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Papers In Press, published online ahead of print August 1, 2009
J. Lipid Res., doi:10.1194/jlr.M800398-JLR200

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Journal of Lipid Research, Vol. 50, 1563-1570, August 2009
Copyright © 2009 by American Society for Biochemistry and Molecular Biology

Characterization of human lysophospholipid acyltransferase 3^[S]

Shilpa Jain^*, Xiaoling Zhang, Preeti J. Khandelwal^*, Aleister J. Saunders^*, Brian S. Cummings and Peter Oelkers^1,*

^* Department of Biology, Drexel University, Philadelphia, PA, 19104
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, GA 30602

¹ To whom correspondence should be addressed. e-mail: oelkersp{at}arcadia.edu

Esterifying lysophospholipids may serve a variety of functions, including phospholipid remodeling and limiting the abundance of bioactive lipids. Recently, a yeast enzyme, Lpt1p, that esterifies an array of lysophospholipids was identified. Described here is the characterization of a human homolog of LPT1 that we have called lysophosphatidylcholine acyltransferase 3 (LPCAT3). Expression of LPCAT3 in Sf9 insect cells conferred robust esterification of lysophosphatidylcholine in vitro. Kinetic analysis found apparent cooperativity with a saturated acyl-CoA having the lowest K_0.5 (5 µM), a monounsaturated acyl-CoA having the highest apparent V_max (759 nmol/min/mg), and two polyunsaturated acyl-CoAs showing intermediate values. Lysophosphatidylethanolamine and lysophosphatidylserine were also utilized as substrates. Electrospray ionization mass spectrometric analysis of phospholipids in Sf9 cells expressing LPCAT3 showed a relative increase in phosphatidylcholine containing saturated acyl chains and a decrease in phosphatidylcholine containing unsaturated acyl chains. Targeted reduction of LPCAT3 expression in HEK293 cells had essentially an opposite effect, resulting in decreased abundance of saturated phospholipid species and more unsaturated species. Reduced LPCAT3 expression resulted in more apoptosis and distinctly fewer lamellipodia, suggesting a necessary role for lysophospholipid esterification in maintaining cellular function and structure.—Jain, S., X. Zhang, P. J. Khandelwal, A. J. Saunders, B. S. Cummings, and P. Oelkers. Characterization of human lysophospholipid acyltransferase 3.

Supplementary key words acyl-CoA • apoptosis • lamellipodia • mass spectrometry • membrane composition • substrate specificity

Abbreviations: AGPAT, 1-acyl-sn-glycerol-3-phosphate acyltransferase; ATYL, acyl-transferase-like; DGAT, diacylglycerol acyltransferase; ESI-MS, electrospray ionization–mass spectrometry; HEK, human embryonic kidney; LPCAT, lysophosphatidylcholine acyltransferase; LPLAT, lysophospholipid acyltransferase; MBOAT, membrane bound o-acyltransferase; MOI, multiplicity of infection; MTT, 3-(4, dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide; NS, non-silencing; PA, phosphatidic acid; PAF, platelet activating factor; PC, phosphatidylcholine; ePC, 1-alkyl 2-acyl phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PI, propidium iodine; PL, glycerophospholipid; PS, phosphatidylserine

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