Title : Mass spectrometric characterization of human
N-acylethanolamine-hydrolyzing acid amidase
Abstract :
- N-Acylethanolamine-hydrolyzing acid amidase ( NAAA ) is a lysosomal enzyme that primarily degrades palmitoylethanolamine (PEA), a lipid amide that inhibits inflammatory responses
- We developed a HEK293 cell line stably expressing the NAAA pro-enzyme (zymogen) and a single step chromatographic purification of the protein from the media
- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF MS analysis of the zymogen (47.7 kDa) treated with peptide-N-glycosidase F ( PNGase F) identified 4 glycosylation sites , and acid cleavage of the zymogen into α- and β- subunits (14.6 and 33.3 kDa) activated the enzyme
- Size exclusion chromatography estimated the mass of the active enzyme as 45 ± 3 kDa, suggesting formation of an α/β heterodimer
- MALDI-TOF MS fingerprinting covered more than 80% of the amino acid sequence , including the N-terminal peptides , and evidence for the lack of a disulfide bond between subunits
- The significance of the cysteine residues was established by their selective alkylation resulting in almost complete loss of activity
- The purified enzyme was kinetically characterized with PEA and a novel fluorogenic substrate, N-(4-methyl coumarin) palmitamide (PAMCA)
- The production of sufficient quantities of NAAA and a high throughput assay could be useful in discovering novel inhibitors and determining the structure and function of this enzyme