Searchable abstracts of presentations at key conferences in endocrinology
Endocrine Abstracts (2003) 5 P219

BES2003 Poster Presentations Steroids (39 abstracts)

Mutagenesis studies on recombinant human 11beta-hydroxysteroid dehydrogenase type 1

EA Walker 1 , SV Hughes 1 , JP Ride 2 , M Hewison 1 & PM Stewart 1

1Division of Medical Sciences, University of Birmingham, Birmingham, UK; 2Department of Biosciences, University of Birmingham, Birmingham, UK.

11beta-hydroxysteroid dehydrogenase type 1 (11B-HSD1) catalyses the inter-conversion of cortisone and cortisol, and has been implicated in the pathogenesis of a number of disorders including insulin resistance and obesity. The enzyme is a glycosylated membrane-bound protein, located in the lumen of the endoplasmic reticulum (ER) and has proved difficult to purify in an active state. Previously, we reported the successful expression and purification of human 11B-HSD1 from E. coli and demonstrated that glycosylation was not required for full enzymatic activity. However, glycosylation may improve protein stability and folding within the ER. Multiple sequence alignments of 11B-HSD1 from mammalian species indicates that 2 of the 3 putative N-glycosylation sites identified in the human sequence are absent in the guinea-pig. Additionally, modelling studies suggest that the amino-acid residues present in the guinea pig sequence at these positions may improve protein stability. Since recent reports by others have suggested that 11B-HSD1 from guinea-pig is more amenable to purification than the human enzyme, we investigated mutating the glycosylation sites within the human 11B-HSD1 to exactly match those present at the same region of the guinea-pig.
Three mutant variants which removed glycosylation signals were constructed and used in our bacterial expression system: NTS123YNR which involved replacing all 3 amino-acid residues at position 123,124 and 125; N162Q, which only involved a single amino-acid change; and the double mutation, NTS123YNR-N162Q. Each was tested for expression levels of recombinant protein by Western blot analysis of bacterial lysates and also for 11B-HSD1 activity. Both reductase and dehydrogenase activity was dramatically decreased by all 3 mutations. N162Q resulted in an ~8-fold reduction in activity compared to wild-type whilst the other 2 mutations completely abolished activity. In addition, expression levels were not improved.
In summary, although glycosylation itself is not required for 11B-HSD1 activity, the residues at these positions are critical for the correct functioning of the human enzyme, and simple replacement by guinea-pig equivalents improves neither protein folding nor activity.

Volume 5

22nd Joint Meeting of the British Endocrine Societies

British Endocrine Societies 

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