Researcher Database

Katsumi Maenaka
Faculty of Pharmaceutical Sciences Molecular Pharmaceutical Sciences Molecular and Cellular Biological Sciences

Researcher Profile and Settings


  • Faculty of Pharmaceutical Sciences Molecular Pharmaceutical Sciences Molecular and Cellular Biological Sciences

Job Title

  • Professor


  • Doctor of Engineering(The University of Tokyo)

J-Global ID

Research Interests

  • 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   

Research Areas

  • Life sciences / Immunology
  • Life sciences / Biophysics
  • Life sciences / Structural biochemistry

Academic & Professional Experience

  • 2010/04 - Today Hokkaido University Professor
  • 2000 - 2002 Assistant Professor, National Institute of Geretics
  • 2002 - 九州大学助教授
  • 2002 - Associate Professor, kyushu university
  • 2000 - 2001 National Institute of Genetics
  • 1997 - 1999 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
  • 1997 - 1999 Postdoctral Fellow (Human Frontier Science Program)
  • 1996 - 1997 日本学術振興会特別研究員
  • 1996 - 1997 Postdoctral Fellow


  •        - 1996  The University of Tokyo
  •        - 1996  The University of Tokyo  Graduate School, Division of Engineering  Chemistry and Biotechnology
  •        - 1991  The University of Tokyo  The Faculty of Engineering
  •        - 1991  The University of Tokyo  Faculty of Engineering  Division of Industrial Chemistry

Association Memberships

  • THE PHARMACEUTICAL SOCIETY OF JAPAN   THE JAPANESE SOCIETY FOR VIROLOGY   THE CRYSTALLOGRAPHIC SOCIETY OF JAPAN   THE JAPAN SOCIETY OF HEPATOLOGY   JAPANESE SOCIETY FOR CHEMICAL BIOLOGY   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   

Research Activities

Published Papers

  • The measles virus V protein binding site to STAT2 overlaps with that of IRF9
    Yuma Nagano, Aoi Sugiyama, Madoka Kimoto, Takuya Wakahara, Yasuyo Noguchi, Xinxin Jiang, Shinya Saijo, Nobutaka Shimizu, Nana Yabuno, Min Yao, Paul Gooley, Gregory Moseley, Takashi Tadokoro, Katsumi Maenaka, Toyoyuki Ose
    J. Virol. in press 2020/06 [Refereed][Not invited]
  • Rika Yamazaki, Atsushi Furukawa, Kouyuki Hirayasu, Kohei Yumoto, Hideo Fukuhara, Hisashi Arase, Katsumi Maenaka
    Journal of Biological Chemistry 2020/05/18 [Refereed][Not invited]
  • 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
    Biochem. Biophys. Res. Commun. in press 2020/05 [Refereed][Not invited]
  • Hiroshi Watanabe, Kimiko Kuroki, Chisato Yamada, Yukari Saburi, Naoyoshi Maeda, Katsumi Maenaka
    Human immunology 81 (4) 186 - 190 2020/04 [Refereed][Not invited]
    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.
  • 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 105631 - 105631 2020/03/22 [Refereed][Not invited]
    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.
  • Takashi Tadokoro, Cassandra M Modahl, Katsumi Maenaka, Narumi Aoki-Shioi
    Toxins 12 (3) 2020/03/12 [Refereed][Not invited]
    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.
  • 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 2020/03/05 [Refereed][Not invited]
    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.
  • 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 2020/01/24 [Refereed][Not invited]
    The human immunodeficiency virus (HIV) accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2-infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The di-leucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2-mediated endocytosis for CD3. The highly conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of simian immunodeficiency virus (SIV) Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide insights into the distinct pathogenesis of HIV-2 infection.
  • Takashi Tadokoro, Mst Lubna Jahan, Yuri Ito, Maino Tahara, Surui Chen, Atsutoshi Imai, Natsumi Sugimura, Koki Yoshida, Mizuki Saito, Toyoyuki Ose, Takao Hashiguchi, Makoto Takeda, Hideo Fukuhara, Katsumi Maenaka
    The FEBS journal 287 (1) 145 - 159 2020/01 [Refereed][Not invited]
    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.
  • 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 2020/01 [Refereed][Not invited]
    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.
  • 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 2019/12/15 [Refereed][Not invited]
    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.
  • 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 1475-6366 2019/12 [Refereed][Not invited]
    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.
  • Atsushi Furukawa, Manami Meguro, Rika Yamazaki, Hiroshi Watanabe, Ami Takahashi, Kimiko Kuroki, Katsumi Maenaka
    International journal of molecular sciences 20 (23) 2019/11/26 [Refereed][Not invited]
    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.
  • Takaya Shiraishi, Masahiro Sakaitani, Satoko Otsuguro, Katsumi Maenaka, Toshiharu Suzuki, Tadashi Nakaya
    International journal of molecular medicine 44 (4) 1574 - 1584 1107-3756 2019/10 [Refereed][Not invited]
    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.
  • 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) 2019/08/19 [Refereed][Not invited]
    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.
  • Masanori Noda, Kentaro Ishii, Mika Yamauchi, Hiroaki Oyama, Takashi Tadokoro, Katsumi Maenaka, Tetsuo Torisu, Susumu Uchiyama
    Journal of pharmaceutical sciences 108 (7) 2323 - 2333 0022-3549 2019/07 [Refereed][Not invited]
    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.
  • 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 0892-6638 2019/04 [Refereed][Not invited]
    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.
  • 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 0021-9258 2019/01/25 [Refereed][Not invited]
    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.
  • 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 1359-7345 2019/01/10 [Refereed][Not invited]
    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.
  • Wataru Kakuguchi, Takao Nomura, Tetsuya Kitamura, Satoko Otsuguro, Kazuhiro Matsushita, Masahiro Sakaitani, Katsumi Maenaka, Kanchu Tei
    Cancer medicine 7 (12) 6269 - 6280 2018/12 [Refereed][Not invited]
    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.
  • 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 2018/10/18 [Refereed][Not invited]
    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.
  • 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 2018/09/27 [Refereed][Not invited]
    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.
  • 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 1931-3128 2018/06/13 [Refereed][Not invited]
    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.
  • 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 0166-3542 2018/03 [Refereed][Not invited]
  • 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 0022-1899 2018/02 [Refereed][Not invited]
  • Atsushi Furukawa, Kosuke Kakita, Tomoki Yamada, Mikihiro Ishizuka, Jiro Sakamoto, Nanao Hatori, Naoyoshi Maeda, Fumina Ohsaka, Takashi Saitoh, Takao Nomura, Kimiko Kuroki, Hisanori Nambu, Hisashi Arase, Shigeki Matsunaga, Masahiro Anada, Toyoyuki Ose, Shunichi Hashimoto, Katsumi Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 292 (51) 21128 - 21136 0021-9258 2017/12 [Refereed][Not invited]
    Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor alpha (PILR alpha) on immune cells. PILR alpha belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)- like family, members of which bind SA. PILR alpha is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILR alpha complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILR alpha binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILR alpha. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILR alpha complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILR alpha and for the rational design of herpes simplex virus-1 entry inhibitors.
  • Yoshito Abe, Seijiro Shioi, Shunsuke Kita, Hikaru Nakata, Katsumi Maenaka, Daisuke Kohda, Tsutomu Katayama, Tadashi Ueda
    FEBS LETTERS 591 (22) 3805 - 3816 1873-3468 2017/11 [Refereed][Not invited]
    The heat shock protein HspQ (YccV) of Escherichia coli has been proposed to participate in the retardation of replication initiation in cells with the dnaA508 allele. In this study, we have determined the 2.5-angstrom- resolution X-ray structure of the trimer of HspQ, which is also the first structure of a member of the YccV superfamily. The acidic character of the HspQ trimer suggests an interaction surface with basic proteins. From these results, we discuss the cellular function of HspQ, including its relationship with the DnaA508 protein.
  • Abe Y, Shioi S, Kita S, Nakata H, Maenaka K, Kohda D, Katayama T, Ueda T
    FEBS letters 591 (22) 3805 - 3816 0014-5793 2017/11 [Refereed][Not invited]
  • 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) 1420-3049 2017/10 [Refereed][Not invited]
    Due to their lower production cost compared with monoclonal antibodies, single-chain variable fragments (scFvs) have potential for use in several applications, such as for diagnosis and treatment of a range of diseases, and as sensor elements. However, the usefulness of scFvs is limited by inhomogeneity through the formation of dimers, trimers, and larger oligomers. The scFv protein is assumed to be in equilibrium between the closed and open states formed by assembly or disassembly of VH and VL domains. Therefore, the production of an scFv with equilibrium biased to the closed state would be critical to overcome the problem in inhomogeneity of scFv for industrial or therapeutic applications. In this study, we obtained scFv clones stable against GA-pyridine, an advanced glycation end-product (AGE), by using a combination of a phage display system and random mutagenesis. Executing the bio-panning at 37 degrees C markedly improved the stability of scFvs. We further evaluated the radius of gyration by small-angle X-ray scattering (SAXS), obtained compact clones, and also visualized open-close dynamics of these scFvs by high-speed atomic force microscopy (HS-AFM), revealing that one of the compact clones was biased to the closed state. Finally, nuclear magnetic resonance (NMR) analysis revealed that peak intensity and line width became homogeneous, supporting that dynamic features and/or formation of oligomers was improved in the thus-obtained clone. These findings should contribute to the future industrial and therapeutic use of scFvs.
  • Naoyoshi Maeda, Katsumi Maenaka
    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES 18 (10) 1422-0067 2017/10 [Refereed][Not invited]
    Matricellular proteins differ from other classical extracellular matrix proteins; for instance, they are transiently expressed as soluble proteins rather than being constitutively expressed in pathological conditions, such as acute viral infections. Accumulating studies have revealed that matricellular proteins, including osteopontin and tenascin-C, both of which interact with integrin heterodimers, are involved in inflammatory diseases, autoimmune disorders, and cancers. The concentrations of these matricellular proteins are elevated in the plasma of patients with certain types of cancers, indicating that they play important roles in oncogenesis. Chronic viral infections are associated with certain cancers, which are distinct from non-viral cancers. Viral oncogenes play critical roles in the development and progression of such cancers. It is vital to investigate the mechanisms of tumorigenesis and, particularly, the mechanism by which viral proteins induce tumor progression. Viral proteins have been shown to influence not only the viral-infected cancer cells, but also the stromal cells and matricellular proteins that constitute the extracellular matrix that surrounds tumor tissues. In this review, we summarize the recent progress on the involvement of matricellular proteins in oncogenic virus-induced cancers to elucidate the mechanism of oncogenesis and consider the possible role of matricellular proteins as therapeutic targets in virus-induced cancers.
  • Naoyoshi Maeda, Chisato Yamada, Ami Takahashi, Kimiko Kuroki, Katsumi Maenaka
    INTERNATIONAL IMMUNOPHARMACOLOGY 50 202 - 207 1567-5769 2017/09 [Refereed][Not invited]
    Human leukocyte antigen (HLA)-G is an immune checkpoint molecule that plays critical roles in immune response and in triggering inhibitory signaling to immune cells such as T cells, natural killer cells, and antigen presenting cells. Thus, the application of HLA-G can be considered for treating immune response-related inflammatory disorders. We have previously reported that treatment with HLA-Gl and HLA-G2 ameliorates the joint swelling associated with collagen-induced arthritis of DBA/1 mice, an animal model for rheumatoid arthritis. In this study, we further investigated the effects of HLA-G1 on atopic dermatitis (AD), the most common inflammatory skin disorder. AD-like lesions were induced with the extract of the house dust mite Dermatophagoides farinae in NC/Nga mice. Continuous administration of HLA-G1 ameliorated the AD-like skin lesions in the mice. Furthermore, production of immunoglobulin E, interleukin (IL)-13, and IL-17A was significantly reduced in HLA-Gl-treated mice, suggesting a Th2/Th17-mediated immune-inhibitory function of HLA-G1 in vivo. Our studies shed light on novel therapeutic strategies with recombinant HLA-G proteins for immune reaction-mediated chronic inflammatory disorders.
  • 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 1465-7392 2017/07 [Refereed][Not invited]
    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.
  • 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 0022-1767 2017/05 [Refereed][Not invited]
    HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and (beta 2-microglobulin (beta 2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the alpha 2 domain-deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown. In this study, we unexpectedly found that HLA-G2 naturally forms a beta 2m-free and nondisulfide-linked homodimer, which is in contrast to the disulfide-bonded beta 2m-associated HLA-G1 homodimer. Furthermore, single-particle analysis, using electron microscopy, revealed that the overall structure and domain organization of the HLA-G2 homodimer resemble those of the HLA class II heterodimer. The HLA-G2 homodimer binds to leukocyte Ig-like receptor B2 with slow dissociation and a significant avidity effect. These findings provide novel insights into leukocyte Ig-like receptor B2-mediated immune regulation by the HLA-G2 isoform, as well as the gene evolution of HLA classes.
  • 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 0960-894X 2017/05 [Refereed][Not invited]
    We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors. (C) 2017 Elsevier Ltd. All rights reserved.
  • Masaki Inoue, Daisuke Ando, Haruhiko Kamada, Shintaro Taki, Mayumi Niiyama, Yohei Mukai, Takashi Tadokoro, Katsumi Maenaka, Taisuke Nakayama, Yuji Kado, Tsuyoshi Inoue, Yasuo Tsutsumi, Shin-ichi Tsunoda
    JOURNAL OF BIOLOGICAL CHEMISTRY 292 (16) 6438 - 6451 0021-9258 2017/04 [Refereed][Not invited]
    Tumor necrosis factor-alpha (TNF) exerts its biological effect through two types of receptors, p55 TNF receptor (TNFR1) and p75 TNF receptor (TNFR2). An inflammatory response is known to be induced mainly by TNFR1, whereas an anti-inflammatory reaction is thought to be mediated by TNFR2 in some autoimmune diseases. We have been investigating the use of an antagonistic TNF mutant (TNFR1-selective antagonistic TNF mutant (R1antTNF)) to reveal the pharmacological effect of TNFR1-selective inhibition as a new therapeutic modality. Here, we aimed to further improve and optimize the activity and behavior of this mutant protein both in vitro and in vivo. Specifically, we examined a trimeric structural fusion of R1antTNF, formed via the introduction of short peptide linkers, as a strategy to enhance bioactivity and molecular stability. By comparative analysis with R1antTNF, the trimeric fusion, referred to as single-chain R1antTNF (scR1antTNF), was found to retain in vitro molecular properties of receptor selectivity and antagonistic activity but displayed a marked increase in thermal stability. The residence time of scR1antTNF in vivo was also significantly prolonged. Furthermore, molecular modification using polyethylene glycol (PEG) was easily controlled by limiting the number of reactive sites. Taken together, our findings show that scR1antTNF displays enhanced molecular stability while maintaining biological activity compared with R1antTNF.
  • 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 1554-8929 2016/11 [Refereed][Not invited]
    The CD1d protein is a nonpolymorphic MHC class I-like protein that controls the activation of natural killer T (NKT) cells through the presentation of self- and foreign-lipid ligands, glycolipids, or phospholipids, leading to the secretion of various cytokines. The CD1d contains a large hydrophobic lipid binding pocket: the A' pocket of CD1d, which recognizes hydrophobic moieties of the ligands, such as long fatty acyl chains. Although lipid protein interactions typically rely on hydrophobic interactions between lipid chains and the hydrophobic sites of proteins, we showed that the small polar regions located deep inside the hydrophobic A' pocket could be used for the modulation of the lipid binding. A series of the ligands, alpha-galactosyl ceramide (alpha-GalCer) derivatives containing polar groups in the acyl chain, was synthesized, and the structure activity relationship studies demonstrated that simple modification from a methylene to an amide group in the long fatty acyl chain, when introduced at optimal positions, enhanced the CD1d recognition of the glycolipid ligands. Formation of hydrogen bonds between the amide group and the polar residues was supported by molecular dynamics (MD) simulations and WaterMap calculations. The computational studies suggest that localized hydrating water molecules may play an important role in the ligand recognition. Here, the results showed that confined polar residues in the large hydrophobic lipid binding pockets of the proteins could be potential targets to modulate the affinity for its ligands.
  • 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 0918-6158 2016/11 [Refereed][Not invited]
    Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILR alpha) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRa were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.
  • Tahara M, Bürckert JP, Kanou K, Maenaka K, Muller CP, Takeda M
    Viruses 8 (11) 2016/11 [Refereed][Not invited]
  • Zhansong Lin, Kimiko Kuroki, Nozomi Kuse, Xiaoming Sun, Tomohiro Akahoshi, Ying Qi, Takayuki Chikata, Takuya Naruto, Madoka Koyanagi, Hayato Murakoshi, Hiroyuki Gatanaga, Shinichi Oka, Mary Carrington, Katsumi Maenaka, Masafumi Takiguchi
    CELL REPORTS 17 (9) 2210 - 2220 2211-1247 2016/11 [Refereed][Not invited]
    Natural killer (NK) cells control viral infection in part through the interaction between killer cell immunoglobulin-like receptors (KIRs) and their human leukocyte antigen (HLA) ligands. We investigated 504 anti-retroviral (ART)-free Japanese patients chronically infected with HIV-1 and identified two KIR/HLA combinations, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03, that impact suppression of HIV-1 replication. KIR2DL2(+) NK cells suppressed viral replication in HLA-C*14:03(+) or HLA-C*12:02(+) cells to a significantly greater extent than did KIR2DL2(-) NK cells in vitro. Functional analysis showed that the binding between HIV-1-derived peptide and HLA-C*14:03 or HLA-C*12:02 influenced KIR2DL2(+) NK cell activity through reduced expression of the peptide-HLA (pHLA) complex on the cell surface (i.e., reduced KIR2DL2 ligand expression), rather than through reduced binding affinity of KIR2DL2 to the respective pHLA complexes. Thus, KIR2DL2/HLA-C*12:02 and KIR2DL2/HLA-C*14:03 compound genotypes have protective effects on control of HIV-1 through a mechanism involving KIR2DL2-mediated NK cell recognition of virusinfected cells, providing additional understanding of NK cells in HIV-1 infection.
  • 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 0198-8859 2016/09 [Refereed][Not invited]
    HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and beta 2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1 month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B. (C) 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
  • Atsutoshi Imai, Takashi Tadokoro, Shunsuke Kita, Masataka Horiuchi, Hideo Fukuhara, Katsumi Maenaka
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 478 (2) 580 - 585 0006-291X 2016/09 [Refereed][Not invited]
    The BacMam system uses modified insect viruses (baculoviruses) as vehicles to efficiently deliver genes for expression in mammalian cells. The technique can be widely applied to large-scale recombinant protein production with appropriate modifications, high-throughput screening platforms for cell-based assays, and the delivery of large genes. The silkworm system is often employed as a rapid and cost-effective approach for recombinant baculovirus generation. Here we have developed the novel BacMam system using silkworm baculovirus, and shown the successful expression of EGFP in mammalian cells. The transduction to mammalian cells via the BacMam system was improved by adding phosphate buffered saline and sodium butyrate to the culture medium and lowering the temperature after viral infection. This study provides an alternative gene delivery system for mammalian cells, which has various potential applications, including efficient native protein production and gene therapy. (C) 2016 Elsevier Inc. All rights reserved.
  • 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 1066-5099 2016/08 [Refereed][Not invited]
    Glioblastoma (GBM), one of the most malignant human cancers, frequently recurs despite multi-modal treatment with surgery and chemo/radiotherapies. GBM-initiating cells (GICs) are the likely cell-of-origin in recurrences, as they proliferate indefinitely, form tumors in vivo, and are resistant to chemo/radiotherapies. It is therefore crucial to find chemicals that specifically kill GICs. We established temozolomide (the standard medicine for GBM)-resistant GICs (GICRs) and used the cells for chemical screening. Here, we identified 1-(3-C-ethynyl-beta-D-ribopentofuranosyl) uracil (EUrd) as a selective drug for targeting GICRs. EUrd induced the death in GICRs more effectively than their parental GICs, while it was less toxic to normal neural stem cells. We demonstrate that the cytotoxic effect of EUrd on GICRs partly depended on the increased expression of uridine-cytidine kinase-like 1 (UCKL1) and the decreased one of 5'-nucleotidase cytosolic III (NT5C3), which regulate uridine-monophosphate synthesis positively and negatively respectively. Together, these findings suggest that EUrd can be used as a new therapeutic drug for GBM with the expression of surrogate markers UCKL1 and NT5C3.
  • Md. Imran Hossain, Shinya Hanashima, Takuto Nomura, Sebastien Lethu, Hiroshi Tsuchikawa, Michio Murata, Hiroki Kusaka, Shunsuke Kita, Katsumi Maenaka
    BIOORGANIC & MEDICINAL CHEMISTRY 24 (16) 3687 - 3695 0968-0896 2016/08 [Refereed][Not invited]
    A novel series of CD1d ligand alpha-galactosylceramides (alpha-GalCers) were synthesized by incorporation of the heavy atoms Br and Se in the acyl chain backbone of alpha-galactosyl-N-cerotoylphytosphingosine. The synthetic analogues are potent CD1d ligands and stimulate mouse invariant natural killer T (iNKT) cells to selectively enhance Th1 cytokine production. These synthetic analogues would be efficient X-ray crystallographic probes to disclose precise atomic positions of alkyl carbons and lipid-protein interactions in KRN7000/CD1d complexes. (C) 2016 Elsevier Ltd. All rights reserved.
  • Maino Tahara, Jean-Philippe Burckert, Kazuhiko Kanou, Katsumi Maenaka, Claude P. Muller, Makoto Takeda
    VIRUSES-BASEL 8 (8) 1999-4915 2016/08 [Refereed][Not invited]
    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.
  • 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 1462-0324 2016/06 [Refereed][Not invited]
    Objective. Phosphatidylserine-dependent, also called aPS-PT, recognizes the phosphati dylserine-prothrombin complex, which is associated with APS. We have previously reported that aPS-PT induces tissue factor (TF) expression on monocytes through the p38 mitogen-activated protein kinase pathway. However, the cell surface interaction between prothrombin and aPS-PT, which is involved in the activation of cell-signalling pathways, has remained unknown. The objective of this study was to identify membrane proteins involved in the binding of prothrombin and aPS-PT to monocyte surfaces as well as the induction of TF expression. Methods. RAW264.7 cells with FLAG-tagged prothrombin were incubated and separated using affinity chromatography with anti-FLAG antibody-conjugated Sepharose beads. Immunopurified proteins were then analysed by an online nano-liquid chromatography-tandem mass spectrometry. The binding between prothrombin and the identified protein, ribophorin II (RPN2), was analysed by ELISA and surface plasmon resonance. To elucidate the role of RPN2 in TF expression, the TF mRNA level in RAW264.7 cells treated with RPN2 small interfering RNA was determined by quantitative real-time PCR (qPCR). Results. RPN2 was identified as a candidate molecule involved in the binding of prothrombin to the cell surface. The binding between prothrombin and RPN2 was confirmed by ELISA and surface plasmon resonance. RAW264.7 cells treated with RPN2 small interfering RNA showed significant reduction of the TF expression mediated by prothrombin and a mouse monoclonal aPS-PT. Conclusion. We identified that RPN2 is one of the prothrombin-binding proteins on monocyte surfaces, suggesting that RPN2 is involved in the pathophysiology of thrombosis in patients with APS.
  • 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 0065-7727 2016/03/13 [Refereed][Not invited]
  • 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  2016/03 [Refereed][Not invited]
  • 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  1742-4690 2015/11 [Refereed][Not invited]
    Background: Adult T-cell leukemia (ATL) is a CD4(+) T-cell neoplasm with a poor prognosis. A previous study has shown that there is a strong correlation between the secreted matricellular protein osteopontin (OPN) level and disease severity in ATL patients. Here, we investigated the role of OPN in ATL pathogenesis and the possible application of anti-OPN monoclonal antibody (mAb) for ATL immunotherapy in NOD/Shi-scid, IL-2Rg(null) (NOG) mice. Results: Subcutaneous inoculation of ATL cell lines into NOG mice increased the plasma level of OPN, which significantly correlated with metastasis of the inoculated cells and survival time. Administration of an SVVYGLR motifrecognizing anti-OPN mAb resulted in inhibition not only of tumor growth but also of tumor invasion and metastasis. The number of fibroblast activating protein-positive fibroblasts was also reduced by this mAb. We then co-inoculated mouse embryonic fibroblasts (MEFs) isolated from wild-type (WT) or OPN knockout mice together with ATL-derived TL-OmI cells into the NOG mice. The mice co-inoculated with WT MEFs displayed a significant decrease in survival relative to those injected with TL-OmI cells alone and the absence of OPN in MEFs markedly improved the survival rate of TL-OmI-inoculated mice. In addition, tumor volume and metastasis were also reduced in the absence of OPN. Conclusion: We showed that the xenograft NOG mice model can be a useful system for assessment of the physiological role of OPN in ATL pathogenesis. Using this xenograft model, we found that fibroblast-derived OPN was involved in tumor growth and metastasis, and that this tumor growth and metastasis was significantly suppressed by administration of the anti-OPN mAbs. Our findings will lead to a novel mAb-mediated immunotherapeutic strategy targeting against the interaction of OPN with integrins on the tumor of ATL patients.
  • 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 0925-2738 2015/10 [Refereed][Not invited]
  • Hajime Yamauchi, Takanori Matsumaru, Tomoko Morita, Susumu Ishikawa, Katsumi Maenaka, Ichigaku Takigawa, Kentaro Semba, Shunsuke Kon, Yasuyuki Fujita
    SCIENTIFIC REPORTS 5 15336  2045-2322 2015/10 [Refereed][Not invited]
    Recent studies have revealed that cell competition can occur between normal and transformed epithelial cells; normal epithelial cells recognize the presence of the neighboring transformed cells and actively eliminate them from epithelial tissues. Here, we have established a brand-new high-throughput screening platform that targets cell competition. By using this platform, we have identified Rebeccamycin as a hit compound that specifically promotes elimination of RasV12-transformed cells from the epithelium, though after longer treatment it shows substantial cytotoxic effect against normal epithelial cells. Among several Rebeccamycin-derivative compounds, we have found that VC1-8 has least cytotoxicity against normal cells but shows the comparable effect on the elimination of transformed cells. This cell competition-promoting activity of VC1-8 is observed both in vitro and ex vivo. These data demonstrate that the cell competition-based screening is a promising tool for the establishment of a novel type of cancer preventive medicine.
  • Hidetaka Akita, Taichi Nakatani, Kimiko Kuroki, Katsumi Maenaka, Kota Tange, Yuta Nakai, Hideyoshi Harashima
    INTERNATIONAL JOURNAL OF PHARMACEUTICS 490 (1-2) 142 - 145 0378-5173 2015/07 [Refereed][Not invited]
    Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold. (C) 2015 Elsevier B.V. All rights reserved.
  • Shintaro Mikuni, Kota Kodama, Akira Sasaki, Naoki Kohira, Hideki Maki, Masaharu Munetomo, Katsumi Maenaka, Masataka Kinjo
    PLOS ONE 10 (7) e0130933  1932-6203 2015/07 [Refereed][Not invited]
    FtsZ is an attractive target for antibiotic research because it is an essential bacterial cell division protein that polymerizes in a GTP-dependent manner. To find the seed chemical structure, we established a high-throughput, quantitative screening method combining fluorescence cross-correlation spectroscopy (FCCS) and surface plasmon resonance (SPR). As a new concept for the application of FCCS to polymerization-prone protein, Staphylococcus aureus FtsZ was fragmented into the N-terminal and C-terminal, which were fused with GFP and mCherry (red fluorescent protein), respectively. By this fragmentation, the GTP-dependent head-to-tail dimerization of each fluorescent labeled fragment of FtsZ could be observed, and the inhibitory processes of chemicals could be monitored by FCCS. In the first round of screening by FCCS, 28 candidates were quantitatively and statistically selected from 495 chemicals determined by in silico screening. Subsequently, in the second round of screening by FCCS, 71 candidates were also chosen from 888 chemicals selected via an in silico structural similarity search of the chemicals screened in the first round of screening. Moreover, the dissociation constants between the highest inhibitory chemicals and Staphylococcus aureus FtsZ were determined by SPR. Finally, by measuring the minimum inhibitory concentration, it was confirmed that the screened chemical had antibacterial activity against Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA).
  • 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 0014-2980 2015/06 [Refereed][Not invited]
    Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended -sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.
  • 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 0022-538X 2015/03 [Refereed][Not invited]
    Mumps virus (MuV) infection induces formation of cytoplasmic inclusion bodies (IBs). Growing evidence indicates that IBs are the sites where RNA viruses synthesize their viral RNA. However, in the case of MuV infection, little is known about the viral and cellular compositions and biological functions of the IBs. In this study, pulldown purification and N-terminal amino acid sequencing revealed that stress-inducible heat shock protein 70 (Hsp72) was a binding partner of MuV phosphoprotein (P protein), which was an essential component of the IB formation. Immunofluorescence and immunoblotting analyses revealed that Hsp72 was colocalized with the P protein in the IBs, and its expression was increased during MuV infection. Knockdown of Hsp72 using small interfering RNAs (siRNAs) had little, if any, effect on viral propagation in cultured cells. Knockdown of Hsp72 caused accumulation of ubiquitinated P protein and delayed P protein degradation. These results show that Hsp72 is recruited to IBs and regulates the degradation of MuV P protein through the ubiquitin-proteasome pathway. IMPORTANCE Formation of cytoplasmic inclusion bodies (IBs) is a common characteristic feature in mononegavirus infections. IBs are considered to be the sites of viral RNA replication and transcription. However, there have been few studies focused on host factors recruited to the IBs and their biological functions. Here, we identified stress-inducible heat shock protein 70 (Hsp72) as the first cellular partner of mumps virus (MuV) phosphoprotein (P protein), which is an essential component of the IBs and is involved in viral RNA replication/transcription. We found that the Hsp72 mobilized to the IBs promoted degradation of the MuV P protein through the ubiquitin-proteasome pathway. Our data provide new insight into the role played by IBs in mononegavirus infection.
  • Hideo Fukuhara, Atsushi Furukawa, Katsumi Maenaka
    STRUCTURE 22 (12) 1694 - 1696 0969-2126 2014/12 [Refereed][Not invited]
    C-type lectin-like receptor 2 (CLEC-2) is a member of the C-type lectin (like) receptor (CLR) family that uses a Ca2+ binding domain to bind specific glycans. However, in this issue of Structure, Nagae and colleagues report on how the structures of CLEC-2 in complex with a glycopeptide podoplanin and a snake venom protein, rhodocytin, show a different mode of binding.
  • 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  2041-1723 2014/12 [Refereed][Not invited]
    Interleukin (IL)-18 is a proinflammatory cytokine that belongs to the IL-1 family and plays an important role in inflammation. The uncontrolled release of this cytokine is associated with severe chronic inflammatory disease. IL-18 forms a signalling complex with the IL-18 receptor alpha (R alpha) and beta (R beta) chains at the plasma membrane, which induces multiple inflammatory cytokines. Here, we present a crystal structure of human IL-18 bound to the two receptor extracellular domains. Generally, the receptors' recognition mode for IL-18 is similar to IL-1 beta; however, certain notable differences were observed. The architecture of the IL-18 receptor second domain (D2) is unique among the other IL-1R family members, which presumably distinguishes them from the IL-1 receptors that exhibit a more promiscuous ligand recognition mode. The structures and associated biochemical and cellular data should aid in developing novel drugs to neutralize IL-18 activity.
  • 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 0021-9258 2014/09 [Refereed][Not invited]
    Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.
  • 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  2045-2322 2014/08 [Refereed][Not invited]
    Protein kinase B (PKB) also known as Akt is involved in many signal transduction pathways. As alterations of the PKB pathway are found in a number of human malignancies, PKB is considered an important drug target for cancer therapy. However, production of sufficient amounts of active PKB for biochemical and structural studies is very costly because of the necessity of using a higher organism expression system to obtain phosphorylated PKB. Here, we report efficient production of active PKB alpha using the BmNPV bacmid expression system with silkworm larvae. Following direct injection of bacmid DNA, recombinant PKB alpha protein was highly expressed in the fat bodies of larvae, and could be purified using a GST-tag and then cleaved. A final yield of approximately 1 mg PKB alpha/20 larvae was recorded. Kinase assays showed that the recombinant PKB alpha possessed high phosphorylation activity. We further confirmed phosphorylation on the activation loop by mass spectrometric analysis. Our results indicate that the silkworm expression system is of value for preparation of active-form PKB alpha with phosphorylation on the activation loop. This efficient production of the active protein will facilitate further biochemical and structural studies and stimulate subsequent drug development.
  • Kimiko Kuroki, Jing Wang, Toyoyuki Ose, Munechika Yamaguchi, Shigekazu Tabata, Nobuo Maita, Seiko Nakamura, Mizuho Kajikawa, Amane Kogure, Takeshi Satoh, Hisashi Arase, Katsumi Maenaka
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111 (24) 8877 - 8882 0027-8424 2014/06 [Refereed][Not invited]
    Paired Ig-like type 2 receptor a (PILR alpha) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILR alpha. Furthermore, we determined the crystal structures of PILR alpha and its complex with an sTn and its attached peptide region. The structures show that PILR alpha exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.
  • 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 0022-538X 2014/05 [Refereed][Not invited]
    Proteolytic cleavage of the hemagglutinin (HA) protein is essential for influenza A virus (IAV) to acquire infectivity. This process is mediated by a host cell protease(s) in vivo. The type II transmembrane serine protease TMPRSS2 is expressed in the respiratory tract and is capable of activating a variety of respiratory viruses, including low-pathogenic (LP) IAVs possessing a single arginine residue at the cleavage site. Here we show that TMPRSS2 plays an essential role in the proteolytic activation of LP IAVs, including a recently emerged H7N9 subtype, in vivo. We generated TMPRSS2 knockout (KO) mice. The TMPRSS2 KO mice showed normal reproduction, development, and growth phenotypes. In TMPRSS2 KO mice infected with LP IAVs, cleavage of HA was severely impaired, and consequently, the majority of LP IAV progeny particles failed to gain infectivity, while the viruses were fully activated proteolytically in TMPRSS2(+/+) wild-type (WT) mice. Accordingly, in contrast to WT mice, TMPRSS2 KO mice were highly tolerant of challenge infection by LP IAVs (H1N1, H3N2, and H7N9) with >= 1,000 50% lethal doses (LD50) for WT mice. On the other hand, a high-pathogenic H5N1 subtype IAV possessing a multibasic cleavage site was successfully activated in the lungs of TMPRSS2 KO mice and killed these mice, as observed for WT mice. Our results demonstrate that recently emerged H7N9 as well as seasonal IAVs mainly use the specific protease TMPRSS2 for HA cleavage in vivo and, thus, that TMPRSS2 expression is essential for IAV replication in vivo.
  • Yuki Satoh, Shinsuke Miki, Toyoyuki Ose, Azusa Oikawa, Katsumi Maenaka, Ryouhei Terauchi, Kozo Asano, Teruo Sone
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 78 (4) 680 - 686 0916-8451 2014/04 [Refereed][Not invited]
    The avirulence gene AVR-Pia of Magnaporthe oryzae, which induces a hypersensitive reaction in rice cultivars containing the resistance gene Pia, was expressed in Escherichia coli. AVR-Pia protein was collected as inclusion bodies, denatured, and refolded. Finally, recombinant AVR-Pia (rAVR-Pia) protein was purified by column chromatography. Infiltration of rAVR-Pia triggered cell browning in the leaves of rice cultivar Aichiasahi (Pia), with accumulation of H2O2 and induction of PR1a expression in rice. On the other hand, these reactions were not observed in Shin-2 (pia) leaves after the same treatment. This observation indicated that rAVR-Pia had the function of an avirulence protein. rAVR-Pia was used for immunization of a rabbit, and anti-AVR-Pia antiserum was prepared. The specificity of this antibody was appraised by detecting native AVR-Pia in the inoculated leaf sheath extract using Western blotting in combination with immunoprecipitation. Native AVR-Pia was successfully detected, and its molecular weight was estimated to be 7.4 kDa, indicating signal peptide cleavage. Additionally, secreted native AVR-Pia was quantified as 3.7 ng/g rice sheath.
  • Atsuko Ibusuki, Kazuhiro Kawai, Shigeru Yoshida, Youhei Uchida, Ayano Nitahara-Takeuchi, Kimiko Kuroki, Mizuho Kajikawa, Toyoyuki Ose, Katsumi Maenaka, Masanori Kasahara, Takuro Kanekura
    JOURNAL OF INVESTIGATIVE DERMATOLOGY 134 (2) 396 - 404 0022-202X 2014/02 [Refereed][Not invited]
    Murine epidermal gamma delta T cells, known as dendritic epidermal T cells (DETCs), survey tissue stress through the invariant T-cell receptor (TCR) and non-clonotypic receptors such as NKG2D. NKG2D signaling via the DAP10-phosphatidylinositol 3-kinase (PI3K) pathway directly stimulates cytotoxicity in natural killer (NK) cells and costimulates CD8(+) T cells to augment TCR signals. In activated murine NK cells, NKG2D signals also via the DAP12-Syk/ZAP70 pathway that triggers both cytotoxicity and cytokine production. It remains controversial whether NKG2D on DETCs is a primary activating receptor or functions only as a costimulatory receptor, and signaling pathways initiated by NKG2D ligation in DETCs have not been analyzed. We show that stimulation of short-term DETC lines with recombinant NKG2D ligands triggers degranulation (exocytosis of cytotoxic granules) via the PI3K-dependent signaling pathway, but does not induce cytokine production or Syk/ZAP70 activation. Coengagement of TCR or Syk/ZAP70 signaling was not crucial for DETC-mediated killing of NKG2D ligand-expressing target cells. Thus, NKG2D can function as a coactivating stress receptor that directly triggers cytotoxicity in DETCs, at least after priming, via the PI3K-dependent, Syk/ZAP70-independent signaling pathway.
  • Atsushi Minami, Toyoyuki Ose, Kyohei Sato, Azusa Oikawa, Kimiko Kuroki, Katsumi Maenaka, Hiroki Oguri, Hideaki Oikawa
    ACS CHEMICAL BIOLOGY 9 (2) 562 - 569 1554-8929 2014/02 [Refereed][Not invited]
    Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 X 10(-5) M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.
  • Ryo Kanda, Yoichi Sutoh, Jun Kasamatsu, Katsumi Maenaka, Masanori Kasahara, Toyoyuki Ose
    PLOS ONE 9 (1) e85875  1932-6203 2014/01 [Refereed][Not invited]
    Jawless vertebrates represented by lampreys and hagfish use variable lymphocyte receptors (VLRs) as antigen receptors to mount adaptive immune responses. VLRs generate diversity that is comparable to immunoglobulins and T-cell receptors by a gene conversion-like mechanism, which is mediated by cytosine deaminases. Currently, three types of VLRs, VLRA, VLRB, and VLRC, have been identified in lampreys. Crystal structures of VLRA and VLRB in complex with antigens have been reported recently, but no structural information is available for VLRC. Here, we present the first crystal structure of VLRC from the Japanese lamprey (Lethenteron japonicum). Similar to VLRA and VLRB, VLRC forms a typical horseshoe-like solenoid structure with a variable concave surface. Strikingly, its N-terminal cap has a long loop with limited sequence variability that protrudes toward the concave surface, which is the putative antigen-binding surface. Furthermore, as predicted previously, its C-terminal cap lacks a highly variable protruding loop that plays an important role in antigen recognition by lamprey VLRA and VLRB. Recent work suggests that VLRC+ lymphocytes in jawless vertebrates might be akin to gamma delta T cells in jawed vertebrates. Structural features of lamprey VLRC described here suggest that it may recognize antigens in a unique manner.
