Identification and characterization of 17β-hydroxysteroid dehydrogenases in the zebrafish, Danio rerio

Molecular and Cellular Endocrinology 215 (2004) 19–30

Identification and characterization of 17␤-hydroxysteroid dehydrogenases in the zebrafish, Danio rerio R. Mindnich, D. Deluca, J. Adamski∗ GSF–National Research Center for Environment and Health, Institute of Experimental Genetics, Ingolstaedter Landstr.1, Neuherberg 85764, Germany

Abstract The 17␤-hydroxysteroid dehydrogenases (17␤-HSDs) are key enzymes in the final steps of steroid hormone synthesis. 17␤-HSD type 1 (HSD17B1) catalyzes the reduction of estrone to estradiol, while type 3 (HSD17B3) performs the conversion of androstenedione to testosterone. Here we present a functional genomics study of putative candidates of these enzymes in the zebrafish. By an in silico screen of zebrafish EST databases we identified three candidate homologs for both HSD17B1 and HSD17B3. Phylogenetic analysis, unique expression patterns (RT-PCR) during embryogenesis and adulthood, as well as activity measurements revealed that one of the HSD17B1 candidates is the zebrafish homolog, while the other two are paralogous photoreceptor-associated retinol dehydrogenases. All three HSD17B3 candidate genes showed nearly identical, ubiquitous expressions in embryogenesis and adult tissues and were identified to be paralogs of HSD17B12 and a yet uncharacterized putative steroid dehydrogenase. Phylogenetic analysis shows that HSD17B3 and HSD17B12 are descendants from a common ancestor. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: 17␤-Hydroxysteroid dehydrogenase; HSD17B1; HSD17B3; HSD17B12; Zebrafish; Embryogenesis; Evolution

1. Introduction The group of 17␤-hydroxysteroid dehydrogenases (17␤-HSD) is defined by their catalytic activity to reduce or oxidize hydroxysteroids, namely androgens and estrogens at position C17 of the steroid backbone (reviewed in Penning, 1997; Peltoketo et al., 1999; Adamski and Jakob, 2001). Up to now, 12 different types of 17␤-HSDs, which differ in substrate specificity and tissue distribution, have been annotated, mainly based on studies in mammals. Despite catalyzing the same enzymatic reaction, most members share only little sequence identity with each other (Krozowski, 1994; Oppermann et al., 1999). Similarities are mainly restricted to conserved residues known to be of structural and functional importance for the group of short-chain dehydrogenases/reductases (SDRs) to which all characterized 17␤-HSDs, except type 5, belong. Many 17␤-HSDs have overlapping substrate spectra but two of them seem to be especially important for the activation of estrogens and androgens: 17␤-HSD type 1 and 17␤-HSD type 3. ∗ Corresponding author. Tel.: +49-89-3187-3155; fax: +49-89-3187-3225. E-mail address: [email protected] (J. Adamski).

17␤-HSD type 1 catalyzes the reduction of estrone to estradiol, the major biologically effective estrogen in mammals. In humans this enzyme is mainly expressed in female steroidogenic tissues like ovaries and placenta but also in other tissues such as breast epithelium, uterus, brain and adipose tissue (Luu-The et al., 1990; Martel et al., 1992). 17␤-HSD type 3 catalyzes the conversion of androstenedione to testosterone which is involved in sexual differentiation. Dysfunction of this enzyme leads to male hermaphroditism (Geissler et al., 1994; Andersson et al., 1996). In concert with its male specific functions it has been reported to be almost exclusively expressed in the testis and to be absent in the ovary (Geissler et al., 1994; Zhang et al., 1996). The most common model organisms to study 17␤-HSD functions are mouse and rat, and comparative physiology suggests a highly similar role of steroid hormones in humans. But investigations of murine 17␤-HSDs have also revealed important differences: rodent HSD17B1, for example, can convert androgens in addition to estrogens (Nokelainen et al., 1996; Puranen et al., 1997) and, in contrast to the human enzyme, is not expressed in the rodent placenta (Nokelainen et al., 1996; Akinola et al., 1998). For some 17␤-HSDs no functional human homolog has yet been cloned, e.g. for rat

