Báo cáo khoa học: Identification of a novel thyroid hormone-sulfating cytosolic sulfotransferase, SULT1 ST5, from zebrafish Molecular cloning, expression, characterization and ontogenic study ppt

Identification of a novel thyroid hormone-sulfating cytosolic sulfotransferase, SULT1 ST5, from zebrafish Molecular cloning, expression, characterization and ontogenic study Shin Yasuda, A. Pavan Kumar, Ming-Yih Liu, Yoichi Sakakibara, Masahito Suiko, Lanzhuang Chen and Ming-Cheh Liu Biomedical Research Center, The University of Texas Health Center, Tyler, USA Sulfation is an important pathway in vivo for the bio- transformation of low molecular mass xenobiotics as well as endogenous compounds [1–3]. Upon sulfation, xenobiotic compounds become more water soluble and can be excreted from the body more easily. For endo- genous compounds such as steroid and thyroid hormones, catecholamines, cholesterol and bile acids, sulfation may be involved in their regulation and homeostasis. In the case of thyroid hormones, sulfa- tion may increase their water solubility and subsequent biliary and urinary excretion [4]. Moreover, 3,3¢,5-tri- iodo-l-Thyronine (l-T 3 ), the major thyroid hormone, loses its affinity for thyroid hormone receptors upon sulfation, and sulfated l-T 3 is subject to accelerated Keywords molecular cloning; sulfotransferase; SULT1; thyroid hormone; zebrafish Correspondence M C. Liu, Biomedical Research Center, The University of Texas Health Center, 11937 U.S. Highway 271, Tyler, TX 75708, USA Fax: +1 903 877 2863 Tel: +1 903 877 2862 E-mail: ming.liu@uthct.edu (Received 7 February 2005, revised 20 April 2005, accepted 25 May 2005) doi:10.1111/j.1742-4658.2005.04791.x By employing RT-PCR in conjunction with 3¢-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (desig- nated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic com- pounds. Its pH optima were 6.0 and 9.0 with 3,3¢,5-triiodo-l-thyronine (l-T 3 ) as substrate and 6.0 with b-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of diva- lent metal cations on the l-T 3 -sulfating activity of SULT1 ST5 revealed that Fe 2+ ,Hg 2+ ,Co 2+ ,Zn 2+ ,Cu 2+ ,Cd 2+ and Pb 2+ exhibited dramatic inhibitory effects, whereas Mn 2+ showed a significant stimulation. Devel- opmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity. Abbreviations D-T 3 , 3,3¢,5-triiodo-D-thyronine; DTT, dithiothreitol; estrone, 1,3,5[10]-estratrinen-3-ol-17-one; IPTG, isopropyl thio-b-D-galactoside; L-Dopa, L-3,4-dihydroxyphenylalanine; L-rT3, 3,3¢,5¢-triiodo-L-thyronine; L-T 3 , 3,3¢,5-triiodo-L-thyronine; L-T 4 , L-thyroxine; PAPS, 3¢-phosphoadenosine-5¢- phosphosulfate; SULT, sulfotransferase. 3828 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS inner ring deiodination, followed by subsequent degra- dation reactions [5]. The enzymes responsible for the sulfation reactions, the cytosolic sulfotransferases (SULTs), catalyze the transfer of a sulfonate group from 3¢-phosphoadeno- sine-5¢-phosphosulfate (PAPS) to the hydroxyl group or amino group of substrate compounds [1–3]. Since the early 1990s, increasing numbers of cytosolic SULTs from different vertebrates have been cloned and sequenced [6,7]. It is now known that all cytosolic SULTs from vertebrates constitute a gene superfamily and, based on amino acid sequence homology, distinct gene families have been further categorized [8]. Two major gene families among them are the phenol SULT family (designated SULT1) and the hydroxysteroid SULT family (designated SULT2) [6–8]. The phenol SULT family consists of at least five subfamilies, phenol SULT (SULT1A), Dopa ⁄ tyrosine (or thyroid hormone) SULT (SULT1B), hydroxyarylamine (or acetylaminofluorene) SULT (SULT1C), tyrosine ester SULT (SULT1D), and estrogen SULT (SULT1E). The hydroxysteroid SULT family currently compri- ses two subfamilies, dehydroepiandrosterone SULT (SULT2A) and cholesterol ⁄ pregnenolone SULT (SULT2B). Despite a considerable amount of work carried out in the past two decades, to a large extent the physio- logical involvement of the various cytosolic SULTs remains unclear. Moreover, only fragmentary informa- tion is available concerning the cell type ⁄ tissue ⁄ organ- specific expression of the different cytosolic SULTs, and very little is known with regard to the ontogeny of these enzymes. To resolve these outstanding issues, a suitable animal model is required. Zebrafish has recently emerged as a popular animal model for a wide range of studies [9,10]. Its advantages, compared with mouse, rat or other vertebrate models, include its small size, the availability of a relatively large number of eggs, rapid development externally of virtually trans- parent embryo, and short generation time. These unique features make the zebrafish an excellent model for systematic studies on the ontogeny of cytosolic SULTs and their tissue- and cell-Type-specific distribu- tion, as well as the physiological relevance of individ- ual cytosolic SULTs. A prerequisite for using zebrafish in these studies, however, is the identification of the various cytosolic SULTs and their biochemical charac- terization. We have recently embarked on the mole- cular cloning of zebrafish cytosolic SULTs [11–14]. Sequence analysis via blast search revealed that the zebrafish cytosolic SULTs we have cloned [11–14] dis- play sequence homology to mammalian cytosolic SULTs. Of the six zebrafish cytosolic SULTs cloned, four fall within the SULT1 gene family [11,12], one belongs to the SULT2 gene family [13], and one appears to be independent of all known SULT gene families [14]. We report here the molecular cloning, expression and characterization of a novel thyroid hormone-sulf- ating cytosolic SULT from zebrafish. Its enzymatic activities toward a variety of endogenous and xeno- biotic compounds including some flavonoids, isoflavo- noids, and other phenolic compounds were examined. Kinetic parameters of the enzyme with thyroid hor- mones and their metabolites as substrates were deter- mined. Moreover, its developmental stage-dependent expression was investigated. Results and Discussion As part of an effort to develop a zebrafish model for investigating, in greater detail, the role of sulfation in the metabolism and homeostasis of thyroid hormones, we identified and characterized a novel zebrafish thy- roid hormone-sulfating SULT in this study. Molecular cloning of the zebrafish cytosolic SULT1 ST5 By employing RT-PCR in conjunction with 3¢-RACE, a full-length cDNA encoding a novel zebrafish cyto- solic SULT was cloned and sequenced. The nucleotide sequence obtained was submitted to the GenBank database under accession no. AY879099. Figure 1 shows the alignment of the deduced amino acid sequence of the newly cloned zebrafish SULT with those of the other four zebrafish SULT1 STs previ- ously identified [11,12]. The open reading frame of the newly cloned SULT encompasses 882 nucleotides and encodes a 293-amino acid polypeptide. Similar to other cytosolic SULTs, the new zebrafish SULT contains sequences resembling the so-called ‘signature seq- uences’ (YPKSGTxW in the N-terminal region and RKGxxGDWKNxFT in the C-terminal region; as underlined in Fig. 1) characteristic of SULT enzymes [7]. Of these two sequences, YPKSGTxW has been demonstrated by X-ray crystallography to be respon- sible for binding to the 5¢-phosphosulfate group of PAPS, a cosubstrate for SULT-catalyzed sulfation reactions [15], and thus designated the 5¢-phosphosul- fate binding (5¢-PSB) motif [16]. The cloned zebrafish SULT also contains the 3¢-phosphate binding (3¢-PB) motif (amino acid residues 187–197; as underlined) responsible for the binding to the 3¢-phosphate group of PAPS [16]. Sequence analysis based on blast search revealed that the deduced amino acid sequence of the S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3829 new zebrafish SULT displays 48 and 46% identity to dog and human SULT1B1, and lower percentage iden- tity to other known SULTs. It is generally accepted that members of the same SULT gene family share at least 45% amino acid sequence identity, and members of subfamilies further divided in each SULT gene fam- ily are > 60% identical in amino acid sequence [6–8]. Based on these criteria, the newly cloned zebrafish SULT appears to belong to the SULT1 gene family, and is tentatively designated zebrafish SULT1 ST5 in accordance with the nomenclature used in the ZFIN database (cf. the dendrogram shown in Fig. 2). Com- pared with known zebrafish SULTs, the newly cloned zebrafish SULT1 ST5 displays 44, 45, 43, and 46% Fig. 1. Alignment of the deduced amino acid sequences of SULT1 ST5 and four known zebrafish SULT1 STs. Two ‘signature sequences’, respectively located in the N- and C-terminal regions, as well as a conserved sequence in the middle region, are underlined. Fig. 