Unique Substrate Specificity of Human Phospholipase A2s

Abstract

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that hydrolyze the sn‐2 acyl‐chain of phospholipids which are the major components of cell membranes. PLA2 produces free fatty acids and lysophospholipids as products which serve as lipid mediators and precursors of bioactive lipids. Among them, the release of ω‐6 and ω‐3 fatty acids including arachidonic acid (AA, ω‐6), eicosapentaenoic acid (EPA, ω‐3), and docosahexaenoic acid (DHA, ω‐3) is a pivotal role of PLA2s because the fatty acids are precursors of various eicosanoids and related bioactive oxylipins responsible for inflammation and resolution. Additionally, in contrast to conventional ester‐linked phospholipids, we also investigated PLA2 specificity toward sn‐1 vinyl ether‐linked phospholipids and phosphatidylinositol phosphates (PIPs) which are involved in various signaling processes.Cytosolic PLA2 (cPLA2), calcium‐independent PLA2 (iPLA2), and secreted PLA2 (sPLA2) are the three major PLA2s that are well studied, and a significant number of PLA2‐related disorders have been reported. Therefore, investigating the substrate specificity of PLA2 toward different phospholipids, can be a key to understanding the etiology of PLA2‐related disorders and developing novel therapeutic targets. Recently, we developed a high‐throughput lipidomics‐based assay to measure PLA2 activity and successfully employed it to investigate the general specificity of PLA2s (1). In the present study, we investigated the specificity of the three major PLA2s toward phospholipids containing ω‐6 and ω‐3 fatty acids in the sn‐2 position, sn‐1 vinyl ethers, and PIPs using this lipidomics‐based assay. Also, we determined the optimal binding of the substrate in the active site of each PLA2 by molecular dynamics (MD) simulations to explain the observed specificity.cPLA2 showed high activity toward AA and EPA, and prefers sn‐1 vinyl ether phospholipids over ester phospholipids. iPLA2 showed the highest activity toward EPA, and no preference was observed between a vinyl ether and an ester. sPLA2 showed a preference for DHA and ester phospholipids. In addition, we determined that although additional phosphates on the inositol headgroup attenuated the activity of all three PLA2s, at least cPLA2 and iPLA2 could hydrolyze PI(4,5)P2. MD simulations revealed that the active site of cPLA2 could precisely bind AA and EPA in the sn‐2 position but not DHA. In summary, the present study revealed a unique and distinct substrate specificity for each of the PLA2s and explained the observed preference of cPLA2 for AA and EPA.Support or Funding InformationSupported by NIH grant GM20501‐43