Báo cáo khoa học: Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family pot

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Analysis of the NADH-dependent retinaldehyde reductaseactivity of amphioxus retinol dehydrogenase enzymesenhances our understanding of the evolution of the retinoldehydrogenase familyDiana Dalfo´, Neus Marque´s and Ricard AlbalatDepartament de Gene`tica, Facultat de Biologia, Universitat de Barcelona, SpainRetinoic acid (RA) regulates critical physiologic pro-cesses in vertebrates, such as anterior–posterior patternformation, cell proliferation, tissue differentiation,morphogenesis, and embryonic development [1]. Themain source of retinoids stems from the enzymaticcleavage of dietary b-carotenes, which produces retin-aldehyde. This, in turn, is reduced to retinol, which issubsequently esterified to retinyl esters and stored inthe liver [2]. Upon demand, these esters can be hydro-lyzed to retinol, which is released into the circulationto be used in target tissues, to undergo oxidation intoretinaldehyde, and to be further transformed into RA.Retinol dehydrogenase and retinaldehyde reductaseactivities are therefore major players in retinoid meta-bolism, making essential contributions to the physio-logic control of RA availability. In vertebrates, retinoldehydrogenase activity has been classically associatedwith two distinct protein families, the short-chainKeywordscephalochordates; chordate evolution;retinaldehyde reductases; retinoic acidmetabolism; retinol dehydrogenasesCorrespondenceR. Albalat, Departament de Gene`tica,Facultat de Biologia, Universitat deBarcelona, Av. Diagonal, 645, 08028Barcelona, SpainFax: +34 934034420Tel: +34 934029009E-mail: ralbalat@ub.eduWebsite: http://www.ub.edu/genetica/indexen.htm(Received 9 March 2007, revised 4 May2007, accepted 30 May 2007)doi:10.1111/j.1742-4658.2007.05904.xIn vertebrates, multiple microsomal retinol dehydrogenases are involved inreversible retinol ⁄ retinal interconversion, thereby controlling retinoid meta-bolism and retinoic acid availability. The physiologic functions of theseenzymes are not, however, fully understood, as each vertebrate form hasseveral, usually overlapping, biochemical roles. Within this context, amphi-oxus, a group of chordates that are simpler, at both the functional andgenomic levels, than vertebrates, provides a suitable evolutionary model forcomparative studies of retinol dehydrogenase enzymes. In a previous study,we identified two amphioxus enzymes, Branchiostoma floridae retinol dehy-drogenase 1 and retinol dehydrogenase 2, both candidates to be thecephalochordate orthologs of the vertebrate retinol dehydrogenaseenzymes. We have now proceeded to characterize these amphioxusenzymes. Kinetic studies have revealed that retinol dehydrogenase 1 andretinol dehydrogenase 2 are microsomal proteins that catalyze the reduc-tion of all-trans-retinaldehyde using NADH as cofactor, a remarkable com-bination of substrate and cofactor preferences. Moreover, evolutionaryanalysis, including the amphioxus sequences, indicates that Rdh genes wereextensively duplicated after cephalochordate divergence, leading to the genecluster organization found in several mammalian species. Overall, our dataprovide an evolutionary reference with which to better understand theorigin, activity and evolution of retinol dehydrogenase enzymes.AbbreviationsAKR, aldo-keto reductase; AR, aldose reductase; CRAD, cis-retinol/androgen dehydrogenase; ER, endoplasmic reticulum; GFP, greenfluorescent 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 3739retinol dehydrogenases [short-chain dehydrogenase ⁄reductase (SDR)-RDHs] and the medium-chainalcohol dehydrogenases [medium-chain dehydrogenase ⁄reductase (MDR)-ADHs] [3,4]. Despite the many bio-chemical studies on these two protein families, the majorenzyme(s) responsible for the in vivo oxidation of retinolremains uncertain. In previous studies, we analyzedthe functionality and evolution of the MDR-ADHfamily [5–8,9]. Here, we focus on the contribution ofSDR-RDH enzymes to retinol ⁄ retinal metabolism.Classically, RDH enzymes have been regarded as acomplex vertebrate group of microsomal proteinsthat catalyze the conversion of retinol to retinaldehydein vitro using NADH as cofactor [4]. However, theRDH family contains many enzymes with diversesubstrate specificities towards cis and trans isomericforms, and, mostly, toward steroids. Hence, attempts todetermine the physiologic contribution of each RDHenzyme to RA metabolism have been impaired by thevariety of enzymes as well as by the overlaps in substraterecognition.Substantial multiplicity and redundancy is also pre-sent in the reductive direction of the pathway. Fourvertebrate protein families have been associated withretinaldehyde reduction. Members of the SDR-RDHgroup such as RDH2, RDH5 and 17b hydroxysteroiddehydrogenase type 9 (17bHSD9) [10–12], non-RDHSDR enzymes, including mouse retinal reductase(RRD), retinal short-chain dehydrogenase/reductase 1(retSDR1), photoreceptor outer segment all-transretinol dehydrogenase (prRDH), retinal reductase 1(RalR1) and pancreas protein 2 (PAN2) [13–17],MDR-ADH forms such as ADH1, ADH4 [18] andamphibian ADH8 [19], and members of the aldo-ketoreductase superfamily, including human aldose reduc-tase (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 physio-logic basis of the RDH and retinaldehyde reductasemultiplicity of vertebrates, analysis of the cephalochor-date amphioxus is invaluable. Cephalochordates areuseful organisms for comparative analyses, as their lowgene complexity and archetypical body plan organiza-tion suggest that they retain many ancient characteris-tics. Amphioxus did not undergo the extensive geneduplications that occurred during early vertebrate evo-lution [22], but rather exhibits an RA-signaling systemand a retinoid content comparable to that of verte-brates [23,24]. In a previous study, we identified twoenzymes, RDH1 and RDH2, that belong to the SDR-RDH group in the species Branchiostoma floridae [25].Here we present experimental data showing that thesetwo enzymes are endoplasmic reticulum (ER)-associ-ated proteins that may participate in retinoid metabo-lism, by catalyzing retinaldehyde reduction. Moreover,phylogenetic analysis indicates that most vertebrateRDHs derive from lineage-specific tandem duplicationsof an ancestral form that may resemble the currentamphioxus enzymes. The novel vertebrate RDHenzymes would have evolved new biochemical activitiesin retinoid and steroid metabolism after cephalochor-date divergence, thereby contributing to the increasedphysiologic complexity of the vertebrate subphylum.ResultsEnzymatic properties of recombinant RDH1 andRDH2Amphioxus RDH1 and RDH2 proteins tagged at theN-terminus with the hemagglutinin (HA) epitope wereproduced in COS-7 cells and purified in the microsom-al fraction. The enzymatic activity of this fraction wasassayed against retinoids. Given that most vertebrateRDHs can catalyze cis-retinol and ⁄ or trans-retinol oxi-dation, these were the substrates initially evaluated.Indeed, mouse RDH1 (kindly provided by J. L. Napoli,University of California, Berkeley, CA, USA) was usedto monitor the retinol oxidation assay. Unexpectedly,the oxidative activity observed for the amphioxusenzymes was below the detection capacity of the assay,< 0.002 nmol (Fig. 1A–C), although a wide range ofconditions were used: from pH 6 to 9, 5–12.5 lmall-trans-retinol, 0.5–2 mm NAD+and NADP+, and10–100 lg of microsomes obtained from independentassays. Negligible activity was also observed when9-cis-retinol was assayed (data not shown). Next, weanalyzed whether RDH1 and RDH2 exhibited reduc-tase activity (Fig. 1D,E). Retinol production in vitroincreased 2.5-fold and 25-fold for RDH1 and RDH2,respectively, in the presence of NADH, as comparedto controls. However, these differences were not detec-ted with NADPH, even though COS cells showedintrinsic NADPH-dependent retinal reductase activity[12]. The specific activity of each amphioxus enzymewas 0.25 nmol of retinolÆmin)1Æ(mg of microsomes))1for RDH1 incubated with 15 lm all-trans-retinal, and1.4 nmol of retinolÆmin)1Æ(mg of microsomes))1forRDH2 with 12.5 lm all-trans-retinal (Table 1). Fur-thermore, to examine whether RDH forms had isomerspecificity, we also assayed the RDH1 and RDH2activities toward 9-cis-retinal. However, only residual9-cis-retinal reductase activity, less than 0.03 nmolÆmin)1Æmg)1, was detected for these two enzymes(Fig. 1F,G). The reaction products were extracted andAmphioxus retinol dehydrogenase enzymes D. Dalfo´et al.3740 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBSanalyzed by RP-HPLC, and the kinetic constants ofthe two RDHs for all-trans-retinal were determined(Fig. 1H,I; Table 1). The apparent Kmvalues ofRDH1 and RDH2 (8.7 lm and 8.9 lm, respectively)were similar, whereas the maximum specific activities(0.3 nmolÆmin)1Æmg)1and 2.3 nmolÆmin)1Æmg)1, respect-ively) and the maximum specific activities ⁄ Kmratios(0.03 and 0.26, respectively) were > 7.5-fold higher forRDH2 than for RDH1. Finally, the apparent cofactorKmvalues of amphioxus enzymes (Fig. 1J,K; Table 1)were 224 lm and 98 lm NADH for RDH1 andRDH2, respectively, whereas no significant activitywas detected with NADPH. This cofactor preference isconsistent with the presence and absence of specificamino acids at certain positions in the amphioxusenzymes (Fig. 2A): both enzymes contain the D and Tresidues at the equivalent positions of cow RDH5 forNADH specificity, and lack any positively chargedamino acid at the corresponding position of the ratRDH2 K64, which may be essential for NADPHpreference [26].Activity of recombinant RDH1 and RDH2in intact cellsAmphioxus RDH1 and RDH2 and mouse RDH1 wereexpressed in COS-7 cells to evaluate their activitieswith retinoids in an intact cell system. In agreementFig. 