0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2003.11.010

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type 6 (Biswas and Russell, 1997) and mouse type 9 (Su et al., 1999). Aside from mammals, steroid hormone action is also being investigated in other vertebrates such as reptiles, amphibians and fish. In teleost fish, the gonadal sex differentiation (reviewed in Nakamura et al., 1998) and the effects of certain steroid hormones on sex reversal (reviewed in Hunter and Donaldson, 1987) have been studied for decades. cDNAs encoding for aromatase have been cloned from several fish and were found to play key roles in the regulation of estradiol synthesis (reviewed in Callard et al., 2001). Important roles for estrogens in ovarian differentiation (Guiguen et al., 1999) and for 11-oxygenated androgens in testicular differentiation (Liu et al., 2000) have been described. But only recently, the first fish 17␤-HSD, the type 1 homolog of the Japanese eel, has been cloned (Kazeto et al., 2000). This enzyme displays a substrate specificity similar to the human protein but seems to differ slightly from any known mammalian HSD17B1 in being expressed not only in female tissues but also in testis. To get more insight into 17␤-HSD activity in fish and its role in sexual development and reproduction we performed a functional genomics study of HSD17B1 and HSD17B3 in the zebrafish, Danio rerio. In this study, we describe the cloning and characterization of several zebrafish candidate genes. We were able to identify the zebrafish 17␤-HSD type 1 and two paralogs of 17␤-HSD type 12 that are compared to the respective mouse homologs concerning their spatial and temporal expression as well as substrate specificity. In addition, we found two closely related genes coding for the photoreceptor-associated retinol dehydrogenase and one putative steroid dehydrogenase of unknown function. A homolog of 17␤-HSD type 3 could not be identified.

2. Materials and methods 2.1. Identification of zebrafish homologs For the identification of zebrafish candidate genes, the protein sequences of mouse 17␤-HSD type 1 and type 3 were compared to the zebrafish EST database at NCBI (http://www.ncbi.nlm.nih.gov) using tblastn. EST sequences were chosen as putative homologs if either they were already annotated as similar to the respective 17␤-HSDs or had an alignment score >80 bits when their complete putative coding sequence was aligned to the respective 17␤-HSDs in the SwissProt database using blastx. 2.2. Identification of full-length cds of the zebrafish candidate genes Clones were obtained from the Resource Center, Primary Database (RZPD) of the German Human Genome Project at the Max-Planck Institute (Berlin, Germany) and verified by sequencing (SequiServe, Vaterstetten, Germany). In cases

where this was not possible or the EST clones from the RZPD did not contain the complete coding sequence (cds) we used the following approaches to obtain complete sequences. 2.2.1. cDNA library screen Based on the available candidate EST sequences, primer pairs were designed (zf 3.1: forward primer 5 -TAACGTGGTGGAGACGCTACAGC-3 , reverse primer 5 ATTCTGGGCAGCACCAGACG-3 ; zf 3.2: forward primer 5 -AGACAATGCAGAGTGCTGCTGG-3 , reverse primer 5 -GCCCTCTGTGAAGTCTGC CTG-3 ) and used for a PCR on cDNA from total adult zebrafish to produce gene-specific probes. The PCR product was purified (PCR Purification Kit, Qiagen) and randomly 32 P-labeled (Prime-It RmT Random Primer Labeling Kit, Stratagene). cDNA libraries of zebrafish liver (MPMGp532) and brain (UCDMp611) from RZPD were screened according to the manufacturers protocol and clones producing significant signals were ordered. In this way, clones containing the full-length cds of zf 3.1 and zf 3.2 could be retrieved (RZPD clones UCDMp611M0914Q2 and MPMGp532F1218Q1, respectively). 2.2.2. Complementation of cds In some cases, clones containing the full-length cds could not be obtained by the above methods. In those cases, we used the sequence information available from the assembly of the candidate ESTs to reconstruct the genes. Clone AI558663 of zf 1.1 lacked about the first 90 bp; the missing piece was amplified from cDNA by PCR and assembled with AI558663 by fusion-PCR: forward primer 5 -ACCGACTTGCATGATCCGC-3 and reverse primer 5 -AGGCCCATGACGCTGCTC-3 were designed according to EST-sequence information of BG307645. Using these primers a specific fragment was amplified by PCR on total cDNA from adult zebrafish and purified by gel extraction (Gel Extraction Kit, Qiagen). 0.24 pmol DNA from a plasmid preparation of clone AI558663 and 0.4 pmol of the PCR-fragment were added to a 100 ␮l PCR reaction and were combined with forward primer 5 -CGCGGATCCATGGCGAGC-3 and reverse primer 5 -CCGGAATTCTCTTCAGTCTGGAGATATGG-3 and Turbo-Pfu polymerase using the following program: 1 cycle 4 95 ◦ C, 2 cycles 30 95 ◦ C 2 50 ◦ C 2 72 ◦ C, 30 cycles 30 95 ◦ C 30 50 ◦ C 2 72 ◦ C. The resulting PCR product, containing the complete coding sequence of zf 1.1, was purified by gel extraction (Gel Extraction Kit, Qiagen). In case of zf 3.3, clone BQ078723 contained a sequence that corresponded to the C-terminus of the protein but comprised only about half of the full-length cds. The complete coding sequence of this gene was not cloned but reconstructed from a contig of available EST sequences (AW595044, AW567518, BG739062 and AW567561) to allow for analysis of the gene structure, protein sequence and phylogeny.