  • 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 0022-538X 2013/11 [Refereed][Not invited]
    Here, we show that human parainfluenza viruses and Sendai virus (SeV), like other respiratory viruses, use TMPRSS2 for their activation. The membrane fusion proteins of respiratory viruses often possess serine and glutamine residues at the P2 and P3 positions, respectively, but these residues were not critical for cleavage by TMPRSS2. However, mutations of these residues affected SeV growth in specific epithelial cell lines, suggesting the importance of these residues for SeV replication in epithelia.
  • Kurimoto E, Kuroki K, Yamaguchi Y, Yagi-Utsumi M, Igaki T, Iguchi T, Maenaka K, Kato K
    Molecular immunology 55 (3-4) 393 - 399 0161-5890 2013/10 [Refereed][Not invited]
  • 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 0027-8424 2013/10 [Refereed][Not invited]
    Mincle [macrophage inducible Ca2+-dependent (C-type) lectin; CLEC4E] and MCL (macrophage C-type lectin; CLEC4D) are receptors for the cord factor TDM (trehalose-6,6'-dimycolate), a unique glycolipid of mycobacterial cell-surface components, and activate immune cells to confer adjuvant activity. Although it is known that receptor-TDM interactions require both sugar and lipid moieties of TDM, the mechanisms of glycolipid recognition by Mincle and MCL remain unclear. We here report the crystal structures of Mincle, MCL, and the Mincle-citric acid complex. The structures revealed that these receptors are capable of interacting with sugar in a Ca2+-dependent manner, as observed in other C-type lectins. However, Mincle and MCL uniquely possess shallow hydrophobic regions found adjacent to their putative sugar binding sites, which reasonably locate for recognition of fatty acid moieties of glycolipids. Functional studies using mutant receptors as well as glycolipid ligands support this deduced binding mode. These results give insight into the molecular mechanism of glycolipid recognition through C-type lectin receptors, which may provide clues to rational design for effective adjuvants.
  • Dimitrios Kontogiannatos, Luc Swevers, Katsumi Maenaka, Enoch Y. Park, Kostas Iatrou, Anna Kourti
    PLOS ONE 8 (9) e73834  1932-6203 2013/09 [Refereed][Not invited]
    Juvenile hormone esterase (JHE) is a carboxylesterase that has attracted great interest because of its critical role in regulating larval to adult transition in insects and other arthropods. Previously, we characterized an ecdysteroid sensitive and juvenile hormone non-susceptible juvenile hormone esterase related gene (SnJHER) in the corn stalk borer, Sesamia nonagrioides. SnJHER was rhythmically up-regulated close to each molt during the corn stalk borer's larval development. In this paper we attempted to functionally characterize SnJHER using several reverse genetics techniques. To functionally characterize SnJHER, we experimented with different dsRNA administration methods, including hemolymph, bacterial or baculovirus-mediated RNA interference, (RNAi). Our findings indicate the potential implication of SnJHER in the developmental programming of Sesamia nonagrioides. It is still unclear whether SnJHER is closely related to the authentic JHE gene, with different or similar biological functions.
  • Hideo Fukuhara, Surui Chen, Shin Takeda, Katsumi Maenaka
    YAKUGAKU ZASSHI-JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN 133 (5) 549 - 559 0031-6903 2013/05 [Refereed][Not invited]
    The genus Morbillivirus includes measles virus, canine distemper virus and rinderpest virus. These are highly contagious and exhibit high mortality. These viruses have the attachment glycoprotein, hemagglutinin (H), at the virus surface, which bind to signaling lymphocyte activation molecule (SLAM) and Nectin 4 as receptors for the entry. However, the molecular mechanism for this entry has been limitedly understood. Here we summarize the current topics, (1) newly identified receptor, Nectin 4, (2) crystal structures of H-receptor complexes and (3) detail biochemical studies of the H-F communication for the entry. These provide insight on the mechanism of morbillivirus entry event and furthermore drug developments.
  • 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 0198-8859 2013/04 [Refereed][Not invited]
    HLA-G, a natural immunosuppressant present in the human placenta during pregnancy, prevents fetal destruction by the maternal immune system. The immunosuppressive effect of HLA-G is mediated by the immune cell inhibitory receptors, LILRB1 and LILRB2. HLA-G forms disulfide-linked dimers by natural oxidation, and the dimer associates with LILRB1/B2 much more strongly than the monomer. Furthermore, the dimer formation remarkably enhanced the LILRB-mediated signaling. In this report, we studied the in vivo immunosuppressive effect of the HLA-G dimer, using the collagen-induced arthritis model mouse. Mice were treated with the HLA-G monomer or dimer intracutaneously at the left foot joint, once or for 5 days, and the clinical severity was evaluated daily in a double-blind study. The HLA-G monomer and dimer both produced excellent anti-inflammatory effects with a single, local administration. Notably, as compared to the monomer, the dimer exhibited significant immunosuppressive effects at lower concentrations, which persisted for about two months. In accordance with this result, a binding study revealed that the HLA-G dimer binds PIR-B, the mouse homolog of the LILRBs, with higher affinity and avidity than the monomer. The HLA-G dimer is expected to be quite useful as an anti-rheumatoid arthritis agent, in small amounts with minimal side effects. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
  • 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 0022-538X 2013/03 [Refereed][Not invited]
    Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.
  • 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 0022-538X 2013/02 [Refereed][Not invited]
    Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.
  • 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 0022-538X 2013/01 [Refereed][Not invited]
    Effective vaccination programs have dramatically reduced the number of measles-related deaths globally. Although all the available data suggest that measles eradication is biologically feasible, a structural and biochemical basis for the single serotype nature of measles virus (MV) remains to be provided. The hemagglutinin (H) protein, which binds to two discrete proteinaceous receptors, is the major neutralizing target. Monoclonal antibodies (MAbs) recognizing distinct epitopes on the H protein were characterized using recombinant MVs encoding the H gene from different MV genotypes. The effects of various mutations on neutralization by MAbs and virus fitness were also analyzed, identifying the location of five epitopes on the H protein structure. Our data in the present study demonstrated that the H protein of MV possesses at least two conserved effective neutralizing epitopes. One, which is a previously recognized epitope, is located near the receptor-binding site (RBS), and thus MAbs that recognize this epitope blocked the receptor binding of the H protein, whereas the other epitope is located at the position distant from the RBS. Thus, a MAb that recognizes this epitope did not inhibit the receptor binding of the H protein, rather interfered with the hemagglutinin-fusion (H-F) interaction. This epitope was suggested to play a key role for formation of a higher order of an H-F protein oligomeric structure. Our data also identified one nonconserved effective neutralizing epitope. The epitope has been masked by an N-linked sugar modification in some genotype MV strains. These data would contribute to our understanding of the antigenicity of MV and support the global elimination program of measles.
  • 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 0042-6822 2013/01 [Refereed][Not invited]
    Recent outbreaks in monkeys have proven that canine distemper virus (CDV) causes diseases in a wide range of mammals. CDV uses SLAM and nectin4 as receptors to replicate in susceptible animals. Here, we show that human nectin4, but not human SLAM, is fully functional as a CDV receptor. The CDV Ac96I strain hardly replicated in nectin4-expressing human epithelial NCI-H358 cells, but readily adapted to grow in them. Unsurprisingly, no amino acid change in the H protein was required for the adaptation. The original Ac96I strain possessed a truncated C protein, and a subpopulation possessing the intact C protein was selected after growth in NCI-H358 cells. Other CDV strains possessing the intact C protein showed significantly higher growth abilities in NCI-H358 cells than the Ac96I strain with the truncated C protein. These findings suggest that the C protein is functional in human epithelial cells and critical for CDV replication in them. (C) 2012 Elsevier Inc. All rights reserved.
  • Rodolfo E. Gamez Sazo, Katsumi Maenaka, Weiyong Gu, Patrick M. Wood, Mary Bartlett Bunge
    BIOMATERIALS 33 (33) 8529 - 8539 0142-9612 2012/11 [Refereed][Not invited]
    One of the most exciting new avenues of research to repair the injured spinal cord is to combine cells for implantation with scaffolds that protect the cells and release growth factors to improve their survival and promote host axonal regeneration. To realize this goal, we fabricated biodegradable, photocurable gelatin tubes and membranes for exploratory in vitro studies. Detailed methods are described for their fabrication with a high gelatin concentration. Gelatin membranes fabricated in the same way as tubes and photo-co-immobilized with rhBDNF or rhNT-3, with or without Schwann cells (SCs), showed an initial burst of neurotrophin release within 24 h, with release diminishing progressively for 21 days thereafter. SCs attained their typical bipolar conformation on membranes without neurotrophins but adhesion, alignment and proliferation were improved with neurotrophins, particularly rhBDNF. When dorsal root ganglion explants were cultured on membranes containing laminin and fibronectin plus both neurotrophins, neurite outgrowth was lengthier compared to combining one neurotrophin with laminin and fibronectin. Thus, these gelatin membranes allow SC survival and effectively release growth factors and harbor extracellular matrix components to improve cell survival and neurite growth. These scaffolds, based on the combination of cross-linked gelatin technology and incorporation of neurotrophins and extracellular matrix components, are promising candidates for spinal cord repair. (C) 2012 Elsevier Ltd. All rights reserved.
  • 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 0004-3591 2012/10 [Refereed][Not invited]
    Objective Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLAB27 normally forms complexes with beta 2-microglobulin (beta 2m) and peptide to form heterotrimers. However, an unusual characteristic of HLAB27 is its ability to form beta 2m-free heavy chain homodimers (HLAB272), which, unlike classic HLAB27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLAB272 to KIR-3DL2positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLAB272 in order to confirm its expression in SpA and to inhibit its proinflammatory properties. Methods We generated monoclonal antibodies by screening a human phage display library positively against B272 and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B272-expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2B272 interactions was tested using cell lines and PBMCs from patients with SpA. Results Monoclonal antibody HD6 specifically recognized recombinant HLAB272 by ELISA and by SPR assay. HD6 bound to cell lines expressing B272. FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLAB272 to KIR-3DL2 and the survival and proliferation of KIR-3DL2positive NK cells. Finally, HD6 inhibited production of the proinflammatory diseaseassociated cytokine interleukin-17 by PBMCs from patients with AS. Conclusion These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.
  • 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 0141-5492 2012/10 [Refereed][Not invited]
    Baculoviral expression systems, including those of Autographa californica multiple nucleopolyhedrovirus Bombyx mori nucleopolyhedrovirus (BmNPV), are used for recombinant protein production. Four B. mori-derived (BmN4, Bm5, Bmc140, and Bme21) cell lines were infected with recombinant BmNPV viruses expressing firefly luciferase or EGFP as reporters under the control of a viral polyhedrin promoter. Bme21 exhibited significantly higher (100-fold) luciferase activity than BmN4 and Bm5. With the EGFP reporter protein, Bme21 cells showed a marked increase in the ratio of EGFP-positive cells, reaching 90 % on day 4 post-infection, while Bm5 and BmN4 cells had a slow increase in the ratio of their EGFP-positive population. The viral titer in a supernatant of Bme21 cell culture increased faster than those of Bm5 and BmN4 cells. This susceptibility indicates that the Bme21 cell line is useful for large-scale protein expression using BmNPV.
  • Ribophorin II Is Involved in the Tissue Factor Expression Mediated by Phosphatidylserine-Dependent Antiprothrombin Antibody On Monocytes.
    Yuichiro Fujieda, Olga Amengual, Yusaku Kanetsuka, Toshio Odani, Kotaro Otomo, Kenji Oku, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Kimiko Kuroki, Katsumi Maenaka, Masaki Matsumoto, Shigetsugu Hatakeyama, Tatsuya Atsumi
    ARTHRITIS AND RHEUMATISM 64 (10) S741 - S741 0004-3591 2012/10 [Refereed][Not invited]
  • Ryuta Arai, Masumi Tsuda, Takuya Watanabe, Toyoyuki Ose, Chikashi Obuse, Katsumi Maenaka, Akio Minami, Yusuke Ohba
    EUROPEAN JOURNAL OF CANCER 48 (15) 2417 - 2430 0959-8049 2012/10 [Refereed][Not invited]
    Synovial sarcoma is an obstinate, high-grade malignancy because of its modest responses to radiotherapy and chemotherapy; the identification of effective therapeutics for this sarcoma is therefore necessary. Inhibition of Src family kinases (SFKs) suppresses the proliferation of synovial sarcoma cells in vitro, as we have previously reported. In this study, to validate the efficacy of Src inhibition in vivo, we employed SU6656, which was originally identified as a specific SFK inhibitor. SU6656 treatment significantly impaired the growth of established, existing tumours formed by synovial sarcoma cells in mice. Tumour cell invasion into the surrounding tissues was also abolished by SU6656. It is noteworthy that SU6656 but not PP2 induced a defect in cleavage furrow formation during cytokinesis, resulting in G2/M accumulation and subsequent apoptosis. Intriguingly, SU6656 abrogated the catalytic activities of Aurora kinases and led to the down-regulation of phosphorylated histone H3 coincidently with p53 accumulation, as did the Aurora kinase inhibitor VX-680. Structural comparison indicated an extensive similarity between the catalytic domains of SFKs and Aurora kinases. The structural analysis also revealed the potential binding mode of SU6656 to the ATP-binding cleft of Aurora B via four hydrogen bonds. SU6656 prevented angiogenesis within the tumours by attenuating vascular endothelial growth factor (VEGF) production by tumour cells and the subsequent chemotaxis of endothelial cells; these effects were the result of the inhibition of SFKs but not Aurora kinases. Based on these results, we hereby report a novel property of SU6656 as a dual inhibitor of SFKs and Aurora kinases, the suppression of both of which effectively abrogates tumour development and the progression of synovial sarcoma in vivo. (C) 2011 Elsevier Ltd. All rights reserved.
  • 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 0022-538X 2012/09 [Refereed][Not invited]
    Canine distemper virus (CDV) uses signaling lymphocyte activation molecule (SLAM), expressed on immune cells, as a receptor. However, epithelial and neural cells are also affected by CDV in vivo. Wild-type CDV strains showed efficient replication with syncytia in Vero cells expressing dog nectin4, and the infection was blocked by an anti-nectin4 antibody. In dogs with distemper, CDV antigen was preferentially detected in nectin4-positive neurons and epithelial cells, suggesting that nectin4 is an epithelial cell receptor for CDV and also involved in its neurovirulence.
  • Joanna Giles, Jackie Shaw, Christopher Piper, Isabel Wong-Baeza, Kirsty McHugh, Anna Ridley, Demin Li, Izabela Lenart, Antony N. Antoniou, Katilin DiGleria, Kimiko Kuroki, Katsumi Maenaka, Paul Bowness, Simon Kollnberger
    JOURNAL OF IMMUNOLOGY 188 (12) 6184 - 6193 0022-1767 2012/06 [Refereed][Not invited]
    Possession of HLA-B27 (B27) strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with beta(2)-microglobulin (beta(2)m) and peptide and (beta 2m free) free H chain (FHC) forms including B27 dimers (termed B27(2)) at the cell surface. In this study, we characterize the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR) B1 and LILRB2 immune receptors biophysically, biochemically, and by FACS staining. LILRB1 bound to B27 heterotrimers with a K-D of 5.3 +/- 1.5 mu M but did not bind B27 FHC. LILRB2 bound to B27(2) and B27 FHC and B27 heterotrimers with K(D)s of 2.5, 2.6, and 22 +/- 6 mu M, respectively. Domain exchange experiments showed that B27(2) bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class 1 FHCs. B27-transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with K(D)s of 15.0 +/- 0.8 and 16.0 +/- 2.0 mu M, respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis. The Journal of Immunology, 2012, 188: 6184-6193.
  • 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 2012/04 [Refereed][Not invited]
  • 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 0929-8665 2012/04 [Refereed][Not invited]
    Measles virus (MV), one of the most contagious agents, infects immune cells using the signaling lymphocyte activation molecule (SLAM) on the cell surface. A complex of SLAM and the attachment protein, hemagglutinin (MV-H), has remained elusive due to the intrinsic handling difficulty including glycosylation. Furthermore, crystals obtained of this complex are either nondiffracting or poorly-diffracting. To solve this problem, we designed a systematic approach using a combination of the following techniques; (1) a transient expression system in HEK293SGnTI(-) cells, (2) lysine methylation, (3) structure-guided mutagenesis directed at better crystal packing, (4) Endo H treatment, (5) single-chain formation for stable complex, and (6) floating-drop vapor diffusion. Using our approach, the receptor-binding head domain of MV-H covalently fused with SLAM was successfully crystallized and diffraction was improved from 4.5 angstrom to a final resolution of 3.15 angstrom. These combinational methods would be useful as crystallization strategies for complexes of glycoproteins and their receptors.
  • 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 (Baltimore, Md. : 1950) 188 (8) 3972 - 3979 0022-1767 2012/04 [Refereed][Not invited]
  • [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 0047-1852 2012/04 [Refereed][Not invited]
  • Kimiko Kuroki, Atsushi Furukawa, Katsumi Maenaka
    FRONTIERS IN MICROBIOLOGY 3 429  1664-302X 2012 [Refereed][Not invited]
    Cell surface receptors are responsible for regulating cellular function on the front line, the cell membrane. Interestingly, accumulating evidence clearly reveals that the members of cell surface receptor families have very similar extracellular ligand-binding regions but opposite signaling systems, either inhibitory or stimulatory. These receptors are designated as paired receptors. Paired receptors often recognize not only physiological ligands but also non-self ligands, such as viral and bacterial products, to fight infections. In this review, we introduce several representative examples of paired receptors, focusing on two major structural superfamilies, the immunoglobulin-like and the C-type lectin-like receptors, and explain how these receptors distinguish self and non-self ligands to maintain homeostasis in the immune system. We further discuss the evolutionary aspects of these receptors as well as the potential drug targets for regulating diseases.
  • Rieko Kojima, Mizuho Kajikawa, Mitsunori Shiroishi, Kimiko Kuroki, Katsumi Maenaka
    JOURNAL OF MOLECULAR BIOLOGY 413 (4) 762 - 772 0022-2836 2011/11 [Refereed][Not invited]
    CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4(+) T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function. (C) 2011 Elsevier Ltd. All rights reserved.
  • [Structures and molecular recognition mechanism of leukocyte immunoglobulin-like receptor family].
    Kuroki K, Maenaka K
    Seikagaku. The Journal of Japanese Biochemical Society 83 (8) 715 - 726 0037-1017 2011/08 [Refereed][Not invited]
  • 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 0273-2289 2011/07 [Refereed][Not invited]
    A single-chain variable fragment antibody against herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D-scFv) has been successfully expressed in the hemolymph of silkworm larvae using a rapid Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. Variable heavy- and light-chain domains were cloned directly from the cDNA of the hybridoma cell line 2C4 and assembled together with flexible peptide linker (Gly(4)Ser)(3) between two domains. The yield of functional 2,4-D-scFv after purification was 640 mu g per 30 ml of hemolymph, which is equivalent to 21.3 mg per liter of hemolymph. The characterization of 2,4-D-scFv using an indirect competitive enzyme-linked immunosorbent assay (icELISA) revealed that it has wide cross-reactivities against 2,4,5-trichlorophenoxyacetic acid (65.5%), 2,4-dichlorophenol (47.9%), and 2,4-dichlorobenzoic acid (26.0%), making it possible to apply 2,4-D-scFv to icELISA for detecting/determining 2,4-D and its metabolites. Judging from its cost and time requirements and its ease of handling, this BmNPV bacmid DNA expression system is more useful for expressing functional scFv than bacterial systems, which frequently require costly and time-consuming refolding.
  • Jun Kamishikiryo, Hideo Fukuhara, Yuki Okabe, Kimiko Kuroki, Katsumi Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 286 (27) 23823 - 23830 0021-9258 2011/07 [Refereed][Not invited]
    Human Th17 cells express high levels of CD161, a member of the killer cell lectin-like receptor (KLR) family (also referred to as NK receptor-P1A (NKRP1A) or KLRB1), as a representative marker. CD161 is also expressed on natural killer (NK) cells and NKT cells. Lectin-like transcript 1 (LLT1), another KLR family member, was recently identified as a ligand for CD161. This interaction may play pivotal roles in the immunomodulatory functions of Th17 cells as well as those of NK and NKT cells. However, the molecular basis for the interaction is poorly understood. Here we show that the extracellular domain of CD161 bound directly to LLT1 with a K(d) of 48 mu M and with the fast kinetics typical of cell-cell recognition receptors. Mutagenesis revealed that the similar membrane-distal beta-sheet and loop regions of both CD161 and LLT1 were utilized for the binding, and notably, these regions correspond to the ligand-binding sites for major histocompatibility complex (MHC)-recognizing KLRs. Furthermore, we found a pair of detrimental mutations for both molecules that restored the binding. These results reveal a new template model for the recognition mode between the KLR family members and provide insights into the molecular mechanism underlying Th17/NK/NKT-mediated immune responses.
  • 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 0021-9258 2011/07 [Refereed][Not invited]
    Binding of class I MHC molecules (MHCI) to an inhibitory receptor, PIR-B, expressed on B cells and myeloid cells provides constitutive cellular inhibition, thus ensuring peripheral tolerance. Recent unexpected findings pointed to a novel inhibitory role of PIR-B in neuritere generation through binding to three axonal outgrowth inhibitors of myelin, including Nogo. Thus, it becomes interesting to determine whether the actions of the inhibitory myelin proteins and MHCI could coexist independently or be mutually exclusive as to the PIR-B-mediated immune and neural cell inhibition. Here, we present data supporting the competition of Nogo-and MHCI-mediated inhibition where they coexist. Kinetic analyses of Nogo and MHCI binding to the whole or a part of the recombinant PIR-B ectodomain revealed that PIR-B binds with higher affinity to Nogo than MHCI and that the MHCI binding only occurred with the N-terminal domains of PIR-B, whereas Nogo binding occurred with either the N- or C-terminal ectodomains. Importantly, kinetic tests indicated that the binding to PIR-B of Nogo and MHCI was competitive. Both endogenous and exogenous Nogo intensified the PIR-B-mediated suppression of interleukin-6 release from lipopolysaccharide-stimulated wildtype, but not PIR-B-deficient, cultured mast cells, indicating that PIR-B mediates Nogo-induced inhibition. Thus, we propose a novel mechanism by which PIR-B-mediated regulation is achieved differentially but competitively via MHCI and Nogo in cells of the immune system.
  • Takao Hashiguchi, Toyoyuki Ose, Marie Kubota, Nobuo Maita, Jun Kamishikiryo, Katsumi Maenaka, Yusuke Yanagi
    NATURE STRUCTURAL & MOLECULAR BIOLOGY 18 (2) 135 - U191 1545-9993 2011/02 [Refereed][Not invited]
    Measles virus, a major cause of childhood morbidity and mortality worldwide, predominantly infects immune cells using signaling lymphocyte activation molecule (SLAM) as a cellular receptor. Here we present crystal structures of measles virus hemagglutinin (MV-H), the receptor-binding glycoprotein, in complex with SLAM. The MV-H head domain binds to a beta-sheet of the membrane-distal ectodomain of SLAM using the side of its beta-propeller fold. This is distinct from attachment proteins of other paramyxoviruses that bind receptors using the top of their beta-propeller. The structure provides templates for antiviral drug design, an explanation for the effectiveness of the measles virus vaccine, and a model of the homophilic SLAM-SLAM interaction involved in immune modulations. Notably, the crystal structures obtained show two forms of the MV-H-SLAM tetrameric assembly (dimer of dimers), which may have implications for the mechanism of fusion triggering.
  • Seiichi Sakamoto, Benyakan Pongkitwitoon, Kaori Sasaki-Tabata, Waraporn Putalun, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto
    ANALYST 136 (10) 2056 - 2063 0003-2654 2011 [Refereed][Not invited]
    A fluorescent single-domain antibody (fluobody), a chimera of a green fluorescent protein (AcGFP) with a single chain variable fragment antibody (scFv), against plumbagin (5-hydorxy-2-methyl-1,4-naphthoquinone; PL) was successfully expressed in the hemolymph of silkworm larvae using a Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system to develop a rapid, simple, and sensitive fluorescence-linked immunosorbent assay (FLISA). In this study, two kinds of fluobody, in which the PL-scFv was fused at the N-terminus (N-fluobody) or C-terminus of AcGFP (C-fluobody), were expressed in silkworm larvae for comparative purposes. Interestingly, both fluobodies expressed in the BmNPV bacmid DNA system retained both of their original functions as an AcGFP and a PL-scFv, although the functions of the N-fluobody were found to be inferior to those of C-fluobody when they were expressed in Escherichia coli. Moreover, an improvement in the limit of quantification for PL measurement was observed in FLISA (24 ng mL(-1)) compared with conventional ELISA (0.2 mu g mL(-1)). Since both the C-fluobody and N-fluobody are useful probes for FLISA and the time-, cost-consuming refolding step required in the conventional bacterial expression system can be avoided when they are expressed in the BmNPV bacmid DNA system, the silkworm expression system is useful for expressing fluobodies when developing FLISA.
  • Kuroki K, Maenaka K
    Methods in molecular biology (Clifton, N.J.) 748 83 - 106 1064-3745 2011 [Refereed][Not invited]
  • Takao Hashiguchi, Katsumi Maenaka, Yusuke Yanagi
    FRONTIERS IN MICROBIOLOGY 2 247  1664-302X 2011 [Refereed][Not invited]
    Measles is one of the most contagious viral diseases, and remains a major cause of childhood morbidity and mortality worldwide. The measles virus (MV), a member of the family Paramyxoviridae, enters cells through a cellular receptor, the signaling lymphocyte activation molecule (SLAM), CD46 or nectin-4. Entry is mediated by two MV envelope glycoproteins, the hemagglutinin (H) and the fusion (F) protein. The H protein mediates receptor attachment, while the F protein causes membrane fusion. The interaction between the H and F proteins is essential to initiate the cell entry process. Recently determined crystal structures of the MV-H protein unbound and bound to SLAM or CD46 have provided insights into paramyxovirus entry and the effectiveness of measles vaccine.
  • Seiichi Sakamoto, Benyakan Pongkitwitoon, Seiko Nakamura, Katsumi Maenaka, Hiroyuki Tanaka, Satoshi Morimoto
    JOURNAL OF BIOCHEMISTRY 148 (3) 335 - 340 0021-924X 2010/09 [Refereed][Not invited]
    A single-chain variable fragment (scFv) antibody against ginsenoside Re (G-Re) have been successfully expressed in the silkworm larvae using Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid DNA system. The baculovirus donor vector for expression of scFv against G-Re (GRe-scFv) was constructed to contain honeybee melittin signal sequence to accelerate secretion of the recombinant GRe-scFv into the haemolymph of silkworm larvae. Functional recombinant GRe-scFv was purified by cation exchange chromatography followed by immobilized metal ion affinity chromatography. The yield of purified GRe-scFv was 6.5 mg per 13 silkworm larvae, which is equivalent to 650 mg/l of the haemolymph, exhibiting extremely higher yield than that expressed in Escherichia coli (1.7 mg/l of culture medium). It was revealed from characterization that GRe-scFv retained similar characteristic of the parental monoclonal antibody (MAb) against G-Re (MAb-4G10), making it possible to develop indirect competitive enzyme-linked immunosorbent assay (icELISA) for quality control of total ginsenosides in various ginsengs. The detectable range for calibration of G-Re by developed icELISA shows 0.05-10 mu g/ml. These results clearly suggested that the silkworm expression system is quite useful for the expression of functional scFv that frequently required time- and cost-consuming re-folding when it expressed in E. coli.
  • Yuka Kawashima, Nozomi Kuse, Hiroyuki Gatanaga, Takuya Naruto, Mamoru Fujiwara, Sachi Dohki, Tomohiro Akahoshi, Katsumi Maenaka, Philip Goulder, Shinichi Oka, Masafumi Takiguchi
    JOURNAL OF VIROLOGY 84 (14) 7151 - 7160 0022-538X 2010/07 [Refereed][Not invited]
    HLA-B*51 alleles are reported to be associated with slow disease progression to AIDS, but the mechanism underlying this association is still unclear. In the present study, we analyzed the effect of HLA-B*5101 on clinical outcome for Japanese hemophiliacs who had been infected with HIV-1 before 1985 and had been recruited in 1998 for this study. HLA-B*5101(+) hemophiliacs exhibited significantly slow progression. The analysis of HLA-B*5101-restricted HIV-1-specific cytotoxic T-lymphocyte (CTL) responses to 4 HLA-B*-restricted epitopes in 10 antiretroviral-therapy (ART)-free HLA-B*5101(+) hemophiliacs showed that the frequency of Pol283-8-specific CD8(+) T cells was inversely correlated with the viral load, whereas the frequencies of CD8(+) T cells specific for 3 other epitopes were positively correlated with the viral load. The HLA-B*5101(+) hemophiliacs whose HIV-1 replication had been controlled for approximately 25 years had HIV-1 possessing the wild-type Pol283-8 sequence or the Pol283-8V mutant, which does not critically affect T-cell recognition, whereas other HLA-B*5101(+) hemophiliacs had HIV-1 with escape mutations in this epitope. The results suggest that the control of HIV-1 over approximately 25 years in HLA-B*5101-positive hemophiliacs is associated with a Pol283-8-specific CD8(+) T-cell response and that lack of control of HIV-1 is associated with the appearance of Pol283-8-specific escape mutants.
  • 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 1347-8613 2010/03 [Refereed][Not invited]
    We have reported that the differentiation-inducing factors (DIFs) DIF-1 and DIF-3, morphogens secreted from Dictyostelium discoideum, inhibit proliferation of several cancer cells via suppression of the Wnt/beta-catenin signaling pathway. However, the target molecules of DIFs involved in the anti-proliferative effects are still unknown. In the present study, DIF-1 tethered resins were synthesized to explore the target molecules of DIFs, and mitochondrial malate dehydrogenase (mMDH) was identified as one of the target molecules. In the in vitro assay, DIF-1 and other analogs including 2-MIDIF-1, DIF-3, and 6-MIDIF-3 were found to be capable of binding to mMDH but not to cytoplasmic MDH. However, only DIF-1 and 2-MIDIF-1 inhibited the enzymatic activity of mMDH. The effects of DIF analogs on ATP content and cell proliferation were then analyzed using HeLa cells. DIF-1 and 2-MIDIF-1 were found to lower the ATP content and both chemicals inhibited HeLa cell proliferation, suggesting that inhibition of mMDH activity affected cell energy production, probably leading to the inhibition of proliferation. These results suggest that the inhibition of mMDH activity by DIF-1 and 2-MIDIF-1 could be one of the mechanisms to induce anti-proliferative effects, independent of the inhibition of the Wnt/beta-catenin signaling pathway.
  • Tatsuya Kato, Mizuho Kajikawa, Katsumi Maenaka, Enoch Y. Park
    APPLIED MICROBIOLOGY AND BIOTECHNOLOGY 85 (3) 459 - 470 0175-7598 2010/01 [Refereed][Not invited]
    Many recombinant proteins have been successfully produced in silkworm larvae or pupae and used for academic and industrial purposes. Several recombinant proteins produced by silkworms have already been commercialized. However, construction of a recombinant baculovirus containing a gene of interest requires tedious and troublesome steps and takes a long time (3-6 months). The recent development of a bacmid, Escherichia coli and Bombyx mori shuttle vector, has eliminated the conventional tedious procedures required to identify and isolate recombinant viruses. Several technical improvements, including a cysteine protease or chitinase deletion bacmid and chaperone-assisted expression and coexpression, have led to significantly increased protein yields and reduced costs for large-scale production. Terminal N-acetyl glucosamine and galactose residues were found in the N-glycan structures produced by silkworms, which are different from those generated by insect cells. Genomic elucidation of silkworm has opened a new chapter in utilization of silkworm. Transgenic silkworm technology provides a stable production of recombinant protein. Baculovirus surface display expression is one of the low-cost approaches toward silkworm larvae-derived recombinant subunit vaccines. The expression of pharmaceutically relevant proteins, including cell/viral surface proteins, membrane proteins, and guanine nucleotide-binding protein (G protein) coupled receptors, using silkworm larvae or cocoons has become very attractive. Silkworm biotechnology is an innovative and easy approach to achieve high protein expression levels and is a very promising platform technology in the field of life science. Like the "Silkroad," we expect that the "Bioroad" from Asia to Europe will be established by the silkworm expression system.
  • Toyoyuki Ose, Kimiko Kuroki, Masaaki Matsushima, Katsumi Maenaka, Izumi Kumagai
    JOURNAL OF BIOCHEMISTRY 146 (5) 651 - 657 0021-924X 2009/11 [Refereed][Not invited]
    In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel -sheet, designated as a platform, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)(5). A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)(3) was bound in the ABC subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these platform residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.
  • Seiko Nakamura, Kimiko Kuroki, Izuru Ohki, Kaori Sasaki, Mizuho Kajikawa, Takuma Maruyama, Masayuki Ito, Yosuke Kameda, Mitsuhiko Ikura, Kazuo Yamamoto, Naoki Matsumoto, Katsumi Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 284 (40) 27327 - 27335 0021-9258 2009/10 [Refereed][Not invited]
    The killer cell lectin-like receptor G1, KLRG1, is a cell surface receptor expressed on subsets of natural killer (NK) cells and T cells. KLRG1 was recently found to recognize E-cadherin and thus inhibit immune responses by regulating the effector function and the developmental processes of NK and T cells. E-cadherin is expressed on epithelial cells and exhibits Ca(2+)-dependent homophilic interactions that contribute to cell-cell junctions. However, the mechanism underlying the molecular recognition of KLRG1 by E-cadherin remains unclear. Here, we report structural, binding, and functional analyses of this interaction using multiple methods. Surface plasmon resonance demonstrated that KLRG1 binds the E-cadherin N-terminal domains 1 and 2 with low affinity (K(d) similar to 7-12 mu M), typical of cell-cell recognition receptors. NMR binding studies showed that only a limited N-terminal region of E-cadherin, comprising the homodimer interface, exhibited spectrum perturbation upon KLRG1 complex formation. It was confirmed by binding studies using a series of E-cadherin mutants. Furthermore, killing assays using KLRG1(+)NK cells and reporter cell assays demonstrated the functional significance of the N-terminal region of E-cadherin. These results suggest that KLRG1 recognizes the N-terminal homodimeric interface of domain 1 of E-cadherin and binds only the monomeric form of E-cadherin to inhibit the immune response. This raises the possibility that KLRG1 detects monomeric E-cadherin at exposed cell surfaces to control the activation threshold of NK and T cells.
  • 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 0006-291X 2009/09/25 [Refereed][Not invited]
    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.
  • Jun Kamishikiryo, Katsumi Maenaka
    CURRENT PHARMACEUTICAL DESIGN 15 (28) 3318 - 3324 1381-6128 2009/09 [Refereed][Not invited]
    Human leukocyte antigen-G (HLA-G) is a non-classical HLA class I molecule, which was first discovered in 1987 by Geraghty and colleagues [1]. While classical HLA class I molecules are expressed on all nucleated cells, the expression of the HLA-G molecule is highly tissue-restricted, such as to placental trophoblast cells. HLA-G binds inhibitory receptors such as leukocyte immunoglobulin-like receptors B1 (LILRB1/ILT2/CD85j) and LILRB2 (ILT4/CD85d), which are widely expressed on immune cells, to suppress a broad range of immune responses [2-4]. Thus, the expression of HLA-G in placenta protects the fetus from the maternal immune system. On the other hand, emerging studies have shown the relevance of the HLA-G molecule in pathologic conditions, such as transplantation rejection, autoimmunity, and cancer. HLA-G has other unique characteristics, in contrast with classical HLA molecules, including the existence of various forms of HLA-G: several splice variants, subunit-deficient conformations, homodimers, and their combinations have been found [5]. In this review, we highlight the molecular basis for the tolerogenic ability of the HLA-G molecule, especially by LILR recognition of various forms of HLA-G. We also discuss the potential clinical applications of HLA-G molecules.
  • 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 0006-291X 2009/07 [Refereed][Not invited]
    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS Using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha Subunit (G(i)alpha) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [(35)S]GTP gamma S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production. (C) 2009 Elsevier Inc. All rights reserved.
  • Mitsunori Shiroishi, Katsumi Maenaka
    PROTEIN AND PEPTIDE LETTERS 16 (4) 447 - 449 0929-8665 2009/04 [Refereed][Not invited]
    Human leukocyte antigen-G (HLA-G) is a nonclassical MHC class I (MHCI) molecule that is expressed mainly on placenta trophoblast cells. Leukocyte Ig-like receptor B2 (LILRB2) is a human inhibitory immune receptor that recognizes HLA-G with a higher affinity than any other MHCI although this interaction is only in the mu M range. The interaction between HLA-G and LILRB2 seems to play a dominant role in the escape of the fetus from the maternal immune response. Here we report the crystallization and x-ray analysis of the LILRB2/HLA-G complex. The extracellular domains of HLA-G and LILRB2 were expressed in Escherichia coli, refolded and purified. The initial crystallization trials using novel PEG-based screening sets provided crystals of the LILRB2/HLA-G complex with 40-50% PEG400 as the precipitant. These crystals belong to space group P3(1)21 (a=b=81.4 angstrom, c=186.7 angstrom, gamma=120 degrees). Dehydration of the crystals by soaking them in a solution containing a higher concentration of PEG400 dramatically improved the resolution and also the mosaicity.
