Expression of 11β-Hydroxysteroid Dehydrogenase Using Recombinant Vaccinia Virus

Abstract

Ligand specificity of the type I steroid receptor is apparently conferred by the activity of 11 beta-hydroxysteroid dehydrogenase. To determine the kinetic properties of this enzyme, rat liver cDNA was expressed in cultured cells using recombinant vaccinia virus. Although this enzyme catalyzes only dehydrogenation when purified from rat liver, the recombinant enzyme obtained from cell lysates catalyzed both 11 beta-dehydrogenation of corticosterone to 11-dehydrocorticosterone and the reverse 11-oxoreduction reaction. At pH 8.5, the first order rate constant Kcat/Km for dehydrogenase activity exceeded that for reductase (63 vs. 38 min-1 x 10(-4], whereas the rate constants for the two reactions were nearly equal (48 vs. 47 min-1 x 10(-4] at pH 7.0. These results are consistent with the previously determined pH optima for these activities in liver microsomes. Removal (with glucose-6-phosphate dehydrogenase) of NADP+ produced by the reductase reaction significantly increased reductase activity. Glycyrrhetinic acid, a known inhibitor of the dehydrogenase reaction, also inhibited the reductase reaction at slightly higher concentrations (50% inhibitory concentration, less than 5 nM for dehydrogenase, 10-20 nM for reductase). Partial inhibition of glycosylation with A1-tunicamycin decreased dehydrogenase activity 50% without affecting reductase activity. The data demonstrate that a single polypeptide catalyzes both dehydrogenation and reduction, although the presence of additional enzyme forms catalyzing one or the other activity has not been ruled out.

Functional Expression, Characterization, and Purification of the Catalytic Domain of Human 11-β-Hydroxysteroid Dehydrogenase Type 1

“…The catalytic domain is glycosylated (13)(14)(15), which is in agreement with a lumenal orientation. Experiments to resolve the importance of glycosylation have also yielded varying results.…”

“…Enzymatic deglycosylation of rabbit (9) and human (13) 11␤-HSD1 has indicated that glycosylation is not important for enzyme activity. However, partial inhibition of glycosylation of the rat enzyme by tunicamycin decreased dehydrogenase activity but not reductase activity (14), and mutation of the rat (15) and human (13) sequences at putative N-glycosylation sites resulted in reduced or abolished activity.…”

The N-terminal Anchor Sequences of 11β-Hydroxysteroid Dehydrogenases Determine Their Orientation in the Endoplasmic Reticulum Membrane

“…These results suggest that glycosylation does not significantly alter the activity of 11␤-HSD1, which is supported by the report of Ozols (9) that endo-␤-N-acetylglucosaminidase H treatment of purified 11␤-HSD1 did not significantly affect activity. On the other hand, other investigators reported decreased 11␤-HSD1 activity following incubation with tunicamycin, an inhibitor of glycosylation, or of proteins containing mutated glycosylation sites (4,64).…”

“…The isoform 11␤-HSD1 is expressed in a wide array of tissues, with highest levels in the liver, from where it was purified originally (2). It catalyzes both the oxidation and reduction of glucocorticoids but acts predominantly as an oxidoreductase, thereby increasing the concentration of active glucocorticoids (3)(4)(5)(6)(7)(8). Studies on the purified protein demonstrated glycosylation and existence of a disulfide bond, suggesting that the bulk of 11␤-HSD1 is oriented to the ER lumen (9).…”

Cloning and Primary Structure of Murine 11β‐Hydroxysteroid Dehydrogenase/Microsomal Carbonyl Reductase

“…Expression of a truncated form lacking the hydrophobic N-terminal part of 11P-HSDl A cDNA, and termed 11P-HSD1 B, resulted in a non-glycosylated, catalytically inactive protein species (Obeid et al, 1993), suggesting a membrane attachment and surrounding as well as glycosylation as important structural features for this class of hydroxysteroid dehydrogenases. Furthermore, tunicamycin-mediated inhibition of glycosylation resulted in a 50 % decrease of the Ilp-dehydrogenase reaction, whereas the 11-0x0 reductase reaction of heterologous expressed 11P-HSDlA rat cDNA was unaffected (Agarwal et al, 1990). The attachment of N-linked sugars to the protein could provide residues necessary for interlintrasubunit H bonding or proper orientation of the holoenzyme within the membrane environment.…”