Glyco

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

Output (sent_index, trigger, protein, sugar, site):

0. glycosylation, , N-acylethanolamine-hydrolyzing acid amidase, -, -
11. N-glycosylation, , -, -, sites
11. N-glycosylation, , NAAA, -, sites
12. N-glycosylation, , -, -, Asn-37, Asn-107, Asn-309, and Asn-333
12. N-glycosylation, , -, -, sites
2. glycosylated, , NAAA, -, -

Output(Part-Of) (sent_index, protein, site):

11. NAAA, sites

*Output_Site_Fusion* (sent_index, protein, sugar, site):

12. N-acylethanolamine-hydrolyzing acid amidase, -, Asn-37, Asn-107, Asn-309, and Asn-333