-antibody interactions, which can facilitate the development of novel stealth polymers that can avoid antibody induction.
  • Enhanced effect of the immunosuppressive soluble HLA-G2 homodimer by site-specific PEGylation.
    Chisato Yamada; Kimiko Kuroki; Naoyoshi Maeda; Hiroshi Watanabe; Ami Takahashi; Katsumi Maenaka
    Scientific reports, 15, 1, 2509, 2509, 2025年01月20日, [国際誌]
    英語, 研究論文(学術雑誌), Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule that has an immunosuppressive effect mediated by binding to immune inhibitory leukocyte immunoglobulin-like receptors (LILR) B1 and LILRB2. A conventional HLA-G isoform, HLA-G1, forms a heterotrimeric complex composed of a heavy chain (α1-α3 domains), β2-microglobulin (β2m) and a cognate peptide. One of the other isoforms, HLA-G2, lacks a α2 domain or β2m to form a nondisulfide-linked homodimer, and its ectodomain specifically binds to LILRB2 expressed in human monocytes, macrophages, and dendritic cells. The administration of the ectodomain of HLA-G2, designated the soluble HLA-G2 homodimer, showed significant immunosuppressive effects in mouse models of rheumatoid arthritis and systemic lupus erythematosus, presumably by binding to a mouse ortholog of LILRB2, paired immunoglobulin-like receptor B. However, the refolded soluble HLA-G2 homodimer used in these studies tends to aggregate and degrade; thus, its stability for clinical use has been a concern. In the present study, we improved the stability of the refolded soluble HLA-G2 homodimer via a site-directed PEGylation method. PEGylation at an original free cysteine residue, Cys42, resulted in increased lyophilization and thermal and serum stability. Furthermore, the PEGylated soluble HLA-G2 homodimer could better suppress atopic symptoms in mice than the non-PEGylated homodimer. These results suggest that PEGylated soluble HLA-G2 homodimers could be candidates for immunosuppressive biologics that specifically target LILRB2-positive myelomonocytic antigen-presenting cells.
  • X-206 exhibits broad-spectrum anti-β-coronavirus activity, covering SARS-CoV-2 variants and drug-resistant isolates.
    Jiei Sasaki; Akihiko Sato; Michihito Sasaki; Iori Okabe; Kota Kodama; Satoko Otsuguro; Kosuke Yasuda; Hirotatsu Kojima; Yasuko Orba; Hirofumi Sawa; Katsumi Maenaka; Yusuke Yanagi; Takao Hashiguchi
    Antiviral research, 106039, 106039, 2024年11月19日, [国際誌]
    英語, 研究論文(学術雑誌), Coronaviruses such as the Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), and SARS-CoV-2, causing MERS, SARS, and Coronavirus disease-19, respectively, are highly pathogenic to humans. Notably, several antiviral drugs against SARS-CoV-2, such as nirmatrelvir and remdesivir, have been approved. However, no approved vaccines or antiviral agents are available for other highly pathogenic β-coronaviruses. In this study, we identified two compounds, thapsigargin and X-206, that exhibit antiviral activities against SARS-CoV, MERS-CoV, and SARS-CoV-2. Notably, both compounds effectively inhibited the cell-to-cell fusion mediated by the Spike proteins of all three β-coronaviruses. X-206 exhibited antiviral activity against nirmatrelvir- and remdesivir-resistant SARS-CoV-2 isolates and SARS-CoV-2 variants, including Delta, BA.5, and XBB.1. Consequently, the mechanism of action of these compounds with anti-β-coronavirus activities may differ from that of the approved direct-acting drugs for SARS-CoV-2, thereby offering potential use as a cocktail with other antivirals, and serving as a chemical basis for developing therapeutic agents against β-coronaviruses in preparation for the next spillover and pandemic.
