Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family ´ ´ Diana Dalfo, Neus Marques and Ricard Albalat ` Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Spain Keywords cephalochordates; chordate evolution; retinaldehyde reductases; retinoic acid metabolism; retinol dehydrogenases Correspondence ` R Albalat, Departament de Genetica, Facultat de Biologia, Universitat de Barcelona, Av Diagonal, 645, 08028 Barcelona, Spain Fax: +34 934034420 Tel: +34 934029009 E-mail: ralbalat@ub.edu Website: http://www.ub.edu/genetica/ indexen.htm (Received March 2007, revised May 2007, accepted 30 May 2007) doi:10.1111/j.1742-4658.2007.05904.x In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol ⁄ retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes We have now proceeded to characterize these amphioxus enzymes Kinetic studies have revealed that retinol dehydrogenase and retinol dehydrogenase are microsomal proteins that catalyze the reduction of all-trans-retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes Retinoic acid (RA) regulates critical physiologic processes in vertebrates, such as anterior–posterior pattern formation, cell proliferation, tissue differentiation, morphogenesis, and embryonic development [1] The main source of retinoids stems from the enzymatic cleavage of dietary b-carotenes, which produces retinaldehyde This, in turn, is reduced to retinol, which is subsequently esterified to retinyl esters and stored in the liver [2] Upon demand, these esters can be hydro- lyzed to retinol, which is released into the circulation to be used in target tissues, to undergo oxidation into retinaldehyde, and to be further transformed into RA Retinol dehydrogenase and retinaldehyde reductase activities are therefore major players in retinoid metabolism, making essential contributions to the physiologic control of RA availability In vertebrates, retinol dehydrogenase activity has been classically associated with two distinct protein families, the short-chain Abbreviations AKR, aldo-keto reductase; AR, aldose reductase; CRAD, cis-retinol/androgen dehydrogenase; ER, endoplasmic reticulum; GFP, green fluorescent protein; HAR, human aldose reductase; HSD, hydroxysteroid dehydrogenase; HSI-AR, human small intestine aldose reductase; MDR, medium-chain dehydrogenase ⁄ reductase; NLS, nuclear localization sequence; PAN2, pancreas protein 2; RA, retinoic acid; RRD, mouse retinal reductase; SDR, short-chain dehydrogenase ⁄ reductase FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3739 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes retinol dehydrogenases [short-chain dehydrogenase ⁄ reductase (SDR)-RDHs] and the medium-chain alcohol dehydrogenases [medium-chain dehydrogenase ⁄ reductase (MDR)-ADHs] [3,4] Despite the many biochemical studies on these two protein families, the major enzyme(s) responsible for the in vivo oxidation of retinol remains uncertain In previous studies, we analyzed the functionality and evolution of the MDR-ADH family [5–8,9] Here, we focus on the contribution of SDR-RDH enzymes to retinol ⁄ retinal metabolism Classically, RDH enzymes have been regarded as a complex vertebrate group of microsomal proteins that catalyze the conversion of retinol to retinaldehyde in vitro using NADH as cofactor [4] However, the RDH family contains many enzymes with diverse substrate specificities towards cis and trans isomeric forms, and, mostly, toward steroids Hence, attempts to determine the physiologic contribution of each RDH enzyme to RA metabolism have been impaired by the variety of enzymes as well as by the overlaps in substrate recognition Substantial multiplicity and redundancy is also present in the reductive direction of the pathway Four vertebrate protein families have been associated with retinaldehyde reduction Members of the SDR-RDH group such as RDH2, RDH5 and 17b hydroxysteroid dehydrogenase type (17bHSD9) [10–12], non-RDH SDR enzymes, including mouse retinal reductase (RRD), retinal short-chain dehydrogenase/reductase (retSDR1), photoreceptor outer segment all-trans retinol dehydrogenase (prRDH), retinal reductase (RalR1) and pancreas protein (PAN2) [13–17], MDR-ADH forms such as ADH1, ADH4 [18] and amphibian ADH8 [19], and members of the aldo-keto reductase superfamily, including human aldose reductase (AR), human small intestine aldose reductase (HSI-AR) and chicken aldo-keto reductase (AKR) [20,21], all catalyze retinaldehyde reduction in vitro To shed light on the evolutionary origin and physiologic basis of the RDH and retinaldehyde reductase multiplicity of vertebrates, analysis of the cephalochordate amphioxus is invaluable Cephalochordates are useful organisms for comparative analyses, as their low gene complexity and archetypical body plan organization suggest that they retain many ancient characteristics Amphioxus did not undergo the extensive gene duplications that occurred during early vertebrate evolution [22], but rather exhibits an RA-signaling system and a retinoid content comparable to that of vertebrates [23,24] In a previous study, we identified two enzymes, RDH1 and RDH2, that belong to the SDRRDH group in the species Branchiostoma floridae [25] Here we present experimental data showing that these 3740 two enzymes are endoplasmic reticulum (ER)-associated proteins that may participate in retinoid metabolism, by catalyzing retinaldehyde reduction Moreover, phylogenetic analysis indicates that most vertebrate RDHs derive from lineage-specific tandem duplications of an ancestral form that may resemble the current amphioxus enzymes The novel vertebrate RDH enzymes would have evolved new biochemical activities in retinoid and steroid metabolism after cephalochordate divergence, thereby contributing to the increased physiologic complexity of the vertebrate subphylum Results Enzymatic properties of recombinant RDH1 and RDH2 Amphioxus RDH1 and RDH2 proteins tagged at the N-terminus with the hemagglutinin (HA) epitope were produced in COS-7 cells and purified in the microsomal fraction The enzymatic activity of this fraction was assayed against retinoids Given that most vertebrate RDHs can catalyze cis-retinol and ⁄ or trans-retinol oxidation, these were the substrates initially evaluated Indeed, mouse RDH1 (kindly provided by J L Napoli, University of California, Berkeley, CA, USA) was used to monitor the retinol oxidation assay Unexpectedly, the oxidative activity observed for the amphioxus enzymes was below the detection capacity of the assay, < 0.002 nmol (Fig 1A–C), although a wide range of conditions were used: from pH to 9, 5–12.5 lm all-trans-retinol, 0.5–2 mm NAD+ and NADP+, and 10–100 lg of microsomes obtained from independent assays Negligible activity was also observed when 9-cis-retinol was assayed (data not shown) Next, we analyzed whether RDH1 and RDH2 exhibited reductase activity (Fig 1D,E) Retinol production in vitro increased 2.5-fold and 25-fold for RDH1 and RDH2, respectively, in the presence of NADH, as compared to controls However, these differences were not detected with NADPH, even though COS cells showed intrinsic NADPH-dependent retinal reductase activity [12] The specific activity of each amphioxus enzyme was 0.25 nmol of retinolỈmin)1Ỉ(mg of microsomes))1 for RDH1 incubated with 15 lm all-trans-retinal, and 1.4 nmol of retinolỈmin)1Ỉ(mg of microsomes))1 for RDH2 with 12.5 lm all-trans-retinal (Table 1) Furthermore, to examine whether RDH forms had isomer specificity, we also assayed the RDH1 and RDH2 activities toward 9-cis-retinal However, only residual 9-cis-retinal reductase activity, less than 0.03 nmolỈ min)1Ỉmg)1, was detected for these two enzymes (Fig 1F,G) The reaction products were extracted and FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes Fig Enzymatic activity of amphioxus RDH1 and RDH2 enzymes The biochemical activity of the microsomal fraction of COS-7 cells transfected with amphioxus HA-RDH1expressing (A, D, F), HA-RDH2-expressing (B, E, G) and mouse Rdh1-expressing (C) constructs was analyzed For oxidative reactions (A–C), the microsomal fraction (15 lg) was incubated with all-trans-retinol (10 lM) and NAD+ (1 mM) at pH 8.0 for 15 at 37 °C For retinal reduction, the microsomal fraction (15 lg) was incubated with 10 lM all-trans-retinal (D, E) or 9-cis-retinal (F, G) and NADH (1 mM) at pH 6.0 for 15 at 37 °C Elution was monitored at 380 nm for retinal detection (A–C) and 325 nm for retinol (D–G) detection The values for all-transretinaldehyde reduction of amphioxus RDH1 (H) and RDH2 (I) were determined at mM NADH using eight concentrations of substrate, from 0.5 to 20 lM and from 0.5 to 15 lM for RDH1 and RDH2, respectively The apparent Km values for cofactor NADH were determined at 15 lM and 12.5 lM alltrans-retinaldehyde for RDH1 (J) and RDH2 (K), respectively, using six concentrations of cofactor, from 0.005 to 1.5 mM Assays were performed with 15 lg of microsomes for 15 at 37 °C Each point represents the average of three replicates analyzed by RP-HPLC, and the kinetic constants of the two RDHs for all-trans-retinal were determined (Fig 1H,I; Table 1) The apparent Km values of RDH1 and RDH2 (8.7 lm and 8.9 lm, respectively) were similar, whereas the maximum specific activities (0.3 nmolỈmin)1Ỉmg)1 and 2.3 nmolỈmin)1Ỉmg)1, respectively) and the maximum specific activities ⁄ Km ratios (0.03 and 0.26, respectively) were > 7.5-fold higher for RDH2 than for RDH1 Finally, the apparent cofactor Km values of amphioxus enzymes (Fig 1J,K; Table 1) were 224 lm and 98 lm NADH for RDH1 and RDH2, respectively, whereas no significant activity was detected with NADPH This cofactor preference is consistent with the presence and absence of specific amino acids at certain positions in the amphioxus enzymes (Fig 2A): both enzymes contain the D and T residues at the equivalent positions of cow RDH5 for NADH specificity, and lack any positively charged amino acid at the corresponding position of the rat RDH2 K64, which may be essential for NADPH preference [26] Activity of recombinant RDH1 and RDH2 in intact cells Amphioxus RDH1 and RDH2 and mouse RDH1 were expressed in COS-7 cells to evaluate their activities with retinoids in an intact cell system In agreement FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3741 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes Table All-trans-retinal activity and kinetic constants of B floridae RDH enzymes compared with those of known vertebrate retinal reductases Values are from this work, B floridae RDH1 (BfRDH1) and BfRDH2, or from the literature [6-10,13,16,17,25] ND, not determined HAR, human aldose reductase All-trans-retinal NADH Specific activity (nmolỈ min)1Ỉmg)1) BfRDH1 BfRDH2 RalR1 PAN2 RRD retSDR1 HAR HIS-AR Chicken AKR RDH2 17bHSD9 RDH5 Maximum specific activity (nmolỈ min)1Ỉmg)1) Km (lM) 0.25 1.4 ND ND ND 0.04 ND ND ND 0.3 2.3 18 27 40 ND 15 193 170 8.7 8.9 0.5 0.08 2.3 ND 10 19 32 0.25 0.17 16 ND ND ND ± ± ± ± ± 0.06 0.5 0.05 1 a ±1 ± 4a ± 15a ND ND ND Km (lM) ± ± ± ± 4.1 3.8 0.05 0.02 ±2 ±4 ±4 224 98 1300 1060 ND ND ND ND ND ± ± ± ± 81 25 200 70 retinal, respectively RDH2 enzymes were more efficient than RDH1 enzymes, and generated 64 and 134 pmol of retinol in the same assay conditions Overall, transfection with RDH1 and RDH2 cDNA increased the level of retinaldehyde reduction by  35% and  50%, respectively, as compared to the mock-transfected cells Mouse RDH1, in contrast, oxidized retinol to retinaldehyde (Fig 3B) but did not support retinaldehyde reduction Indeed, mouse RDH1 decreased the amount of retinol generated in the assays with retinaldehyde incubation (data not shown), suggesting that the substrate used by this enzyme was the retinol generated from retinaldehyde by endogenous COS-7 cell reductase activity Intracellular localization ND ND ND kcat values in min)1 a with the biochemical analysis of the microsomal-purified enzymes, amphioxus RDH1 and RDH2 catalyzed the reduction of all-trans-retinaldehyde into all-transretinol in intact cells (Fig 3A), whereas RDH1- and RDH2-transfected cells showed no differences from mock-transfected cells in the generation of all-transretinaldehyde from all-trans-retinol (Fig 3B) Mocktransfected COS-7 cells reduced all-trans-retinaldehyde, indicating that the cells harbor reductases Transfection with amphioxus RDH1 cDNA produced a net 47 pmol and 99 pmol of retinol per mg of total protein after h of incubation with 10 lm and 20 lm of COS-7 cells were transiently transfected with constructs expressing the amphioxus RDH1 and RDH2 enzymes fused to several peptides: HA epitope, green fluorescent protein (GFP) and b-galactosidase In agreement with the RDH1 and RDH2 purification in the microsomal fraction (Fig 2D), immunostaining of cells expressing HA-RDH1 and HA-RDH2 proteins revealed a typical pattern of ER-associated proteins, with no nuclear staining or plasma membrane localization observed (Fig 2B,C) GFP fusion was used to visualize the intracellular localization in living cells, thereby avoiding any artefacts caused by the cell fixation process RDH2 fused to GFP either at the C-terminus (RDH21)335-GFP) or at the N-terminus (GFP-RDH21)335) of the enzyme (Fig 2E,I) exhibited a pattern that overlapped with the ER-Tracker Blue White DPX, which was used as a specific ER marker in living cells (Fig 2F,J) The subcellular localization of the RDH2 enzyme (RDH21)335) fused to the b-galactosidase protein was also consistent with a typical pattern of ER-associated proteins (Fig 2M) Fig ER subcellular localization of amphioxus RDH1 and RDH2 proteins (A) Sequence alignment of amphioxus RDH1 and RDH2 enzymes Amino acid substitutions are shown and identities are represented by dashes The active site (YTVAK) and the cofactor-binding motifs are marked in bold The D43, T67 and A69 residues, involved in cofactor specificity, are indicated by asterisks Flanking the N-terminal hydrophobic segment, the LERGR motif is underlined Arrows indicate the truncated RDH2 forms fused to GFP or to b-galactosidase proteins (B, C) Immunostaining with an antibody to HA of COS-7 cells transfected with constructs encoding HA-RDH1 and HA-RDH2, respectively, and examined using confocal microscopy (D) Western blot of the pellets after 13 000 g (lanes and 4) and 100 000 g centrifugations (lanes and 3, microsomal fractions) of homogenates of COS cells transfected with HA-RDH1 (lanes and 2) and HA-RDH2 (lanes and 4) (E–L) In vivo localization of RDH2 in the ER of the cells COS-7 cells were transfected with constructs encoding RDH21)335-GFP (E), RDH21)28GFP (G), RDH21)58-GFP (H), GFP-RDH21)335 (I), GFP-RDH2295)335 (K) and GFP (L) ER-tracker Blue White DPX marker was used to specifically visualize the ER in living cells (F, J) (M–R) Localization of RDH2-b-galactosidase chimeric proteins COS-7 cells were transfected with constructs encoding the full-length (RDH21)335) (M) and four C-terminal truncated RDH2 forms (RDH21)229, RDH21)165, RDH21)137 and RDH21)58) (N–Q, respectively) fused to the NLS-b-galactosidase protein, and with the pb-galactosidase-N2 empty vector (R) The pb-galactosidase-N2 vector contains a nuclear localization sequence (NLS) 5¢ to the LacZ gene The SV40 NLS localizes the b-galactosidase codified by the empty vector to the nucleus Cells were immunostained with an antibody to b-galactosidase and examined using a Zeiss Axiophot fluorescence microscope 3742 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ D Dalfo et al To analyze the contribution of protein domains to the ER anchorage, the localization of the full-length enzyme was compared with those of five truncated forms The pattern of the full-length construct was essentially identical to that of the C-terminal truncated Amphioxus retinol dehydrogenase enzymes RDH2 forms (RDH21)229, RDH21)165, RDH21)137, and RDH21)58 RDH21)28) fused either to b-galactosidase (Fig 2N–Q) or to GFP (Fig 2G,H), whereas the b-galactosidase and GFP controls showed a signal, mainly in the nucleus (Fig 2L,R) The observation FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3743 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes ER of transfected COS cells, but showed a diffuse signal similar to that of the GFP control (compare Fig 2K,L) Evolution of the RDH group Fig Synthesis of retinol and retinaldehyde in transfected COS-7 cells expressing amphioxus RDH enzymes (A) Different concentrations (5, 10 and 20 lM) of all-trans-retinaldehyde