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Physiol. Genomics 34: 256-264, 2008. First published June 10, 2008; doi:10.1152/physiolgenomics.90234.2008
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Received 1 May 2008; accepted in final form 4 June 2008.
Physiological Genomics 34:256-264 (2008)
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Call For Papers: Comparative Genomics

Functional and structural characterization of the zebrafish Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1)

Daniel Markovich 1, Alessandro Romano 2, Carlo Storelli 2 and Tiziano Verri 2

1 Molecular Biology Lab, School of Biomedical Sciences, University of Queensland, St. Lucia, Australia
2 Department of Biological and Environmental Sciences and Technologies, University of Salento (formerly University of Lecce), Lecce, Italy


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sulfate plays an essential role during growth, development and cellular metabolism. In this study, we characterized the function and structure of the zebrafish (Danio rerio) Na+-sulfate cotransporter 1 (NaS1) cDNA and gene (slc13a1). Zebrafish NaS1 encodes a protein of 583 amino acids with 13 putative transmembrane domains. Expression of zebrafish NaS1 protein in Xenopus oocytes led to Na+-sulfate cotransport, which was significantly inhibited by thiosulfate, selenate, molybdate, and tungstate. Zebrafish NaS1 transport kinetics were: Vmax = 1,731.670 ± 92.853 pmol sulfate/oocyte·hour and Km = 1.414 ± 0.275 mM for sulfate and Vmax = 307.016 ± 32.992 pmol sulfate/oocyte·hour, Km = 24.582 ± 4.547 mM and n (Hill coefficient) = 1.624 ± 0.354 for sodium. Zebrafish NaS1 mRNA is developmentally expressed in embryos from day 1 postfertilization and in the intestine, kidney, brain, and eye of adult zebrafish. The zebrafish NaS1 gene slc13a1 contains 15 exons spanning 8,716 bp. Characterization of the zebrafish NaS1 contributes to a greater understanding of sulfate transporters in a well-defined genetic model and will allow the elucidation of evolutionary and functional relationships among vertebrate sulfate transporters.

sulfate; transporter; gene expression; Xenopus laevis oocytes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
INORGANIC SULFATE (SO42–) is a physiologically important ion involved in many metabolic and cellular processes, including production of sulfated proteoglycans, biosynthesis of sulfated hormones, detoxification, and elimination of endogenous compounds and xenobiotics by sulfonation (20). Essentially, SO42– is required for life, and reduced blood sulfate levels (hyposulfatemia) have been identified in several human disorders including autism, Alzheimer's, Parkinson's, motor neurone disease, and liver cirrhosis (1, 12, 35). Sulfate is obtained from the diet, and its homeostasis is maintained primarily through renal tubular mechanisms (20). Since sulfate is a hydrophilic anion, it requires membrane transporters to enter or exit cells (20). Two functionally and structurally distinct families of sulfate transporters exist: the Na+-dependent SO42– transporters (Na+-sulfate cotransporters), which belong to the solute-linked carrier gene family 13 (SLC13), and the Na+-independent SO42– transporters (sulfate/anion exchangers), which belong to the SLC26 gene family (21, 23). Previously, we have cloned the human (15) and rodent Na+-sulfate cotransporter 1 (NaS1) (2, 22) sulfate transporters, whose genes have been classified as SLC13A1 (23). Mammalian NaS1 proteins are expressed on the brush border membranes of the renal proximal tubule, where they mediate the first step of sulfate reabsorption (20). Recently, using a gene targeted approach, we generated a NaS1 null mouse (6), which has reduced serum sulfate levels (hyposulfatemia), confirming NaS1 is essential for maintaining sulfate homeostasis in mice. Functional sulfate transport studies have been performed in nonmammalian eukaryotes, including birds (9, 30), mussels (8), and fish (7, 27, 29, 31), but little is known about the molecular structures of these transporters. Several fish sulfate transporters have been recently characterized. These include the rainbow trout (Oncorhynchus mykiss) sulfate/anion transporter 1 (Sat1; rainbow trout SLC26A1) (13), the zebrafish prestin (zPres; zebrafish SLC26A5) (33), and the Japanese eel (Anguilla japonica) NaS1 (ajSlc13a1) and Sat1 (ajSlc26a1) transporters (26), whose functional activities closely resemble those of the mammalian orthologs (2, 15, 22). In this study, we have characterized the function and structure of the zebrafish (Danio rerio) NaS1 cDNA and gene (slc13a1).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
All procedures for zebrafish used throughout this study were conducted according to the Italian Guidelines for Animal Care (D.L. 116/92) in application of the European Communities Council Directives (86/609/ECC), with all efforts to minimize animal sufferings and the number of animals necessary to collect reliable scientific data. All experiments with Xenopus laevis conformed to the guidelines of the University of Queensland Animal Ethics Committee.

Fish breeding and embryo collection.
Adult zebrafish were purchased from local pet stores and maintained at the Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. To collect fish larvae, adult zebrafish were bred by natural crosses in a male to female ratio of 2:1, according to standard procedures (36). Eggs were collected within 2 h of fertilization and maintained at 28.5°C in embryo water (0.6 mg/l Instant Ocean sea salts; Aquarium Systems, Sarrebourg, France). Staging of the embryos was based on morphological criteria, following the published classification (14).

