UC San Diego

Human phospholipase A₂s (PLA₂) constitute a superfamily of enzymes that hydrolyze the sn-2 acyl-chain of glycerophospholipids, producing lysophospholipids and free fatty acids. Each PLA₂ enzyme type contributes to specific biological functions based on its expression, subcellular localization, and substrate specificity. Among the PLA₂ superfamily, the cytosolic cPLA₂ enzymes, calcium-independent iPLA₂ enzymes, and secreted sPLA₂ enzymes are implicated in many diseases, but a central issue is the preference for double-bond positions in polyunsaturated fatty acids (PUFAs) occupying the sn-2 position of membrane phospholipids. We demonstrate that each PLA₂ has a unique preference between the specific omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 arachidonic acid (AA), which are the precursors of most proinflammatory and anti-inflammatory or resolving eicosanoids and related oxylipins. Surprisingly, we discovered that human cPLA₂ selectively prefers AA, whereas iPLA₂ prefers EPA, and sPLA₂ prefers DHA as substrate. We determined the optimal binding of each phospholipid substrate in the active site of each PLA₂ to explain these specificities. To investigate this, we utilized recently developed lipidomics-based LC-MS/MS and GC/MS assays to determine the sn-2 acyl chain specificity in mixtures of phospholipids. We performed μs timescale molecular dynamics (MD) simulations to reveal unique active site properties, especially how the precise hydrophobic cavity accommodation of the sn-2 acyl chain contributes to the stability of substrate binding and the specificity of each PLA₂ for AA, EPA, or DHA. This study provides the first comprehensive picture of the unique substrate selectivity of each PLA₂ for omega-3 and omega-6 fatty acids.