  • Structural basis for receptor-binding domain mobility of the spike in SARS-CoV-2 BA.2.86 and JN.1
    Hisano Yajima; Yuki Anraku; Yu Kaku; Kanako Terakado Kimura; Arnon Plianchaisuk; Kaho Okumura; Yoshiko Nakada-Nakura; Yusuke Atarashi; Takuya Hemmi; Daisuke Kuroda; Yoshimasa Takahashi; Shunsuke Kita; Jiei Sasaki; Hiromi Sumita; Keita Matsuno; Naganori Nao; Hirofumi Sawa; Keita Mizuma; Jingshu Li; Izumi Kida; Yume Mimura; Yuma Ohari; Shinya Tanaka; Masumi Tsuda; Lei Wang; Yoshikata Oda; Zannatul Ferdous; Kenji Shishido; Hiromi Mohri; Miki Iida; Takasuke Fukuhara; Tomokazu Tamura; Rigel Suzuki; Saori Suzuki; Shuhei Tsujino; Hayato Ito; Naoko Misawa; Ziyi Guo; Alfredo A. Hinay; Kaoru Usui; Wilaiporn Saikruang; Spyridon Lytras; Keiya Uriu; Ryo Yoshimura; Shusuke Kawakubo; Luca Nishumura; Yusuke Kosugi; Shigeru Fujita; Jarel Elgin M.Tolentino; Luo Chen; Lin Pan; Wenye Li; Maximilian Stanley Yo; Kio Horinaka; Mai Suganami; Mika Chiba; Kyoko Yasuda; Keiko Iida; Adam Patrick Strange; Naomi Ohsumi; Shiho Tanaka; Eiko Ogawa; Tsuki Fukuda; Rina Osujo; Kazuhisa Yoshimura; Kenji Sadamas; Mami Nagashima; Hiroyuki Asakura; Isao Yoshida; So Nakagawa; Kazuo Takayama; Rina Hashimoto; Sayaka Deguchi; Yukio Watanabe; Yoshitaka Nakata; Hiroki Futatsusako; Ayaka Sakamoto; Naoko Yasuhara; Tateki Suzuki; Yukari Nakajima; Takashi Irie; Ryoko Kawabata; Kaori Sasaki-Tabata; Terumasa Ikeda; Hesham Nasser; Ryo Shimizu; M. S. T. Monira Begum; Michael Jonathan; Yuka Mugita; Sharee Leong; Otowa Takahashi; Takamasa Ueno; Chihiro Motozono; Mako Toyoda; Akatsuki Saito; Anon Kosaka; Miki Kawano; Natsumi Matsubara; Tomoko Nishiuchi; Jiri Zahradnik; Prokopios Andrikopoulos; Miguel Padilla-Blanco; Aditi Konar; Jumpei Ito; Katsumi Maenaka; Kei Sato; Takao Hashiguchi
    Nature Communications, 15, 1, Springer Science and Business Media LLC, 2024年10月07日
    英語, 研究論文(学術雑誌), Since 2019, SARS-CoV-2 has undergone mutations, resulting in pandemic and epidemic waves. The SARS-CoV-2 spike protein, crucial for cellular entry, binds to the ACE2 receptor exclusively when its receptor-binding domain (RBD) adopts the up-conformation. However, whether ACE2 also interacts with the RBD in the down-conformation to facilitate the conformational shift to RBD-up remains unclear. Herein, we present the structures of the BA.2.86 and the JN.1 spike proteins bound to ACE2. Notably, we successfully observed the ACE2-bound down-RBD, indicating an intermediate structure before the RBD-up conformation. The wider and mobile angle of RBDs in the up-state provides space for ACE2 to interact with the down-RBD, facilitating the transition to the RBD-up state. The K356T, but not N354-linked glycan, contributes to both of infectivity and neutralizing-antibody evasion in BA.2.86. These structural insights the spike-protein dynamics would help understand the mechanisms underlying SARS-CoV-2 infection and its neutralization.
  • Identification of BAY61-3606 Derivatives With Improved Activity in Splicing Modulation That Induces Inclusion of Cassette Exons Similar to the Splicing Factor 3B Subunit 1 Mutation.
    Takanori Matsumaru; Toshiki Iwamatsu; Kana Ishigami; Makoto Inai; Wataru Kanto; Ayumi Ishigaki; Atsushi Toyoda; Satoshi Shuto; Katsumi Maenaka; Shinichi Nakagawa; Hiroshi Maita
    Chemical biology & drug design, 104, 4, e70002, 2024年10月, [国際誌]
    英語, 研究論文(学術雑誌), Splicing modulation by a small compound offers therapeutic potential for diseases caused by splicing abnormality. However, only a few classes of compounds that can modulate splicing have been identified. We previously identified BAY61-3606, a multiple kinase inhibitor, as a compound that relaxes the splicing fidelity at the 3' splice site recognition. We have also reported the synthesis of derivatives of BAY61-3606. In this study, we tested those compounds for their splicing modulation capabilities and identified two contrasting compounds. These compounds were further investigated for their effects on the whole transcriptome, and analysis of changes in transcription and splicing revealed that the highly active derivative in the splicing reporter assay also showed significantly higher activity in modulating the splicing of endogenously expressed genes. Particularly, cassette exon inclusion was highly upregulated by this compound, and clustering analysis revealed that these effects resembled those in splicing factor 3b subunit 1 (SF3B1) K700E mutant cells but contrasted with those of the splicing inhibitor H3B-8800. Additionally, a group of serine/arginine-rich (SR) protein genes was identified as representatively affected, likely via modulation of poison exon inclusion. This finding could guide further analysis of the mode of action of these compounds on splicing, which could be valuable for developing drugs for diseases associated with splicing abnormalities.
  • Molecular and structural insights into SARS-CoV-2 evolution: from BA.2 to XBB subvariants.
    Hisano Yajima; Tomo Nomai; Kaho Okumura; Katsumi Maenaka; Jumpei Ito; Takao Hashiguchi; Kei Sato
    mBio, e0322023, 2024年09月16日, [国際誌]
    英語, 研究論文(学術雑誌), Due to the incessant emergence of various SARS-CoV-2 variants with enhanced fitness in the human population, controlling the COVID-19 pandemic has been challenging. Understanding how the virus enhances its fitness during a pandemic could offer valuable insights for more effective control of viral epidemics. In this manuscript, we review the evolution of SARS-CoV-2 from early 2022 to the end of 2023-from Omicron BA.2 to XBB descendants. Focusing on viral evolution during this period, we provide concrete examples that SARS-CoV-2 has increased its fitness by enhancing several functions of the spike (S) protein, including its binding affinity to the ACE2 receptor and its ability to evade humoral immunity. Furthermore, we explore how specific mutations modify these functions of the S protein through structural alterations. This review provides evolutionary, molecular, and structural insights into how SARS-CoV-2 has increased its fitness and repeatedly caused epidemic surges during the pandemic.
  • Chlamydia trachomatis L2 434/Bu readily activates glycolysis under hypoxia for efficient metabolism.
    Ruiyu Li; Saicheng Zhang; Satoko Otsuguro; Manabu Nagao; Akira Matsuda; Jeewan Thapa; Torahiko Okubo; Katsumi Maenaka; Hideaki Higashi; Hiroyuki Yamaguchi
    Biochemical and biophysical research communications, 736, 150461, 150461, 2024年08月06日, [国際誌]
    英語, 研究論文(学術雑誌), To understand why Chlamydia trachomatis (Ct) (L2/434/Bu) favors hypoxia, we examined the dynamics of infected cells using a glycolysis-related PCR array and metabolomic analysis, along with the perturbation of nucleotide synthesis. Our findings revealed that, compared to normoxia, hypoxia with infection significantly and selectively upregulates the expression of genes related to glycolysis, glycogen degradation, and the pentose phosphate pathway. Furthermore, hypoxia induced a significant decrease in metabolite levels, particularly methionine-related metabolites, independent of infection, indicating efficient metabolism under hypoxia. Additionally, the perturbation of nucleotide synthesis with adenosine derivatives impaired Ct growth. Collectively, our results suggest that Ct favors a hypoxic environment with efficient metabolism, in which Ct readily activates glycolysis responsible for stable nucleotide synthesis as well as ATP supply.
  • Glycan-shielded homodimer structure and dynamical features of the canine distemper virus hemagglutinin relevant for viral entry and efficient vaccination.
