title: N-Glycosylation during translation is essential for human arylacetamide deacetylase enzyme activity.
abstract: Human arylacetamide deacetylase (AADAC) can hydrolyze clinical drugs such as flutamide, phenacetin, and rifamycins. AADAC is a glycoprotein, but the role of glycosylation remains unclear. In the present study, we investigated the effect of glycosylation on AADAC enzyme activity. Immunoblot analysis of mutant AADACs that contained an asparagine (N, Asn) to glutamine (Q, Gln) substitution at either residue 78 or 282 (N78Q or N282Q) showed a different migration compared with the wild-type protein. A mutant AADAC that contained N to Q substitutions at both residue 78 and 282 (N78Q/N282Q) showed a similar migration to AADAC in human liver microsomes (HLM) treated with endoglycosidase H (Endo H), which produces deglycosylated proteins. This result indicated that AADAC was glycosylated at both N78 and N282. Mutant types of AADAC with the N282Q and the N78Q/N282Q substitutions showed dramatically lower phenacetin hydrolase activity than did the wild-type protein. The treatment of wild-type AADAC-expressing HuH-7 cells with tunicamycin, which produces unglycosylated protein, decreased AADAC enzyme activity. However, the treatment of the HLM with Endo H caused no decrease of AADAC activity. Thus, the oligosaccharide chain, per se, was not important for AADAC activity in the mature form. The mutant types of AADAC containing the N282Q and the N78Q/N282Q substitutions were not detected by immunoblotting analysis after non-reducing SDS-PAGE, suggesting that the glycosylation of AADAC at N282 was important for proper protein folding. Overall, this study found that the translational, but not post-translational, N-glycosylation of AADAC plays a crucial role in regulating AADAC enzyme activity.
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Kyotaka
authors.initials: K
authors.lastname: Muta
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan. Electronic address: tatsuki@p.kanazawa-u.ac.jp.
authors.forename: Tatsuki
authors.initials: T
authors.lastname: Fukami
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Nakajima
authors.affiliation: Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Yokoi
mesh_terms: D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation / Q000235:genetics; D006031:Glycosylation; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis / Q000235:genetics
chemical_list: C569302:AADAC protein, human; D002265:Carboxylic Ester Hydrolases
keywords: AADAC; Arylacetamide deacetylase; CES; DMSO; ER; Endo H; Enzyme activity; GPT; GlcN-Ac-1-phosphate transferase; HLM; N-Glycosylation; PNGase F; Phenacetin; Protein folding; SNP; arylacetamide deacetylase; carboxylesterase; dimethyl sulfoxide; endoglycosidase H; endoplasmic reticulum; human liver microsomes; peptide: N-glycosidase F; single nucleotide polymorphism; α-Glc; α-glucosidase