  • Hathairat Thananchai, Tariro Makadzange, Katsumi Maenaka, Kimiko Kuroki, Yanchun Peng, Chris Conlon, Sarah Rowland-Jones, Tao Dong
    AIDS 23 (2) 189 - 193 0269-9370 2009/01 [Refereed][Not invited]
    Objectives: The HIV-1 Nef protein selectively downregulates human leukocyte antigen (HLA)-A and HLA-B but not HLA-C molecules on the Surface of infected cells. This allows HIV-infected cells to evade recognition by most cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. We investigated the recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope SFNCGGEFF (SF9) and its variant SFNCGGEFL (SL9) by T cells and NK receptors. Design and method: Recognition of HIV-1 gp120 peptides (SF9 and SL9) by T-cell clones was measured by staining with HLA-Cw4-peptide tetrameric complexes and cytolytic assays using target cell pulsed with either peptides. KIR2DL1 binding to these two peptides was measured using surface plasmon resonance and tetramer staining of an NK cell line. Result: CTLs could recognize SF9 better than the variant SL9, as shown by both tetramer staining and cytolytic assays. Intriguingly, an HLA-Cw4 tetramer folded with the 'escape' variant SL9 could bind to KIR2DL1 on NK cell lines with higher affinity than HLA-Cw4-SF9. The binding of KIR2DL1 to its ligand results in inhibition of NK cell function. Our results indicate that the HIV-1 gp120 variant peptide SL9 could potentially escape both from NK cell and CTL recognition by increasing its affinity for KIR2DL1 binding. Conclusion: These data suggest that HIV-1 can acquire mutations that are capable of escaping from both CTL and NK cell recognition, a phenomenon we have termed 'double escape'. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins
  • Jun Okada, Tatsuo Maruyama, Konomi Motomura, Kimiko Kuroki, Katsumi Maenaka, Masafumi Sakono, Masahiro Goto
    CHEMICAL COMMUNICATIONS (46) 7197 - 7199 1359-7345 2009 [Refereed][Not invited]
    We employed a urease-catalyzed reaction to gradually remove a high concentration of a chaotropic agent (urea) from a denatured protein solution and demonstrated that efficient protein refolding can be achieved by the urease-catalyzed reaction, without large-volume dilution.
  • 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 1073-6085 2008/10 [Refereed][Not invited]
    The recombinant protein expression by Bombyx mori nucleopolyhedrovirus (BmNPV) infecting silkworm larvae or pupae may endow us with a potent system for the production of large eukaryotic proteins. However, the screening of silkworm strains ideally suited to this method has scarcely been conducted. In the present study, we injected recombinant BmNPV containing a reporter gene, luciferase or DsRed, into hemocoel of fifth instar larvae of selected 12 silkworm strains. Among them, the strain d17 is found to be the highest in reporter expression from the intrinsic polyhedrin promoter of Autographa californica NPV or the silkworm actin A3 promoter. These results suggest that the d17 strain is highly permissive for BmNPV replication and is the most likely candidate of a "factory" for large-scale expression using the BmNPV bacmid system.
  • 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 1466-4879 2008/04 [Refereed][Not invited]
    Leukocyte immunoglobulin-like receptors (LILRs) are inhibitory, stimulatory or soluble receptors encoded within the leukocyte receptor complex. Some LILRs are extensively polymorphic, and exhibit evidence for balancing selection and association with disease susceptibility. LILRA2 (LIR7/ILT1) is an activating receptor highly expressed in inflammatory tissues, and is involved in granulocyte and macrophage activation. In this study, we examined the association of LILRA2 and adjacently located LILRA1 with systemic lupus erythematosus (SLE), rheumatoid arthritis ( RA) and microscopic polyangiitis (MPA). Polymorphism screening detected a LILRA2 SNP ( rs2241524 G > A) that disrupts splice acceptor site of intron 6. Case-control association studies on 273 Japanese SLE, 296 RA, 50 MPA and 284 healthy individuals revealed increase of genotype A/A in SLE (12.1%, odds ratio ( OR) 1.82, 95% confidence interval (CI) 1.02-3.24, P=0.041) and in MPA (16.0%, OR 2.52, 95% CI 1.07-5.96, P=0.049) compared with healthy individuals (7.0%). The risk allele caused an activation of a cryptic splice acceptor site that would lead to a novel LILRA2 isoform lacking three amino acids in the linker region (Delta 419-421). Flow cytometry indicated that this isoform was expressed on the surface of monocytes. These findings suggested that LILRA2 Delta 419-421 isoform encoded by the splice site SNP may play a role in SLE and MPA.
  • Aoki N, Sakiyama A, Kuroki K, Maenaka K, Kohda D, Deshimaru M, Terada S
    Biochimica et biophysica acta 1784 (4) 621 - 628 0006-3002 2008/04 [Refereed][Not invited]
  • Shigekazu Tabata, Kimiko Kuroki, Jing Wang, Mizuho Kajikawa, Ikuo Shiratori, Daisuke Kohda, Hisashi Arase, Katsumi Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 283 (14) 8893 - 8901 0021-9258 2008/04 [Refereed][Not invited]
    Paired Ig-like type 2 receptors ( PILRs) are one of the paired receptor families, which consist of two functionally opposite members, inhibitory ( PILR alpha) and activating ( PILR beta) receptors. PILRs are widely expressed in immune cells and recognize the sialylated O-glycosylated ligand CD99, which is expressed on activated T cells, to regulate immune responses. To date, their biophysical properties have not yet been examined. Here we report the affinity, kinetic, and thermodynamic analyses of PILR-CD99 interactions using surface plasmon resonance ( SPR) together with site-directed mutagenesis. The SPR analysis clearly demonstrated that inhibitory PILR alpha can bind to CD99 with low affinity ( K-d similar to 2.2 mu M), but activating PILR beta binds with similar to 40 times lower affinity ( K-d similar to 85 mu M). In addition to our previous mutagenesis study ( Wang, J., Shiratori, I., Saito, T., Lanier, L. L., and Arase, H. ( 2008) J. Immunol. 180, 1686 - 1693), the SPR analysis showed that PILR alpha can bind to each Ala mutant of the two CD99 O-glycosylated sites ( Thr-45 and Thr-50) with similar binding affinity to wild-type CD99. This indicated that both residues act as independent and equivalent PILR alpha binding sites, consistent with the highly flexible structure of CD99. On the other hand, it is further confirmed that PILR beta can bind the T50A mutant, but not the T45A mutant, indicating a recognition difference between PILR alpha and PILR beta. Kinetic studies demonstrated that the PILR-CD99 interactions show fast dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses revealed that the PILR alpha-CD99 interaction is enthalpically driven with a large entropy loss (-T Delta S = 8.9 kcal.mol(-1)), suggesting the reduction of flexibility upon complex formation. This is in contrast to the entropically driven binding of selectins to sugar-modified ligands involved in leukocyte rolling and infiltration, which may reflect their functional differences.
  • 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 0021-9258 2008/04 [Refereed][Not invited]
    The interaction of measles virus with its receptor signaling lymphocytic activation molecule (SLAM) controls cell entry and governs tropism. We predicted potential interface areas of the measles virus attachment protein hemagglutinin to begin the investigation. We then assessed the relevance of individual amino acids located in these areas for SLAM-binding and SLAM-dependent membrane fusion, as measured by surface plasmon resonance and receptor-specific fusion assays, respectively. These studies identified one hemagglutinin protein residue, isoleucine 194, which is essential for primary binding. The crystal structure of the hemagglutinin-protein localizes Ile-194 at the interface of propeller blades 5 and 6, and our data indicate that a small aliphatic side chain of residue 194 stabilizes a protein conformation conducive to binding. In contrast, a quartet of residues previously shown to sustain SLAM-dependent fusion is not involved in binding. Instead, our data prove that after binding, this quartet of residues on propeller blade 5 conducts conformational changes that are receptor-specific. Our study sets a structure-based stage for understanding how the SLAM-elicited conformational changes travel through the H-protein ectodomain before triggering fusion protein unfolding and membrane fusion.
  • 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 0166-0934 2008/04 [Refereed][Not invited]
    Measles virus (MV) enters cells by binding to the signaling lymphocyte activation molecule (also called CD150) on the cell surface, and thus shows the lymphotropism and immunosuppressive effects. The head domain (residues Asp(149) to Arg(617)) of the MV hemagglutinin (MV-H), the attachment protein, was produced using a transient expression system in HEK293T cells. The purified MV-H protein was heterogeneous because of a variety of complex-sugar modifications. The complex-sugar-type MV-H was crystallized successfully, and the crystals belonged to the space group P41212 with the unit cell dimension of a = b = 134 angstrom, c = 100 angstrom, but diffracted only to 3.0 A resolution. MV-H was also expressed in HEK293SGnTI(-) cells lacking the N-acetylglucosaminyltransferase I activity, which render N-linked glycans of the proteins restricted and homogeneous, producing the oligomannose, Man(5)GlcNAc(2). The native and selenomethionyl derivative proteins of the oligomannose-type MV-H were crystallized, and the native crystals well diffracted to 2.6 angstrom resolution. Thus, homogeneous sugar modification may be useful for improved crystallization of heavily sugar-modified viral envelope proteins. (C) 2008 Elsevier B.V. All rights reserved.
  • Tetsuya Hori, Masahiro Okada, Katsumi Maenaka, Tatsuo Fukagawa
    MOLECULAR BIOLOGY OF THE CELL 19 (3) 843 - 854 1059-1524 2008/03 [Refereed][Not invited]
    We previously identified a multisubunit complex (CENP-H/I complex) in kinetochores from human and chicken cells. We showed that the CENP-H/I complex is divided into three functional classes. In the present study, we investigated CENP-O class proteins, which include CENP-O, -P, -Q, -R, and -50 (U). We created chicken DT40 cell knockouts of each of these proteins, and we found that all knockout lines were viable, but that they showed slow proliferation and mitotic defects. Kinetochore localization of CENP-O, -P, -Q, and -50 was interdependent, but kinetochore localization of these proteins was observed in CENP-R -deficient cells. A coexpression assay in bacteria showed that CENP-O, -P, -Q, and -50 proteins form a stable complex that can associate with CENP-R. Phenotype analysis of knockout cells showed that all proteins except for CENP-R were required for recovery from spindle damage, and phosphorylation of CENP-50 was essential for recovery from spindle damage. We also found that treatment with the proteasome inhibitor MG132 partially rescued the severe mitotic phenotype observed in response to release from nocodazole block in CENP-50-deficient cells. This suggests that CENP-O class proteins are involved in the prevention of premature sister chromatid separation during recovery from spindle damage.
  • Two novel NKG2D ligands of the mouse H60 family with differential expression patterns and binding affinities to NKG2D.
    Takada A, Yoshida S, Kajikawa M, Miyatake Y, Tomaru U, Sakai M, Chiba H, Maenaka K, Kohda D, Fugo K, Kasahara M
    Journal of immunology (Baltimore, Md. : 1950) 180 (3) 1678 - 1685 0022-1767 2008/02 [Refereed][Not invited]
  • Mayumi Igura, Nobuo Maita, Jun Kamishikiryo, Masaki Yamada, Takayuki Obita, Katsumi Maenaka, Daisuke Kohda
    EMBO JOURNAL 27 (1) 234 - 243 0261-4189 2008/01 [Refereed][Not invited]
    Asn-glycosylation is widespread not only in eukaryotes but also in archaea and some eubacteria. Oligosaccharyltransferase (OST) catalyzes the co-translational transfer of an oligosaccharide from a lipid donor to an asparagine residue in nascent polypeptide chains. Here, we report that a thermophilic archaeon, Pyrococcus furiosus OST is composed of the STT3 protein alone, and catalyzes the transfer of a heptasaccharide, containing one hexouronate and two pentose residues, onto peptides in an Asn-X-Thr/ Ser-motif-dependent manner. We also determined the 2.7-angstrom resolution crystal structure of the C-terminal soluble domain of Pyrococcus STT3. The structure-based multiple sequence alignment revealed a new motif, DxxK, which is adjacent to the well-conserved WWDYG motif in the tertiary structure. The mutagenesis of the DK motif residues in yeast STT3 revealed the essential role of the motif in the catalytic activity. The function of this motif may be related to the binding of the pyrophosphate group of lipid-linked oligosaccharide donors through a transiently bound cation. Our structure provides the first structural insights into the formation of the oligosaccharide-asparagine bond.
  • Shigekazu Tabata, Kimiko Kuroki, Nobuo Maita, Jing Wang, Ikuo Shiratori, Hisashi Arase, Daisuke Kohda, Katsumi Maenaka
    Human paired immunoglobulin-like (Ig-like) type 2 receptor alpha (PILR alpha) is a type I membrane protein that is mainly expressed in immune-related cells such as monocytes, granulocytes and dendritic cells. PILR alpha can suppress the functions of such immune cells because it has the immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region, which recruits the phosphatase Src homology-2 (SH2) domain-containing protein tyrosine phosphatase 2 (SHP-2) to inhibit phophorylations induced by activation signals. The extracellular region of human PILR alpha comprises one immunoglobulin superfamily V-set domain and a stalk region. The V-set domain (residues 13-131) of human PILR alpha was overexpressed in Escherichia coli as inclusion bodies, refolded by rapid dilution and purified. The PILR alpha protein was successfully crystallized at 293 K using the sitting-drop vapour-diffusion method. The crystals diffracted to 1.3 angstrom resolution at SPring-8 BL41XU; they belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 40.4, b = 45.0, c = 56.9 angstrom, and contain one molecule per asymmetric unit.
  • 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 0027-8424 2007/12 [Refereed][Not invited]
    Measles still remains a major cause of childhood morbidity and mortality worldwide. Measles virus (MV) vaccines are highly successful, but the mechanism underlying their efficacy has been unclear. Here we report the crystal structure of the MV attachment protein, hemagglutinin, responsible for MV entry. The receptor-binding head domain exhibits a cubic-shaped beta-propeller structure and forms a homodimer. N-linked sugars appear to mask the broad regions and cause the two molecules forming the dimer to tilt oppositely toward the horizontal plane. Accordingly, residues of the putative receptor-binding site, highly conserved among MV strains, are strategically positioned in the unshielded area of the protein. These conserved residues also serve as epitopes for neutralizing antibodies, ensuring the serological monotype, a basis for effective MV vaccines. Our findings suggest that sugar moieties in the MV hemagglutinin critically modulate virus-receptor interaction as well as antiviral antibody responses, differently from sugars of the HIV gp120, which allow for immune evasion.
  • Daisuke Kohda, Masaki Yamada, Mayumi Igura, Jun Kamishikiryo, Katsumi Maenaka
    GLYCOBIOLOGY 17 (11) 1175 - 1182 0959-6658 2007/11 [Refereed][Not invited]
    We developed a new in vitro assay for oligosaccharyltransferase (OST), which catalyzes the transfer of preassembled oligosaccharides on lipid carriers onto asparagine residues in polypeptide chains. The asparagine residues reside in the sequon, Asn-X-Thr/Ser, where X can be any amino acid residue except Pro. We demonstrate the potency of our assay using the OST from yeast. In our method, polyacrylamide gel electrophoresis is used to separate the glycopeptide products from the peptide substrates. The substrate peptide is fluorescently labeled and the formation of glycopeptides is analyzed by fluorescence gel imaging. Two in vitro OST assay methods are now widely used, but both the methods depend on previous knowledge of the oligosaccharide moiety: One method uses lectin binding as the separation mechanism and the other method uses biosynthetically or chemoenzymatically synthesized lipid-linked oligosaccharides as donors. N-linked protein glycosylation is found in all three domains of life, but little is known about the N-glycosylation in Archaea. Thus, our new assay, which does not require a priori knowledge of the oligosaccharides, will be useful in such cases. Indeed, we have detected the OST activity in the membrane fraction from a hyperthermophilic archaeon, Pyrococcus furiosus.
  • Takashi Saitoh, Mayumi Igura, Takayuki Obita, Toyoyuki Ose, Rieko Kojima, Katsumi Maenaka, Toshiya Endo, Daisuke Kohda
    EMBO JOURNAL 26 (22) 4777 - 4787 0261-4189 2007/11 [Refereed][Not invited]
    Most mitochondrial proteins are synthesized in the cytosol and imported into mitochondria. The N-terminal presequences of mitochondrial-precursor proteins contain a diverse consensus motif (phi chi chi phi phi, phi is hydrophobic and chi is any amino acid), which is recognized by the Tom20 protein on the mitochondrial surface. To reveal the structural basis of the broad selectivity of Tom20, the Tom20 presequence complex was crystallized. Tethering a presequence peptide to Tom20 through a disulfide bond was essential for crystallization. Unexpectedly, the two crystals with different linker designs provided unique relative orientations of the presequence with respect to Tom20, and neither configuration could fully account for the hydrophobic preference at the three hydrophobic positions of the consensus motif. We propose the existence of a dynamic equilibrium in solution among multiple states including the two bound states. In accordance, NMR N-15 relaxation analyses suggested motion on a sub-millisecond timescale at the Tom20-presequence interface. We suggest that the dynamic, multiple-mode interaction is the molecular mechanism facilitating the broadly selective specificity of the Tom20 receptor toward diverse mitochondrial presequences.
  • Mayumi Igura, Nobuo Maita, Takayuki Obita, Jun Kamishikiryo, Katsumi Maenaka, Daisuke Kohda
    Oligosaccharyltransferase catalyzes the transfer of preassembled oligosaccharides onto asparagine residues in nascent polypeptide chains. The STT3 subunit is thought to bear the catalytic site. The C-terminal domain of the STT3 protein of Pyrococcus furiosus was expressed in Escherichia coli cells. STT3 protein prepared from two different sources, the soluble fraction and the inclusion bodies, produced crystals that diffracted to 2.7 angstrom. During crystallization screening, cocrystals of P. furiosus STT3 with an E. coli 50S ribosomal protein, L7/ L12, were accidentally obtained. This cross-species interaction is not biologically relevant, but may be used to design a built-in polypeptide substrate for the STT3 crystals.
  • Kirniko Kuroki, Katsurni Maenaka
    EUROPEAN JOURNAL OF IMMUNOLOGY 37 (7) 1727 - 1729 0014-2980 2007/07 [Refereed][Not invited]
    HLA-G is a non-classical human MHC class I molecule, which has several characteristics distinct from classical MHC, such as low polymorphism and restricted tissue distribution. HLA-G is expressed on placenta, thymus and some tumors. At the maternal-fetal interface, trophoblasts do not express major classical MHC class I molecules (MHCI), HLA-A and -B, to prevent normal T cell responses. Instead, HLA-G is expressed and can suppress a wide range of immune responses by binding to inhibitory immune cell surface receptors, such as leukocyte Ig-like receptor (LILR) B1 and LILRB2. HLA-G exists in various forms, including beta 2m-associated or -free disulfide-linked dimers that can be expressed either at the cell surface or in soluble form. However, until recently the physiological role of these different molecular forms has been unclear. In this issue of the European Journal of Immunology, one article demonstrates that the disulfide-linked homodimer of beta 2m-associated HLA-G is the major fraction expressed by trophoblast cells. The HLA-G dimer modulates the function of LILRB1-expressing antigen-presenting cells by principally binding to LILRB1. On the other hand, another recent report showed that beta 2m-free disulfide-linked HLA-G dimers are produced by villous cytotrophoblast cells. Taken together, these results provide strong evidence in support of the hypothesis that HLA-G dimers play a role in immune suppression at the maternal-fetal interface. Further in-depth investigation will help to clarify the precise mechanism of HLA-G receptor recognition and signaling in vivo and the role of these interactions in successful reproduction.