were added to COS-7 cells expressing RDH1 (black bars) and RDH2 (white bars) Retinol was extracted from the cells and analyzed by HPLC The bars represent the net retinol production per mg of total protein after h of incubation with the three substrate concentrations (B) Retinol oxidation in transfected cells was also evaluated for RDH1, RDH2 and mouse RDH1 (gray bars), which was used as a positive control of the reaction The bars represent the net retinal production per mg of total protein after h of incubation with 5, 10 and 20 lM all-trans-retinol that nuclear or cytosolic staining was not increased for any construct suggested that the N-terminal segment would be sufficient to target and anchor the protein to the ER membranes Furthermore, we analyzed the contribution of the C-terminal end to ER localization We fused the last 41 amino acids of the amphioxus RDH2 enzyme (a region equivalent to the reported C-terminal segment of mouse RDH1 [27]) to GFP: GFP-RDH2295)335 The protein did not localize to the tblastn comparisons showed that the sequences most similar to the amphioxus enzymes were those of the vertebrate RDHs (E-value ¼ 2e-75 and 1e-71 with Pan troglodytes, similar to sterol ⁄ retinol dehydrogenase, for RDH1 and RDH2, respectively) In the phylogenetic analysis, amphioxus RDH branched outside a clade comprising the ‘classic’ SDR-RDH1 ⁄ ⁄ ⁄ ⁄ ⁄ ⁄ ⁄ (RDH1–7 ⁄ 9) members, which includes six human enzymes [similar-RDH2, RDH4, orphan short-chain dehydrogenase/reductase (SDR-O), RDH, RDH5 and dehydrogenase/reductase member (DHRS9)], eight rat forms (similar-RDH1, RDH2, similar-RDH2, RDH3, SDR-O, 17b-HSD19, RDH5 and DHRS9) and 11 mouse proteins [RDH1, RDH9, RDH6, truncatedRDH, similar-RDH, cis-retinol/androgen dehydrogenase (CRAD)-L, RDH7, SDR-O, 17b-HSD19, RDH5 and DHRS9] (Fig 4A) Except for DHRS9, the genes encoding these enzymes were not spread over several chromosomes, but rather clustered in the human genome at 12q13–14 and in the syntenic regions of rat and mouse chromosomes and 10, respectively [28] (Fig 4B) DHRS9 genes are located in human chromosome 2, rat chromosome and mouse chromosome 2, which would be paralogous to chromosomes 12, and 10, respectively [29] The overall analysis, examining the topology of the phylogenetic tree and the position of each gene inside the cluster, was informative regarding the orthology relationships of the distinct enzymes, and allowed us to define five RDH classes (Fig 4A) It is of note that other vertebrate SDRs (some reported as RDH enzymes), such as RDH8, RDH10, RDH11, RDH12, RDH13, RDH14, similar to epidermal retinal Fig (A) Phylogenetic relationship of the RDH1–7 ⁄ and other retinoid ⁄ steroid active SDR forms from human (Hs), rat (Rn) and mouse (Mm) genomes with the amphioxus RDH enzymes A neighbor-joining tree was generated with the CLUSTALX program, and confidence in each node was assessed by 1000 bootstrap replicates The RDH1–7 ⁄ cluster comprises all the vertebrate sequences that group with the amphioxus enzymes Additional enzymes involved in retinoid ⁄ steroid metabolism appear to be distantly related (less than 55% of sequence identity in the region used for the tree reconstruction, data not shown), and were therefore considered to be members of distinct SDR groups The bootstrap values defining each group are shown (black numbers) Internally, the RDH1–7 ⁄ enzymes grouped into five classes, I–V The bootstrap values defining each class are shown (red numbers) (B) Structural organization of the human, rat and mouse RDH clusters using the Map Viewer website from NCBI The name of the each Rdh gene (black boxes) is indicated Alternative names for each gene are listed in supplementary Table S2 Orthology relationships among genes of several species are indicated (continuous lines) Notice that Rdh5 genes are in the same chromosome but outside the RDH clusters (dotted line), and that Dhrs9 genes are located in distinct chromosomes Genes flanking the RDH sequences are also depicted (green boxes) TAC3, tachykinin 3; KIAA0352 (ZBTB29), zinc finger and BTB domain containing 39; ADMR, adrenomedullin receptor; PRIM1, primase polypeptide 1; NACA, nascent-polypeptide-associated complex alpha-polypeptide; CD63, CD63 antigen; BLOC1S1, biogenesis of lysosome-related organelles complex-1, subunit 1; ABCB11, ATP-binding cassette, subfamily B, member 11; LRP2, low-density lipoprotein receptor-related protein 3744 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes A B FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3745 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes dehydrogenase (RaDH9), epidermal retinal dehydrogenase (eRaDH2), retSDR1, DHRS4, 17b-HSD11, 17b-HSD12, 11b-HSD11, 11b-HSD12 and short-chain dehydrogenase/reductase 10 isoform B (SCDR10B), branched outside the vertebrate–cephalochordate clade (Fig 4A) and were located in diverse nonparalogous mammalian chromosomes These enzymes would be therefore distantly related to the RDH1–7 ⁄ forms and should be considered members of separate enzyme families Indeed, the position of the amphioxus enzymes in the phylogenetic tree implied that these families are ancient, pre-dating the emergence of the chordate phylum Discussion The biochemical characterization revealed that the amphioxus RDH enzymes catalyzed all-trans-retinal reduction (Table 1, Figs and 3) Unfortunately, comparison with other RDHs was limited to rat RDH2, mouse 17bHSD9 and RDH5, as the reductase capacity of most vertebrate RDHs has not been assayed Thus, we also compared amphioxus data with those from other non-RDH vertebrate retinal reductases, such as retSDR1, RalR1, PAN2, RRD, human AR, HSI, and chicken AR (Table 1), although the comparison was also hindered by the variety of assay conditions used We have shown that amphioxus enzymes show isomer preference, trans versus cis retinal forms, as occurs with the vertebrate RalR1, PAN2, RRD, retSDR1, prRDH, HSI and chicken AKR enzymes Moreover, although retinol ⁄ retinal interconversion is a reversible reaction, neither amphioxus RDH1 or RDH2 showed significant activity towards retinol in the in vitro assays with microsomal proteins or in the intact cell systems This strict preference towards the reductive direction has been reported for other retinoid-active enzymes (e.g the vertebrate retinal reductases RRD [13], HAR [20,21] and prRDH [15]), whereas other enzymes (RalR1 [30], PAN2 [17], HSI and chicken AKR [20,21]) also catalyze retinol oxidation, albeit with considerably lower efficiency The specific activities towards all-trans-retinal of amphioxus enzymes were 0.25 nmolỈmin)1Ỉmg)1 for RDH1 and 1.4 nmolỈmin)1Ỉ mg)1 for RDH2; these were 6.3-fold and 23-fold higher, respectively, than that reported for retSDR1 (0.04 nmolỈmin)1Ỉmg)1), a photoreceptor enzyme that reduces all-trans-retinal in the visual cycle [14] The specific activity of RDH2 was 5.6-fold and 10.8-fold higher than that of rat RDH2 (0.25 nmolỈmin)1Ỉmg)1) [10] and mouse 17bHSD9 (0.13 nmolỈmin)1Ỉmg)1) [12], respectively, whereas the activity of amphioxus RDH1 was comparable to those of these enzymes In contrast, 3746 the amphioxus enzymes showed lower retinaldehyde reductase efficiency than some vertebrate enzymes The specific activity of mouse RDH5 with all-trans-retinal (16 nmolỈmin)1Ỉmg)1 [11]) was higher than that of either RDH1 or RDH2; RalR1 [30], PAN2 [17] and RRD [13] showed lower Km and higher maximum specific activity values for all-trans-retinal (Table 1) and therefore higher maximum specific activity ⁄ Km ratios, which are a measure of the catalytic effectiveness of the enzymes; the AKR members (human AR, HSI and chicken AKR) [20,21] showed similar Km values but higher maximum specific activities (Table 1), which also implied higher maximum specific activity ⁄ Km ratios, i.e greater effectiveness, for the vertebrate AKR than for the cephalochordate forms Overall, these data support our finding that amphioxus RDH shows retinal reductase activity within the range reported for diverse vertebrate enzymes The most significant difference between the amphioxus and the other retinal reductases was, nevertheless, their preference for the NADH cofactor To our knowledge, these are the first SDR retinaldehyde reductases reported to use NADH instead of NADPH Conventionally, cofactor preference had been directly related to the oxidative or reductive direction of the reaction Therefore, it was assumed that oxidative RDHs would be NADH-dependent, whereas NADPH enzymes would catalyze the reductive reaction This hypothesis was based on the ratios between the oxidized and reduced forms of the coenzymes [31] It appears, however, that cofactor ratios vary greatly among organs and cell types, and that the redox status can be greatly influenced by external factors [32] Noticeably, amphioxus enzymes have the capacity to reduce retinaldehyde to retinol in intact cells (Fig 3), suggesting that the endogenous NADH level in COS-7 cells is enough to allow this reduction Our data support the contention that coenzyme preference does not necessarily constrain the direction of the reaction In fact, several RDHs (e.g human, mouse and bovine RDH10 [33] and human RDH-E2 [32]) prefer NADP to NAD as a cofactor Structurally, most of the cytosolic SDR enzymes are composed of 250–280 amino acid residues [34,35], whereas the membrane-associated SDR enzymes are extended at both the N-terminal and C-terminal ends by up to about 350 amino acids [36,37] Amphioxus RDH1 and RDH2 were 332 and 335 amino acids long, respectively, and their subcellular localization in transfected COS-7 cells concurred with that of ER-associated proteins The observation that nuclear or cytosolic staining was not increased for any C-terminal truncated constructs indicated that the N-terminal FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ D Dalfo et al segment was sufficient to target and anchor the protein to the ER membranes As the shortest segment was only 28 amino acids long (from the initial methionine to the LERGR motif), it can be assumed that the signaling sequence for ER localization falls in this region of the protein In addition to the targeting function, signal sequences are also crucial in protein topogenesis, as they participate in the final cytosolic ⁄ lumenal orientation The most prominent determinant of signal orientation is the distribution of charged amino acids at either end of the hydrophobic sequence According to the ‘positive-inside’ rule, the most positively charged flanking transmembrane segment is usually found on the cytosolic side of the membrane [38,39] Amphioxus RDH did not show positively charged amino acids at the N-terminus of the signaling sequence, but rather contained the L24ERGR motif at the C-terminus of the hydrophobic sequence, thereby resembling the R19ERQV, R19ERKV, R19VRQV and R19DRQ(S ⁄ C) sequences of a number of vertebrate RDHs [40] This motif would predict, therefore, a cytosolic orientation of the amphioxus enzymes For several RDH enzymes, the C-terminal transmembrane segment forms a hydrophobic helix-turnhelix that is sufficient to retain them in the ER, e.g CRAD1 [41] We fused the last 41 amino acids of the amphioxus RDH2 enzyme (a region equivalent to the reported C-terminal segment of mouse RDH1 [27]) to GFP This protein did not localize to the ER of transfected COS cells (Fig 2K), and therefore the C-terminal end of amphioxus RDH2 was not sufficient for ER targeting Amphioxus RDH2 was structurally more similar to the enzymes that rely exclusively on the N-terminal hydrophobic segment as membrane anchor (e.g mouse RDH1 [27], human 11bHSD1 [42], human 11bHSD2 [43] and human RalR1 [16]) than to the other RDHs such as bovine RDH5 [44], mouse RDH4 [45] and mouse CRAD1 [41,46], which would be anchored to both the N-terminal and C-terminal hydrophobic segments Finally, evolutionary analysis including the amphioxus enzymes highlighted the relevance of using evolutionary criteria rather than biochemical classifications for gene nomenclature and family description The phylogenetic tree and the genomic organization now permit a proper definition of the vertebrate RDH1–7 ⁄ group and reveal an internal classification of mammalian RDH1–7 ⁄ enzymes into five classes, pointing to recurrent gene tandem duplications as the most likely mechanism for the cluster organization of the Rdh genes In a recent study [47], Belyaeva & Kedishvili proposed a model for the evolution of the vertebrate RDH1–7 ⁄ group (referred to as the RDOH-like SDR Amphioxus retinol dehydrogenase enzymes group in their article) On the basis of a comparative genomic and phylogenetic analysis that included several vertebrate species, these authors suggested that early in vertebrate evolution, an initial tandem duplication of the Rdh ancestor gave rise to the ‘Dhrs9 ⁄ Rdhl– 11-cis-RDH-homolog’ cluster The 11-cis-RDH-homolog gene was afterwards duplicated by a mechanism that implied translocation of the new copy to another region of the genome to generate the 11-cis-RDH ⁄ Rdh5 gene Later on, the 11-cis-RDH ⁄ Rdh5 gene underwent several tandem duplication events in its new chromosomal location, which led to the appearance of the current RDH cluster in tetrapods However, an alternative evolutionary model is possible (Fig 5) We hypothesize that an initial tandem duplication of an Rdh ancestor gave rise to a two-gene cluster, which was further duplicated, probably as a result of the genome duplication events that took place during early vertebrate evolution [22] During fish evolution, one gene was lost, leading to the ‘Rdh5 (AAH97151) + Dhrs9 (Rdhllike) + Rdhl’ combination currently found in zebrafish [47] In amphibians and mammals, extra tandem duplications produced the RDH clusters found in Xenopus, human, mouse, rat, dog and cow [47] (Fig 4B) Eventually, the mammalian Rdhl ⁄ CX410306 ortholog was lost The closer phylogenetic relationship of RDH5 ⁄ 11cis-RDH to Rdhl ⁄ CX410306 enzymes than to RDH4SDR-O-RDH forms [47] is consistent with this model Furthermore, the observation that the Ddrs9 genes and the Rdh5-RDH cluster are located in paralogous chromosomes also supports our hypothesis In conclusion, the analysis of amphioxus enzymes contributes to improving our understanding of the functional complexity of vertebrate gene families regarding retinoid metabolism However, to date, no convincing enzymes for retinol oxidation have been found among cephalochordate RDH members The full genome sequence of amphioxus, currently being released, will allow comprehensive searches for novel candidates, which may also have relevant physiologic roles in the retinoid pathway of vertebrates Experimental procedures Expression of HA-RDH, GFP-RDH, and b-galactosidase-RDH proteins To produce RDH1 and RDH2 proteins tagged at the N-terminus with the HA epitope, the full-length coding sequences of the Rdh1 and Rdh2 genes were PCR-amplified (oligonucleotides 1–2, and 3–4, respectively; the oligonucleotide sequences used in this study are provided in supplementary Table S1) from plasmids containing the Rdh1 and FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3747 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes Fig Hypothetical model of RDH1–7 ⁄ group evolution leading to the current vertebrate multiplicity Fish and amphibian arrangements are those described for Danio rerio and Xenopus tropicalis in [47] Rdh2 cDNAs and cloned in the pACT2 vector (Clontech, Mountain View, CA, USA) The HA-tagged Rdh1 and Rdh2 coding fragments were released from the pACT2 vector and cloned into the pCDNA3 vector (Invitrogen, Carlsbad, CA, USA) To fuse amphioxus RDH2 either at the N-terminus or C-terminus of the GFP, the full-length coding region, two C-terminal truncated RDH2 forms and one N-terminal truncated RDH2 form were PCR-amplified (RDH21)335, oligonucleotides and 6, amino acids 1–335, full-length; RDH21)58, oligonucleotides and 7, amino acids 1–58, truncated just after the cofactor-binding sequence GXXXGXG; RDH21)28, oligonucleotides and 8, amino acids 1–28, truncated after the LERGR motif; RDH2295)335, oligonucleotides and 6, amino acids 295–335) and cloned into the pEGFP-N2 and pEGFP-C2 vectors (Clontech) To produce both the full-length and the four C-terminal truncated RDH2 enzymes fused at the N-terminus of b-galactosidase, the coding regions of Rdh2 were generated by PCR amplification: RDH21)335 (oligonucleotides 10–11; amino acids 1–335, full-length), RDH21)229 (oligonucleotides 10–12; amino acids 1–229, lacking the C-terminal end), RDH21)165 (oligonucleotides 10–13; amino acids 1–165, truncated just before the active site, YXXXK), RDH21)137 (oligonucleotides 10–14; amino acids 1–137, truncated after the GLVNNAG region), and RDH21)58 (oligonucleotides 10–15; amino acids 1–58, truncated just 3748 after the cofactor-binding sequence GXXXGXG) The design of these constructs was based on the predicted transmembrane segments of the RDH2 enzyme given by the tmpred [48], das [49] and hmmtop [50] programs (data not shown) The five PCR fragments were cloned in the pbGalN2 vector, in frame at the 5¢ end of the coding region of LacZ This vector expresses b-galactosidase protein fused to a nuclear localization sequence (NLS) driven by the strong human cytomegalovirus immediate early promoter, and was created by cloning the NLS-LacZ gene from the PSP-1.72b-galactosidase plasmid [51] into a pEGFP-N2 vector from which the Gfp coding sequence had been removed The SV40 NLS localizes b-galactosidase to the nucleus All the constructs were verified by sequencing For subcellular localization experiments, COS-7 cells (African green monkey kidney cells; ECACC, Porton Down, Wiltshire, UK) were grown in DMEM with GlutaMAX II (Invitrogen) and 4500 mgỈL)1 d-glucose, supplemented with 10% fetal bovine serum, 100 mL)1 penicillin G and 100 lgỈmL)1 streptomycin in a 5% CO2 humidified atmosphere at 37 °C Cells were seeded on glass coverslips into 12-well plates (5 · 104 cells per well) and transfected 24 h later with 0.5 lg of purified plasmid DNA per well, using 2.3 lL of FuGene6 (Roche, Basel, Switzerland) Cells were transfected with constructs encoding HA-RDH1, HA-RDH2, and the full-length and the four C-terminal FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS ´ D Dalfo et al truncated RDH2-b-galactosidase proteins Twenty-four hours later, cells were fixed with 4% formaldehyde for 15 at room temperature, and permeabilized with methanol for at room temperature Nonspecific binding was blocked with 1% BSA for h at room temperature Cells were then incubated for h at room temperature with a : 200 dilution of rabbit anti-HA serum (Sigma-Aldrich, St Louis, MO, USA) or with a : 1000 dilution of rabbit anti-b-galactosidase serum (ICN, Costa Mesa, CA, USA) Cells were then incubated with a : 200 dilution of Rhodamine Red-X-conjugated donkey anti-(rabbit IgG) (Molecular Probes, Eugene, OR, USA) for h at room temperature in the dark After each incubation with antibody, cells were washed twice in NaCl ⁄ Pi · for at room temperature The analyses of HA-RDH1 and HA-RDH2 constructs were performed with a SP2 Leica confocal microscope (Leica Microsystems GmbH, Wetzlar, Germany), and those of RDH2-b-galactosidase constructs were done with a Zeiss Axiophot fluorescence microscope (Carl Zeiss, Oberkochen, Germany) GFP fusion proteins were used to visualize the intracellular localization in living cells Twenty-four hours after transfection with Rdh21)335-pEGFP-N2, Rdh21)58-pEGFPN2, Rdh21)28-pEGFP-N2, pEGFP-C2-RDH21)335 and pEGFP-C2-RDH2295)335 constructs (RDH21)335-GFP, RDH21)58-GFP, RDH21)28-GFP, GFP-RDH21)335 and GFP-RDH2295)335, respectively), GFP localization was analyzed with a Leica DMIL fluorescence microscope Alternatively, transfected cells on coverslips were rinsed with NaCl ⁄ Pi · and incubated with a lm prewarmed solution of ER-Tracker Blue White DPX (Molecular Probes) for 30 at 37 °C Coverslips were rinsed and mounted using a drop of Vectashield H-1000 (Vector Laboratories, Burlingame, CA, USA) Analyses were done with a Zeiss Axiophot fluorescence microscope Enzymatic activity and kinetic constants To analyze the enzymatic activity of HA-RDH1 and HA-RDH2 proteins, COS-7 cells were seeded into Petri dishes (1 · 106 cells per plate), and transfected 24 h later with 2.4 lg of plasmid DNA and 14.4 lL of FuGene6 (Roche) Control experiments were performed with cells transfected with equal amounts of the empty vector After 72 h of transfection with HA-constructs, COS-7 cells were collected by centrifugation, at 400 g, Eppendorf centrifuge 5702, rotor 5702R (Eppendorf, Hamburg, Germany), resuspended in 20 mm Hepes, 150 mm KCl, mm EDTA, 10% sucrose and mm dithiothreitol (pH 7.5), and homogenized by sonication for (Sonifier 250; Branson, Danburg, CT, USA) Debris and unbroken cells were removed by two centrifugation steps at 13 000 g for 15 at °C, Eppendorf minispin The microsomes were subsequently collected by ultracentrifugation at 100 000 g for h at °C (Optima TL ultracentri- Amphioxus retinol dehydrogenase enzymes fuge, rotor TLS55; Beckman Coulter Inc., Fullerton, CA, USA), and resuspended in 0.1 m potassium phosphate, 0.1 mm EDTA, 0.1 mm dithiothreitol and 20% glycerol Protein concentrations were determined by the Bradford method (Bio-Rad, Hercules, CA, USA), using BSA as standard Microsomes were stored at ) 80 °C until analysis For western blot analysis, proteins were resolved by 12.5% SDS ⁄ PAGE and transferred onto a nitrocellulose membrane Nonspecific binding was blocked with 10% nonfat milk and 10% NaCl ⁄ Pi · 10 The membrane was incubated with a : 1000 dilution of anti-HA mouse serum (BAbCO, Richmond, CA, USA) for h at room temperature, washed three times with MPT (0.5% nonfat milk, 10% NaCl ⁄ Pi · 10 and 0.05% Tween-20) for at room temperature, and then incubated with a : 3000-fold dilution of peroxidase-conjugated sheep anti-(mouse IgG) The filters