From the Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, the Department of Medicine, University of Oklahoma Health Sciences Center, and the Oklahoma Center for Medical Glycobiology, Oklahoma City, Oklahoma 73104
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INTRODUCTION |
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Niehrs and Huttner (10) reported the purification of TPST from bovine adrenal medulla in 1990 and William et al. (11) reported the purification of TPST from rat submandibular glands in 1997. Mouse and human cDNAs encoding TPST-1 were first isolated in our laboratory using amino acid sequences obtained from a purified rat liver TPST (12). Subsequently, we and others identified a second member of the gene family, TPST-2, based on its high degree of homology to TPST-1 (13, 14). Each TPST cDNA encodes a protein with type II transmembrane topology with a short N-terminal cytoplasmic domain, a single 17-residue transmembrane domain, and a luminal catalytic domain (Fig. 2). TPST-1 and -2 are of similar size (370 377 residues), and each has two N-glycosylation sites and six conserved luminal cysteine residues. Two structural motifs found in cytosolic and membrane-bound sulfotransferases are conserved in TPST-1 and -2. In the known sulfotransferase crystal structures, these motifs are involved in binding of the 5'-and 3'-phosphate groups of the reaction product 3',5'-ADP and are designated the 5'-PSB and 3'-PB motifs, respectively (15). The predicted amino acid sequences of human and mouse TPST-1 are
96% identical. Human and mouse TPST-2 have a similar degree of identity. Human and mouse TPST-1 are
65 67% identical to human and mouse TPST-2, respectively. Multiple sequence alignments of TPSTs from various species show that the membrane-proximal portion of the luminal domain is poorly conserved. This
40-amino acid segment likely represents a "stem" region that may be dispensable for catalysis, analogous to that found in many glycosyltransferases. The human TPST1 and TPST2 genes are on 7q11.21 and 22q12.1, respectively, whereas the mouse Tpst1 (Mouse Genome Informatics accession number MGI:1298231) and Tpst2 (MGI:1309516) genes are both on chromosome 5,
18.5 Mb apart. There is no evidence for the existence of additional mouse or human TPST genes in genomic or expressed sequence tag (EST) data bases.
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Species and Tissue Distribution of Tyrosine O-Sulfation |
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Searches of the EST data base reveals cDNAs encoding TPST-1 and/or TPST-2 orthologs in many other vertebrate (rat, dog, cow, pig, chicken, zebrafish, fugu, channel catfish, and African clawed frog) and invertebrate species (Caenorhabditis elegans, Drosophila melanogaster, Anopheles gambiae, Ciona intestinalis, Halocynthia roretzi, and Schistosoma japonicum).3 It is interesting to note that D. melanogaster has only a single TPST gene, unlike most other species, including C. elegans, which have two TPST genes. Tyrosine-sulfated proteins (20) and TPST activity (21) have been described in several plant species, but no plant TPST orthologs have yet been identified, and none is apparent in the completed Arabidopsis thaliana genome. Furthermore, no tyrosine-sulfated proteins, TPST activity, or putative TPST orthologs have been described in prokaryotes or in yeast.
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Substrate Specificity of Tyrosylprotein Sulfotransferases |
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The location of sulfation has been unambiguously defined in several proteins. Based on the amino acid sequences flanking known tyrosine O-sulfation sites, coupled with in vitro studies on the sulfation of various synthetic peptides, it is evident that there is no sequon for tyrosine O-sulfation per se. Although consensus features for tyrosine O-sulfation have been described, some proteins known to be tyrosine-sulfated do not fulfill these features (16). Nevertheless, it is clear that the dominant characteristic of known sulfation sites is that there are generally between 3 and 4 acidic amino acids within ±5 residues of the sulfotyrosine.
The SwissProt Group at the Swiss Institute of Bioinformatics has developed a software tool, called Sulfinator,5 that predicts tyrosine O-sulfation sites in proteins (22). Although the positive predictive value of this algorithm is uncertain, it can be used to estimate the number of tyrosine-sulfated proteins in a genomic scale dataset. Twenty-six percent of the 5421 mouse proteins in the SwissProt data base were predicted to have one or more tyrosine O-sulfation sites by the Sulfinator program. Data base entries were examined, and those proteins that lacked a predicted signal peptide, signal-anchor, and/or transmembrane domain were excluded, as were proteins in which the predicted sulfation site(s) is cytosolic in orientation, leaving 380 positive predictions which constitute 7% of the original 5421 proteins.6 This suggests that as many as 2,100 mouse proteins might be tyrosine-sulfated, assuming that the mouse genome encodes 30,000 proteins. Thus, it is very likely that we are only beginning to appreciate the complexity of the substrate repertoire of these enzymes.
A great deal of interest in the field has recently focused on the role of tyrosine O-sulfation in G-protein-coupled receptor (GPCR) function after CCR5, a major HIV co-receptor, was shown to be tyrosine-sulfated (23). Site-directed mutagenesis and chlorate inhibition studies showed that sulfation of one or more tyrosine residues in the N-terminal extracellular domain of CCR5 are required for optimal binding of MIP-1/CCL3, MIP-1
/CCL4, and RANTES/CCL5 and for optimal HIV co-receptor function. CCR5 mutants in which all four tyrosine residues in the N-terminal extracellular domain were changed to phenylalanine have
100-fold weaker affinity for MIP-1
/CCL3 and RANTES/CCL5. Other studies have shown that synthetic sulfotyrosine-containing peptides modeled on the N-terminal extracellular domain of CCR5, but not comparable phosphotyrosine-containing peptides, bound to the gp120-CD4 complex and inhibited HIV entry into cells (24). Likewise, mutagenesis studies indicate that sulfation of tyrosine residue(s) in the N-terminal extracellular domains of CXCR4, CCR2B, CX3CR1, C5a receptor, and the thyroid-stimulating hormone receptor is an important requirement for optimal binding of SDF-1
/CXCL12, MCP-1/CCL2, fractalkine/CX3CL1, C5a, and thyroid-stimulating hormone, respectively (2529). In general, these studies have shown that ligand binding to mutated GPCRs or undersulfated wild-type GPCRs is between 5- and 100-fold weaker than binding to the native receptors. A comparison of the primary sequences of the known chemokine receptors shows that their N-terminal domains are highly acidic and contain one or more tyrosine residues. The fact that this is the dominant feature of known tyrosine sulfation sites suggests that many, and perhaps all, of the chemokine receptors may be sulfated. Chemokine receptors and their cognate ligands play crucial roles in innate and adaptive immunity, hematopoeisis, angiogenesis, tumor growth, and metastasis (30). Thus, the possibility that chemokine receptors as a class may require tyrosine sulfation for optimal chemokine binding has broad pathophysiological implications.
It is not known whether TPST-1 and -2 have distinct structural requirements for efficient sulfation of macromolecular substrates. However, in vitro studies using synthetic peptide acceptors indicate some differences in substrate preferences. We reported that peptides modeled on the sulfation sites of human C4 chain and heparin cofactor II are more efficiently sulfated by TPST-1 than by TPST-2 (13). In addition, Seibert et al. studied sulfation of a peptide modeled on the N-terminal domain of human CCR5 that has four potential tyrosine O-sulfation sites (31). By determining the time course of appearance of various reaction products, they showed that each enzyme sulfated the peptide in a sequential fashion. Tyr14 and Tyr15 were sulfated first, followed by Tyr10 and then Tyr3. However, TPST-1 clearly preferred Tyr14 over Tyr15 as the initial sulfation site, whereas TPST-2 preferred Tyr15 over Tyr14. These studies also suggest that TPST-1 and -2 are not processive enzymes.
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Regulation of Tyrosine O-Sulfation |
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It is possible that tyrosine O-sulfation might be modulated by the action of sulfatases. However, several lines of evidence argue that efficient mechanisms to desulfate tyrosine sulfate in intact proteins or tyrosine sulfate itself do not exist inside the cell or in the extracellular milieu. Dodgson et al. (33) reported that after intraperitoneal administration in rats, tyrosine [35S]sulfate was metabolized by deamidation, but the metabolites were excreted without loss of the sulfate ester. Furthermore, Jones et al. (34) reported that after intraperitoneal or intravenous injection of [35S]sulfate-labeled fibrinopeptide B in the rabbit, virtually all the injected radiolabel was recovered in the urine as free tyrosine [35S]sulfate or its deamidated metabolites. Tyrosine sulfate is also a normal constituent of human urine (28 mg/day) (35). Thus, even after degradative proteolysis, presumably in the lysosome, the sulfate ester of tyrosine O-sulfate is surprisingly stable. This is consistent with observations that tyrosine O-sulfate is a very poor substrate for mammalian sulfatases (36).
Several tyrosine-sulfated peptides and proteins have been purified from native sources, and the location and stoichiometry of sulfation have been determined. However, in most cases these proteins/peptides were isolated using bioassays to follow their purification and/or affinity chromatographic methods that depended on the presence of the sulfate esters, thus precluding an unbiased assessment of stoichiometry in vivo. Two exceptions are notable because they were clearly purified from an extracellular source, unlike most of the others, which were purified from tissues, and they were isolated using methods that were independent of the sulfation state of the protein. The activation peptide of human plasma-derived factor IX is stoichiometrically sulfated at Tyr155 (37). Human glycoprotein Ib purified from human platelets has three tyrosine O-sulfation sites. Sulfation at Tyr278 and Tyr279 is near stoichiometric (>85%), whereas at Tyr276 sulfation is substoichiometric (
50%) (38). Furthermore, cholecystokinin circulates in multiple stoichiometrically sulfated forms in human plasma each with a common C-terminal heptapeptide amide sequence -Tyr(SO3)-Met-Gly-Trp-Met-Asp-Phe-NH2 (39). In addition, Blombäck et al. (40) showed that the circulating half-lives of [35S]sulfatelabeled fibrinopeptide B and intact fibrinogen in the rabbit are indistinguishable. These data indicate the absence of an efficient protein-tyrosine sulfatase activity in the extracellular space. Taken together these observations indicate that tyrosine O-sulfation is irreversible in vivo.
