cDNA cloning and characterization of human and mouse Ca2+-independent phosphatidylethanolamine N-acyltransferases
cDNA cloning and characterization of human and mouse Ca2+-independent phosphatidylethanolamine N-acyltransferases
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DOI：
10.1016/j.bbalip.2008.09.006
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发表时间：
2009-01-01
影响因子：
4.8
通讯作者：
Ueda, Natsuo
中科院分区：
文献类型：
--
作者：
Jin, Xing-Hua;Uyama, Toru;Ueda, Natsuo
通过磷脂酰乙醇胺 (PE) 的 N-酰化形成 N-酰基磷脂酰乙醇胺是生物活性 N-酰基乙醇胺（包括内源性大麻素 anandamide 和抗炎物质 N-棕榈酰乙醇胺）生物合成途径的第一步。我们最近克隆了一种能够催化该反应的大鼠酶，并将该酶称为Ca2+非依赖性N-酰基转移酶（iNAT）。在这里，我们报告了人类和小鼠 iNAT 的 cDNA 克隆和表征。我们从人和小鼠睾丸中克隆了 iNAT 同源 cDNA，并在 COS-7 细胞中过表达它们。纯化的重组蛋白从磷脂酰胆碱的sn-1和sn-2位上提取酰基，并催化PE的N-酰化以及磷脂酶A(1)/A(2)样水解。 iNAT 活性主要在可溶性组分而非颗粒组分中检测到，并且 Ca2+ 仅略微增加 iNAT 活性。这些结果证明人类和小鼠同源物具有 iNAT 的功能。至于iNAT的器官分布，人类睾丸和胰腺以及小鼠睾丸的表达水平迄今为止最高，表明其在特定器官中的生理重要性。此外，诱变研究表明大鼠 iNAT 的 His-154 和 Cys-241 在催化中发挥关键作用，并且 N 末端结构域在膜关联或蛋白质-蛋白质相互作用中可能发挥作用。 (C) 2008 Elsevier B.V. 保留所有权利。
The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca2+-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A(1)/A(2)-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca2+. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein-protein interaction. (C) 2008 Elsevier B.V. All rights reserved.