RECOMBINANT SNAKE DISINTEGRINS USED FOR MAMMALIAN INTEGRIN STUDY

Abstract

Rhodostomin, containing 68 amino acid residues with six pairs of disulfide bonds, can be isolated from the venom of Agkistrodon (Calloselasma) rhodostoma and belongs to a group of disintegrins that have a high potency and inhibit platelet aggregation. This review focuses on the application of genetically engineered rhodostomins in the study of the integrins of platelets and cell lines. An artificial gene encoding rhodostomin was first obtained by ligation of synthetic oligonucleotides followed by PCR amplification. Next, it was fused downstream of glutathione S-transferase (GST) to produce GST-rhodostomin. The GST-rhodostomin produced by E. coli was easily purified by one-step affinity column. The biological activity of GST-rhodostomin is as potent as the native rhodostomin, indicating that the recombinant rhodostomin has the correct folding, and this exposes its Arg-Gly-Asp (RGD) domain, the major functional motif binding to integrin receptors. In addition to the RGD domain, the flanking sequences of RGD also play a role in direct binding to the integrin αIIbβ3 of platelets and this has been revealed by mutagenesis studies. Without inside-out signaling stimulation, platelets’ calcium oscillation and FAK phosphorylation are induced via GST-rhodostomin directly by its binding to platelets. By plating rat bone marrows on a GST-rhodostomin coated plate, we were able to separate and enrich osteoblast progenitor cells from mixed types of cells. This suggests that GST-rhodostomin has potential in the enrichment of certain type of cells in vitro.