title: The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2gamma: identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling.
abstract: Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2gamma (iPLA2gamma) 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 iPLA2gamma hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2gamma effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2gamma 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 iPLA2gamma. 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 iPLA2gamma-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2gamma 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 iPLA2gamma 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 iPLA2gamma-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.
authors.affiliation: Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
authors.forename: Wei
authors.initials: W
authors.lastname: Yan
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Jenkins
authors.forename: Xianlin
authors.initials: X
authors.lastname: Han
authors.forename: David J
authors.initials: DJ
authors.lastname: Mancuso
authors.forename: Harold F
authors.initials: HF
authors.lastname: Sims
authors.forename: Kui
authors.initials: K
authors.lastname: Yang
authors.forename: Richard W
authors.initials: RW
authors.lastname: Gross
mesh_terms: D000818:Animals; D002384:Catalysis; D003001:Cloning, Molecular / Q000379:methods*; D015777:Eicosanoids / Q000096:biosynthesis; D054513:Group IV Phospholipases A2; D006801:Humans; D008244:Lysophosphatidylcholines / Q000096:biosynthesis*; D013058:Mass Spectrometry; D009206:Myocardium / Q000201:enzymology; D010741:Phospholipases A / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D015290:Second Messenger Systems; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: C503491:2-arachidonoyllysophosphatidylcholine; D015777:Eicosanoids; D008244:Lysophosphatidylcholines; D010741:Phospholipases A; D054513:Group IV Phospholipases A2