2. Classification of zebrafish SULT1 ST5 on the basis of deduced amino acid sequen- ce. The dendrogram shows the degree of amino acid sequence homology among cytosolic SULTs. References for individual SULTs are given in [18]. h, Human; m, mouse; zf, zebrafish. The dendrogram was gen- erated based on the Greedy algorithm [31, 32]. A novel zebrafish cytosolic sulfotransferase S. Yasuda et al. 3830 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS amino acid sequence identity to, respectively, zebrafish SULT1 ST1, 2, 3, and 4 previously reported [11,12]. Expression, purification, and characterization of recombinant zebrafish cytosolic SULT1 ST5 The coding region of zebrafish SULT1 ST5 cDNA was subcloned into pGEX-2TK, a prokaryotic expression vector, for the expression of recombinant enzyme in Escherichia coli. Recombinant zebrafish SULT1 ST5, purified from the E. coli extract, migrated at  34 kDa position upon SDS ⁄ PAGE (not shown). This is in agreement with the calculated molecular mass (34 452 Da) based on its deduced amino acid sequence. Purified zebrafish SULT1 ST5 was subjected to func- tional characterization with respect to its enzymatic properties. A pilot experiment showed that the enzyme exhibited strong activity toward b-naphthol. A pH- dependence experiment subsequently performed showed that the enzyme displayed maximum activity at pH 6.0, with b-naphthol as substrate (Fig. 3A). With l-T 3 as substrate, zebrafish SULT1 ST5 was active over a broader pH range, with optimal activities observed at, respectively, 6.0 and 9.0 (Fig. 3B). Whether the differ- ent pH-dependence profiles with b-naphthol and l-T 3 as substrates are due to their structural differences (b- naphthol being a neutral compound and l-T 3 , with its amino and carboxyl groups, being a charged molecule) or, in fact, reflect distinct catalytic mechanisms remains to be clarified. Several endogenous and xenobiotic compounds were tested as substrates for the enzyme, and the activity data obtained are given in Table 1. Interestingly, among the endogenous substrates, zebra- fish SULT1 ST5 showed sulfating activities toward only thyroid hormones and their metabolites, including l-T 3 , 3,3¢,5-triiodo-d-thyronine (d-T 3 ), 3,3¢ ,5¢-triiodo- l-thyronine (l-rT 3 ), l-Thyroxine (l-T 4 ), and l-Thyro- nine. The enzyme also exhibited activities toward some of the xenobiotic compounds tested, including chloro- genic acid, kaempferol, gallic acid, genistein, b-naph- thol, catechin, caffeic acid, daidzein, butylated hydroxy anisole, quercetin, myricetin, n-propyl gallate, and p-nitrophenol. These latter activities are in line with this new enzyme being a member of the SULT1 (phenol SULT) gene family. It is worthwhile pointing out that human and dog thyroid hormone SULTs (SULT1B1) have also been shown to display activities toward xeno- biotic phenolic compounds such as b-naphthol, and p-nitrophenol [17–19]. It should also be pointed out that, of the five zebrafish SULTs previously reported [11–14], SULT1 ST1, 2, and 3 also exhibited consider- able activities toward l-T 3 and l-T 4 [11,12]. Unlike the SULT1 ST5 identified in this study, however, zebrafish SULT1 ST1, 2, and 3 were also found to be active toward several other endogenous compounds including dopamine, 1,3,5[10]-estratrinen-3-ol-17-one (estrone), l-3,4-dihydroxyphenylalanine (l-Dopa), and dehydro- epiandrosterone [11,12]. SULT1 ST5 therefore appears to be the only zebrafish enzyme known, to date, that displays substrate specificity exclusively for thyroid hormones and their metabolites. It will be interesting to investigate whether SULT1 ST5 plays a unique and important role in the metabolism and homeostasis of thyroid hormones in vivo. To investigate in more detail the sulfation of thyroid hormones and their metabolites, the kinetics of sulfa- tion of these compounds by zebrafish SULT1 ST5 was examined. Data obtained were processed using the 0 10 20 30 40 3456789101112 pH n( ytivitcA cificepS)gm/nim/lom 0 20 40 60 80 100 3456789101112 pH n( ytivitcA cificepS)gm/nim/lom B A Fig. 3. pH dependence of the sulfating activity of zebrafish SULT1 ST5 with (A) b-naphthol and (B) L-T 3 as substrates. The enzy- matic assays were carried out under standard assay conditions as described under Experimental procedures, using different buffer systems as indicated. The data represent calculated mean values derived from three experiments. S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3831 excel program to generate the best fitting trendlines for the Lineweaver–Burk double-reciprocal plots. Table 2 shows the kinetic constants determined for the sulfation of thyroid hormones and their metabolites, as well as b-naphthol. It appeared that the K m values for the thyroid hormone⁄ metabolites, except l-Thyronine, were of the same order of magnitude, indicating com- parable affinities of the enzyme for these substrates. V max values showed smaller variations, with the lowest activity for l-T 4 . Catalytic efficiency of the enzyme, as reflected by V max ⁄ K m , appeared to be comparable with l-T 3 , d-T 3 , l-rT 3 or l-Thyronine as substrates, and sig- nificantly lower with l-T 4 as substrate. It is worth mentioning that human and rat thyroid hormone- sulfating SULT1B1 also display K m values (approxi- mately 40 lm) for l-T 3 [18,19] comparable with that (38.7 lm) of zebrafish SULT1 ST5. With b-naphthol as substrate, zebrafish SULT1 ST5 showed V max and K m values comparable with those determined for l-Thyronine. Despite these similar kinetic parameters, however, whether the same catalytic mechanism is involved with b-naphthol and thyroid hormones as substrates remains to be clarified. As discussed earlier, b-naphthol is a neutral compound and thyroid hor- mones, by contrast, are charged molecules. Moreover, the enzyme showed distinct pH-dependence profiles with these two kinds of substrates (Fig. 3). Previous studies performed in our laboratory revealed that the sulfating activity of human cytosolic SULTs could be dramatically inhibited by certain divalent metal cations [20–22]. As an aquatic animal, zebrafish is a good model for studying the effects of divalent metal cations. To investigate the inhibitory ⁄ stimulatory effects of divalent metal cations on the sulfating activity of ze- brafish SULT1 ST5, enzymatic assays using l-T 3 as the substrate were carried out in the absence or presence of various divalent metal cations at a concentration of 1mm. As a control for the counter ion, Cl – , parallel assays in the presence 2 mm NaCl were also performed. Results obtained are compiled in Table 3. The degrees of inhibition or stimulation were calculated by compar- ing the activities determined in the presence of metal cations with the activities determined in the absence of metal cations. It was noted that, among the 10 divalent metal cations tested, Fe 2+ ,Hg 2+ ,Co 2+ ,Zn 2+ ,Cu 2+ , Cd 2+ and Pb 2+ exhibited profound inhibitory effects Table 1. Specific activities of zebrafish SULT1 ST5 with endogenous and xenobiotic compounds as substrates. Specific activity refers to nmol substrate sulfatedÆmin )1 Æmg )1 purified enzyme. Data represent means ± SD derived from three experiments. ND, Specific activity determined is lower than the detection limit (estimated to be  0.01 nmolÆmin )1 Æmg protein )1 ). Endogenous compounds Specific activity (nmolÆmin )1 Æmg )1 ) Xenobiotic compounds Specific activity (nmolÆmin )1 Æmg )1 ) 3,3¢,5-Triiodo- D-Thyronine (D-T 3 ) 24.32 ± 1.47 Chlorogenic acid 23.52 ± 0.51 L-Thyronine 17.74 ± 1.20 Kaempferol 14.52 ± 0.73 3,3¢,5-Triiodo- L-Thyronine (L-T 3 ) 17.39 ± 0.78 Gallic acid 14.26 ± 0.25 3,3¢,5¢-Triiodo- L-Thyronine (L-rT 3 ) 11.62 ± 0.48 Genistein 12.71 ± 0.17 L-Thyroxine (L-T 4 ) 4.31 ± 0.14 b-Naphthol 12.26 ± 0.16 17b-Estradiol ND b Catechin 11.47 ± 0.41 Estrone ND Caffeic acid 9.95 ± 0.17 4-Androstene-3,17-dione ND Daidzein 9.87 ± 0.58 Cholesterol ND Butylated hydroxy anisole 8.38 ± 0.31 Corticosterone ND Quercetin 7.88 ± 0.33 Cortisone ND Myricetin 5.38 ± 0.19 Dehydroepiandrosterone ND n-Propyl gallate 5.32 ± 0.03 D-Dopa ND p-Nitrophenol 4.76 ± 0.11 L-Dopa ND b-Napthylamine 0.44 ± 0.03 Dopamine ND Bisphenol A 0.37 ± 0.02 Hydrocortisone ND p-Octylphenol 0.27 ± 0.03 17a-Hydroxy progesterone ND Epigallocatechin gallate ND 17a-Hydroxy pregnenolone ND Butylated hydroxy toluene ND Pregnenolone ND Diethylstilbestrol ND Progesterone ND Epicatechin ND Table 2. Kinetic constants of zebrafish SULT1 ST5 with L-T 3 , D-T 3 , L-rT 3 , L-thyronine, and b-naphthol as substrates. Data shown repre- sent means ± SD derived from three determinations. Substrate V max (min )1 ) K m (lM) V max ⁄ K m L-T 3 28.8 ± 2.5 38.7 ± 5.9 0.74 D-T 3 35.6 ± 3.4 27.7 ± 2.8 1.29 L-rT 3 14.6 ± 0.6 17.1 ± 0.7 0.85 L-T 4 6.6 ± 0.6 44.5 ± 6.7 0.15 L-Thyronine 41.9 ± 2.3 114.2 ± 12.8 0.37 b-Naphthol 32.3 ± 3.7 97.7 ± 8.9 0.33 A novel zebrafish cytosolic sulfotransferase S. Yasuda et al. 3832 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS on the sulfating activity of the zebrafish SULT, whereas Mn 2+ showed a significant stimulation. Addition of EDTA (at 2 mm concentration) restored the sulfating activity of SULT1 ST5 in the cases of Zn 2+ ,Cd 2+ , and Pb 2+ . In contrast, the inhibition by Fe 2+ ,Hg 2+ ,Co 2+ , and Cu 2+ appeared to be irreversible. It should be poin- ted out, however, that these divalent metal cations were tested at a 1 mm concentration. Whether the divalent metal cations as environmental pollutants may enter zebrafish and accumulate to high enough levels to exert inhibitory or stimulatory effects on the SULT, thereby disrupting the homeostasis of thyroid hormones, remain to be clarified. Developmental stage-dependent expression of zebrafish thyroid hormone-sulfating cytosolic SULT1 ST5 In view of its thyroid hormone-sulfating activity, an important question is whether expression of the newly identified SULT1 ST5 correlates with the development of the thyroid hormone endocrine system of the zebra- fish. To gain insight into this, RT-PCR was used to examine the expression of mRNA encoding the thyroid hormone-sulfating SULT1 ST5 at different develop- mental stages. As shown in Fig. 4A, no expression was detected in unfertilized eggs and during the early phase of embryonic development. A low level of expression of zebrafish SULT1 ST5 was observed at the beginning of the hatching period during embryogenesis, and this gradually increased to a high level of expression throughout the larval stage and into maturity. Interest- ingly, previous studies have revealed that it is during the hatching period when primary organ systems inclu- ding the thyroid gland are formed [23]. The develop- mental expression of the four zebrafish SULT1 STs previously identified were also examined (Fig. 4A). For SULT1 ST1, a low level of message was detected in unfertilized eggs and in embryos immediately following fertilization. Throughout the cleavage period, blastula period, and the early part of gastrula period, however, the message encoding SULT1 ST1 could not be detec- ted. Thereafter, the expression started and increased to a high level during the larval stage onto maturity. For SULT1 ST2, no expression was detected in unfertilized eggs and during embryonic development. The expres- sion appeared in 1- to 4-week-old larvae, and, intrigu- ingly, decreased considerably in adult zebrafish. For SULT1 ST3, a significant level of its coding message was detected in unfertilized eggs. During embryonic development, there appeared to be an initial decrease in expression until the end of the segmentation period Table 3. Effects of divalent metal cations on the sulfating activity of zebrafish SULT1 ST5 with L-T 3 as the substrate. Data represent means ± SD derived from three determinations. Data shown in par- entheses are percentage of the activity determined for the control without divalent cation or EDTA. Divalent cation tested Specific activity (nmolÆmin )1 Æmg )1 ) Divalent cation only (1 m M) Divalent cation + EDTA (1 mM cations + 2 mM EDTA) Control 27.4 ± 2.3 (100%) 29.0 ± 0.4 (106%) FeCl 2 1.9 ± 0.1 (6.9%) 1.9 ± 0.1 (6.9%) HgCl 2 2.0 ± 0.4 (7.3%) 3.8 ± 0.2 (13.9%) CoCl 2 2.3 ± 0.7 (8.4%) 2.6 ± 0.2 (9.5%) ZnCl 2 1.7 ± 0.5 (6.2%) 31.5 ± 0.2 (115%) CuCl 2 3.0 ± 0.4 (10.9%) 1.9 ± 0.4 (6.9%) CdCl 2 1.7 ± 0.1 (6.2%) 27.6 ± 1.5 (101%) MnCl 2 30.9 ± 3.4 (113%) 32.1 ± 1.2 (117%) CaCl 2 29.7 ± 1.3 (108%) 29.2 ± 1.1 (107%) MgCl 2 31.9 ± 0.4 (116%) 25.9 ± 2.8 (94.5%) Pb(CH 3 COO) 2 1.4 ± 0.1 (5.1%) 27.2 ± 1.0 (99.2%) NaCl a 29.8 ± 1.9 (109%) 31.0 ± 4.3 (113%) a Tested at a 2 mM concentration as a control for the counter ion, Cl – . A B Fig. 4. Developmental stage-dependent expression of zebrafish SULTs. (A) RT-PCR analysis of the expression of SULT1 ST5 and previously identified zebrafish SULT1 STs at different stages during embryogenesis and larval development onto maturity. Final PCR mixtures were subjected to 2% agarose electrophoresis. Samples analyzed in lanes 1–14 correspond to unfertilized zebrafish eggs, 0, 1, 3, 6, 12, 24, 48, and 72-h zebrafish embryos, 1, 2, 3, 4-week-old zebrafish larvae, and 3-month-old zebrafish. The PCR products cor- responding to different zebrafish SULT1 ST cDNAs, visualized by ethidium bromide staining, are marked by arrows. (B) RT-PCR ana- lysis of the expression of zebrafish b-actin at the same develop- mental stages as those described in (A). S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3833 (24 h post fertilization), which then increased to a high level of expression throughout the rest of the embry- onic development and the larval stage onto maturity. A low level of message encoding SULT1 ST4 was detected in unfertilized eggs, indicating its presence as a maternal transcript. No SULT1 ST4 message, how- ever, was detected in the embryos until the segmen- tation period (12–24 h post fertilization); it then gradually increased to a high level of expression throughout the rest of the embryonic development and the larval stage onto maturity. The physiological impli- cations of the differential expression of the various SULTs mentioned above remain to be clarified. In contrast to the developmental stage-dependent expres- sion of the SULT1 isoform 5, b-actin, a housekeeping protein, was found to be expressed throughout the entire developmental process (Fig. 4B). To summarize, we identified a thyroid hormone-sulf- ating cytosolic SULT that may be involved in meta- bolism and homeostasis of thyroid hormones and their metabolites in zebrafish. This study is part of an over- all effort to obtain a complete repertoire of the cyto- solic SULT enzymes present in zebrafish. As pointed out earlier, the identification of the various cytosolic SULTs and their biochemical characterization is a pre- requisite for using the zebrafish as a model for a sys- tematic investigation on fundamental issues regarding cytosolic SULTs. More work is warranted in order to achieve this goal. Experimental procedures Materials p-Nitrophenol, dopamine, l-Dopa, d-Dopa, b-naphthol, b-naphthylamine, aprotinin, thrombin, l-Thyronine, l-T 3 , d-T 3 , l-rT 3 , l-T 4 , estrone, 17b-estradiol, bisphenol A, 4-octylphenol, daidzein, kaempferol, caffeic acid, genistein, myricetin, quercetin, gallic acid, chlorogenic acid, catechin, epicatechin, epigallocatechin gallate, n-propyl gallate, dehy- droepiandrosterone, ATP, SDS, Mes, Mops, Hepes, Taps, Ches, Caps, Trizma base, dithiothreitol (DTT), and isopro- pyl thio-b-d-galactoside (IPTG) were products of Sigma Chemical Company (St. Louis, MO, USA). TRI Reagent was from Molecular Research Center, Inc, (Cincinnati, CH, USA). Unfertilized zebrafish eggs and zebrafish embryos and larvae at different developmental stages were prepared by Scientific Hatcheries (Huntington Beach, CA, USA). Total RNA from a 3-month-old zebrafish was prepared as des- cribed previously [12]. Taq DNA polymerase was a product of Promega (Madison, WI, USA). Takara Ex Taq DNA polymerase and 3¢-Full RACE Core Kit were purchased from PanVera Corp ⁄ Invitrogen (Carlsbad, CA, USA). T 4 DNA ligase and BamHI restriction endonuclease were from New England Biolabs (Beverly, MA, USA). Oligonucleotide primers were synthesized by MWG Biotech (Highpoint, NC, USA). pSTBlue-1 AccepTor Vector Kit and BL21 (DE3) competent cells were purchased from Novagen (Madison, WI, USA). Prestained protein molecular mass standard was from Life Technologies (Grand Island, NY, USA). First- strand cDNA Synthesis Kit, pGEX-2TK glutathi- one S-Transferase (GST) gene fusion vector, GEX-5¢-and GEX-3¢ sequencing primers, and glutathione Sepharose 4B were products of Amersham Biosciences (Piscataway, NJ, USA). Recombinant human bifunctional ATP sulfurylase ⁄ adenosine 5¢-phosphosulfate kinase was prepared as des- cribed previously [24]. Cellulose TLC plates were products of EM Science (Gibbstown, NJ, USA). Carrier-free sodium [ 35 S]sulfate, Ecolume scintillation cocktail, cortisone, corti- costerone, 4-androstene-3,17-dione, hydrocortisone, prog- esterone, pregnenolone, 17a-OH progesterone, and 17a-OH pregnenolone were from ICN Biomedicals (Costa Mesa, CA, USA). All other reagents were of the highest grades commer- cially available. cDNA cloning of zebrafish cytosolic SULT1 ST5 By searching the EST database, a zebrafish cDNA (Gen- Bank accession no. BI884567) encoding the 5¢-region of a putative cytosolic SULT was identified. To obtain the 3¢-coding region and the untranslated sequence further downstream, 3¢-RACE was performed using the Takara-3¢- Full RACE Core Kit. First-strand cDNA was synthesized using AMV reverse transcriptase with zebrafish total RNA as the