Characterization of a Microsomal Retinol Dehydrogenase Gene from Amphioxus: Retinoid Metabolism Before Vertebrates

Chemico-Biological Interactions 130–132 (2001) 359–370 www.elsevier.com/locate/chembiont

Characterization of a microsomal retinol dehydrogenase gene from amphioxus: retinoid metabolism before vertebrates

Diana Dalfo´, Cristian Can˜estro, Ricard Albalat, Roser Gonza`lez-Duarte * Departament de Gene`tica, Facultat de Biologia, Uni6ersitat de Barcelona, A6. Diagonal, 645, E-08028, Barcelona, Spain

Abstract

Amphioxus, a member of the subphylum Cephalochordata, is thought to be the closest living relative to vertebrates. Although these animals have a vertebrate-like response to retinoic acid, the pathway of retinoid metabolism remains unknown. Two different enzyme systems — the short chain dehydrogenase/reductases and the cytosolic medium-chain alcohol dehydrogenases (ADHs) — have been postulated in vertebrates. Nevertheless, recent data show that the vertebrate-ADH1 and ADH4 retinol-active forms originated after the divergence of cephalochordates and vertebrates. Moreover, no data has been gathered in support of medium-chain retinol active forms in amphioxus. Then, if the cytosolic ADH system is absent and these animals use retinol, the microsomal retinol dehydrogenases could be involved in retinol oxidation. We have identiﬁed the genomic region and cDNA of an amphioxus Rdh gene as a preliminary step for functional characterization. Besides, phylogenetic analysis supports the ancestral position of amphioxus Rdh in relation to the vertebrate forms. © 2001 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Retinol dehydrogenase; Retinoid metabolism; Amphioxus

* Corresponding author. Fax: +34-93-4110969. E-mail address: [email protected] (R. Gonza`lez-Duarte).

1. Introduction

Genome analysis in model organisms support an increasing number of gene families and family members. Duplications of many classes of genes, often interacting in gene networks, allowed the emergence of new developmental control mechanisms at the origin of vertebrates. One such mechanism, the retinoic acid (RA) system, is believed to be involved in patterning the body plan through the regulation of developmental control genes (reviewed in [1,2]). Evidence that RA controls Hox gene expression supports the hypothesis of its endogenous role in establishing the anterior-posterior (AP) axis (reviewed in [3]). However, this AP pattern regulation is not restricted to vertebrates but constitutes a chordate innovation [4], as amphioxus (subphylum Cephalochordata) and ascidians (subphylum Urochordata) also show a vertebrate- like response to RA [5–8]. Amphioxus is thought to be the closest living relative to vertebrates. Cephalochordate gene complexity and body plan organization have led to the assumption that amphioxus are ‘archetypal’ organisms [9,10]. Excess RA affects amphioxus development by extending anteriorly the expression domain of Hox-1 in the nerve cord and compressing and shifting forward the Pax-1 domain [5]. Vitamin A (retinol) is metabolized to retinoic acid through the intermediate retinal. Although the RA gene transcription regulation by binding to RAR or RXR transcription factors has been established, the conversion of retinol to RA remains to be elucidated. Retinol oxidation, the rate limiting step in the synthesis of RA, is catalyzed in 6itro by distinct classes of cytosolic medium-chain alcohol dehydrogenases (ADHs) [11] as well as different microsomal short-chain dehydrogenase/reductases (SDR) (reviewed in [12]). Despite intensive work on the analysis on cofactors preference for the oxidation/reduction steps (NAD+ vs. NADP+) and the physiolog- ical status of retinol (unbound vs. bound retinol to cellular retinol binding protein, CRBP) a clear picture of retinoid metabolism has not emerged. On the other hand, recent data [13] suggest that no MDR (medium-chain dehydrogenase/reductases)– ADH enzymes other than ADH class 3 (glutathione-dependent formaldehyde dehydrogenase), considered a non-retinol metabolizing enzyme, are present in cephalochordates. Thus, if these animals metabolize retinol, an SDR-retinol dehydrogenase microsomal enzyme (RDH) could be involved in the first oxidation step of retinal production. We here report the nucleotide coding sequence and the deduced protein structure of the RDH enzyme in two amphioxus species, Branchiostoma lanceolatum (BlRdh) and Branchiostoma floridae (BfRdh) as a first step for functional analysis. The exon–intron architecture of BlRdh together with the phylogenetic analysis based on the amino acid sequence suggest that amphioxus Rdh is orthologous to the vertebrate forms.

