Fusogenic Activity of PLA2-IIA and SMase in PEG-Mediated Membrane Fusion

Abstract

Phospholipase A2-IIA (PLA2-IIA) and sphingomyelinase (SMase) have been shown to induce fusion of membranes both in vivo and in vitro. The fusogenic potential of these two enzymes has been observed, for example, during neurotransmitter release. It is also believed that the products of hydrolysis of these two enzymes induce a reduction in the free energy cost of the fusion event. Assessment of the biophysical mechanisms by which these enzymes propel membrane fusion may provide relevant information for a variety of biological scenarios including neurotransmitter release. Poly(ethylene glycol)-(PEG-) mediated fusion of small unilamellar vesicles (SUVs) was studied in the presence of PLA2-IIA and SMase at varying temperatures between 17C and 40C. Lipid composition was dioleoylphosphatidylcholine (DOPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), porcine brain sphingomyelin (SM), and cholesterol (CH) in a ratio of 35:30:15:20. Content mixing (CM), lipid mixing (LM) and leakage (L) time courses were obtained and globally fitted to a 3 or 4-state sequential vesicle fusion model introduced by Weinberg and Lentz (Weinberg and Lentz, Biophys. J., 2007, 92; 4012). Based on the global fits, rate constants for the transitions between states, and LM, CM and L occurrence probabilities are obtained. We observe that both LM and CM are enhanced by enzyme activity in SUVs. In addition, the exogenous addition of hydrolysis products also promotes LM and CM in the absence of the enzymes. A careful study of the temperature dependent plots is used to explore alterations in the energy barriers between intermediate states. These findings may shed light into the biological mechanisms wherein specific vesicle lipid structure and enzyme catalysis become interrelated to induce membrane fusion.