were washed again three times with MPT, and antibody binding to HA-RDH1 and HA-RDH2 proteins was visualized with an ECL western blotting analysis system (Amersham Biosciences, Little Chalfont, UK) Oxidative and reductive activities of RDH1 and RDH2 proteins were determined using commercial trans and cis isomers of retinoids (Sigma-Aldrich), except for 9-cis-retinol, which was synthesized by chemical reduction of 9-cis´ retinal with NaBH4 (kindly provided by X Pares, Universi` tat Autonoma de Barcelona, Bellaterra, Spain) Purity was checked by HPLC Stock solutions of retinoids were prepared in ethanol Ethanol-dissolved retinoids were solubilized in the reaction buffer by a 10 sonication in the presence of equimolar delipidated BSA (Sigma-Aldrich) Concentrations of ethanol in the reaction mixture did not exceed 1% Retinoid concentrations were determined on the basis of the corresponding extinction coefficients at the appropriate wavelengths in an aqueous buffer, as previously described [52] Catalytic activity was assayed in 90 mm potassium phosphate and 40 mm KCl at pH 6.0 for reductive activity, and at pH 8.0 for oxidative activity in siliconized Eppendorf tubes Reactions were started by the addition of cofactor and carried out for 15 at 37 °C in 0.5 mL The amount of protein used in the reaction mixture was 15 lg The reaction was terminated by the addition of an equal volume of cold methanol supplemented with 20 lm butylated hydroxytoluene Retinoids were extracted using solid-phase extraction on a Waters Oasis HLB Extraction cartridge (Waters, Milford, MA, USA), following the manufacturer’s instructions, and analyzed using a Waters Alliance HPLC System The elution was monitored on a Waters 2695 Alliance ⁄ PDA Waters 2996 at 380 nm for retinal isomers and 325 nm for retinol isomers Retinoids were separated using an RP-HPLC column (Kromasil 100 C18 lm 25 · 0.46 cm; Teknokroma, Sant Cugat ` del Valles, Spain) with acetonitrile ⁄ acetate ammonium 1% (85 : 15) as mobile phase The flow rate was 1.8 mLỈmin)1 Under these conditions, elution times were as follows: 10.9 for 9-cis-retinol, 11.4 for all-trans-retinol, FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3749 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes 14.8 for 9-cis-retinal, and 15.3 for all-trans-retinal Retinoids were quantitated by comparing their peak areas with a calibration curve constructed from peak areas of a series of standards The peak detection limit was about pmol of retinoid The apparent Km values for the reduction of all-trans-retinal were determined at mm NADH using eight concentrations of substrate (from 0.5 to 20 lm and from 0.5 to 15 lm for RDH1 and RDH2, respectively) The apparent Km values for cofactor NADH were determined at 15 lm and 12.5 lm of all-trans-retinal for RDH1 and RDH2, respectively, using six concentrations of cofactor, from 0.005 to 1.5 mm Kinetic analyses were performed with 15 lg of protein The background level of product formed in control reactions using microsomes from COS-7 cells transfected with an empty vector was taken into account and subtracted from each experimental data point Kinetic constants obtained by nonlinear Marquardt’s regression analysis were calculated from at least three independent experiments using the commercial grafit curve-fitting software (Erithacus Software Ltd, Horley, UK) and expressed as means ± SD To measure activity in intact COS-7 cells, 24 h after transfection the medium was replaced with fresh medium containing delipidated BSA (5 mgỈmL)1) and all-transretinol or all-trans-retinal at three concentrations (5, 10 or 20 lm) After h of incubation, cells were lysed, and retinoids were extracted and analyzed by RP-HPLC as described above Evolutionary analysis Accession numbers for all the sequences used in this study are provided in supplementary Table S2 Protein sequence alignments and a neighbor-joining tree were generated with clustalx [53] and drawn with the treeviewppc program [54] Confidence in each node was assessed by 1000 bootstrap replicates The N-terminal and C-terminal regions of several SDR enzymes were highly variable in length and sequence, and produced unreliable alignments These regions were therefore excluded from the phylogenetic analyses, and only alignments from amino acids 30–293, referred to the amphioxus sequences, were considered Human similar-RDH2, rat similar-RDH1 and similarRDH2, and mouse truncated RDH and similar-RDH sequences were excluded from the phylogenetic analysis, as they were pseudogenes and ⁄ or partial RDH sequences The genomic organization of the RDH group was deduced using the Map Viewer website from NCBI: http:// www.ncbi.nlm.nih.gov/mapview/ Acknowledgements ´ ´ We thank X 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http://www.blackwell-synergy.com Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS ... paralogous chromosomes also supports our hypothesis In conclusion, the analysis of amphioxus enzymes contributes to improving our understanding of the functional complexity of vertebrate gene families... The overall analysis, examining the topology of the phylogenetic tree and the position of each gene inside the cluster, was informative regarding the orthology relationships of the distinct enzymes, ... comparable to those of these enzymes In contrast, 3746 the amphioxus enzymes showed lower retinaldehyde reductase efficiency than some vertebrate enzymes The specific activity of mouse RDH5 with