基本情報

所属

• 薬学研究院　創薬科学部門　生体機能科学分野

職名

• 准教授

学位

• 博士（保健学）（東京大学大学院）

ホームページURL

http://kaken.nii.ac.jp/d/r/90553313.ja.html

科研費研究者番号

• 90553313

J-Global ID

• 201301093635778260

研究キーワード

• ウイルスペプチド   抗体医薬品   構造解析   分子免疫学   バイオ医薬品   遺伝子多型産物   HIV Nef   HIV-2   免疫チェックポイント   LILR   KIR   免疫制御   HLA   表面プラズモン共鳴法   細胞表面受容体   蛋白質間相互作用   

研究分野

• ライフサイエンス / ゲノム生物学
• ライフサイエンス / 薬系分析、物理化学
• ライフサイエンス / 免疫学
• ライフサイエンス / 医化学
• ライフサイエンス / 生物物理学
• ライフサイエンス / 機能生物化学
• ライフサイエンス / 構造生物化学

担当教育組織

•  学士課程　薬学部
•  修士課程　生命科学院
•  博士課程　生命科学院

職歴

• 2018年04月 - 現在 北海道大学大学院薬学研究院 准教授
• 2010年07月 - 2018年03月 北海道大学大学院薬学研究院 助教
• 2010年04月 - 2010年06月 北海道大学大学院薬学研究院 日本学術振興会特別研究員(PD)
• 2008年04月 - 2010年03月 九州大学生体防御医学研究所 日本学術振興会特別研究員(PD)
• 2004年04月 - 2008年03月 九州大学生体防御医学研究所 学術研究員
• 2002年04月 - 2004年03月 東京大学大学院医学系研究科 日本学術振興会特別研究員(DC2)

学歴

•         - 2004年03月   東京大学大学院医学系研究科国際保健学専攻
•         - 1999年03月   東京理科大学理工学部応用生物科学科

所属学協会

• 日本組織適合性学会   日本免疫学会   日本リウマチ学会   日本生化学会   日本分子生物学会   日本薬学会   

研究活動情報

論文

• Impact of Micropolymorphism Outside the Peptide Binding Groove in the Clinically Relevant Allele HLA-C*14 on T Cell Responses in HIV-1 Infection.

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

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

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

  Kimiko Kuroki, Hideo Fukuhara, Takashi Tadokoro, Katsumi Maenaka

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

  On the immune cell surface, many immune receptors are expressed and modulate the inhibitory or activating signals to control the immune responses. Recently, some of these receptors have been categorized as immune checkpoint receptors and targeted for cancer immunity or autoimmune diseases. To analyze the weak and fast binding typical for immune receptor-ligand interactions, a real-time surface plasmon resonance (SPR) technique is useful. However, it sometimes becomes difficult to optimize the immobilization conditions and appropriate controls. Considering that receptor orientation is relevant for achieving function on the cell surface, it is important to immobilize ligand proteins using specific tags at the membrane proximal end to avoid steric hindrance and structural changes in specific binding regions. Here we introduce a sensor chip, Sensor Chip CAP (Cytiva), which enables reversible and orientation-controlled immobilization of biotinylated ligands, resulting in a significant cost-effective method. We further show preparation methods of several biotinylated immune receptor proteins for SPR analysis, which are also useful for structural and other functional analyses.
• カルシウムイオンによるERp57-CNX複合体の構造機能調節

  谷川 雄哉, 金村 進吾, 伊藤 大, 林 雨曦, 松崎 元紀, 黒木 喜美子, 山口 宏, 前仲 勝実, 李 映昊, 稲葉 謙次, 奥村 正樹

  日本生化学会大会プログラム・講演要旨集 94回 [3T14m - 245)] (公社)日本生化学会 2021年11月
• Ca2+ Regulates ERp57-Calnexin Complex Formation

  Yuya Tanikawa, Shingo Kanemura, Dai Ito, Yuxi Lin, Motonori Matsusaki, Kimiko Kuroki, Hiroshi Yamaguchi, Katsumi Maenaka, Young-Ho Lee, Kenji Inaba, Masaki Okumura

  Molecules 26 10 2853 - 2853 2021年05月11日 [査読有り][招待有り]
   

  ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.
• HLA-G-LILRB 複合体構造を基にした機能解析

  黒木, 喜美子, 松原, 永季, 神田, 諒, 宮下, 尚之, 白石, 充典, 福永, 裕子, 上敷領, 淳, 福永, 淳, 福原, 秀雄, 廣瀬, 薫, Hunt, Joan S, 杉田, 有治, 喜多, 俊介, 尾瀬, 農之, 前仲, 勝実

  福山大学薬学部研究年報 = Annual report of the Faculty of Pharmacy & Pharmaceutical Sciences, Fukuyama University 38 35 - 36 福山大学薬学部 2020年12月25日
• Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.

  Hiroki Kusaka, Shunsuke Kita, Takashi Tadokoro, Kouki Yoshida, Yoshiyuki Kasai, Harumi Niiyama, Yukari Fujimoto, Shinya Hanashima, Michio Murata, Shigeru Sugiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka

  Protein expression and purification 172 105631 - 105631 2020年08月 [査読有り][通常論文]
   

  CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and β2-microglobulin (β2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with β2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 μg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.
• Therapeutic effects of soluble human leukocyte antigen G2 isoform in lupus-prone MRL/lpr mice.

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

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

  Human leukocyte antigen (HLA)-G, a non-classical HLA class I molecule, has one of the splicing isoforms, HLA-G2, which lacks one domain (α2) and forms a non-covalent homodimer. HLA-G2 is expressed on placental cells, regulatory T cells, tumor cells, and virus-infected cells, and is involved in immunosuppression. The major isoform of HLA-G, HLA-G1, binds to leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2, on the contrary, HLA-G2 binds to only LILRB2. We previously reported that HLA-G2 bound LILRB2 more strongly than HLA-G1 and also to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs. Furthermore, HLA-G2 showed immunosuppressive effects in both collagen-induced arthritis (CIA) and atopic dermatitis-like model mice. In this study, we examine in vivo effects of HLA-G2 in systemic lupus erythematosus (SLE) model mice. HLA-G2 showed the suppression of the typical SLE symptoms such as serum anti-dsDNA antibody level and urinary albumin index. Furthermore, HLA-G2 tended to downregulate B-lymphocyte stimulator (BLyS) production. This is the first observation of the immunosuppressive effects of HLA-G2 isoform in SLE model mice, suggesting that HLA-G2 could be a useful therapeutic agent for SLE.
• Structure of HIV-2 Nef Reveals Features Distinct from HIV-1 Involved in Immune Regulation.

  Kengo Hirao, Sophie Andrews, Kimiko Kuroki, Hiroki Kusaka, Takashi Tadokoro, Shunsuke Kita, Toyoyuki Ose, Sarah L Rowland-Jones, Katsumi Maenaka

  iScience 23 1 100758 - 100758 2020年01月24日 [査読有り][通常論文]
   

  The human immunodeficiency virus (HIV) accessory protein Nef plays a major role in establishing and maintaining infection, particularly through immune evasion. Many HIV-2-infected people experience long-term viral control and survival, resembling HIV-1 elite control. HIV-2 Nef has overlapping but also distinct functions from HIV-1 Nef. Here we report the crystal structure of HIV-2 Nef core. The di-leucine sorting motif forms a helix bound to neighboring molecules, and moreover, isothermal titration calorimetry demonstrated that the CD3 endocytosis motif can directly bind to HIV-2 Nef, ensuring AP-2-mediated endocytosis for CD3. The highly conserved C-terminal region forms a α-helix, absent from HIV-1. We further determined the structure of simian immunodeficiency virus (SIV) Nef harboring this region, demonstrating similar C-terminal α-helix, which may contribute to AP-1 binding for MHC-I downregulation. These results provide insights into the distinct pathogenesis of HIV-2 infection.
• Structural and Functional Basis for LILRB Immune Checkpoint Receptor Recognition of HLA-G Isoforms.

  Kimiko Kuroki, Haruki Matsubara, Ryo Kanda, Naoyuki Miyashita, Mitsunori Shiroishi, Yuko Fukunaga, Jun Kamishikiryo, Atsushi Fukunaga, Hideo Fukuhara, Kaoru Hirose, Joan S Hunt, Yuji Sugita, Shunsuke Kita, Toyoyuki Ose, Katsumi Maenaka

  Journal of immunology (Baltimore, Md. : 1950) 203 12 3386 - 3394 2019年12月15日 [査読有り][通常論文]
   

  Human leukocyte Ig-like receptors (LILR) LILRB1 and LILRB2 are immune checkpoint receptors that regulate a wide range of physiological responses by binding to diverse ligands, including HLA-G. HLA-G is exclusively expressed in the placenta, some immunoregulatory cells, and tumors and has several unique isoforms. However, the recognition of HLA-G isoforms by LILRs is poorly understood. In this study, we characterized LILR binding to the β2-microglobulin (β2m)-free HLA-G1 isoform, which is synthesized by placental trophoblast cells and tends to dimerize and multimerize. The multimerized β2m-free HLA-G1 dimer lacked detectable affinity for LILRB1, but bound strongly to LILRB2. We also determined the crystal structure of the LILRB1 and HLA-G1 complex, which adopted the typical structure of a classical HLA class I complex. LILRB1 exhibits flexible binding modes with the α3 domain, but maintains tight contacts with β2m, thus accounting for β2m-dependent binding. Notably, both LILRB1 and B2 are oriented at suitable angles to permit efficient signaling upon complex formation with HLA-G1 dimers. These structural and functional features of ligand recognition by LILRs provide novel insights into their important roles in the biological regulations.
• Evaluation of the Reactivity and Receptor Competition of HLA-G Isoforms toward Available Antibodies: Implications of Structural Characteristics of HLA-G Isoforms.

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

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

  The human leucocyte antigen (HLA)-G, which consists of seven splice variants, is a tolerogenic immune checkpoint molecule. It plays an important role in the protection of the fetus from the maternal immune response by binding to inhibitory receptors, including leukocyte Ig-like receptors (LILRs). Recent studies have also revealed that HLA-G is involved in the progression of cancer cells and the protection from autoimmune diseases. In contrast to its well characterized isoform, HLA-G1, the binding activities of other major HLA-G isoforms, such as HLA-G2, toward available anti-HLA-G antibodies are only partially understood. Here, we investigate the binding specificities of anti-HLA-G antibodies by using surface plasmon resonance. MEM-G9 and G233 showed strong affinities to HLA-G1, with a nM range for their dissociation constants, but did not show affinities to HLA-G2. The disulfide-linker HLA-G1 dimer further exhibited significant avidity effects. On the other hand, 4H84 and MEM-G1, which can be used for the Western blotting of HLA-G isoforms, can bind to native HLA-G2, while MEM-G9 and G233 cannot. These results reveal that HLA-G2 has a partially intrinsically disordered structure. Furthermore, MEM-G1, but not 4H84, competes with the LILRB2 binding of HLA-G2. These results provide novel insight into the functional characterization of HLA-G isoforms and their detection systems.
• キャリア支援キックオフシンポジウム〜ニーズを探る〜 キャリアパス関連アンケート結果の報告

  黒木 喜美子, 石塚 敏, 王寺 典子, 坂本 慎太郎, 奥平 裕子, 藤井 明美, 宮川 卓, 成瀬 妙子

  MHC: Major Histocompatibility Complex 26 2Suppl. 57 - 57 (一社)日本組織適合性学会 2019年09月
• キャリア支援キックオフシンポジウム〜ニーズを探る〜 ダイバーシティ関連アンケート結果の報告

  王寺 典子, 石塚 敏, 奥平 裕子, 坂本 慎太郎, 黒木 喜美子, 藤井 明美, 宮川 卓, 成瀬 妙子

  MHC: Major Histocompatibility Complex 26 2Suppl. 58 - 58 (一社)日本組織適合性学会 2019年09月
• Specificity of Morbillivirus Hemagglutinins to Recognize SLAM of Different Species.

  Hideo Fukuhara, Yuri Ito, Miyuki Sako, Mizuho Kajikawa, Koki Yoshida, Fumio Seki, Mwila Hilton Mwaba, Takao Hashiguchi, Masa-Aki Higashibata, Toyoyuki Ose, Kimiko Kuroki, Makoto Takeda, Katsumi Maenaka

  Viruses 11 8 2019年08月19日 [査読有り][通常論文]
   

  Measles virus (MV) and canine distemper virus (CDV) are highly contagious and deadly, forming part of the morbillivirus genus. The receptor recognition by morbillivirus hemagglutinin (H) is important for determining tissue tropism and host range. Recent reports largely urge caution as regards to the potential expansion of host specificities of morbilliviruses. Nonetheless, the receptor-binding potential in different species of morbillivirus H proteins is largely unknown. Herein, we show that the CDV-H protein binds to the dog signaling lymphocyte activation molecule (SLAM), but not to the human, tamarin, or mouse SLAM. In contrast, MV-H can bind to human, tamarin and dog SLAM, but not to that of mice. Notably, MV binding to dog SLAM showed a lower affinity and faster kinetics than that of human SLAM, and MV exhibits a similar entry activity in dog SLAM- and human SLAM-expressing Vero cells. The mutagenesis study using a fusion assay, based on the MV-H-SLAM complex structure, revealed differences in tolerance for the receptor specificity between MV-H and CDV-H. These results provide insights into H-SLAM specificity related to potential host expansion.
• Crystal structure of the complex between venom toxin and serum inhibitor from Viperidae snake.

