Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase
Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase
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DOI：
10.1074/jbc.m413473200
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发表时间：
2005-03-25
影响因子：
4.8
通讯作者：
Ueda, N
中科院分区：
文献类型：
--
作者：
Tsuboi, K;Sun, YX;Ueda, N
生物活性N-酰基乙醇胺，包括花生四烯酸酰胺（内源性大麻素）和N-棕榈酰乙醇胺（抗炎和神经保护物质），通过脂肪酸酰胺水解酶水解为脂肪酸和乙醇胺。此外，我们发现另一种酰胺水解酶仅在酸性pH下催化相同的反应，并且我们从大鼠肺中纯化了它（上田，N.，Yamanaka，K.，和Yamamoto，S.（2001）J.Biol.Chem.276，35552 - 35557）。在这里，我们报告的互补DNA克隆和功能表达的酶被称为“N-酰基乙醇胺水解酸性酰胺酶（NAAA）”从人类，大鼠和小鼠。推测的一级结构表明，NAAA与脂肪酸酰胺水解酶没有同源性，但属于胆酰甘氨酸水解酶家族。人NAAA基本上是相同的基因产物，已注意到类似于酸性神经酰胺酶，但缺乏神经酰胺水解活性。在HEK293细胞中过表达的重组人NAAA水解各种N-酰基乙醇胺，其中N-棕榈酰乙醇胺是最具反应性的底物。最有趣的是，用NAAA也检测到非常低的神经酰胺水解活性，并且用酸性神经酰胺酶观察到N-月桂酰乙醇胺水解活性。通过衣霉素和糖苷内切酶的测定，发现NAAA是一种糖蛋白。此外，该酶在pH 4.5下被蛋白水解加工成较短的形式，但在pH 7.4下不被蛋白水解加工成较短的形式。绿色荧光蛋白-NAAA融合蛋白的表达分析显示在HEK293细胞中呈溶酶体样分布。mRNA在大鼠的器官分布显示其广泛分布，在肺中表达最高。这些结果表明，NAAA是一种新的N-酰基乙醇胺水解酶，显示出与酸性神经酰胺酶的结构和功能相似性。
Bioactive N-acylethanolamines, including anandamide (an endocannabinoid) and N-palmitoylethanolamine (an anti-inflammatory and neuroprotective substance), are hydrolyzed to fatty acids and ethanolamine by fatty acid amide hydrolase. Moreover, we found another amidohydrolase catalyzing the same reaction only at acidic pH, and we purified it from rat lung (Ueda, N., Yamanaka, K., and Yamamoto, S. (2001) J. Biol. Chem. 276, 35552-35557). Here we report complementary DNA cloning and functional expression of the enzyme termed "N-acylethanolamine-hydrolyzing acid amidase (NAAA)" from human, rat, and mouse. The deduced primary structures revealed that NAAA had no homology to fatty acid amide hydrolase but belonged to the choloylglycine hydrolase family. Human NAAA was essentially identical to a gene product that had been noted to resemble acid ceramidase but lacked ceramide hydrolyzing activity. The recombinant human NAAA overexpressed in HEK293 cells hydrolyzed various N-acylethanolamines with N-palmitoylethanolamine as the most reactive substrate. Most interestingly, a very low ceramide hydrolyzing activity was also detected with NAAA, and N-lauroylethanolamine hydrolyzing activity was observed with acid ceramidase. By the use of tunicamycin and endoglycosidase, NAAA was found to be a glycoprotein. Furthermore, the enzyme was proteolytically processed to a shorter form at pH 4.5 but not at pH 7.4. Expression analysis of a green fluorescent protein-NAAA fusion protein showed a lysosome-like distribution in HEK293 cells. The organ distribution of the messenger RNA in rats revealed its wide distribution with the highest expression in lung. These results demonstrated that NAAA is a novel N-acylethanolamine-hydrolyzing enzyme that shows structural and functional similarity to acid ceramidase.