Plasmids.
The cDNA clone containing the full coding sequence of zebrafish NaS1 (GenBank accession no. BC058313) in the pCMV-SPORT6.1 (pCMV-SPORT6.1/zfNaS1) was purchased from Geneservice (Cambridge, UK; IMAGE clone no. 6793065).The zebrafish NaS1 insert was completely sequenced to confirm identity of the cDNA clone.

In silico analysis.
PROSITE 19.7 computational tools (http://www.expasy.org/prosite/) were employed to scan for potential N-glycosylation, cAMP/cGMP-dependent protein kinase, and protein kinase C recognition sequences. TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) was used to predict the putative transmembrane domains (TMDs). Clustal W 1.82 (http://www.ebi.ac.uk/clustalw/) was used to align zebrafish NaS1 full coding sequence with the full coding sequences of selected fish (Japanese eel) and mammalian (mouse, rat, and human) NaS1. GenBank accession numbers of NaS1 coding sequences used for comparison were: BC058313 (zebrafish NaS1), AB111926 (Japanese eel NaS1; Ref. 26), L19102 (rat NaS1; Ref. 22), NM_019481 (mouse NaS1; Ref. 2), AF260824 (human NaS1; Ref. 15). Clustal W 1.82 was also used to align zebrafish NaS1 amino acid sequence with the amino acid sequences of Japanese eel, mouse, rat, and human NaS1 proteins. GenBank accession numbers of NaS1 proteins used for comparison were: AAH58313 (zebrafish NaS1), BAD22605 (Japanese eel NaS1; Ref. 26), AAA41677 (rat NaS1; Ref. 22), NP_062354 (mouse NaS1; Ref. 2), AAG60583 (human NaS1; Ref. 15). The genomic structure of zebrafish slc13a1 was deduced by comparing contig CH211-245M13 (GenBank accession no. BX322610.5) with the zebrafish slc13a1 cDNA sequence. Annotation was carried out by the genome viewer/annotation software Artemis (release 4) (32).

Xenopus laevis oocytes and transport measurements.
Mature Xenopus laevis females were purchased from the African Xenopus Facility C.C. (Noordhoek, South Africa) and kept under standard conditions as previously described (4). Stage V and VI oocytes from X. laevis were maintained at 18°C in modified Barth's solution [88 mM NaCl, 1 mM KCl, 0.82 mM MgSO4, 0.4 mM CaCl2, 0.33 mM Ca(NO3)2, 2.4 mM NaHCO3, 10 mM HEPES/Tris pH 7.4, gentamicin sulfate 20 mg/l]. Zebrafish NaS1 cRNA was synthesized in vitro using the mMESSAGE mMACHINE SP6 kit (Ambion, Applied Biosystems, Scoresby, VIC, Australia) according to the manufacturer's protocol. Briefly, the transcription mixture was added to either 1 µg of NotI linearized pCMV-SPORT6.1/zfNaS1 plasmid DNA. The reaction was incubated at 37°C for 2 h, followed by a 15 min incubation with 2 units RNase-free deoxyribonuclease I. The 50 µl reaction of cRNA was precipitated with 25 µl 7.5 M LiCl/75 mM EDTA overnight at –20°C. cRNA was resuspended in 15 µl of nuclease-free water, checked on a gel, and used directly for injection into X. laevis oocytes at 5–10 ng/µl. Oocytes were injected with either 50 nl water (control) or 5 ng zebrafish NaS1 cRNA using a Nanojet automatic injector (Drummond Scientific, Broomall, PA). Uptake of [35S]-sulfate was performed on day 3 postinjection in the presence (100 mM NaCl) or absence (100 mM choline chloride) of sodium, using groups of 10 oocytes per individual data point. Briefly, for the initial uptake studies (refer to the legend to Fig. 4A for details) oocytes were washed at room temperature for 2 min in Na+-free (100 mM choline chloride, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES/Tris pH 7.5) or Na+-containing solution A (100 mM NaCl, 2 mM KCl, 1 mM CaCl2,1 mM MgCl2, 10 mM HEPES/Tris pH 7.5), and then placed for 1 h into 100 µl of Na+-free or Na+-containing solution A containing 0.1 mM K2SO4 and 20 µCi/ml 35SO42– (New England Nuclear Radiochemicals, PerkinElmer Life and Analytical Sciences, Melbourne, VIC, Australia). For substrate specificity experiments, 1 h uptakes of 0.1 mM K2SO4 (20 µCi/ml 35SO42–) were measured in (previously rinsed) oocytes in Na+-containing solution A, in the absence or presence of 1 mM test inhibitors (see legend to Fig. 4B for details). In kinetic experiments, 1 h sulfate uptakes were measured in Na+-containing solution A in the presence of increasing Na2SO4 concentrations (0.01–25 mM; 20–30 µCi/ml 35SO42–) (see legend to Fig. 4C for details). In Na+ activation experiments, 1 h uptakes of 0.5 mM K2SO4 (35SO42– 25 µCi/ml) were measured in the absence or presence of different concentrations of Na+ (5–100 mM), obtained by mixing Na+-free and Na+-containing solution A as appropriate (see legend to Fig. 4D for details). To stop the uptake reactions, the oocytes were washed three times with Na+-free or Na+-containing ice cold solution A as appropriate. Then oocytes were lysed with 1% SDS, dissolved in scintillant (Emulsifier Safe, Canberra Packard, Mount Waverley, VIC, Australia), and counted by liquid scintillation spectrometry. Data are means ± SE of n number of experiments. Statistically significant differences (P < 0.05) were determined by the Student's t-test for paired or nonpaired data as appropriate.

