Human 11 beta-hydroxysteroid dehydrogenase 1/carbonyl reductase: additional domains for membrane attachment?

11 beta -Hydroxysteroid dehydrogenase type 1 (11 beta -HSD 1) is a membrane integrated glycoprotein, which physiologically performs the interconversio n of active and inactive glucocorticoid hormones and which also participate s in xenobiotic carbonyl compound detoxification. Since 11 beta -HSD 1 is f ixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon exp ression experiments in Escherichia coil, 11 beta -HSD 1 turns out to be har dly soluble without detergents. This study describes attempts to increase t he solubility of 11 beta -HSD I via mutagenesis experiments by generating s everal truncated forms expressed in E. coli and the yeast Pichia pastoris. Furthermore, we investigated if the codon for methionine 31 in human 11 bet a -HSD 1 could serve as an alternative start codon, thereby leading to a so luble form of the enzyme, which lacks the membrane spanning segment. Our re sults show that deletion of the hydrophobic membrane spanning domain did no t alter the solubility of the enzyme. In contrast, the enzyme remained boun d to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed i n E. call nor with that expressed in P. pastoris. Hydrophobicity plots prov ed the hydrophobic nature of 11 beta -HSD 1 and indicated the existence of additional membrane attachment sites within its primary structure. (C) 2001 Elsevier Science Ireland Ltd. All rights reserved.