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J. Biol. Chem., Vol. 280, Issue 29, 26669-26679, July 22, 2005

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The Highly Selective Production of 2-Arachidonoyl Lysophosphatidylcholine Catalyzed by Purified Calcium-independent Phospholipase A₂

IDENTIFICATION OF A NOVEL ENZYMATIC MEDIATOR FOR THE GENERATION OF A KEY BRANCH POINT INTERMEDIATE IN EICOSANOID SIGNALING^*

Wei Yan, Christopher M. Jenkins, Xianlin Han, David J. Mancuso, Harold F. Sims, Kui Yang, and Richard W. Gross¶||

From the Division of Bioorganic Chemistry and Molecular Pharmacology, Departments of Internal Medicine, Chemistry, and ^¶Molecular Biology & Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A₂ (iPLA₂) isoform in Sf9 cells, purification of the N-terminal His-tagged enzyme by affinity chromatography, and the identification of its remarkable substrate selectivity that results in the highly selective generation of 2-arachidonoyl lysophosphatidylcholine. Mass spectrometric analyses demonstrated that purified iPLA₂ hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA₂ effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA₂ with 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine resulted in the rapid release of palmitic acid and the selective accumulation of 2-arachidonoyl lysophosphatidylcholine (LPC), which was not metabolized further by iPLA₂. The putative regiospecificity of the 2-arachidonoyl LPC product was authenticated by its diagnostic fragmentation pattern during tandem mass spectrometric analysis. To identify the physiological relevance of iPLA₂-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA₂ and similarly identified the selective accumulation of 2-arachidonoyl LPC. Furthermore, tandem mass spectrometric analysis demonstrated that 2-arachidonoyl LPC is a natural product in human myocardium, a tissue in which iPLA₂ expression is robust. Because 2-arachidonoyl LPC represents a key branch point intermediate that can potentially lead to a variety of bioactive molecules in eicosanoid signaling (e.g. arachidonic acid, 2-arachidonoylglycerol), these results have uncovered a novel eicosanoid selective pathway through iPLA₂-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.

Received for publication, March 2, 2005 , and in revised form, May 5, 2005.

^* This work was supported by National Institutes of Health Grants 5P01HL05728-08 and 5R01HL41250-12. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

S The on-line version of this article (available at http://www.jbc.org) contains additional text and Figs. S1 and S2.

^|| To whom correspondence should be addressed: Division of Bioorganic Chemistry and Molecular Pharmacology, Washingtion University School of Medicine, 660 South Euclid Ave., Campus Box 8020, St. Louis, MO 63110. Tel.: 314-362-2690; Fax: 314-362-1402; E-mail: rgross{at}wustl.edu.

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