template in conjunction with an oligo(dT)-3 sites adaptor primer. Afterwards, PCR was carried out using an oligonucleotide (5¢-CCATGGAAACAGTATCTGGAGA GG-3¢) designed based on the sequence determined for the above-mentioned zebrafish SULT cDNA and a 3 sites adaptor primer as the primer pair with the first-strand cDNA as the template. Amplification conditions were 2 min at 94 °C and 25 cycles of 30 s at 94 °C, 30 s at 59 °C, and 5 min at 72 °C, followed by a 5 min extension at 72 °C. The reaction mixture was analyzed by agarose electrophoresis. A discrete PCR product detected was iso- lated and subcloned into pSTBlue-1 cloning vector and sub- jected to nucleotide sequencing [25]. The nucleotide and deduced amino acid sequences of the cDNA were analyzed using blast search for sequence homology to known cyto- solic SULTs. RT-PCR was subsequently employed to amplify the complete coding region of this novel zebrafish SULT. With zebrafish total RNA as the template and oligo(dT) as the primer, the first-strand cDNA was syn- thesized using the First-Strand cDNA Synthesis Kit (Amersham Biosciences). Using sense (5¢-CGCGGATCC ATGAGCCGGAGAACAAGCGAAACT-3¢) and antisense (5¢-CGCGGATCCTTATATAGTGAAGCGTATTGGAAG A novel zebrafish cytosolic sulfotransferase S. Yasuda et al. 3834 FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS AGGACA-3¢) oligonucleotide primers designed based on 5¢- and 3¢-coding sequences determined as mentioned above, a PCR in a 100 lL reaction mixture was carried out under the action of EX Taq DNA polymerase, with zebra- fish first-strand cDNA prepared as the template. Amplifica- tion conditions were 2 min at 94 °C and 20 cycles of 94 °C for 35 s, 60 °C for 40 s, 72 °C for 1 min. The final reaction mixture was applied onto a 1.2% agarose gel, separated by electrophoresis, and visualized by ethidium bromide stain- ing. The PCR product band detected was excised from the gel, and the DNA therein was isolated by spin filtration. Purified PCR product was subjected to BamHI restriction and cloned into BamHI-restrictd pGEX-2TK vector, and verified for autheticity by nucleotide sequencing [25]. Bacterial expression and purification of the recombinant zebrafish cytosolic SULT1 ST5 pGEX-2TK harboring cloned zebrafish SULT cDNA was transformed into competent BL21 (DE3) cells. Transformed cells were grown to D 600  0.6 in 1 L Luria–Bertani med- ium supplemented with 60 lgÆmL )1 ampicillin, and induced with 0.1 mm IPTG. After an overnight induction at room temperature, cells were collected by centrifugation and homogenized in 25 mL ice-cold lysis buffer (10 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA) using an Aminco French Press. Twenty microliters of 10 mgÆmL )1 aprotinin (a protease inhibitor) was added to the crude homogenate. The crude homogenate was subjected to cen- trifugation at 10 000 g for 15 min at 4 °C. The supernatant collected was fractionated using 2.5 mL of glutathione Sepharose, and the bound GST fusion protein was treated with 3 mL of a thrombin digestion buffer (50 mm Tris ⁄ HCl, pH 8.0, 150 mm NaCl, and 2.5 mm CaCl 2 ) containing 5 unitÆmL )1 bovine thrombin. Following 20 min incubation at room temperature with constant agitation, the prepar- ation was subjected to centrifugation. The recombinant zebrafish SULT present in the supernatant collected was analyzed with respect to its enzymatic properties. Enzymatic assay The sulfating activity of the purified zebrafish SULT was assayed using [ 35 S]PAPS as the sulfate donor. The standard assay mixture, with a final volume of 25 lL, contained 50 mm Taps buffer (pH 8.0), 14 lm [ 35 S]PAPS (15 CiÆ mmol )1 ), 1 mm DTT, and 50 lm of substrate. The reaction was started by the addition of the enzyme, allowed to pro- ceed for 3 min at 28 °C, and terminated by heating at 100 °C for 2 min. The precipitates formed were cleared by centrifugation, and the supernatant was subjected to the analysis of [ 35 S]sulfated product using the previously devel- oped TLC procedure [26], with n-butanol ⁄ isopropanol ⁄ 88% formic acid ⁄ water (3 : 1 : 1 : 1, v ⁄ v ⁄ v ⁄ v) as the solvent sys- tem. To examine the pH dependence, different buffers (50 mm Mes at 5.5, 6.0, or 6.5; Mops at 6.5, 7.0, or 7.5; Taps at 7.5, 8.0 8.5 or 9.0; Ches at 9.0, 9.5, or 10.0; and Caps at 10.0, 10.5, or 11.0), instead of 50 mm Taps (pH 8.0), were used in the reactions, with 500 lm b-naph- thol or 200 lm L-T 3 as