  Narumi Shioi, Takashi Tadokoro, Seijiro Shioi, Yuki Okabe, Haruki Matsubara, Shunsuke Kita, Toyoyuki Ose, Kimiko Kuroki, Shigeyuki Terada, Katsumi Maenaka

  The Journal of biological chemistry 294 4 1250 - 1256 2019年01月25日 [査読有り][通常論文]
   

  Venomous snakes have endogenous proteins that neutralize the toxicity of their venom components. We previously identified five small serum proteins (SSP-1-SSP-5) from a highly venomous snake belonging to the family Viperidae as inhibitors of various toxins from snake venom. The endogenous inhibitors belong to the prostate secretory protein of 94 amino acids (PSP94) family. SSP-2 interacts with triflin, which is a member of the cysteine-rich secretory protein (CRISP) family that blocks smooth muscle contraction. However, the structural basis for the interaction and the biological roles of these inhibitors are largely unknown. Here, we determined the crystal structure of the SSP-2-triflin complex at 2.3 Å resolution. A concave region centrally located in the N-terminal domain of triflin is fully occupied by the terminal β-strands of SSP-2. SSP-2 does not bind tightly to the C-terminal cysteine-rich domain of triflin; this domain is thought to be responsible for its channel-blocker function. Instead, the cysteine-rich domain is tilted 7.7° upon binding to SSP-2, and the inhibitor appears to sterically hinder triflin binding to calcium channels. These results help explain how an endogenous inhibitor prevents the venomous protein from maintaining homeostasis in the host. Furthermore, this interaction also sheds light on the binding interface between the human homologues PSP94 and CRISP-3, which are up-regulated in prostate and ovarian cancers.
• Structural and thermodynamic analyses reveal critical features of glycopeptide recognition by the human PILRα immune cell receptor.

  Furukawa A, Kakita K, Yamada T, Ishizuka M, Sakamoto J, Hatori N, Maeda N, Ohsaka F, Saitoh T, Nomura T, Kuroki K, Nambu H, Arase H, Matsunaga S, Anada M, Ose T, Hashimoto S, Maenaka K

  The Journal of biological chemistry 292 51 21128 - 21136 2017年12月 [査読有り][通常論文]
   

  Before entering host cells, herpes simplex virus-1 uses its envelope glycoprotein B to bind paired immunoglobulin-like type 2 receptor alpha (PILR alpha) on immune cells. PILR alpha belongs to the Siglec (sialic acid (SA)-binding immunoglobulin-like lectin)- like family, members of which bind SA. PILR alpha is the only Siglec member to recognize not only the sialylated O-linked sugar T antigen (sTn) but also its attached peptide region. We previously determined the crystal structure of PILR alpha complexed with the sTn-linked glycopeptide of glycoprotein B, revealing the simultaneous recognition of sTn and peptide by the receptor. However, the contribution of each glycopeptide component to PILR alpha binding was largely unclear. Here, we chemically synthesized glycopeptide derivatives and determined the thermodynamic parameters of their interaction with PILR alpha. We show that glycopeptides with different sugar units linking SA and peptides (i.e. "GlcNAc-type" and "deoxy-GlcNAc-type" glycopeptides) have lower affinity and more enthalpy-driven binding than the wild type (i.e. GalNAc-type glycopeptide). The crystal structures of PILR alpha complexed with these glycopeptides highlighted the importance of stereochemical positioning of the O4 atom of the sugar moiety. These results provide insights both for understanding the unique O-glycosylated peptide recognition by the PILR alpha and for the rational design of herpes simplex virus-1 entry inhibitors.
• Therapeutic application of human leukocyte antigen-G1 improves atopic dermatitis-like skin lesions in mice

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

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

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

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

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

  HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and (beta 2-microglobulin (beta 2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the alpha 2 domain-deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown. In this study, we unexpectedly found that HLA-G2 naturally forms a beta 2m-free and nondisulfide-linked homodimer, which is in contrast to the disulfide-bonded beta 2m-associated HLA-G1 homodimer. Furthermore, single-particle analysis, using electron microscopy, revealed that the overall structure and domain organization of the HLA-G2 homodimer resemble those of the HLA class II heterodimer. The HLA-G2 homodimer binds to leukocyte Ig-like receptor B2 with slow dissociation and a significant avidity effect. These findings provide novel insights into leukocyte Ig-like receptor B2-mediated immune regulation by the HLA-G2 isoform, as well as the gene evolution of HLA classes.
• Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection

  Naoyoshi Maeda, Atsushi Furukawa, Kosuke Kakita, Masahiro Anada, Shunichi Hashimoto, Shigeki Matsunaga, Kimiko Kuroki, Toyoyuki Ose, Akihisa Kato, Jun Arii, Yasushi Kawaguchi, Hisashi Arase, Katsumi Maenaka

  BIOLOGICAL & PHARMACEUTICAL BULLETIN 39 11 1897 - 1902 2016年11月 [査読有り][通常論文]
   

  Herpes simplex virus type 1 (HSV-1) is a causative agent for a variety of diseases. Although antiherpetic drugs such as acyclovir have been developed to inhibit virus replication through interaction with DNA kinases, their continuous administration leads to an increase in the frequency of drug-resistant HSV-1, which is an important clinical issue that requires urgent solution. Recently, we reported that the sialylated O-linked sugar T antigen (sTn) and its attached peptide region (O-glycosylated sTn peptide) derived from the HSV-1 glycoprotein B (gB) protein inhibited HSV-1 infection by specifically targeting paired immunoglobulin-like type 2 receptor alpha (PILR alpha) in vitro. In this study, to further identify novel inhibitors of gB-mediated HSV-1 infection in vitro, we established a cell-based fusion assay for rapid drug screening. Chinese hamster ovary (CHO) cells were transfected with expression plasmids for HSV-1 gB, gD, gH, and gL, and T7 RNA polymerase, and were designated as the effector cells. The CHO-K1 cells stably expressing PILRa were transfected with the expression plasmid for firefly luciferase under the T7 promoter, and were designated as the target cells. The effector and target cells were co-cultured, and luminescence was measured when both cells were successfully fused. Importantly, we found that cell-to-cell fusion was specifically inhibited by O-glycosylated sTn peptide in a dose dependent manner. Our results suggested that this virus-free cell-based fusion assay system could be a useful and promising approach to identify novel inhibitors of gB-mediated HSV-1 infection, and will aid in the development of antiviral therapeutic strategies for HSV-1-associated diseases.
• HIV-1 Control by NK Cells via Reduced Interaction between KIR2DL2 and HLA-C^∗12:02/C^∗14:03.

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

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

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

  Ami Takahashi, Kimiko Kuroki, Yuki Okabe, Yoshiyuki Kasai, Naoki Matsumoto, Chisato Yamada, Toshiyuki Takai, Toyoyuki Ose, Shigeyuki Kon, Tadashi Matsuda, Katsumi Maenaka

  HUMAN IMMUNOLOGY 77 9 754 - 759 2016年09月 [査読有り][通常論文]
   

  HLA-G is involved in maternal-fetal immune tolerance and is reported to be a natural tolerogenic molecule. Seven-spliced isoforms including dimeric and beta 2m-free forms have been identified. The major isoform, HLA-G1 (and its soluble type HLA-G5), binds to the inhibitory immune receptors, leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2. We previously reported that HLA-G1 also binds to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs, and had a significant immunosuppressive effect in collagen-induced arthritis (CIA) mice. Although HLA-G2 and its soluble form HLA-G6 bind specifically to LILRB2, its functional characteristics are largely unknown. In this study, we report the significant immunosuppressive effect of HLA-G2 dimer in CIA mice. Surface plasmon resonance analysis revealed a specific interaction of HLA-G2 with PIR-B. CIA mice were administered HLA-G2 protein subcutaneously once in the left footpad and clinical severity was evaluated in a double-blind study. A single administration of HLA-G2 maintained a suppressive effect for over 1 month. These results suggested that the HLA-G2 protein might be a useful biopharmaceutical for the treatment of rheumatoid arthritis by binding to inhibitory PIR-B. (C) 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
• Ribophorin II is involved in the tissue factor expression mediated by phosphatidylserine-dependent antiprothrombin antibody on monocytes

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

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

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

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

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

  Efficient DNA carriers are needed as a gene medication for curing brain disorders. In the present study, the function of a neutral lipid envelope-type nanoparticle (LNP) encapsulating pDNA was evaluated after intracerebroventricular administration. The lipid envelope was composed of a series of SS-cleavable and pH-activated lipid like materials (ssPalm) including myristic acid, vitamin A and vitamin E in the hydrophobic scaffold (LNPssPalmM, LNPssPalmA, LNPssPalmE, respectively). The LNPssPalmA and LNPssPalmE were extensively distributed in the corpus callosum, and then gene expression occurred mainly astrocytes in this region, while not in LNPssPalmM. The recombinant human ApoE3-dependent enhancement of the uptake into an astrocyte-derived cell line (KT-5) was observed in LNPssPalmA and LNPssPalmE. Thus, ApoE in the brain plays a key role in the cellular uptake of these particles by astrocytes, and this uptake is dependent on the structure of the hydrophobic scaffold. (C) 2015 Elsevier B.V. All rights reserved.
• Crystal structure of extracellular domain of human lectin-like transcript 1 (LLT1), the ligand for natural killer receptor-P1A

  Shunsuke Kita, Haruki Matsubara, Yoshiyuki Kasai, Takaharu Tamaoki, Yuki Okabe, Hideo Fukuhara, Jun Kamishikiryo, Elena Krayukhina, Susumu Uchiyama, Toyoyuki Ose, Kimiko Kuroki, Katsumi Maenaka

  EUROPEAN JOURNAL OF IMMUNOLOGY 45 6 1605 - 1613 2015年06月 [査読有り][通常論文]
   

  Emerging evidence has revealed the pivotal roles of C-type lectin-like receptors (CTLRs) in the regulation of a wide range of immune responses. Human natural killer cell receptor-P1A (NKRP1A) is one of the CTLRs and recognizes another CTLR, lectin-like transcript 1 (LLT1) on target cells to control NK, NKT and Th17 cells. The structural basis for the NKRP1A-LLT1 interaction was limitedly understood. Here, we report the crystal structure of the ectodomain of LLT1. The plausible receptor-binding face of the C-type lectin-like domain is flat, and forms an extended -sheet. The residues of this face are relatively conserved with another CTLR, keratinocyte-associated C-type lectin, which binds to the CTLR member, NKp65. A LLT1-NKRP1A complex model, prepared using the crystal structures of LLT1 and the keratinocyte-associated C-type lectin-NKp65 complex, reasonably satisfies the charge consistency and the conformational complementarity to explain a previous mutagenesis study. Furthermore, crystal packing and analytical ultracentrifugation revealed dimer formation, which supports a complex model. Our results provide structural insights for understanding the binding modes and signal transduction mechanisms, which are likely to be conserved in the CTLR family, and for further rational drug design towards regulating the LLT1 function.
• Structural basis for simultaneous recognition of an O-glycan and its attached peptide of mucin family by immune receptor PILRα.

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

  Proceedings of the National Academy of Sciences of the United States of America 111 24 8877 - 8882 2014年06月 [査読有り][通常論文]
   

  Paired Ig-like type 2 receptor a (PILR alpha) recognizes a wide range of O-glycosylated mucin and related proteins to regulate broad immune responses. However, the molecular characteristics of these recognitions are largely unknown. Here we show that sialylated O-linked sugar T antigen (sTn) and its attached peptide region are both required for ligand recognition by PILR alpha. Furthermore, we determined the crystal structures of PILR alpha and its complex with an sTn and its attached peptide region. The structures show that PILR alpha exhibits large conformational change to recognize simultaneously both the sTn O-glycan and the compact peptide structure constrained by proline residues. Binding and functional assays support this binding mode. These findings provide significant insight into the binding motif and molecular mechanism (which is distinct from sugar-recognition receptors) by which O-glycosylated mucin proteins with sTn modifications are recognized in the immune system as well as during viral entry.
• NKG2D triggers cytotoxicity in murine epidermal γδ T cells via PI3K-dependent, Syk/ZAP70-independent signaling pathway.

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

  The Journal of investigative dermatology 134 2 396 - 404 2014年02月 [査読有り][通常論文]
   

  Murine epidermal gamma delta T cells, known as dendritic epidermal T cells (DETCs), survey tissue stress through the invariant T-cell receptor (TCR) and non-clonotypic receptors such as NKG2D. NKG2D signaling via the DAP10-phosphatidylinositol 3-kinase (PI3K) pathway directly stimulates cytotoxicity in natural killer (NK) cells and costimulates CD8(+) T cells to augment TCR signals. In activated murine NK cells, NKG2D signals also via the DAP12-Syk/ZAP70 pathway that triggers both cytotoxicity and cytokine production. It remains controversial whether NKG2D on DETCs is a primary activating receptor or functions only as a costimulatory receptor, and signaling pathways initiated by NKG2D ligation in DETCs have not been analyzed. We show that stimulation of short-term DETC lines with recombinant NKG2D ligands triggers degranulation (exocytosis of cytotoxic granules) via the PI3K-dependent signaling pathway, but does not induce cytokine production or Syk/ZAP70 activation. Coengagement of TCR or Syk/ZAP70 signaling was not crucial for DETC-mediated killing of NKG2D ligand-expressing target cells. Thus, NKG2D can function as a coactivating stress receptor that directly triggers cytotoxicity in DETCs, at least after priming, via the PI3K-dependent, Syk/ZAP70-independent signaling pathway.
• Allosteric Regulation of Epoxide Opening Cascades by a Pair of Epoxide Hydrolases in Monensin Biosynthesis

  Atsushi Minami, Toyoyuki Ose, Kyohei Sato, Azusa Oikawa, Kimiko Kuroki, Katsumi Maenaka, Hiroki Oguri, Hideaki Oikawa

  ACS CHEMICAL BIOLOGY 9 2 562 - 569 2014年02月 [査読有り][通常論文]
   

  Multistep catalysis of epoxide hydrolase/cyclase in the epoxide opening cascade is an intriguing issue in polyether biosynthesis. A pair of structurally homologous epoxide hydrolases was found in gene clusters of ionophore polyethers. In the epoxide opening reactions with MonBI and MonBII involved in monensin biosynthesis, we found that MonBII and catalytically inactive MonBI mutant catalyzed two-step reactions of bisepoxide substrate analogue to afford bicyclic product although MonBII alone catalyzed only the first cyclization. The X-ray crystal structure of MonBI dimers suggested the importance of the KSD motif in MonBI/MonBI interaction, which was further supported by gel filtration chromatography of wild-type MonBI and mutant MonBI. The involvement of the KSD motif in heterodimer formation was confirmed by in vitro assay. Direct evidence of MonBI/MonBII interaction was obtained by native mass spectrometry. Its dissociation constant was determined as 2.21 X 10(-5) M by surface plasmon resonance. Our results suggested the involvement of an allosteric regulation mechanism by MonBI/MonBII interaction in monensin skeletal construction.
• 1P005 Lectin-like transcript 1 (LLT1)の構造解析とLLT1-CD161 複合体のモデル作製(蛋白質 : 構造,ポスター,第52回日本生物物理学会年会(2014年度))