2. Materials and methods

2.1. Genomic DNA and library screenings

Branchiostoma lanceolatum were kindly provided by the Laboratoire Arago D. Dalfo´etal. / Chemico-Biological Interactions 130–132 (2001) 359–370 361

(Observatoire Oceánologique de Banyuls-Sur-Mer, France). The animals were kept at −70°C until use. Total genomic DNA was isolated using the guanidine isothiocyanate method [14] with minor modifications [15]. Genomic DNA of B. lanceolatum (250 ng) was amplified with 2.5 U Taq polymerase (Biotherm) using two 17-mer degenerate oligonucleotides (5 pmol each) deduced from sequence alignment of the mammalian retinol dehydrogenases, namely 5% TGYGA- YWSNGGNTTYGG 3% and 5% GCRTTRTTNACNARNCC 3% (from nucleotide positions 2477 to 2493 and 2900 to 2884 of the human Rdh sequence: AF037062). After 2 min at 94°C and 40 cycles at 94°C for 1 min, 58°C for 1 min and 72°C for 1 min plus 5 min at 72°C, the PCR product was cloned in a pUC18 vector and sequenced. The PCR fragment was used to screen the B. lanceolatum genomic library. A two-animal B. lanceolatum genomic library was constructed with Lambda FIX-II/XhoI partial fill-in vector. The probe was labeled with [a-32P]dCTP by random-hexamer priming. High stringency hybridization was carried out in phosphate/SDS solution [16] at 65°C overnight. Filters were washed at 65°C for 1×5 min and 3×15 min in 2×SSC, 0.1% SDS. DNA fragments from positive recombinant phages were isolated, subcloned into a pUC18 vector, characterized by restriction mapping and sequenced in an ABI-Prism 377 automated DNA sequencer from PE Biosystems. The same probe as above plus a PCR fragment, which contained exon 4 of BlRdh were then used to screen a B. floridae 6–20 h — embryos cDNA library in Lambda Zap II, kindly provided by J. Langeland [17]. Hybridizations and washes were performed as above but at 55°C. Positive clones were isolated and characterized by sequence analysis.

2.2. Southern blot analysis

Total genomic DNA from several B. lanceolatum animals was isolated as above. Ten micrograms of each animal were digested by PstI and resolved on 0.9% agarose gels, one animal per lane, and transferred to nylon membranes. Southern blots were hybridized with a 32P-labeled probe containing BlRdh exon 4. Hybridiza- tions and washes were performed as above but at 55°C.

2.3. Sequence alignment and e6olutionary tree

Sequences data were taken from GenBank (proteins database) and SWISS- PROT. The accession number of sequences used in the present analysis are as follows: retinol dehydrogenases: (1) RDH H. sapiens (AAC72923), (2) RDH4 H. sapiens (NP–003699), (3) RDH homolog H. sapiens (NP–005762), (4) RDH5 H. sapiens (NP–002896), (5) 11-cis-RDH B. taurus (CAA57715), (6) 11-cis-RDH M. musculus (CAA66347), (7) CRAD1 M. musculus (AAB97166), (8) CRAD2 M. musculus (AAC40159), (9) RDH1 R. nor6egicus (AAB07997), (10) RDH2 R. nor6egicus (AAC52316), (11) RDH3 R. nor6egicus (AAB07995), (12) RDH C. elegans (CAA98465), (13) RDH B. lanceolatum (AF283542), (14) RDH1 B. floridae (AF283540), (15) RDH2 B. floridae (AF283541), (16) RDH4 D. melanogaster 362 D. Dalfoétal. / Chemico-Biological Interactions 130–132 (2001) 359–370

