探討Rhodostomin 的XRGD Motif 在辨識整合蛋白上所扮演的角色

Abstract

Integrins are a large family of α β heterodimeric receptors on cell surface. There are 24 integrins have been identified in mammals, and they mediate cell to cell and extraceullar matrix (ECM) interactions and regulate cell processes, including adhesion, migration, proliferation, and apoptosis. Many members of the integrin family recognize an Arg-Gly-Asp (RGD) motif within their ligands. Disintegrins are a family of RGD-containing proteins isolated from snake venoms and are the most potent integrin antagonists. It is reported that the residues flanking the RGD motif of integrin ligands are important for regulating integrin recognition. For example, it has been demonstrated that the mutation of the residue N-terminal to the RGD motif from proline to alanine on disintegrins can enhance their recognition of integrin α5β1. In this study I used rhodostomin (Rho) as the scaffold to study the role of the residue in the XRGD motif in recognizing integrins. Rho is a disintegrin that contain 68 amino acid including 6 disulfide bonds and a PRGDMP sequence at the positions of 48-53. In this study I have successfully expressed and purified seventeen XRGD mutants in the P. pastoris expression system. According to the results of cell adhesion and platelet aggregation assays, I found that the residue N-terminal to the RGD motif of Rho plays an important role for their integrin recognition. The mutations on the residue N-terminal to the RGD motif in inhibiting integrins αvβ3, α5β1, and αIIbβ3 have affinity changed of 0.6-3.2 folds, 0.2-18 folds, and 0.7-30 folds, respectively. These findings showed that the residue N-terminal to the RGD motif of Rho was more sensitive to integrin α5β1. We also found that Rho mutants containing acidic residues (D and E) N-terminal to the RGD motif selectively inhibited integrin αvβ3, and Rho mutant containing the GRGD motif selectively inhibited integrins αvβ3 and α5β1. The results of this study will serve as the basis for designing integrins αvβ3 and α5β1-specific disintegrins.