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

  生物物理 54 1 S141  一般社団法人 日本生物物理学会 2014年
• Structural and functional mosaic nature of MHC class I molecules in their peptide-free form

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

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

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

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

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

  HLA-G, a natural immunosuppressant present in the human placenta during pregnancy, prevents fetal destruction by the maternal immune system. The immunosuppressive effect of HLA-G is mediated by the immune cell inhibitory receptors, LILRB1 and LILRB2. HLA-G forms disulfide-linked dimers by natural oxidation, and the dimer associates with LILRB1/B2 much more strongly than the monomer. Furthermore, the dimer formation remarkably enhanced the LILRB-mediated signaling. In this report, we studied the in vivo immunosuppressive effect of the HLA-G dimer, using the collagen-induced arthritis model mouse. Mice were treated with the HLA-G monomer or dimer intracutaneously at the left foot joint, once or for 5 days, and the clinical severity was evaluated daily in a double-blind study. The HLA-G monomer and dimer both produced excellent anti-inflammatory effects with a single, local administration. Notably, as compared to the monomer, the dimer exhibited significant immunosuppressive effects at lower concentrations, which persisted for about two months. In accordance with this result, a binding study revealed that the HLA-G dimer binds PIR-B, the mouse homolog of the LILRBs, with higher affinity and avidity than the monomer. The HLA-G dimer is expected to be quite useful as an anti-rheumatoid arthritis agent, in small amounts with minimal side effects. (C) 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
• Distinct HIV-1 Escape Patterns Selected by Cytotoxic T Cells with Identical Epitope Specificity

  Yuichi Yagita, Nozomi Kuse, Kimiko Kuroki, Hiroyuki Gatanaga, Jonathan M. Carlson, Takayuki Chikata, Zabrina L. Brumme, Hayato Murakoshi, Tomohiro Akahoshi, Nico Pfeifer, Simon Mallal, Mina John, Toyoyuki Ose, Haruki Matsubara, Ryo Kanda, Yuko Fukunaga, Kazutaka Honda, Yuka Kawashima, Yasuo Ariumi, Shinichi Oka, Katsumi Maenaka, Masafumi Takiguchi

  JOURNAL OF VIROLOGY 87 4 2253 - 2263 2013年02月 [査読有り][通常論文]
   

  Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.
• 3P006 HLA-G2/G6アイソフォームの単粒子構造解析(01A.蛋白質:構造,ポスター,日本生物物理学会年会第51回(2013年度))

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

  生物物理 53 1 S212  一般社団法人 日本生物物理学会 2013年
• Ribophorin II Is Involved in the Tissue Factor Expression Mediated by Phosphatidylserine-Dependent Antiprothrombin Antibody On Monocytes.

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

  ARTHRITIS AND RHEUMATISM 64 10 S741 - S741 2012年10月 [査読有り][通常論文]
  
• Inhibiting HLA-B27 homodimer-driven immune cell inflammation in spondylarthritis

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

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

  Objective Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLAB27 normally forms complexes with beta 2-microglobulin (beta 2m) and peptide to form heterotrimers. However, an unusual characteristic of HLAB27 is its ability to form beta 2m-free heavy chain homodimers (HLAB272), which, unlike classic HLAB27, bind to killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). Binding of HLAB272 to KIR-3DL2positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLAB272 in order to confirm its expression in SpA and to inhibit its proinflammatory properties. Methods We generated monoclonal antibodies by screening a human phage display library positively against B272 and negatively against B27 heterotrimers. Specificity was tested by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) assay, and fluorescence-activated cell sorting (FACS) analysis of B272-expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR-3DL2B272 interactions was tested using cell lines and PBMCs from patients with SpA. Results Monoclonal antibody HD6 specifically recognized recombinant HLAB272 by ELISA and by SPR assay. HD6 bound to cell lines expressing B272. FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLAB272 to KIR-3DL2 and the survival and proliferation of KIR-3DL2positive NK cells. Finally, HD6 inhibited production of the proinflammatory diseaseassociated cytokine interleukin-17 by PBMCs from patients with AS. Conclusion These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.
• HLA-B27 Homodimers and Free H Chains Are Stronger Ligands for Leukocyte Ig-like Receptor B2 than Classical HLA Class I

  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 2012年06月 [査読有り][通常論文]
   

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

  Kimiko Kuroki, Atsushi Furukawa, Katsumi Maenaka

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

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

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

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

  CD160 was recently identified as a T cell coinhibitory molecule that interacts with the herpesvirus entry mediator (HVEM) on antigen-presenting cells to deliver a potent inhibitory signal to CD4(+) T cells. HVEM also binds to the coinhibitory receptor BTLA (B- and T-lymphocyte attenuator) and the costimulatory receptor LIGHT (which is homologous to lymphotoxins, exhibits inducible expression, and competes with the herpes simplex virus glycoprotein D for HVEM, a receptor expressed by T lymphocytes, or TNFSF14), thus regulating the CD160/BTLA/LIGHT/HVEM signaling pathway. To date, the detailed properties of the formation of these complexes, especially HVEM binding to the newly identified receptor CD160, and the relationship of CD160 with BTLA and LIGHT are still unclear. We performed N-terminal sequencing and a mass spectrometric analysis, which revealed that the extracellular domain of CD160 exists primarily in the monomeric form. The surface plasmon resonance analysis revealed that CD160 binds directly to the cysteine-rich domain 1-3 of HVEM with a similar affinity to, but slower dissociation rate than, that of BTLA. Notably, CD160 competed with BTLA for binding to HVEM; in contrast, LIGHT did not affect HVEM binding to either CD160 or BTLA. The results of a mutagenesis study of HVEM also suggest that the CD160 binding region on HVEM was slightly different from, but overlapped with, the BTLA binding site. Interestingly, an anti-CD160 antibody exhibiting antiangiogenic properties blocked CD160/HVEM binding. These results provide insight into the molecular architecture of the CD160/BTLA/LIGHT/HVEM signaling complex that regulates immune function. (C) 2011 Elsevier Ltd. All rights reserved.
• Molecular Basis for LLT1 Protein Recognition by Human CD161 Protein (NKRP1A/KLRB1)

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

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

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

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

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

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

  Kuroki K, Maenaka K

  Methods in molecular biology (Clifton, N.J.) 748 83 - 106 2011年 [査読有り][通常論文]
  
• Structures and molecular recognition for leukocyte immunoglobulin-like receptor family

  Kimiko Kuroki, Katsumi Maenaka

  Seikagaku 83 8 715 - 726 2011年 [査読有り][通常論文]
  
• Importance of the Hydrogen Bonding Network Including Asp52 for Catalysis, as Revealed by Asn59 Mutant Hen Egg-white Lysozymes

  Toyoyuki Ose, Kimiko Kuroki, Masaaki Matsushima, Katsumi Maenaka, Izumi Kumagai

  JOURNAL OF BIOCHEMISTRY 146 5 651 - 657 2009年11月 [査読有り][通常論文]
   

  In the catalysis of sugar hydrolysis by hen egg-white lysozyme, Asp52 is thought to stabilize the reaction intermediate. This residue is involved in the well-ordered hydrogen bonding network including Asn46, Asp48, Ser50 and Asn59 on the anti-parallel -sheet, designated as a platform, on which the substrate sugar sits. To reveal the role of this hydrogen bonding network in the hydrolysis, we characterized Asn59 mutants by biochemical and crystallographic studies. Surprisingly, the introduction of only a methylene group by the Asn59Gln mutation markedly reduced the bacteriolytic activity and abolished the hydrolytic activity towards the synthetic substrate, PNP-(GlcNAc)(5). A similar result was also obtained with the Asn59Asp mutant. The crystal structure of the Asn59Asp mutant in complex with the substrate analogue revealed that, as in the wild-type, the (GlcNAc)(3) was bound in the ABC subsites. The reduced activity would be caused by subtle changes in the side-chain orientations as well as the electrostatic characteristics of Asp59, resulting in the rearrangement of the hydrogen bonding network of the platform. These results suggest that the precise locations of these platform residues, maintained by the well-ordered hydrogen bonding network, are crucial for efficient hydrolysis.
• Molecular Basis for E-cadherin Recognition by Killer Cell Lectin-like Receptor G1 (KLRG1)

  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 2009年10月 [査読有り][通常論文]
   

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

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

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

  Immune cell surface receptors are directly involved in human diseases, and thus represent major drug targets. However, it is generally difficult to obtain sufficient amounts of these receptors for biochemical and structural studies because they often require posttranslational modifications, especially sugar modification. Recently, we have established a bacmid expression system for the baculovirus BmNPV, which directly infects silkworms, an attractive host for the large-scale production of recombinant sugar-modified proteins. Here we produced the human immune cell surface receptor, killer cell Ig-like receptor 2DL1 (KIR2DL1), by using the BmNPV bacmid expression system, in silkworms. By the direct injection of the bacmid DNA, the recombinant KIR2DL1 protein was efficiently expressed, secreted into body fluids, and purified by Ni(2+) affinity column chromatography. We further optimized the expression conditions, and the final yield was 0.2mg/larva. The sugar profiling revealed that the N-linked sugars of the purified protein comprised very few components, two paucimannose-type oligosaccharides, Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc and Manalpha1-6Manbeta1-4GlcNAcbeta1-4(Fucalpha1-6)GlcNAc. This revealed that the protein product was much more homogeneous than the complex-sugar type product obtained by mammalian cell expression. The surface plasmon resonance analysis demonstrated that the purified KIR2DL1 protein exhibited specific binding to the HLA-Cw4 ligand. Moreover, the CD spectrum showed the proper secondary structure. These results clearly suggested that the silkworm expression system is quite useful for the expression of cell surface receptors that require posttranslational modifications, as well as for their structural and binding studies, due to the relatively homogeneous N-linked sugar modifications.
• Reciprocal recognition of an HLA-Cw4-restricted HIV-1 gp120 epitope by CD8(+) T cells and NK cells

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

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

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

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

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

  We employed a urease-catalyzed reaction to gradually remove a high concentration of a chaotropic agent (urea) from a denatured protein solution and demonstrated that efficient protein refolding can be achieved by the urease-catalyzed reaction, without large-volume dilution.
• Serotriflin, a CRISP family protein with binding affinity for small serum protein-2 in snake serum

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

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

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

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

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

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

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

  Biochimica et biophysica acta 1784 4 621 - 628 2008年04月 [査読有り][通常論文]
  
• Biophysical characterization of O-glycosylated CD99 recognition by paired ig-like type 2 receptors

  Shigekazu Tabata, Kimiko Kuroki, Jing Wang, Mizuho Kajikawa, Ikuo Shiratori, Daisuke Kohda, Hisashi Arase, Katsumi Maenaka

  JOURNAL OF BIOLOGICAL CHEMISTRY 283 14 8893 - 8901 2008年04月 [査読有り][通常論文]
   

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

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

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

  Measles virus (MV) enters cells by binding to the signaling lymphocyte activation molecule (also called CD150) on the cell surface, and thus shows the lymphotropism and immunosuppressive effects. The head domain (residues Asp(149) to Arg(617)) of the MV hemagglutinin (MV-H), the attachment protein, was produced using a transient expression system in HEK293T cells. The purified MV-H protein was heterogeneous because of a variety of complex-sugar modifications. The complex-sugar-type MV-H was crystallized successfully, and the crystals belonged to the space group P41212 with the unit cell dimension of a = b = 134 angstrom, c = 100 angstrom, but diffracted only to 3.0 A resolution. MV-H was also expressed in HEK293SGnTI(-) cells lacking the N-acetylglucosaminyltransferase I activity, which render N-linked glycans of the proteins restricted and homogeneous, producing the oligomannose, Man(5)GlcNAc(2). The native and selenomethionyl derivative proteins of the oligomannose-type MV-H were crystallized, and the native crystals well diffracted to 2.6 angstrom resolution. Thus, homogeneous sugar modification may be useful for improved crystallization of heavily sugar-modified viral envelope proteins. (C) 2008 Elsevier B.V. All rights reserved.
• 3P-015 MHC分子を構成するβ2ミクログロブリンのNMR構造解析(蛋白質・物性(3),第46回日本生物物理学会年会)

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

  生物物理 48 S130  一般社団法人 日本生物物理学会 2008年
• Expression, crystallization and preliminary X-ray diffraction analysis of human paired Ig-like type 2 receptor alpha (PILR alpha)

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

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

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

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

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

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

  Kimiko Kuroki, Katsurni Maenaka

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

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

  Toyoyuki Ose, Nicolas Soler, Linda Rasubala, Kimiko Kuroki, Daisuke Kohda, Dominique Fourmy, Satoko Yoshizawa, Katsumi Maenaka

  STRUCTURE 15 5 577 - 586 2007年05月 [査読有り][通常論文]
   

  Selenocysteine (Sec) is the "21st" amino acid and is genetically encoded by an unusual incorporation system. The stop codon UGA becomes a Sec codon when the selenocysteine insertion sequence (SECIS) exists downstream of UGA. Sec incorporation requires a specific elongation factor, SeIB, which recognizes tRNA(Sec) via use of an EF-Tu-like domain and the SECIS mRNA hairpin via use of a C-terminal domain (SeIB-C). SeIB functions in multiple translational steps: binding to SECIS mRNA and tRNA(Sec), delivery of tRNA(Sec) onto an A site, GTP hydrolysis, and release from tRNA and mRNA. However, this dynamic mechanism remains to be revealed. Here, we report a large domain rearrangement in the structure of SeIB-C complexed with RNA. Surprisingly, the interdomain region forms new interactions with the phosphate backbone of a neighboring RNA, distinct from SECIS RNA binding. This SeIB-RNA interaction is sequence independent, possibly reflecting SeIB-tRNA/-rRNA recognitions. Based on these data, the dynamic SeIB-ribosome-mRNA-tRNA interactions will be discussed.
• Detection of weak ligand interactions of leukocyte Ig-like receptor B1 by fluorescence correlation spectroscopy

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

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

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

  Naoyuki Tsuchiya, Chieko Kyogoku, Risa Miyashita, Kimiko Kuroki

  CURRENT MEDICINAL CHEMISTRY 14 4 431 - 439 2007年 [査読有り][通常論文]
   

  A large number of molecules in the immune system are encoded by multigene families. These genes are rich in pairs of activating and inhibitory receptors that share the same ligands, thereby playing a crucial role in immunoregulation. Furthermore, multigene families tend to be highly polymorphic. Thus, multigene families are strong candidates for containing genes that enhance susceptibility to immune system-related diseases. Here, we review studies from our group, as well as other investigators, on three multigene families that belong to the immunoglobulin (1g) - like receptor superfamily: Fey receptor (FCGR), killer cell Ig-like receptor (KIR) and leukocyte Ig-like receptor (LILR) families. FCGR genes have been implicated in susceptibility to systemic lupus erythematosus (SLE). In FCGR2B encoding an inhibitory receptor expressed in B cells, monocytes and dendritic cells, a polymorphism within the transmembrane region, Ile232Thr, was identified and found to be associated with susceptibility to SLE in three Asian populations. Functional analyses revealed that SLE-associated Fc gamma RIIb-232Thr was less efficient in entering the membrane lipid raft, and exhibited reduced inhibitory potential against B cell receptor signaling. Although the frequency of this polymorphism was low in Caucasians, another polymorphism within the promoter region was reported to be associated with SLE. KIR/HLA combinations have been shown to be associated with various autoimmune and infectious diseases. Recently, LILR families have also been found to be highly polymorphic, and association with several diseases has been identified. These results emphasize the role of multigene families in the diversity of human immune response and susceptibility to diseases.
• Structural basis for recognition of the nonclassical MHC molecule HLA-G by the leukocyte Ig-like receptor B2 (LILRB2/LIR2/ILT4/CD85d)

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

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

  HLA-G is a nonclassical MHC class I (MHCI) molecule that can suppress a wide range of immune responses in the maternal-fetal interface. The human inhibitory immune receptors leukocyte lg-like receptor (LILR) B1 [also called LIR1, Ig-like transcript 2 (ILT2), or CD85j] and LILRB2 (LIR2/ILT4/CD85d) preferentially recognize HLA-G. HLA-G inherently exhibits various forms, including beta(2)-microglobulin (beta(2)m)-free and disulfide-linked dimer forms. Notably, LILRB1 cannot recognize the beta(2)m-free form of HLA-G or HLA-B27, but LILRB2 can recognize the beta(2)m-free form of HLA-B27. To date, the structural basis for HLA-G/LILR recognition remains to be examined. Here, we report the 2.5-A resolution crystal structure of the LILRB2/HLA-G complex. LILRB2 exhibits an overlapping but distinct MHCI recognition mode compared with LILRB1 and dominantly recognizes the hydrophobic site of the HLA-G alpha 3 domain. NMR binding studies also confirmed these LILR recognition differences on both conformed (heavy chain/peptide/beta(2)m) and free forms of beta(2)m. Binding studies using beta(2)m-free MHCIs revealed differential beta(2)m-dependent LILR-binding specificities. These results suggest that subtle structural differences between LILRB family members cause the distinct binding specificities to various forms of HLA-G and other MHCIs, which may in turn regulate immune suppression.
• Crystal structure of the human monocyte-activating receptor, "Group 2" leukocyte Ig-like receptor A5 (LILRA5/LIR9/ILT11)

  Mitsunori Shiroishi, Mizuho Kajikawa, Kimiko Kuroki, Toyoyuki Ose, Daisuke Kohda, Katsumi Maenaka

  JOURNAL OF BIOLOGICAL CHEMISTRY 281 28 19536 - 19544 2006年07月 [査読有り][通常論文]
   

  Human leukocyte Ig-like receptor B1 (LILRB1) and B2 (LILRB2) belong to "Group 1" receptors and recognize a broad range of major histocompatibility complex class I molecules (MHCIs). In contrast, "Group 2" receptors show low similarity with LILRB1/B2, and their ligands remain to be identified. To date, the structural and functional characteristics of Group 2 LILRs are poorly understood. Here we report the crystal structure of the extracellular domain of LILRA5, which is an activating Group2 LILR expressed on monocytes and neutrophils. Unexpectedly, the structure showed large changes in structural conformation and charge distribution in the region corresponding to the MHCI binding site of LILRB1/B2, which are also distinct from killer cell Ig-like receptors and F alpha receptors. These changes probably confer the structural hindrance for the MHCI binding, and their key amino acid substitutions are well conserved in Group 2 LILRs. Consistently, the surface plasmon resonance and flow cytometric analyses demonstrated that LILRA5 exhibited no affinities to all tested MHCIs. These results raised the possibility that LILRA5 as well as Group 2 LILRs do not play a role in any MHCI recognition but could possibly bind to non-MHCI ligand(s) on the target cells to provide a novel immune regulation mechanism.
• Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer

  M Shiroishi, K Kuroki, T Ose, L Rasubala, Shiratori, I, H Arase, K Tsumoto, Kumagai, I, D Kohda, K Maenaka

  JOURNAL OF BIOLOGICAL CHEMISTRY 281 15 10439 - 10447 2006年04月 [査読有り][通常論文]
   

  HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1: 2 (HLA-G dimer: receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.
• 1P139 BmNPV expression system; Application to human natural killer cell receptors(4. Protein engineering,Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

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

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

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

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

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

  生物物理 46 2 S165  一般社団法人 日本生物物理学会 2006年
• 1P027 Structual and biochemical studies of MHC class I recognition by human Leukocyte Ig-like receptors(1. Protein structure and dynamics (I),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

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

  生物物理 46 2 S153  一般社団法人 日本生物物理学会 2006年
• Entropically driven MHC class I recognition by human inhibitory receptor leukocyte Ig-like receptor B1 (LILRB1/ILT2/CD85j)

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

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

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

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

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

  Leukocyte immunoglobulin-like receptor subfamily B member I (LILRB1/LIR1/ILT2) is an inhibitory receptor broadly expressed on leukocytes and recognizes HLA-class I and human cytomegalovirus UL18. LILRB1 is encoded within the leukocyte receptor complex on 19q13.4, previously implicated to be a susceptibility region to systemic lupus erythematosus (SLE). In this study, we screened for polymorphisms of LILRB1 and examined their association with SLE and rheumatoid arthritis (RA). In the 5' portion of LILRB1, three haplotypes containing four non-synonymous substitutions within the ligand-binding domains and two single nucleotide polymorphisms within the promoter region were identified and designated as PE01-03. In the 3' portion, two haplotypes (CY01, 02) containing a non-synonymous substitution of the cytoplasmic region were identified. CY01 and 02 did not co-segregate with PE01-03. Significant association with susceptibility to SLE or RA was not observed; however, among the subjects not carrying RA-associated HLA-DRB1 shared epitope (SE), LILRB1.PE01/01 diplotype was significantly associated with RA (odds ratio 2.05, P = 0.019 and Pc = 0.038). Gross difference was not observed in the crystal structures, thermostabilities and binding affinities to HLA-class I ligands among LILRB1.PE01-03 haplotype products; however, surface expression of LILRB1 was significantly decreased in lymphocytes and monocytes from the carriers of PE01 haplotype. These findings demonstrated that LILRB1 is highly polymorphic and is associated with susceptibility to RA in HLA-DRB1 SE negative subjects, possibly by insufficient inhibitory signaling in leukocytes. In addition, these observations suggested that the polymorphisms of LILR family members may be substantially involved in the diversity of human immune responses.
• Association of a functional CD19 polymorphism with susceptibility to systemic sclerosis

  N Tsuchiya, K Kuroki, M Fujimoto, Y Murakami, TF Tedder, K Tokunaga, K Takehara, S Sato

  ARTHRITIS AND RHEUMATISM 50 12 4002 - 4007 2004年12月 [査読有り][通常論文]
   

  Objective. CD19 is overexpressed in B cells from patients with systemic sclerosis (SSc), and plays a crucial role not only for autoantibody production, but also for skin fibrosis in mouse models. We previously reported the association of a GT repeat polymorphisim in the 3'-untranslated region (3'-UTR) of CD19 with human systemic lupus erythematosus. In this study, we examined whether CD19 polymorphisms are associated with genetic susceptibility to SSc. Methods. A case-control association study was performed for CD19 polymorphisms, -499G>T in the promoter region and a GT repeat polymorphism in the 3'-UTR, in 134 patients with SSc and 96 healthy individuals recruited at Kanazawa University. CD19 expression levels in the peripheral blood naive and memory B cells from SSc patients were examined by 2-color How cytometry. Results. Carrier frequencies of the -499T allele in the promoter (odds ratio [OR] 2.18, 95% confidence interval [95% CI] 1.31-3.86, P = 0.003) and of the (GT)(14) allele in the 3'-UTR (OR 1.86, 95% CI 1.05-3.28, P = 0.03) were significantly increased in SSc patients compared with healthy controls. Association was particularly evident in patients with limited cutaneous SSc with anticentromere antibodies. These alleles were in linkage disequilibrium, but the -499T allele seemed to play a primary role. CD19 expression levels in peripheral blood B cells were significantly elevated in both naive (P = 0.0029) and memory (P = 0.0022) B cells from patients with SSc who had the -499T allele as compared with those without the -499T allele. Conclusion. The CD19 -499>T polymorphism is associated with higher CD19 expression in B cells, and with susceptibility to SSc.
• Molecular genetic analyses of human NKG2C (KLRC2) gene deletion

  R Miyashita, N Tsuchiya, K Hikami, K Kuroki, T Fukazawa, M Bijl, CGM Kallenberg, H Hashimoto, T Yabe, K Tokunaga

  INTERNATIONAL IMMUNOLOGY 16 1 163 - 168 2004年01月 [査読有り][通常論文]
   

  Human NKG2A, NKG2C and NKG2E genes are located on 12p13 in the NK gene complex. We recently identified deletion of NKG2C in a Japanese population. This study was performed to identify the breakpoint, and to examine the association of NKG2C deletion with susceptibility to rheumatoid arthritis and systemic lupus erythematosus. The location of the breakpoint was determined to be 1.5-1.8 kb telomeric from the 3' end of NKG2A by comparing sequences of the intergenic segments upstream and downstream of the NKG2C gene in the common haplotype with the intergenic sequence between NKG2A and NKG2E in the deletion haplotype. Based on this information, a genotyping system was developed. The frequency of NKG2C deletion haplotype was 20.2% in Japanese and 20.0% in Dutch populations. The frequency of homozygous deletion was 4.1% in Japanese and 3.8% in Dutch. Evidence for an association with rheumatic diseases was not detected. These results indicated that NKG2C deletion is commonly present in Japanese and Dutch, suggesting that NKG2C is not essential for survival and reproduction, and is not associated with rheumatic diseases.
• Polymorphisms of human CD19 gene: possible association with susceptibility to systemic lupus erythematosus in Japanese

  K Kuroki, N Tsuchiya, BP Tsao, JM Grossman, T Fukazawa, K Hagiwara, H Kano, M Takazoe, T Iwata, H Hashimoto, K Tokunaga

  GENES AND IMMUNITY 3 S21 - S30 2002年10月 [査読有り][通常論文]
   

  CD19 regulates the signaling for B lymphocyte development, activation and proliferation. In mice, CD19 deficiency and overexpression were shown to result in hypogammaglobulinemia and autoantibody production, respectively. In the present study, we screened for the polymorphisms of CD19, and examined the detected polymorphisms for the association with rheumatoid arthritis (RA), Crohn's disease and systemic lupus erythematosus (SLE). Two SNPs, c.705G>T (P235P and IVS14-30C>T, were decreased (P = 0.0096 and P = 0.028, respectively), in SLE A GT repeat polymorphism, c.*132(GT)(12-18), was detected within the 3'-untranslated region, and individuals with greater than or equal to 15 times repeat was significantly increased in the independent two groups of Japanese SLE patients (P = 0.011 and P = 0.035, respectively); the overall difference between total SLE and controls was striking (P = 0.0061). No association was observed for RA and Crohn's disease. In addition, no variations other than the common polymorphisms were detected in four patients with common variable immunodeficiency, the phenotype of which resembles CD19 deficient mice. In Caucasian SLE families, this GT repeat polymorphism was rare. CD19 mRNA level in the isolated peripheral blood B lymphocytes was lower in individuals possessing (GT)(15-18) alleles compared with those without these alleles, both in controls and in SLE patients; however, the difference did not reach statistical significance. These results suggested that either the slight reduction in the CD19 mRNA level associated with the elongation of GT repeat, or an allele of another locus in linkage disquilibrium with CD19 (GT)(15-18), may be associated with susceptibility to SLE in Japanese.

その他活動・業績

• 【免疫系多重遺伝子ファミリー多様性と疾患】免疫系多重遺伝子ファミリー分子LILRBの構造解析と疾患

  黒木 喜美子 炎症と免疫 30 (5) 390 -393 2022年08月 

  近年、免疫反応の調節にかかわる細胞表面受容体が創薬ターゲットとして注目され、抗体医薬品から中分子、低分子医薬品の開発が精力的に進められている。一方で、細胞外領域の配列相同性が高い多重遺伝子群からなる受容体を特異的に、かつ、有効に制御することは、予期せぬ副作用を避けるために必須であり、そのためには立体構造をもとにした疾患との機能的関連を正確に理解する必要がある。(著者抄録)
• 免疫抑制性タンパク質HLA-G2の新規受容体探索と結合親和性評価

  渡邊 紘士, 黒木 喜美子, 前仲 勝実 日本薬学会年会要旨集 142年会 26L -pm03S 2022年03月
• 物理系薬学部会シンポジウム～若い力で推進する物理系薬学～ 免疫抑制タンパク質HLA-Gの機能・構造解析

  黒木 喜美子 日本薬学会年会要旨集 142年会 S01 -4 2022年03月
• 免疫抑制性タンパク質HLA-G2の新規受容体探索と結合親和性評価

  渡邊 紘士, 黒木 喜美子, 前仲 勝実 日本薬学会年会要旨集 142年会 26L -pm03S 2022年03月
• 免疫系受容体の弱く速い結合を介したリガンド認識機構—Ligand recognition mechanisms by immune system receptors via weak and fast binding—特集 物質共生の観点からDDSを考える

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

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

  渡邊紘士, 黒木喜美子, 前仲勝実 Drug Delivery System 37 (2) 2022年
• カルシウムイオンによるERp57-CNX複合体の構造機能調節

  谷川 雄哉, 金村 進吾, 伊藤 大, 林 雨曦, 松崎 元紀, 黒木 喜美子, 山口 宏, 前仲 勝実, 李 映昊, 稲葉 謙次, 奥村 正樹 日本生化学会大会プログラム・講演要旨集 94回 [3T14m -245)] 2021年11月
• 全身性エリテマトーデスモデルマウスにおけるHLA-G2蛋白質の発症抑制効果の検証

  渡邊 紘士, 黒木 喜美子, 山田 千聖, 佐分利 由香里, 高橋 愛実, 前田 直良, 前仲 勝実 日本薬学会年会要旨集 141年会 27V08 -am09S 2021年03月
• 免疫抑制分子HLA-G2特異的新規モノクローナル抗体の作製

  赤岩 愛記, 黒木 喜美子, 引地 和馬, 古川 敦, 前田 直良, 前仲 勝実 日本薬学会年会要旨集 141年会 29P01 -105S 2021年03月
• Ca²⁺によるERp57-CNX複合体の構造機能調節メカニズムの解明

  金村進吾, 谷川雄哉, 伊藤大, LIN Yuxi, 松崎元紀, 黒木喜美子, 山口宏, 前仲勝実, LEE Young-Ho, 稲葉謙次, 奥村正樹 日本分子生物学会年会プログラム・要旨集(Web) 44th 2021年
• 毒ヘビ由来Ca²⁺チャネル阻害型神経毒と毒ヘビ血清蛋白質の複合体構造

  田所高志, 岡部由紀, 松原永季, 喜多俊介, 尾瀬農之, 黒木喜美子, 前仲勝実, 寺田成之, 塩井(青木)成留実 日本結晶学会年会講演要旨集 2021 2021年
• 【抑制化受容体と免疫応答】抑制化受容体の構造

  渡邊 紘士, 黒木 喜美子, 前仲 勝実 炎症と免疫 29 (1) 2 -7 2020年12月 

  近年、抑制化受容体は免疫チェックポイント阻害薬の標的として注目され、抗体医薬品を中心に創薬開発・臨床試験が精力的に実施されている。一方で、抑制化受容体のなかには、相同性の高いファミリーを形成する受容体や、一つの受容体が非自己リガンドを含む多様な分子種を認識するものも存在する。これらの受容体に対する創薬開発においては、リガンド認識機構を構造的に理解したうえで、目的に応じた阻害薬をデザインすることが必須となる。(著者抄録)
• CD1dとアミド基導入抗原複合体のX線結晶構造解析

  喜多 俊介, 日下 裕規, 井貫 晋輔, Md. Imran Hossain, 花島 慎弥, 田所 高志, 新山 真由美, 杉山 成, 相羽 俊彦, 尾瀬 農之, 黒木 喜美子, 深瀬 浩一, 村田 道雄, 藤本 ゆかり, 前仲 勝実 日本結晶学会 講演要旨集 2019年11月 [査読無し][通常論文]
  
• キャリア支援キックオフシンポジウム〜ニーズを探る〜 キャリアパス関連アンケート結果の報告

  黒木 喜美子, 石塚 敏, 王寺 典子, 坂本 慎太郎, 奥平 裕子, 藤井 明美, 宮川 卓, 成瀬 妙子 MHC: Major Histocompatibility Complex 26 (2Suppl.) 57 -57 2019年09月 [査読無し][通常論文]
  
• キャリア支援キックオフシンポジウム〜ニーズを探る〜 ダイバーシティ関連アンケート結果の報告

  王寺 典子, 石塚 敏, 奥平 裕子, 坂本 慎太郎, 黒木 喜美子, 藤井 明美, 宮川 卓, 成瀬 妙子 MHC: Major Histocompatibility Complex 26 (2Suppl.) 58 -58 2019年09月 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dの結晶構造解析

  喜多俊介, 日下裕規, HOSSAIN Md. Imran, 花島慎弥, 井貫晋輔, 田所高志, 新山真由美, 杉山成, 相羽俊彦, 相羽俊彦, 尾瀬農之, 黒木喜美子, 深瀬浩一, 藤本ゆかり, 村田道雄, 前仲勝実 量子ビームサイエンスフェスタ(Web) 2018 2019年
• 【リウマチ・膠原病におけるHLA研究の進展】免疫制御分子HLA-Gとリウマチ性疾患

  黒木 喜美子 炎症と免疫 26 (6) 484 -488 2018年10月 [査読無し][通常論文]
   

  Human Leukocyte Antigen(HLA)-Gは、非古典的ヒト主要組織適合性複合体の一つである。おもに白血球上に発現し、免疫チェックポイント受容体としても知られる抑制性受容体Leukocyte Immunoglobulin-like Receptor(LILR)B1、B2に結合することで、免疫抑制機能を発揮する。とくに妊娠時の胎盤では、胎児が発現するHLA-Gが母体免疫細胞上のLILRB1、LILRB2を介して母体免疫を制御し、妊娠成立に寄与している。筆者らは、多型性の低い生体内免疫制御分子であるHLA-Gのうち、スプライシングによりドメインを欠損するにもかかわらず、主要なHLA-G1アイソフォームの機能を補完するHLA-G2アイソフォームに着目し、過剰な免疫を制御する抗炎症タンパク質製剤としての応用を目指している。(著者抄録)
• 脂質抗原提示分子CD1dによる抗原認識機構の解析

  日下 裕規, 喜多 俊介, 大野 祐介, 木原 章雄, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 日本生化学会大会プログラム・講演要旨集 91回 [3P -090] 2018年09月 [査読無し][通常論文]
  
• HLAと疾患感受性 多様な免疫抑制タンパク質HLA-Gの機能・構造解析

  黒木 喜美子 MHC: Major Histocompatibility Complex 25 (2Suppl.) 64 -64 2018年09月 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dによる抗原認識機構の解析

  日下 裕規, 喜多 俊介, 大野 祐介, 木原 章雄, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 日本生化学会大会プログラム・講演要旨集 91回 [3P -090] 2018年09月 [査読無し][通常論文]
  
• 脂質抗原提示分子CD1dによる抗原認識における種差

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

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

  田所高志, 塩井(青木, 成留実, 岡部由紀, 松原永季, 喜多俊介, 尾瀬農之, 黒木喜美子, 前仲勝実, 寺田成之 日本蛋白質科学会年会プログラム・要旨集 18th 90 2018年05月23日 [査読無し][通常論文]
  
• HIV-2 Nefタンパク質のX線結晶構造

  平尾 憲吾, 黒木 喜美子, Andrews Sophie, 尾瀬 農之, Rowland-Jones Sarah, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [2P -0091] 2017年12月 [査読無し][通常論文]
  
• ヒト免疫受容体NKRP1AによるリガンドLLT1の分子認識機構

  田所 高志, 喜多 俊介, 松原 永季, 笠井 宣征, 玉置 貴晴, 岡部 由紀, 日下 裕規, 石山 夢美, 福原 秀雄, 上敷領 淳, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [2P -0185] 2017年12月 [査読無し][通常論文]
  
• 免疫補助制御分子CD160とリガンドHVEMの分子認識機構に向けた研究

  岩森 美樹, 黒木 喜美子, 齊藤 貴士, 阿部 千紘, 小島 理恵子, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [3P -0103] 2017年12月 [査読無し][通常論文]
  
• PEG化タンパク質精製法の確立に向けた新規構造化PEG分子の設計と機能解析

  山田 千聖, Wawro Adam, 黒木 喜美子, 高橋 愛実, 村岡 貴博, 金原 数, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [3P -0167] 2017年12月 [査読無し][通常論文]
  
• HLA-Cw12拘束性HIV-1ペプチドに対する宿主免疫受容体認識機構

  山下 諒也, 黒木 喜美子, 渡邊 洋介, 阪田 竜馬, 村越 勇人, 赤星 智寛, 滝口 雅文, 前仲 勝実 生命科学系学会合同年次大会 2017年度 [1P -0139] 2017年12月 [査読無し][通常論文]
  
• 単純ヘルペスウイルス由来糖蛋白質がヒト受容体を認識するメカニズム

  尾瀬農之, 石塚幹広, 古川敦, 黒木喜美子, 前仲勝実 日本結晶学会年会講演要旨集 2017 47 2017年11月23日 [査読無し][通常論文]
  
• 分泌タンパク質ANGPTL2を介したシグナル伝達を阻害可能な低分子化合物探索

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

  山崎莉佳, 古川敦, 平安恒幸, 黒木喜美子, 荒瀬尚, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 17th 64 2017年05月22日 [査読無し][通常論文]
  
• CD1dの一本鎖化による安定化と結晶構造解析

  日下裕規, 喜多俊介, HOSSAIN Imuran Md, 花島慎弥, 井貫晋輔, 新山真由美, 杉山成, 尾瀬農之, 黒木喜美子, 藤本ゆかり, 村田道雄, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 17th 120 2017年05月22日 [査読無し][通常論文]
  
• HLAの立体構造と免疫制御受容体の分子認識機構

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

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

  前仲 勝実, 喜多 俊介, 黒木 喜美子 MHC: Major Histocompatibility Complex 23 (2Suppl.) 63 -63 2016年10月 [査読無し][通常論文]
   

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

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

  HLAは非常に遺伝子多型性が高く,多数の遺伝子ファミリーを形成することによって多重性も獲得し,自己・非自己認識を担っている糖タンパク質である。通常,幅広い抗原由来のペプチドをT細胞へ提示するが,さらに,様々な免疫制御受容体との相互作用を介して免疫応答を多面的に調節し,個体の恒常性を維持していることが明らかになってきた。このようなHLAが持つ多面的機能の理解には,X線結晶構造解析による立体構造の決定や物理化学的な相互作用解析が大きな貢献を果たしてきた。本稿では,HLAの分子構造から特にHLAクラスIと受容体群との分子認識機構に着目し,どのようにHLAが免疫反応を制御しているかを概説するとともに,疾患との関連を考察する。(著者抄録)
• アンジオポエチン様因子Angptl2の結晶構造解析および免疫受容体LILRB2との相互作用解析

  荒牧峻彦, 黒木喜美子, 門松毅, 寺田和豊, 尾池雄一, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 104 2016年05月19日 [査読無し][通常論文]
  
• 可溶性HLA‐Gの製剤化に向けて

  田所高志, 可野巧, 市川聡, 松田彰, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 155 2016年05月19日 [査読無し][通常論文]
  
• 免疫疾患患者血清中のHLA‐Gの定量に向けた抗HLA‐G抗体の特性評価

  目黒愛実, 古川敦, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 16th 81 2016年05月19日 [査読無し][通常論文]
  
• HLA‐Cw12拘束性HIV‐1由来ペプチドの探索

  山下諒也, 黒木喜美子, 渡邊洋介, 小柳円, 滝口雅文, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 39th ROMBUNNO.1P‐0042 (WEB ONLY) 2016年 [査読無し][通常論文]
  
• PEG化タンパク質の精製に向けた新規PEG化試薬の設計

  山田 千聖, Wawro Adam, 黒木 喜美子, 高橋 愛実, 村岡 貴博, 金原 数, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [2P0459] -[2P0459] 2015年12月 [査読無し][通常論文]
  
• (プロ)レニン受容体の調製と機能評価系の構築

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

  喜多 俊介, 田所 高志, 松原 永季, 笠井 宣征, 玉置 貴晴, 岡部 由紀, 日下 裕規, 石山 夢美, 福原 秀雄, 上敷領 淳, Krayukhina Elena, 内山 進, 尾瀬 農之, 黒木 喜美子, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 88回・38回 [3T17p -02(3P0328)] 2015年12月 [査読無し][通常論文]
  
• Crystal structure of human CD1d-β2m produced by silkworm-baculovirus expression system

  Kita S, Kusaka H, Yoshida K, Kasai Y, Niiyama M, Hanashima S, Sugiyama S, Murata M, Kuroki K, Maenaka K CD1-MR1-2015 2015年11月 [査読無し][通常論文]
  
• 構造生物学が推進する医薬基盤とその応用 抑制型免疫受容体PILRαによるヘルペスウイルス1型由来O型糖鎖およびペプチド配列の同時認識と創薬

  尾瀬農之, 古川敦, 黒木喜美子, 前仲勝実 医学のあゆみ 254 (8) 559 -565 2015年08月22日 [査読無し][通常論文]
  
• THE IMMUNOSUPPRESSIVE EFFECT OF HLA-G2/G6 HOMODIMER IN COLLAGEN-INDUCED ARTHRITIS MICE

  Ami Takahashi, Kimiko Kuroki, Katsumi Maenaka TISSUE ANTIGENS 86 (2) 78 -78 2015年08月 [査読無し][通常論文]
  
• 【構造生物学が推進する医薬基盤とその応用】抑制型免疫受容体PILRαによるヘルペスウイルス1型由来O型糖鎖およびペプチド配列の同時認識と創薬

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

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

  可野巧, 田所高志, 市川聡, 松田彰, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 115 2015年05月26日 [査読無し][通常論文]
  
• 毒ヘビがもつ自己の毒に対する内在性阻害タンパク質Small Serum Proteinの機能と構造解析

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

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

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

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

  日下裕規, 喜多俊介, 吉田康貴, 笠井宜征, 新山真由美, 杉山成, 村田道雄, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 89 2015年05月26日 [査読無し][通常論文]
  
• アンジオポエチン様因子Angptl2の発現系構築と免疫受容体LILRB2との相互作用解析

  荒牧峻彦, 黒木喜美子, 門松毅, 寺田和豊, 尾池雄一, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 15th 138 2015年05月26日 [査読無し][通常論文]
  
• 単純ヘルペスウイルスがペア型レセプターPILRαを利用するメカニズム

  尾瀬農之, 黒木喜美子, 山口宗親, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実 物構研サイエンスフェスタ要旨集 3rd 79 2015年 [査読無し][通常論文]
  
• 単純ヘルペスウイルス1型感染阻害物質の探索

  前田直良, 古川敦, 坂本二郎, 黒木喜美子, 尾瀬農之, 柿田浩輔, 穴田仁洋, 橋本俊一, 有井潤, 加藤哲久, 川口寧, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 62nd 273 2014年10月31日 [査読無し][通常論文]
  
• 【HLAのブレイクスルー-臨床応用と疾患研究の進歩】HLAクラスI受容体-LILR-のあらたな知見

  黒木 喜美子 医学のあゆみ 251 (4) 311 -316 2014年10月 [査読無し][通常論文]
   

