Abstract
Basic snake venom PLA2 from Naja nigricollis (N-PLA2) shows cytotoxicity toward several cell types. We herein show that N-PLA2 induces aggregation and lysis of negative and zwitterionic charged vesicles via a non-enzymatic mechanism, using turbidity measurement, transmission electron microscopy, quasi-elastic laser light scattering and lipid mixing assays. The results show that the N-PLA2-induced aggregation process, at temperatures near that of the gel to liquid-crystalline phase transition of 10% DMPS and pure DMPC vesicles can ultimately convert the smaller vesicles into large aggregates in the presence of EDTA, followed by their lysis into small lipid particles of the order of 15 nm. Anionic phosphatidylserine is seen to promote the PLA2-induced aggregation of vesicles but kinetically inhibits the lytic action of N-PLA2 on pure zwitterionic phosphatidylcholine vesicles. In contrast PLA2 from Naja naja atra (A-PLA2) does not exhibit such an activity mechanism. Analysis of the data, indicates the possibility of a new phospholipid-binding region from residues 54-76 consisting a cationic amphiphilic region which is located on the opposite side of the interface adsorption surface on PLA2. The penetration of N-PLA2 into phospholipid membrane analyzed by the binding to Monomyristoyl phosphatidylcholine micelles and Langmuir monolayer measurement indicates the presence of an extended hydrophobic patch in N-PLA2, in addition to the positively charged clusters. Gr II PLA2 and human sPLA2 have been found to specifically bind to heparin and heparin-like molecules on the cell surface. Herein we found that heparin can bind to N-PLA2 and induce its aggregation, which can potentiate the binding and penetration of N-PLA2 into membrane bilayer. It is well-known that cardiotoxins (CTXs) and phospholipase A2 (PLA2) from snake venom act synergistically on biological membranes, but the molecular mechanism responsible for the observed effect remains unknown. In this report acidic PLA2 from Naja naja atra which by itself lacks enzymatic and aggregation/fusion activity, was found to enhance the CTX’s (I, III and V from Naja naja atra) induced aggregation/fusion activity of sphingomyelin vesicles. Based on the structural aspects and cross-linking experiments we propose that electrostatic interaction between CTX and PLA2 is a possible mechanism underlying the mutual synergism. Cardiotoxin (CTX) from snake venom is a small basic membrane-active peptide has been reported to activate phospholipase A2 (PLA2) activity on acidic phospholipid vesicles and biological membrane. In this report, CTXs (CTX I, III and V from Naja naja atra) were found to enhance the binding of PLA2 from Naja naja atra by shorten the lag phase and increasing the initial rate of PLA2 hydrolysis to zwitterionic DMPC vesicles. By comparative studies of 11 CTXs purified from Naja naja tara, Naja mossambica mossambica and Naja nigricollis with their respect to activation of PLA2. All CTXs can shorten the lag phase and increasing the initial rate of PLA2 enzyme. Interestingly, the high sequence homology of melittin and phospholipase A2–activating protein (PLAP) was found with CTX (Table 1). This is evident upon comparing the amino acid sequences near the tip of loop II of CTX and the M region of PLAP (aa 260-280).Cobra venom cardiotoxins (CTXs), are a group of homologous proteins, are known to consist of 60-62 amino acid residues and three extended β－sheet loops. The CTXs induced aggregation/fusion activity of sphinomyelin large unilamellar vesicles (SM-LUVs?100nm) was found to follow a sigmoid curve as a function of CTXs concentration implicating cooperativity between CTXs at 39.4℃. By comparative studies of 11 CTXs with their respect to theirs interaction with SM-LUVs indicates acid residues of CTX molecules, eg., Glu-17, Asp-42, and Asp-59, located in the highly conserved region fall on the opposite side of three loops which are though to constitute the putative phospholipid binding sites, indicating an important role on cooperativity between CTXs. Comparative CTX Tr with M1, which has subtitution of Asp-59 by Asn-59, shows higher aggregation /fusion activity. Carboxylate group modified CTX M4 at Glu-17 and Asp-42 with carbodiimide (semicarbazide) result in a decreased aggregation/fusion activity. There seems to be no significant conformation change of modified CTX M4 as evidenced by the CD spectra. Moreover, Acidic PLA2 from Naja naja atra which by itself lacks enzymatic and aggregation/fusion activity of LUVs, was found to enhance the native CTX M4 as well as modified M4 induced aggregation/fusion activity and thus the binding of CTX to sphingomyelin vesicles.