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RESEARCH COMMUNICATION |
a Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 31062 Toulouse, France
b LIIPAT, Institute of Pathology, University of Oslo, Rikshospitalet, Oslo, Norway
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Key Words: ATP sulfurylase • APS kinase • endothelium • lymphocyte recirculation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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2–3(SO4–6)Galß1–4(Fuc1–3)GlcNAc] and 6-sulfo-sLex [Sia2–3Galß1–4(Fuc1–3)(SO4–6) GlcNAc] (16). Studies with chlorate, a selective inhibitor of ATP sulfurylase (ATP sulfate adenylyltransferase), the first enzyme in the sulfate activation pathway, have previously revealed that ATP sulfurylase activity is absolutely required for sulfation of L-selectin ligands in HEVs (13, 14). ATP sulfurylase catalyzes the production of adenosine-5'-phosphosulfate (APS) from sulfate and ATP and provides APS substrate to APS kinase (ATP adenosine-5'-phosphosulfate 3'-phosphotransferase), which transfers a phosphate group from ATP to APS to yield PAPS (adenosine 3'-phosphate 5'-phosphosulfate), the activated sulfate donor used by all sulfotransferases (17–20). Sulfation of L-selectin counterreceptors is also likely to require a sulfate transporter (21) to allow efficient sulfate incorporation into HEVs (22), a PAPS translocase (23) to transport PAPS into the Golgi, and one or more membrane-associated sulfotransferases (24) to transfer sulfate from PAPS to HEV sialomucins. Although the transfer of sulfate from PAPS to HEV sialomucins by sulfotransferases appears to be the most specific step in the pathway, sulfation of HEV sialomucins may also be controlled at earlier steps. For example, the observation that PAPS production is the rate-limiting step for sulfation in other systems (20, 21, 25, 26) suggests that modulation of PAPS synthesis and availability in HEVs could play a major role in the control of HEV sialomucins sulfation.
To further characterize the molecular mechanisms controlling lymphocyte recruitment, we have started to isolate the genes encoding HEV enzymes involved in sulfation of L-selectin ligands. To isolate human HEV cDNAs encoding ATP sulfurylase, we screened an HEV cDNA library (27) with a human expressed sequence tag (EST) probe homologous to plants ATP sulfurylase cDNAs (28, 29). This strategy allowed us to isolate a novel 2.5 kilobase (kb) HEV cDNA encoding human PAPS synthetase, a multifunctional enzyme containing both ATP sulfurylase and APS kinase domains, which is sufficient for PAPS synthesis. A likely murine homologue of human PAPS synthetase has previously been cloned from mouse brain (30). Our results significantly extend this latter work by showing that PAPS synthetase is expressed in human tonsil, MECA-79+ HEV-derived endothelial cells and is the target of chlorate, data that link PAPS synthetase to the sulfation and functional activity of L-selectin ligands in HEVs. Our study also reveals the wide tissue distribution of PAPS synthetase mRNA in the human body, suggesting that human PAPS synthetase may play a key role in the sulfation not only of HEV sialomucins, but also of many other molecules including leukocyte PSGL-1, the ligand for P-selectin (31–33), proteoglycans, hormones, drugs, and xenobiotics (18–20).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Metabolic labeling and analysis of PAPS production by high-voltage paper electrophoresis
Chinese hamster ovary (CHO) dhfr cells (ATCC CRL-9096) transfected with expression vector pcDNA3 (Invitrogen Corporation, San Diego, Calif.) or expression plasmids encoding PAPS synthetase (pcDNA3-hPAPSS) and DRA sulfate transporter (pCDNA3-hDRA), using lipofectamine (Gibco-BRL), were trypsinized the day after transfection and transferred to 12-well plates in complete medium (MEM with 10% calf serum). After 48 h, cells were sulfate-depleted by incubation in sulfate-free BME medium (Sigma, St. Louis, Mo.) for 30 min and then pulsed for 15 min with 100 µCi 35S sodium sulfate (carrier-free; specific activity 1050–1600 Ci/mmol; DuPont-New England Nuclear, Boston, Mass.) in 250 µl serum-free BME. After labeling, supernatants were discarded, cells were washed twice in serum-free BME, and harvested by trypsinization. 35S-labeled cells were lysed in 20 µl cell extraction buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-Cl pH 7.4, 0.1 mM PMSF). Aliquots (10 µl) of the cell extracts were then spotted onto Whatman 3MM paper and subjected to high-voltage paper electrophoresis for 30 min at 1500 V and 100 mA (paper length, 27 cm) in H2O/pyridine/acetic acid (493/5/2) buffer, pH 5.3, at 4°C. After electrophoresis, the paper was dried and radiolabeled compounds were visualized by autoradiography. 35S-PAPS (specific activity 1 Ci/mmol; DuPont-New England Nuclear) was used as a calibration standard.
Chlorate inhibition of PAPS production
Forty-eight hours after transfection, CHO cells that had been transfected with expression vectors pcDNA3 or pCDNA3-hPAPS synthetase were starved for 30 min in sulfate-free BME medium supplemented with fresh 10 mM sodium chlorate (Merck, Nogent sur Marne, France). Sulfate-depleted cells were then labeled for 15 min with 400 µCi/ml 35S-labeled sodium sulfate in the continuous presence of 10 mM sodium chlorate. Control labelings of the same transfected cells were made in chlorate-free medium. Extracts of the cells labeled in the presence or absence of chlorate were then analyzed for PAPS production by high-voltage paper electrophoresis and autoradiography.
`Virtual' and standard Northern blots
The Capfinder PCR (polymerase chain reaction) cDNA synthesis kit (Clontech, Palo Alto, Calif.) was used to generate high yields of full-length cDNAs from 1 µg total RNA of cultured human tonsil, HEV-derived endothelial cells (HEVEC) that were 98% MECA-79+ on day 2 and < 1% MECA-79+ by day 8 (Baekkevold et al., unpublished results). For `virtual' Northern blots, HEVEC cDNAs (0.2 µg per lane) were electrophoresed on a 1% agarose gel, transferred onto nylon membranes, and hybridized with 32P-labeled PAPS synthetase or hevin cDNA probes at 42°C overnight in 50% formamide, 5x Denhardt's solution, 5x SSC, 0.5% SDS, 50 µg/ml tRNA, and 50 µg/ml herring sperm DNA. The membranes were then washed with 1x SSC, 0.1% SDS at room temperature (2x15') and at 65°C (2x15'), and exposed to Kodak XAR-5 film at -70°C for 24 (Hevin) or 72 h (PAPS synthetase). For standard Northern blot analysis, blots of poly (A) RNA from multiple human tissues (Clontech) were hybridized at 65°C overnight in 5x SSC, 10x Denhardt's solution, 2% SDS, 50 µg/ml herring sperm DNA, and 50 µg/ml yeast tRNA, washed with 1x SSC, 0.1% SDS at 65°C (3x20'), and exposed, with two intensifying screens, to Kodak XAR-5 film at -70°C for 18 or 36 h. Full-length PAPS synthetase (2.5 kb BamHI-KpnI fragment) and hevin (2.6 kb EcorI-XhoI fragment) cDNAs were 32P labeled by random priming and used as probes in the Northern blots.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Fig. 1A).