  • Kaori Sasaki, Toyoyuki Ose, Naoaki Okamoto, Katsumi Maenaka, Taku Tanaka, Hisao Masai, Mihoko Saito, Tsuyoshi Shirai, Daisuke Kohda
    EMBO JOURNAL 26 (10) 2584 - 2593 0261-4189 2007/05 [Refereed][Not invited]
    In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3' end of a nascent leading DNA strand in the stalled replication forks. The crystal structures in complexes with oligonucleotides and the combination of fluorescence correlation spectroscopy and mutagenesis reveal that the N-terminal domain of PriA possesses a binding pocket for the 3'-terminal nucleotide residue of DNA. The interaction with the deoxyribose 3'-OH is essential for the 3'-terminal recognition. In contrast, the direct interaction with 3'-end nucleobase is unexpected, considering the same affinity for oligonucleotides carrying the four bases at the 3' end. Thus, the N-terminal domain of PriA recognizes the 3'end base in a base-non-selective manner, in addition to the deoxyribose and 5'-side phosphodiester group, of the 3''-terminal nucleotide to acquire both sufficient affinity and non-selectivity to find all of the stalled replication forks generated during DNA duplication. This unique feature is prerequisite for the proper positioning of the helicase domain of PriA on the unreplicated double-stranded DNA.
  • Toyoyuki Ose, Nicolas Soler, Linda Rasubala, Kimiko Kuroki, Daisuke Kohda, Dominique Fourmy, Satoko Yoshizawa, Katsumi Maenaka
    STRUCTURE 15 (5) 577 - 586 0969-2126 2007/05 [Refereed][Not invited]
    Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SeIB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SeIB-C). SeIB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SeIB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SeIB-RNA interaction is sequence independent, possibly reflecting SeIB-tRNA/-rRNA recognitions. Based on these data, the dynamic SeIB-ribosome-mRNA-tRNA interactions will be discussed.
  • Kimiko Kuroki, Sayoko Kobayashi, Mitsunori Shiroishi, Mizuho Kajikawa, Naoaki Okamoto, Daisuke Kohda, Katsumi Maenaka
    JOURNAL OF IMMUNOLOGICAL METHODS 320 (1-2) 172 - 176 0022-1759 2007/03 [Refereed][Not invited]
    Fluorescence correlation spectroscopy (FCS) can directly and quickly detect the translational diffusion of individual fluorescence-labeled molecules in solutions. Although FCS analyses for protein-protein interactions have been performed, the very weak interactions generally observed in cell-cell recognition of the immune system have not been examined in detail. Here, we report the FCS analysis for low-affinity and fast-kinetic binding (K-d greater than mu M range) of the human inhibitory immune cell surface receptor, leukocyte immunoglobulin-like receptor B1 (LILRB1), to its ligands, MHC (major histocompatibility complex) class I molecules (MHCIs) by using the single-molecule FCS detection system which requires only a small amount of sample. Since the random labeling technique for LILRB1 disturbed the MHCI binding, we performed site-specific labeling of LILRB1 by introducing a cysteine residue at the C-terminus, which could be covalently attached with the fluorescence reagent, Alexa647. This technique can be applied to other type I membrane receptors. The low-affinity binding of LILRB1-Alexa647 to MHCIs (HLA-Cw4, and -G1) was detected by FCS, even though non-labeled MHCIs were only twice as big as the labeled LILRB1. Their dissociation constants (7.5 mu M (HLA-Cw4) and 5.7 mu M (HLA-G1)) could be determined and were consistent with surface plasmon resonance (SPR) data. These results indicate that the single-molecule FCS detection system is capable of analyzing the binding characteristics of immune cell surface receptors even in difficult cases such as (1) small amount of protein samples, (2) small difference in molecular weight and (3) weak affinity. Therefore, it is a powerful tool for characterization and high throughput inhibitor screening of a wide variety of cell-cell recognition receptors involved in immunologically relevant events. (c) 2006 Elsevier B.V. All rights reserved.
  • Cutting Edge: Allele-specific and peptide-dependent interactions between KIR3DL1 and HLA-A and HLA-B.
    Thananchai H, Gillespie G, Martin MP, Bashirova A, Yawata N, Yawata M, Easterbrook P, McVicar DW, Maenaka K, Parham P, Carrington M, Dong T, Rowl, Jones S
    Journal of immunology (Baltimore, Md. : 1950) 178 (1) 33 - 37 0022-1767 2007/01 [Refereed][Not invited]
  • 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 0027-8424 2006/10 [Refereed][Not invited]
    HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte lg-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including beta(2)-microglobulin (beta(2)m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the beta(2)m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the beta(2)m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-A resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCI recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G alpha 3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/beta(2)m) and free forms of beta(2)m. Binding studies using beta(2)m-free MHCIs revealed differential beta(2)m-dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCIs, which may in turn regulate immune suppression.
  • MHC class I-like MILL molecules are beta(2)-microglobulin-associated, GPI-anchored glycoproteins that do not require TAP for cell surface expression
    Mizuho Kajikawa, Tomohisa Baba, Utano Tomaru, Yutaka Watanabe, Satoru Koganei, Sachiyo Tsuji-Kawahara, Naoki Matsumoto, Kazuo Yamamoto, Masaaki Miyazawa, Katsumi Maenaka, Akihiro Shizu, Masanori Kasahara
    JOURNAL OF IMMUNOLOGY 177 (5) 3108 - 3115 0022-1767 2006/09 [Refereed][Not invited]
    MILL (MHC class I-like located near the leukocyte receptor complex) is a family of MHC class I-like molecules encoded outside the MHC, which displays the highest sequence similarity to human MICA/B molecules among known class I molecules. In the present study, we show that the two members of the mouse MILL family, MILL1 and MILL2, are GPI-anchored glycoproteins associated with beta(2)-microglobulin (beta(2)m) and that cell surface expression of MILL1 or MILL2 does not require functional TAP molecules. MILL1 and MILL2 molecules expressed in bacteria could be refolded in the presence of beta(2)m, without adding any peptides. Hence, neither MILL1 nor MILL2 is likely to be involved in the presentation of peptides. Immunohistochemical analysis revealed that MILL1 is expressed in a subpopulation of thymic medullary epithelial cells and a restricted region of inner root sheaths in hair follicles. The present study provides additional evidence that MILL is a class I family distinct from MICA/B.
  • Mitsunori Shiroishi, Mizuho Kajikawa, Kimiko Kuroki, Toyoyuki Ose, Daisuke Kohda, Katsumi Maenaka
    JOURNAL OF BIOLOGICAL CHEMISTRY 281 (28) 19536 - 19544 0021-9258 2006/07 [Refereed][Not invited]
    Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and F alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.
  • 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 0175-7598 2006/06 [Refereed][Not invited]
    Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.
  • Shiroishi M, Kohda D, Maenaka K
    Biochimica et biophysica acta 1764 (5) 985 - 988 0006-3002 2006/05 [Refereed][Not invited]
  • 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 0021-9258 2006/04 [Refereed][Not invited]
    HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1: 2 (HLA-G dimer: receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.
  • Sasaki K, Ose T, Tanaka T, Mizukoshi T, Ishigaki T, Maenaka K, Masai H, Kohda D
    Biochimica et biophysica acta 1764 (1) 157 - 160 0006-3002 2006/01 [Refereed][Not invited]
  • 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 0022-2836 2006/01 [Refereed][Not invited]
    The human inhibitory receptor, leukocyte immunoglobulin (Ig)-like receptor B1 (also called Ig-like transcript (ILT) 2, CD85j), is broadly expressed on leukocytes. LILRB1 binds to a wide range of major histocompatibility complex class I molecules (MHCIs) and transduces negative signals that can, for example, prevent killing of MHO-expressing cells. Here we report the kinetic, thermodynamic, NMR and crystallographic analyses of MHCl recognition by LILRB1. Kinetic studies demonstrated that LILRB1 binds to MHCIs with fast association and dissociation rates, typical of cell-cell recognition receptors. Thermodynamic analyses showed that LILRB1-MHCI interactions are entropically driven (-T Delta S = -9.4 similar to-6.6 kcal mol(-1)) with low heat capacity changes (Delta C-p= -0.22 similar to-0.10 kcal mol(-1) K-1). The crystal structures of LILRB1 in the different crystal forms exhibited variation in the elbow angle between the two N-terminal Ig-like domains, indicating interdomain flexibility Consistently, NMR analysis provided the direct evidence of the conformational changes of LILRB1 upon the MHCI binding. These findings suggest that LILRB1-MHCI interactions, while involving some conformational adjustment, are not accompanied by a very large reduction in conformational flexibility at the binding interface. This mode of binding is distinct from '' Induced-fit '' binding, which is associated with large reductions in conformational flexibility and would be suitable for rapid engagement of MHCIs to enable fast monitoring of the expression level of MHCIs on target cells. (c) 2005 Elsevier Ltd. All rights reserved.
  • 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 0964-6906 2005/08 [Refereed][Not invited]
    Leukocyte immunoglobulin-like receptor subfamily B member I (LILRB1/LIR1/ILT2) is an inhibitory receptor broadly expressed on leukocytes and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated to be a susceptibility region to systemic lupus erythematosus (SLE). In this study, we screened for polymorphisms of LILRB1 and examined their association with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four non-synonymous substitutions within the ligand-binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a non-synonymous substitution of the cytoplasmic region were identified. CY01 and 02 did not co-segregate with PE01-03. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 diplotype was significantly associated with RA (odds ratio 2.05, P = 0.019 and Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1 was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.
  • K Maenaka, K Fukushi, H Aramaki, Y Shirakihara
  • M Igura, T Ose, T Obita, C Sato, K Maenaka, T Endo, D Kohda
  • L Rasubala, D Fourmy, T Ose, D Kohda, K Maenaka, S Yoshizawa
  • Yoshizawa S, Rasubala L, Ose T, Kohda D, Fourmy D, Maenaka K
    Nature structural & molecular biology 12 (2) 198 - 203 1545-9993 2005/02 [Refereed][Not invited]
  • M Vales-Gomez, M Shiroishi, K Maenaka, HT Reyburn
    JOURNAL OF VIROLOGY 79 (4) 2251 - 2260 0022-538X 2005/02 [Refereed][Not invited]
    Human cytomegalovirus carries a gene, UL18, that is homologous to cellular major histocompatibility complex (MHC) class I genes. Like MHC class I molecules, the protein product of the UL18 gene associates with beta2-microglobulin, and the stability of this complex depends on peptide loading. UL18 protein binds to ILT2 (CD85j), an inhibitory receptor present on B cells, monocytes, dendritic cells, T cells, and NK cells that also recognizes classical and nonclassical MHC molecules. These observations suggest that UL18 may play a role in viral immune evasion, but its real function is unclear. Since this molecule has similarity with polymorphic MHC proteins, we explored whether the UL18 gene varied between virus isolates. We report here that the UL18 gene varies significantly between virus isolates: amino acid substitutions were found in the predicted (alpha1, alpha2, and alpha3 domains of the UL18 protein molecule. We also studied the ability of several variant UL18 proteins to bind to the ILT2 receptor. All of the variants tested bound to ILT2, but there were marked differences in the affinity of binding to this receptor. These differences were reflected in functional assays measuring inhibition of the cytotoxic capacity of NK cells via interaction with ILT2. In addition, the variants did not bind other members of the CD85 family. The implications of these data are discussed.
  • T Motohashi, T Shimojima, T Fukagawa, K Maenaka, EY Park
    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 326 (3) 564 - 569 0006-291X 2005/01 [Refereed][Not invited]
    Silkworm is one of the most attractive hosts for large-scale production of eukaryotic proteins as well as recombinant baculoviruses for gene transfer to mammalian cells. The bacmid system of Autographa californica nuclear polyhedrosis virus (AcNPV) has already been established and widely used. However, the AcNPV does not have a potential to infect silkworm. We developed the first practical Bombyx mori nuclear polyhedrosis virus bacmid system directly applicable for the protein expression of silkworm. By using this system, the green fluorescence protein was successfully expressed in silkworm larvae and pupae not only by infection of its recombinant virus but also by direct injection of its bacmid DNA. This method provides the rapid protein production in silkworm as long as 10 days, is free from biohazard, thus will be a powerful tool for the future production factory of recombinant eukaryotic proteins and baculoviruses. (C) 2004 Elsevier Inc. All rights reserved.
  • 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 0006-291X 2005/01 [Refereed][Not invited]
    PriB is not only an essential protein necessary for the replication restart on the collapsed and disintegrated replication fork, but also an important protein for assembling of primosome onto PhiX174 genomic DNA during replication initiation. Here we report a 2.0-Angstrom-resolution X-ray structure of a biologically functional form of PriB from Escherichia coli. The crystal structure revealed that despite a low level of primary sequence identity, the PriB monomer, as well as the dimeric form, are structurally identical to the N-terminal DNA-binding domain of the single-stranded DNA-binding protein (SSB) from Escherichia coli, which possesses an oligonucleotides-binding-fold. The oligonucleotide-PriB complex model based on the oligonucleotides-SSB complex structure suggested that PriB had a DNA-binding pocket conserved in SSB from Escherichia coli and might bind to single-stranded DNA in the manner of SSB. Furthermore, surface plasmon resonance analysis and fluorescence measurements demonstrated that PriB binds single-stranded DNA with high affinity, by involving tryptophan residue. The significance of these results with respect to the functional role of PriB in the assembly of primosome is discussed. (C) 2004 Published by Elsevier Inc.
  • T Kamura, K Maenaka, S Kotoshiba, M Matsumoto, D Kohda, RC Conaway, JW Conaway, KI Nakayama
    GENES & DEVELOPMENT 18 (24) 3055 - 3065 0890-9369 2004/12 [Refereed][Not invited]
    The ECS (Elongin B/C-Cul2/Cul5-SOCS-box protein) complex is a member of a family of ubiquitin ligases that share a Cullin-Rbx module. SOCS-box proteins recruit substrates to the ECS complex and are linked to Cullin-Rbx via Elongin B/C. VHL has been implicated as a SOCS-box protein, but lacks a C-terminal sequence (downstream of the BC box) of the SOCS box. We now show that VHL specifically interacts with endogenous Cul2-Rbx1 in mammalian cells, whereas SOCS-box proteins associate with Cul5-Rbx2. We also identify LRR-1 and FEM1B as proteins that share a region of homology with VHL (the VHL box, including the BC box and downstream residues) and associate with Cul2-Rbx1. ECS complexes can thus be classified into two distinct protein assemblies, that is, those that contain a subunit with a VHL box (composed of the BC box and a downstream Cul2 box) that interacts with Cul2-Rbx1, and those that contain a subunit with a SOCS box (BC box and downstream Cul5 box) that interacts with Cul5-Rbx2. Domain-swapping analyses showed that the specificity of interaction of VHL-box and SOCS-box proteins with Cullin-Rbx modules is determined by the Cul2 and Cul5 boxes, respectively. Finally, RNAi-mediated knockdown of the Cul2-Rbx1 inhibited the VHL-mediated degradation of HIF-2alpha, whereas knockdown of Cul5-Rbx2 did not affect it. These data suggest that the functions of the Cul2-Rbx1 and Cul5-Rbx2 modules are distinct.
  • H Wada, N Matsumoto, K Maenaka, K Suzuki, K Yamamoto
    EUROPEAN JOURNAL OF IMMUNOLOGY 34 (1) 81 - 90 0014-2980 2004/01 [Refereed][Not invited]
    The human non-classical MHC class I molecule HLA-E is a ligand for both an inhibitory NK cell receptor (CD94/NKG2A) and an activating receptor (CD94/NKG2d). To identify HLA-E surface recognized by both receptors, especially to determine if both receptors recognize the same epitope, we made a series of individually Ala-substituted HLA-E proteins and analyzed their binding to CD94/NKG2A or CD94/NKG2C. Eight HLA-E mutations that significantly impaired HLA-E binding to CD94/NKG2A are all found in the top of alpha1/alpha2 domain of HLA-E. These results suggest that CD94/NKG2A binds a HLA-E surface equivalent to a NKG2D binding site on MICA. Of the eight mutations that impaired HLA-E binding to CD94/ NKG2A, six significantly impaired HLA-E binding to CD94/NKG2C suggesting that CD94/ NKG2C also binds a similar surface of HLA-E. Unexpectedly, the two HLA-E mutations (D69A and H155A) selectively abrogated HLA-E binding to CD94/NKG2A, not largely affected CD94/NKG2C. These results indicate that a mostly shared, but partly distinct set of HLA-E residues is discriminated by the two receptors.
  • Uemura Y, Senju S, Fujii S, Iwai LK, Maenaka K, Tabata H, Kanai T, Chen YZ, Nishimura Y
    Modern rheumatology 13 (3) 205 - 214 1439-7595 2003/09 [Refereed][Not invited]
  • 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 0027-8424 2003/07 [Refereed][Not invited]
    Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (K-d = 2-45 muM), some interesting differences were observed. ILT2 generally bound with a 2- to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3- to 4-fold higher affinity than to classical MHCIs, suggesting that ILT/HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8(+) T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.
  • NR Zaccai, K Maenaka, T Maenaka, PR Crocker, R Brossmer, S Kelm, EY Jones
    STRUCTURE 11 (5) 557 - 567 0969-2126 2003/05 [Refereed][Not invited]
    The Siglec family of receptors mediates cell surface interactions through recognition of sialylated glycoconjugates. The crystal structure of the N-terminal immunoglobulin-like domain of the Siglec sialoadhesin (SnD1) in complex with 2,3-sialyllactose has informed the design of sialic acid analogs (sialosides) that bind Siglecs with significantly enhanced affinities and specificities. Binding assays against sialoadhesin (Sn; Siglec-1), CD22 (Siglec-2), and MAG (Siglec-4) show a 10- to 300-fold reduction in IC50 values (relative to methyl-alpha-Neu5Ac) for three sialosides bearing aromatic group modifications of the glycerol side chain: Me-alpha-9-N-benzoyi-amino-9-deoxy-Neu5Ac (BENZ), Me-alpha-9-N-(naphthyl-2-carbonyl)-amino-9-deoxy-Neu5Ac (NAP), and Me-alpha-9-N-(biphenyl-4-carbonyl)-amino-9-deoxy-Neu5Ac (BIP). Crystal structures of these sialosides in complex with SnD1 suggest explanations for the differences in specificity and affinity, providing further ideas for compound design of physiological and potentially therapeutic relevance.
  • Systematic analysis of the combinatorial nature of epitopes recognized by TCR leads to identification of mimicry epitopes for glutamic acid decarboxylase 65-specific TCRs.
    Uemura Y, Senju S, Maenaka K, Iwai LK, Fujii S, Tabata H, Tsukamoto H, Hirata S, Chen YZ, Nishimura Y
    Journal of immunology (Baltimore, Md. : 1950) 170 (2) 947 - 960 0022-1767 2003/01 [Refereed][Not invited]
  • 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 0907-4449 2002/10 [Refereed][Not invited]
    PhoB is a transcriptional factor that activates more than 30 genes of the pho regulon in response to phosphate starvation. Crystals of its C-terminal domain (PhoBC) were obtained in two forms. The first crystal form, obtained from phosphate solution, belongs to space group P2(1), with unit-cell parameters a = 30.7, b = 105.9, c = 30.9 Angstrom, beta = 110.3degrees. The second form, crystallized from PEG solution, belongs to the same space group, but has a smaller unit cell (a = 30.6, b = 37.5, c = 44.4 Angstrom, beta = 109.4degrees). Crystals of selenomethionyl-derivatized PhoBC were obtained using the conditions for the second crystal form. Diffraction data from wild-type PhoBC (2.0 Angstrom resolution) and MAD data sets from selenomethionyl-derivative PhoBC (3.0 Angstrom resolution) have been collected at 100 K with a synchrotron-radiation source. MAD data analysis is in progress.