1. Enzymatic activity of amphioxusRDH1 and RDH2 enzymes. The biochemicalactivity of the microsomal fraction of COS-7cells transfected with amphioxus HA-RDH1-expressing (A, D, F), HA-RDH2-expressing(B, E, G) and mouse Rdh1-expressing (C)constructs was analyzed. For oxidative reac-tions (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 min at37 °C. For retinal reduction, the microsomalfraction (15 lg) was incubated with 10 lMall-trans-retinal (D, E) or 9-cis-retinal (F, G)and NADH (1 mM) at pH 6.0 for 15 min at37 °C. Elution was monitored at 380 nm forretinal detection (A–C) and 325 nm for ret-inol (D–G) detection. The values for all-trans-retinaldehyde reduction of amphioxus RDH1(H) and RDH2 (I) were determined at 1 mMNADH using eight concentrations of sub-strate, from 0.5 to 20 lM and from 0.5 to15 lM for RDH1 and RDH2, respectively.The apparent Kmvalues for cofactor NADHwere determined at 15 lM and 12.5 lM all-trans-retinaldehyde for RDH1 (J) and RDH2(K), respectively, using six concentrations ofcofactor, from 0.005 to 1.5 mM. Assayswere performed with 15 lg of microsomesfor 15 min at 37 °C. Each point representsthe average of three replicates.D. Dalfo´et al. Amphioxus retinol dehydrogenase enzymesFEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3741with the biochemical analysis of the microsomal-puri-fied enzymes, amphioxus RDH1 and RDH2 catalyzedthe reduction of all-trans-retinaldehyde into all-trans-retinol in intact cells (Fig. 3A), whereas RDH1- andRDH2-transfected cells showed no differences frommock-transfected cells in the generation of all-trans-retinaldehyde from all-trans-retinol (Fig. 3B). Mock-transfected COS-7 cells reduced all-trans-retinaldehyde,indicating that the cells harbor reductases. Transfec-tion with amphioxus RDH1 cDNA produced a net47 pmol and 99 pmol of retinol per mg of total proteinafter 1 h of incubation with 10 lm and 20 lm ofretinal, respectively. RDH2 enzymes were more effi-cient than RDH1 enzymes, and generated 64 and134 pmol of retinol in the same assay conditions.Overall, transfection with RDH1 and RDH2 cDNAincreased the level of retinaldehyde reduction by 35% and  50%, respectively, as compared to themock-transfected cells. Mouse RDH1, in contrast,oxidized retinol to retinaldehyde (Fig. 3B) but did notsupport retinaldehyde reduction. Indeed, mouse RDH1decreased the amount of retinol generated in the assayswith retinaldehyde incubation (data not shown), sug-gesting that the substrate used by this enzyme was theretinol generated from retinaldehyde by endogenousCOS-7 cell reductase activity.Intracellular localizationCOS-7 cells were transiently transfected with con-structs expressing the amphioxus RDH1 and RDH2enzymes fused to several peptides: HA epitope, greenfluorescent protein (GFP) and b-galactosidase. Inagreement with the RDH1 and RDH2 purification inthe microsomal fraction (Fig. 2D), immunostaining ofcells expressing HA-RDH1 and HA-RDH2 proteinsrevealed a typical pattern of ER-associated proteins,with no nuclear staining or plasma membrane localiza-tion observed (Fig. 2B,C). GFP fusion was used tovisualize the intracellular localization in living cells,thereby avoiding any artefacts caused by the cellfixation process. RDH2 fused to GFP either at theC-terminus (RDH21)335-GFP) or at the N-terminus(GFP-RDH21)335) of the enzyme (Fig. 2E,I) exhibiteda pattern that overlapped with the ER-Tracker BlueWhite DPX, which was used as a specific ER markerin living cells (Fig. 2F,J). The subcellular localizationof the RDH2 enzyme (RDH21)335) fused to the b-ga-lactosidase protein was also consistent with a typicalpattern of ER-associated proteins (Fig. 2M).Table 1. All-trans-retinal activity and kinetic constants of B. floridaeRDH enzymes compared with those of known vertebrate retinalreductases. Values are from this work, B. floridae RDH1 (BfRDH1)and BfRDH2, or from the literature [6-10,13,16,17,25]. ND, notdetermined. HAR, human aldose reductase.All-trans-retinal NADHSpecificactivity(nmolÆmin)1Æmg)1)Maximumspecificactivity(nmolÆmin)1Æmg)1)Km(lM)Km(lM)BfRDH1 0.25 0.3 ± 0.06 8.7 ± 4.1 224 ± 81BfRDH2 1.4 2.3 ± 0.5 8.9 ± 3.8 98 ± 25RalR1 ND 18 ± 0.05 0.5 ± 0.05 1300 ± 200PAN2 ND 27 ± 1 0.08 ± 0.02 1060 ± 70RRD ND 40 ± 1 2.3 NDretSDR1 0.04 ND ND NDHAR ND 15 ± 1a10 ± 2 NDHIS-AR ND 193 ± 4a19 ± 4 NDChickenAKRND 170 ± 15a32 ± 4 NDRDH2 0.25 ND ND ND17bHSD9 0.17 ND ND NDRDH5 16 ND ND NDakcatvalues in min)1.Fig. 2. 