Title : Proteolytic activation and glycosylation of
N-acylethanolamine-hydrolyzing acid amidase , a lysosomal
enzyme involved in the endocannabinoid metabolism
Abstract :
- N-acylethanolamine-hydrolyzing acid amidase ( NAAA ) is a lysosomal enzyme hydrolyzing bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine
- Previously, we suggested that NAAA is glycosylated and proteolytically cleaved
- Here, we investigated the mechanism and significance of the cleavage of human NAAA overexpressed in human embryonic kidney 293 cells
- Western blotting with anti- NAAA antibody revealed that most of NAAA in the cell homogenate was the cleaved 30-kDa form
- However, some of NAAA were released outside the cells and the extracellular enzyme was mostly the uncleaved 48-kDa form
- When incubated at pH 4 .5, the 48-kDa form was time-dependently converted to the 30-kDa form with concomitant increase in the N-palmitoylethanolamine-hydrolyzing activity
- The purified 48-kDa form was also cleaved and activated
- However, the cleavage did not proceed at pH 7.4 or in the presence of p-chloromercuribenzoic acid
- The mutant C126S was resistant to the cleavage and remained inactive
- These results suggested that this specific proteolysis is a self-catalyzed activation step
- We next determined N-glycosylation sites of human NAAA by site-directed mutagenesis addressed to asparagine residues in six potential N-glycosylation sites
- The results exhibited that Asn-37, Asn-107, Asn-309, and Asn-333 are actual N-glycosylation sites
- The glycosylation appeared to play an important role in stabilizing the enzyme protein