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2.3. Gene structure The full-length coding sequence of each zebrafish candidate gene was aligned against the zebrafish WGS database at NCBI using blastn. The resulting alignments revealed the exon–intron boundaries. Where necessary, boundaries were adjusted in accordance to the splice site consensus sequence (exon)–GT(intron)AG–(exon). 2.4. Phylogenetic analysis The data sets were created by retrieving related sequences from three different sources: a psi-blast (Altschul et al., 1997) of the mouse HSD17B1 and HSD17B3 protein sequences against the non-redundant protein database at NCBI; the BLink-link of the mouse HSD17B1 and HSD17B3 protein entries in the NCBI database (NP 034605 and NP 032317, respectively); a translated blast (tblastn) of the zebrafish 17␤-HSD candidates against EST databases of Ciona intestinalis and Caenorhabditis elegans. Sequences were aligned by ClustalW (Thompson et al., 1994; http://www2.ebi.ac.uk/clustalw) and the alignment monitored and manually edited in BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Phylogenetic analyses were conducted with MEGA version 2.1 (Kumar et al., 2001 and references therein, http://www.megasoftware.net) using Maximum Parsimony. Accession numbers of selected sequences: HSD17B1: human (NP 000404.1), marmoset monkey (AAG01115.2), rabbit (AAK20951.1), cow (AAF73061.1), mouse (NP 034605.1), rat (NP 036983.1), eel (JC7561), chicken (BAA19567.1); prRDH: cow (NP 776592.1), human (NP 056540.1), mouse (XP 134689.1), rat (XP 233743.1); microbial oxidoreductases: L. lactics (NP 267465.1), X. axonopodis (NP 640892.1), S. pombe (NP 592771.1), E. coli (CAD30682.2), S. coelicolor (NP 639619.1), X. fastidiosa (NP 299361.1), E. cloacae (AAK11695.1), M. loti a (NP 104075.1), M. loti b (NP 103540.1); human SD (NP 113651.3), C. intestinalis 33 (BW274894.1), C. elegans HSD17B: (a) (NP 506449.1), (b) (NP 507092.1), (c) (NP 506448.1), (d) (NP 498386.2); C. elegans SDR (NP 505205.1); D. melanogaster RE48687p (AAN71421.1), D. discoideum HP (AAM08487.1), D. melanogaster CG1444 (NP 572420.1), HSD17B12: duck (O57314), Xenopus (AAH41194.1), mouse (NP 062631.1), human (NP 057226.1); HSD17B3: human (NP 000188.1), rat (NP 446459.1), mouse (NP 032317.1); C. intestinalis 32 (AL669220.1), P. falciparum Kik1 (NP 702865.1); plant 3-keto-acyl-reductases: B. napus (AAO43448.1), A. thaliana (AAB82765.1), Z. mays (AAB82767.1), H. vulgare (AAB82766.1). 2.5. RNA preparation 2.5.1. Developmental stages Zebrafish embryos were staged in accordance to Kimmel et al. (1995) collected in 1.5 ml tubes and immediately frozen

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in liquid nitrogen after removal of medium. RNA extraction was performed using the RNeasy Mini Kit (Qiagen). Between 20 and 35 embryos were homogenized in QBT buffer using a syringe and a 20 G needle and subsequently processed according to the manufacturer’s protocol. 2.5.2. Adult For total RNA from adult zebrafish, one male fish (AB wild-type strain) was homogenized in liquid nitrogen using a mortar and pestle. About 150 mg of the homogenized tissue was subjected to RNA-extraction using the RNeasy Midi Kit (Qiagen). Organs of adult, fertile AB wild-type zebrafish (from 3 months up to 2 years) were dissected under the microscope, transferred to 1.5 ml tubes and frozen in liquid nitrogen. Composition of organ samples: brain (whole), gonads (total), skin (without scales), muscle (skeletal muscle), liver (mainly first and third lobe), spleen (total), kidney (excluding most of the interrenal), heart (whole), intestine (total) and eyes (whole). For preparation of RNA several organs were pooled and homogenized in TRIzol using a rotor–stator. RNA was extracted by addition of 0.2 volume of chloroform, vortexing for 15 s, incubation for 3 min at room temperature and centrifugation for 15 min at 4 ◦ C at maximum speed in a table-top Eppendorf centrifuge. 0.53 volumes of ethanol were added to the supernatant while slowly vortexing. The solution was applied to a pre-equilibrated column of the RNeasy Midi Kit (Qiagen) and further processed according to the manufacturer’s protocol. Amount and quality of the total RNA were assessed by spectrophotometry and gel electrophoresis, respectively. Residual genomic DNA was digested by addition of RQ1 RNase-free DNase I (Promega) according to manufacturer’s protocol and enzyme and buffer afterwards removed with the RNeasy Mini Kit. 2.6. Expression analysis by RT-PCR One microgram or the amount yielded from about 20 embryos (developmental stages shield to tailbud) were transcribed into cDNA using the First Strand cDNA Synthesis Kit (MBI Fermentas) and poly dT-primers in a total volume of 20 ␮l. One microliter cDNA was added to a total volume of 20 ␮l reaction (1.5 mM MgCl2 , 1 ␮l lab-made Taq-polymerase) and the PCR run on a Robo-Cycler (Stratagene) with 1 cycle 3 95 ◦ C, 35 cycles 30 95 ◦ C, 30 55 ◦ C, 1 72 ◦ C. Quality of the cDNA was first tested with primers for zebrafish actin: forward (5 -CTGGTTGTTGACAACGGATCCG-3 ) and reverse (5 -CAGACTCATCGTACTCCTGCTTGC3 ). Expression of the various 17␤-HSD candidates was investigated using the following primers in the standard PCR reaction: zf 1.1 forward: 5 CAGAAAGTGGTGCTGATCACCGGCTGC-3 , reverse: 5 -AGGCCCATGACGCTGCTC-3 ; zf 1.2 forward: 5 -