The longest cDNA we isolated contains a single, long open reading frame of 1872 nt that translates into a putative 624 amino acid protein with a calculated molecular mass of 70.9 kDa. In vitro translation of the isolated cDNA in rabbit reticulocyte lysate resulted in the production of a protein with an apparent molecular mass of 75 kDa, which is similar to the predicted molecular mass (data not shown). Analysis of the predicted protein sequence for the presence of specific motifs revealed the existence from amino acid residues 59 to 66 (GLSGAGKT) of an ATP/GTP binding site motif (P-loop) that confers strictly to the consensus P-loop sequence GXXXXGK(TS) (34). Comparison of the predicted protein sequence with sequences from plant ATP sulfurylases (28, 29) shows that the carboxy-terminal part of the human protein (aa 241–627) exhibits, respectively, 58 and 59% identity with ATP sulfurylases ( Fig. 1B, C) from A. thaliana (GenBank N°U05218; N°U06276; N°U06275) and S. tuberosum (potato) (GenBank N°X75041; N°X79053). Using the BLAST program, a search was conducted of the National Center for Biotechnology Information nonredundant protein database that confirmed the similarity of the human sequence with plants ATP sulfurylases (35). This search also revealed lower but significant homology with yeast and fungi ATP sulfurylases (25–28% sequence identity over 320–370 amino acids). These sequence comparisons strongly suggest that the HEV cDNA we have isolated encodes a bona fide human ATP sulfurylase. Surprisingly, BLAST searches also revealed striking similarities between the first 225 amino acids of the human sequence and APS kinases from different species including plants, yeast, and bacteria (Fig 1B). The homology with sequences from bacteria is impressive ( Fig. 1D). For instance, the human sequence displays more than 50% identity with APS kinases from Mycobacterium tuberculosis (GenBank N°Z73419) and Escherichia coli (GenBank N°M86936). The similarity of the human sequence with both ATP sulfurylases and APS kinases suggests that the cDNA we have isolated encodes a bifunctional ATP sulfurylase-APS kinase or PAPS synthetase. The gene corresponding to this human PAPS synthetase cDNA appears to have evolved through the fusion of separate genes encoding ATP sulfurylase and APS kinase in plants, yeast, fungi, and bacteria ( Fig. 1B). While this work was in progress, two other groups reported the identification of similar bifunctional ATP sulfurylase-APS kinase cDNAs in mouse and the marine worm Urechis caupo (30, 36). The human protein exhibits, respectively, 97 and 71% sequence identity with the mouse (GenBank N°U34883) and marine worm (GenBank N°L39001) proteins. In addition, blast searches revealed the presence of a putative PAPS synthetase in the nematode Caenorhabditis elegans (GenBank N°Z68880), which displays 57% identity with the human sequence in 625 amino acid overlap. These latter results suggest that PAPS synthetase genes encoding bifunctional ATP sulfurylase-APS kinase enzymes are likely to be found in most animals.
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Expression of human PAPS synthetase mRNA in MECA-79+ HEV endothelial cells
To confirm the expression of PAPS synthetase mRNA in HEV endothelial cells, we used RNA from cultured HEVEC. The detailed purification and characterization of the HEVEC will be described elsewhere (Baekkevold et al., unpublished results). Cultured HEVEC are 98% MECA-79 positive 2 days after purification from human tonsils, but have completely lost MECA-79 antigen expression by 8 days of culture, indicating a possible down-regulation of the HEV enzymes involved in the biosynthesis of this sulfation-dependent epitope. Due to the limited amount of starting material available (less than 1 µg total RNA), we assessed PAPS synthetase mRNA expression by using virtual instead of standard Northern blots. For this purpose, PCR-generated full-length cDNAs were prepared from HEVEC (cultured for 2 or 8 days) total RNA, electrophoresed on an agarose gel, transferred to nylon filters, and hybridized with cDNA probes corresponding to PAPS synthetase or hevin, a secreted protein abundantly expressed in human tonsil HEV endothelial cells (27). Two bands at 2.5 and 2.3 kb were detected with the PAPS synthetase probe in 2- and 8-day HEVEC samples ( Fig. 2).
The 2.5 kb band corresponds to the size of the full-length PAPS synthetase cDNA whereas the 2.3 kb species is likely to be due to annealing of the oligo-dT primer to an internal stretch of adenines (sequence AAAAAAAGAAAAAAA) found 230 nt upstream of the PAPS synthetase polyadenylation signal (
Fig. 1A), since more than two-thirds of the cDNA clones isolated from the HEV library we have sequenced were initiated at this artefactual site. With the hevin probe, a single major band of 2.6 kb, corresponding to the size of the hevin mRNA, was detected in the HEVEC samples ( Fig. 2). These results indicate that PAPS synthetase, similar to hevin, is expressed in human tonsil, MECA-79+ HEV-derived endothelial cells (day 2) and that its expression is maintained even after MECA-79 down-regulation (day 8).
