Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity

Abstract

An acidic phospholipase A2 enzyme (NnPLA2-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA2-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA2-I cognate complex to vimentin as compared to the binding of individual NnPLA2-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA2-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA2-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA2-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (KD) values of −12.86 kcal/mol and 3.67 × 10−10 M, respectively; however, NnPLA2-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.