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