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

• Project/Area Number: 07557335
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 試験
• Research Field: Bacteriology (including Mycology)
• Research Institution: Kurume 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: ¥2,300,000 (Direct Cost: ¥2,300,000); Fiscal Year 1996: ¥2,300,000 (Direct Cost: ¥2,300,000)
• Keywords: diphtheria 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.

Research Products

• [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
• [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
• [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
• [Publications] Tsuneoka,M.: "c-myc activates RCC1 gene expression through E-box elements." Oncogene. (in press).
• [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)
• [Publications] Nakamura,Y.: "Expression and distribution of CD9 in myelin of the central and perpheral nervous systems." Am.J.Pathol.149. 575-583 (1996)
• [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)
• [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)
• [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)
• [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)
• [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)
• [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)