  Human leukocyte antigen(HLA)はさまざまな疾患との関連が報告される。非常に多様性の高い分子であり、主要な受容体であるT細胞受容体との相互作用が古くから注目されてきた。一方で、T細胞以外でも、NK細胞上のkiller cell immunoglobulin(Ig)-like receptor(KIR)ファミリー、CD94/NKG2ファミリーや骨髄系細胞上のleukocyte Ig-like receptor(LILR)ファミリーなど多数のペア型受容体がHLAクラスIを認識し、免疫系細胞の活性化を制御している。このように、多数の受容体がHLAクラスIを認識する意義と重要性について、受容体の特異性や分子認識機構の解析から疾患発症メカニズムの解明まで幅広く精力的な研究が進められてきた。同時に、有核細胞に広く発現する古典的HLAクラスI分子(HLA-A、B、C)だけでなく、局所発現する非古典的HLAクラスI分子のひとつHLA-Gが抑制性LILR受容体を介して伝達する免疫抑制機能についても注目されてきている。(著者抄録)
• 自己免疫疾患に関わるHLAクラスI分子による免疫制御の分子基盤

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

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

  Kita S, Kusaka H, Yoshida K, Kasai Y, Niiyama M, Sugiyama S, Murata M, Kuroki K, Maenaka K ICCBM15 2014年09月 [査読無し][通常論文]
  
• Structural Analysis for the Interaction of sialic T antigen glycopeptide of HSV-1 with Entry Receptor PILR alpha

  Takao Nomura, Jiro Sakamoto, Fumina Oosaka, Kosuke Kakita, Atsushi Furukawa, Masahiro Anada, Shunichi Hashimoto, Kimiko Kuroki, Toyoyuki Ose, Hisashi Arase, Takashi Saitoh, Katsumi Maenaka PROTEIN SCIENCE 23 230 -230 2014年07月 [査読無し][通常論文]
  
• 免疫学 HLA-Gによるコラーゲン誘導性関節炎抑制効果

  黒木 喜美子 医学のあゆみ 249 (12) 1261 -1262 2014年06月 [査読無し][通常論文]
  
• 「構造生物学と免疫」MHCクラスI受容体の構造生物学

  黒木喜美子, 前仲勝実 JSI Newsl 22 (2) 22 2014年04月09日 [査読無し][通常論文]
  
• 単純ヘルペスウイルスがペア型レセプターPILRαを利用する分子基盤

  尾瀬農之, 黒木喜美子, 山口宗親, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実 日本結晶学会年会講演要旨集 2014 110 2014年 [査読無し][通常論文]
  
• NMRによるヒトCD160細胞外ドメインの立体構造解析

  阿部千紘, 黒木喜美子, 小島理恵子, 齊藤貴士, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0723 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• 合理的HIVワクチン設計のためのgp120エピトープ探索系の確立に向けて

  阪田竜馬, 黒木喜美子, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0722 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• HEK293細胞を用いたHLA‐Cw12拘束性HIV‐1由来免疫制御ペプチドの探索

  渡邊洋介, 黒木喜美子, 小柳円, 滝口雅文, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 37th 1P-0721 (WEB ONLY) 2014年 [査読無し][通常論文]
  
• 単純ヘルペスウイルス1型(HSV‐1)のglycoprotein B(gB)とmyelin associated glycoprotein(MAG)との相互作用解明

  青木亨丞, 福原秀雄, 黒木喜美子, 武田森, 末永忠広, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 61st 329 2013年10月29日 [査読無し][通常論文]
  
• 【細胞表面受容体】構造的特徴:1回膜貫通型 受容体の遺伝子:KIR,LILR,PILR 免疫系細胞表面のペア型受容体

  黒木 喜美子, 前仲 勝実 生体の科学 64 (5) 490 -491 2013年10月 [査読無し][通常論文]
  
• HIV由来ペプチドライブラリーを用いたHLA‐Cw12拘束性免疫制御ペプチドの同定

  渡邊洋介, 黒木喜美子, 小柳円, 滝口雅文, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 36th 3P-0758 (WEB ONLY) 2013年 [査読無し][通常論文]
  
• HIV由来変異ペプチドによる宿主免疫の二重逃避機構

  黒木 喜美子, 前仲 勝実 臨床免疫・アレルギー科 58 (2) 210 -216 2012年08月 [査読無し][通常論文]
  
• 麻疹ウイルス及び犬ジステンパーウイルスH蛋白質の構造学的研究

  尾瀬農之, 橋口隆生, 酒匂幸, 伊藤由梨, 福原秀雄, 黒木喜美子, 柳雄介, 竹田誠, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 12th 83 2012年05月31日 [査読無し][通常論文]
  
• 機能および構造解析に向けた細胞表面蛋白質の調製法

  福原秀雄, 橋口隆生, 黒木喜美子, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 12th 19 2012年05月31日 [査読無し][通常論文]
  
• ヒト免疫制御受容体LILRB1とリガンドHLA‐Gの複合体の結晶構造解析

  黒木喜美子, 松原永季, 神田諒, 上敷領淳, 尾瀬農之, 福永裕子, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 35th 1P-0060 (WEB ONLY) 2012年 [査読無し][通常論文]
  
• 各臓器移植についての拒絶反応の解説・移植の新しい治療法の紹介(第5回) NKレセプターとHLAの最前線

  黒木 喜美子, 北辻 千展, 前仲 勝実 MHC: Major Histocompatibility Complex 18 (3) 215 -233 2011年12月 [査読無し][通常論文]
   

  ナチュラルキラー(NK)細胞上には多数のNKレセプターが存在し、自然免疫で中心的な役割を果たすNK細胞の傷害活性を制御している。ヒトNKレセプターは、構造上免疫グロブリン(Ig)様レセプターに属するkiller cell immunoglobulin(Ig)-like receptor群、leukocyte Ig-like receptor群およびC型レクチン様レセプターに属するNKG2/CD94群に分類され、いずれも活性型と抑制型が存在するペア型レセプターの一員である。抑制型NKレセプターの多くはMHCクラスIをリガンドとして認識し、正常な自己細胞に対してNK細胞が傷害しないように制御しているが、活性型NKレセプターのリガンドについては未だ不明な点も多い。本稿では特にMHCクラスIをリガンドとするNKレセプターに焦点を当て、その機能、構造、疾患との関連について最近の知見を概説する。(著者抄録)
• gamma/deltaT細胞とNK細胞 ヒトCD161(NKRP1A/KLRB1)とリガンドLLT1の分子認識機構(Molecular basis for LLT1 recognition by human CD161 (NKRP1A/KLRB1))

  黒木 喜美子, 上敷領 淳, 福原 秀雄, 岡部 由紀, 前仲 勝実 日本免疫学会総会・学術集会記録 40 77 -77 2011年11月 [査読無し][通常論文]
  
• Leukocyte Immunoglobulin-Like Receptor(LILR)ファミリーの分子認識

  黒木 喜美子, 前仲 勝実 生化学 83 (8) 715 -726 2011年08月 [査読無し][通常論文]
   

  Leukocyte Immunoglobulin(Ig)-Like Receptor(LILR)は、細胞外にIg様ドメインを二つまたは四つ持ち、免疫系の細胞に広く発現するペア型受容体ファミリーであり、細胞内ドメインの構造から活性型、抑制型、分泌型の3種類に分類される。1997年以降、現在までに11種類の機能的LILRタンパク質が同定されており、疾患と発現量または遺伝子多型との関連が多数報告されているにも関わらず、その機能・構造・リガンド特異性については未だ不明な点が多い。本稿では特にリガンドとして主要組織適合性複合体(MHC)クラスI分子を認識する抑制性受容体LILRB1とLILRB2に焦点を当て、LILRファミリーの立体構造およびリガンドとの分子認識機構について、筆者らの研究成果を中心に最近の知見を概説する。(著者抄録)
• 生命現象を担う生体分子に着目した創薬と臨床応用 多様な形態を有するHLA-G蛋白質とヒト免疫制御受容体との分子認識

  黒木 喜美子, 福永 裕子, 上敷領 淳, 白石 充典, 尾瀬 農之, 前仲 勝実 日本薬学会年会要旨集 131年会 (1) 188 -188 2011年03月 [査読無し][通常論文]
  
• 抑制型レセプターCD160によるHVEM認識の分子基盤

  小島理恵子, 梶川瑞穂, 白石充典, 黒木喜美子, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 34th 2T7PI-2 (WEB ONLY) 2011年 [査読無し][通常論文]
  
• 免疫抑制分子HLA‐Gダイマーの局所投与による関節炎抑制効果の検討

  黒木喜美子, 岡部由紀, 廣瀬薫, 福永裕子, 小柳悟, 大戸茂弘, 前仲勝実 日本分子生物学会年会プログラム・要旨集(Web) 34th 2P-0444 (WEB ONLY) 2011年 [査読無し][通常論文]
  
• ヒト免疫制御受容体LILRB1とリガンドHLA‐Gの複合体の結晶構造解析

  松原永季, 黒木喜美子, 上敷領淳, 尾瀬農之, 前仲勝実 日本結晶学会年会講演要旨集 2011 95 2011年 [査読無し][通常論文]
  
• HIV由来変異ペプチドによるKIR2DL1を介した免疫逃避機構の分子基盤

  黒木喜美子, 上敷領淳, 尾瀬農之, THANANCHAI Hathairat, MAKADZANGE Tariro, ROWLAND‐JONES Sarah, DONG Tao, 前仲勝実 日本結晶学会年会講演要旨集 2011 94 2011年 [査読無し][通常論文]
  
• HIV由来ペプチドライブラリーを用いたHLA-Cw4拘束性免疫制御ペプチドの同定

  黒木 喜美子, 福永 裕子, 尾瀬 豊之, 前仲 勝実 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集 83回・33回 1P -1023 2010年12月 [査読無し][通常論文]
  
• HSV‐1のエンベロープ蛋白質gBペプチドおよびペア型レセプターPILRαの複合体結晶構造解析

  尾瀬農之, 山口宗親, WAN Jing, 黒木喜美子, 田畑栄一, 真板宣夫, 中村聖子, 梶川瑞穂, 白鳥行大, 佐藤毅史, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 58th 420 2010年10月15日 [査読無し][通常論文]
  
• NKおよびクラスI認識受容体研究の最前線 クラスI認識受容体の構造とリガンド認識

  黒木 喜美子, 福永 裕子, 上敷領 淳, 白石 充典, 尾瀬 農之, 前仲 勝実 MHC: Major Histocompatibility Complex 17 (2) 136 -136 2010年08月 [査読無し][通常論文]
  
• 新規CRISP蛋白質の発見と毒蛇血清蛋白質SSP‐2との相互作用領域の解析

  塩井(青木, 成留実, 清村康子, 黒木喜美子, 前仲勝実, 寺田成之 日本蛋白質科学会年会プログラム・要旨集 10th 73 2010年05月15日 [査読無し][通常論文]
  
• LILR1/HLA‐GとLILR82/HLA‐G複合体の結合機構の違い

  宮下尚之, 黒木喜美子, 前仲勝実, 杉田有治 日本蛋白質科学会年会プログラム・要旨集 10th 66 2010年05月15日 [査読無し][通常論文]
  
• 活性化型受容体LILRA1‐MHC相互作用を介した免疫反応制御の分子基盤

  上敷領淳, 黒木喜美子, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 10th 163 2010年05月15日 [査読無し][通常論文]
  
• 細胞表面において生体防御に関わる分子認識の構造生物学

  福原秀雄, 橋口隆生, 黒木喜美子, 白石充典, 佐々木香織, 梶川瑞穂, 尾瀬農之, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 10th 32 2010年05月15日 [査読無し][通常論文]
  
• ペア型レセプターPILRα及びHSV‐1のエンベロープ蛋白質gB模倣ペプチド複合体結晶構造

  尾瀬農之, 山口宗親, 黒木喜美子, 田畑栄一, 真板宣夫, 梶川瑞穂, 中村聖子, WANG Jing, 佐藤毅史, 荒瀬尚, 前仲勝実 日本結晶学会年会講演要旨集 2010 97 2010年 [査読無し][通常論文]
  
• T細胞補助シグナル CD160-HVEM相互作用を中心としたT細胞制御機構の分子基盤

  小島 理恵子, 梶川 瑞穂, 白石 充典, 黒木 喜美子, 前仲 勝実 日本免疫学会総会・学術集会記録 39 161 -161 2009年11月 [査読無し][通常論文]
  
• Paired Immunoglobulin(Ig)Like type 2 Receptor(PILR)αによるGlycoprotein B(gB)認識機構の解明

  山口宗親, 黒木喜美子, 田畑栄一, 真板宣夫, 梶川瑞穂, 尾瀬農之, 中村聖子, 王静, 佐藤毅, 荒瀬尚, 前仲勝実 日本ウイルス学会学術集会プログラム・抄録集 57th 149 2009年10月01日 [査読無し][通常論文]
  
• BmNPVバクミドを用いたヒト膜タンパク質受容体のカイコ個体での生産

  梶川瑞穂, 佐々木香織, 黒木喜美子, 橋口隆生, 岡部由紀, 福原秀雄, 竹田誠, 柳雄介, 脇本義太郎, 豊岡勝, 武田茂樹, 本橋智子, 霜島司, PARK Enoch Y, 前仲勝実 日本生物工学会大会講演要旨集 61st 281 -281 2009年08月25日 [査読無し][通常論文]
  
• CD160‐HVEM相互作用を中心としたT細胞活性制御の分子基盤

  小島理恵子, 梶川瑞穂, 白石充典, 黒木喜美子, 前仲勝実 日本分子生物学会年会講演要旨集 32nd (Vol.1) 63 2009年 [査読無し][通常論文]
  
• Association of LILRA2 (ILT1, LIR7) splice site polymorphism with systemic lupus erythematosus and microscopic polyangiitis (vol 9, page 214, 2008)

  K. Mamegano, K. Kuroki, R. Miyashita, M. Kusaoi, S. Kobayashi, K. Matsuta, K. Maenaka, M. Colonna, S. Ozaki, H. Hashimoto, Y. Takasaki, K. Tokunaga, N. Tsuchiya GENES AND IMMUNITY 9 (7) 650 -650 2008年10月 [査読無し][通常論文]
  