(AAF59214), (17) CRAD1 D. melanogaster (AAF58586). 11-ß-Hydroxysteroid dehydrogenases: (18) 11-ß-HSD-2 H. sapiens (AAB48544), (19) 11-ß-HSD-2 B. taurus (AAC26137), (20) 11-ß-HSD-2 Mus sp. (AAC60711), (21) 11-ß-HSD-2 R. nor6egicus (AAA87007), (22) 11-ß-HSD-2 O. cuniculus (AAA86387), (23) 11-ß- HSD-2 E. caballus (AAD31185), (24) 11-ß-HSD O. aries (AAA93156), (25) 11-ß- HSD-2 O. aries (AAB50810). 17-ß-Hydroxysteroid dehydrogenases: (26) 17-ß-HSD-2 H. sapiens (AAC41917), (27) 17-ß-HSD-2 M. musculus (P51658), (28) 17-ß-HSD-6 R. nor6egicus (AAB88253), (29) 17-ß-HSD-2 R. nor6egicus (CAA62617). ß-Hydroxybutirate dehydrogenases: (30) 3-OH-BDH H. sapiens (AAA58352), (31) D-ß-OH-BDH R. nor6egicus (AAB59684), (32) D-ß-OH-BDH C. elegans (AAC46535). Others: (33) ADH/ribitolDH (A) C. elegans (AAB69884), (34) ADH/ribitolDH (B) C. elegans (AAB54122), (35) Oxidoreductase H. sapiens a (AAB67236), (36) 3- -HSD H. sapiens (NP–003716). Amino acid sequence alignments were generated with the Clustal X program [18] and the percentage similarities were calculated using the set of programs in the Lasergene package (DNASTAR, Madison, WI, USA). Synonymous and non-synonymous nucleotide substitutions were estimated with Nei and Gojobori method [19] using the MEGA (Molecular Evolutionary Genetic Analysis) package. A neighbor-joining tree was constructed from the aminoacid alignment with the Clustal X program and drawn with the TreeViewPPC program [20].

3. Results

3.1. Cloning and sequence analysis

The amplified PCR fragment was cloned and sequenced to verify that it corre- sponded to BlRdh. Screening of the B. lanceolatum genomic library with this PCR probe gave 12 positive plaques. Restriction analysis showed that all the phages overlapped the same genomic region. Two phages were further analyzed. Phage 5.11 contained the promoter region plus exons 1–3, whereas phage 11.121 included exons 2–6 and downstream sequences. The coding sequence and the intron–exon boundaries of BlRdh were deduced from the B. floridae cDNA sequence. The screening of the B. floridae library was performed with the same probe as above plus a PCR fragment which contained exon 4 of BlRdh (Materials and Methods). Two distinct phages containing BfRdh cDNA sequences were isolated: BfRdh1 and BfRdh2. The B. floridae clones contained different full-length Rdh-encoding sequences of 1497 and 1567 bp, respectively. The ORFs of BfRdh1 and BfRdh2 were 996 and 1005 bp and the encoded proteins of 332 and 335 aa, respectively. At the nucleotide level, 15.5% (38 out of 245) of the synonymous positions and 5.8% (44 out of 759) of the non-synonymous sites differed. Also, the two clones differed markedly in sequence over their 5% untranslated region (UTR). Attempts to find an optimal alignment between the two 5% UTR sequences using the Clustal X program were unsuccessful, those being dependent on the gap penalty. Overall, these data support two different Rdh genes in B. floridae. D. Dalfoétal. / Chemico-Biological Interactions 130–132 (2001) 359–370 363

3.2. Southern blot analysis

B. lanceolatum genomic DNA from single adult animals was digested with PstI, resolved on agarose gels, one animal per lane, and probed with BlRdh exon 4 (Fig. 1). Only one band was detected in lanes 4 and 5 and lanes 1, 2 and 3 showed two. A closer examination of the banding pattern revealed that although band sizes were close to 2.0 kb (5 out of 6), they were rarely of identical size. These data argue in favor of a single-copy highly polymorphic gene sequence.

3.3. Exon–intron structure of amphioxus Rdh

The exon/intron organization of BlRdh was established by comparison of the genomic clone with the B. ﬂoridae cDNA. The coding region of BlRdh appeared distributed in six exons. The exon/intron architecture of vertebrate (human and mouse), amphioxus (B. lanceolatum) and a putative C. elegans Rdh were compared (Fig. 2A). Relative intron positions could be aligned although invertebrates showed a higher intron number and of smaller size than vertebrates. For instance, vertebrate exons 2 and 3 had been split into two different exons each in amphioxus. When the amphioxus Rdh sequence was compared with other SDR members, high homology was found to ß-hydroxybutyrate dehydrogenases (ß-OH-BDH), 17-ß-hydroxysteroid dehydrogenases type 2 (17-ß-HSD-2) and 11-ß-hydroxysteroid dehydrogenases type 2 (11-ß-HSD-2). These enzymes together with the RDHs cluster separately from all other hydroxysteroid dehydrogenases [21]. However, their genomic structures vary and the exon-intron architecture of ß-OH-BDH, 17-ß- HSD-2 and 11-ß-HSD-2 differs from that of the Rdh genes (Fig. 2B).

