Tyrosine O-sulfation promotes proteolytic processing of progastrin.

Tyrosine O-sulfation is a common post-translational modification of secretory and membrane proteins. The biological function of sulfation is known in only a few proteins, where it appears to enhance protein-protein interactions. Based on known sequences around sulfated tyrosines, a consensus sequence for prediction of target tyrosines has been proposed. However, some proteins are tyrosine sulfated at sites that deviate from the proposed consensus. Among these is progastrin. It is possible that the deviation explains the incomplete sulfation characteristic for bioactive gastrin peptides. In order to test this hypothesis, we have performed site-directed mutagenesis of the gastrin gene followed by heterologous expression in an endocrine cell line. The results show that substitution of the alanyl residue immediately N-terminal to the sulfated tyrosine with an acidic amino acid promotes the sulfation of gastrin peptides. Hence, the study supports the proposed consensus sequence for tyrosine sulfation. Importantly, however, the results also reveal that complete sulfation increases the endoproteolytic maturation of progastrin. Thus, our study suggests an additional function for tyrosine sulfation of possible general significance.     

Tyrosine Sulfation of Chemokine Receptor CCR2 Enhances Interactions with Both Monomeric and Dimeric Forms of the Chemokine Monocyte Chemoattractant Protein-1 (MCP-1)

Chemokine receptors are commonly post-translationally sulfated on tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of tyrosine sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.     

Identification of tyrosine sulfation in extracellular leucine-rich repeat proteins using mass spectrometry

Multiple and variable tyrosine sulfation in extracellular class II leucine-rich repeat proteins/proteoglycans were characterized by mass spectrometry. The sulfogroup on tyrosine is labile and is released from peptides under normal mass spectrometric conditions. Thus, special approaches must be considered in order to identify this modification. By using a combination of mass spectrometry studies operating in negative and positive ion mode, tyrosine sulfation could be identified. In positive mode, the peptides normally appeared non-sulfated, whereas in negative mode a mixture of sulfated and non-sulfated species was observed. A combination of peptides released by different proteinases was used to obtain details on the locations of sulfate groups. Multiple tyrosine sulfates were observed in the N-terminal region of fibromodulin (up to 9 sites), osteoadherin (up to 6 sites), and lumican (2 sites). Osteoadherin contains two additional sulfated tyrosine residues close to its C terminus. We also identified an error in the published sequence of bovine fibromodulin, resulting in the replacement of Thr37 by Tyr37-Gly38, thus increasing its homology with its human counterpart.     

Identification of the site of sulfation of the fourth component of human complement

The alpha-chain of the fourth component of complement (C4) contains tyrosine sulfate (Karp, D.R. (1983) J. Biol. Chem. 258, 12745-12748). Here we have determined the site and stoichiometry of sulfation of C4 secreted by the human hepatoma-derived cell line Hep G2. C4 was labeled with [35S]sulfate and isolated from culture medium by immunoprecipitation. C4 digested with trypsin and chymotrypsin and analyzed by reverse-phase high-performance liquid chromatography contained a single sulfate-labeled peptide. Digestion of C4 with trypsin alone yielded two major sulfate-labeled peptides, suggesting that there may be some sequence variability in C4 near the site of sulfation. Sequential Edman degradation of tryptic peptides labeled with [3H]tyrosine and [35S]sulfate detected tyrosine residues at positions 5, 13, 16, and 18. Chymotrypsin cleaved 5 residues off the NH2-terminal end of tryptic peptides, yielding a peptide with tyrosine at positions 8, 11, and 13. Comparison of the position of tyrosine residues with the reported sequence of C4 identified the sites of sulfation as tyrosine residues at positions 738, 741, and 743 in the alpha-chain of C4. All 3 of these tyrosine residues appeared to be sulfated. When sulfation of C4 was partially inhibited by addition of catechol to culture medium, three different forms of the peptide were resolved by high-performance liquid chromatography, consistent with peptides containing 1, 2, or 3 sulfates. Comparison of the quantities of tyrosine and tyrosine sulfate in C4 which had been labeled with [3H]tyrosine and digested with Pronase also indicated that C4 contained an average of 2-3 residues of tyrosine sulfate/molecule. These results suggest that the biologically active form of the protein is sulfated.     

