Mutations in putative glycosylation sites of rat 11β-hydroxysteroid dehydrogenase affect enzymatic activity

Anil K. Agarwal, Tomoatsu Mune, Carl Monder, Perrin C. White

Research output: Contribution to journalArticle

44 Scopus citations

Abstract

11 β-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.

Original languageEnglish (US)
Pages (from-to)70-74
Number of pages5
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Volume1248
Issue number1
DOIs
StatePublished - Apr 5 1995

Keywords

  • 11β-Hydroxysteroid dehydrogenase
  • Alcohol dehydrogenase, short chain
  • Glycosylation
  • Site-directed mutagenesis

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology

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