    Hideo Fukuhara; Kohei Yumoto; Miyuki Sako; Mizuho Kajikawa; Toyoyuki Ose; Mihiro Kawamura; Mei Yoda; Surui Chen; Yuri Ito; Shin Takeda; Mwila Mwaba; Jiaqi Wang; Takao Hashiguchi; Jun Kamishikiryo; Nobuo Maita; Chihiro Kitatsuji; Makoto Takeda; Kimiko Kuroki; Katsumi Maenaka
    eLife, 12, 2024年07月24日, [国際誌]
    英語, 研究論文(学術雑誌), Canine distemper virus (CDV) belongs to morbillivirus, including measles virus (MeV) and rinderpest virus, which causes serious immunological and neurological disorders in carnivores, including dogs and rhesus monkeys, as recently reported, but their vaccines are highly effective. The attachment glycoprotein hemagglutinin (CDV-H) at the CDV surface utilizes signaling lymphocyte activation molecule (SLAM) and Nectin-4 (also called poliovirus-receptor-like-4; PVRL4) as entry receptors. Although fusion models have been proposed, the molecular mechanism of morbillivirus fusion entry is poorly understood. Here, we determined the crystal structure of the globular head domain of CDV-H vaccine strain at 3.2 Å resolution, revealing that CDV-H exhibits a highly tilted homodimeric form with a six-bladed β-propeller fold. While the predicted Nectin-4-binding site is well conserved with that of MeV-H, that of SLAM is similar but partially different, which is expected to contribute to host specificity. Five N-linked sugars covered a broad area of the CDV-H surface to expose receptor-binding sites only, supporting the effective production of neutralizing antibodies. These features are common to MeV-H, although the glycosylation sites are completely different. Furthermore, real-time observation using high-speed atomic force microscopy revealed highly mobile features of the CDV-H dimeric head via the connector region. These results suggest that sugar-shielded tilted homodimeric structure and dynamic conformational changes are common characteristics of morbilliviruses and ensure effective fusion entry and vaccination.
  • Thermostability and binding properties of single-chained Fv fragments derived from therapeutic antibodies.
    Takashi Tadokoro; Harumi Tsuboi; Kota Nakamura; Tetsushi Hayakawa; Reo Ohmura; Izumi Kato; Masaki Inoue; Shin-Ichi Tsunoda; Sayaka Niizuma; Yukari Okada; Satoko Otsuguro; Katsumi Maenaka
    Protein science : a publication of the Protein Society, 33, 7, e5084, 2024年07月, [国際誌]
    英語, 研究論文(学術雑誌), Small antibody fragments have recently been used as alternatives to full-length monoclonal antibodies in therapeutic applications. One of the most popular fragment antibodies is single-chain fragment variables (scFvs), consisting of variable heavy (VH) and variable light (VL) domains linked by a flexible peptide linker. scFvs have small molecular sizes, which enables good tissue penetration and low immunogenicity. Despite these advantages, the use of scFvs, especially for therapeutic purpose, is still limited because of the difficulty to regulate the binding activity and conformational stability. In this study, we constructed and analyzed 10 scFv fragments derived from 10 representatives of FDA-approved mAbs to evaluate their physicochemical properties. Differential scanning calorimetry analysis showed that scFvs exhibited relatively high but varied thermostability, from 50 to 70°C of melting temperatures, and different unfolding cooperativity. Surface plasmon resonance analysis revealed that scFvs fragments that exhibit high stability and cooperative unfolding likely tend to maintain antigen binding. This study demonstrated the comprehensive physicochemical properties of scFvs derived from FDA-approved antibodies, providing insights into antibody design and development.
  • 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, Elsevier BV, 2024年03月07日, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • 中分子創薬が直面する課題とその克服に向けて
    宮地 弘幸; 前仲 勝実
    薬学雑誌, 144, 5, 527, 528, 公益社団法人 日本薬学会, 2024年, [国内誌]
    日本語, 研究論文(学術雑誌)
  • Symposium Review 実験及びシミュレーションのための統合データベースの開発—Development of Integrated Database for Experiments and Simulations
    山田 一作; 木村 直貴; 野村 尚生; 乙黒 聡子; 宮地 弘幸; 重田 育照; 前仲 勝実
    薬学雑誌 = Journal of the Pharmaceutical Society of Japan / 日本薬学会 編, 144, 5, 539, 543, 東京 : 日本薬学会, 2024年, [国内誌]
    日本語, 研究論文(学術雑誌), Researchers collect data and use various methods to organize it. Ensuring the reliability and reproducibility of data is crucial, and collaboration across different research fields is on the rise. However, when there is geographical distance, sharing data becomes a challenging task. Therefore, there is a need for the development of a mechanism for sharing data on the web. We have developed an integrated database to facilitate the sharing and management of research data, particularly focusing on small molecules. The integrated database serves as a platform for centralizing data related to small molecules, including their chemical structures, wet lab experimental data, simulation data, and more. It has been constructed as a web application, offering features such as library management for small molecules, registration and viewing of wet lab experiment results, generation of initial conformations for simulations, and data visualization. This enables researchers to efficiently share their research data and collaborate seamlessly, whether within their research group or via cloud-based access that allows project and team members to connect from anywhere. This integrated database plays a critical role in connecting wet lab experiments and simulations, enabling researchers to cross-reference and analyze experimental data comprehensively. It serves as an essential tool to advance research and foster idea generation.
