Discovery and characterization of Ca2+-independent phosphatidylethanolarnine N-acyltransferase generating the anandarnide precursor and its congeners
Discovery and characterization of Ca2+-independent phosphatidylethanolarnine N-acyltransferase generating the anandarnide precursor and its congeners
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DOI：
10.1074/jbc.m606369200
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发表时间：
2007-02-09
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;Okamoto, Yasuo;Ueda, Natsuo
N-酰基磷脂酰乙醇胺（NAPE）是生物活性N-酰基乙醇胺的前体，包括内源性大麻素anandamide。在动物组织中，NAPE是通过甘油磷脂的sn-1位的脂肪酰基链转移到磷脂酰乙醇胺（PE）的氨基上形成的，该反应被认为是N-酰基乙醇胺合成中的主要限速步骤。然而，Ca 2+依赖性，膜相关的N-酰基转移酶（NAT）负责这一反应尚未被克隆。在这项研究中，NAT的卵磷脂-视黄醇酰基转移酶（LRAT）的功能相似性的基础上，我们研究了一个可能的PE N-酰化活性在两个大鼠LRAT同源蛋白。在COS-7细胞中过表达后，一种名为大鼠LRAT样蛋白（RLP）-1的蛋白质催化放射性酰基从磷脂酰胆碱（PC）转移到PE，导致放射性NAPE的形成。然而，RLP-1的活性主要是在胞浆中，而不是膜部分检测到的，几乎没有刺激的Ca 2+。此外，RLP-1对作为酰基供体的PC的sn-1和sn-2位置没有显示出选择性，因此可以从2-花生四烯酸酰基-PC和PE生成N-花生四烯酸酰基-PE（花生四烯酸酰胺前体）。与此相反，在相同的测定条件下，部分纯化的NAT从大鼠脑是高度钙依赖性的，膜相关的，并具体的SN-1-酰基的PC。RLP-1 mRNA主要表达于大鼠睾丸组织中，睾丸胞液具有RLP-1样活性。这些结果表明，RLP-1可以作为PE N-酰基转移酶发挥作用，与已知的Ca 2+依赖性NAT在催化上是可区分的。
N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-acyltransferase (NAT) responsible for this reaction has not yet been cloned. In this study, on the basis of the functional similarity of NAT to lecithin-retinol acyltransferase (LRAT), we examined a possible PE N-acylation activity in two rat LRAT homologous proteins. Upon overexpression in COS-7 cells, one protein, named rat LRAT-like protein (RLP)-1, catalyzed transfer of a radioactive acyl group from phosphatidylicholine (PC) to PE, resulting in the formation of radioactive NAPE. However, the RLP-1 activity was detected mainly in the cytosolic rather than membrane fraction and was little stimulated by Ca2+. Moreover, RLP-1 did not show selectivity with respect to the sn-1 and sn-2 positions of PC as an acyl donor and therefore could generate N-arachidonoyl-PE (anandamide precursor) from 2-arachidonoyl-PC and PE. In contrast, under the same assay conditions, partially purified NAT from rat brain was highly Ca2+-dependent, membrane-associated, and specific for the sn-1-acyl group of PC. RLP-1 mRNA was expressed predominantly in testis among various rat tissues, and the testis cytosol exhibited an RLP-1-like activity. These results reveal that RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca2+-dependent NAT.