Reverse transcription-polymerase chain reaction.
Total RNA was isolated from either tissues of adult animals or 1–6 days postfertilization (dpf) embryos, using the TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Reverse transcription was performed in the presence of oligo(dT)12-18 with the SuperScript III First-Strand Synthesis System for reverse transcription-polymerase chain reaction (RT-PCR, Invitrogen) using 5 µg total RNA and following the manufacturer's instructions. Two microliters of the resulting cDNA products were used to perform PCR with Platinum Taq DNA polymerase (Invitrogen) in the presence of specific pairs of primers derived from zebrafish slc13a1 cDNA sequence (forward: 5'-CCACATTATCCGGAACATCC-3', starting at nucleotide 758; reverse: 5'-ACCTCCACCAACAAGCAGAC-3', starting at nucleotide 1293; Fig. 1). Analogously, to assess RNA quality and efficiency of the reverse transcription step, 2 µl of cDNA products were used to perform PCR in presence of zebrafish β-actin-specific primers (GenBank accession no. NM_131031; forward: 5'-CGTGACATCAAGGAGAAGCT-3', starting at nucleotide 681; reverse: 5'-ATCCACATCTGCTGGAAGGT-3', starting at nucleotide 1123). After a denaturing step at 94°C for 2 min, PCR amplification was performed for cycles of denaturation, annealing and extension respectively, as follows: for slc13a1, 94°C/45 s, 61°C/45 s, 72°C/30 s (35 cycles); for β-actin, 94°C/30 s, 50°C/60 s, 72°C/60 s (35 cycles). RT-PCR products were separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide, and their identity was confirmed by cloning and sequencing.


Figure 1
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  		Fig. 1. Nucleotide and predicted amino acid sequence of zebrafish Na+-sulfate cotransporter 1 (NaS1). Numbers on the left refer to the nucleotide (top row) and amino acid (bottom row) positions. Nucleotides are numbered, starting from the first ATG initiation codon within a strong Kozak consensus sequence. ***Stop codon. A polyadenylation signal is double underlined. In the amino acid sequence, putative transmembrane domains are underlined and named I–XIII. Potential protein kinase C phosphorylation sites at the cytoplasmic surface (white boxes) and a potential extracellular N-glycosylation site (gray box) are indicated.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Zebrafish NaS1 sequence analysis.
Zebrafish NaS1 cDNA is 2,441 bp long, with six nucleotides (nt) of 5'-untranslated region (UTR), an open reading frame of 1,752 bp and 689 nt of 3'-UTR (Fig. 1). The polyadenylation signal (AATAAA) starts at position 2383. Translation of the zebrafish NaS1 cDNA produces a protein of 583 amino acids having 13 putative TMDs with an intracellular NH2 terminus and an extracellular COOH terminus, as predicted for the mammalian NaS1 proteins (20). Based on this model, seven potential intracellular protein kinase C phosphorylation sites (Ser193, Thr215, Ser224, Thr321, Thr327, Ser399, Thr412) and one extracellular N-glycosylation site (Asn579) are predicted in the zebrafish NaS1 protein (Fig. 2). Only one protein kinase C phosphorylation site (Thr321), between TMDs VIII and IX, and one N-glycosylation site (Asn579), near the COOH terminus, are conserved with other NaS1 orthologs (human, rat, mouse, and Japanese eel) whose functional significance was previously assessed (2, 15, 22, 26) (Fig. 2). At the cDNA level, zebrafish NaS1 shares 59% nucleotide sequence identity with mouse NaS1, 60% with rat and human NaS1, and 66% with the Japanese eel NaS1. At the amino acid level, zebrafish NaS1 shares 55% sequence identity with mouse, rat, and human NaS1 proteins and 59% with the Japanese eel NaS1 (Fig. 2). The zebrafish NaS1 gene (slc13a1) contains 15 exons, with the translation initiation site on exon 1 (Fig. 3). Zebrafish slc13a1 exon sizes vary from 40 to 188 bp, with the exception of exon 15 having 786 bp, which contains the TGA stop codon and the entire 3'-UTR (Table 1). Nucleotide sequences at the intron/exon boundaries of zebrafish slc13a1 conform to the GT/AG rule for the intron donor and acceptor splice sites (Table 1). Zebrafish slc13a1 is localized on chromosome 25 and is 8.7 kb in length, being ~3 kb larger than the Japanese pufferfish Takifugu rubripes (fugu) slc13a1 (5.6 kb), but much smaller than the Western clawed frog Xenopus tropicalis (25 kb), chicken Gallus gallus (30.3 kb), and mammalian (rat, mouse, and human) (77.5–86.4 kb) NaS1 genes (Table 2). The reason for the differences in gene sizes is the presence of larger introns (up to 21 kb) in the mammalian genes compared with smaller introns in zebrafish slc13a1 (Table 1). Comparative analysis at the genomic level revealed highest nucleotide sequence identity among vertebrate slc13a1 in exons 3, 4, 12, 13, and 14, and lowest identities in exons 1, 5, 6, 9, and 11, whereas no significant sequence identity was found in intronic sequences (for a detailed graphical representation of zebrafish slc13a1 vs. fugu, Western clawed frog, chicken, rat, mouse, and human NaS1 genes, see Supplemental Fig. S1).1


Figure 2
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  		Fig. 2. Amino acid alignment of human, mouse, rat, Japanese eel, and zebrafish NaS1 proteins. Multiple sequence alignment was generated using Clustal W 1.82 using the tool at the www.ebi.ac.uk/clustalw web site with the following (default) parameters: Matrix = Gonnet 250, Gap Open = 10, End Gaps = –1, Gap Extension = 0.2, Gap Distances = 4. Putative transmembrane domains (I–XIII; reference sequence: zebrafish NaS1) are indicated. Potential phosphorylation ({blacktriangledown}, protein kinase C; •, protein kinase A) and N-glycosylation (Y) sites are indicated and underlined along the sequence where found. The proposed "sodium:sulfate symporter signature" motif (PROSITE pattern: PS01271; amino acid residues 512-528 in zebrafish NaS1) is highlighted in black; the underlined valine (V) along the predicted zebrafish sequence does not fall into the canonical signature.

 

Figure 3
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  		Fig. 3. Structure of the zebrafish NaS1 gene [solute-linked carrier gene family 13a1 (slc13a1)] and its relationships to the zebrafish NaS1 mRNA and predicted protein structure. Exons of the zebrafish slc13a1 are depicted as black vertical bars, widths drawn to scale. In the middle section, the relative contribution of each exon to the spliced mRNA transcript is shown. Grey regions indicate the untranslated 5' and 3' sequences. The marked base pair position of the intron/exon boundaries refer to the translation start. The superimposition of the exon boundaries with the predicted secondary structure of the NaS1 protein is shown in the bottom part. {blacktriangledown}, exon/intron boundaries; the numbered grey boxes represent the predicted 13 membrane spanning domains.

 

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  		Table 1. DNA sequences at the exon/intron boundaries of the zebrafish slc13a1 gene

 

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  		Table 2. Gene ID, chromosome location, and length of SLC13A1 genes

 
Zebrafish NaS1 function.
Zebrafish NaS1 transport function was characterized in Xenopus oocytes (Fig. 4). Zebrafish NaS1 led to a strong (>100-fold) induction of sulfate transport in the presence of sodium (NaCl), with a weaker (but significant) sulfate uptake ({approx}17-fold) in the absence of sodium (choline chloride) when compared with water-injected (control) oocytes (Fig. 4A). Zebrafish NaS1-mediated Na+-sulfate cotransport was significantly inhibited by thiosulfate, selenate, molybdate, and tungstate (in order of potency), whereas oxalate, phosphate, succinate, citrate, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid produced no significant changes compared with the control (no inhibitor) group (Fig. 4B). Typical Michaelis-Menten saturation for SO42– transport was observed by zebrafish NaS1, with a calculated Km (substrate concentration yielding one-half maximal velocity) for SO42– of 1.414 ± 0.275 mM and a Vmax (maximal velocity) for SO42– of 1,731.670 ± 92.853 pmol SO42–/oocyte·hour (Fig. 4C). Zebrafish NaS1-induced SO42– uptake showed Na+ saturation, and using the generalized Hill equation led to the following parameters: Km for Na+ of 24.582 ± 4.547 mM, Vmax for Na+ of 307.016 ± 32.992 pmol SO42–/oocyte·hour, and a Hill coefficient n of 1.624 ± 0.354 (Fig. 4D), suggesting a stoichiometry of ~2 Na+ ions for one SO42– ion.


Figure 4
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  		Fig. 4. Functional characterization of zebrafish NaS1 protein in Xenopus oocytes. A: zebrafish NaS1-induced sulfate transport in Xenopus oocytes. Oocytes were injected with either 50 nl of water or 50 nl of zebrafish NaS1 cRNA (5 ng/oocyte). Three days later, transport of 0.1 mM K2SO4 (20 µCi/ml 35SO42–) was measured in a Na+-containing (100 mM NaCl) or Na+-free (100 mM choline chloride) solution. Uptake rates are: water 7.38 ± 2.52 pmol sulfate/oocyte·hour vs. zebrafish NaS1 (zfNaS1) 755.44 ± 82.52 pmol sulfate/oocyte·hour (NaCl groups); water 2.2 ± 1.56 pmol sulfate/oocyte·hour vs. zebrafish NaS1 36.94 ± 5.64 pmol sulfate/oocyte·hour (Choline Cl groups). *Significance vs. control (water groups) for 95% probability (P < 0.05). B: inhibition of zebrafish NaS1-induced sulfate transport in Xenopus oocytes. Oocytes were injected with either 50 nl of water or 50 nl of zebrafish NaS1 cRNA (5 ng/oocyte). Three days later, transport of 0.1 mM K2SO4 (20 µCi/ml 35SO42–) was measured in a Na+-containing (100 mM NaCl) solution in the absence (control) or presence of various inhibitors: thiosulfate (1 mM), selenate (1 mM), molybdate (1 mM), tungstate (1 mM), oxalate (1 mM), phosphate (1 mM), succinate (1 mM), citrate (1 mM), and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS, 1 mM). Uptake values represent means ± SE for 7–10 oocytes per condition and are representative of 3 similar experiments. *Significance vs. control for 95% probability (P < 0.05). C: sulfate concentration dependence of zebrafish NaS1-induced Na+-sulfate cotransport in Xenopus oocytes. Oocytes were injected with either 50 nl of water or 50 nl zebrafish NaS1 cRNA (5 ng/oocyte). Three days later, transport was measured in the presence of 100 mM NaCl at different Na2SO4 concentrations (0.01–25 mM; 20–30 µCi/ml 35SO42–). Uptake values represent means ± SE for 7–10 oocytes per condition and are representative of 3 similar experiments. The curve was fitted to a Michaelis-Menten equation using nonlinear regression. D: sodium concentration dependence of zebrafish NaS1-induced Na+-sulfate cotransport in Xenopus oocytes. Oocytes were injected with either 50 nl of water or 50 nl zebrafish NaS1 cRNA (5 ng/oocyte). Three days later, transport of 0.1 mM K2SO4 (25 µCi/ml 35SO42–) was measured in the presence of different concentrations of Na+ (0–100 mM NaCl, replaced by choline chloride). Uptake values represent means ± SE for 7–10 oocytes per condition and are representative of 3 similar experiments. The curve was fitted to a generalized Hill equation using nonlinear regression.

 
Zebrafish NaS1 expression in adult tissues and embryos.
Using RT-PCR, zebrafish NaS1 mRNA was amplified in adult zebrafish intestine, kidney, brain, and eye, but not in gill, spleen, or skeletal muscle (Fig. 5A). By RT-PCR, zebrafish NaS1 mRNA was detected as early as embryonic day 1 postfertilization and up until the early larval stages of day 6 postfertilization (Fig. 5B).


Figure 5
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  		Fig. 5. Zebrafish NaS1 mRNA expression. A: tissue distribution in adult zebrafish fish. RT-PCR was performed using equal amounts of total RNA (1 µg) isolated from adult zebrafish tissues, using either zebrafish slc13a1- or zebrafish β-actin-specific primers (see MATERIALS AND METHODS). H2O indicates no RNA in RT (negative control). Data are representative of 3 similar experiments. B: Zebrafish NaS1 expression in zebrafish embryos. RT-PCR was performed using equal amounts of total RNA (2 µg) isolated from 1–6 days postfertilization (dpf) zebrafish embryos, using either zebrafish slc13a1- or zebrafish β-actin-specific primers (see MATERIALS AND METHODS). H2O indicates no RNA in RT (negative control). Data are representative of 3 similar experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we have structurally and functionally characterized the cyprinid teleost zebrafish (D. rerio) NaS1 sulfate transporter, which represents an excellent molecular tool for studying the evolutionary and functional relationships among sulfate transporters in vertebrates. The zebrafish NaS1 protein shares a high overall identity with previously characterized NaS1 transporters (55–59%) in mammals (2, 15, 22) and the Japanese eel (A. japonica) (26). Zebrafish slc13a1 is similar in size to the Japanese pufferfish T. rubripes (fugu) slc13a1, but much smaller than the frog, chicken, and mammalian slc13a1. Zebrafish slc13a1 contains 15 exons, which is conserved in all mammalian, bird, amphibian, and teleost fish slc13a1 (see Supplemental Fig. S1). The highest sequence identity amongst vertebrate slc13a1 is in exons 3 and 4, which encode amino acids that span the second half of the first intracellular loop to the first half of the second intracellular loop, and exons 12, 13, and 14, which encode the second half of the fourth intracellular loop to the first half of the sixth (last) intracellular loop, respectively. Furthermore, exon 1 in all slc13a1 contains the 5'-UTR and the initiation codon. The major differences in the genomic organization of vertebrate slc13a1 is in the length of introns, with exons being relatively similar in length. Homology searches between gene sequences of such evolutionary distant species as fish and mammals may offer a fast and reliable method of phylogenetic footprinting (25) for detection and functional analysis of known and novel putative conserved regulatory sequences (cis-regulatory elements) and/or enhancers. Due to their phylogenetic position, fish represent a useful tool to focus on amino acid residues, motifs, conserved regions, and protein domains that are relevant to the general function of proteins in vertebrates or, conversely, to the determination of species-specific phenotypes. Our comparative analysis of NaS1 amino acid sequences from two fish (zebrafish and Japanese eel) and three mammalian (human, mouse, and rat) species has allowed the recognition of a number of highly conserved amino acids (and the exclusion of others), simply on the basis of the overall sequence alignment. In both fish and mammalian NaS1 proteins, a single highly conserved NH2-glycosylation site exists close to its COOH terminus (Asn591 in human and rat, Asn590 in mouse, Asn615 in Japanese eel, and Asn579 in zebrafish). This residue, when mutated in human NaS1, significantly reduces its Vmax for SO42– (16). The presence of a highly conserved protein kinase C phosphorylation motif (close to TMD IX) found in all fish and mammalian NaS1 sequences (Thr423 in human and rat, Thr422 in mouse, Thr445 in Japanese eel, and Thr412 in zebrafish) and a large protein kinase C phosphorylation motif-rich area (in the intracellular loop between TMD IV and V) would merit further analysis. The function of mammalian NaS1 proteins has already been investigated by single amino acid substitution analysis to define relevant structural/functional motifs or domains (16, 17, 19, 28). Using comparative analysis, we have confirmed identity or conservative substitutions along the vertebrate series of all those amino acids centered around Ser260 in human NaS1 (namely, Thr257-Gly258-Thr259-Ser260-Thr261-Asn262-Leu263), for which mutational analysis has established a significant effect on function (17). This seven-residue stretch is conserved in all sequences, except in zebrafish where a serine (Ser256) is found instead of the first threonine. However, at the cytoplasmic face of TMD VI, the conserved Ser288 of the human and rat NaS1 and Ser287 of mouse NaS1 correspond to cysteines in the fish proteins (i.e., Cys320 of the Japanese eel and Cys287 of the zebrafish NaS1). This finding disagrees with this residue being conserved in all SLC13 transporters (17) and indirectly proposes a secondary role in the transport function of the protein. Interestingly, at the outer face of TMD VI, the only two amino acid residues (Phe285 and Pro290) that surround Ser288 in human NaS1 are conserved in both fish and mammalian sequences, but when mutated, these residues produce maximal loss of function (17). Taken together, these findings strengthen the predictive potency of our fish-to-mammal comparative analysis. Such comparative approaches might help in rationalizing the selection of the most suitable amino acid residues to target for site-directed mutagenesis, to study structure-function relationships of NaS1 proteins.