3.4. Phylogenetic analysis

The phylogeny of the RDHs and related enzymes was established from protein structure comparisons. The amphioxus RDHs clustered with the vertebrate enzymes and does not group specifically with any one type of vertebrate Rdh proteins (Fig. 3). The C. elegans proteins deduced from genomic sequences group together and are not significantly closer to any particular protein. The dendogram is consistent with an ancestral position for the amphioxus RDHs. Finally, a dupli- cated RDH appeared restricted to the B. floridae lineage.

4. Discussion

4.1. Genomic organization of amphioxus Rdh

Increasing evidence supports a retinoic acid signaling pathway in the common ancestor of vertebrates and pre-vertebrates [4–8]. Therefore, a metabolic pathway for RA synthesis could be expected in urochordates and cephalochordates. Al- 364 D. Dalfo´etal. / Chemico-Biological Interactions 130–132 (2001) 359–370 though the enzyme system could differ in each subphylum, conservation of the main steps throughout evolution seems plausible. The cephalochordate–vertebrate split preceded the expansion of the MDR–ADH family that originated the different vertebrate ADH classes, including the ADH1 and ADH4 retinol-active forms [22]. As no evidence has been gathered in support of an MDR-type enzyme in amphioxus other than ADH3, a microsomal short chain dehydrogenase/reductase system could well be responsible for retinol conversion to retinal.

Fig. 1. Southern blot of genomic DNA from single B. lanceolatum animals, PstI restricted and probed with BlRdh exon 4. Molecular weight markers are shown. D. Dalfo´etal. / Chemico-Biological Interactions 130–132 (2001) 359–370 365

Fig. 2. Exon-intron structure. (A) Exon (boxed) and intron (non-boxed) distribution of Rdh genes. Exon number is shown in Roman.The number below the exon boxes refers to the last amino acid of each exon. Intron sizes are drawn to scale whereas unknown intron sizes are shown as dotted lines. Although some amino acid numbers differ, the exon/intron boundaries of Rdh remain unchanged. (B) Comparison of the exon–intron structures for Rdh, 17-b-HSD-2, 11-b-HSD-2, b-OH-BDH and Adh/Ribitoldh genes. Sequence data were taken from GenBank (nucleotides database). Accession numbers of the compared sequences are as follows: Rdh H. sapiens (AF037062), 11-cis-Rdh M. musculus (X97752), Rdh B. lanceolatum (AF283542), Rdh C. elegans (Z74032), 17-b-HSD-2 H. sapiens (L40796), 11-b-HSD-2 H. sapiens (U27317 and U27318), b-OH-BDH C. elegans (U23455) and Adh/Ribitoldh C. elegans (AF022968). 366 D. Dalfo´etal. / Chemico-Biological Interactions 130–132 (2001) 359–370

Fig. 3. Phylogenetic relationship of the RDH proteins and ß-OH-BDH, 17-ß-HSD-2 and 11-ß-HSD-2 constructed by neighbour joining. Percentage bootstrap support for each node is shown only where it reaches 80% or above. No root has been assumed in the analysis. The branch lengths are proportional to the divergence between the sequences. The tree indicates a close relationship of amphioxus and vertebrate Rdh genes. The duplications for different vertebrates Rdhs probably occurred after the separation of the amphioxus and vertebrate lineages. The four C. elegans enzymes cluster together and they are not signiﬁcantly closer to any particular protein group.

We have characterized the genomic and the cDNA sequences of B. lanceolatum and B. floridae Rdh, a good candidate to perform the first step of retinol oxidation. Besides, exon–intron structure (Fig. 2) and protein sequence comparisons (Fig. 4) have shown the close relationship of RDH with vertebrate RDHs, whereas other hydroxysteroid active enzymes such as ß-OH-BDH, 11-ß-HSD-2 and 17-ß-HSD-2 appear more distantly related (Fig. 3). The mammalian vision-related RDHs cluster separately from the other RDH enzymes. Amphioxus RDHs show slightly lower sequence similarity to the vision-related forms than to the other vertebrate RDHs: 40–43% and 42–49%, respectively. Finally, two Drosophila Rdh DNA sequences, named CRAD1 and RDH4, appear more related to 17-ß-HSD-2 and 11-ß-HSD-2 than to any other compared sequence. From the Rdh structure, intron size increases and reduction in the number of exons are predicted during the cephalochordate– vertebrate transition. Far from being restricted to Rdh the same trend has been reported in other amphioxus genes, such as Adh3 [22] and Presenilin (our data). Alignment of the deduced amphioxus RDH proteins exhibited features of the short chain dehydrogenase/reductase enzyme family: the putative consensus sequence for cofactor binding, G(X3)GXG (at Gly 41 for BlRdh and BfRdh); the D. Dalfoétal. / Chemico-Biological Interactions 130–132 (2001) 359–370 367

