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Structure and Expression of a New Complementary DNA Encoding the almost Exclusive 3β-Hydroxysteroid Dehydrogenase/Δ 5 -Δ 4 -lsomerase in Human Adrenals and Gonads

1991; Oxford University Press; Volume: 5; Issue: 8 Linguagem: Inglês

10.1210/mend-5-8-1147

1944-9917

Éric Rhéaume, Yves Lachance, Huifen Zhao, Nathalie Breton, Marline Dumont, Yvan de Launoit, Claude Trudel, Van Luu‐The, Jacques Simard, Fernand Labrie,

Biochemical Acid Research Studies

The 3β-hydroxysteroid dehydrogenase/Δ5-Δ4 isomerase (3βHSD) enzyme catalyzes the oxidation and isomerization of Δ5−3β-hydroxysteroid precursors into Δ4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3βHSD catalyzes the interconversion of 3β-hydroxy- and 3-keto-5α-androstane steroids. Clinical observations in patients with 3βHSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3βHSD cDNA (type I) as probe suggested the existence of multiple related 3βHSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type II) encoding 3βHSD after screening of a human adrenal λgt22A library. The nucleotide sequence of 1676 basepairs of human 3βHSD type II cDNA predicts a protein of 371 amino acids with a calculated molecular mass of 41,921 daltons, which displays 93.5% and 96.2% homology with human placental type I and rhesus macaque ovary 3βHSD deduced proteins, respectively. To characterize and compare the kinetic properties of the two isoenzymes, plasmids derived from pCMV and containing type I or type II 3βHSD fulllength cDNA inserts were transiently expressed in HeLa human cervical carcinoma cells. In vitro incubation with NAD+ and 3H-labeled pregnenolone or dehydroepiandrosterone shows that the type I protein possesses a 3βHSD/Δ5-Δ4 isomerase activity higher than type II, with respective Km values of 0.24 vs. 1.2 μm for pregnenolone and 0.18 vs. 1.6 μm for dihydroepiandrosterone, while the specific activity of both types is equivalent. Moreover, incubation in the presence of NADH of homogenates from cells transfected with type I or type II 3βHSD indicates that dihydrotestosterone is converted into 5α-androstane- 3β,17β-diol, with Km values of 0.26 and 2.7 μm, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3βHSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3βHSD mRNA species in the placenta and skin and represent the predominantly expressed species in mammary gland tissue. The present data show for the first time that adrenals and gonads express a type of 3βHSD isoenzyme that is distinct from the type expressed in the placenta. Detection of 3βHSD gene expression in nonclassical steroidogenic tissues such as skin and mammary gland suggests that 3βHSD is likely to play an important role in the intracrine formation of sex steroids in peripheral target tissues. Characterization of type II 3βHSD, in addition to permitting studies of the regulation of its tissue-specific expression, offers the opportunity of elucidating the molecular basis of classical and nonclassical 3βHSD deficiencies, the second most common cause of congenital adrenal hyperplasia and the predicted most common genetic disorder in women with signs of androgen excess, respectively.