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 aremarked in bold. The D43, T67 and A69 residues, involved in cofactor specificity, are indicated by asterisks. Flanking the N-terminal hydropho-bic 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, andexamined using confocal microscopy. (D) Western blot of the pellets after 13 000 g (lanes 2 and 4) and 100 000 g centrifugations (lanes 1and 3, microsomal fractions) of homogenates of COS cells transfected with HA-RDH1 (lanes 1 and 2) and HA-RDH2 (lanes 3 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)28-GFP (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 specific-ally visualize the ER in living cells (F, J). (M–R) Localization of RDH2-b-galactosidase chimeric proteins. COS-7 cells were transfected withconstructs encoding the full-length (RDH21)335) (M) and four C-terminal truncated RDH2 forms (RDH21)229, RDH21)165, RDH21)137andRDH21)58) (N–Q, respectively) fused to the NLS-b-galactosidase protein, and with the pb-galactosidase-N2 empty vector (R). The pb-galac-tosidase-N2 vector contains a nuclear localization sequence (NLS) 5¢ to the LacZ gene. The SV40 NLS localizes the b-galactosidase codifiedby the empty vector to the nucleus. Cells were immunostained with an antibody to b-galactosidase and examined using a Zeiss Axiophotfluorescence microscope.Amphioxus retinol dehydrogenase enzymes D. Dalfo´et al.3742 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBSTo analyze the contribution of protein domains tothe ER anchorage, the localization of the full-lengthenzyme was compared with those of five truncatedforms. The pattern of the full-length construct wasessentially identical to that of the C-terminal truncatedRDH2 forms (RDH21)229, RDH21)165, RDH21)137,and RDH21)58RDH21)28) fused either to b-galactosi-dase (Fig. 2N–Q) or to GFP (Fig. 2G,H), whereas theb-galactosidase and GFP controls showed a signal,mainly in the nucleus (Fig. 2L,R). The observationD. Dalfo´et al. Amphioxus retinol dehydrogenase enzymesFEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3743that nuclear or cytosolic staining was not increased forany construct suggested that the N-terminal segmentwould be sufficient to target and anchor the protein tothe ER membranes. Furthermore, we analyzed thecontribution of the C-terminal end to ER localization.We fused the last 41 amino acids of the amphioxusRDH2 enzyme (a region equivalent to the reportedC-terminal segment of mouse RDH1 [27]) to GFP:GFP-RDH2295)335. The protein did not localize to theER of transfected COS cells, but showed a diffusesignal similar to that of the GFP control (compareFig. 2K,L).Evolution of the RDH grouptblastn comparisons showed that the sequences mostsimilar to the amphioxus enzymes were those of thevertebrate RDHs (E-value ¼ 2e-75 and 1e-71 with Pantroglodytes, similar to sterol ⁄ retinol dehydrogenase, forRDH1 and RDH2, respectively). In the phylogeneticanalysis, amphioxus RDH branched outside a cladecomprising the ‘classic’ SDR-RDH1 ⁄ 2 ⁄ 3 ⁄ 4 ⁄ 5 ⁄ 6 ⁄ 7 ⁄ 9(RDH1–7 ⁄ 9) members, which includes six humanenzymes [similar-RDH2, RDH4, orphan short-chaindehydrogenase/reductase (SDR-O), RDH, RDH5 anddehydrogenase/reductase member 9 (DHRS9)], eightrat forms (similar-RDH1, RDH2, similar-RDH2,RDH3, SDR-O, 17b-HSD19, RDH5 and DHRS9) and11 mouse proteins [RDH1, RDH9, RDH6, truncated-RDH, similar-RDH, cis-retinol/androgen dehydrogen-ase (CRAD)-L, RDH7, SDR-O, 17b-HSD19, RDH5and DHRS9] (Fig. 4A). Except for DHRS9, the genesencoding these enzymes were not spread over severalchromosomes, but rather clustered in the human gen-ome at 12q13–14 and in the syntenic regions of ratand mouse chromosomes 7 and 10, respectively [28](Fig. 4B). DHRS9 genes are located in human chromo-some 2, rat chromosome 3 and mouse chromosome 2,which would be paralogous to chromosomes 12, 7 and10, respectively [29]. The overall analysis, examiningthe topology of the phylogenetic tree and the positionof each gene inside the cluster, was informative regard-ing the orthology relationships of the distinct enzymes,and allowed us to define five RDH classes (Fig. 4A). Itis of note that other vertebrate SDRs (some reportedas RDH enzymes), such as RDH8, RDH10, RDH11,RDH12, RDH13, RDH14, similar to epidermal retinalFig. 