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ATGTAACTGACCAGCAATCTATACTTGATGC-3 , reverse: 5 -GGAGTCTGCGCTTCCATCG-3 ; zf 1.3 forward: 5 -AGGCTCCGGTTCTCCGGTCAG-3 , reverse: 5 -CATCCAGACTGAGACCCTCCACCG-3 ; zf 3.1 forward: 5 -CGGTGCACTTATCACGGCCT CGC-3 , reverse: 5 -ATTCTGGGCAGCACCAGACG-3 ; zf 3.2 forward: 5 -AGACAATGCAGAGTGCTGCTGG-3 , reverse: 5 -GCCCTCTGTGA AGTCTGCCTG-3 ; zf 3.3 forward: 5 -CAATGGAAATCTGATGAGGGCGAG-3 , reverse: 5 -CAGATTCAATCTTAGGATAGATGTCCACAGATC-3 . 2.7. Cloning and expression of recombinant proteins Full-length coding sequences of all three zebrafish candidate genes for 17␤-HSD type 1 were cloned into a modified pGEX 2T PL2 vector and expression of the recombinant protein was performed as described in Leenders et al. (1996). Bacteria were harvested by centrifugation, resuspended in lysis buffer (PBS containing 0.1 mg/ml lysozyme and protease-inhibitors), lysed by five freeze/thaw cycles and genomic DNA digested by addition of 1 U benzonase (Sigma) and MgCl2 to a final concentration of 5 mM. Samples were centrifuged to separate soluble and insoluble proteins. The pellet fraction was resuspended in an equal amount of lysis buffer. 10 ␮l of both fractions were analyzed by SDS-PAGE and Coomassie staining. 2.8. Enzymatic measurements Since the recombinant proteins were nearly always found in the pellet fraction, 10 ␮l of this fraction was subjected to activity measurements. Catalytic activity towards estrone and estradiol was assessed as described in Leenders et al. (1996). For measuring the interconversion of androgens, the same method was used but the buffer changed to 100 mM sodium phosphate buffer, pH 7.4 with final concentrations of 18.7 nM testosterone or 13.5 nM androstenedione. All steroids were from NEN.

3. Results 3.1. Identification of putative zebrafish-homologs of 17β-HSD type 1 and 3 The Blast-based searches of the zebrafish EST-database at NCBI revealed six sequences that could be putative homologs of 17␤-HSD type 1 and 3, each represented by multiple, non-identical ESTs. According to the chronology in which the clones were identified they were provisionally named zf 1.1, zf 1.2 and zf 1.3 for putative 17␤-HSD type 1 and zf 3.1, zf 3.2 and zf 3.3 for putative 17␤-HSD type 3 homologs, respectively. The level of amino acid identity to the mouse genes differed from 30% to nearly 60% depending on the part of the protein to which the EST sequence