Functional analysis of mammalian NaS1 transporters has led to a general scheme of sulfate transport and to the idea that such transport may occur in virtually all vertebrates according to the basic design (in terms of mode of transport, kinetics, cation and membrane potential dependence, electrogenicity, substrate specificity, protein sorting to the membrane, etc.) defined in the mammalian systems (for recent review, see Ref. 21). This concept basically comes from the lack of transport information in nonmammalian vertebrates. Recently, sulfate transport has been demonstrated for the teleost fish NaS1 protein, but no kinetic characterization (Km and Vmax) of the transport properties of this protein has been determined (26). Our functional characterization of the zebrafish NaS1 in Xenopus oocytes has revealed that the zebrafish transporter closely resembles the mammalian proteins in terms of both affinity and transport capacity, although a Km for sulfate of ~1.4 mM is slightly higher than the Km values reported for the mammalian proteins (ranging between 0.1 and 0.6 mM) (21). The zebrafish NaS1 lower affinity for sulfate may be due to its higher capacity for sulfate transport, with zebrafish Vmax being at least one order of magnitude higher than mammalian Vmax values (21), whereas the Na+ interaction and inhibition pattern are in close agreement with the mammalian NaS1 proteins. Like the rodent NaS1, and unlike the human protein, the zebrafish NaS1-induced Na+-sulfate cotransport in Xenopus oocytes is not inhibited by citrate or succinate. Also, like all mammalian orthologs, and unlike the Japanese eel protein, zebrafish NaS1 is not significantly inhibited by oxalate. Taken together, these data extend the general model of Na+-dependent sulfate transport via NaS1 to all vertebrates, although slight differences in transport function exist among species.

Zebrafish NaS1 mRNA is developmentally expressed starting from day 1 postfertilization, which correlates with recent in situ hybridization data showing NaS1 mRNA expression in the proximal straight tubule of the embryonic pronephros (18, 34, 37). In these studies, NaS1 mRNA expression was restricted to pronephric structures for the first 6 dpf and was not detected in other tissues/organs of the developing embryo, being in agreement with our RT-PCR data showing pronephric NaS1 mRNA expression. In the adult fish, zebrafish NaS1 mRNA was expressed in the kidney, intestine, brain, and eye, which correlates with the expression profiles suggested by the analysis of the expressed sequence tags counts (UniGene Dr.83210; kidney ≥ intestine > brain). NaS1 mRNA expression in the adult zebrafish is broader than in the adult Japanese eel, where NaS1 mRNA expression (as assessed by Northern blotting) was restricted to the kidneys of freshwater-adapted fish only (26). The species-specific characteristic of NaS1 mRNA expression in adult teleost fish tissues parallels the tissue distribution data in adult mammals. As assessed by Northern blotting and/or RT-PCR, human NaS1 mRNA is found in the kidney (15), whereas rat NaS1 mRNA is detected in the kidney and small intestine (22), and mouse NaS1 mRNA expressed in the kidney, duodenum/jejunum, ileum, and colon, with weaker signals in the cecum, testis, adrenal gland, and adipose tissue (2). Therefore, the overall picture that emerges from the NaS1 tissue distribution across vertebrates leads to the proposal that NaS1 is 1) invariably expressed in the kidney and 2) variably expressed, according to the species, in other organs, such as the intestine (and possibly the brain), where it may play important roles in sulfate homeostasis. In regards to the teleost fish, body ion regulation is achieved by integrated ion and water transport in the gills, kidney, and intestine (11). In zebrafish, a freshwater carnivorous fish, the presence of NaS1 in both kidneys and intestines of adult fish would suggest a role in sulfate homeostasis, as confirmed in mammals by NaS1 null mice (6). Freshwater teleosts actually face water loading and ion loss through their permeable body surface and accordingly eliminate excess water and absorb solutes (11). Therefore, the presence of a NaS1 Na+-sulfate cotransporter at the apical membrane of kidney tubular cells would facilitate sulfate reabsorption from the glomerular filtrate to the blood and thereby represent the mechanism to maintain blood sulfate concentrations within a physiological range, whereas its presence in the intestine could contribute to other physiological needs, such as cellular metabolism during growth, ensuring significant sulfate absorption from the dietary sources. Interestingly, the absence of NaS1 in gills (Fig. 5A), an organ that plays a major role in sodium, chloride, and calcium ion absorption processes in freshwater fish, including zebrafish (3, 5, 10, 24), may suggest that sulfate homeostasis does not involve gills or that another sulfate transporter may be present in the gills of zebrafish.

