Purification, Functional Characterization And Cloning Of An Antimicrobial Phospholipase A₂ From Bungarus Fasciatus Venom

Phospholipase A₂ (PEA2; EC 3.1.1.4) proteins are a diverse family of lipolytic enzymes that specifically catalyze the hydrolysis of fatty acid ester bonds at position 2 of 1,2-diacyl-sn-3-phosphoglycerides to produce free fatty acids and lysophospholipids (Kini, 1997). PLA₂s from snake venoms display several biological effects, including platelet aggregation activity (Takagi et al., 1988), inhibitory effect on platelet aggregation induced by ADP, collagen and arachidonic acid in both human whole blood and platelet-rich plasma in a dose-dependent manner (Min Zhou Huang et al., 1997), bactericidal activities, neurotoxic activities (Veridiana et al., 2004), antitumoral effect (Patricia et al., 2004), anticoagulant activities (Robin et al., 2003).In this experiment, we first report the purification and characterization of a PLA₂ from Bungarus fasciatus venom from southeast of China belonging to group IA. Further more a complete nucleotide sequence and partial characterization of the new PLA₂s were identified. The PLA₂ purified from Bungarus fasciatus venom by gel filtration chromatography, cation-exchange chromatography and high-performance liquid chromatography, selected the faction by antimicrobial activity. This novel PLA₂ isolated from Bungarus fasciatus venom displayed bactericidal activity against Escherichia coli and Staphylococcus aureus. In addition it showed anti-tumor activity as well as anticoagulation activity but no platelet aggregation enhance or inhibit effect induce by ADP, weak hemolytic activity. We have determined the N-terminal amino acid sequence. According to the sequence that reported previously, we cloned the full length of cDNA successfully.In the present report, we obtained 15 complete nucleotide sequences of phospholipase A₂ (PLA₂) from the cDNA library of Bungarus fasciatus venom glands with a special N-terminal primer. These homologys with the open reading frame length of 435bp, encoded 10 different PLA₂ enzymes included a signal peptide and the mature protein with 27 and 118 amino acid residues, respectively. They shared more than 75% similarity with each other, which should be classified into group IA type PLA₂. It suggested that the Bungarus fasciatus venom was the abundant resource of PLA₂. On the other hand, we compared these PLA₂ from Bungarus fasciatus venom with other ones from variety of snake venoms, the multiple sequences blast result showed that the signal peptides were more conserved than the mature peptides and the number of nucleotide substitutions nonsynonymous sites were equal to synonymous sites, implying that neutral selection evolution occurred in this group of PLA₂.This is the first report on the isolation and identification of a cDNA encoding a complete PLA₂ from Bungarus fasciatus venom, exhibiting bactericidal effects. We further cloned a serial of complete nucleotide sequences from Bungarus fasciatus venom. Hoping this report will give light to more deep researches.