Gene Structure, Expression Analysis, and Membrane Topology of RDH4

Retinoids are chromophores involved in vision, transcriptional regulation, and cellular differentiation. Members of the short chain alcohol dehydrogenase/reductase superfamily catalyze the transformation of retinol to retinal. Here, we describe the identification and properties of three enzymes from a novel subfamily of four retinol dehydrogenases (RDH11-14) that display dualsubstrate specificity, uniquely metabolizing all-trans-and cis-retinols with C 15 pro-R specificity. RDH11-14 could be involved in the first step of all-trans-and 9-cis-retinoic acid production in many tissues. RDH11-14 fill the gap in our understanding of 11-cis-retinal and all-trans-retinal transformations in photoreceptor (RDH12) and retinal pigment epithelial cells (RDH11). The dualsubstrate specificity of RDH11 explains the minor phenotype associated with mutations in 11-cisretinol dehydrogenase (RDH5) causing fundus albipunctatus in humans and engineered mice lacking RDH5. Furthermore, photoreceptor RDH12 could be involved in the production of 11-cis-retinal from 11-cis-retinol during regeneration of the cone visual pigments. These newly identified enzymes add new elements to important retinoid metabolic pathways that have not been explained by previous genetic and biochemical studies.Retinoids are indispensable light-sensitive elements of vision and also serve as essential modulators of cellular differentiation and proliferation in diverse cell types, including those comprising the epithelium and immune system. Retinoids modulate the growth of both normal and malignant cells through their binding to retinoid receptors. All-trans-retinoic acid signals through specific interactions with the nuclear retinoic acid receptors, whereas its isomer, 9-cis-retinoic acid, is a high affinity ligand of retinoic acid receptors and retinoid X receptors. In the retina, light-dependent photoisomerization of 11-cis-retinylidene to the all-transretinylidene moiety of rod and cone photoreceptors is a key reaction that triggers visual * This research was supported by National Institutes of Health Grants EY08061, CA75173, and DK59125, a grant from Research to Prevent Blindness, Inc. (to the Department of Ophthalmology, University of Washington), and by the E. K. Bishop Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. S The on-line version of this article (available at http://www.jbc.org) contains Supplemental Figs. 1 and 2 and Tables 1-3. § These authors contributed equally to this work. § § Recipient of a Research to Prevent Blindness Senior Investigator Award. To whom correspondence should be addressed: Dept. of Ophthalmology, University of Washington, Seattle,; E-mail: palczews@u.washington.edu. Transformations of retinoids occur mostly through enzymatic or photochemical reactions, although they readily isomerize non-enzymatically to thermodynamic equilibriu...

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“…1C). This gene structure is different from other SDR superfamily RDHs expressed in the eye (30)(31)(32).…”

Section: Rdh11-14 Sequence Analyses and Genementioning

confidence: 57%

Dual-substrate Specificity Short Chain Retinol Dehydrogenases from the Vertebrate Retina

Haeseleer¹,

Jang²,

Imanishi³

et al. 2002

Journal of Biological Chemistry

184

227

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“…Each well was transfected with 100 ng of receptor Gal4-RXR plasmid, 100 ng of reporter MH100-tk-luc plasmid (24), and 200 ng of internal control plasmid encoding ␤-galactosidase. In addition, 100 ng of each plasmid encoding murine CRAD1 (13), CRAD2 (14), a CRADlike enzyme, CRADL, 2 RDH5 (17,25) or murine ADH4 (a kind gift from Dr. Gregg Duester) were co-transfected with 100 ng of expression plasmid encoding retinal dehydrogenase 1 (Raldh1) or Raldh2 in various combinations. Original retinal dehydrogenase plasmids were kind gifts from Dr. Joseph Napoli.…”

Section: Methodsmentioning

confidence: 99%

“…For affinity purification, the rabbit antisera were passed over columns containing the CRAD1-glutathione S-transferase fusion protein immobilized on CNBr-activated Sepharose, and bound Ig was eluted and characterized. The rabbit antibodies to RDH5 have been described previously (17), and the rabbit antiserum to human ADH4 was a kind gift from Dr. Jan-Olov Höög.…”