In conclusion, the zebrafish NaS1 Na+-sulfate cotransporter has been structurally and functionally characterized with respect to its transport function and expression in adult fish and embryos. Information on zebrafish slc13a1 gene and protein structure and function has been presented, also in context of a comparative analysis among vertebrates. Zebrafish NaS1 contributes greatly toward understanding sulfate transport in a well-established developmental animal model, both for the perspective of using zebrafish for analyzing its physiological responses to changes in sulfate levels and other environmental factors, as well as for studying protein structure/function relationships and its relevance to human diseases with mutations in the NaS1 gene.


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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
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This work was supported by the Australian National Health and Medical Research Council and grants from the University of Salento (formerly University of Lecce; Fondi ex-60%, 2004–2007) and from the Apulian Region (Cod. Cip PS_070 and Cod. Cip PE_062).


   FOOTNOTES
 
Address for reprint requests and other correspondence: D. Markovich, School of Biomedical Sciences, Univ. of Queensland, St. Lucia, QLD 4072, Australia (e-mail: d.markovich{at}uq.edu.au).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 The online version of this article contains supplemental material. Back


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

  1. Alam Z, Coombes N, Waring RH, Williams AC, Steventon GB. Platelet sulphotransferase activity, plasma sulphate levels and sulphation capacity in patients with migraine and tension headache. Cephalalgia 17: 761–764, 1997.[CrossRef][Web of Science][Medline]
  2. Beck L, Markovich D. The mouse Na(+)-sulfate cotransporter gene Nas1. Cloning, tissue distribution, gene structure, chromosomal assignment, and transcriptional regulation by vitamin D. J Biol Chem 275: 11880–11890, 2000.[Abstract/Free Full Text]
  3. Boisen A, Amstrup J, Novak I, Grosell M. Sodium and chloride transport in soft water and hard water acclimated zebrafish (Danio rerio). Biochim Biophys Acta 1618: 207–218, 2003.[Medline]
  4. Colman A. Translation of eukaryotic messenger RNA in Xenopus oocytes. In: Transcription and Translation: A Practical Approach. edited by Hames BD, Higgins SJ. Oxford, UK: IRL, 1984, p. 271–302.
  5. Craig P, Wood C, McClelland G. Gill membrane remodeling with soft-water acclimation in zebrafish (Danio rerio). Physiol Genomics 30: 53–60, 2007.[Abstract/Free Full Text]
  6. Dawson PA, Beck L, Markovich D. Hyposulfatemia, growth retardation, reduced fertility and seizures in mice lacking a functional NaSi-1 gene. Proc Natl Acad Sci USA 100: 13704–13709, 2003.[Abstract/Free Full Text]
  7. Dickman KG, Renfro JL. Primary culture of flounder renal tubule cells: transepithelial transport. Am J Physiol Renal Fluid Electrolyte Physiol 251: F424–F432, 1986.[Abstract/Free Full Text]
  8. Dietz TH, Udoetok AS, Cherry JS, Silverman H, Byrne RA. Kidney function and sulfate uptake and loss in the freshwater bivalve Toxolasma texasensis. Biol Bull 199: 14–20, 2000.[Abstract]
  9. Dudas PL, Renfro JL. Transepithelial sulfate transport by avian renal proximal tubule epithelium in primary culture. Am J Physiol Regul Integr Comp Physiol 283: R1354–R1361, 2002.[Abstract/Free Full Text]
  10. Evans D, Piermarini P, Choe K. The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97–177, 2005.[Abstract/Free Full Text]
  11. Evans DH. Osmotic and ion regulation. In: The Physiology of Fishes, edited by Evans D. Boca Raton, FL: CRC, 1993, p. 315–341.
  12. Heafield M, Fearn S, Steventon G, Waring R, Williams A, Sturman S. Plasma cysteine and sulphate levels in patients with motor neurone, Parkinson's and Alzheimer's disease. Neurosci Lett 110: 216–220, 1990.[CrossRef][Web of Science][Medline]
  13. Katoh F, Tresguerres M, Lee KM, Kaneko T, Aida K, Goss GG. Cloning of rainbow trout SLC26A1: involvement in renal sulfate secretion. Am J Physiol Regul Integr Comp Physiol 290: R1468–R1478, 2006.[Abstract/Free Full Text]
  14. Kimmel C, Ballard W, Kimmel S, Ullmann B, Schilling T. Stages of embryonic development of the zebrafish. Dev Dyn 203: 253–310, 1995.[Web of Science][Medline]
