Evaluation of Enzymatic Activity of Various HSD17B3 Mutants Using Androgen Receptor-Mediated Transactivation

17β-Hydroxysteroid dehydrogenases (17β-HSDs, HSD17B) catalyze the reduction of 17-ketosteroids and the oxidation of 17β-hydroxysteroids to regulate the production of sex steroids. Among HSD17B family, 17β-HSD type 3 (HSD17B3) is expressed in testicular Leydig cells and contributes to development of male sexual characteristics by converting androstenedione (A4) to testosterone (T). Mutations of HSD17B3 genes cause a 46,XY disorder of sexual development (46,XY DSD) as a result of low T production. Therefore, the evaluation of HSD17B3 enzymatic activity is important for understanding and diagnosing this disorder. Although various amino acid substitutions of HSD17B3 have been reported in previous studies, the enzymatic activities of these proteins were often not defined. This is probably due to the difficulties that such enzymatic activities have been evaluated by quantifying the conversion of A4 into T using radioactive isotopes and liquid chromatography-mass spectrometry-mass spectrometry (LC-MS/MS). We adapted a method that easily evaluates enzymatic activity of HSD17B3 proteins by quantifying the conversion from A4 to T using androgen receptor (AR)-mediated transactivation. HEK293 cells were transfected to express human HSD17B3, and incubated medium containing A4. Depending on the incubation time with HSD17B3-expressing cells, the culture media progressively increased luciferase activities in CV-1 cells, transfected with the AR expression vector and androgen-responsive reporter. These luciferase activities reflected T concentrations in culture media defined by LC-MS/MS. This system is also applicable to detect the conversion of A4 to T by HSD17B1 and HSD17B5. In addition, it can evaluate the conversion of 11-ketoandrostenedione to 11-ketotestosterone by HSD17B family. Establishment of HEK293 cells expressing various missense mutations in the HSD17B3 gene with (N74T, A188V, M197K, A200V,, V225M, H271R) or without (V31I, E67K and G289S) the manifestation of 46,XY DSD revealed that this system is effective to evaluate the enzymatic activities of mutant proteins. A188V, A200V, V225M and H271R mutations completely inactivated the enzymatic function. N74T and M197K mutations showed some residual activities. In contrast, V31I, E67K and G289S substitutions showed similar activities as the wild-type protein.