Mutations in putative glycosylation sites of rat 11 beta-hydroxysteroid dehydrogenase affect enzymatic activity.
11 beta-hydroxysteroid dehydrogenase (11-HSD) catalyzes the interconversion of corticosterone and 11-dehydrocorticosterone in rats, or cortisol and cortisone in humans. The 'liver' or 'Type I' isozyme is a widely distributed glycoprotein that utilizes NADP+ as a co-factor. To study the role of glycosylation in maintaining enzymatic activity, we introduced mutations into the two potential N-linked glycosylation sites (asparagine-X-serine, residues 158-160 and 203-205) predicted from the rat cDNA sequence. Mutagenesis was performed by a PCR based technique, and wild-type (WT) and mutant cDNAs were expressed in Chinese hamster ovary cells after cloning into the pCMV4 vector. At each putative glycosylation site, asparagine (N) was changed to glutamine (Q) or aspartic acid (D), and serine (S) changed to alanine (A). All three modifications of the first site (N158Q, N158D, S160A) had minimal (75-100% of WT) effects on dehydrogenase activity and caused a mild (50-75% of WT) decrease in reductase activity. In contrast, mutations at the second site had marked effects, with N203Q and N203D completely abolishing both dehydrogenase and reductase activities and S205A decreasing both activities to about 20% of WT. The double mutation of S160A and S205A also abolished all activity, even though the enzyme carrying each mutation alone was, at least, partially active. The results suggest that N203 (which is highly but not completely conserved in short chain dehydrogenase enzymes) is essential for activity of 11-HSD. N-linked glycosylation may be necessary for full activity or stability of the enzyme.
Agarwal
A K
AK
Division of Pediatric Endocrinology, University of Texas Southwestern Medical Center, Dallas 75235-9063, USA.
Mune
T
T
Monder
C
C
White
P C
PC
D042842:11-beta-Hydroxysteroid Dehydrogenases; D000581:Amidohydrolases; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006031:Glycosylation; D006913:Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D051381:Rats; D016415:Sequence Alignment; D014162:Transfection
D006913:Hydroxysteroid Dehydrogenases; D042842:11-beta-Hydroxysteroid Dehydrogenases; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase