cDNA Cloning and Characterization of a New Human Microsomal NAD+-dependent Dehydrogenase that Oxidizes All-trans-retinol and 3α-Hydroxysteroids

Abstract

We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/ reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat alltrans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (<73% identity). The mRNA for RoDH-4 is abundant in adult liver, where it is translated into RoDH-4 protein, which is associated with microsomal membranes, as evidenced by Western blot analysis. Significant amounts of RoDH-4 message are detected in fetal liver and lung. Recombinant RoDH-4, expressed in microsomes of Sf9 insect cells using BacoluGold Baculovirus system, oxidizes all-trans-retinol and 13-cis-retinol to corresponding aldehydes and oxidizes the 3␣-hydroxysteroids androstane-diol and androsterone to dihydrotestosterone and androstanedione, respectively. NAD ؉ and NADH are the preferred cofactors, with apparent K m values 250 -1500 times lower than those for NADP ؉ and NADPH. All-trans-retinol and 13-cis-retinol inhibit RoDH-4 catalyzed oxidation of androsterone with apparent K i values of 5.8 and 3.5 M, respectively. All-transretinol bound to cellular retinol-binding protein (type I) exhibits a similar K i value of 3.6 M. Unliganded cellular retinol-binding protein has no effect on RoDH activity. Citral and acyclic isoprenoids also act as inhibitors of RoDH-4 activity. Ethanol is not inhibitory. Thus, we have identified and characterized a sterol/retinol-oxidizing short chain dehydrogenase/reductase that prefers NAD ؉ and recognizes all-trans-retinol as substrate. RoDH-4 can potentially contribute to the biosynthesis of two powerful modulators of gene expression: retinoic acid from retinol and dihydrotestosterone from 3␣-androstane-diol.Short chain alcohol dehydrogenases/reductases are either cytosolic or membrane-bound enzymes with a subunit molecular mass of 25-35 kDa that utilize a vast variety of substrates, including steroids and prostaglandins (1). Recently, this family of enzymes has expanded to include the retinol-oxidizing dehydrogenases (2-6). Retinol dehydrogenases are involved in the biosynthesis of all-trans-retinoic acid, the activating ligand for a family of nuclear receptors (7). All-trans-retinoic acid is produced from all-trans-retinol in two oxidative steps: all-transretinol is oxidized to all-trans-retinal and then further to alltrans-retinoic acid. Retinol dehydrogenases catalyze the ratelimiting step: the oxidation of retinol to retinaldehyde (8). Although the effects of retinoic acid on gene transcription and regulation have been intensively studied during the last decade, the exact enzymes that synthesize this morphogen and the mechanisms that regulate its production in tissues are not fully understood. Enzymatic activity capable of oxidizing retinol to retinaldehyde is readily detected in the cytosolic and microsomal fractions of total cell homogenates (9). The cytosol...

Kinetic Analysis of Human Enzyme RDH10 Defines the Characteristics of a Physiologically Relevant Retinol Dehydrogenase

“…RDH10-His 6 was expressed in Sf9 cells using the Baculovirus expression system (Orbigen Inc., San Diego, CA) as described previously for other retinoid-active microsomal SDRs (18 -20). Microsomes containing RDH10 were isolated by differential centrifugation, resuspended in 90 mM potassium phosphate, pH 7.4, 40 mM potassium chloride (reaction buffer), supplemented with 1 mM dithiothreitol and 20% glycerol, and stored in small aliquots at Ϫ80°C (20).…”

“…The corresponding proteins could not be detected in HEK293 cells by immunoblotting using available antisera (20,29), therefore, the efficiency of siRNA pools specific for RoDH4 and RL-HSD was tested using stably transfected cell lines overexpressing RoDH4 and RL-HSD (21,30). Both siRNA pools decreased the levels of their respective proteins by Ͼ50% after 3 days of incubation (Fig.…”

Characterization of a Novel Airway Epithelial Cell-specific Short Chain Alcohol Dehydrogenase/Reductase Gene Whose Expression Is Up-regulated by Retinoids and Is Involved in the Metabolism of Retinol

“…Liver hepatocytes and stellate cells need retinol dehydrogenases because of their critical role in retinoid metabolism (57). The fact that multiple retinol dehydrogenases are expressed in the liver (17,22,58,54) suggests a need for this apparent redundancy. Therefore, we speculate that hRDH-TBE also serves a specific, if not essential, role in the retinoid metabolism in the airway.…”

Evidence for the Expression of Alcohol Dehydrogenase Class I Gene in Rat Uterus and Its Up-regulation by Progesterone

“…These mice were viable and fertile without obvious phenotype under the condition of normal diet. It was reported that there existed microsomal retinol dehydrogenases that are members of the short-chain dehydrogenase/reductase enzyme family [47][48][49][50]. One or more members of the short-chain dehydrogenase/reductase family may function physiologically in the conversion of retinal to RA, hence allowing these knockout mice to grow and reproduce normally.…”