4. (A) Phylogenetic relationship of the RDH1–7 ⁄ 9 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 theCLUSTALX program, and confidence ineach node was assessed by 1000 bootstrap replicates. The RDH1–7 ⁄ 9 cluster comprises all the vertebrate sequences that group with theamphioxus enzymes. Additional enzymes involved in retinoid ⁄ steroid metabolism appear to be distantly related (less than 55% of sequenceidentity in the region used for the tree reconstruction, data not shown), and were therefore considered to be members of distinct SDRgroups. The bootstrap values defining each group are shown (black numbers). Internally, the RDH1–7 ⁄ 9 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 clus-ters using the Map Viewer website from NCBI. The name of the each Rdh gene (black boxes) is indicated. Alternative names for each geneare listed in supplementary Table S2. Orthology relationships among genes of several species are indicated (continuous lines). Notice thatRdh5 genes are in the same chromosome but outside the RDH clusters (dotted line), and that Dhrs9 genes are located in distinct chromo-somes. Genes flanking the RDH sequences are also depicted (green boxes). TAC3, tachykinin 3; KIAA0352 (ZBTB29), zinc finger and BTBdomain containing 39; ADMR, adrenomedullin receptor; PRIM1, primase polypeptide 1; NACA, nascent-polypeptide-associated complexalpha-polypeptide; CD63, CD63 antigen; BLOC1S1, biogenesis of lysosome-related organelles complex-1, subunit 1; ABCB11, ATP-bindingcassette, subfamily B, member 11; LRP2, low-density lipoprotein receptor-related protein 2.Fig. 3. Synthesis of retinol and retinaldehyde in transfected COS-7cells expressing amphioxus RDH enzymes. (A) Different concentra-tions (5, 10 and 20 lM) of all-trans-retinaldehyde were added toCOS-7 cells expressing RDH1 (black bars) and RDH2 (white bars).Retinol was extracted from the cells and analyzed by HPLC. Thebars represent the net retinol production per mg of total proteinafter 1 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 positivecontrol of the reaction. The bars represent the net retinal produc-tion per mg of total protein after 1 h of incubation with 5, 10 and20 lM all-trans-retinol.Amphioxus retinol dehydrogenase enzymes D. Dalfo´et al.3744 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBSABD. Dalfo´et al. Amphioxus retinol dehydrogenase enzymesFEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3745dehydrogenase 2 (RaDH9), epidermal retinal dehydro-genase 2 (eRaDH2), retSDR1, DHRS4, 17b-HSD11,17b-HSD12, 11b-HSD11, 11b-HSD12 and short-chaindehydrogenase/reductase 10 isoform B (SCDR10B),branched outside the vertebrate–cephalochordate clade(Fig. 4A) and were located in diverse nonparalogousmammalian chromosomes. These enzymes would betherefore distantly related to the RDH1–7 ⁄ 9 forms andshould be considered members of separate enzymefamilies. Indeed, the position of the amphioxus enzy-mes in the phylogenetic tree implied that these familiesare ancient, pre-dating the emergence of the chordatephylum.DiscussionThe biochemical characterization revealed that theamphioxus RDH enzymes catalyzed all-trans-retinalreduction (Table 1, Figs 1 and 3). Unfortunately, com-parison with other RDHs was limited to rat RDH2,mouse 17bHSD9 and RDH5, as the reductase capacityof most vertebrate RDHs has not been assayed. Thus,we also compared amphioxus data with those fromother non-RDH vertebrate retinal reductases, such asretSDR1, RalR1, PAN2, RRD, human AR, HSI, andchicken AR (Table 1), although the comparison wasalso hindered by the variety of assay conditions used.We have shown that amphioxus enzymes show iso-mer preference, trans versus cis retinal forms, as occurswith the vertebrate RalR1, PAN2, RRD, retSDR1,prRDH, HSI and chicken AKR enzymes. Moreover,although retinol ⁄ retinal interconversion is a reversiblereaction, neither amphioxus RDH1 or RDH2 showedsignificant activity towards retinol in the in vitro assayswith microsomal proteins or in the intact cell systems.This strict preference towards the reductive directionhas 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 withconsiderably lower efficiency. The specific activitiestowards all-trans-retinal of amphioxus enzymes were0.25 nmolÆmin)1Æmg)1for RDH1 and 1.4 nmolÆmin)1Æmg)1for RDH2; these were 6.3-fold and 23-foldhigher, respectively, than that reported for retSDR1(0.04 nmolÆmin)1Æmg)1), a photoreceptor enzyme thatreduces all-trans-retinal in the visual cycle [14]. Thespecific activity of RDH2 was 5.6-fold and 10.8-foldhigher than that of rat RDH2 (0.25 nmolÆmin)1Æmg)1)[10] and mouse 17bHSD9 (0.13 nmolÆmin)1Æmg)1) [12],respectively, whereas the activity of amphioxus RDH1was comparable to those of these enzymes. In contrast,the amphioxus enzymes showed lower retinaldehydereductase efficiency than some vertebrate enzymes. Thespecific activity of mouse RDH5 with all-trans-retinal(16 nmolÆmin)1Æmg)1[11]) was higher than that ofeither RDH1 or RDH2; RalR1 [30], PAN2 [17] andRRD [13] showed lower Kmand higher maximum spe-cific activity values for all-trans-retinal (Table 1) andtherefore higher maximum specific