PID15, a novel 6 kDa secreted peptide, mediates Naja naja venom phospholipase A(2) induced apoptosis in isolated human peripheral lymphocytes

BACKGROUND: Snake venoms are a complex mixture of active principles mainly peptides and proteins also including amino acids, nucleotides, free lipids, carbohydrates and metallic elements bound to proteins that interfere in several biological systems. In this study, we aimed to understand the mode of action of the apoptosis inducing ability of Naja naja venom phospholipase A(2) (NV-PLA(2)) using isolated human peripheral lymphocytes. RESULTS: Human peripheral lymphocytes when incubated with Naja naja venom phospholipase A(2) (NV-PLA(2)) induced up to 68% DNA fragmentation. The dialysed conditioned media obtained by incubating lymphocytes with NV-PLA(2) at 15(th) min induced 44% DNA fragmentation, referred to as cmlp-active. Cmlp-active showed 20.5% increased protein concentration than the corresponding control condition media cmlp-c-15. Test for creatine kinase activity in cmlp-active proved negative and negligible amount of lactate dehydrogenase did not show significant DNA fragmentation. Fractionation of cmlp-active on Sephadex G-25 showed two peaks, major peak induced 38% DNA fragmentation, which was further rechromatographed on Sephadex G-25. The single peak obtained was named PID15 (Phospholipase A( 2 )Induced DNA fragmentation factor secreted at 15( th ) min). Q-Tof MS/MS analysis of PID-15 showed it is a 6 kDa peptide. PID15 sequence analysis gave 40 amino acids in the following order, msilpcknvs iwvikdtaas dkevvlgsdr aikflylatg. The homology search for the sequence revealed it to be an Apoptosis Inducing Factor (AIF). CONCLUSION: Results indicate that the secretion of PID15 is dependent on concentration of NV-PLA(2) treatment, incubation time and also on temperature and the probable membrane origin of PID15 and not of cytosolic origin with apoptosis inducing ability.