Human 11β-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 interconversion of active and inactive glucocorticoid hormones and which also participates in xenobiotic carbonyl compound detoxification. Since 11 beta -HSD 1 is fixed to the endoplasmic reticulum (ER) with a N-terminal membrane spanning domain, the enzyme is very difficult to purify in an active state. Upon expression experiments in Escherichia coli, 11 beta -HSD 1 turns out to be hardly soluble without detergents. This study describes attempts to increase the solubility of 11 beta -HSD I via mutagenesis experiments by generating several truncated forms expressed in E. coli and the yeast Pichia pastoris. Furthermore, we investigated if the codon for methionine 31 in human 11 beta -HSD 1 could serve as an alternative start codon, thereby leading to a soluble form of the enzyme, which lacks the membrane spanning segment. Our results show that deletion of the hydrophobic membrane spanning domain did not alter the solubility of the enzyme. In contrast, the enzyme remained bound to the ER membrane even without the N-terminal membrane anchor. However, activity could not be found, neither with the truncated protein expressed in E. call nor with that expressed in P. pastoris. Hydrophobicity plots proved 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.