Lipidomics of phospholipase A reveals exquisite specificity in macrophages.

Phospholipase A (PLA) constitutes a superfamily of enzymes that hydrolyze phospholipids at their sn-2 fatty acyl position. Our laboratory has demonstrated that PLA enzymes regulate membrane remodeling and cell signaling by their specificity toward their phospholipid substrates at the molecular level. Recent in vitro studies show that each type of PLA, including Group IVA cytosolic PLA (cPLA), Group V secreted PLA (sPLA), Group VIA calcium independent PLA (iPLA) and Group VIIA lipoprotein-associated PLA, also known as platelet-activating factor acetyl hydrolase, can discriminate exquisitely between fatty acids at the sn-2 position. Thus, these enzymes regulate the production of diverse PUFA precursors of inflammatory metabolites. We now determined PLA specificity in macrophage cells grown in cell culture, where the amounts and localization of the phospholipid substrates play a role in which specific phospholipids are hydrolyzed by each enzyme type. We used PLA stereospecific inhibitors in tandem with a novel UPLC-MS/MS-based lipidomics platform to quantify more than a thousand unique phospholipid molecular species demonstrating cPLA, sPLA, and iPLA activity and specificity toward the phospholipids in living cells. The observed specificity follows the in vitro capability of the enzymes and can reflect the enrichment of certain phospholipid species in specific membrane locations where particular PLA's associate. For assaying, we target 20:4-PI for cPLA, 22:6-PG for sPLA and 18:2-PC for iPLA. These new results provide great insight into the physiological role of PLA enzymes in cell membrane remodeling and could shed light on how PLA enzymes underpin inflammation and other lipid-related diseases.