Expression and localization of the 20α-hydroxysteroid dehydrogenase (HSD) enzyme in the reproductive tissues of the cynomolgus monkey Macaca fascicularis.
ABSTRACT This study was conducted to characterize and functionally analyze the monkey 20α-hydroxysteroid dehydrogenase (20α-HSD) in the ovary, placenta, and oviduct. We focused on 20α-HSD mRNA expression and protein localization in monkey reproductive tissues and the molecular characterization of the promoter region. Reverse transcription-polymerase chain reaction (RT-PCR) monkey 20α-HSD mRNA was more strongly detected in the ovary at pre-ovulation than in the placenta and oviduct at pre-parturition. The mRNA was approximately 1.2kb in size and the expression was high in the ovary, which was the same as the RT-PCR result. We also produced His tagged 20α-HSD proteins by using an Escherichia coli expression system. In a western blot for the 20α-HSD protein, only 1 band of approximately 37-kDa was detected in the ovary, oviduct tissue, and recombinant protein produced in the Chinese hamster ovary (CHO) cell line. However, in the placenta, additional 2 bands (35 and 39 kDa) were detected. Immunohistochemical analyses suggested that the monkey 20α-HSD protein was localized mainly in the syncytiotrophoblast of the placenta and the isthmus cells of the oviduct. According to promoter analyses with the enhanced green fluorescent protein (EGFP) gene, the monkey 20α-HSD promoter was efficiently expressed in the CHO-K1 cell line; however, the promoter was not expressed in bovine fetal fibroblast (bFF) cell. Taken together, our study showed that the 20α-HSD mRNA and protein are coordinately expressed in the ovary at pre-ovulation and in the placenta and oviduct at pre-parturition. Therefore, monkey 20α-HSD in the placenta, ovary and oviduct plays an important role in the estrous cycle, pregnancy, and parturition.
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ABSTRACT: The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.Biochemical Journal 10/1997; 326 ( Pt 3):625-36. · 4.65 Impact Factor
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ABSTRACT: Japanese monkey liver contains multiple forms of dihydrodiol dehydrogenase with 3(20)alpha-hydroxysteroid dehydrogenase activity. Here we have purified the major and minor forms (DD1 and DD4) of the enzyme from Cynomolgus monkey liver, and isolated cDNA species for the two enzyme forms by reverse transcription-PCR. The cDNAs encoded proteins comprising of 323 amino acids, in which the sequence identity between DD1 and DD4 was 83%. The sequences deduced from the cDNAs for DD1 and DD4 perfectly matched the partial sequences of peptides derived from the respective enzymes. We also isolated the cDNAs for DD1 and DD4 of Japanese monkey liver, which had almost identical amino acid sequences with those of the respective enzymes of Cynomolgus monkey liver. The monkey DD1s and DD4s showed the highest sequence identity (94%) with AKR1C1 and AKR1C4, respectively, of four isoenzymes of human 3(20)alpha-hydroxysteroid dehydrogenase, which belongs to the aldo-keto reductase family. The substrate specificity and inhibitor sensitivity of the purified recombinant Cynomolgu monkey DD1 and Japanese monkey DD4 were also essentially identical to those of the recombinant AKR1C1 and AKR1C4, respectively, indicating that DD1 and DD4 are homologues of human AKR1C1 and AKR1C4, respectively. The mRNA for DD1 was detected only in liver, kidney, intestine and adrenal gland among Japanese monkey tissues, and that for DD4 was expressed in liver and kidney. These tissue distribution patterns differ from those of human AKR1C1 and AKR1C4, which are expressed ubiquitously and liver-specific, respectively. In addition, no mRNA for an enzyme corresponding to another isoenzyme (AKR1C2) of the human enzyme was detected in livers of the two monkey strains. The results suggest a difference in the metabolism of steroids and xenobiotics mediated by 3(20)alpha-hydroxysteroid dehydrogenase isoenzymes between monkeys and humans.Drug Metabolism and Pharmacokinetics 02/2002; 17(4):348-56. · 2.07 Impact Factor
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ABSTRACT: Two major and two minor forms of dihydrodiol dehydrogenase with similar molecular weights of around 36000 were purified from monkey liver cytosol. All the forms oxidized trans-dihydrodiols of benzene and naphthalene and reduced aromatic aldehydes, but showed differences in charge, specificity for other substrates and inhibitor sensitivity. One major (pI 8.7) and one minor (pI 7.9) form of the enzyme exhibited high activity for alicyclic alcohols and sensitivity to o-phenanthroline. The other major form (pI 6.2) oxidized 3 alpha-hydroxysteroids and was inhibited by dexamethasone and indomethacin, whereas the other minor form (pI 5.8) showed high reductase activity for aldehydes including D-glucuronate and sensitivity to barbital and sorbinil, and cross-reacted with human aldehyde reductase. The results indicate that the multiple forms of monkey liver dihydrodiol dehydrogenase are indanol dehydrogenases, 3 alpha-hydroxysteroid dehydrogenase and aldehyde reductase.CHEMICAL & PHARMACEUTICAL BULLETIN 11/1989; 37(10):2852-4. · 1.56 Impact Factor