Effect of disulfide bond on the conformation and anticoagulant activity of an Arg-Gly-Asp motif displayed on a mutant insulin protein framework |
| |
Authors: | Jian Liu Xing-Wen Yang Wei-Qiang Chen Jian-Guo Tang |
| |
Affiliation: | (1) National Laboratory of Protein Engineering and Plant Genetic Engineering, College of life Sciences, Peking University, Peking, China;(2) Institute of Medicinal Plants Development, Chinese Academy of Medical Sciences and Peking Union Medical College, Peking, China |
| |
Abstract: | Protein engineering techniques were employed to graft the known anticoagulant Arg-Gly-Asp (RGD) motif-containing sequences onto the surface of a mutant, inactive insulin framework. To probe the effect of a disulfide bond on the resultant anticoagulant activity, a native RGD-containing sequence from disintegrin dendroaspin, CFTPRGDMPGPYC, as well as a modified sequence, SFTPRGDMPGPYS, were each examined. The peptide was placed between the C-terminal of the B chain and the N-terminal of the A chainand connected with B27 and A1 residues of the inactive insulin that lacks the characteristic intramolecular A6-11 disulfide bondwithin the A chain. The two RGD-containing insulin genes were over-expressed in E. coli, and purified and designatedas RGD-Cys-Ins and RGD-Ser-Ins, respectively. Their amino acid compositions and mass data were in good agreement with those ofexpected. The RGD-Cys-Ins showed inhibition of platelet aggregation with an IC50 of 3 M, while the latter was3.5-fold less active. The in vivo assay also indicatedthat the RGD-Cys-Ins had a higher activity in prolonging the bleeding time in mice than RGD-Ser-Ins. Both RGD-Cys-Ins and RGD-Ser-Ins retained about 25% of the proinsulin immunoactivity and had almost no insulin receptor binding activity. The results indicate the necessity for the RGD motif to be conformationally constrained for it to elicit a greater anticoagulant activity. |
| |
Keywords: | bleeding time disulfide bond inhibition of platelet aggregation insulin protein frame peptide display RGD motif |
本文献已被 SpringerLink 等数据库收录! |
|