Cited by 362Adrenal Hormones and DisordersSexual Differentiation and DisordersSteroid Chemistry and Biochemistry

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

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Rhéaume, Eric, Lachance, Yves, Zhao, Hui-Fen, Breton, Nathalie, Dumont, Marline, Launoit, Yvan de, Trudel, Claude, Luu-The, Van +2 more·1991·Molecular Endocrinology

Abstract

The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase (3 beta HSD) enzyme catalyzes the oxidation and isomerization of delta 5-3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus leading to the formation of all classes of steroid hormones. In addition, 3 beta HSD catalyzes the interconversion of 3 beta-hydroxy- and 3-keto-5 alpha-androstane steroids. Clinical observations in patients with 3 beta HSD deficiency as well as our recent data obtained by Southern blot analysis using a human placental 3 beta HSD cDNA (type I) as probe suggested the existence of multiple related 3 beta HSD isoenzymes. We now report the isolation and characterization of a second type of cDNA clone (arbitrarily designated type II) encoding 3 beta HSD after screening of a human adrenal lambda gt22A library. The nucleotide sequence of 1676 basepairs of human 3 beta HSD 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 3 beta HSD 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 3 beta HSD full-length 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 3 beta HSD/delta 5-delta 4 isomerase activity higher than type II, with respective Km values of 0.24 vs. 1.2 microM for pregnenolone and 0.18 vs. 1.6 microM 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 3 beta HSD indicates that dihydrotestosterone is converted into 5 alpha-androstane-3 beta, 17 beta-diol, with Km values of 0.26 and 2.7 microM, respectively. Ribonuclease protection assay using type I- and type II-specific cRNA probes revealed that type II transcripts are the almost exclusive 3 beta HSD mRNA species in the human adrenal gland, ovary, and testis, while type I transcripts correspond to the almost exclusive 3 beta HSD 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 3 beta HSD isoenzyme that is distinct from the type expressed in the placenta.(ABSTRACT TRUNCATED AT 400 WORDS)

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  1. [34]34. de Peretti E, Forest MG, Feit JP, David M 1980 Endocrine studies in two children with male pseudohermaphroditism due to 3/3-hydroxysteroid (3/3HSD) dehydrogenase defect. In: Genazzani AR, Thijssen JHH, Siiteri PK (eds) Adrenal Androgens. Raven Press, New York, pp 141-145
  2. [66]66. Kingston RE 1987 Transfection of DNA into eukaryotic cells. In: Ausubel EM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (eds) Current Protocols in Molecular Biology. Wiley and Sons, New York, pp 9.11 - 9.16
  3. [70]70. Gilman M 1989 Rybonuclease protection assay. In: Au- subel FM, Brent R, Kingston RE, Moore DD, Seidman TA, Smith TA, Struhl K (eds) Current Protocols in Molecular Biology. Wiley and Sons, New York, pp 4.7.1-4.7.8

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Authors
Rhéaume, E., Lachance, Y., Zhao, H.-F., Breton, N., Dumont, M., Launoit, Y. D., Trudel, C., Luu-The, V., Simard, J., & Labrie, F.
Affiliations
Université Laval (CA)
Venue
Molecular Endocrinology
Published
1991-08
Volume
5
Issue
8
Pages
1147-1157
ISSN
  • 0888-8809 (ISSN-L)
  • 1944-9917
OpenAlex
W2110661248
Cited by
362
References
62
Citations by year
2026: 3 · 2025: 7 · 2024: 8 · 2023: 6 · 2022: 5 · 2021: 3 · 2020: 2 · 2019: 8 · 2018: 7 · 2017: 7 · 2016: 8 · 2015: 8 · 2014: 11 · 2013: 11 · 2012: 9
FWCI
12.25
Type
journal article
Language
en
Open access
Locations
  • Molecular Endocrinology (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Fonds de Recherche du Québec - Santé
  • Medical Research Council
Topics
  • Health Sciences › Medicine › Endocrinology, Diabetes and Metabolism › Adrenal Hormones and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sexual Differentiation and Disorders
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Steroid Chemistry and Biochemistry
Keywords
cDNA cloning, adrenal gland, gonads, isozymes, enzyme kinetics, dihydrotestosterone, steroidogenesis
MeSH terms
Adrenal Glands, Amino Acid Sequence, Animals, Base Sequence, Cattle, DNA, Female, HeLa Cells, Humans, Isoenzymes, Macaca mulatta, Male, Molecular Sequence Data, Multienzyme Complexes, Ovary, Plasmids, Progesterone Reductase, RNA, Messenger, Steroid Isomerases, Testis, Transfection, Restriction Mapping, Gene Expression, Rats

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