Structure and Function of Retinol Dehydrogenases of the Short Chain Dehydrogenase/Reductase Family

Molecular Aspects of Medicine 24 (2003) 403–409 www.elsevier.com/locate/mam

Structure and function of retinol dehydrogenases of the short chain dehydrogenase/reductase family Martin Lideen, Kristian Tryggvason, Ulf Eriksson * Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden

Abstract All-trans-retinol is the common precursor of the active retinoids 11-cis-retinal, all-trans- retinoic acid (atRA) and 9-cis-retinoic acid (9cRA). Genetic and biochemical data supports an important role of the microsomal members of the short chain dehydrogenases/reductases (SDRs) in the ﬁrst oxidative conversion of retinol into retinal. Several retinol dehydrogenases of this family have been reported in recent years. However, the structural and functional data on these enzymes is limited. The prototypic enzyme RDH5 and the related enzyme CRAD1 have been shown to face the lumen of the endoplasmic reticulum (ER), suggesting a compartmentalized synthesis of retinal. This is a matter of debate as a related enzyme has been proposed to have the opposite membrane topology. Recent data indicates that RDH5, and presumably other members of the SDRs, occur as functional homodimers, and need to interact with other proteins for proper intracellular localization and catalytic activity. Further analyses on the compartmentalization, membrane topology, and functional properties of microsomal retinol dehydrogenases, will give important clues about how retinoids are pro- cessed. Ó 2003 Elsevier Ltd. All rights reserved.

Abbreviations: atRA, all-trans-retinoic acid; 9cRA, 9-cis-retinoic acid; ER, endoplasmic reticulum; RalDH, retinal dehydrogenase; RAR, retinoic acid receptor; RDH, retinol dehydrogenase; RPE, retinal pigment epithelium; RXR, retinoid X receptor; SDR, short chain dehydrogenase/reductase

Keywords: Retinal; Retinal pigment epithelium; Retinol; Retinol dehydrogenase; SDR

* Corresponding author. Tel.: +46-8-728-7109; fax: +46-8-332812. E-mail address: [email protected] (U. Eriksson).

1. Introduction

Retinoids (vitamin A derivatives) are essential for a wide range of physiological processes, including embryonic development, reproduction, vision, immune responses, and diﬀerentiation and maintenance of various epithelia. 11-Cis-retinal serves as the chromophore of the visual pigments in vertebrate retinas, and the hormonal retinoids, all-trans-retinoic acid (atRA) and 9-cis-retinoic acid (9cRA), regulate the non-visual functions via activation of two classes of nuclear retinoid receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Recent results suggest that retinoids are also involved in the maintenance of circadian ry- thms, mediated by specialized photoreceptor cells (Lucas et al., 2003; Van Gelder et al., 2003). Retinol is converted into retinal and retinoic acid, by one or two rounds of oxidation, respectively. Two classes of enzymes have been implicated in the ﬁrst oxidation of retinol into retinal, the microsomal members of the shortchain dehydrogenases/reductases (SDRs), and the cytosolic medium chain alcohol dehydrogenases (ADHs) (Duester, 2000; Napoli, 1999). However, not much is known about the contribution of the individual enzymes to retinoid biosynthesis in vivo, nor is it clear how they function at the cell biological level, i.e. in terms of compartmentalization of the enzymes and their substrates, and how they function in concert with other proteins. Here we review recent data on the structure-function relation- ships of retinol dehydrogenases of the short chain dehydrogenase/reductase (SDR) family.

2. Retinol dehydrogenases of the short chain reductase/dehydrogenase family

In recent years, several retinol dehydrogenases (RDHs) have been added to the family of SDRs. In common to the SDRs is a well conserved domain structure with an N-terminal coenzyme––binding motif (consensus GXXXGXG) and an active site sequence (consensus YXXXK). Substrates for the SDR family of enzymes include steroids, fatty acids, and lipid alcohols, such as retinol. Several isozymes able to oxidize various isomers of retinol have been described, displaying different tissue distribution and substrate specificities in vitro. The first retinol dehydrogenase of the SDR family to be identified and cloned, was the bovine 11-cis-retinol dehydrogenase (RDH5), abundantly expressed in the retinal pigment epithelium (RPE) of the eye (Simon et al., 1995). Mutations in the RDH5 gene has been shown to cause fundus albipunctatus, a rare form of stationary night blindness, characterized by a delay in the regeneration of cone and rod photopig- ments, and accumulation of white spots in the retina (Yamamoto et al., 1999). This provided the first genetic evidence that members of the SDR family are involved in retinoid metabolism in vivo, and that RDH5 plays a crucial role in the production of 11-cis-retinal. However, the photoreceptor cells ultimately do recover, indicating that other enzymes or biochemical pathways can produce 11-cis-retinal at a slower rate than normal. Likewise, studies on RDH5-deficient mice show that alternative M. Liden et al. / Molecular Aspects of Medicine 24 (2003) 403–409 405 pathways must exist (Driessen et al., 2000). These mice have a normal dark adaptation and show no signs of fundus albipuntcatus, and only at high bleaching levels delayed dark adaptation can be detected. Recently, a new subfamily of dual-substrate enzymes (RDH11-14) were described, which could account for this redun- dancy in mice (Haeseleer et al., 2002). An all-trans-retinol dehydrogenase, denoted RDH10, has also been identified in the RPE, although its function remains to be clarified (Wu et al., 2002). In the photoreceptor outer segments, two enzymes, retSDR1 and prRDH, have been implicated in the reduction of all-trans-retinal into all-trans-retinol, which is the rate limiting step in the visual cycle (Haeseleer et al., 1998; Rattner et al., 2000). In addition to 11-cis-retinol, RDH5 oxidizes 9-cis-retinol. Taken together with the extra-ocular expression pattern, it was suggested early on that the enzyme may have dual functions, catalyzing chromophore production in the eye, and the first oxidative step in 9cRA synthesis at other sites (Romert et al., 1998). However, the mild phenotype seen in RDH5-deficient mice does not support an essential role of the enzyme in 9cRA biogenesis in mice (Driessen et al., 2000). The related enzymes, cis- retinol/androgen dehydrogenase 1-3 (CRAD1-3), efficiently metabolize 9-cis and/or all-trans-retinol, and are highly expressed in the liver, kidney, and lung (Chai et al., 1997; Su et al., 1998; Zhuang et al., 2002). RoDH1, RoDH2 and RoDH3, cloned from rat liver (Chai et al., 1995a,b; Chai et al., 1996), and their human homologues, RoDH4 and RoDH-like 3a-HSD (Gough et al., 1998; Kedishvili et al., 2001), have been shown to metabolize all-trans-retinol, as well as 3a-hydroxysteroids. Yet another SDR, active with all-trans and cis-retinols, as well as steroids, is the mRDH1 identified in mouse (Zhang et al., 2001).

3. Structure and function of RDHs

Biochemical data on the structure, topology and localization of the RDHs has only been reported for a few of the RDHs. RDH5 and CRAD1 are attached to the endoplasmic reticulum (ER) membrane by two membrane anchoring helices, with the bulk of the enzyme, including the co-factor binding and the active site, facing the lumenal side, and a C-terminal tail of 6–7 amino acids protruding into the cytosol (Fig. 1). This is evidenced by a number of independent observations, i.e. by pro- teinase K protection of wild type and HA epitope-tagged RDH5; Endo H treatement of N-glycosylated mutants; immunofluorescence at conditions of selective membrane permeabilization; and electron microscopy (Simon et al., 1999; Tryggvason et al., 2001, and unpublished data). However, other authors have come to different con- clusions regarding the related enzyme RoDH1/RoDH4 (Belyaeva et al., 2003; Lapshina et al., 2003; Wang et al., 2001). There is experimental evidence that the cytosolic tail is of functional importance in vivo. Deletion of the cytosolic tail in murine RDH5 and CRAD1, abolishes en- zymatic activity in a cellular reporter assay (Fig. 2), but not in vitro, and mislocalizes the enzyme intracellularly (Tryggvason et al., 2001). These results may reflect the need for interactions with other components for proper localization and catalytic 406 M. Liden et al. / Molecular Aspects of Medicine 24 (2003) 403–409

Fig. 1. A molecular model of RDH5. (A) Schematic model showing the topology of microsomal RDHs. (B) A molecular model of dimeric RDH5, generated based on the coordinates of the related enzyme 17b- hydroxysteroid dehydrogenase. Residues 27-287 were ﬁt into the known structure, and the N- and C- terminal membrane-anchoring segments (residues 1–26, and 288–317, respectively), have been added to the model.

A β-carotene B Retinyl Esters RDH RalDH 9-cis retinol9-cis retinal 9-cis retinoic acid

11-cis Retinol all-trans Retinol 9-cis Retinol Cytosol Visual cycle Nucleus GAL4 RXR 11-cis Retinal all-trans Retinal 9-cis Retinal

GAL4x4 TK Luciferase

all-trans Retinoic Acid 9-cis Retinoic Acid

Fig. 2. Metabolic pathways of retinoids, and the principles of the cell reporter assay. (A) All-trans-retinol, derived from dietary retinoids, is the ultimate precursor for the biologically active retinoids 11-cis-retinal, all-trans-retinoic acid and 9-cis-retinoic acid. (B) A reporter system that can be used to study the function of retinol dehydrogenases in intact cells. JEG3 cells are cotransfected with plasmids encoding retinol and retinal dehydrogenases (RDH and RalDH), as well as reporter plasmids encoding a Gal4-RXR (retinoid X receptor) fusion protein and a fusion of Gal4 DNA response elements with a TK-promoter driven luciferase gene. By addition of 9-cis-retinol as substrate for RDH, 9cRA is ultimately produced, thus activating transcription of the luciferase gene. The luciferase activity can be quantified luminometrically, and used as a measure of the activities of the retinoid metabolizing enzymes. activity. A common structural feature of SDRs is homodimer and tetramer formation (Joornvall€ et al., 1995). RDH5, and presumably CRAD1, occur as functional homodimers, as indicated by genetic and biochemical data. A patient with fundus albipunctatus was found to be compound heterozygote, expressing one active RDH5 mutant (A294P) and one inactive (R280H), leading to a trans-dominant negative M. Liden et al. / Molecular Aspects of Medicine 24 (2003) 403–409 407 effect on the active mutant exerted by the inactive mutant. Cross-linking data and molecular modelling suggested that RDH5 occurs as functional homodimers. Taken together, this implied that formation of inactive heterodimers of the RDH5 mutants caused the phenotype seen in this patient (Lideen et al., 2001). The fact that RDH5 and CRAD1 are facing the lumen of the ER has several important functional implications. For instance, the cofactor NAD+, required for the oxidative reaction, must either be transported from the cytosol, which is rich in cofactor, or produced in the lumen by as yet unknown pathways. Measurements in isolated microsomes confirm that sufficient concentration of NAD+ is present in the lumen (Bublitz and Lawler, 1987), and the milieu in the lumen is oxidative (Hwang et al., 1992), which would favor retinol oxidation. Likewise, the substrates need to be transported into the lumen of the ER by some means, and the product 9-cis-retinal is somehow translocated to the cytosol where retinal dehydrogenases (RalDHs) cata- lyze the formation of 9cRA. Since the pathway of 9cRA biosynthesis can be reconstituted in different cell lines, using the reporter system mentioned above, these functions may be carried out by general cellular mechanisms, in common to many cell types. Interestingly, the cytosolic enzyme ADH4, reported to oxidize 9-cis-retinol in vitro, was much less efficient than the microsomal enzymes in transfected cells, despite the fact that it is localized in the same compartment as the retinal dehydrogenase (Tryggvason et al., 2001). Perhaps the lumenal milieu is necessary for efficient retinol oxidation in vivo, and cytosolic enzymes may have other functions, possibly acting as retinal reductases.

4. Interactions of RDHs with other components involved in retinoid metabolism

As discussed above, the requirement of an intact cytosolic tail in RDH5 and CRAD1 for function in intact cells implies that interaction with other protein(s) is necessary for correct localization and/or catalytic activity. The nature and identity of such binding proteins remains to be determined. Interactions of RDH5 with other proteins have been shown in a few cases, although the exact functions of these are unknown. For instance, RDH5 was ﬁrst noticed through its association with RPE65 (Baavik et al., 1992). This protein is highly expressed in the RPE, localized to both the plasma membrane and the ER membranes, and has been implicated in various events of retinoid metabolism, including retinol uptake (Baavik et al., 1992), and isomerization (Redmond et al., 1998). Another RPE protein reported to interact with RDH5 is the retinal G protein-coupled receptor (RGR) (Chen et al., 2001). This retinochrome-like opsin mediates photoismerization of its bound all-trans-retinal into 11-cis-retinal, and RDH5 is proposed to reduce the product into 11-cis-retinol, although the function of this backward reaction is unclear.

5. Concluding remark

In summary, more data on the expression, compartmentalization and membrane topolgy of microsomal retinol dehydrogenases, as well as functional characterization 408 M. Liden et al. / Molecular Aspects of Medicine 24 (2003) 403–409 in intact cells and animals, should clarify the role of the individual enzymes in retinoid biosynthesis.

Acknowledgements

We thank Prof. Bengt Persson for contributing work on the molecular model of RDH5. This work was supported by The Swedish Research Council and Karolinska Institutet.

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