Truncation of the N- and C-terminal regions of the human 11 beta-hydroxysteroid dehydrogenase type 2 enzyme and effects on solubility and bidirectional enzyme activity

The 11 beta-hydroxysteroid dehydrogenase type II enzyme (11 beta HSD2) endows specificity on the mineralocorticoid receptor by metabolising glucocorticoids. Sequence comparisons with other microsomal proteins showed the strongly preferred topology of a lumenal pentapeptide followed by three transmembrane helices with residues beyond Ala(73) on the cytoplasmic side of the membrane, suggesting that 11 beta HSD2 is anchored to the endoplasmic reticulum by the N-terminal region. However, deletion of the N-terminus (11 beta HSD2 Delta N) and expression of the construct in mammalian cells showed that the enzyme remained bound to the microsomal fraction, indicating that other regions are also involved in membrane anchoring. Crosslinking studies and nonreducing SDS-PAGE demonstrated that 11 beta HSD2 is a non-covalently linked dimer. Deletion of the non-conserved C-terminal region (11 beta HSD2 Delta C) resulted in an enzyme with a K-m of 215 nM for cortisol in whole cell assays, while 11 beta HSD2 and 11 beta HSD2 Delta N displayed a K-m of 62 and 74 nM, respectively. In homogenates 11 beta HSD2 and 11 beta HSD2 Delta C displayed maximal activity at 140 mM NaCl or KCl, but showed a marked decrease in enzyme activity with increasing salt. 11 beta HSD2 was more stable than 11 beta HSD2 Delta C in the presence of NaSCN suggesting that the C-terminal region plays a role in enzyme stability. There was no detectable activity in homogenates containing 11 beta HSD2 Delta N, while 11 beta HSD2 Delta C and 11 beta HSD2 displayed a K-m of 135 and 46 nM, respectively. Although 11 beta HSD2 is conventionally considered a unidirectional dehydrogenase all constructs converted 11-dehydrodexamethasone to dexamethasone in whole cell assays, providing an explanation for the potency of the synthetic glucocorticoid in the face of a powerful inactivator of natural glucocorticoids. (C) 1997 Elsevier Science Ireland Ltd.