Books etc

  • 植田 正, 前仲 勝実 (Joint editor)
    南江堂 2018/01 (ISBN: 4524403515) 278
  • 津本 浩平, 植田 正, 前仲 勝実 (Joint translation)
    南江堂 2016/02 (ISBN: 4524268642) 451
  • 千田 俊哉, 前仲 勝実 
    Springer 2016 (ISBN: 9784431560289)
  • 植田正, 前仲勝実 (Joint work)
    南江堂 2012/09 (ISBN: 4524402969) 258
  • 植田 正, 前仲 勝実 (Joint editor)
    南江堂 2007/10 (ISBN: 4524402462) 238
  • Molecular recognition by Ig-like receptors, KIRs and Fc-gammma-Rs. In Activating and Inhibitory Immunoglobulin-Like Receptors (Eds. M.D.Cooper, T.Takai and J.V.Ravetch)
    Spring-Verlag Tokyo,Inc. 2001
  • Molecular recognition by Ig-like receptors, KIRs and Fc-gammma-Rs. In Activating and Inhibitory Immunoglobulin-Like Receptors
    Eds. M.D.Cooper, T.Takai and J.V.Ravetch 2001
  • リゾチーム-酵素および抗原のモデルタンパク質として-
    シリーズ分子生物学6 構造生物学(三浦謹一郎編)


  • CD85レセプター群の分子認識
    2000 -2005
  • Molecular recognition of CD85 receptors
    2000 -2005
  • Fcレセプター群の分子認識
    1998 -2005
  • Melcular recongnition of Fc receptors
    1998 -2005


Industrial Property Rights

Awards & Honors

  • 2015 北海道大学 北海道大学総長賞研究奨励賞
    受賞者: 前仲 勝実
  • 2014 北海道大学 北海道大学総長賞教育奨励賞
    受賞者: 前仲 勝実
  • 2008 日本免疫学会 日本免疫学会研究奨励賞
    受賞者: 前仲 勝実

Research Grants & Projects

  • Molecular basis for cell entry of canine distemper virus
    Date (from‐to) : 2015/04 -2018/03 
    Author : MAENAKA Katsumi
  • 文部科学省:科学研究費補助金(基盤研究(B))
    Date (from‐to) : 2010 -2012 
    Author : 前仲 勝実
  • 文部科学省:科学研究費補助金(新学術領域研究(研究領域提案型))
    Date (from‐to) : 2010 -2012 
    Author : 前仲 勝実
  • 免疫系細胞表面レセプター群の分子認識
    Date (from‐to) : 1997 -2010
  • Molecular recongnition of cell surface receptors in immune system
    Grant-in-Aid for Scientific Research
    Date (from‐to) : 1997 -2010
  • 文部科学省:科学研究費補助金(特定領域研究)
    Date (from‐to) : 2008 -2009 
    Author : 前仲 勝実
  • 文部科学省:科学研究費補助金(特定領域研究)
    Date (from‐to) : 2008 -2009 
    Author : 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...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2007 -2009 
    Author : Katsumi MAENAKA
    Cell surface antigens have various forms depending on states of cells, whose structural characteristics can be recognized by paired immune receptors to control immune responses. Here, in order to clarify the molecular basis for these events, we performed the ligand binding and structural analyses of PILRs and KLRG1. PILRs bind to its ligand, CD99, with sugar- and peptide-dependent manner, furthermore, KLRG1 discriminate the monomer and the dimer configurations of the E-cadherin. These results provided important insights on the immune regulation by paired immune cell receptors.
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(特定領域研究)
    Date (from‐to) : 2003 -2008 
    Author : Daisuke KOHDA, Katsumi MAENAKA, Takashi SAITOH
  • 文部科学省:科学研究費補助金(特定領域研究)
    Date (from‐to) : 2006 -2007 
    Author : 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...
  • 文部科学省:科学研究費補助金(特定領域研究)
    Date (from‐to) : 2005 -2006 
    Author : 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...
  • 文部科学省:科学研究費補助金(特定領域研究)
    Date (from‐to) : 2005 -2006 
    Author : 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., ...
  • Ministry of Education, Culture, Sports, Science and Technology:Grants-in-Aid for Scientific Research(基盤研究(B))
    Date (from‐to) : 2004 -2006 
    Author : Daiske KOHDA, 前仲 勝実
    Structure-function studies of protein domains are always very productive in a sense that one study on a representative protein domain makes it possible to infer the structures and functions of many family proteins that contain the protein domain. The structure determination of SH2 and SH3 domains in the early 1990s represents the first good example. We determined the first three-dimensional structure of the PX domain. The present study aimed at the systematic detection of the interaction of the PX domains with the SH3 domains in yeast,. Since many PX and SH3 domains are encoded in eukaryoti...
  • 文部科学省:科学研究費補助金(若手研究(B))
    Date (from‐to) : 2004 -2005 
    Author : 前仲 勝実
  • 文部科学省:科学研究費補助金(萌芽研究)
    Date (from‐to) : 2004 -2005 
    Author : 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...
  • 文部科学省:科学研究費補助金(特定領域研究)
    Date (from‐to) : 2003 -2004 
    Author : 神田 大輔, 前仲 勝実
  • 文部科学省:科学研究費補助金(奨励研究(A), 若手研究(B))
    Date (from‐to) : 2001 -2002 
    Author : 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:科学研究費補助金(特定領域研究(C))
    Date (from‐to) : 2000 -2000 
    Author : 前仲 勝実
    <背景と目的>ゲノム解析の進展により人間の病理に関わる多くの細胞表面レセプター群が見出されてくると、現在全体の約1/3を占めている免疫グロブリン(Ig)様ドメインを持つレセプターを統合的に解析することが重要になってくる。本研究では最近免疫系細胞に幅広く見出されてきた免疫レセプター抑制性モチーフ(ITIM)を細胞内ドメインにもつヒト抑制性免疫レセプタースーパーファミリー(Inhibitory-receptor superfamily,以下IRSと省略する)の多くがIg様ドメインを持ち、また多様なリガンドを認識するため、統合的な蛋白質間分子認識データを収集する格好の標的であることに着目した。我々はIRSの中から、主要組織適合性抗原(MHC)を認識するKiller cell Ig-like receptor(KIR)やIg-like transcript(ILT)、更には抗体Fc部位を認識するFcγRについてリガンド分子認識機構の機能解析及びX線結晶構造解析を行うことを目的とした。<検討結果と考察>本年度は、KIRのMHCに対する分子認識に関して2つのIg様ドメインを細胞外にもつKIR2Dのこれまでの解析を踏まえ、更に3つのIg様ドメインを持つKIR3DのリガンドMHC、HLA-B51の結晶構造解析及び活性型KIR2Dの機能解析を行った。決定したHLA-B51の立体構造から、KIR...
  • ファージディスプレイ系の改良
  • 免疫系レセプターに対する阻害剤開発
  • Improvement of Phage display system
  • Development of inhibitors for immunoreceptors

Educational Activities

Teaching Experience

  • Advanced Biostatistics Drug Discovery ScienceAdvanced Biostatistics Drug Discovery Science Hokkaido University
  • Special Lecture on Drug Discovery Science II (Biocamp)Special Lecture on Drug Discovery Science II (Biocamp) Hokkaido University
  • Special Lecture on Drug Discovery Science I (Life innovation seminar)Special Lecture on Drug Discovery Science I (Life innovation seminar) Hokkaido University
  • Special lecture of the basics of biomedicineSpecial lecture of the basics of biomedicine Hokkaido University
  • Special lecture of state-of-art drug developmentSpecial lecture of state-of-art drug development Hokkaido University
  • Pharmaceutical SciencePharmaceutical Science Hokkaido University
  • Physical Chemistry IIPhysical Chemistry II Hokkaido University
  • Physical Chemistry IPhysical Chemistry I Hokkaido University
  • 生命科学研究
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • Pharmaceutical Science
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
    キーワード : 神経薬理学、薬物送達学、免疫・感染学、放射性薬品化学、生体イメージング
  • 生命科学実習
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : biopharmaceuticals, nucleic acid pharmaceuticals, antibody pharmaceuticals, cancer, autoimmune disease, viral infectious disease, gene therapy, low-molecular compounds, compound library, organic synthesis, in silico screening, conformation analysis, acceptor, membrane protein, drug metabolism, pharmacokinetics
  • 生命科学論文講読Ⅰ
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • Inter-Graduate School Classes(General Subject):Humanities and Social Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 医薬品開発、商業的開発、スタートアップビジネス
  • 生命科学論文講読Ⅱ
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 生命科学院
  • Inter-Graduate School Classes(General Subject):Natural and Applied Sciences
    開講年度 : 2018
    課程区分 : 修士課程
    開講学部 : 大学院共通科目
    キーワード : 創薬、ドラッグデザイン、化合物スクリーニング、バリデーション
  • 生命科学特別研究
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 生命科学院
  • Physical Chemistry II
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
    キーワード : 熱力学、平衡、物質の状態、溶液化学、電気化学
  • 生命科学文献講読
    開講年度 : 2018
    課程区分 : 博士後期課程
    開講学部 : 生命科学院
  • Physical Chemistry I
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
    キーワード : 化学結合、量子化学、分光学、電子スペクトル、振動スペクトル、回転スペクトル、核磁気共鳴(NMR)、X線結晶構造解析等、機器分析
  • 卒業研究準備実習Ⅰ
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 卒業研究準備実習Ⅱ
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬学論文講読演習Ⅰ
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬学論文講読演習Ⅱ
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬学論文講読演習Ⅲ
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬学総合演習
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬学卒業研究
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬科学演習
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬科学論文講読演習
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部
  • 薬科学卒業研究
    開講年度 : 2018
    課程区分 : 学士課程
    開講学部 : 薬学部

Campus Position History

  • 2013年4月1日 
  • 2013年4月1日 
  • 2014年4月1日 
  • 2016年4月1日 
  • 2018年4月1日 

Position History

  • 2013年4月1日 
  • 2013年4月1日 
  • 2014年4月1日 
  • 2016年4月1日 
  • 2018年4月1日 

Committee Membership

  • 2002 -2003   Japanese Society for Biophysics   Associate editor   Japanese Society for Biophysics

Social Contribution

Social Contribution

Social Contribution

  • 北大道新セミナー
    Date (from-to) : 2019/05/23-2019/05/23
    Role : Lecturer

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