activity ⁄ Kmratios,which are a measure of the catalytic effectiveness ofthe enzymes; the AKR members (human AR, HSI andchicken AKR) [20,21] showed similar Kmvalues buthigher maximum specific activities (Table 1), whichalso implied higher maximum specific activity ⁄ Kmratios, i.e. greater effectiveness, for the vertebrateAKR than for the cephalochordate forms. Overall,these data support our finding that amphioxus RDHshows retinal reductase activity within the range repor-ted for diverse vertebrate enzymes.The most significant difference between the amphi-oxus and the other retinal reductases was, nevertheless,their preference for the NADH cofactor. To ourknowledge, these are the first SDR retinaldehydereductases reported to use NADH instead of NADPH.Conventionally, cofactor preference had been directlyrelated to the oxidative or reductive direction of thereaction. Therefore, it was assumed that oxidativeRDHs would be NADH-dependent, whereas NADPHenzymes would catalyze the reductive reaction. Thishypothesis was based on the ratios between the oxid-ized and reduced forms of the coenzymes [31]. Itappears, however, that cofactor ratios vary greatlyamong organs and cell types, and that the redox statuscan be greatly influenced by external factors [32].Noticeably, amphioxus enzymes have the capacity toreduce retinaldehyde to retinol in intact cells (Fig. 3),suggesting that the endogenous NADH level in COS-7cells is enough to allow this reduction. Our data sup-port the contention that coenzyme preference does notnecessarily constrain the direction of the reaction. Infact, several RDHs (e.g. human, mouse and bovineRDH10 [33] and human RDH-E2 [32]) prefer NADPto NAD as a cofactor.Structurally, most of the cytosolic SDR enzymes arecomposed of 250–280 amino acid residues [34,35],whereas the membrane-associated SDR enzymes areextended at both the N-terminal and C-terminal endsby up to about 350 amino acids [36,37]. AmphioxusRDH1 and RDH2 were 332 and 335 amino acids long,respectively, and their subcellular localization in trans-fected COS-7 cells concurred with that of ER-associ-ated proteins. The observation that nuclear orcytosolic staining was not increased for any C-terminaltruncated constructs indicated that the N-terminalAmphioxus retinol dehydrogenase enzymes D. Dalfo´et al.3746 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBSsegment was sufficient to target and anchor the proteinto the ER membranes. As the shortest segment wasonly 28 amino acids long (from the initial methionineto the LERGR motif), it can be assumed that the sign-aling sequence for ER localization falls in this regionof the protein. In addition to the targeting function,signal sequences are also crucial in protein topogenesis,as they participate in the final cytosolic ⁄ lumenal orien-tation. The most prominent determinant of signal ori-entation is the distribution of charged amino acids ateither end of the hydrophobic sequence. According tothe ‘positive-inside’ rule, the most positively chargedflanking transmembrane segment is usually found onthe cytosolic side of the membrane [38,39]. AmphioxusRDH did not show positively charged amino acids atthe N-terminus of the signaling sequence, but rathercontained the L24ERGR motif at the C-terminusof the hydrophobic sequence, thereby resemblingthe R19ERQV, R19ERKV, R19VRQV andR19DRQ(S ⁄ C) sequences of a number of vertebrateRDHs [40]. This motif would predict, therefore, acytosolic orientation of the amphioxus enzymes.For several RDH enzymes, the C-terminal trans-membrane segment forms a hydrophobic helix-turn-helix that is sufficient to retain them in the ER, e.g.CRAD1 [41]. We fused the last 41 amino acids of theamphioxus RDH2 enzyme (a region equivalent to thereported C-terminal segment of mouse RDH1 [27]) toGFP. This protein did not localize to the ER of trans-fected COS cells (Fig. 2K), and therefore the C-ter-minal end of amphioxus RDH2 was not sufficient forER targeting. Amphioxus RDH2 was structurallymore similar to the enzymes that rely exclusively onthe N-terminal hydrophobic segment as membraneanchor (e.g. mouse RDH1 [27], human 11bHSD1 [42],human 11bHSD2 [43] and human RalR1 [16]) than tothe other RDHs such as bovine RDH5 [44], mouseRDH4 [45] and mouse CRAD1 [41,46], which wouldbe anchored to both the N-terminal and C-terminalhydrophobic segments.Finally, evolutionary analysis including the amphi-oxus enzymes highlighted the relevance of using evolu-tionary criteria rather than biochemical classificationsfor gene nomenclature and family description. Thephylogenetic tree and the genomic organization nowpermit a proper definition of the vertebrate RDH1–7 ⁄ 9group and reveal an internal classification of mamma-lian RDH1–7 ⁄ 9 enzymes into