Endocrine Abstracts

3beta-hydroxysteroid dehydrogenase (3beta-HSD) expression is essential for the synthesis of all classes of steroid hormones, converting delta⁵-3beta-hydroxysteroids into hormonally active delta⁴-3-ketosteroids in NAD⁺-dependent reactions. A variety of 3beta-HSD isoforms have been described in primate, rodent and other mammals, often exhibiting distinct kinetic properties, preferential subcellular localization, as well as alternative substrate specificity demonstrable in some isoforms. In addition, inactivity of certain of the described mutations of the human type II 3beta-HSD gene arises from an unstable mutant enzyme rather than a catalytically compromised enzyme.

We sought to establish whether 3beta-HSD fused to either green fluorecent protein (GFP) or glutathione-S-transferase (GST) domains retains typical 3beta-HSD activity and, in particular, whether any increased stability of mutant enzyme is attainable.

Non-steroidogenic HEK 293T and CHO-K1 cells were transiently transfected with pCMV-based vectors containing cDNAs encoding human type II 3beta-HSD fused N-terminally with GFP or GST domains, or C-terminally with a GFP domain. Additionally, cDNA constructs were made of GFP and GST domains separately linked to the N-terminal of the human 3beta-HSD type II L6F mutant that appears much less stable than the wild-type enzyme. Subsequently the transfected cells were incubated with radiolabelled pregnenolone (1 micromole/L) for various periods up to 24 hours. Typical 3beta-HSD activity was associated with the various fusion constructs expressed. Western blotting using antibody raised against recombinant human type II 3beta-HSD was used to confirm the presence and molecular size of the expressed fusions. Cytoplasmic expression of the GFP-fusions was established with fluorescent microscopy.

Our findings demonstrate that GFP and GST fusions with 3beta-HSDs retain not only GFP and GST activities but also apparently typical 3beta-HSD activities. Furthermore, such fusions also provide increased stability to HSDs known to demonstrate reduced stability in certain cellular environs. (Supported, in part, by MRC Programme Grant G0000066)