N-Glycosylation plays a role in protein folding of human UGT1A9.
UDP-glucuronosyltransferases (UGTs) catalyze the glucuronidation of a variety of xeno/endobiotics. UGTs are type I membrane proteins of the endoplasmic reticulum (ER) with a glycosylated luminal domain. In the present study, we investigated the role of N-glycosylation in the function of human UGT1A9. Mutation analysis at the potential N-glycosylation sites at residues 71, 292, and 344 (from asparagine to glutamine) revealed that all of them were glycosylated, but the extent of glycosylation and/or size of the glycan differed. In comparison with the wild-type, these mutants showed decreased enzyme activities in parallel with the extent of the band shift in Western blot analysis. To evaluate the role of glycosylation in the enzyme activity, we produced unglycosylated UGT1A9 by treating HEK293 cells transiently transfected with expression plasmid with tunicamycin. The unglycosylated UGT1A9 was almost inactive, which was not an indirect effect of ER stress. To the contrary, the deglycosylated UGT1A9, which was produced by the treatment with Endo H under the non-denaturing condition, showed the same enzyme kinetics as the control. These results suggest that the glycosylation that occurs during translation is important for the folding of UGT1A9. The thermal stability analysis of the mutated and deglycosylated UGT1A9 proteins supported the findings. In conclusion, we found that the N-glycosylation has an important role in the folding of UGT1A9.
Nakajima
Miki
M
Kanazawa University, Kakuma-machi, Japan. nmiki@kenroku.kanazawa-u.ac.jp
Koga
Toshihisa
T
Sakai
Haruko
H
Yamanaka
Hiroyuki
H
Fujiwara
Ryoichi
R
Yokoi
Tsuyoshi
T
D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007212:Indolizines / Q000494:pharmacology; D017510:Protein Folding; D014415:Tunicamycin / Q000494:pharmacology; D000090264:UDP-Glucuronosyltransferase 1A9; D054836:Indolizidines
D014453:Glucuronosyltransferase; D007212:Indolizines; D014415:Tunicamycin; D000090264:UDP-Glucuronosyltransferase 1A9; C000715591:UGT1A9 protein, human; C037806:castanospermine; D054836:Indolizidines