Fig. 4. Alignment of amphioxus, vertebrate and C. elegans retinol-associated SDR sequences. Strictly conserved residues are shown on a black background and highly conserved residues (\90%) on a grey background. 368 D. Dalfo´etal. / Chemico-Biological Interactions 130–132 (2001) 359–370

active site consensus sequence, Y(X3)K (at Tyr 182 and 183 for BlRdh and BfRdh, respectively); and the LXNNAG (at Leu 144 for BlRdh and BfRdh) segment, all strictly conserved in the SDR enzymes [23]. A second Y(X3)K motif (at Tyr 203), present in the rat RDH enzymes [24] and some other mammalian proteins, is also shared by both B. ﬂoridae proteins. Moreover, although the search in amphioxus for membrane-spanning domains in RDH using the HMMTOP [25] and TMPred [26] programs has provided a variable number of domains, our data (data not shown) support that the amphioxus enzyme is an integral membrane protein [27].

4.2. Gene copy number

Our data are consistent with Rdh being a highly polymorphic single-copy gene in the B. lanceolatum species. Again, evidence in favor of highly polymorphic genes in amphioxus has been gathered for Adh3 and Presenilin associated with sequence repeats found at intron positions, near exon boundaries. Allelic variations cannot account for the differences between the two B. ﬂoridae cDNA sequences. Amino acid differences (12.8%) are of the same order as for the three rat isoenzymes when compared with each other (2.2–17.7%) [24]. Moreover, the unsuccessful attempts to align the 5%UTR also points to the presence of two closely related Rdh genes in B. ﬂoridae.

4.3. Phylogenetic relationship of RDH enzymes

The amphioxus sequences are closer to the vertebrate retinol dehydrogenases than to the C. elegans sequences or ß-OH-BDH, 17-ß-HSD and 11-ß-HSD (Fig. 3). The phylogenetic analysis suggests that all mammalian RDHs evolved from an ancestor common to cephalochordates and vertebrates. Moreover, the two B. floridae Rdhs are equally divergent with respect to B. lanceolatum, an indication that the duplication was restricted to the first species. Finally, the C. elegans ORF similar to retinol dehydrogenases suggest that C. elegans metabolizes retinoids. However, the RA-response of protostomes is different from that of vertebrates [4]. Therefore, the functional meaning of these enzymatic activities remains to be elucidated. It has been proposed that there is a single ancestor for the 17-ß-HSD-2, 11-ß-HSD-2, ß-OH-BDH and the retinol dehydrogenases [21]. According to our data, the duplication event of the ancestral gene that originated the hydroxysteroid and the retinol enzymes predated the cephalochordate–vertebrate divergence. The fact that adrenal and sex steroid receptors arose before vertebrates, in urochordates or cephalochordates [28] and that retinoic acid receptors have also been found in amphioxus [29] and ascidians [8] suggest a parallel evolution of the metabolic pathways and the signal transduction mechanisms for the acquisition of new chordate features. The characterization of the amphioxus Rdh gene opens the way to retinoid metabolism analysis avoiding the widespread gene duplication and functional redundancy present in vertebrates. D. Dalfoétal. / Chemico-Biological Interactions 130–132 (2001) 359–370 369

Acknowledgements

This work was supported by grants from DGICYT (Ministerio de Educacioń y Cultura, Spain, PB96-0220, UE98-0014), EU contract (BIO4 CT 97-2123), a FPI fellowship to D.D. from the MEC (Ministerio de Educacioń y Cultura) and a FPI fellowship to C.C from the CIRIT (Generalitat de Catalunya). We are indebted to the Serveis Cientı´fico-Te`cnics (UB) for DNA sequences analysis. We thank R. Rycroft for revising the English version of this manuscript.

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