Section: Proteinase K Assay Sds-page Immunoblotting Rt-pcr Analysimentioning

confidence: 99%

“…Polyclonal rabbit antisera to CRAD1 were generated by immunizing rabbits with a glutathione S-transferase fusion protein containing amino acids 19 -288 of CRAD1, essentially as described for the production of rabbit antisera to RDH5 (17). For affinity purification, the rabbit antisera were passed over columns containing the CRAD1-glutathione S-transferase fusion protein immobilized on CNBr-activated Sepharose, and bound Ig was eluted and characterized.…”

Section: Proteinase K Assay Sds-page Immunoblotting Rt-pcr Analysimentioning

confidence: 99%

“…RDH5 is a homodimeric integral membrane protein anchored to the lipid bilayer via an aminoterminal signal anchor peptide and a carboxyl-terminal transmembrane segment. The catalytic domain of the enzyme is lumenal and only the extreme carboxyl-terminal 6 -8 amino acid residues reside in the cytosol (16,17) (the RDH4 enzyme has been renamed to RDH5 to indicate that it is the murine counterpart of RDH5). Genetic evidence for a role of the microsomal RDHs in ocular retinoid metabolism comes from the identification of mutations in RDH5 in patients suffering from fundus albipunctatus (19,20).…”

mentioning

confidence: 99%

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Biosynthesis of 9-cis-Retinoic Acidin Vivo

Tryggvason

Romert

Eriksson

2001

Journal of Biological Chemistry

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Retinoic acid is generated by a two-step mechanism. First, retinol is converted into retinal by a retinol dehydrogenase, and, subsequently, retinoic acid is formed by a retinal dehydrogenase. In vitro, several enzymes are suggested to act in this metabolic pathway. However, little is known regarding their capacity to contribute to retinoic acid biosynthesis in vivo. We have developed a versatile cell reporter system to analyze the role of several of these enzymes in 9-cis-retinoic acid biosynthesis in vivo. Using a Gal4-retinoid X receptor fusion proteinbased luciferase reporter assay, the formation of 9-cisretinoic acid from 9-cis-retinol was measured in cells transfected with expression plasmids encoding different combinations of retinol and retinal dehydrogenases. The results suggested that efficient formation of 9-cisretinoic acid required co-expression of retinol and retinal dehydrogenases. Interestingly, the cytosolic alcohol dehydrogenase 4 failed to efficiently catalyze 9-cis-retinol oxidation. A structure-activity analysis showed that mutants of two retinol dehydrogenases, devoid of the carboxyl-terminal cytoplasmic tails, displayed greatly reduced enzymatic activities in vivo, but were active in vitro. The cytoplasmic tails mediate efficient endoplasmic reticulum localization of the enzymes, suggesting that the unique milieu in the endoplasmic reticulum compartment is necessary for in vivo activity of microsomal retinol dehydrogenases.Retinol (vitamin A) and its derivatives are essential dietary compounds needed in a variety of physiological processes, e.g. embryonic development, reproduction, cell differentiation, postnatal growth, maintenance of the immune system, and vision (1-3). The metabolically active retinoids are 9-cis-retinoic acid (9cRA) 1 and all-trans-retinoic acid (atRA) in extraocular tissues, acting as ligands for two classes of nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) (4). In ocular tissues, 11-cis-retinal serves as the chromophore of the visual pigments (2).Most cells obtain all-trans-retinol as the main source of retinoids, and it is known that activation of retinol into the active metabolites occur via tissue-specific isomerization and oxidation reactions. First, 9-cis-, 11-cis-, or all-trans-retinols are oxidized by retinol dehydrogenases (RDHs) into the corresponding retinals. For generation of the two isomers of RA, 9-cis-and all-trans-retinal are then further oxidized by a class of retinal dehydrogenases (Raldhs) (for reviews, see Refs. 5 and 6). It has been suggested that oxidation of the two isomers of retinol are the rate-limiting factors in the pathways generating 9cRA and atRA.Two classes of RDHs have been implicated in oxidation of the different stereo isomers of retinol, i.e. microsomal members of the short chain alcohol dehydrogenase/reductases, and the cytosolic medium chain alcohol dehydrogenases (ADHs). Several members of both classes of enzymes can oxidize different stereo isomers of retinol in vitro. However,...

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