matched (not shown). In addition, annotations in GenBank about the function of the candidate genes were not conclusive. All three candidates for 17␤-HSD type 1 were annotated as similar to this protein. As it was not clear whether this might be due to the existence of paralogous HSD17B1 in zebrafish all three candidate genes were selected for further characterization. In case of the putative homologs of 17␤-HSD type 3, none was annotated to be significantly similar to this protein but instead to the putative steroid dehydrogenase Kik-1 which is also named 17␤-HSD type 12. Since we found evidence that type 3 and 12 are very closely related (see below) we took all putative zebrafish HSD17B3 orthologs in consideration. Where possible, clones were ordered directly from RZPD and sequenced. In other cases, the cds was complemented as described in Section 2. The full-length cds of the following candidate genes have been submitted to GenBank at NCBI: zf 1.1: AY306007 (zfprRDH2), zf 1.2: AY306005 (zfHSD17B1), zf 1.3: AY306006 (zfprRDH1). 3.2. Characterization by exon structure It is obvious that exon sizes and structures are highly informative characters to assess homology relationships at levels of high sequence divergence, because they are highly conserved and yet show very little tendency for convergent evolution; this effect is also apparent in paralogous genes. Therefore, we inspected these characteristics in all zebrafish candidate genes in comparison to mouse HSD17B1, HSD17B3 and HSD17B12. The characteristic structure of 17␤-HSD type 1 consists of six exons with specific sizes as depicted in Fig. 1A. In principle, this structure seems to be conserved for all three zebrafish candidates as exon–intron boundaries are strictly conserved (Fig. 2A). Changes in exon sizes appear in exons 1, 2, 5 and 6 and take place in a triplet-size manner. In comparison to the mouse genes exon 2 is slightly shortened by six nucleotides whereas exon 5 is increased by 9 nucleotides in zf 1.1 and zf 1.3, and by 12 nucleotides in zf 1.2. Considering the exact size matches of zf 1.1 and zf 1.3 in exons 2, 5 and 6 these zebrafish genes are more similar to each other than to zf 1.2. As the last exon does not harbor any functionally important residues variations in size may not influence the functionality of the protein. However, the first exon contains the co-factor binding site and therefore zf 1.2, that matches the size of HSD17B1 in the first exon, seems to be closer related to the 17␤-HSD type 1 than the other two zebrafish candidate genes. The exon structure of 17␤-HSD type 3 comprises eleven exons with defined sizes (Fig. 1B). A very similar structure is shared by 17␤-HSD type 12 which differs from type 3 mainly in a slightly smaller exon 5 (−3 bp) and an exon 6 increased by 9 bp. As was the case for 17␤-HSD type 1 exon–intron boundaries in 17␤-HSD type 3 are conserved (Fig. 2B) whereas differences occur only inside of exons by addition or deletion of codons. The only exception seems

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Fig. 1. Gene structure of the zebrafish candidate genes in comparison to mouse 17␤-HSD type 1 and type 3. The gene structure of mouse 17␤-HSD type 1 (A) and type 3 (B) are depicted in the first row with exon sizes given in nucleotides. The zebrafish genes and in (B) also mouse HSD17B12 are shown in comparison to HSD17B1 and HSD17B3: “–“ indicates a matched size, “+” addition and “−“ deletion of the given number of nucleotides. Exons of the individual mouse genes are in scale whereas the space between exons does not reflect the respective intron size.

to be the border intron 5/exon 6 for mouse 17␤-HSD type 3 where the sequence alignment suggests a gap of nine nucleotides right at the beginning of the exon. On the other hand, the first five amino acids of this exon do not seem to be conserved so that this gap may as well be shifted to the right. Comparison of the structure of the zebrafish candidates with 17␤-HSD type 3 and 12 demonstrate that they fit the pattern of exon sizes very well. The size variations themselves do not take place randomly but are restricted to certain exons, namely exon 1, 4, 5, 6 and 11. Zf 3.1 and zf 3.3 share identical sizes in all but the first exon and are more similar to each other than to zf 3.2. In addition, exon 5 and 6 are different from HSD17B3 but exactly reflect the situation in HSD17B12 and hence, zf 3.1 and zf 3.3 seem to be closer related to the 17␤-HSD type 12. The zf 3.2 also shares the size of exon 6 with HSD17B12 but in exon 5 resembles HSD17B3. Another variation is the increase of the size of exon 4 by three nucleotides which is only present in zf 3.2 and not the mouse genes. Thus, while zf 3.1 and zf 3.3 are clearly paralogs highly similar to HSD17B12, it is not clear whether zf 3.2 is closer related to HSD17B3 or HSD17B12. 3.3. Protein level: homologies and conserved features Amino acid identity was used as a criterion for the selection of homologous genes but varied highly even for the same candidate gene due to fact that the available EST sequences resembled only parts of the zebrafish genes. Therefore, the completely reconstructed protein sequences of the zebrafish and mouse enzymes were again compared at the protein level for their degree of sequence identity (Table 1). In addition, we looked at the presence of residues known to be of functional importance.

For the 17␤-HSD type 1 candidates (Table 1A) it becomes clear that zf 1.1 and zf 1.3 are more similar to each other (70% identity) as compared to zf 1.2 (41%) or the mouse protein (38 and 39%, respectively). Zf 1.2 shares the highest identity (51%) to mouse HSD17B1 and hence, seems to be the best candidate for a type 1 homolog. A similar pattern is found in case of the 17␤-HSD type 3 candidates (Table 1B). Here, zf 3.1 and zf 3.3 share 67% amino acid identity and seem to be closer related to each other than to HSD17B3 (40%), HSD17B12 (60%) or zf 3.2 (∼45%). None of the zebrafish genes shows a significantly increased identity to HSD17B3. Instead, zf 3.1 and zf 3.3 share apparent sequence identity to HSD17B12. Comparison of zf 3.2 to all other genes of this group always led to Table 1 Amino acid sequence percentage identity between murine HSD17B1 and HSD17B3/12 and their respective zebrafish paralogs Enzyme

zf 1.1

zf 1.2

zf 1.3

mHSD17B1

(A) 17␤-HSD type 1 zf 1.1 100 zf 1.2 41 zf 1.3 70 mHSD17B1 38

100 41 51

100 39

100

Enzyme

zf 3.2

zf 3.3

mHSD17B3

mHSD17B12

100 40 60

100 41

100

zf 3.1

(B) 17␤-HSD type 3 and 12 zf 3.1 100 zf 3.2 46 100 zf 3.3 67 44 mHSD17B3 40 45 mHSD17B12 60 47

Sequences were compared by pairwise Blast. The different degrees of sequence identity indicate non-uniform relations between the mouse gene and the putative zebrafish homologs.

