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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online March 5, 2003 as doi:10.1096/fj.02-0787fje. |
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Laboratoire de Biologie Vasculaire, Institut de Pharmacologie et de Biologie Structurale du CNRS, 205, 31077 Toulouse, France; and
* ENDOCUBE, La Pyrénéenne, BP2701, 31312 Labège Cedex, France
2Correspondence: Laboratoire de Biologie Vasculaire, Institut de Pharmacologie et de Biologie Structurale, CNRS UMR5089, 205 route de Narbonne, 31077 Toulouse, France. E-mail: Jean-Philippe.Girard{at}ipbs.fr
SPECIFIC AIMS
Lymphocyte emigration from the blood into most secondary lymphoid organs and chronically inflamed tissues occurs at the level of high endothelial venules (HEV). A unique characteristic of HEV endothelial cells (HEVEC) is their capacity to incorporate large amounts of sulfate into sialomucin-type counter-receptors for the lymphocyte homing receptor L-selectin. We have previously shown that sulfate uptake into HEVEC is mediated by two distinct functional classes of sulfate transporters: Na+-coupled transporters and sulfate/anion exchangers. Here, we report the molecular characterization from human HEVEC of SLC26A11, a novel Na+-independent sulfate transporter from the SLC26 sulfate/anion exchanger family.
PRINCIPAL FINDINGS
1. Molecular cloning of SLC26A11, a novel member of the SLC26 sulfate/anion exchanger family
To identify novel human sulfate transporters, we used the TBLASTN program to search the GenBank human EST database with the sequence of Saccharomyces cerevisiae high-affinity sulfate transporter SUL2 (GenBank Acc. NP013193). This strategy led to the discovery of an EST (GenBank Acc. BE312666) encoding a novel member of the SLC26 sulfate/anion exchanger family (Table 1
), designated SLC26A11, which exhibits 55% homology with yeast SUL2. Analysis of SLC26A11 mRNA expression by RT-PCR analysis revealed that HEVECs express this novel member of the SLC26 family. To assemble a full-length SLC26A11 cDNA, we used the initial EST sequence as the starting point of an exhaustive search of overlapping ESTs. This resulted in the assembly of a 2901 bp SLC26A11 cDNA encoding a 606 amino acid protein, which was verified by cloning and sequencing SLC26A11 cDNAs from human HEVEC and kidney. Prosite and Pfam databases searches revealed that SLC26A11 contains three motifs characteristic of the SLC26 sulfate/anion exchanger family: the Prosite SLC26 sulfate transporter signature (PS01130), the Pfam sulfate transporter domain (Pf00916), and the sulfate transporter and anti-Sigma factor domain (STAS; Pf01740). Sequence alignments indicated that SLC26A11 is the most divergent member of the human SLC26 sulfate/anion exchanger family, being more closely related to sulfate transporters from yeast and plants than to other members of the human SLC26 family.
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2. SLC26A11 functions as a sodium-independent sulfate transporter
The close homology of SLC26A11 protein to previously reported sulfate transporters, including lower eukaryote sulfate transporters and human sulfate transporters of the SLC26 sulfate/anion exchanger family, suggested a related transport function for human SLC26A11. Therefore, we used the baculovirus expression system to test whether SLC26A11 functions as a sulfate transporter. As found previously, we observed that noninfected Sf9 insect cells or Sf9 cells infected with baculovirus expressing the GFP or wild-type baculovirus express an endogenous sodium-independent sulfate transporter (Fig. 1
) that is sensitive to the anion exchanger inhibitor 4,4'-diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS). We found that Sf9 cells infected with a recombinant SLC26A11 baculovirus incorporate about three times more sulfate than those infected with control baculoviruses in the same conditions (Fig. 1)
. The observed sulfate transport activity was independent of the presence of Na+ in the uptake buffer. In contrast, SLC26A11-mediated sulfate uptake was efficiently inhibited by DIDS (Fig. 1)
. Together, these data show that SLC26A11 is a novel Na+-independent, DIDS-sensitive sulfate transporter from the SLC26 sulfate/anion exchanger family.
