Cloning of a cDNA for a Second Retinol Dehydrogenase Type II

Chai, Xin‐Sheng; Zhai, Yan; Popescu, Gabriela; Napoli, Joseph L.

doi:10.1074/jbc.270.47.28408

Cited by 95 publications

(56 citation statements)

References 39 publications

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“…2). A very strong signal was observed in human liver, similar to the expression patterns of rat all-trans-retinol dehydrogenases (3,4,12). In addition, relatively high levels of hybridizing message were detected in fetal liver and lung (Fig.…”

Section: Resultsmentioning

confidence: 53%

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

Gough

VanOoteghem

Sint

et al. 1998

Journal of Biological Chemistry

109

118

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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...

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Section: Resultsmentioning

confidence: 53%

“…Recently, this family of enzymes has expanded to include the retinol-oxidizing dehydrogenases (2)(3)(4)(5)(6). Retinol dehydrogenases are involved in the biosynthesis of all-trans-retinoic acid, the activating ligand for a family of nuclear receptors (7).…”

mentioning

confidence: 99%

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

Gough

VanOoteghem

Sint

et al. 1998

Journal of Biological Chemistry

109

118

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“…1). Mutants RD (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) or RD(1-30), which lack the first 18 and 30 amino acid residues of Rdh1, respectively, did not localize to the ER. Surprisingly both distributed in the mitochondria fraction with cytochrome c and localized in mitochondria in intact cells (Figs.…”

Section: Resultsmentioning

confidence: 99%

Elements in the N-terminal Signaling Sequence That Determine Cytosolic Topology of Short-chain Dehydrogenases/Reductases

Zhang

Napoli

2004

Journal of Biological Chemistry

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High affinity, retinoid-specific binding proteins chaperone retinoids to manage their transport and metabolism. Proposing mechanisms of retinoid transfer between these binding proteins and membrane-associated retinoid-metabolizing enzymes requires insight into enzyme topology. We therefore determined the topology of mouse retinol dehydrogenase type 1 (Rdh1) and cis-retinoid androgen dehydrogenase type 1 (Crad1) in the endoplasmic reticulum of intact mammalian cells. The properties of Rdh1 were compared with a chimera with a luminal signaling sequence (11␤-hydroxysteroid dehydrogenase (11␤-HSD1)(1-41)/Rdh1(23-317); the green fluorescent protein (GFP) fusion proteins Rdh1(1-22)/ GFP, Crad1(1-22)/GFP, and 11␤-HSD1(1-41)/GFP; and signaling sequence charge difference mutants using confocal immunofluorescence, antibody access, proteinase K sensitivity, and deglycosylation assays. An N-terminal signaling sequence of 22 residues, consisting of a hydrophobic helix ending in a net positive charge, anchors Rdh1 and Crad1 in the endoplasmic reticulum facing the cytoplasm. Mutating arginine to glutamine in the signaling sequence did not affect topology. Inserting one or two arginine residues near the N terminus of the signaling sequence caused 28-95% inversion from cytoplasmic to luminal, depending on the net positive charge remaining at the C terminus of the signaling sequence; e.g. the mutant L3R,L5R,R16Q,R19Q,R21Q faced the lumen. Experiments with N-and C-terminal epitope-tagged Rdh1 and molecular modeling indicated that a hydrophobic helix-turn-helix near the C terminus of Rdh1 (residues 289 -311) projects into the cytoplasm. These data provide insight into the features necessary to orient type III (reverse signal-anchor) proteins and demonstrate that Rdh1, Crad1, and other short-chain dehydrogenases/reductases, which share similar N-terminal signaling sequences such as human Rdh5 and mouse Rdh4, orient with their catalytic domains facing the cytoplasm.Retinol requires conversion into all-trans-retinoic acid to fulfill the vitamin A function of regulating gene expression that begins shortly after conception and continues throughout vertebrate life (1, 2). Retinoid-active SDRs 1 recognize the major physiological form of retinol, retinol bound with CRBP, to generate retinal for irreversible conversion into all-trans-retinoic acid (3). The most active of the retinol dehydrogenases include mouse Rdh1 and its orthologs, rat Rodh1 and -2 and human RDH-E/RoDH4 (4 -8). rRodh2 e.g. has lower apparent K m values with holo-CRBP than with unbound retinol and catalyzes retinal synthesis in the presence of excess apoCRBP. Dehydrogenation of retinol in the presence of excess CRBP with concentrations of both ligand and binding protein in the micromolar range indicates that the reaction proceeds through the SDR accessing CRBP-bound retinol because the CRBP-retinol complex has a K d value ϳ0

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“…Two classes of unrelated enzymes have been implicated in the oxidation of retinol, the classical cytosolic medium chain alcohol dehydrogenases (reviewed in ref. 19) and recently identified microsomal members of the short chain alcohol dehydrogenase͞ reductase (SDR) superfamily (20)(21)(22)(23)(24). Enzymes from both groups are able to oxidize retinol in vitro but the relative role of the different enzymes in retinol oxidation in vivo is still enigmatic.…”

mentioning

confidence: 99%

The identification of a 9- cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9- cis retinoic acid

Romert

Tuvendal

Simon

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membranebound enzyme is a member of the short-chain alcohol dehydrogenase͞reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.

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Cloning of a cDNA for a Second Retinol Dehydrogenase Type II

Cited by 95 publications

References 39 publications

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

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

Elements in the N-terminal Signaling Sequence That Determine Cytosolic Topology of Short-chain Dehydrogenases/Reductases

The identification of a 9- cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9- cis retinoic acid

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