Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:591-599
© 1996 American Heart Association, Inc.
Articles |
Purification, Properties, Sequencing, and Cloning of a Lipoprotein-Associated, Serine-Dependent Phospholipase Involved in the Oxidative Modification of Low-Density Lipoproteins
From SmithKline Beecham Pharmaceuticals, Departments of Mechanistic Enzymology (D.G.T., H.F.B.), Protein Chemistry (C.S.), and Vascular Biology (K.M, C.H.M.), Welwyn, Hertfordshire, and Department of Biotechnology (S.Q.J.R., G.M.P.L., I.S.G.), Harlow, UK, and Human Genome Sciences, Rockville, Md (H.L.).
Correspondence to Dr Colin Macphee, Vascular Biology, SmithKline Beecham Pharmaceuticals, The Frythe, Welwyn, Herts AL6 9AR, UK.
Abstract A novel LDL-associated phospholipase A2 (LDL-PLA2) has been purified to homogeneity from human LDL obtained from plasma apheresis. This enzyme has activity toward both oxidized phosphatidylcholine and platelet activating factor (PAF). A simple purification procedure involving detergent solubilization and affinity and ion exchange chromatography has been devised. Vmax and Km for the purified enzyme are 170 µmol·min-1·mg-1 and 12 µmol/L, respectively. Extensive peptide sequence from LDL-PLA2 facilitated identification of an expressed sequence tag partial cDNA. This has led to cloning and expression of active protein in baculovirus. A lipase motif is also evident from sequence information, indicating that the enzyme is serine dependent. Inhibition by diethyl p-nitrophenyl phosphate and 3,4-dichloroisocoumarin and insensitivity to EDTA, Ca2+, and sulfhydryl reagents confirm that the enzyme is indeed a serine-dependent hydrolase. The protein is extensively glycosylated, and the glycosylation site has been identified. Antibodies to this LDL-PLA2 have been raised and used to show that this enzyme is responsible for >95% of the phospholipase activity associated with LDL. Inhibition of LDL-PLA2 before oxidation of LDL reduces both lysophosphatidylcholine content and monocyte chemoattractant ability of the resulting oxidized LDL. Lysophosphatidylcholine production and monocyte chemoattractant ability can be restored by addition of physiological quantities of pure LDL-PLA2.
Key Words: phospholipase A2 • LDL • oxidation • PAF-AH
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