Molecular characterization of a novel lysosomal enzyme degrading the anti-inflammatory lipid mediator N-acylethanolamine

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N-Acylethanolamines (NAEs) represent a class of endogenous bioactive lipids generated from glycerophospholipids, and they include N-palmitoylethanolamine (an anti-inflammatory substance) and N-arachidonoylethanolamine (anandamide, an endogenous ligand of cannabinoid receptor). It is generally accepted that NAEs are hydrolyzed to free fatty acids and ethanolamine by the catalysis of fatty acid amide hydrolase (FAAH) acting principally at alkaline and neutral pH. Several years ago, our laboratory found another enzyme catalyzing the same reaction only at acidic pH. However, the molecular cloning and detailed analysis of this enzyme, termed “N-acylethanolamine-hydrolyzing acid amidase (NAAA)”, have not yet been performed. In the present study, we purified the enzyme from rat lung, and cloned its cDNA from human, mouse, and rat. The primary structures revealed that NAAA had no homology to FAAH, but revealed 33-35% amino acid identity to lysosomal acid ceramidase, which hydrolyzes ceramide to free fatty acids and sphingosine at acidic pH. Recombinant human NAAA hydrolyzed various NAEs, and exhibited the highest activity toward N-palmitoylethanolamine. In addition, a low ceramide-hydrolyzing activity was detected with NAAA. We also observed a lysosome-like distribution of NAAA-green fluorescence protein fusion protein, as expressed in HEK293 cells. The organ distribution of mRNA and enzyme activity in rats revealed its wide distribution with the highest level in lung. Interestingly, macrophages expressed NAAA abundantly, suggesting a role of this enzyme in inflammation. By using an NAAA-selective inhibitor which we developed, we showed that NAAA and FAAH cooperatively degraded NAEs in macrophages. Taken together, our results indicated that NAAA is a novel lysosomal enzyme having structural and functional similarity to acid ceramidase, and functions as a second NAE hydrolase in mammalian tissues.

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