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Elucidation of molecular mechanisms of signal-transduction in multi-functional proteins and its application for drug design

Research Project

Project/Area Number 04403025
Research Category

Grant-in-Aid for General Scientific Research (A)

Allocation TypeSingle-year Grants
Research Field Physical pharmacy
Research InstitutionUniversity of Tokyo

Principal Investigator

KATO Koichi (1994)  University of Tokyo, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (20211849)

荒田 洋治 (1992-1993)  東京大学, 薬学部, 教授 (40011499)

Co-Investigator(Kenkyū-buntansha) ARATA Yoji  Water Research Institute, Director, 所長 (40011499)
加藤 晃一  東京大学, 薬学部, 助手 (20211849)
Project Period (FY) 1992 – 1994
Project Status Completed (Fiscal Year 1994)
Budget Amount *help
¥37,200,000 (Direct Cost: ¥37,200,000)
Fiscal Year 1994: ¥7,100,000 (Direct Cost: ¥7,100,000)
Fiscal Year 1993: ¥10,100,000 (Direct Cost: ¥10,100,000)
Fiscal Year 1992: ¥20,000,000 (Direct Cost: ¥20,000,000)
KeywordsImmunoglobulin / Nuclear magnetic resonance / Stable-isotope labeling / Antigen-binding site / IgG-binding protein / Hinge region / Dynamical structure / Multi-functional protein / 糖鎖 / 部位特異的変異 / 免疫複合体 / 抗原-抗体反応 / 動的高次溝造
Research Abstract

We developed stable-isotope-assisted NMR strategies for structural analyzes of big systems in order to elucidate molecular mechanisms of signal transduction of multi-functional proteins. This project was mainly aimed at elucidation of the principles for the constructions of the functional sites in immunoglobulin molecules especially responsible for antigen recognition, effector functions, and the mediation of both functions.
1. Binding sites on IgG molecules of a variety of ligands, e.g., antigens and bacterial IgG-binding proteins, were identified. Conformations of the ligands in the complexes were determined.
2. Conformational changes of IgG molecules induced upon ligand-binding were detected on the basis of the chemical shift data. Dynamical structures of the antigen-binding sites were revealed on the basis of the H-D exchange and the relaxation data.
3. On the basis of the ^<13>C NMR data, it was revealed that the hinge region of IgG has a mosaic structure with a heterogeneous nature of flexibility. This flexibility allows the Fc portion to be mobile even in huge immune complexes.
4. By use of an IgG mutant that lacks the N-linked oligosaccharides attached to thd Fc portion, it was revealed that these sugar chains have crucial roles in the construction of local structures in the C_H2 domain that are responsible for expression of the effector functions.

Report

(4 results)
  • 1994 Annual Research Report   Final Research Report Summary
  • 1993 Annual Research Report
  • 1992 Annual Research Report
  • Research Products

    (43 results)

All Other

All Publications (43 results)

