MUTATIONS IN PUTATIVE GLYCOSYLATION SITES OF RAT 11-BETA-HYDROXYSTEROID DEHYDROGENASE AFFECT ENZYMATIC-ACTIVITY

11 beta-hydroxysteroid dehydrogenase (11-HSD) catalyzes the interconve rsion of corticosterone and 11-dehydrocorticosterone in rats, or corti sol and cortisone in humans. The 'liver' or 'Type I' isozyme is a wide ly distributed glycoprotein that utilizes NADP(+) as a co-factor. To s tudy the role of glycosylation in maintaining enzymatic activity, we i ntroduced mutations into the two potential N-linked glycosylation site s (asparagine-X-serine, residues 158-160 and 203-205) predicted from t he rat cDNA sequence. Mutagenesis was performed by a PCR based techniq ue, and wild-type (WT) and mutant cDNAs were expressed in Chinese hams ter ovary cells after cloning into the pCMV4 vector. At each putative glycosylation site, asparagine (N) was changed to glutamine (Q) or asp artic acid (D), and serine (S) changed to alanine (A). All three modif ications 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 seco nd 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 mut ation alone was, at least, partially active. The results suggest that N203 (which is highly but not completely conserved in short chain dehy drogenase enzymes) is essential for activity of 11-HSD. N-linked glyco sylation may be necessary for full activity or stability of the enzyme .