TY - JOUR
T1 - The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2γ
T2 - Identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling
AU - Yan, Wei
AU - Jenkins, Christopher M.
AU - Han, Xianlin
AU - Mancuso, David J.
AU - Sims, Harold F.
AU - Yang, Kui
AU - Gross, Richard W.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2005/7/22
Y1 - 2005/7/22
N2 - Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2γ (iPLA 2γ) 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 iPLA2γ hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2γ effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2γ 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 iPLA 2γ. 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 iPLA2γ-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2γ 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 iPLA2γ 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 iPLA2γ-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.
AB - Herein, we report the heterologous expression of the human peroxisomal 63-kDa calcium-independent phospholipase A2γ (iPLA 2γ) 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 iPLA2γ hydrolyzed saturated or monounsaturated aliphatic groups readily from either the sn-1 or sn-2 positions of phospholipids. In addition, purified iPLA2γ effectively liberated arachidonic acid from the sn-2 position of plasmenylcholine substrates. In contrast, incubation of iPLA2γ 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 iPLA 2γ. 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 iPLA2γ-mediated 2-arachidonoyl LPC production utilizing naturally occurring membranes, we incubated purified rat hepatic peroxisomes with iPLA2γ 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 iPLA2γ 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 iPLA2γ-mediated 2-arachidonoyl LPC production to amplify and diversify the repertoire of biologic lipid second messengers in response to cellular stimulation.
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U2 - 10.1074/jbc.M502358200
DO - 10.1074/jbc.M502358200
M3 - Article
C2 - 15908428
AN - SCOPUS:22844452820
VL - 280
SP - 26669
EP - 26679
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 29
ER -