  • [Publications] H.Takanashi et al.: "Dynamical structure of the antibody combining site as studied by ^< 1>H-^<15>N shift correlation NMR spectroscopy" Biochemistry. 31. 2464-2468 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Odaka et al.: "Isotope-edited nuclear magnetic resonance study of Fv fragment of anti-dansyl mouse monoclonal antibody:Recognition of the dansyl hapten" Biochemistry. 31. 10686-10691 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Gouda et al.: "Three-dimensional solution structure of the B domain of stapnylococcal protein A:Comparison of the solution and crystal structure" Biochemistry. 31. 9665-9672 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.kim et al.: "^<13>C-NMR spectral analysis of the structures of mouse immunoglobulin GI carrying allotypes a and j" J.Immunol.Methoas. 153. 223-227 (1992)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] R.Mizutani et al.: "Effector functions of a mouse IgG that lacks the entire C_Hl domain" J.Immunol.150. 131-138 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] K.Kato et al.: "^<13>C NMR study of the mode of interaction in solution of the B fragment of staphylococcal protein A and Fc fragments of mouse immunoglobulin G" FEBS Lett.328. 49-54 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] R.Mizutani et al.: "Molecular structural studies of effector functions of a mouse immunog:obulin G that lacks the entire C_<11>l domain:Small-angle X-ray scattering,nanosecond fluoresccncc depolarization and stable isotope-aided NMR analyses" Mol.Immunol.30. 1665-1669 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] T.Nakayama et al.: "A multinuclear NMR study of the affinity maturation of anti-NP mouse monoclonal antibodies:Comparison of antibody combining sites of primary response antibody NIG9 and secondary response antibody 3B62" Biochemistry. 32. 13961-13968 (1993)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim et al.: "Dynamical structure of the hinge region of immunoglobulin G as studied by ^<13>C nuclear magnetic resonance spectroscopy" J.Mol.Biol.236. 300-309 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim et al.: "O-glycosylation in hinge region of mouse immunoglobulin G2b" J.Biol.Chem.269. 12345-12350 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim et al.: "Application of ^<13>C NMR spectroscopy to paratope mapping for larger antigen-Fab complexes" FEBS Lett.346. 246-250 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Takahashi et al.: "Role of the domain-domain interaction in the construction of the antigen combining site:A comparative study by ^1H-^<15>N shift correlation NMR spectroscopy of the Fv and Fab fragments of anti-dansyl mouse monoclonal antibody" J.Mol.Biol.243. 494-503 (1994)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] K.Kato et al.: "A model for the complex between protein G and an antibody Fc fragment in solution" Structure. 3. 79-85 (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Y.Yamaguchi et al.: "Proteolytic fragmentation with high specificity of mouse immunoglobulin G:Mapping of proteolytic cleavage sites in the hinge region" J.Immunol.Methods. in press. (1995)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Takahashi, E.Suzuki, I.Shimada, and Y.Arata: "Dynamical structure of the antibody combining site as studied by ^1H-^<15>N shift correlation NMR spectroscopy" Biochemistry. 31. 2464-2468 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] A.Odaka, J.I.Kim, H.Takahashi, I.Shimada, and Y.Arata: "Isotope-edited nuclear magnetic resonance study of Fv frgament of anti-dansyl mouse monoclonal antibody : Recognition of the dansyl hapten" Biochemistry. 31. 10686-10691 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Gouda, H.Torigoe, A.Saito, M.Sato, Y.Arata, and I.Shimada: "Three-dimensional solucion structure of the B domain of staphylococcal protein A : Comparison of the solution and crystal structure" Biochemistry. 31. 9665-9672 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim, K.Kato, A.Higuchi, N.Nomura, H.Noguchi and Y.Arata: "^<13>C-NMR spectral analysis of the structures of mouse immunoglobulin G1 carrying allotypes a and j" J.Immunol.Methods. 153. 223-227 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] R.Mizutani, T.Igarashi, T.Tanaka, I.Shimada, and Y.Arata: "Effector functions of a mouse IgG that lacks the entire C_H1 domain" J.Immunol.150. 131-138 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] K.Kato, H.Gouda, W.Takaha, A.Yoshino, C.Matsunaga, and Y.Arata: "^<13>C NMR study of the mode of interaction in solution of the B fragment of staphylococcal protein A and Fc fragments of mouse immunoglobulin G" FEBS Lett.328. 49-54 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] R.Mizutani, C.Matsunaga, T.Kouyama, M.Sato, Y.Katsube, K.Kato, I.Shimada, and Y.Arata: "Molecular structural studies of effector functions of a mouse immunoglobulin G that lacks the entire C_H1 domain : Small-angle X-ray scattering, nanosecond fluorescence depolarization and stable isotope-aided NMR analyzes" Mol.Immunol.30. 1665-1669 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] T.Nakayama, Y.Arata, and I.Shimada: "A multinuclear NMR study of the affinity maturation of anti-NP mouse monoclonal antibodies : Comparison of antibody combining sites of primary response antibody N1G9 and secondary response antibody 3B62" Biochemistry. 32. 13961-13968 (1993)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim, C.Matsunaga, A.Yoshino, K.Kato, and Y.Arata: "Dynamical structure of the hinge region of immunoglobulin G as studied by ^<13>C nuclear magnetic resonance spectroscopy" J.Mol.Biol.236. 300-309 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim, Y.Yamaguchi, K.Masuda, C.Matsunaga, K.Yamamoto, T.Irimura, N.Takahashi, K.Kato, and Y.Arata: "O-Glycosylation in hinge region of mouse immunoglobulin G2b" J.Biol.Chem.269. 12345-12350 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim, K.Kato, S.Yamato, T.Igarashi, C.Matsunaga, H.Ohtsuka, A.Higuchi, N.Nomura, H.Noguchi, and Y.Arata: "Application of ^<13>C NMR spectroscopy to paratope mapping for larger antigen-Fab complexes" FEBS Lett.346. 246-250 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Takahashi, H.Tamura, N.Shimba, I.Shimada, and Y.Arata: "Role of the domain-domain interaction in the construction of the antigen combining site : A comparative study by ^1H-^<15>N shift correlation NMR spectroscopy of the Fv and Fab fragments of anti-dansyl mouse monoclonal antibody" J.Mol.Biol.243. 494-503 (1994)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] K.Kato, L.-Y.Lian, I.L.Barsukov, J.P.Derrick, H.Kim, R.Tanaka, A.Yoshino, M.Shiraishi, I.Shimada, Y.Arata, and G.C.K.Roberts: "A model for the complex between protein G and an antibody Fc fragment in solution" Structure. 3. 79-85 (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] Y.Yamaguchi, H.Kim, K.Kato, K.Masuda, I.Shimada, and Y.Arata: "Proteolytic fragmentation with high specificity of mouse immunoglobulin G : Mapping of proteolytic cleavage sites in the hinge region" J.Immunol.Methods. (in press). (1995)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      1994 Final Research Report Summary
  • [Publications] H.Kim et al.: "Dynamical structure of the hinge region of immunoglobulin G as studied by ^<13>C nuclear magnetic resonance spectroscopy" J.Mol.Biol.236. 300-309 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] H.Kim et al.: "O-Glycosylation in hinge region of mouse immunoglobulin G2b" J.Biol.Chem.269. 12345-12350 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] H.Kim et al.: "Application of ^<13>C NMR spectroscopy to paratope mapping for larger antigen-Fab complexes" FEBS Lett.346. 246-250 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] H.Takahashi et al.: "Role of the domain-domain interaction in the construction of the antigen combining site" J.Mol.Biol.243. 494-503 (1994)

