Abstract
We have identified a retinol dehydrogenase (cRDH) that catalyzes the oxidation of 9-cis- but not all-trans-retinol and proposed that this enzyme plays an important role in synthesis of the transcriptionally active retinoid, 9-cis-retinoic acid. There is little information regarding either the biochemical properties of cRDH or how its 9-cis-retinol substrate is formed. We now report studies of the properties and expression of human and mouse cRDH and of the characteristics and location of the murine cRDH gene. Additionally, we report mouse hepatic 9-cis-retinol concentrations and demonstrate that 9-cis-retinol is formed in a time- and protein-dependent manner upon incubation of all-trans-retinol with cell homogenate. Human and mouse cRDH display similar substrate specificities for cis-isomers of retinol and retinaldehyde. Moreover, human and mouse cRDH show marked sensitivity to inhibition by 13-cis-retinoic acid, with both being inhibited by approximately 50% by 0.15 μm 13-cis-retinoic acid (for substrate concentrations of 10 μm). Lesser inhibition is seen for 9-cis- or all-trans-retinoic acids. Immunoblot analysis using antiserum directed against human cRDH demonstrates cRDH expression in several tissues from first trimester human fetuses, indicating that cRDH is expressed early in embryogenesis. Adult mouse brain, liver, kidney, and to a lesser extent small intestine and placenta express cRDH. The murine cRDH gene consists of at least 5 exons and spans approximately 6 kb of genomic DNA. Backcross analysis mapped the mouse cRDH gene to the most distal region of chromosome 10. ▪ Taken together, these data extend our understanding of the properties of cRDH and provide additional support for our hypothesis that cRDH may play an important role in 9-cis-retinoic acid formation.—Gamble, M. V., E. Shang, R. P. Zott, J. R. Mertz, D. J. Wolgemuth, and W. S. Blaner. Biochemical properties, tissue expression, and gene structure of a short chain dehydrogenase/reductase able to catalyze cis-retinol oxidation.
Highlights
We have identified a retinol dehydrogenase that catalyzes the oxidation of 9-cis- but not alltrans-retinol and proposed that this enzyme plays an important role in synthesis of the transcriptionally active retinoid, 9-cis-retinoic acid
Immunoblot analysis showed that the antiserum recognizes strongly and recombinant human cisretinol dehydrogenase (cRDH) as a 32 kDa protein (Fig. 1)
Because the size of the protein recognized by the antiserum separated on the SDS-PAGE gel corresponds to a protein with a mass of 32 kDa, the mass of human cRDH predicted by its cDNA, it would appear that cRDH does not undergo substantial post-translational modification upon expression in CHO cells and further, that such modification is not essential for cRDH activity
Summary
We have identified a retinol dehydrogenase (cRDH) that catalyzes the oxidation of 9-cis- but not alltrans-retinol and proposed that this enzyme plays an important role in synthesis of the transcriptionally active retinoid, 9-cis-retinoic acid. Backcross analysis mapped the mouse cRDH gene to the most distal region of chromosome 10 Taken together, these data extend our understanding of the properties of cRDH and provide additional support for our hypothesis that cRDH may play an important role in 9-cisretinoic acid formation.—Gamble, M. Biochemical properties, tissue expression, and gene structure of a short chain dehydrogenase/reductase able to catalyze cis-retinol oxidation. As members of the RXR family of receptors are able to serve as partners in forming heterodimers with the vitamin D receptor, the thyroid hormone receptors, the peroxisomal proliferator activator receptors, and several other ligand dependent transcription factors, 9-cis-retinoic acid likely plays an important role in regulating a broad spectrum of hormonally responsive genes [4]. Based on the relatively large number of enzymes that can catalyze retinol oxidation, it would appear likely that there is redundancy in the enzymatic machinery needed for retinoic acid formation
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