Toward a framework for sulfoproteomics: Synthesis and characterization of sulfotyrosine-containing peptides

Tyrosine sulfation is one of the most common post-translational modifications in secreted and transmembrane proteins and a key modulator of extracellular protein-protein interactions. Several proteins known to be tyrosine sulfated play important roles in physiological processes, and in some cases a direct link between protein function and tyrosine sulfation has been established. In blood coagulation, tyrosine sulfation of factor VIII is required for efficient binding of von Willebrand factor; in leukocyte adhesion, tyrosine sulfation of the P-selectin glycoprotein ligand-1 mediates high-affinity binding to P-selectin; and in leukocyte chemotaxis, tyrosine sulfation of chemokine receptors is required for optimal interaction with chemokine ligands. Furthermore, tyrosine sulfation has been implicated in several infectious diseases. In particular, tyrosine sulfation of the HIV-1 co-receptor CCR5 is required for viral entry into host cells and tyrosine sulfation of the Duffy antigen/receptor for chemokines is crucial for erythrocyte invasion by the malaria parasite plasmodium vivax. Despite increasing interest in tyrosine sulfation in recent years, the sulfoproteome still remains largely unexplored. To date, only a relatively small number of sulfotyrosine-containing peptides and proteins have been identified, and a specific role for tyrosine sulfation has not been established for most of these. Here, we provide an overview of the biology and enzymology of tyrosine sulfation and discuss recent developments in preparative and analytical methods that are central to sulfoproteome research.     

Characterization of a novel modification of a CHO-produced mAb: Evidence for the presence of tyrosine sulfation

Herein we describe the investigation of a Chinese hamster ovary (CHO)-expressed human mAb molecule found partially modified by a +80 Da adduct. This mass difference, suggestive of a single sulfation or phosphorylation addition, was observed by mass analysis of the intact and reduced molecule by mass spectrometry (MS). The modification was located on tyrosine 31 (Y31) of the light chain in the complementarity-determining region 1 by liquid chromatography (LC)-MS peptide mapping and electron transfer dissociation fragmentation. The complete loss of the 80 Da modification moiety during collision induced dissociation fragmentation suggested this modification could not be a tyrosine phosphorylation. Treatment of the mAb with alkaline phosphatase confirmed our hypothesis. Western blot experiment using anti-tyrosine sulfation antibody and LC retention time correlation with corresponding synthetic sulfated peptides further confirmed the identification of tyrosine sulfation on the light chain. The unique sequence motif with neighboring acidic amino acids and local secondary structure might play a role to make Y31 a substrate residue for sulfation. This type of modification, to our knowledge, has not been previously reported for CHO-produced human IgG antibodies.     

Glucagon-like peptide-1(1-37) can enhance blood glucose homeostasis in mice

Tyrosyl O-sulfation is a common posttranslational derivatization of proteins that may also modify regulatory peptides. Among these are members of the cholecystokinin (CCK)/gastrin family. While sulfation of gastrin peptides is without effect on the bioactivity, O-sulfation is crucial for the cholecystokinetic activity (i.e. gallbladder emptying) of CCK peptides. Accordingly, the purification of CCK as a sulfated peptide was originally monitored by its gallbladder emptying effect. Since then, the dogma has prevailed that CCK peptides are always sulfated. The dogma is correct in a semantic context since the gallbladder expresses only the CCK-A receptor that requires sulfation of the ligand. CCK peptides, however, are also ligands for the CCK-B receptors that do not require ligand sulfation. Consequently, unsulfated CCK peptides may act via CCK-B receptors. Since in vivo occurrence of unsulfated products of proCCK with an intact α-amidated C-terminal tetrapeptide sequence (-Trp-Met-Asp-PheNH(2)) has been reported, it is likely that unsulfated CCK peptides constitute a separate hormone system that acts via CCK-B receptors. This review discusses the occurrence, molecular forms, and possible physiological as well as pathophysiological significance of unsulfated CCK peptides.     