    • Related Report
      1994 Annual Research Report
  • [Publications] Y.Yamaguchi etal.: "Proteolytic fragmentation with high specificity of mouse immunoglobulin G:Mapping of proteolytic cleavage sites in the hinge region" J.Immunol.Methods. (印刷中). (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] K.Kato et al.: "A model for the complex between protein G and an antibody Fc fragment in solution" Structure. (印刷中). (1995)

    • Related Report
      1994 Annual Research Report
  • [Publications] H.Kim et al.: "Dynamical structure of the hinge region of immunoglobulin G as studied by ^<13>C nuclear magnetic resonance spectroscopy" J.Mol.Biol.(印刷中). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] T.Nakayama et al.: "A multinuclear NMR study of the affinity maturation of anti-NP mouse monoclonal antibodies:Comparison of antibody combining sites of primary response antibody N1G9 and secondary response antibody 3B62" Biochemistry. (印刷中). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] R.Mizutani et al.: "Molecular structural studies of effector functions of a mouse immunoglobulin G that lacks the entire C_H1 domain:Small angle X-ray scattering,nanosecond fluorescence depolarization,and stable isotope-aided NMR analyses" Mol.Immunol.(印刷中). (1994)

    • Related Report
      1993 Annual Research Report
  • [Publications] K.Kato et al.: "^<13>C NMR study of the mode of interaction in solution of the B fragment of Staphylococcal protein A and the Fc fragments of mouse immunoglobulin G" FEBS Lett.328. 49-54 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] R.Mizutani et al.: "Effector functions of a mouse IgG that lacks the entire C_H1 domain:C1q binding and complement fixation in the absence of antigen" J.Immunol.150. 131-138 (1993)

    • Related Report
      1993 Annual Research Report
  • [Publications] 金 夏亨: "^<13>C-NMR spectral analysis of the structures of mouse immunoglobulin Gl carrying allortypes a and j" J.Immunological Methods. 153. 223-227 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 合田 浩明: "Three-dimensional solution structre of the B domain of staphylococcal protein A:comparisons of solution and crystal structures" Biochemistry. 31. 9665-9672 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 尾高 麻乃: "Isotope-edited nucler magnetixc resonance study of Fv fragment of anti-dansyl mouse monoclonal antibody:recognition of the dansyl hapten" Biochemistry. 31. 10686-10691 (1992)

    • Related Report
      1992 Annual Research Report
  • [Publications] 高橋 栄夫: "Dynamical structure of the antibody combining site as studied by ^1H-^<15>N shift correlation NMR spectroscopy" Biochemistry. 31. 2464-2468 (1992)

    • Related Report
      1992 Annual Research Report

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Published: 1992-04-01   Modified: 2016-04-21  

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