~ Pergamon ft. Steroid Biochem. Molec. Biol. Vol. 55, No. 5/6, pp. 457-464, 1995 Copyright © 1995 Elsevier Science Ltd 0960-0760(95)00194-8 Printed in Great Britain. All rights reserved 0960-0760/95 $9.50 + 0.00 The Human l lfl-Hydroxysteroid Dehydrogenase Type II Enzyme: Comparisons with Other Species and Localization to the Distal Nephron Z. Krozowski, 1. A. L. Albistonfl V. R. Obeyesekere, 1 R. K. Andrews 2 and R. E. Smith 1 1Laboratory of Molecular Hypertension and 2 Vascular Biology Laboratory, Baker Institute of Medical Research, Prahran 3181, Australia Effective glucocorticoid inactivation is currently thought to be an indispensable feature of mineralo- corticoid target cells. The enzyme 11/~-hydroxysteroid dehydrogenase (11fl-HSD) inactivates gluco- corticoids and prevents them from binding to the non-selective mineralocorticoid receptor. In the kidney it is the NAD dependent high affinity isoform (11fl-HSD2) which is thought to endow specificity on the receptor. The recent cloning of the human, sheep and rabbit 11fl-HSD2 enzymes permits a comparison of the enzyme from the three species. Human and rabbit enzymes are 87% identical and of similar length, while the human and sheep enzymes have only 75% identity. The last 12 residues in all three species were found to be highly divergent, but most of the ovine dishomology can be accounted for by the deletion of a single nucleotide toward the C-terminus of the protein resulting in a shift in reading frame generating a protein 27 residues longer than the human isoform. Numerous other deletions were also observed in this region of the sheep cDNA sequence. Further- more, the rabbit cDNA also displayed a large degree of dishomology with the human sequence a short distance downstream from the termination codon. Conserved overlapping cytoplasmic translocation signals; were observed in all three species, suggesting a topology whereby the enzyme is anchored into the endoplasmic reticulum by multiple hydrophobic regions in the N-terminus and the bulk of the 11fl-HSD2 peptide is sited in the cytoplasm. A polyclonal antibody generated against the C-terminus of human 11fl-HSD2 was used to localize the enzyme within the kidney. A high level of immunoreactivity was observed in distal tubules and collecting ducts, localizing the enzyme to the same part of the nephron as the mineralocorticoid receptor. Moderate levels of staining were also seen in vascular smooth muscle cells. These results support the notion that 11fl-HSD2 is an autocrine protector of the mineralocorticoid receptor and that it plays an important role in cardiovascular homeostatic mechanisms. J. Steroid Biochem. Molec. Biol., Vol. 55, No. 5/6, pp. 457-464, 1995 INTRODUCTION The study of the reguh,tion of active hormone concen- trations at the level of the target cell has recently become an important subject. In the kidney glucocorti- coids are inactivated [1,2] by the enzyme llft- hydroxysteroid dehydrogenase (llfl-HSD) allowing aldosterone to occupy the non-selective mineralocorti- Proceedings of the Workshop on the Molecular and Cell Biology of Hydroxysteroid Dehydrogenases, Hannover, Germany, 19-22 April 1995. *Correspondence to Z. Krozowski. coid receptor [3]. A low affinity isoform of the enzyme (llfl-HSD1) exists in the liver where it performs the reverse reaction, producing active glucocorticoid by converting cortisone to cortisol. In the distal tubule of the kidney, however, a high affinity unidirectional l lf-HSD isoform (llf-HSD2) converts glucocorti- colds to their inactive ll-keto metabolites [4]. Inhi- bition of 1lfl-HSD activity by licorice leads to sodium retention, hypokalemia and elevated blood pressure [5], symptoms similar to those observed in the congenital syndrome of apparent mineralocorticoid excess (AME) where patients also display low cortisone to cortisol 457