  • The human immune checkpoint molecule, HLA-G2, induces tolerance in monocytes and dendritic cells via upregulation of PD-L1
    Ami Takahashi; Kimiko Kuroki; Naoyoshi Maeda; Mie Nieda; Katsumi Maenaka
    2023年10月18日
  • 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, [国際誌]
    英語, 研究論文(学術雑誌), 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.
  • Unprecedented spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions
    Hideo Fukuhara; Hisham M. Dokainish; Shunsuke Kita; Koshiro Tabata; Akira Takasu; Juha T. Huiskonen; Yuki Anraku; Toshiya Senda; David I. Stuart; Michihito Sasaki; Yasuko Orba; Yasuhiko Suzuki; Hirofumi Sawa; Katsumi Maenaka
    2023年10月11日
  • 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国内誌]
    英語, 研究論文(学術雑誌), 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, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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, Elsevier BV, 2023年06月, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌)
  • 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
    Research Square Platform LLC, 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, MDPI AG, 2022年08月03日, [国際誌]
    英語, 研究論文(学術雑誌), 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, (一社)日本生物物理学会, 2022年08月
    英語
  • 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, (一社)日本生物物理学会, 2022年08月
    英語
  • 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国内誌]
    英語, 研究論文(学術雑誌), 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, [国内誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, Elsevier BV, 2021年08月, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, Wiley, 2021年04月21日
    研究論文(学術雑誌)
  • 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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
    日本語
  • 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
    日本語
  • 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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, American Society for Microbiology, 2020年08月17日, [査読有り], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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, [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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, Wiley Blackwell, 2017年11月01日, [査読有り]
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国内誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • HIV-1 Control by NK Cells via Reduced Interaction between KIR2DL2 and HLA-C12:02/C14:03.
    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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • Structures and Functions of MHC-like Proteins
    Shunsuke Kita; Katsumi Maenaka
    Encyclopedia of Immunobiology, 2, 271, 278, Elsevier Inc., 2016年04月27日, [査読有り]
    英語, 論文集(書籍)内論文
  • 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]
  • 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]
    英語, 研究論文(学術雑誌)
  • 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, Springer Japan, 2016年01月01日, [査読有り]
    英語, 論文集(書籍)内論文
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • モルビリウイルス属の細胞侵入機構
    福原 秀雄; 陳 甦芮; 武田 森; 前仲 勝実
    薬学雑誌, 133, 5, 549, 559, 2013年05月, [査読有り], [国内誌]
    日本語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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]
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • [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], [国内誌]
    日本語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国内誌]
    日本語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国内誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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
    英語
  • 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
    英語
