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.
authors: Center for Drug Discovery, Northeastern University , Boston, Massachusetts 02115, United States.
authors: Jay M
authors: JM
authors: West
authors: Nikolai
authors: N
authors: Zvonok
authors: Kyle M
authors: KM
authors: Whitten
authors: Jodianne T
authors: JT
authors: Wood
authors: Alexandros
authors: A
authors: Makriyannis
mesh_terms: D000577:Amides; D000581:Amidohydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D063388:Endocannabinoids; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004983:Ethanolamines; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010169:Palmitic Acids; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D000577:Amides; D063388:Endocannabinoids; D004792:Enzyme Precursors; D004983:Ethanolamines; D010169:Palmitic Acids; D021122:Protein Subunits; C005958:palmidrol; D000581:Amidohydrolases; C523108:NAAA protein, human; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase