Analyses of Flanking Amino Acid Residues Around the RGD-motif of Rhodostomin for Interaction with Integrin aIIbb3

Chih-Pei Chang, Jui-Chin Chang, Hsin-Hou Chang, and Szecheng J. Lo

 

Institute of Microbiology and Immunology

National Yang-Ming University

Taipei, Taiwan 11221, R.O.C.

 

 

ABSTRACT

 

Rhodostomin (RHO), an RGD-containing peptide isolated from snake venom, has been demonstrated to inhibit platelet aggregation through interaction of integrin aIIbb3, but it lacks direct evidence of RHO-integrin aIIbb3 binding. A synthetic RHO gene was fused with glutathione S-transferase (GST) and the GST-RHO protein was expressed in a large quantity in E. coli, which can be purified by a single step of affinity column. GST-RHO pull-down assays were then developed, coupling with a biotin-avidin-HRP-ECL detection, to demonstrate the direct binding of RHO to integrin aIIbb3. To further analyze the importance of amino acids flanking the RGD sequence of RHO, 10 alanine-insertion mutants were created. The pull-down assay showed that 4 alanine-insertion mutants upstream to the RGD motif and 3 insertions downstream to RGD reduced integrin aIIbb3 binding activity mildly. In contrast, two insertions immediately next to RGD and one insertion prior to the 57Cys nearly lost binding activity to aIIbb3 as the RGE mutant. Results of platelet aggregation inhibition and platelet adhesion assays by insertion mutants were consistent with results of pull-down assay, suggesting that the exact integrin-interaction region of RHO is Arg-Gly-Asp-Met-Pro (49RGDMP53). When the RGD flanking sequence of RHO (46RIPRGDMPD54) was substituted by RPTRGDCDL and NWKRGDCDL, which are from human MDC-15 (D15) and its derivative (D15/NWK), respectively, two chimera showed almost no activity to aIIbb3 by pull-down and platelet aggregation inhibition. These results suggest that integrin aIIbb3 is not the native receptor for MDC-15 and further support that the RGDMP sequence of RHO is important for aIIbb3 binding.