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Role of Tyrosine O-Sulfation in Protein Function |
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Until recently all of the accumulated data on the role of tyrosine O-sulfation in protein function has been derived from in vitro studies. In most cases in which a role for sulfation in the function of a protein has been defined, that function has been decreased in the absence of tyrosine O-sulfation but not absent. The only evidence that decreased tyrosine O-sulfation may have functional consequences in vivo comes from cases of mild to moderate hemophilia A that are due to missense mutations in the factor VIII gene that result in a Tyr1680 Phe substitution in the von Willebrand factor binding site at the junction of the B and A3 domain (46). Sulfation of Tyr1680 in factor VIII is required for optimal binding to von Willebrand factor, which acts as a carrier protein for factor VIII in plasma, thereby increasing the circulating half-life of factor VIII in vivo (47). Thus, this mutation may directly explain the mild to moderate hemophilia in these patients.
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Emerging Insights from Tpst1 and Tpst2 Knock-out Mice |
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In contrast to Tpst1/ mice, the growth of male and female Tpst2/ mice was severely delayed. The maximum difference in body weight occurred at 4 weeks of age at which time the body weight of both male and female homozygotes was on average 20% below that of wild-type littermates. However, Tpst2/ mice attained normal body weights at 10 weeks of age and otherwise appeared healthy to 12 months of age. A careful examination of the reproductive performance of Tpst2/ mice revealed a severe defect in male but not female reproductive performance. In 20 different matings between Tpst2/ males and either Tpst2/ or Tpst2+/ females, only a single pup was sired by a Tpst2/ male over a 2-month breeding period, despite the detection of vaginal plugs in most of the mating partners. Our preliminary analysis indicates that Tpst2/ males have normal gonadal function, as assessed by normal testicular weights and serum follicle-stimulating hormone, luteinizing hormone, and testosterone levels. Epididymal sperm counts, sperm morphology, sperm motility, and testicular histology are normal. In addition, we observed no gross or histological abnormalities in the male reproductive tract. These data indicate that defective fertility of Tpst2/ males may involve abnormalities in sperm transport, sperm capacitation, and/or fertilization per se. These possibilities are currently being investigated.
Many basic questions about tyrosine O-sulfation and the enzymes that catalyze its formation remain unanswered. Little is known about how TPST expression is regulated and how the two TPST isoenzymes differ with respect to substrate specificity. There is also not any information regarding the relative abundance of TPST-1 and -2 in cells. Targeted disruption of the Tpst1 and Tpst2 genes in mice result in distinct phenotypic effects. Currently we cannot directly link the phenotypic effects of TPST-1 or TPST-2 deficiency to defective sulfation of any known TPST substrate(s). Nevertheless, these data offer compelling evidence that the substrate repertoire of this enzyme system is substantially more complex than currently appreciated. Whether the distinct phenotypic effects observed reflect differences in substrate specificity, cellular distribution, and/or relative abundance of the two isoenzymes is not clear and will require further investigation. Nevertheless, these data demonstrate that the two TPST isoenzymes have distinct biological roles and underscore the importance of tyrosine O-sulfation in several physiological processes that had not been previously appreciated, including growth, regulation of body weight, and reproductive physiology.
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FOOTNOTES |
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* This minireview will be reprinted in the 2003 Minireview Compendium, which will be available in January, 2004. This work was supported by Grant HL63152 from the United States Public Health Service.
The on-line version of this article (available at http://www.jbc.org) contains Supplemental Table I.
To whom correspondence should be addressed: Oklahoma Medical Research Foundation, 825 NE 13th St., Mailstop 45, Oklahoma City, OK 73104. Tel.: 405-271-7314; Fax: 405-271-7417; E-mail: kevin-moore{at}omrf.ouhsc.edu.
1 The abbreviations used are: TPST, tyrosylprotein sulfotransferase; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; EST, expressed sequence tag data base; PSGL-1, P-selectin glycoprotein ligand-1; GPCR, G-proteincoupled receptor; HIV, human immunodeficiency virus; RANTES, regulated on activation normal T cell expressed and secreted; APS, adenosine phosphosulfate.
2 Y. B. Ouyang and K. L. Moore, unpublished observations.
3 We have sequenced several additional TPST cDNAs and deposited the sequences in GenBankTM, including D. melanogaster TPST (AY124548
[GenBank]
), H. roretzi TPST-2 (AY079190
[GenBank]
), Ictalurus punctatus TPST-2 (AF510735
[GenBank]
), Danio rerio TPST-1 (AF510736
[GenBank]
), and D. rerio TPST-2 (AF510737
[GenBank]
).
4 D. Corbeil and W.B. Huttner, personal communication.
5 www.expasy.org/tools/sulfinator/.
6 K. L. Moore, unpublished observation.
7 Y. B. Ouyang, J. Deng, and K. L. Moore, unpublished observations.
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REFERENCES |
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