Expression and characterization of human group V phospholipase A2

Abstract

Group V phospholipase A₂ (GV-PLA₂) has been shown to be involved in signal transduction and inflammatory processes in cellular studies, but the physical and biochemical properties of this important enzyme have been unclear. We report the over-expression and characterization of GV-PLA₂. The GV-PLA₂ cDNA was synthesized from human heart polyA⁺ mRNA by RT-PCR, and an expression construct containing the GV-PLA₂ was established. After expression in Escherichia coli cells, the protein was solubilized and purified to homogeneity in a single step using nickel affinity chromatography. The purified GV-PLA₂ protein was folded to form active enzyme. The recombinant GV-PLA₂ has an absolute requirement for Ca²⁺ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with sonicated vesicles as substrate. GV-PLA₂ preferentially hydrolyzes phosphatidylethanolamine (PE) vesicles compared to phosphatidylcholine (PC) vesicles. However, hydrolysis of PC and PE is equivalent in mixed vesicles of the phospholipids. The fatty acid preference of GV-PLA₂ is linoleoyl>palmitoyl>arachidonyl with a PC head group and sonicated vesicles. 3-(3-Actamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), a potent inhibitor of human group IIA PLA₂, strongly inhibits GV-PLA₂ with an IC₅₀ value of about 36 nM which is comparable to its inhibition of group IIA PLA₂.