N-glycosylation is not essential for enzyme activity of 11beta-hydroxysteroid dehydrogenase type 2.
11Beta-hydroxysteroid dehydrogenase (11beta-HSD) catalyzes the oxidation of cortisol and corticosterone to cortisone and 11-dehydrocorticosterone, respectively. NAD-dependent 11beta-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 11beta-HSD (11beta-HSD2). First, cultured human colonic epithelial cells (T84 cells), which express native 11beta-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 11beta-HSD2 protein was the same size in cells expressing either the wild-type or the N394D mutant. Likewise, the molecular mass of the 11beta-HSD2 protein in T84 cells was not altered by treatment with peptide-N-glycosidase F or tunicamycin. We conclude that human 11beta-HSD2 is not a N-glycoprotein and N-glycosylation is not essential for the expression of enzyme activity.
Kyossev
Z N
ZN
Division of Nephrology, University of Arkansas for Medical Sciences, and The John L. McClellan Veterans Affairs Hospital, Little Rock 72205, USA.
Reeves
W B
WB
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000581:Amidohydrolases; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation / Q000187:drug effects; D006801:Humans; D006913:Hydroxysteroid Dehydrogenases / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014162:Transfection; D014407:Tumor Cells, Cultured; D014415:Tunicamycin / Q000494:pharmacology
D007527:Isoenzymes; D014415:Tunicamycin; D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase