Cholesterol Content And Domain Formation As Regulators Of PLA2-IIA Activity In Anionic Membranes

Abstract

Phospholipase A2 type IIA (PLA2-IIA) hydrolyses the sn-2 position of glycerolipids to produce free fatty acids and lysolipids. The enzyme presents a strong affinity towards membranes enriched in anionic lipids, and lipid-packing is known to influence the ability of PLA2-IIA to extract lipids from the membrane. This leads to an interrelation between membrane structure, and enzyme activity. In this study we evaluate the activity of PLA2-IIA on unilamellar vesicles composed of either 1- palmitoyl-2-oleoyl phosphatidylglycerol (POPG) or dimyristoyl - phosphatidylglycerol (DMPG), in combination with cholesterol (Chol) and sphyngomyelin (SM). We expose the vesicles to PLA2-IIA and monitor the activity at 37°C, where both POPG and DMPG are in the liquid-disordered phase in their pure form. For DMPG/Chol or POPG/Chol the results show that adding cholesterol alone inhibits PLA2-IIA activity. However, this effect is more accentuated in DMPG vesicles compared to POPG vesicles. We attribute this difference to a closer interaction of cholesterol with the saturated acyl chains of DMPG, leading to tighter lipid packing and a reduced hydrolysis rate. In the second part of the study, for POPG and DMPG samples in which we included cholesterol and sphyngomyelin in equal molarities, we detect high hydrolytic activity in a wider range of compositions for a given Chol/PG ratio compared to samples without SM. We propose that a strong affinity between SM and cholesterol, related to liquid-ordered domain formation, leads to a depletion of cholesterol from the PG-rich regions, maintaining a high PLA2-IIA hydrolysis rate. In the third part of our study, we focus on changes in rigidity of the membranes after exposure to PLA2-IIA based on Laurdan General Polarization measurements. We find a general increase in rigidity following the hydrolytic burst in POPG binary and ternary systems.