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3. Coexpression of SLC26A11 and SLC26A2 in human HEVEC
To analyze expression of SLC26 family sulfate transporters in HEVEC from human tonsils, we performed RT-PCR analysis using oligonucleotides specific for each SLC26 family member. We included all the SLC26 family members (Table 1)
that have been found to exhibit significant sulfate transport activity (SLC26A1, SLC26A2, SLC26A3, SLC26A8, SLC26A9, and SLC26A11), with the exception of SLC26A7, which is expressed in human HEVEC but has only very low sulfate transport activity. We found that SLC26A11 and SLC26A2/DTDST are expressed in tonsillar HEVEC at levels comparable to those found in kidney. SLC26A1, SLC26A3, and SLC26A8 mRNA expression was not detectable in HEVEC. These results indicate that HEVEC coexpress transcripts for two distinct sulfate transporters of the SLC26 sulfate/anion exchanger family: SLC26A11 and SLC26A2/DTDST.
4. The human SLC26A11 gene is a candidate for hereditary hearing loss diseases
A BLAST search of the draft human genome sequence database at NCBI using SLC26A11 cDNA or protein sequences as baits revealed a genomic hit from the Homo Sapiens chromosome 17 working draft sequence (GenBank Acc. AC123764) that covered the whole SLC26A11 cDNA. Using this genomic sequence, we identified two UniSTS in the SLC26A11 gene (UniSTS:97442 and UniSTS:176842) indicating that the SLC26A11 gene is located on human chromosome 17 at 17q25, very close to microsatellite marker D17S784. Three distinct hereditary hearing loss diseases loci have been mapped to chromosome 17q25. These include DFNA20 and DFNA26, two nonsyndromic forms of sensorineural hearing loss, and USH1G, an Usher type I syndrome that shows, in addition to a congenital hearing loss, late-onset retinitis pigmentosa. Since two members of the SLC26 gene family (SLC26A4/Pendrin and SLC26A5/Prestin) have already been shown to be important for the function of the inner ear, the SLC26A11 gene is an attractive candidate for the hearing loss diseases DFNA20, DFNA26 and USH1G.
CONCLUSIONS AND SIGNIFICANCE
Here, we report the cloning from human HEVEC of SLC26A11, a novel member of the SLC26 sulfate/anion exchanger family. Functional expression studies in Sf9 insect cells showed that SLC26A11 is a novel Na+-independent sulfate transporter. The SLC26A11 sulfate transport activity (
3-fold enhancement compared with the control) observed was similar to that previously seen in the same expression system for the sulfate transporter SLC26A3/DRA. Studies with the anion exchanger inhibitor DIDS revealed that similar to what has been found for other SLC26 family members, SLC26A11-mediated sulfate transport is inhibited in the presence of DIDS.
Analysis of SLC26 sulfate transporter expression by RT-PCR revealed that SLC26A11 and SLC26A2/DTDST are coexpressed in human HEVEC. In contrast, HEVEC appears to lack expression of the other sulfate transporters of the SLC26 family—SLC26A1, SLC26A3, and SLC26A8. This suggests that together with SLC26A2/DTDST, SLC26A11 is likely to be an important mediator of the DIDS-sensitive component of sulfate uptake into HEVEC (Fig. 2
). Sulfate incorporation into HEVEC is the first essential step in the HEV sulfation pathway that contributes significantly to the extensive sulfation of L-selectin counter-receptors by providing high levels of sulfate to PAPS synthetase and HEV sulfotransferases. By modulating sulfate incorporation into HEVEC, SLC26A11 together with SLC26A2/DTDST and the Na+-coupled transporter SLC13A4/SUT1 could therefore play a major role in the regulation of lymphocyte migration through HEV in organized lymphoid tissues and through HEV-like vessels at sites of chronic inflammation. Future studies are required to determine the relative contribution of SLC26A11, SLC26A2/DTDST, and SLC13A4/SUT1 to sulfate incorporation into HEV in vivo. Nevertheless, our results demonstrate that SLC26A11 is a novel Na+-independent sulfate transporter that is likely to play a key role in sulfation of L-selectin counter-receptors in HEV.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0787fje; to cite this article, use FASEB J. (March 5, 2003) 10.1096/fj.02-0787fje 
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