Molecular Characterization of N-Acylethanolamine-hydrolyzing Acid Amidase, a Novel Member of the Choloylglycine Hydrolase Family with Structural and Functional Similarity to Acid Ceramidase

Abstract

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.

Endocannabinoid Signaling in Autism

“…However, the degradation of AEA to AA and ethanolamine is mainly due to 2 fatty acid amide hydrolases (FAAH and FAAH-2) [9,10]. When FAAH and FAAH-2 are inhibited, N-acylethanolaminehydrolyzing acid amidase cleaves AEA in an alternate route [11,12]. The main enzymes responsible for AEA metabolism are reported in Fig.…”

PUFA-derived endocannabinoids: an overview

“…Other enzymes involved in anandamide hydrolysis are N-acylethanolamine acid amidase (71) and FAAH-2 (70) , this latter being an isozyme of FAAH-1 with about 20% sequence identity at the amino acid level, mainly expressed in human subjects, but not in rodents (70) . N-acylethanolamine acid amidase is an N-glycosylated protein, localised in the lysosomes or the Golgi apparatus with an optimal pH of 4·5-5 (71)(72)(73)(74) . FAAH-2 is more effective at metabolizing oleamide than anandamide or other N-acyl-ethanolamines.…”

The synthesis and biological evaluation of para-substituted phenolic N-alkyl carbamates as endocannabinoid hydrolyzing enzyme inhibitors

“…affecting the inhibition potencies toward both enzymes; with short N-ethyl group (9) the IC 50 values against FAAH was 15 mM, whereas with longer N-n-hexyl (11) and N-dodecyl (12) Replacement of the open chain N-alkyl groups with bulky N-tert-butyl group (13) reduced the inhibitory potencies toward FAAH and MGL (IC 50 s; 7.0 mM and >100 mM, respectively). This is probably due to steric hindrance near the carbamate group, thus reducing crucial interactions within the active sites of the enzymes.…”

“…The uptake assay using RBL2H3 basophilic leukaemia cells was undertaken as described previously [75] using tritium-labelled arachidonoylethanolamide [arachidonoyl 5,6,8,9,11,12,14, H] (American Radiolabeled Chemicals Inc., St Louis, MO, USA). The cells were preincubated with compound 26 and/or URB597 for 10 min at 37 C prior to addition of [ 3 H]AEA (assay concentration 100 nM) and incubation for a further 10 min at 37 C. In these cells, the uptake of AEA is driven by its subsequent FAAH-catalysed hydrolysis to arachidonic acid [76], and the difference between the observed activity in the absence and presence of an FAAH inhibitor like URB597 represents the component of uptake due to FAAH.…”

“…AEA is also hydrolyzed by other enzymes such as FAAH-2, a second FAAH isoform [10], and N-acylethanolaminehydrolyzing acid amidase (NAAA) [11]. FAAH has a broad substrate selectivity, and it has been well characterized both pharmacologically and biochemically.…”