Cloning and characterization of novel disintegrins from Agkistrodon halys venom

Snake venom disintegrins act as potent inhibitors of platelet aggregation. In this report, we isolated genes encoding novel members of disintegrins through the screening of Agkistrodon Italys venom gland cDNA library. Subsequent characterization of positives revealed the presence of distinct disintegrins named salmosin1, 2, and 3, each containing a characteristic RGD/KGD sequence essential for the binding to integrins, Whereas salmosin1 was identical to previously described salmosin purified from A. halys venom, salmosin2 and salmosin3 were predicted to be a novel, 73 amino acid protein with a KGD sequence, and an 80 amino acid protein with an additional 7th disulfide bond, respectively. Taken together, this is the first report describing 3 unique disintegrins, namely, salmosin1 with RGD, salmosin2 with KGD and salmosin3 with 7 disulfide bonds are found in a single species of venom. Subsequently, to compare the platelet aggregation inhibitory potential of the recombinant protein with that of natural protein, salmosin1 was expressed in E. coli and purified to homogeneity, Recombinant and natural salmosin1 inhibited the binding of alpha(IIb)beta(3) to fibrinogen with an almost identical IC50 value of 2.2 nM and 4.5 nM respectively. Moreover, recombinant salmosin1 displayed an IC50 value approximately 5-fold lower than flavoridin, which was previously described as the most potent venom disintegrin so far. In conclusion, we identified 3 disintegrins with distinct properties through the molecular cloning approach and found that the recombinant salmosin1 retained one of the most potent alpha(IIb)beta(3) antagonist activity.