The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2γ

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, Richard W. Gross

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)26669-26679
Number of pages11
JournalJournal of Biological Chemistry
Volume280
Issue number29
DOIs
StatePublished - Jul 22 2005
Externally publishedYes

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Calcium-Independent Phospholipase A2
Lysophosphatidylcholines
Eicosanoids
Arachidonic Acid
Affinity chromatography
Sf9 Cells
Peroxisomes
Palmitic Acid
Phospholipases A2
Second Messenger Systems
Substrates
Biological Products
Affinity Chromatography
Purification
Rats
Phospholipids
Myocardium
Protein Isoforms
Tissue
Membranes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The highly selective production of 2-arachidonoyl lysophosphatidylcholine catalyzed by purified calcium-independent phospholipase A2γ : Identification of a novel enzymatic mediator for the generation of a key branch point intermediate in eicosanoid signaling. / Yan, Wei; Jenkins, Christopher M.; Han, Xianlin; Mancuso, David J.; Sims, Harold F.; Yang, Kui; Gross, Richard W.

In: Journal of Biological Chemistry, Vol. 280, No. 29, 22.07.2005, p. 26669-26679.

Research output: Contribution to journalArticle

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abstract = "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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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

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AU - Sims, Harold F.

AU - Yang, Kui

AU - Gross, Richard W.

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