Differentiating human phospholipase A2's activity toward phosphatidylinositol, phosphatidylinositol phosphate and phosphatidylinositol bisphosphate

Phospholipase A₂'s (PLA₂'s) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA₂ Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA₂'s, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA₂^'s toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P₂). The in vitro assay revealed that Group IVA cPLA₂ (GIVA cPLA₂) showed relatively high activity toward PI and PI(4)P among the tested PLA₂'s; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA₂ and GVIA iPLA₂ showed detectable activity toward PI(4,5)P₂, but it appeared to be a poorer substrate for all of the PLA₂'s tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA₂ contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA₂ can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA₂ may best account for the PI and PIP hydrolysis in living cells.