  • 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
    英語
  • 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
    英語
  • 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
    英語
  • 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
    英語
  • 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], [国際誌]
    英語, 研究論文(学術雑誌), 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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], [国際誌]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(国際会議プロシーディングス)
  • 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]
    英語, 研究論文(学術雑誌)
  • 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]
    英語, 研究論文(学術雑誌)
■ その他活動・業績
■ 書籍等出版物
■ 主な担当授業
  • 卒業研究準備実習Ⅰ, 2024年, 学士課程, 薬学部
  • 生命医薬科学概論, 2024年, 修士課程, 生命科学院
  • 卒業研究準備実習Ⅱ, 2024年, 学士課程, 薬学部
  • 大学院共通授業科目(一般科目):自然科学・応用科学, 2024年, 修士課程, 大学院共通科目
  • 薬学論文講読演習Ⅰ, 2024年, 学士課程, 薬学部
  • 大学院共通授業科目(一般科目):自然科学・応用科学, 2024年, 修士課程, 大学院共通科目
  • 薬学論文講読演習Ⅱ, 2024年, 学士課程, 薬学部
  • 大学院共通授業科目(一般科目):複合領域, 2024年, 修士課程, 大学院共通科目
  • 薬学論文講読演習Ⅲ, 2024年, 学士課程, 薬学部
  • 大学院共通授業科目(一般科目):自然科学・応用科学, 2024年, 修士課程, 大学院共通科目
  • 薬学総合演習, 2024年, 学士課程, 薬学部
  • 医療薬学特論, 2024年, 修士課程, 生命科学院
  • 薬学卒業研究, 2024年, 学士課程, 薬学部
  • 物理化学Ⅰ, 2024年, 学士課程, 薬学部
  • 薬科学演習, 2024年, 学士課程, 薬学部
  • 物理化学Ⅱ, 2024年, 学士課程, 薬学部
  • 薬科学論文講読演習, 2024年, 学士課程, 薬学部
  • 専門横断科目(一般科目):複合領域, 2024年, 学士課程, 専門横断科目
  • 薬科学卒業研究, 2024年, 学士課程, 薬学部
  • 生命科学研究, 2024年, 修士課程, 生命科学院
  • 生命科学実習, 2024年, 修士課程, 生命科学院
  • 生命科学論文講読Ⅰ, 2024年, 修士課程, 生命科学院
  • 生命科学論文講読Ⅱ, 2024年, 修士課程, 生命科学院
  • 生命科学特別研究, 2024年, 博士後期課程, 生命科学院
  • 生命科学文献講読, 2024年, 博士後期課程, 生命科学院
  • 臨床薬学特別研究, 2024年, 博士後期課程, 生命科学院
  • 臨床薬学論文講読Ⅰ, 2024年, 博士後期課程, 生命科学院
  • 臨床薬学論文講読Ⅱ, 2024年, 博士後期課程, 生命科学院
  • 臨床薬学論文執筆演習, 2024年, 博士後期課程, 生命科学院
■ 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
  • HLAと薬剤に着目した自己免疫性水疱症の発症機序の解明
    科学研究費助成事業
    2025年04月01日 - 2028年03月31日
    氏家 英之; 前仲 勝実; 村松 憲; 伊東 孝政
    日本学術振興会, 基盤研究(B), 北海道大学, 25K02670
  • ウイルス免疫逃避に対抗する液性免疫記憶の進化機序の解明
    革新的先端研究開発支援事業(AMED-CREST)
    2022年10月 - 2028年03月
    高橋宜聖; 山下和男; 前仲勝実; 黒田大祐
    AMED, 免疫記憶の理解とその制御に資する医療シーズの創出, 研究分担者, 23gm1810004
  • マテリアル・シンバイオシスのための生命物理化学
    科学研究費助成事業 学術変革領域研究(A)
    2020年11月19日 - 2025年03月31日
    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。
    日本学術振興会, 学術変革領域研究(A), 長崎大学, 20H05871
  • 細胞表面蛋白質の弱い分子認識の定量化・構造解析
    科学研究費助成事業 学術変革領域研究(A)
    2020年11月19日 - 2025年03月31日
    前仲 勝実; 望月 慎一
    日本学術振興会, 学術変革領域研究(A), 北海道大学, 20H05873
  • 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
    科学研究費助成事業 基盤研究(A)
    2019年04月01日 - 2024年03月31日
    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。
    日本学術振興会, 基盤研究(A), 北海道大学, 19H01171
  • 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
    科学研究費助成事業 挑戦的研究(萌芽)
    2018年06月29日 - 2020年03月31日
    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。
    日本学術振興会, 挑戦的研究(萌芽), 北海道大学, 18K19278
  • 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
    科学研究費助成事業 挑戦的萌芽研究
    2016年04月01日 - 2018年03月31日
    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。
    日本学術振興会, 挑戦的萌芽研究, 熊本大学, 16K15321
  • 毒ヘビ神経毒を標的とした咬傷治療薬創製の基盤研究
    科学研究費助成事業 若手研究(B)
    2016年04月01日 - 2018年03月31日
    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。
    日本学術振興会, 若手研究(B), 福岡大学, 16K18880
  • 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
    科学研究費助成事業 基盤研究(A)
    2015年04月01日 - 2018年03月31日
    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。
    日本学術振興会, 基盤研究(A), 北海道大学, 15H02384
  • 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
    科学研究費助成事業 基盤研究(B)
    2015年04月01日 - 2018年03月31日
    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。
    日本学術振興会, 基盤研究(B), 鹿児島大学, 15H04729
  • 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
    科学研究費補助金(基盤A)
    2015年04月 - 2018年03月
    前仲 勝実