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Functional expression of the human PAPS synthetase HEV cDNA in CHO cells
To confirm that the isolated cDNA encodes a bifunctional ATP sulfurylase-APS kinase enzyme that is sufficient for PAPS production, we performed functional expression studies in CHO cells. The human PAPS synthetase (PAPSS) cDNA was placed under the control of the strong CMV promoter in the expression vector pcDNA3. CHO cells were transiently transfected with this pcDNA3-hPAPSS expression construct or pcDNA3 expression vector alone, and intracellular PAPS production was analyzed by high-voltage paper electrophoresis after metabolic labeling with 35S sodium sulfate ( Fig. 3).
High-voltage paper electrophoresis allows clear separation of 35S-PAPS from 35S-sulfated proteins and proteoglycans that stay at the origin (17). Extracts from CHO cells transfected with pcDNA3 expression vector alone exhibit low levels of PAPS production ( Fig. 3, lane 2). In contrast, CHO cells transfected with the human PAPS synthetase expression construct (pcDNA3- hPAPSS) synthesize very high levels of PAPS ( Fig. 3, lane 3). Although expression of human PAPS synthetase in CHO cells resulted in high levels of PAPS production, it did not enhance the sulfation of proteins and proteoglycans, suggesting that PAPS synthesis is not the rate-limiting step for sulfation in these cells. We then tested whether PAPS synthetase activity may be limited by the levels of intracellular sulfate (20). We cotransfected CHO cells with the human expression construct pcDNA3-hPAPSS together with an expression construct for the human DRA sulfate transporter, a member of the superfamily of transporters with 12 membrane-spanning domains that mediates high levels of sulfate incorporation into cells (37). Coexpression of human PAPS synthetase with this sulfate transporter resulted in the same levels of PAPS production as after expression of PAPS synthetase alone ( Fig. 3, lane 4). These results indicate that activity of the human PAPS synthetase in the transfected CHO cells is unlikely to be limited by sulfate availability.
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Human PAPS synthetase is sensitive to chlorate inhibition
Treatment of lymph nodes with chlorate has previously been shown to abrogate synthesis of sulfated L-selectin ligands in HEVs (13, 14). Chlorate competes with sulfate for the binding to ATP sulfurylase, the first enzyme in the pathway of PAPS synthesis (38). We hypothesized that the ATP sulfurylase part of the PAPS synthetase we have characterized may be the target for chlorate inhibition in HEVs. To determine whether human PAPS synthetase is sensitive to chlorate, we analyzed PAPS synthetase activity in CHO cells in the presence of chlorate ( Fig. 4).
We found that chlorate treatment of cells transfected with the human PAPS synthetase expression construct pcDNA3-hPAPSS completely abolishes PAPS production (compare lanes 3 and 5). These results indicate that the human PAPS synthetase, encoded by the transfected HEV cDNA, is a target for chlorate inhibition. Chlorate also inhibits the endogenous ATP sulfurylase activities from CHO cells, since cells treated with chlorate show a profound undersulfation of proteins and proteoglycans ( Fig. 4, lanes 4 and 5). The functional expression studies performed in CHO cells indicate that the human HEV cDNA we isolated encodes a potent PAPS synthetase, likely to be the target for chlorate inhibition in HEVs.
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PAPS synthetase mRNA is widely distributed in human tissues
To determine whether expression of the human PAPS synthetase we have characterized is restricted to HEVs or can also be found in other cells or tissues, we examined expression of PAPS synthetase mRNA in 12 different human tissues ( Fig. 5).
