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An attempt to form mutant CRM197 proteins of diphtheria toxin with neutralizing activity to heregulin.

Research Project

Project/Area Number 07557335
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section試験
Research Field Bacteriology (including Mycology)
Research InstitutionKurume University

Principal Investigator

MEKADA Eisuke  Institute of Life Sciense, Kurume University, Professor, 分子生命科学研究所, 教授 (20135742)

Co-Investigator(Kenkyū-buntansha) MITAMURA Toshihide  Research Institute for Microbial Diseases. Osaka University, Assistant Professor, 微生物病研究所, 助手 (80268846)
UMATA Toshiyuki  Institute of Life Sciense, Kurume University, Assistant Professor, 分子生命科学研究所, 助手 (30213482)
IWAMOTO Ryo  Institute of Life Sciense, Kurume University, Assistant Professor, 分子生命科学研究所, 助手 (10213323)
Project Period (FY) 1995 – 1996
Project Status Completed (Fiscal Year 1996)
Budget Amount *help
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1996: ¥2,300,000 (Direct Cost: ¥2,300,000)
Keywordsdiphtheria toxin / HB-EGF / EGF receptor / heregulin / CRM197 / 腫瘍増殖因子抑制物質
Research Abstract

We have found that diphtheria toxin (DT) bind to the EGF-like domain of diphtheria toxin receptor/membrane-anchored form of heparin-binding EGF-like growth factor (proHB-EGF). DT,or its non-toxic mutant CRM197, inhibits the binding of HB-EGF to EGF receptor, resulting in inhibition of mitogenic activity of HB-EGF.The aim of this work is to produce mutant forms of CRM197 of diphtheria toxin with neutralizing activity to heregulin. We have obtained the following results.
(1) Analysis of the DT-binding site on the EGF-like domain of HB-EGF
To form heregulin-binding CRM197, DT-binding site on EGF-like domain of HB-EGF was precisely determined. We introduced single point mutations for 10 amino acids within the EGF-like domain. Mutation at F115, L127 or E141 greatly decreased the DT-binding activityof HB-EGF,but mutations for the remaining 7 amino acids did not show large effect.
(2) Construction of three-dimentional structure model of the EGF-like domain of HB-EGF
Three-dimentional structure model of the EGF-like domain of HB-EGF was constructed based on the NMR data of EGF and TGFalpha. The model indicated that amino acid residues F115, L127 and E141 exist on the same side of the molecule, suggesting that HB-EGF interacts with DT at this side.
These results is critical to construct heregulin-binding CRM197. Further studies are in progress.

Report

(3 results)
  • 1996 Annual Research Report   Final Research Report Summary
  • 1995 Annual Research Report
  • Research Products

    (12 results)

All Other

All Publications (12 results)

  • [Publications] Tsuneoka,M.: "c-myc activates RCC1 gene expression through E-box elements." Oncogene. (in perss).

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Nakagawa,T.: "Amino-terminal Processing of cell surface heparin-binding EGF-like growth factor upregulates its juxtacrine but not its pafacrine growth factor activity." J.Biol.Chem.271. 30858-30863 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Nakamura,Y.: "Expression and distribution of CD9 in myelin of the central and peripheral nervous systems." Am.J.Pathol.149. 575-583 (1996)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      1996 Final Research Report Summary
  • [Publications] Tsuneoka,M.: "c-myc activates RCC1 gene expression through E-box elements." Oncogene. (in press).

    • Related Report
      1996 Annual Research Report
  • [Publications] Nakagawa,T.: "Amino-terminal processing of cell surface heparin-binding EGF-like growth factor upregulates its juxtacrine but not its paracrine growth factor activity." J.Biol.Chem.271. 30858-30863 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Nakamura,Y.: "Expression and distribution of CD9 in myelin of the central and perpheral nervous systems." Am.J.Pathol.149. 575-583 (1996)

    • Related Report
      1996 Annual Research Report
  • [Publications] Mitamura,T.: "Diphtheria toxin binds to the EGF-like domain of human heparin-binding EGF-like growth factor/diphtheria toxin receptor, and inhibits specifically its mitogenic activity." J.Biol.Chem.270. 1015-1019 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Higashiyama,S.: "The membrane protein CD9/DRAP27 potentiates the growth factor activity of the membrane-anchored heparin-binding EGF-like growth facor (HB-EGF)." J.Cell Biol.128. 929-938 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Nakamura,K.: "Membrane anchored heparin-binding EGF-like growth factor and DRAP27/CD9 form a complex with integrin 3 l at cell-cell contact sites." J.Cell Biol.129. 1691-1705 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Goishi,K.: "Phorbol ester induces the rapid processing of cell surface heparin-binding EGF-like growth factor; Conversion from juxtacrine to paracrine growth factor activity." Mol.Biol.Cell. 6. 967-980 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Shishido,Y.: "Heparin-like molecules on the cell surface potentiate binding of diphtheria toxin to the diphtheria toxin receptor/membrane-anchored heparin-binding EGF-like growth factor." J.Biol.Chem.270. 29578-29585 (1995)

    • Related Report
      1995 Annual Research Report
  • [Publications] Mekada,E.: "Handbook of natural toxins: Volume 8, Bacterial toxins and virulence factors in disease" Moss,J.,Iglewski,B.,Vaughan,M.& Tu,A.T.(Marcel Dekker Inc.,New York), 15 (1995)

    • Related Report
      1995 Annual Research Report

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Published: 1996-04-01   Modified: 2016-04-21  

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