• ウイルス認識の分子基盤 麻疹ウイルスHタンパク質のX線結晶構造解析

  橋口 隆生, 梶川 瑞穂, 真板 宣夫, 竹田 誠, 黒木 喜美子, 佐々木 香織, 柳 雄介, 前仲 勝実 補体シンポジウム講演集 45 61 -61 2008年07月 [査読無し][通常論文]
  
• BmNPVバクミドシステムによるヒト免疫細胞表面レセプターの発現と糖鎖解析

  佐々木香織, 梶川瑞穂, 矢木宏和, 近藤幸子, 黒木喜美子, 本橋智子, 霜島司, PARK Enoch Y, 加藤晃一, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 8th 98 2008年05月23日 [査読無し][通常論文]
  
• 免疫学 HLA-G 副作用の少ない生物学的製剤の可能性

  黒木 喜美子, 前仲 勝実 医学のあゆみ 224 (11) 874 -875 2008年03月 [査読無し][通常論文]
  
• BmNPVバクミドシステムによるヒト免疫細胞表面レセプターの発現と糖鎖解析

  佐々木香織, 梶川瑞穂, 矢木宏和, 近藤幸子, 黒木喜美子, 本橋智子, 霜島司, PARK Enoch Y, 加藤晃一, 前仲勝実 生化学 4P-0214 2008年 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig‐Like type2 Receptor(PILR)の構造解析

  田畑栄一, 黒木喜美子, 梶川瑞穂, 白鳥行大, 荒瀬尚, 神田大輔, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 7th 79 2007年05月07日 [査読無し][通常論文]
  
• エントロピー駆動型の受容体-リガンド相互作用 免疫細胞表面受容体LILR(ILT/LIR/CD85)群の分子認識

  白石 充典, 黒木 喜美子, 神田 大輔, 前仲 勝実 生物物理 47 (2) 93 -99 2007年03月 [査読無し][通常論文]
  
• 難治性血管炎に関する調査研究 日本人顕微鏡的多発血管炎の疾患感受性遺伝子に関する研究

  土屋尚之, 宮下リサ, 豆ケ野剛一, 徳永勝士, 川崎綾, 黒木喜美子, 前仲勝実, COLONNA Marco, 小林茂人, 橋本博史, 尾崎承一 難治性血管炎に関する調査研究 平成18年度総括・分担研究報告書 43 -48 2007年 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig‐Like type2 Receptor(PILR)のCD99認識機構の分子基盤

  田畑栄一, 黒木喜美子, 梶川瑞穂, WANG Jing, 荒瀬尚, 神田大輔, 前仲勝実 生化学 1P-0043 2007年 [査読無し][通常論文]
  
• Molecular basis for LILR recognition of HLA-G and its dimer

  M. Shiroishi, K. Kuroki, T. Ose, L. Rasubala, I. Shiratori, H. Arase, D. Kohda, K. Maenaka TISSUE ANTIGENS 68 (4) CP6 -CP6 2006年10月 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig‐Like type2Receptor(PILR)の構造解析

  田畑栄一, 黒木喜美子, 白鳥行大, 荒瀬尚, 神田大輔, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 6th 96 2006年03月31日 [査読無し][通常論文]
  
• BmNPVバクミドシステムを用いたカイコ個体でのヒト免疫細胞表面レセプターKIRの大量発現

  佐々木香織, 梶川瑞穂, 黒木喜美子, 本橋智子, 霜島司, 朴龍洙, 神田大輔, 前仲勝実 日本蛋白質科学会年会プログラム・要旨集 6th 62 2006年03月31日 [査読無し][通常論文]
  
• NK細胞抑制型レセプターKLRG1(Killer Cell Lectin‐Like Receptor G1)によるKLRG1リガンド認識の構造基盤

  中村聖子, 黒木喜美子, 佐々木香織, 丸山拓馬, 伊藤昌之, 山本一夫, 松本直樹, 神田大輔, 前仲勝実 日本分子生物学会年会講演要旨集 28th 572 2005年11月25日 [査読無し][通常論文]
  
• 白血球抑制性受容体LILRB1とMHCクラスIとの蛍光相関分光法(FCS)およびNMRを用いた相互作用解析

  黒木喜美子, 白石充典, RASUBALA Linda, 小林佐代子, 梶川瑞穂, 田畑栄一, 福永裕子, 岡本直明, 神田大輔, 前仲勝実 日本分子生物学会年会講演要旨集 28th 774 2005年11月25日 [査読無し][通常論文]
  
• ペア型レセプターPaired Ig-Like Type2 Receptor(PILR)のリガンド認識機構の構造基盤

  田畑 栄一, 黒木 喜美子, 白鳥 行大, 荒瀬 尚, 神田 大輔, 前仲 勝美 日本免疫学会総会・学術集会記録 35 96 -96 2005年11月 [査読無し][通常論文]
  
• Killer cell lectin-like receptor G1(KLRG1)が認識するKLRG1リガンド上領域の同定

  丸山 拓馬, 松本 直樹, 伊藤 昌之, 中村 聖子, 黒木 喜美子, 前仲 勝実, 山本 一夫 日本免疫学会総会・学術集会記録 35 97 -97 2005年11月 [査読無し][通常論文]
  
• NK細胞抑制型レセプターKLRG1(Killer Cell Lectin-Like Recepter G1)によるKLRG1リガンド認識の構造基盤

  中村 聖子, 黒木 喜美子, 佐々木 香織, 丸山 拓馬, 伊藤 昌之, 山本 一夫, 松本 直樹, 神田 大輔, 前仲 勝実 日本免疫学会総会・学術集会記録 35 98 -98 2005年11月 [査読無し][通常論文]
  
• HLA-GダイマーのLILR/LIR/ILTレセプターを介した強いシグナル伝達とその構造基盤

  前仲 勝実, 白石 充典, 黒木 喜美子, リンダ・ラズバラ, 白鳥 行大, 荒瀬 尚, 神田 大輔 日本免疫学会総会・学術集会記録 35 99 -99 2005年11月 [査読無し][通常論文]
  
• 白血球抑制性受容体LILRB1とMHCクラスIとのFCSおよびNMRを用いた相互作用解析

  黒木 喜美子, 白石 充典, リンダ・ラスバラ, 小林 佐代子, 梶川 瑞穂, 田畑 栄一, 福永 裕子, 岡本 直明, 神田 大輔, 前仲 勝実 日本免疫学会総会・学術集会記録 35 99 -99 2005年11月 [査読無し][通常論文]
  
• Association of a functional CD19 polymorphism with susceptibility to systemic sclerosis (vol 50, pg 4002, 2004)

  N Tsuchiya, TF Tedder, M Fujimoto, K Takehara, K Kuroki, K Tokunaga, Y Murakami, S Sato ARTHRITIS AND RHEUMATISM 52 (5) 1619 -1619 2005年05月 [査読無し][通常論文]
  
• 関節リウマチ(RA)関連Leukocyte Immunoglobulin-like Receptor(LILR)B1ハプロタイプの構造・発現解析

  黒木 喜美子, 白石 充典, リンダ・ラスバラ, 土屋 尚之, 神田 大輔, 徳永 勝士, 前仲 勝実 日本リウマチ学会総会・学術集会・国際リウマチシンポジウムプログラム・抄録集 49回・14回 211 -211 2005年04月 [査読無し][通常論文]
  
• ヒト免疫細胞受容体ILT/LIR/CD85ファミリーのリガンド認識の分子的基盤

  白石充典, 黒木喜美子, 小島恵理子, 津本浩平, 熊谷泉, 神田大輔, 前仲勝実 日本分子生物学会年会プログラム・講演要旨集 27th 354 2004年11月25日 [査読無し][通常論文]
  
• 関節リウマチ(RA)関連Leukocyte Immunoglobulin-like Receptor(LIR)1ハプロタイプの構造・発現解析

  黒木 喜美子, 土屋 尚之, 白石 充典, リンダ・ラズバラ, 山下 由美, 小池 隆夫, 神田 大輔, 徳永 勝士, 前仲 勝実 日本免疫学会総会・学術集会記録 34 162 -162 2004年11月 [査読無し][通常論文]
  
• ヒトCD19多型と強皮症との関連

  土屋 尚之, 黒木 喜美子, 藤本 学, Tedder Thomas F., 徳永 勝士, 佐藤 伸一 日本免疫学会総会・学術集会記録 34 284 -284 2004年11月 [査読無し][通常論文]
  
• Association of functional CD19 promoter polymorphism with susceptibility to systemic sclerosis

  N Tsuchiya, K Kuroki, Y Murakami, M Fujimoto, TF Tedder, K Tokunaga, K Takehara, S Sato ARTHRITIS AND RHEUMATISM 50 (9) S125 -S125 2004年09月 [査読無し][通常論文]
  
• 白血球免疫グロブリン様受容体LILRA1(LIR6)遺伝子多型と日本人SLEとの関連

  黒木 喜美子, 土屋 尚之, 松多 邦雄, 深沢 徹, 十字 猛, 橋本 博史, 徳永 勝士 日本リウマチ学会総会・学術集会・国際リウマチシンポジウムプログラム・抄録集 48回 175 -175 2004年03月 [査読無し][通常論文]
  
• Distinct associations of the leukocyte immunoglobulin-like receptor (LIR)1 and LIR6 polymorphisms with susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).

  K Kuroki, N Tsuchiya, K Maenaka, L Rasubala, M Shiroishi, Y Yamashita, K Matsuta, T Fukazawa, D Kohda, T Koike, T Juji, H Hashimoto, K Tokunaga ARTHRITIS AND RHEUMATISM 48 (9) S197 -S197 2003年09月 [査読無し][通常論文]
  
• Molecular genetic analyses of human NKG2C gene deletion.

  R Miyashita, N Tsuchiya, K Hikami, K Kuroki, T Fukazawa, M Bijl, CGM Kallenberg, H Hashimoto, T Yabe, K Tokunaga ARTHRITIS AND RHEUMATISM 48 (9) S473 -S473 2003年09月 [査読無し][通常論文]
  
• ヒトNKG2C遺伝子欠失の分子遺伝学的解析

  宮下 リサ, 土屋 尚之, 氷上 光輝, 黒木 喜美子, 徳永 勝士 リウマチ 43 (2) 319 -319 2003年03月 [査読無し][通常論文]
  
• ヒトNKG2-C遺伝子欠失の分子遺伝学的解析

  宮下 リサ, 土屋 尚之, 黒木 喜美子, 屋部 登志雄, 徳永 勝士 日本免疫学会総会・学術集会記録 32 136 -136 2002年10月 [査読無し][通常論文]
  
• Association of leukocyte immunoglobulin-like receptor 1 (LIR1, ILT2, LILRB1) polymorphism with susceptibility to rheumatoid arthritis in Japanese.

  K Kuroki, N Tsuciuya, K Matsuta, T Fukazawa, T Juji, H Hashimoto, K Tokunaga ARTHRITIS AND RHEUMATISM 46 (9) S550 -S551 2002年09月 [査読無し][通常論文]
  
• 【膠原病研究の最近の進歩】リウマチ・膠原病のゲノム解析

  黒木 喜美子, 土屋 尚之 アレルギー・免疫 9 (9) 997 -1005 2002年08月 [査読無し][通常論文]
   

  膠原病に含まれる疾患の病因は未だ不明な点が多いものの,遺伝要因と環境要因の両方が発症に寄与している多因子疾患であると考えられている.その遺伝的な病因解明のために,疾患感受性遺伝子の同定を目的とした連鎖解析研究,関連解析研究が数多く行われてきたが,HLA以外に,多くの集団において共通に疾患との関連が確認された感受性遺伝子は数少ない.主要な膠原病に関する遺伝子解析の現状について紹介した
• SLEの病因・病態と治療戦略 全身性エリテマトーデス疾患感受性遺伝子の検討

  土屋 尚之, 京極 千恵子, 黒木 喜美子, 氷上 光輝, 川崎 綾, 深沢 徹, 橋本 博史, 徳永 勝士 リウマチ 42 (2) 237 -237 2002年03月 [査読無し][通常論文]
  
• ヒトCD19遺伝子3'非翻訳領域内反復配列多型と日本人SLE感受性との関連

  黒木 喜美子, 土屋 尚之, 深沢 徹, 橋本 博史, 徳永 勝士 リウマチ 42 (2) 367 -367 2002年03月 [査読無し][通常論文]
  
• 炎症性疾患と遺伝子多型 候補遺伝子アプローチによるヒト全身性エリテマトーデス疾患感受性遺伝子の検討

  土屋 尚之, 京極 千恵子, 黒木 喜美子, 川崎 綾, 氷上 光輝, 深沢 徹, 橋本 博史, 徳永 勝士 炎症・再生 21 (4) 432 -432 2001年07月 [査読無し][通常論文]
  
• 【膠原病・リウマチと妊娠,出産】慢性関節リウマチ,全身性エリテマトーデスの遺伝素因

  黒木 喜美子, 土屋 尚之 リウマチ科 26 (1) 39 -44 2001年07月 [査読無し][通常論文]
  
• Significant association between CD19 polymorphism and systemic lupus erythematosus.

  K Tokunaga, K Kuroki, N Tsuchiya, A Yamaguchi, BP Tsao, JM Grossman, T Fukazawa, H Hashimoto AMERICAN JOURNAL OF HUMAN GENETICS 67 (4) 353 -353 2000年10月 [査読無し][通常論文]
  
• Association of CD19 polymorphism with susceptibility to systemic lupus erythematosus

  K Kuroki, N Tsuchiya, A Yamaguchi, BP Tsao, JM Grossman, T Fukazawa, H Hashimoto, K Tokunaga ARTHRITIS AND RHEUMATISM 43 (9) S55 -S55 2000年09月 [査読無し][通常論文]
  
• Association of CD19 polymorphism with susceptibility to systemic lupus erythematosus.