Prediction of tyrosine sulfation sites in animal viruses

Post-translational modification of proteins by tyrosine sulfation enhances the affinity of extracellular ligand-receptor interactions important in the immune response and other biological processes in animals. For example, sulfated tyrosines in polyomavirus and varicella-zoster virus may help modulate host cell recognition and facilitate viral attachment and entry. Using a Position-Specific-Scoring-Matrix with an accuracy of 96.43%, we analyzed the possibility of tyrosine sulfation in all 1517 animal viruses available in the Swiss-Prot database. From a total of 97,729 tyrosines, we predicted 5091 sulfated tyrosine sites from 1024 viruses. Our site predictions in hemagglutinin of influenza A, VP4 of rotavirus, and US28 of cytomegalovirus strongly suggest an important link between tyrosine sulfation and viral disease mechanisms. In each of these three viral proteins, we observed highly conserved amino acid sequences surrounding predicted sulfated tyrosine sites. Tyrosine sulfation appears to be much more common in animal viruses than is currently recognized.     

Analysis of the substrate specificity of tyrosylprotein sulfotransferase using synthetic peptides

Tyrosylprotein sulfotransferase (TPST) catalyzes the sulfation of proteins at tyrosine residues. We have analyzed the substrate specificity of TPST from bovine adrenal medulla with a novel assay, using synthetic peptides as substrates. The peptides were modeled after the known, or putative, tyrosine sulfation sites of the cholecystokinin precursor, chromogranin B (secretogranin I) and vitronectin, as well as the tyrosine phosphorylation sites of alpha-tubulin and pp60src. Varying the sequence of these peptides, we found that (i) the apparent Km of peptides with multiple tyrosine sulfation sites decreased exponentially with the number of sites; (ii) acidic amino acids were the major determinant for tyrosine sulfation, acidic amino acids adjacent to the tyrosine being more important than distant ones; (iii) a carboxyl terminally located tyrosine residue may be sulfated. Moreover, TPST catalyzed the sulfation of a peptide corresponding to the tyrosine autophosphorylation site of pp60v-src (Tyr-416) but not of a peptide corresponding to the non-autophosphorylation site of pp60c-src (Tyr-527). These results experimentally define structural determinants for the substrate specificity of TPST and show that this enzyme and certain autophosphorylating tyrosine kinases have overlapping substrate specificities in vitro.     

The hormonogenic tyrosine 5 of porcine thyroglobulin is sulfated

Our previous results showed that sulfated tyrosines of thyroglobulin (Tg), the molecular support of thyroid hormonosynthesis, are involved in the hormonogenic process. Moreover, the consensus sequence required for tyrosine sulfation is present in most of the hormonogenic sites. These observations suggest that tyrosine sulfation might play a critical role in the hormonogenic process. In this paper we studied the putative sulfation of tyrosine 5 contained in the preferential hormonogenic site. Porcine thyrocytes were cultured with thyrotropin but without iodide to preserve the sulfation state of tyrosine 5 and then incubated or not with [35S]sulfate. Secreted Tg was purified and submitted to peptide sequence analysis which confirmed the known peptide sequence of the NH(2) extremity of Tg:NIFEYQV. The treatment of [35S]sulfate-labeled Tg by leucine aminopeptidase, which sequentially digested its amino-terminal extremity, released the same amino acids and further analysis by thin layer chromatography showed that the tyrosine was sulfated. We concluded that tyrosine 5 is sulfated but the role of sulfate group in the hormonogenic process remains to be elucidated.     

Post-translational modification of protein by tyrosine sulfation: active sulfate PAPS is the essential substrate for this modification.