substrate. For the kinetic studies on the sulfation of l-T 3 , d-T 3 , l-rT 3 , l-T 4 , l-Thyronine, and b-naphthol, varying concentrations of these substrate com- pounds and 50 mm Taps buffer at pH 8.0 were used. To determine the stimulatory ⁄ inhibitory effects of divalent metal cations, enzymatic assays in the presence or absence of different divalent metal cations, at 1 mm concentration, were performed under standard conditions described above, with 200 lm L-T 3 as substrate. Analysis of the developmental stage-dependent expression of zebrafish cytosolic SULT1 ST5 RT-PCR was employed to investigate the developmental stage-dependent expression of the zebrafish cytosolic Table 4. Oligonucleotide primers used for PCR amplifications in the analysis of developmental stage-dependent expression of zebrafish cyto- solic SULT1 STs. The sense and antisense oligonucleotide primer sets listed were verified by BLAST search to be specific for the target zebra- fish SULT or b-actin nucleotide sequence. Target sequence Sense and antisense oligonucleotide primers SULT1 ST1 Sense: 5¢-AGTTCAACAAGGAACTGCAGGACGTGTTTG-3¢ Antisense: 5¢-CACATGGCTATAAAATGGTTACATCTGTGT-3¢ SULT1 ST2 Sense: 5¢-TATGTAGGAGCTACAAGAAACATTGAAGGC-3¢ Antisense: 5¢-CAATTCTTACTAGCTGCAGGGAGGGTTGGT-3¢ SULT1 ST3 Sense: 5¢-GAATTGGCCCTAATTTGCACATTAAAGATA-3¢ Antisense: 5¢-GCCTGAAGTTTTTGGTTCACAGTGAAATTT-3¢ SULT1 ST4 Sense: 5¢-ACACTCTGAAGGGGAATTAGGATTAAGAAA-3¢ Antisense: 5¢-CTGACTATACAAGGCTGTGTGCCACAAAAC-3¢ SULT1 ST5 Sense: 5¢-GAAAACACATCACGTACCCTCCCTCTCTGCG-3¢ Antisense: 5¢-ACATCATGGTATATTATTCATTTAGCTGACACTTT-3¢ b-Actin Sense: 5¢-ATGGATGAGGAAATCGCTGCCCTGGTC-3¢ Antisense: 5¢-TTAGAAGCACTTCCTGTGAACGATGGA-3¢ S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3835 SULTs. Total RNAs from zebrafish embryos and larvae at different developmental stages were isolated using the TRI Reagent based on the manufacturer’s instructions. Aliquots containing 5 lg each of the total RNA preparations were used for the synthesis of the first-strand cDNA using the First-Strand cDNA Synthesis Kit (Amersham Biosciences), according to manufacturer’s instructions. One-microliter aliquots of the 33 lL first-strand cDNA solutions prepared were used as the template for the subsequent PCR amplifi- cation. PCRs were carried out in 25 lL reaction mixtures using EX Taq DNA polymerase, in conjunction with gene- specific sense and antisense oligonucleotide primers (Table 4). Amplification conditions were 2 min at 94 °C fol- lowed by 40 cycles of 30 s at 94 °C, 40 s at 60 °C, and 1 min at 72 °C. The final reaction mixtures were applied onto a 1.2% agarose gel, separated by electrophoresis, and visualized by ethidium bromide staining. As a control, PCR amplification of the sequence encoding zebrafish b-actin was concomitantly performed using the above-mentioned first-strand cDNAs as templates, in conjunction with gene- specific sense and antisense oligonucleotide primers (Table 3) designed based on the reported zebrafish b-actin nucleotide sequence (GenBank accession no. AF057040). Miscellaneous methods [ 35 S]PAPS was synthesized from ATP and carrier-free [ 35 S]sulfate using the bifunctional human ATP sulfury- lase ⁄ APS kinase and its purity determined as previously described [27,28]. The [ 35 S]PAPS synthesized was then adjusted to the required concentration and specific activ- ity by the addition of cold PAPS. SDS ⁄ PAGE was per- formed on 12% polyacrylamide gels using the method of Laemmli [29]. 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Anal Biochem 72, 248–254. 31 Brodskii LI, Ivanov VV, Kalaidzidis Ya L, Leontovich AM, Nikolaev VK, Feranchuk S.I & Drachev VA (1995) GeneBee-NET:Internet-based server for analyz- ing biopolymers structure. Biochemistry (Moscow) 60, 923–928. 32 Nikolaev VK, Leontovich AM, Drachev VA & Brodsky LI (1997) Building multiple alignment using iterative analyzing biopolymers structure dynamic improvement of the initial motif alignment. Biochemistry (Moscow) 62, 578–582. S. Yasuda et al. A novel zebrafish cytosolic sulfotransferase FEBS Journal 272 (2005) 3828–3837 ª 2005 FEBS 3837 . Sense: 5¢-ACACTCTGAAGGGGAATTAGGATTAAGAAA-3¢ Antisense: 5¢-CTGACTATACAAGGCTGTGTGCCACAAAAC-3¢ SULT1 ST5 Sense: 5¢-GAAAACACATCACGTACCCTCCCTCTCTGCG-3¢ Antisense:. Sense: 5¢-AGTTCAACAAGGAACTGCAGGACGTGTTTG-3¢ Antisense: 5¢-CACATGGCTATAAAATGGTTACATCTGTGT-3¢ SULT1 ST2 Sense: 5¢-TATGTAGGAGCTACAAGAAACATTGAAGGC-3¢ Antisense: