N-glycosylation is not essential for enzyme activity of 11β- hydroxysteroid dehydrogenase type 2

11β-hydroxysteroid dehydrogenase (11βHSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11β-HSD is expressed at high levels in the distal nephron and contributes to mineralocorticoid specificity in that region. The present studies determined whether N-glycosylation is necessary for the activity of NAD-dependent 11β-HSD (11β-HSD2). First, cultured human colonic epithelial cells (T₈₄ cells), which express native 11β-HSD2 activity, were grown in medium with and without tunicamycin, an inhibitor of N-glycosylation. Tunicamycin had no effect on the enzyme activity. Next, the only putative N-glycosylation site (Asn₃₉₄-Leu₃₉₅-Ser₃₉₆) of the cloned human kidney enzyme was eliminated by site-directed mutagenesis. Chinese hamster ovary (CHO) cells transfected with either the wild-type or the mutant cDNA construct showed no difference in the expressed enzyme activity, and Western blot analysis showed that the 11β-HSD2 protein was the same size in cells expressing either the wild-type or the N₃₉₄D mutant. Likewise, the molecular mass of the 11β-HSD2 protein in T₈₄ cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11β-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.