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Fig. 2. Comparison between amino acid sequences of mouse 17␤-HSD type 1 and 3 and their putative zebrafish homologs. (A) 17␤-HSD type 1, (B) 17␤-HSD type 3. Residues conserved in all aligned sequences are shaded; dashes indicate gaps to facilitate better alignment. Elements typical for SDRs and of functional importance are indicated as follows: horizontal bar: co-factor binding site motiv (TG∗∗∗ G∗ G); stars: catalytic site; dots: structurally conserved residues. Vertical dotted lines represent exon boundaries; where a border was positioned within the codon the amino acid was added to the exon containing the majority of the codon’s nucleotides.

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intermediate values of 44–47% which allows no decision as to where zf 3.2 may belong. Fig. 2 shows the alignment of the zebrafish and mouse protein sequences indicating specific conserved residues. In both cases typical features of the short-chain dehydrogenase/reductase family to which 17␤-HSD type 1 and 3 belong are present in all six zebrafish candidates. Essential amino acids of the co-factor binding site, active center and NAG-structural motiv (Oppermann et al., 1996; Filling et al., 2002) are completely conserved. 3.4. Phylogenetic context Investigation of the zebrafish genes on the genomic and protein level substantiated their close relation to 17␤-HSD type 1 and 3 or 12. Phylogenetic analyses were carried out to elucidate their position in a more complex context encompassing more proteins to reveal relationships that might be missed by restricted pairwise comparisons. For 17␤-HSD type 1 the dendrogramm shows a deep trifurcation dividing three large groups of enzymes (Fig. 3A): the family of 17␤-HSD type 1, the photoreceptor-associated retinol dehydrogenases (also known as prRDH and RDH 8) and the outgroup of bacterial oxidoreductases. The positions of the zebrafish proteins are supported by high bootstrap values, and only zf 1.2 is a member of the 17␤-HSD type 1 group. In this group it matches closely to the eel homolog and is also positioned in vicinity to the chicken protein which shows that the sequence-based tree of the proteins indeed reflects the evolutionary relationship of the organisms, as is expected for true orthologs. The zf 1.1 and zf 1.3 fall into the group of photoreceptor-associated retinol dehydrogenases where they appear as paralogs. As a result, zf 1.2 appears to be the zebrafish homolog of HSD17B1 whereas zf 1.1 and zf 1.3 are both homologs of the closely related group of photoreceptor-associated retinol dehydrogenases (Fig. 3A). In the case of 17␤-HSD type 3, the phylogenetic tree (Fig. 3B) is much more complex. Some groups are supported by high bootstrap values while about a third of the sequences do not fit into any of these. The groups of 17␤-HSD type 3 and 12 appear strictly separated with none of the zebrafish candidates as a member of the 17␤-HSD type 3 group. Instead, zf 3.1 and zf 3.3 show up as orthologs of 17␤-HSD 12. Zf 3.2 belongs to neither 17␤-HSD type 3 nor type 12 but is a member of a poorly characterized group containing proteins, e.g. from Drosophila melanogaster and C. intestinalis, as well as an uncharacterized steroid dehydrogenase-like sequence from human. 3.5. Expression during early development and in adult organs Genes exhibit specific expression patterns that correlate with their in vivo function. To gather more information about the putative functions of the zebrafish 17␤-HSD candidate genes their expression was monitored by RT-PCR during embryogenesis and in organs of adult fish.

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All zebrafish 17␤-HSD type 1 candidates showed unique expression patterns (Fig. 4A). Zf 1.1 mRNA was present in every monitored developmental stage and also in every male and female adult organ. No differences between the sexes or between steroidogenic and non-steroidogenic tissues could be seen. For zf 1.2 no expression could be detected either during embryogenesis or in male adult fish. Instead, a strong signal was produced by female gonads in addition to some other female organs. In the case of zf 1.3 expression started at about 21 somite-stage and was present from then on, probably coinciding with the onset of eye differentiation. In the adult fish clear signals were produced in brain and eye in addition to male and female gonads. In contrast to this, all zebrafish candidate genes for 17␤-HSD type 3 show highly homogenous expression patterns (Fig. 4B). With few exceptions, mRNA of all three genes seems to be present throughout development and in all adult male and female organs. A notable exception is the absence of zf 3.2 mRNA in the shield stage, and extremely weak signals for zf 3.3 in the early developmental stages from shield to tailbud. In general, zf 3.3 expression in some adult organs was only barely detectable. 3.6. Activity of 17β-HSD type 1 candidates For all known 17␤-HSD type 1 genes so far characterized a high substrate affinity to estrone and estradiol has been demonstrated. In vitro, the human enzyme readily catalyzes the interconversion of estrone to estradiol; in vivo, reduction is probably favored over oxidation (Dumont et al., 1992). Hence, we investigated the ability of all three 17␤-HSD type 1 candidates to catalyze estrogen conversion. When bacterially expressed recombinant proteins were examined for 17␤-HSD type 1 activity we found clear differences between the zebrafish candidates (Fig. 5). Reduction of estrone to estradiol as well as vice versa was readily catalyzed by zf 1.2. Under the given conditions nearly 100% of the substrate was metabolized. In contrast, none of the other two candidate proteins showed any activity towards estrone or estradiol. These findings are in agreement with the phylogenetic analysis suggesting zf 1.1 and zf 1.3 to convert retinoids and not estrogens. In addition, we measured conversion of androgens, a reaction additionally catalyzed by the mouse HSD17B1 (Nokelainen et al., 1996). None of the zebrafish proteins was able to catalyze the reduction of androstenedione to testosterone or vice versa. For all types of conversion, we also monitored activity of bacterially expressed GST from insert-lacking pGEX vectors and found no product formation (data not shown). 4. Discussion 4.1. Identification and properties of zebrafish 17β-HSD type 1 An in silico screen of zebrafish EST-databases with mouse HSD17B1 led to the identification of three possible ze-

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Fig. 3. Phylogenetic analysis of the position of zebrafish candidate genes in comparison to 17␤-HSD type 1 and 3. (A) 17␤-HSD type 1, (B) 17␤-HSD type 3. Phylogenetic trees were calculated by means of Maximum Parsimony algorithm; numbers indicate percentage of bootstrap support of a 1000 pseudoreplicates. Names of functionally or evolutionary related groups are given if feasible. Outgroup in (A) microbial oxidoreductases; (B) plant 3-keto-acyl reductases. HP, hypothetical protein; prRDH, photoreceptor-associated retinol dehydrogenase; SD, steroid dehydrogenase; SDR, short-chain dehydrogenase/reductase. GenBank accession numbers of all sequences are listed in Section 2.

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Fig. 4. RT-PCR analysis of expression during zebrafish embryogenesis and in adult organs of zebrafish 17␤-HSD type 1 and type 3 candidates. (A) 17␤-HSD type 1, (B) 17␤-HSD type 3. Gel photographs show the observed distribution of RT-PCR signals for each individual zebrafish gene. Signal strength is primarily not considered to give quantitative information. hpf: hours post-fertilization.

brafish homologs that were characterized by bioinformatic as well as laboratory methods. All results point to that the candidate zf 1.2 is the zebrafish ortholog of 17␤-HSD 1 (zfHSD17B1). zfHSD17B1 in correspondence with other type 1 17␤-HSDs is a member of the SDR family. This is supported by the conservation of functionally important amino acids. It specifically converts estrone to estradiol and vice versa, but does not catalyze the conversion of androgens, in contrast to the mouse and rat homologs (Puranen et al., 1997). The substrate specificity towards estrone and estradiol is in common with that of the human (Luu-The et al., 1995), chicken (Wajima et al., 1999) and eel (Kazeto et al., 2000) 17␤-HSD 1. Up to now, the androgen converting activity has been described only for the rodent 17␤-HSD type 1 enzymes and might be considered a characteristic restricted to this taxon. Expression of HSD17B1 is most prominent in females where it is very high in ovaries. In zebrafish, we found expression exclusively in female adult fish and most prominent in ovaries in addition to skin, muscle,

heart and eyes, which seems to reflect the situation in mammals. This is in contrast to the japanese eel, where HSD17B1 mRNA was detected only in ovaries and testis (Kazeto et al., 2000). During embryogenesis up to 84 hpf (hours post-fertilization) we could not find any HSD17B1 expression, but responsiveness to estrogen in zebrafish starts already at about 24 hpf a time-point where as well expression of P450aromB begins to increase strongly (Kishida and Callard, 2001). A similiar pattern can be observed in mouse. Here, aromatase mRNA was detected at 11 dpc in brain (Harada and Yamada, 1992) and at 17 dpc in testis (Greco and Payne, 1994) but HSD17B1 expression at these stages is absent (Mustonen et al., 1997). In this context, estradiol may rather be synthesized from aromatizable androgens than by reduction of estrone. This pathway may also be used for estradiol production in male zebrafish as we could not detect any HSD17B1 expression in male tissues.