  K Kuroki, N Tsuchiya, A Yamaguchi, BP Tsao, JM Grossman, T Fukazawa, H Hashimoto, K Tokunaga ARTHRITIS AND RHEUMATISM 43 (9) S325 -S325 2000年09月 [査読無し][通常論文]
  
• ヒトCD19遺伝子の変異解析

  黒木 喜美子, 土屋 尚之, 徳永 勝士 リウマチ 40 (2) 469 -469 2000年04月 [査読無し][通常論文]
  

特許

• 特許第7245541号:PD-L1抑制剤、及びPD-L1抑制剤のスクリーニング方法  

  前仲 勝実, 黒木 喜美子, ▲高▼橋 愛実  国立大学法人北海道大学  202303006514021663
• 特開2021-080171:改変タンパク質、医薬品、炎症性疾患の予防又は治療剤及び改変タンパク質の製造方法  2021年05月27日

  前仲 勝実, 黒木 喜美子, 前田 直良, 山田 千聖, ▲高▼橋 愛実  国立大学法人北海道大学  202103010354669885
• 特許第6654781号:KIR2DS1に対するモノクローナル抗体  

  前仲 勝実, 黒木 喜美子, 米田 宏, 福原 秀雄  国立大学法人北海道大学  202003009251852929
• WO2019-177053:全身性エリテマトーデス又は全身性エリテマトーデスに起因して生じる疾患の予防又は治療剤及び膠原病の予防又は治療剤  2019年09月19日

  前仲 勝実, 黒木 喜美子, 前田 直良, 山田 千聖, ▲高▼橋 愛実  国立大学法人北海道大学  202103009615801818
• WO2019-177054:腫瘍予防又は治療剤、PD-L1抑制剤、腫瘍予防又は治療剤のスクリーニング方法及びPD-L1抑制剤のスクリーニング方法  2019年09月19日

  前仲 勝実, 黒木 喜美子, ▲高▼橋 愛実  国立大学法人北海道大学  202103010157614670
• WO2019-054409:HLAタンパク質に相互作用する物質のスクリーニング方法及びスクリーニング用キット  2019年03月21日

  前仲 勝実, 田所 高志, 野村 尚生, 黒木 喜美子, 可野 巧  国立大学法人北海道大学  202003014201864539
• WO2016-031936:KIR2DS1に対するモノクローナル抗体  2016年03月03日

  前仲 勝実, 黒木 喜美子, 米田 宏, 福原 秀雄  国立大学法人北海道大学  201703012187382811
• 特開2015-140322:関節リウマチまたはその関連疾患の予防または治療剤  2015年08月03日

  前仲 勝実, 黒木 喜美子, 高橋 愛実  国立大学法人北海道大学  201503017468526054
• 特許第5637981号:関節リウマチまたはその関連疾患の予防または治療剤  2014年10月31日

  前仲 勝実, 黒木 喜美子, 大戸 茂弘, 小柳 悟  国立大学法人北海道大学  201403053538317146
• WO2010-101192:関節リウマチまたはその関連疾患の予防または治療剤  2010年09月10日

  前仲 勝実, 黒木 喜美子, 大戸 茂弘, 小柳 悟  国立大学法人九州大学  201203086087695096
• 特開2009-106163:核酸配列、ベクター、形質転換体、製造方法、及び、核酸配列プライマー  2009年05月21日

  前仲 勝実, 黒木 喜美子, 神田 大輔, 梶川 瑞穂, 白石 充典  国立大学法人九州大学  200903052598489217

共同研究・競争的資金等の研究課題

• 免疫抑制分子HLA-Gアイソフォームを特異的に機能阻害する腫瘍治療抗体医薬品開発

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2022年04月 -2025年03月 

  代表者 : 黒木 喜美子
• HLA-G2を中心とした多様なリガンド群の免疫制御受容体との分子認識機構の解明

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2018年04月 -2021年03月 

  代表者 : 黒木 喜美子

   

  本申請の目的は、多様な分子形態で存在する生体内免疫抑制蛋白質HLA-Gのうち、ドメインをひとつ欠損するにも関わらず、受容体LILRB2と結合し、通常型HLAG1を補う活性を発揮するHLA-G2アイソフォームを中心とした多様なLILRB2リガンド群との分子認識機構について、詳細な相互作用解析、複合体の立体構造解析を進め、構造的観点から明らかにすることである。その得られた知見をもとに、特にHLA-G2－LILRB2相互作用を標的とした創薬スクリーニングを実施する。 今年度は、昨年度に引き続き、HLA-G2とLILRB2の相互作用部位についての情報を集めるために、分子表面に点在する特定のアミノ酸残基を修飾することにより結合面を決定する系を確立した。点在するアミノ酸残基に変異を導入することにより、修飾の異なるHLA-G2、LILRB2それぞれの組換え蛋白質が調製できた。それらを用いて野生型の結合相手となる分子との結合実験をすることにより、LILRB2とHLA-G2お互いの結合面決定に向けた解析を開始した。安定した結果を得るために修飾法のさらなる最適化が重要となることが示唆されたが、初期データを得ることができており、複合体結晶化などの構造解析による結合部位が困難である、 他のLILRB2リガンドの相互作用面決定に応用できる手法となると期待している。 さらに、HLA-G2とLILRB2の相互作用を標的とした創薬に向けたスクリーニング、in vivoでのHLA-G2機能評価を実施し、多面的に研究を進めることができている。
• 強直性脊椎炎病因蛋白質HLA-B27の受容体LILRB2を介した発症機構の解明

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2013年04月 -2015年03月 

  代表者 : 黒木 喜美子

   

  本研究の目的は、HLA-B27が通常のHLA-クラスIには見られない重鎖ホモダイマーとして細胞表面に発現することに着目し、受容体との認識機構解明、創薬および治療へとつながる分子レベルでの解析を目指すことである。 本年度は、HLA-B27重鎖ホモダイマーの受容体として、これまでに複合体の構造解析に用いていたLILRB2に加え、近年新たに同定されたKIR3DL2を用いた構造解析を行った。KIR3DL2はHEK293細胞で細胞外ドメインを発現させた。KIR3DL2とHLA-B27重鎖ホモダイマーをモル比１：１で混合し、結晶化スクリーニングを試行した。今年度内に解析可能なデータは得られなかったが、今後も引き続き異常型ホモダイマーの受容体を介した分子認識機構の解明に向けて、構造解析を継続する予定である。 また、HLA-B27、B27重鎖ホモダイマーを用いて、シグナル伝達制御可能な化合物スクリーニング系の確立に向けて、複数の手法を試みた。その結果、通常のHLA-B27分子は示差走査熱量分析法や示差走査蛍光測定法のいずれの手法でも分子の変性点が2点あるものの、測定条件の最適化を行えば、スクリーニングの系として用いることが可能であると予想された。また、HLA-B27重鎖ホモダイマーはペプチドを提示していないため、化合物がペプチド溝に結合することにより、ペプチドレパートリーが変化し、異常な免疫反応を誘導する可能性は低いと考えたが、DMSO含有条件下での適した測定系の確立には至らなかった。今後、細胞を用いたアッセイ系についても検討していく予定である。
• ドメイン欠損型HLA-G6アイソフォームと受容体LILRB2の立体構造解析

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2013年04月 -2015年03月 

  代表者 : 黒木 喜美子

   

  HLA-G6アイソフォームの立体構造解析およびin vivoでの機能解析を行った。まず、電子顕微鏡解析によるHLA-G6単独の立体構造解析を行い、ジスルフィド結合を介さないホモ二量体形成が示唆される分子像を得ることに成功した。また、in vivoでのHLA-G6アイソフォームの免疫抑制能を調べるために、CIAマウスを用いた関節炎抑制効果の有無について検討した。その結果、HLA-G6がマウス受容体に結合し、関節炎抑制効果を持つことを明らかにした。
• 強直性脊椎炎病因蛋白質ＨＬＡ－Ｂ２７ホモ二量体の立体構造解析

  日本学術振興会：科学研究費助成事業 新学術領域研究(研究領域提案型)

  研究期間 : 2011年04月 -2013年03月 

  代表者 : 黒木 喜美子

   

  本研究の目的は、B27が通常のHLAクラスIには見られない重鎖ホモダイマーとして細胞表面上に発現することに着目し、受容体との認識機構を解明し、創薬および治療へとつながる分子レベルでの解析を目指すことである。 申請者は昨年度に引き続き、B27ホモダイマーおよびLILR受容体との相互作用について蛋白質レベルでの解析を行った。まず、オクスフォード大学の共同研究者とともに、通常のHLA-B27には結合しない、B27ホモダイマー特異的な抗体の作製を行った。今後、この抗体をB27ホモダイマーの精製や蛋白質構造の安定化等に用いていきたいと考えている。 また、B27ホモダイマー単独および受容体LILRB2との複合体の立体構造解析を複数の手法で行った。X線結晶構造解析においては、B27ホモダイマーはペプチドを提示していないことが分かったため、ペプチドを加えずに調製したダイマー蛋白質を用いて多数の結晶化スクリーニングを試行したが、良質な結晶は得られなかった。B27ホモダイマーはβ2mを伴わないため、分子構造がゆるく、結晶化が困難である可能性が考えられたため、受容体LILRB2との複合体の結晶化を試みた。両タンパク質の混合条件を検討し、pH9でモル比1:1で混合すると凝集体を形成しにくいことが分かり、結晶化スクリーニングを試した。その結果、複数の微結晶を得たが、LILRB2単独の結晶もしくは良質でない結晶だった。今後引き続き条件検討を行う予定である。同時に電子顕微鏡解析を進めており、HLA-B27ホモダイマーが予想していたよりは均一の粒子状の蛋白質であることが分かった。今後画像解析を進め、分子全体のドメイン配向を明らかにしたいと考えている。また、NMR解析によってもLILRB2上のB27ホモダイマー相互作用部位を明らかにするために、LlRB2単独のNMRスペクトルの解析も継続する予定である。
• ドメイン欠損型HLA-G6アイソフォームと免疫制御受容体LILR群の分子認識

  日本学術振興会：科学研究費助成事業 若手研究(B)

  研究期間 : 2011年 -2012年 

  代表者 : 黒木 喜美子

   

  HLA-G6およびLILR受容体との相互作用について、蛋白質レベルでの解析を行った。まず、複数の発現コンストラクトを構築し、大腸菌を用いた封入体巻き戻しの系を用いて、解析に適した安定なHLA-G6蛋白質を調製することに成功した。また、HLA-G6単独および受容体LILRB2との複合体の立体構造解析を複数の手法(X線結晶構造解析、電子顕微鏡解析、NMR解析)で行った。
• リウマチ性疾患因子HLA-B27と免疫制御受容体LILR群の分子認識

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  研究期間 : 2008年 -2010年 

  代表者 : 福原 喜美子, 黒木 喜美子

   

  ヒト細胞表面抗原HLA-B27の異常型β2mフリーホモ二量体(B27_2)が、免疫制御受容体LILR群との相互作用により、リウマチ性自己免疫疾患のひとつである強直性脊椎炎(AS)を引き起こす分子認識機構を、立体構造解析の観点から明らかにすることを目的としている。 今年度はまず、分子動力学的なLILRB2とβ2mフリーのHLAの結合シミュレーションの結果、昨年度得られたK121、Dl22に加え、新たにQ115、D118、R14、E229、D196、E232、D227-E232、V248、V194-E198がLILRB2と相互作用していることが分かった(理研・杉田、宮下博士との共同研究)。そのうち、特にR14、D118、E232はβ2mフリーのHLAの時にのみ相互作用する残基であることが分かった。このシミュレーション結果をもとに、重要な相互作用残基の同定を目指し、実験的に表面プラスモン共鳴法で解析するためにサンプルを準備した。 また、結晶化に関しては、共同研究先(Oxford大学・Paul博士ら)が作製したB27_2特異的抗体を用いたB27_2の精製および抗体との複合体の結晶化を目指して、サンプル供与・条件の準備を引き続き行っている。ペプチド依存的にLILRB2との結合能が6倍程度変化すると報告のあったHLA-B27/ペプチド-LILRB2複合体の結晶構造解析に関しては、昨年度に引き続き、LILRB2:B272=2:1で混合したサンプルの結晶化条件の最適化(pH、塩の種類、塩濃度、PEG濃度)を進めたが、目立った改善は認められず、解析に十分な分解能を得られていない。 今後も引き続き、リウマチ性疾患因子HLA-B27と免疫制御受容体LILR群の分子認識の解明を目指した研究を行う予定である。
• 免疫系の複合疾患感受性遺伝子の同定に向けての研究

  日本学術振興会：科学研究費助成事業 特別研究員奨励費

  研究期間 : 2002年 -2003年 

  代表者 : 黒木 喜美子

   

  前年度、RA、SLEを対象とした関連研究において検出したLIR1多型とRAとの有意な関連は、LIR1と連鎖不平衡にある他の近傍の遺伝子によるものである可能性も考えられた。そのため、隣接して存在するLIR6、LIR5遺伝子について、同様に多型解析、関連研究を行った。LIR6遺伝子の多型スクリーニングの結果、計24箇所のSNPsが日本人において主要な2種のハプロタイプを形成することが見出された。このうち、マイナーハプロタイプLIR6.02陽性率のSLEにおける有意な減少が見出された(P=0.02,OR=0.64)。次に、LIR5について解析を行った。多型スクリーニングにより、計29箇所の多型が検出された。LIR1、LIR6遺伝子とは対照的に、日本人における主要なハプロタイプを決定できなかった。ゆえに、RA、SLEを対象とした関連研究は、機能的に重要であると予想される多型について行った。その結果、最終的に-965G>A多型、Gアリル頻度のRA群における有意な増加(P=0.0005,OR=1.60)とc.-306C>T多型、TアリルのSLE群における有意な減少(P=0.005,OR=0.37)が検出された。RAと有意な関連を示したLIR1.EC01/.EC01とLIR5-965G間、SLEと有意な関連を示したLIR6.02とLIR5 c.-306T間にはそれぞれ有意な連鎖不平衡が認められた。そのため、それぞれの多型部位で検出された疾患との有意な関連が、遺伝的に独立なものか遺伝子座間関連解析により検討した。その結果、LIR5-965Gは独立にRA感受性に関与し、LIR1.EC01/.EC01もまたその可能性が高いことが示唆された。一方、SLE感受性においてはLIR5 c.-306Tが一義的な感受性遺伝子でありLIR6.02との関連は連鎖不平衡による二次的なものであることが示唆された。 RAとの有意な関連が認められた多型によるリガンドとの結合能の変化の有無を調べるために、九州大学生体防御医学研究所ワクチン開発構造生物学分野研究室において研究に従事した。(5月21日〜6月3日、7月15日〜17日、8月18日〜20日)

教育活動情報

主要な担当授業