five classes, pointing torecurrent gene tandem duplications as the most likelymechanism for the cluster organization of the Rdhgenes. In a recent study [47], Belyaeva & Kedishviliproposed a model for the evolution of the vertebrateRDH1–7 ⁄ 9 group (referred to as the RDOH-like SDRgroup in their article). On the basis of a comparativegenomic and phylogenetic analysis that included sev-eral vertebrate species, these authors suggested thatearly in vertebrate evolution, an initial tandem duplica-tion of the Rdh ancestor gave rise to the ‘ Dhrs9 ⁄ Rdhl–11-cis-RDH-homolog’ cluster. The 11-cis-RDH-homologgene was afterwards duplicated by a mechanism thatimplied translocation of the new copy to anotherregion of the genome to generate the 11-cis-RDH ⁄ Rdh5gene. Later on, the 11-cis-RDH ⁄ Rdh5 gene underwentseveral tandem duplication events in its new chromoso-mal location, which led to the appearance of the cur-rent RDH cluster in tetrapods. However, an alternativeevolutionary model is possible (Fig. 5). We hypothesizethat an initial tandem duplication of an Rdh ancestorgave rise to a two-gene cluster, which was furtherduplicated, probably as a result of the genome duplica-tion events that took place during early vertebrate evo-lution [22]. During fish evolution, one gene was lost,leading to the ‘Rdh5 (AAH97151) + Dhrs9 (Rdhl-like)+Rdhl’ combination currently found in zebrafish[47]. In amphibians and mammals, extra tandem dupli-cations produced the RDH clusters found in Xenopus,human, mouse, rat, dog and cow [47] (Fig. 4B). Even-tually, the mammalian Rdhl ⁄ CX410306 ortholog waslost. The closer phylogenetic relationship of RDH5 ⁄ 11-cis-RDH to Rdhl ⁄ CX410306 enzymes than to RDH4-SDR-O-RDH forms [47] is consistent with this model.Furthermore, the observation that the Ddrs9 genes andthe Rdh5-RDH cluster are located in paralogous chro-mosomes also supports our hypothesis.In conclusion, the analysis of amphioxus enzymescontributes to improving our understanding of thefunctional complexity of vertebrate gene familiesregarding retinoid metabolism. However, to date, noconvincing enzymes for retinol oxidation have beenfound among cephalochordate RDH members. Thefull genome sequence of amphioxus, currently beingreleased, will allow comprehensive searches for novelcandidates, which may also have relevant physiologicroles in the retinoid pathway of vertebrates.Experimental proceduresExpression of HA-RDH, GFP-RDH, andb-galactosidase-RDH proteinsTo produce RDH1 and RDH2 proteins tagged at theN-terminus with the HA epitope, the full-length codingsequences of the Rdh1 and Rdh2 genes were PCR-amplified(oligonucleotides 1–2, and 3–4, respectively; the oligonucleo-tide sequences used in this study are provided in supple-mentary Table S1) from plasmids containing the Rdh1 andD. Dalfo´et al. Amphioxus retinol dehydrogenase enzymesFEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3747Rdh2 cDNAs and cloned in the pACT2 vector (Clontech,Mountain View, CA, USA). The HA-tagged Rdh1 andRdh2 coding fragments were released from the pACT2 vec-tor and cloned into the pCDNA3 vector (Invitrogen, Carls-bad, CA, USA). To fuse amphioxus RDH2 either at theN-terminus or C-terminus of the GFP, the full-lengthcoding region, two C-terminal truncated RDH2 forms andone N-terminal truncated RDH2 form were PCR-amplified(RDH21)335, oligonucleotides 5 and 6, amino acids 1–335,full-length; RDH21)58, oligonucleotides 5 and 7, aminoacids 1–58, truncated just after the cofactor-bindingsequence GXXXGXG; RDH21)28, oligonucleotides 5 and8, amino acids 1–28, truncated after the LERGR motif;RDH2295)335, oligonucleotides 9 and 6, amino acids295–335) and cloned into the pEGFP-N2 and pEGFP-C2vectors (Clontech). To produce both the full-length and thefour C-terminal truncated RDH2 enzymes fused at theN-terminus of b-galactosidase, the coding regions of Rdh2were generated by PCR amplification: RDH21)335(oligonu-cleotides 10–11; amino acids 1–335, full-length), RDH21)229(oligonucleotides 10–12; amino acids 1–229, lacking theC-terminal end), RDH21)165(oligonucleotides 10–13; aminoacids 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 justafter the cofactor-binding sequence GXXXGXG). Thedesign of these constructs was based on the predicted trans-membrane segments of the RDH2 enzyme given by thetmpred [48], das [49] and hmmtop [50] programs (data notshown). The five PCR fragments were cloned in the pbGal-N2 vector, in frame at the 5¢ end of the coding region ofLacZ. This vector expresses b-galactosidase protein fusedto a nuclear localization sequence (NLS) driven by thestrong human cytomegalovirus immediate early promoter,and was created by cloning the NLS-LacZ gene from thePSP-1.72b-galactosidase plasmid [51] into a pEGFP-N2vector from which the Gfp coding sequence had beenremoved. The SV40 NLS localizes b-galactosidase to thenucleus. All the constructs were verified by sequencing.For subcellular localization experiments, COS-7 cells(African green monkey kidney cells; ECACC, PortonDown, Wiltshire, UK) were grown in DMEM with Gluta-MAX II (Invitrogen) and 4500 mgÆL)1d-glucose, supple-mented with 10% fetal bovine serum, 100 UÆmL)1penicillinG and 100 lgÆmL)1streptomycin in a 5% CO2humidifiedatmosphere at 37 °C. Cells were seeded on glass coverslipsinto 12-well plates (5 · 104cells per well) and transfected24 h later with 0.5 lg of purified plasmid DNA per well,using 2.3 lL of FuGene6 (Roche, Basel, Switzerland). Cellswere transfected with constructs encoding HA-RDH1,HA-RDH2, and the full-length and the four C-terminalFig. 5. Hypothetical model of RDH1–7 ⁄ 9group evolution leading to the current ver-tebrate multiplicity. Fish and amphibianarrangements are those described for Daniorerio and Xenopus tropicalis in [47].Amphioxus retinol dehydrogenase enzymes D. Dalfo´et al.3748 FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS[...]... topology of 11-cis retinol dehydrogenase in the retinal pigment epithelium suggest a compartmentalized synthesis of 11-cis retinaldehyde J Cell Sci 112, 549–558 Romert A, Tuvendal P, Tryggvason K, Dencker L & Eriksson U (2000) Gene structure, expression analysis, and membrane topology of RDH4 Exp Cell Res 256, 338–345 Tryggvason K, Romert A & Eriksson U (2001) Biosynthesis of 9-cis retinoic acid in vivo: the. .. genomic data: the evolution of the MDR-ADH family Heredity 95, 184–197 10 Imaoka S, Wan J, Chow T, Hiroi T, Eyanagi R, Shigematsu H & Funae Y (1998) Cloning and characterization of the CYP2D1-binding protein, retinol dehydrogenase Arch Biochem Biophys 353, 331–336 11 Driessen CA, Winkens HJ, Kuhlmann ED, Janssen AP, van Vugt AH, Deutman AF & Janssen JJ (1998) The visual cycle retinol dehydrogenase: ... Short-chain dehydrogenases ⁄ reductases FEBS Journal 274 (2007) 3739–3752 ª 2007 The Authors Journal compilation ª 2007 FEBS 3751 ´ D Dalfo et al Amphioxus retinol dehydrogenase enzymes 36 37 38 39 40 41 42 43 44 45 46 47 (SDR): the 2002 update Chem Biol Interact 143–144, 247–253 Filling C, Wu X, Shafqat N, Hult M, Martensson E, Shafqat J & Opperman UC (2001) Subcellular targeting analysis of SDR-type... phase The flow rate was 1.8 mLÆmin)1 Under these conditions, elution times were as follows: 10.9 min for 9-cis -retinol, 11.4 min 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 min for 9-cis-retinal, and 15.3 min for all-trans-retinal Retinoids were quantitated by comparing their... Biosynthesis of 9-cis retinoic acid in vivo: the roles of different retinol dehydrogenases and a structure activity analysis of microsomal retinol dehydrogenases J Biol Chem 276, 19253–19258 Belyaeva OV & Kedishvili NY (2006) Comparative genomic and phylogenetic analysis of short-chain dehydro- 3752 48 49 50 51 52 53 54 genases ⁄ reductases with dual retinol ⁄ sterol substrate specificity Genomics 88, 820–830... 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 min 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... constructed from peak areas of a series of standards The peak detection limit was about 2 pmol of retinoid The apparent Km values for the reduction of all-trans-retinal were determined at 1 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... amplification of the different constructs Table S2 Accession numbers of the sequences used in this study This material is available as part of the online article from 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... aldose reductase are efficient retinal reductases: consequences for retinoid metabolism Biochem J 373, 973–979 ` Holland PW, Garcia-Fernandez J, Williams NA & Sidow A (1994) Gene duplications and the origins of vertebrate development Development Suppl 43, 125–133 ´ ` Dalfo D, Albalat R, Molotkov A, Duester G & Gonzalez Duarte R (2002) Retinoic acid synthesis in the pre- Amphioxus retinol dehydrogenase enzymes. .. determine cytosolic topology of short-chain dehydrogenases ⁄ reductases Studies with retinol dehydrogenase type 1 and cis -retinol ⁄ androgen dehydrogenase type 1 J Biol Chem 279, 51482–51489 Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer S, Scott G, Steffen D, Worley KC, Burch PE et al (2004) Genome sequence of the Brown Norway rat yields insights into mammalian evolution Nature 428, . Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution. whereas the activity of amphioxus RDH1was comparable to those of these enzymes. In contrast, the amphioxus enzymes showed lower retinaldehyde reductase
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