Guinea pig 11β-hydroxysteroid dehydrogenase type 1:: primary structure and catalytic properties

The 11 beta-hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) enzyme is responsible for the interconversion of glucocorticoids and their inactive metabolites, and thus modulates the intracellular level of bioactive glucocortisoids. The present study was designed to clone and characterize 11 beta-HSD1 in the guinea pig, a laboratory animal known for resistance to glucocorticoids. The cDNA encoding guinea Pie 11 beta-HSD1 was cloned by a modified 3'-RACE (rapid amplification of cDNA ends) protocol using the hepatic RNA as template. The cloned cDNA encodes a protein of 300 amino acids that shares 71 to 74% sequence identity with other known mammalian 11 beta-HSD1 plateins. Sequence comparison analysis revealed that the deduced guinea pig 11 beta-HSD1 was longer. by eight amino acids at the C terminus, than those of other mammals. Moreover, one of the two absolutely conserved consensus sites for N-glycosylation was absent. To examine the functional significance of these structural changes, we also characterized 11 beta-HSD1 activity in the hepatic microsomes. Although the guinea pig hepatic enzyme was NADP(H)-dependent and reversible, it displayed equal affinity fur cortisol and cortisone (apparent K-m fur both substrates was 3 mu M). This is in marked contrast to 11 beta-HSD1 in other rnnmmals whose affinity for cortisone is approximately 10 times higher than thar for cortisol (apparent K-m of 0.3 vs. 3.0 mu M). The apparent lower affinity of the guinea pig enzyme for cortisone would suggest that the intracellular bioformation of cortisol from circulating cortisone may he less efficient in this species. Northern blot analysis and RT-PCR revealed that the mRNA Tot 11 beta-HSD1 was widely expressed in the adult guinea pig but at low amounts. In conclusion, the present study has identified distinct features in the deduced primary structure and catalytic function of 11 beta-HSD1 in the guinea pig. Thus, the guinea pig provides a useful model in which the structural determinants of catalytic function of 11 beta-HSD1 may be: studied. (C) 2000 Elsevier Science Inc. All rights reserved.