A single mRNA species of 2.5 kb, corresponding to the size of the PAPS synthetase cDNA (2511 bp), is detected in most tissues examined including testis, pancreas, kidney, thymus, prostate, ovary, small intestine, and colon. Although expressed at lower levels, the PAPS synthetase mRNA is also detected, on longer exposures of the blots, in peripheral blood leukocytes ( Fig. 5, lane 14) and liver (data not shown), an important site of sulfation in the body. The wide pattern of PAPS synthetase mRNA expression in the human body can also be deduced from the presence in dbEST and TIGR EST databases of homologous EST (expressed sequence tags) coming from many different tissue sources, including brain (12 ESTs), lung (3 ESTs), uterus (3 ESTs), embryo (3 ESTs), liver (2 ESTs), colon (2 ESTs), prostate gland (2 ESTs), white blood cells (1 EST), and pancreas (1 EST). The presence of PAPS synthetase mRNAs or ESTs in many tissues of the human body that do not contain HEVs indicates that PAPS synthetase expression is not restricted to HEVs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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Fig. 6)
will provide a better understanding of the mechanisms controlling the recruitment and migration of lymphocytes through HEVs. We have begun to isolate these genes and have succeeded in cloning a novel cDNA expressed in human tonsil, MECA-79+ HEV-derived endothelial cells that encodes PAPS synthetase, a bifunctional ATP sulfurylase/APS kinase sufficient for PAPS production. Sequence comparisons indicate that, similar to its likely homologues in the mouse and marine worm (30, 36), the human PAPS synthetase gene evolved through the fusion of separate genes encoding ATP sulfurylase and APS kinase in lower organisms. By facilitating the transfer or `channeling' of the APS intermediate between the two active sites, the physical association of ATP sulfurylase and APS kinase activities on a single bifunctional protein may allow us to overcome two severe obstacles to PAPS synthesis: the instability of APS and the considerable energy barrier to APS formation by ATP sulfurylase (18, 30). This is likely to result in a more efficient sulfate activation pathway in higher eukaryotes than in lower organisms.
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Northern blot and database searches show that PAPS synthetase mRNA is widely distributed in human tissue. These findings extend current knowledge about tissue distribution of PAPS synthetase, which was only known to be expressed in mouse brain (30), and suggest that PAPS synthetase may be involved in all sulfation reactions in the human body (mucins, proteoglycans, hormones, drugs, etc.). In agreement with this possibility, all clones identified in the HEV cDNA library by moderate stringency hybridization (51 independent clones) correspond to a unique PAPS synthetase cDNA, and we did not find any evidence in GenBank, dbEST, or TIGR-EST databases for the existence of other human PAPS synthetase or ATP sulfurylase sequences, which, if they exist, should have been detected due to the strong evolutionary conservation of ATP sulfurylases. Therefore, although we cannot completely exclude at this time the existence of another PAPS synthetase gene exhibiting a highly restricted expression pattern, all the evidence we have obtained so far (cloning and sequencing data, database searches, expression data) points to the existence in humans of a single widely expressed gene encoding a bifunctional PAPS synthetase, containing both ATP sulfurylase and APS kinase activities, that is responsible for PAPS synthesis in most tissues of the body. These findings are surprising with regard to what happens in plants (A. thaliana) or bacteria (Rhizobium melitoti)—for example, where there are at least three different genes encoding ATP sulfurylase activity (25, 28).
PAPS synthetase and sulfation of L-selectin ligands in high endothelial venules
Functional expression studies in CHO cells reveal that human PAPS synthetase activity is sensitive to chlorate, a potent inhibitor of protein sulfation (38), which has previously been found to abrogate L-selectin recognition of HEV sialomucins GlyCAM-1 and CD34 (13, 14). Modulation of PAPS levels in HEVs by PAPS synthetase is likely to play a key role in the control of L-selectin ligands sulfation and functional activity ( Fig. 6). Experiments with chlorate have already shown that reduction of PAPS levels in HEVs results in profound undersulfation of HEV sialomucins CD34 and GlyCAM-1 and abrogation of L-selectin recognition (13, 14). Evidence for a regulatory role of PAPS synthesis in the control of sulfation has also been provided in other systems (20, 21, 25, 26). For example, the production of PAPS, rather than a default of sulfotransferase activity, has been shown to be the limiting step in the sulfation of lipo-oligosaccharide signals that control Rhizobium-legume symbiosis (25, 26). In this latter case, reduced PAPS production was due to limiting ATP sulfurylase and APS kinase activities (26). In other circumstances, PAPS synthesis has been found to be limited by inorganic sulfate availability. In chondrodysplasias, for instance, mutations in the DTD sulfate transporter gene cause a sulfate uptake defect that results in greatly reduced PAPS production and profound undersulfation of proteoglycans in cartilage (21). These sulfation defects in DTD genetic diseases clearly show that transporters, mediating sulfate incorporation at the plasma membrane, can control the degree of sulfation of proteoglycans or glycoproteins. The capacity of the HEV endothelium to incorporate large amounts of sulfate (22) and thus to provide high levels of inorganic sulfate substrate to PAPS synthetase may therefore contribute significantly to the extensive sulfation of O-linked carbohydrates from HEV sialomucins (16). In addition to PAPS synthesis, sulfation of L-selectin ligands in HEVs may also be controlled at the level of PAPS availability and sulfotransferase activity ( Fig. 6). Although a PAPS translocase that transports PAPS from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus has recently been partially purified (23), its molecular characterisitics have not yet been reported. Similarly, little is known about the sulfotransferases involved in sialomucin sulfation. The large number of distinct sulfated structures and the presence of these structures on a limited number of glycoproteins in a given cell or tissue indicate that the sulfotransferases that transfer sulfate to glycoprotein oligosaccharides are both numerous and highly specific (24). The presence of distinct sulfated sLex structures on GlyCAM-1 suggests the existence of at least two different HEV sulfotransferases involved in the transfer of sulfate to the 6 position of galactose and N-acetylglucosamine residues (16).
Other roles of PAPS synthetase in human tissues
The wide expression pattern of PAPS synthetase in the human body supports the possibility that it has other roles in addition to its role in sulfation of L-selectin ligands in HEVs. For example, PAPS synthetase, which is expressed in leukocytes, may be involved in sulfation of PSGL-1, the leukocyte mucin-like glycoprotein ligand for P-selectin. Sulfation of a tyrosine residue in the amino-terminal part of PSGL-1 has recently been shown to be essential, in conjunction with sLex presented on O-linked glycans, for high-affinity P-selectin binding (31–33). A requirement of PAPS synthetase for tyrosine sulfation of PSGL-1 is likely since PSGL-1 sulfation has been shown to be sensitive to chlorate inhibition (32, 33). However, it remains to be seen whether PAPS synthetase activity or expression levels are regulated in leukocytes and whether this may play a role in the control of PSGL-1 recognition by P-selectin. Similarly, the role of PAPS synthetase in regulating sulfation in other human tissues, such as the liver, where sulfation plays important physiological roles, remains to be characterized. The molecular cloning of the human PAPS synthetase cDNA may help to further define the regulation of PAPS synthesis in human tissues and the importance of PAPS in determining sulfation activity in various biological processes.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Correspondence: Laboratoire de Biologie Moléculaire Eucaryote du CNRS, 118 route de Narbonne, 31062 Toulouse, France. E-mail: girard{at}ibcg.biotoul.fr
3 Abbreviations: APS, adenosine-5'-phosphosulfate; bp, base pair; CHO, Chinese hamster ovary; EST, expressed sequence tag; HEV, high endothelial venule; HEVEC, HEV endothelial cells; kb, kilobase; PAPS, 3'-phosphoadenosine-5'-phosphosulfate; ATCC, American Type Culture Collection; PCR, polymerase chain reaction; ATP sulfurylase, ATP sulfate adenylyltransferase; APS kinase, ATP adenosine-5'-phosphosulfate 3'-phosphotransferase; PAPSS, PAPS synthetase; SDS, sodium dodecyl sulfate.
Received for publication November 26, 1997. Accepted for publication January 15, 1998.
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