2002 Fiscal Year Annual Research Report

構造情報に基づく細胞性免疫システムにおける特異的分子認識機構の解明

Research Project

Project/Area Number	13033002
Research Institution	Tohoku University
Principal Investigator	熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)
Co-Investigator(Kenkyū-buntansha)	津本浩平東北大学, 大学院・工学研究科, 助教授 (90271866)
Keywords	結晶構造解析 / TCR-HLA相互作用 / ILT-HLA相互作用 / 表面プラズモン共鳴 / 等温滴定型熱量測定 / 受容体 / 細胞性免疫 / 自然免疫
Research Abstract	本研究は、細胞性免疫において鍵となる相互作用である、TCR-HLA相互作用、あるいは自然免疫系で近年見出され、その性質が明らかになりつつあるILT-HLA相互作用について、焦点を絞り、分子認識データを立体構造に基づいて収集し、その分子認識能における特異性・親和性を創出する分子機構を明らかにすることを目的としている。大量調製したTCRとHLAクラスIについて、その熱力学的解析を行い,抗体類似であるがエントロピー損が大きく親和性が低いことを明らかにした.現在,その複合体の結晶化を継続して行っている。ペプチドを変化させたものを用いて同様の結晶化を目指した。ヒトImmunoglobulin-like transcript (ILT) 2 (CD85a)がT細胞やNK細胞など比較的広く分布しているのに対し、ILT4(CD85d)は単球系細胞に選択的に発現する細胞表面受容体である。本研究ではILT2およびILT4の細胞外ドメインD1、D2からなる断片を作製し、HLAに対する相互作用を、BIAcore2000を用いて分子レベルで解析した。ILT2、ILT4共に封入体から巻き戻し反応を行うことで可溶性分子として調製した。相互作用解析にあたり、フローセルにビオチン化したHLA分子を固定化し、その上からILT分子の溶液を流した。相互作用解析の結果ILT4がHLA-A11、B35、Cw7、G1に対して広く結合することを確認し(Kd=4〜40μM)、中でも非古典的MHCであるHLA-Gに対して最も強く相互作用することが分かった(Kd=4μM)。またILT2はHLA-Gに対してILT4よりも強い親和性を示した(Kd=1μM)。これらの結晶構造解析を行うべく,結晶化を試みている.

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] Michihiro Nakamura, et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay : application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)
[Publications] Shin-ichi Takemura, et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunotherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. 51. 33-44 (2002)
[Publications] Michihiro Nakamura, et al.: "Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin-Fluorescein Conjugate"Analytical Biochemistry. 304. 231-235 (2002)
[Publications] ARUTO UENO, et al.: "T-cell Immunotherapy for Human MK-1-Expressing Tumors Using a Fusion Protein of the Superantigen SEA and Anti-MK-1 scFv Antibody"ANTICANCER RESEARCH. 22. 769-776 (2002)
[Publications] MASAHIRO KAWAHARA, et al.: "Selection of Highly Productive Mammalian Cells Based on an Inducible Growth Advantage Using an Antibody/Receptor Chimera"JOURNAL OF BIOSCIENCE AND BIOENGINEERING. 93. 399-404 (2002)
[Publications] Michihiro Nakamura, et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. 520. 77-80 (2002)
[Publications] Hideki Watanabe, et al.: "Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens"Biochemical and Biophysical Research Communications. 295. 31-36 (2002)
[Publications] Shinobu Honzawa, et al.: "Chiral Recognition in the Binding of Helicenediamine to Double Strand DNA : Interactions Between Low Molecular Weight Helical Compounds and a Helical Polymer"Bioorganic & Medicinal Chemistry. 10. 3213-3218 (2002)
[Publications] Kouhei Tsumoto, et al.: "Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4 :"FEBS Letters. 520. 77-82 (2002)
[Publications] Shusaku Tsumoto, et al.: "Analysis of amino-acid sequences by statistical technique"Information Sciences. 145. 205-214 (2002)
[Publications] Ryutaro Asano, et al.: "Antitumor activity of Interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli"FEBS Letters. 528. 70-76 (2002)
[Publications] Ryutaro Asano, et al.: "Efficient construction of a diabody using a refolding system anti-carcinoembryonic antigen recombinant antibody fragment"Journal of Biochemistry. 123. 903-909 (2002)
[Publications] Kouhei Tsumoto, et al.: "Mini Review : Practical considerations in refolding proteins from inclusion bodies"Protein Expression and Purification. (印刷中). (2003)
[Publications] Akiko Yokota, et al.: "The role of hydrogen bonding via interfacial water molecules in antigen-antibody complexation : The HyHEL-10-HEL interaction"The Journal of Biological Chemistry. (印刷中). (2003)
[Publications] Michihiro Nakamura, et al.: "A morphological study of filamentous phage infection of Escherichia coli using biotinylated phages"FEBS Letters. (印刷中). (2003)
[Publications] Mitsuo Umetsu, et al.: "How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system : Spectroscopic evidence for highly efficient refolding of a single-chain"The Journal of Biological Chemistry. (印刷中). (2003)
[Publications] Masahiro Kawahara, et al.: "Bypassing antibiotic selection : positive screening of genetically modified cells with an antigen-dependent proliferation switch"Nucleic Acids Research. (印刷中). (2003)
[Publications] Izumi Kumagai, et al.: "Structural consequences of target site-directed functional alteration of an antibody : The case of anti-lysozyme antibody, HyHEL-10"The Journal of Biological Chemistry. (印刷中). (2003)
[Publications] 津本, 熊谷(分担): "コンビナトリアルサイエンスの新展開"シーエムシー出版. 400 (2002)
[Publications] 熊谷, 津本(分担): "新タンパク質科学実験法第3巻構造機能解析(上)"東京化学同人. 200 (2003)
[Publications] 津本, 熊谷(分担): "化学フロンティア【○!8】コンビナトリアル・バイオエンジニアリング"化学同人. 200 (2003)

2002 Fiscal Year Annual Research Report

構造情報に基づく細胞性免疫システムにおける特異的分子認識機構の解明

Principal Investigator

熊谷 泉 東北大学, 大学院・工学研究科, 教授 (10161689)

Research Products

[Publications] Michihiro Nakamura, et al.: "Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay : application of selected VH to histochemistry"Journal of Immunological Methods. 261. 65-72 (2002)

[Publications] Shin-ichi Takemura, et al.: "A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunotherapy for bile duct carcinoma"Cancer Immunology Immunotherapy. 51. 33-44 (2002)

[Publications] Michihiro Nakamura, et al.: "Detection of Biotinylated Proteins in Polyacrylamide Gels Using an Avidin-Fluorescein Conjugate"Analytical Biochemistry. 304. 231-235 (2002)

[Publications] ARUTO UENO, et al.: "T-cell Immunotherapy for Human MK-1-Expressing Tumors Using a Fusion Protein of the Superantigen SEA and Anti-MK-1 scFv Antibody"ANTICANCER RESEARCH. 22. 769-776 (2002)

[Publications] MASAHIRO KAWAHARA, et al.: "Selection of Highly Productive Mammalian Cells Based on an Inducible Growth Advantage Using an Antibody/Receptor Chimera"JOURNAL OF BIOSCIENCE AND BIOENGINEERING. 93. 399-404 (2002)

[Publications] Michihiro Nakamura, et al.: "The effect of an agglutogen on virus infection : biotinylated filamentous phages and avidin as a model"FEBS Letters. 520. 77-80 (2002)

[Publications] Hideki Watanabe, et al.: "Selection of human antibody fragments on the basis of stabilization of the variable domain in the presence of target antigens"Biochemical and Biophysical Research Communications. 295. 31-36 (2002)

[Publications] Shinobu Honzawa, et al.: "Chiral Recognition in the Binding of Helicenediamine to Double Strand DNA : Interactions Between Low Molecular Weight Helical Compounds and a Helical Polymer"Bioorganic & Medicinal Chemistry. 10. 3213-3218 (2002)

[Publications] Kouhei Tsumoto, et al.: "Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4 :"FEBS Letters. 520. 77-82 (2002)

[Publications] Shusaku Tsumoto, et al.: "Analysis of amino-acid sequences by statistical technique"Information Sciences. 145. 205-214 (2002)

[Publications] Ryutaro Asano, et al.: "Antitumor activity of Interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli"FEBS Letters. 528. 70-76 (2002)

[Publications] Ryutaro Asano, et al.: "Efficient construction of a diabody using a refolding system anti-carcinoembryonic antigen recombinant antibody fragment"Journal of Biochemistry. 123. 903-909 (2002)

[Publications] Kouhei Tsumoto, et al.: "Mini Review : Practical considerations in refolding proteins from inclusion bodies"Protein Expression and Purification. (印刷中). (2003)

[Publications] Akiko Yokota, et al.: "The role of hydrogen bonding via interfacial water molecules in antigen-antibody complexation : The HyHEL-10-HEL interaction"The Journal of Biological Chemistry. (印刷中). (2003)

[Publications] Michihiro Nakamura, et al.: "A morphological study of filamentous phage infection of Escherichia coli using biotinylated phages"FEBS Letters. (印刷中). (2003)

[Publications] Mitsuo Umetsu, et al.: "How additives influence the refolding of immunoglobulin-folded proteins in a stepwise dialysis system : Spectroscopic evidence for highly efficient refolding of a single-chain"The Journal of Biological Chemistry. (印刷中). (2003)

[Publications] Masahiro Kawahara, et al.: "Bypassing antibiotic selection : positive screening of genetically modified cells with an antigen-dependent proliferation switch"Nucleic Acids Research. (印刷中). (2003)

[Publications] Izumi Kumagai, et al.: "Structural consequences of target site-directed functional alteration of an antibody : The case of anti-lysozyme antibody, HyHEL-10"The Journal of Biological Chemistry. (印刷中). (2003)

[Publications] 津本, 熊谷(分担): "コンビナトリアルサイエンスの新展開"シーエムシー出版. 400 (2002)

[Publications] 熊谷, 津本(分担): "新タンパク質科学実験法 第3巻構造機能解析(上)"東京化学同人. 200 (2003)

[Publications] 津本, 熊谷(分担): "化学フロンティア【○!8】 コンビナトリアル・バイオエンジニアリング"化学同人. 200 (2003)

熊谷泉東北大学, 大学院・工学研究科, 教授 (10161689)

[Publications] 熊谷, 津本(分担): "新タンパク質科学実験法第3巻構造機能解析(上)"東京化学同人. 200 (2003)

[Publications] 津本, 熊谷(分担): "化学フロンティア【○!8】コンビナトリアル・バイオエンジニアリング"化学同人. 200 (2003)