Yan Wei, Jenkins Christopher M, Han Xianlin, Mancuso David J, Sims Harold F, Yang Kui, Gross Richard W
Division of Bioorganic Chemistry and Molecular Pharmacology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
J Biol Chem. 2005 Jul 22;280(29):26669-79. doi: 10.1074/jbc.M502358200. Epub 2005 May 20.
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.
在此，我们报道了人过氧化物酶体63 kDa钙非依赖性磷脂酶A2γ（iPLA2γ）同工型在Sf9细胞中的异源表达，通过亲和层析纯化N端带有His标签的酶，并鉴定了其显著的底物选择性，该选择性导致2-花生四烯酰溶血磷脂酰胆碱的高选择性生成。质谱分析表明，纯化的iPLA2γ可轻易地从磷脂的sn-1或sn-2位水解饱和或单不饱和脂肪族基团。此外，纯化的iPLA2γ能有效地从缩醛磷脂胆碱底物的sn-2位释放花生四烯酸。相比之下，将iPLA2γ与1-棕榈酰-2-花生四烯酰-sn-甘油-3-磷酸胆碱一起孵育，会导致棕榈酸迅速释放，并选择性积累2-花生四烯酰溶血磷脂酰胆碱（LPC），而iPLA2γ不会进一步代谢该产物。2-花生四烯酰LPC产物的推定区域特异性通过串联质谱分析中的诊断性裂解模式得到验证。为了确定利用天然膜的iPLA2γ介导的2-花生四烯酰LPC生成的生理相关性，我们将纯化的大鼠肝过氧化物酶体与iPLA2γ一起孵育，并同样鉴定出2-花生四烯酰LPC的选择性积累。此外，串联质谱分析表明，2-花生四烯酰LPC是人心肌中的天然产物，而iPLA2γ在该组织中大量表达。由于2-花生四烯酰LPC代表一个关键的分支点中间体，可能会导致类花生酸信号通路中产生多种生物活性分子（如花生四烯酸、2-花生四烯酰甘油），这些结果揭示了一条新的类花生酸选择性途径，即通过iPLA2γ介导的2-花生四烯酰LPC生成，以放大和多样化生物脂质第二信使的种类，从而响应细胞刺激。