    文部科学省, 研究代表者, 競争的資金
  • 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
    科学研究費助成事業 新学術領域研究(研究領域提案型)
    2010年04月01日 - 2016年03月31日
    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。
    日本学術振興会, 新学術領域研究(研究領域提案型), 奈良先端科学技術大学院大学, 22121001
  • 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
    科学研究費助成事業 新学術領域研究(研究領域提案型)
    2010年04月01日 - 2015年03月31日
    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。
    日本学術振興会, 新学術領域研究(研究領域提案型), 北海道大学, 研究代表者, 競争的資金, 22121007
  • HIV変異ペプチドによる免疫逃避機構の構造基盤
    科学研究費助成事業 基盤研究(B)
    2010年 - 2012年
    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。
    日本学術振興会, 基盤研究(B), 北海道大学, 研究代表者, 競争的資金, 22370043
  • 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
    1997年 - 2010年
    競争的資金
  • Molecular recongnition of cell surface receptors in immune system
    Grant-in-Aid for Scientific Research
    1997年 - 2010年
    競争的資金
  • 非古典的MHC細胞表面抗原Millの立体構造解析
    科学研究費助成事業 特定領域研究
    2008年 - 2009年
    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。
    日本学術振興会, 特定領域研究, 九州大学, 研究代表者, 競争的資金, 20057020
  • 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
    科学研究費助成事業 特定領域研究
    2008年 - 2009年
    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。
    日本学術振興会, 特定領域研究, 九州大学, 研究代表者, 競争的資金, 20060018
  • 多様な細胞表面抗原を認識するペア型免疫系受容体群
    科学研究費助成事業 基盤研究(B)
    2007年 - 2009年
    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。
    日本学術振興会, 基盤研究(B), 九州大学, 研究代表者, 競争的資金, 19370042
  • 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
    科学研究費補助金(特定領域研究)
    2003年 - 2008年
    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.
    文部科学省, 特定領域研究, 九州大学, 連携研究者, 競争的資金, 15083205
  • 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
    科学研究費助成事業 特定領域研究
    2006年 - 2007年
    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。
    日本学術振興会, 特定領域研究, 九州大学, 研究代表者, 競争的資金, 18060032
  • セレノシステイン特異的伸長因子SelBの結晶構造解析
    科学研究費助成事業 特定領域研究
    2005年 - 2006年
    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。
    日本学術振興会, 特定領域研究, 九州大学, 研究代表者, 競争的資金, 17026031
  • ペア型レセプターPILRの結晶構造解析
    科学研究費助成事業 特定領域研究
    2005年 - 2006年
    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。
    日本学術振興会, 特定領域研究, 九州大学, 研究代表者, 競争的資金, 17047032
  • 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
    科学研究費助成事業 基盤研究(B)
    2004年 - 2006年
    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.
    日本学術振興会, 基盤研究(B), 九州大学, 連携研究者, 競争的資金, 16370050
  • NK細胞活性化レセプターCD160の分子認識解明
    科学研究費助成事業 若手研究(B)
    2004年 - 2005年
    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。
    日本学術振興会, 若手研究(B), 九州大学, 研究代表者, 競争的資金, 16770078
  • 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
    科学研究費助成事業 萌芽研究
    2004年 - 2005年
    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.
    日本学術振興会, 萌芽研究, 九州大学, 連携研究者, 競争的資金, 16657045
  • 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
    科学研究費補助金(特定領域研究)
    2003年 - 2004年
    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...
    文部科学省, 特定領域研究, 九州大学, 連携研究者, 競争的資金, 15032239
  • 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
    科学研究費補助金(奨励研究(A), 若手研究(B))
    2001年 - 2002年
    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...
    文部科学省, 奨励研究(A), 若手研究(B), 国立遺伝学研究所->九州大学, 研究代表者, 競争的資金, 13780541
  • 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
    科学研究費補助金(特定領域研究(C))
    2000年 - 2000年
    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...
    文部科学省, 特定領域研究(C), 国立遺伝学研究所, 研究代表者, 競争的資金, 12208045
  • ファージディスプレイ系の改良
    競争的資金
  • 免疫系レセプターに対する阻害剤開発
    競争的資金
  • Improvement of Phage display system
    競争的資金
  • Development of inhibitors for immunoreceptors
    競争的資金
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  • 北大道新セミナー
    2019年05月23日 - 2019年05月23日
    講師