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genes evolved from a common ancestor and still share high similarities on exon structure and sequence level it is not clear whether their substrate specificity has already diverged together with expression pattern and physiological function. 4.3. Relation between HSD17B3 and HSD17B12/properties of the paralogous zebrafish HSD17B12 genes

Fig. 5. Conversion of steroids by the recombinant proteins of the zebrafish candidate genes. Columns depict the percentage of substrate converted by the given enzyme after 30 min incubation as described in materials and methods. Each data point was measured at least twice. Results indicate a strong estrone–estradiol converting activity for zf 1.2. A, androstenedione; T, testosterone; E1, estrone; E2, estradiol.

4.2. Photoreceptor-associated retinol dehydrogenases in comparison to 17β-HSD1 Of the three candidate genes for HSD17B1 two could be identified as homologs of the photoreceptor-associated retinol dehydrogenase and accordingly were named zfprRDH1 (zf 1.3) and zfprRDH2 (zf 1.1). Up to now, 14 different retinol dehydrogenases (RDHs) have been annotated and the functions of most of them described (reviewed in Napoli, 1999). All family members share the ability to convert specific retinoids but sequence identity among the various proteins is rather low. Structurally, RDHs belong to the group of SDRs as do most of the 17␤-HSDs. Phylogenetic studies have already described the close relation of some members of the RDH and 17␤-HSD families (Rattner et al., 2000; Baker, 2001) suggesting evolution from a common ancestor. The connection is also demonstrated by the fact that some enzymes originally identified as RDHs can also convert steroids and vice versa (Napoli, 1999). In case of the two identified zebrafish RDHs substrate specificity might be restricted to retinoids, as we could not detect any activity towards estrone or androstenedione. Their putative retinoid-directed activity was inferred from the bovine homolog that converts all-trans-retinal to all-trans-retinol but no steroids were tested for this mammalian enzyme (Rattner et al., 2000). In addition to phylogenetic relations, exon-structure identities and primary sequence homology the zfprRDH1 also showed expression patterns that fit the putative function of a retinol dehydrogenase involved in the vision process. During embryogenesis, expression starts in late somitogenesis at the time when eye and brain undergo enhanced development. But in contrast to the bovine enzyme, expression in adulthood is not exclusively found in eyes but as well in brain and gonads. This suggests that its function in zebrafish is not restricted to vision. This might also be the case for zfprRDH2, the paralog of zfprRDH1, that is widely expressed during embryogenesis and in adult tissues. Although our analysis shows that both

The aim of the present study was to identify the zebrafish homologs of HSD17B1 and HSD17B3, the mammalian key regulators of androgen and estrogen activity. Of the three HSD17B3 candidate genes examined none was found to be the genuine zebrafish ortholog of mammalian HSD17B3. Instead, two putative zebrafish HSD17B12 homologs (zf 3.1 and zf 3.3) were identified. The function of HSD17B12 in any species still has to be elucidated as the gene was only identified based on sequence similarity. Our identification of the two zebrafish genes and the existence of an avian homolog indicate an evolutionarily conserved and important physiological function of HSD17B12. Our results indicate a close relation of HSD17B12 to HSD17B3 which has not been reported before. On the protein sequence level, about 40% of residues are identical which is significantly higher than for convergently evolved 17␤-HSDs. This conclusion is also supported by the detailed phylogenetic analysis (Fig. 3B) which detects a clear sister group relation between HSD17B3 and HSD17B12. In addition, investigation of the gene structure was found to be an extremely useful method to assess homology relationships in silico. In the case of HSD17B3 and HSD17B12 isoforms conservation of intron-positions and several identical exon sizes (Exon 2, 3, 7, 8, 9 and 10) confirm the close relationship of the two groups. The sizes of exon 5 and 6 seem to be characteristic for each group (68 and 36 bp for HSD17B3 and 65 and 45 bp for HSD17B12, respectively). In comparison, a similar relation exists between the type 2 isozymes of 17␤- and 11␤-HSDs which share only about 45% amino acid identity, but the sizes of all “internal” exons (i.e. excluding the first and last exon) are strictly conserved in human, mouse and rat. Despite this apparent close relation, both enzymes have characteristic divergent substrate specificities and catalyze oxidation at different positions of the steroid backbone (Wu et al., 1993; Brown et al., 1996). Therefore, HSD17B3 and HSD17B12 might as well display dissimilar substrate preferences and there is currently no indication that the type 12 isozyme has any 17␤-HSD activity at all. We can already show some differences as both zebrafish putative HSD17B12 are expressed throughout embryogenesis and a variety of adult organs in both sexes which is reflected in the in silico Northern analysis of the mouse and human gene (data not shown). HSD17B3 however was not detected during mouse development and in adulthood is exclusively present in the testis (Mustonen et al., 1997). Identification of the zebrafish HSD17B3 homolog and characterization of HSD17B12 enzyme properties will be necessary to understand the function and evolution of these genes in vertebrates.

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Acknowledgements We thank Dr. Rainer Breitling for help with the phylogenetic analysis and would also like to thank him and Dr. Gabriele Möller for helpful discussion of the manuscript. Part of this work was supported by a DFG grant to J. Adamski.

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