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

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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, phyloge- netic 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). 0009-2797/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0009-2797(00)00261-1 360 D. Dalfo´etal. / Chemico-Biological Interactions 130–132 (2001) 359–370 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 dehydroge- nases (ADHs) [11] as well as different microsomal short-chain dehydrogenase/reduc- tases (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 dehydro- genase 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 Oce´anologique 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. Dalfo´etal. / 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-syn- onymous 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.
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