Phospholipase A2 with Platelet Aggregation Inhibitor Activity from Austrelaps superbus Venom: Protein Purification and cDNA Cloning

Abstract

Four phospholipase A2 (PLA2) enzymes (Superbins a, b, c, and d) with varying platelet aggregation inhibitor activities have been purified from Austrelaps superbus by a combination of gel filtration, ion-exchange, and reversed-phase high-pressure liquid chromatography. Purity and homogeneity of the superbins have been confirmed by high-performance capillary zone electrophoresis and mass spectrometry. The electron spray ionization mass spectrometry data showed that their molecular masses range from 13,140 to 13,236 Da. Each of the proteins has been found to be basic and exhibit varying degrees of PLA2 activity. They also displayed different platelet aggregation inhibitory activities. Superbin a was found to possess the most potent inhibitory activity with an IC50 of 9.0 nM, whereas Superbin d was found to be least effective with an IC50 of 3.0 μM. Superbins b and c were moderately effective with IC50 values of 0.05 and 0.5 μM, respectively. The amino-terminal sequencing confirmed the identity of these superbins. cDNA cloning resulted in the identification of 17 more PLA2 isoforms in A. superbus venom. It has also provided complete information on the precursor PLA2. The precursor PLA2 contained a 27-amino-acid signal peptide and 117- to 125-amino-acid PLA2 (molecular mass ranging from 13,000 to 14,000 Da). Two of these PLA2 enzymes resembled more closely (87%) Superbin a in structure. Two unique PLA2 enzymes containing an extra pancreatic loop also have been identified among the isoforms.