  15. Lee A, Beck L, Markovich D. The human renal sodium sulfate cotransporter (SLC13A1; hNaSi-1) cDNA and gene: organization, chromosomal localization, and functional characterization. Genomics 70: 354–363, 2000.[CrossRef][Web of Science][Medline]
  16. Li H, Pajor AM. Mutagenesis of the N-glycosylation site of hNaSi-1 reduces transport activity. Am J Physiol Cell Physiol 285: C1188–C1196, 2003.[Abstract/Free Full Text]
  17. Li H, Pajor AM. Serines 260 and 288 are involved in sulfate transport by hNaSi-1. J Biol Chem 278: 37204–37212, 2003.[Abstract/Free Full Text]
  18. Liu Y, Pathak N, Kramer-Zucker A, Drummond I. Notch signaling controls the differentiation of transporting epithelia and multiciliated cells in the zebrafish pronephros. Development 134: 1111–1122, 2007.[Abstract/Free Full Text]
  19. Markovich D. Molecular regulation and membrane trafficking of mammalian renal phosphate and sulphate transporters. Eur J Cell Biol 79: 531–538, 2000.[CrossRef][Web of Science][Medline]
  20. Markovich D. Physiological roles and regulation of mammalian sulfate transporters. Physiol Rev 81: 1499–1533, 2001.[Abstract/Free Full Text]
  21. Markovich D, Aronson PS. Specificity and regulation of renal sulfate transporters. Annu Rev Physiol 69: 361–375, 2007.[CrossRef][Web of Science][Medline]
  22. Markovich D, Forgo J, Stange G, Biber J, Murer H. Expression cloning of rat renal Na+/SO4(2-) cotransport. Proc Natl Acad Sci USA 90: 8073–8077, 1993.[Abstract/Free Full Text]
  23. Markovich D, Murer H. The SLC13 gene family of sodium sulphate/carboxylate cotransporters. Pflügers Arch 447: 594–602, 2004.[CrossRef][Web of Science][Medline]
  24. Marshall WS, Grosell M. Ion transport and osmoregulation in fish. In: The Physiology of Fishes, edited by Evans D. Boca Raton, FL: CRC, 2006, p. 177–230.
  25. Müller F, Blader P, Strähle U. Search for enhancers: teleost models in comparative genomic and transgenic analysis of cis regulatory elements. Bioessays 24: 564–572, 2002.[CrossRef][Web of Science][Medline]
  26. Nakada T, Zandi-Nejad K, Kurita Y, Kudo H, Broumand V, Kwon CY, Mercado A, Mount DB, Hirose S. Roles of Slc13a1 and Slc26a1 sulfate transporters of eel kidney in sulfate homeostasis and osmoregulation in freshwater. Am J Physiol Regul Integr Comp Physiol 289: R575–R585, 2005.[Abstract/Free Full Text]
  27. Pelis RM, Renfro JL. Active sulfate secretion by the intestine of winter flounder is through exchange for luminal chloride. Am J Physiol Regul Integr Comp Physiol 284: R380–R388, 2003.[Abstract/Free Full Text]
  28. Regeer RR, Nicke A, Markovich D. Quaternary structure and apical membrane sorting of the mammalian NaSi-1 sulfate transporter in renal cell lines. Int J Biochem Cell Biol 39: 2240–2251, 2007.[CrossRef][Web of Science][Medline]
  29. Renfro J, Pritchard J. Sulfate transport by flounder renal tubule brush border: presence of anion exchange. Am J Physiol Renal Fluid Electrolyte Physiol 244: F488–F496, 1983.[Abstract/Free Full Text]
  30. Renfro JL, Clark NB, Metts RE, Lynch MA. Sulfate transport by chick renal tubule brush-border and basolateral membranes. Am J Physiol Regul Integr Comp Physiol 252: R85–R93, 1987.[Abstract/Free Full Text]
  31. Renfro JL, Dickman KG. Sulfate transport across the peritubular surface of the marine teleost renal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 239: F143–F148, 1980.[Abstract/Free Full Text]
  32. Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajendream M, Barrell B. Artemis: sequence visualization and annotation. Bioinformatics 16: 944–955, 2000.[Abstract/Free Full Text]
  33. Schaechinger TJ, Oliver D. Nonmammalian orthologs of prestin (SLC26A5) are electrogenic divalent/chloride anion exchangers. Proc Natl Acad Sci USA 104: 7693–7698, 2007.[Abstract/Free Full Text]
  34. Thisse B, Pfumio S, Fürthauer M, Loppin B, Heyer V, Degrave A, Woehl R, Lux A, Steffan T, Charbonnier X, Thisse C. Expression of the zebrafish genome during embryogenesis. http://zfin.org.
  35. Waring RH, Ngong JM, Klovrza LV, Green S, Sharp H. Biochemical parameters in autistic children. Dev Brain Dysfunct 10: 40–43, 1997.
  36. Westerfield M. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio). Eugene, OR: University of Oregon Press, 1995.
  37. Wingert R, Selleck R, Yu J, Song H, Chen Z, Song A, Zhou Y, Thisse B, Thisse C, McMahon A, Davidson A. The cdx genes and retinoic acid control the positioning and segmentation of the zebrafish pronephros. PLoS Genet 3: 1922–1938, 2007.[Web of Science][Medline]




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