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Journal of Lipid Research, Vol. 46, 1517-1525, July 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Endothelial lipase releases saturated and unsaturated fatty acids of high density lipoprotein phosphatidylcholine

M. Gauster^*, G. Rechberger, A. Sovic^*, G. Hörl^*, E. Steyrer^*, W. Sattler^* and S. Frank^1,*

^* Institute of Molecular Biology and Biochemistry, Center of Molecular Medicine, Medical University Graz, Harrachgasse 21/III, Graz A-8010, Austria
Institute of Molecular Biology, Biochemistry and Microbiology, University of Graz, Heinrichstrasse 31a, Graz A-8010, Austria

Published, JLR Papers in Press, April 16, 2005. DOI 10.1194/jlr.M500054-JLR200

¹ To whom correspondence should be addressed. e-mail: sasa.frank{at}meduni-graz.at

We assessed the ability of endothelial lipase (EL) to hydrolyze the sn-1 and sn-2 fatty acids (FAs) from HDL phosphatidylcholine. For this purpose, reconstituted discoidal HDLs (rHDLs) that contained free cholesterol, apolipoprotein A-I, and either 1-palmitoyl-2-oleoylphosphatidylcholine, 1-palmitoyl-2-linoleoylphosphatidylcholine, or 1-palmitoyl-2-arachidonylphosphatidylcholine were incubated with EL- and control (LacZ)-conditioned media. Gas chromatography analysis of the reaction mixtures revealed that both the sn-1 (16:0) and sn-2 (18:1, 18:2, and 20:4) FAs were liberated by EL. The higher rate of sn-1 FA cleavage compared with sn-2 FA release generated corresponding sn-2 acyl lyso-species as determined by MS analysis. EL failed to release sn-2 FA from rHDLs containing 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine, whose sn-1 position contained a nonhydrolyzable alkyl ether linkage.

The lack of phospholipase A₂ activity of EL and its ability to liberate [¹⁴C]FA from [¹⁴C]lysophosphatidylcholine (lyso-PC) led us to conclude that EL-mediated deacylation of phosphatidylcholine (PC) is initiated at the sn-1 position, followed by the release of the remaining FA from the lyso-PC intermediate. Thin-layer chromatography analysis of cellular lipids obtained from EL-overexpressing cells revealed a pronounced accumulation of [¹⁴C]phospholipid and [¹⁴C]triglyceride upon incubation with 1-palmitoyl-2-[1-¹⁴C]linoleoyl-PC-labeled HDL₃, indicating the ability of EL to supply cells with unsaturated FAs.

Abbreviations: DMEM, Dulbecco's modified Eagle's medium; EL, endothelial lipase; EL-Ad, adenovirus encoding human endothelial lipase; FA, fatty acid; FCS, fetal calf serum; FFA, free fatty acid; GC, gas chromatography; HDL-PC, HDL phosphatidylcholine; LacZ-Ad, adenovirus encoding ß-galactosidase; lyso-PC, lysophosphatidylcholine; MOI, multiplicity of infection; PAPC, 1-palmitoyl-2-arachidonylphosphatidylcholine; PL, phospholipid; plasmalogen-PC, 1-O-1'-hexadecenyl-2-arachidonoylphosphatidylcholine; PLPC, 1-palmitoyl-2-linoleoylphosphatidylcholine; POPC, 1-palmitoyl-2-oleoylphosphatidylcholine; TG, triglyceride; TLC, thin-layer chromatography

Supplementary key words phospholipases • lysophospholipases • phospholipids • polyunsaturated fatty acid • reconstituted high density lipoprotein

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