In vitro tyrosine sulfation of recombinant proteins would be a valuable tool in converting those proteins expressed in prokaryotic vectors to their natural form. For this purpose tyrosylprotein sulfotransferase (TPST), the enzyme responsible for tyrosine sulfation of proteins, was characterized from a bovine liver Golgi preparation. TPST was active in a acidic environment with a pH optimum of 6.25, and displayed a stimulation by the Mn2+, with the optimum activity in the presence of 5mM MnCl2. TPST was able to sulfate recombinant hirudin variant 1 (rHV-1) expressed in Escherichia coli and the C-terminal hirudin fragment 54-65 but not the N-terminal hirudin fragment 1-15 by using 3'-phosphoadenosine 5'-phosphosulfate (PAPS), indicating its specificity for the naturally sulfated tyrosine 63. Comparison of the reaction kinetics on synthetic peptides showed that the bovine liver TPST has a higher affinity and reaction rates for those peptides with a aspartyl residue on the N-terminal side of the tyrosine when compared with a glutamyl residue.     

A novel sulfotransferase sulfates tyrosine-containing peptides and proteins

The novel sulfotransferase (M.W. 315 kDa) obtained from Eubacterium A-44 catalyzed the sulfation of tyrosine residues of peptides and proteins such as kyotorphin, enkephalin, cholecystokinin-8 (non-sulfated form), trypsin inhibitor and insulin. Also, the enzyme sulfated tyrosine residues of protein fractions purified from Eubacterium A-44.     

In vivo expression and stoichiometric sulfation of the artificial protein sulfophilin, a polymer of tyrosine sulfation sites.

To gain insight into the structural requirements for tyrosine sulfation in vivo, we have constructed and expressed an artificial gene encoding a polypeptide substrate for tyrosylprotein sulfotransferase. This gene codes for a protein, referred to as sulfophilin, which consists of a 12-times repeated heptapeptide unit corresponding to the identified tyrosine sulfation site of chromogranin B (secretogranin I), Glu-Glu-Pro-Glu-Tyr-Gly-Glu. The gene was fused to the signal sequence of secretogranin II to direct the sulfophilin protein to the secretory pathway. Stable expression of the artificial gene in NIH 3T3 cells resulted in the secretion of sulfated sulfophilin. Analysis of the stoichiometry of sulfation revealed that each of the 12 tyrosyl residues in sulfophilin was sulfated. Remarkably, up to 50% of the total protein-bound tyrosine sulfate secreted by the cells was contained in sulfophilin. The results indicate that the structural information contained in the heptapeptide motif is sufficient for stoichiometric tyrosine sulfation to occur in the living cell.     

Expression, tyrosine sulfation, and secretion of yolk protein 2 of Drosophila melanogaster in mouse fibroblasts

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A., Lottspeich, F., and Huttner, W. B. (1988) J. Biol. Chem. 263, 14925-14929), we have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 (YP2) of Drosophila melanogaster. In the present report, tyrosine sulfation of this protein was examined after expression in a heterologous mammalian cell system. Mouse fibroblasts, transfected with Drosophila YP2 genomic DNA inserted into the eucaryotic expression vector pSV2, secreted the fly protein in sulfated form. Analyses of Drosophila YP2 produced by the mouse cells showed that the features of sulfation of this protein were identical to those previously determined for YP2 isolated from flies. YP2 secreted from mouse fibroblasts was found to be exclusively sulfated on tyrosine residues. The stoichiometry of tyrosine sulfation was approximately 1 mol of sulfate/mol of YP2. Sulfate was linked to the same tyrosine residue as in YP2 isolated from flies, tyrosine 172. These results show that essential parameters of the tyrosine sulfation reaction are very similar in insects and mammals and thus highly conserved in evolution.     

Revealing the functional roles of tyrosine sulfation using synthetic sulfopeptides and sulfoproteins

The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine sulfation is a PTM that has been shown to be a key regulator of extracellular protein–protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of tyrosine sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation. In this account, we highlight recent advances in the prediction and detection of tyrosine sulfation and outline the need for continued innovation in this area. We also discuss the utility of emerging solid-phase synthesis and peptide ligation strategies for accessing libraries of homogeneously sulfated peptides and proteins to help reveal functional aspects of the sulfoproteome.     

Sulfation of two tyrosine-residues in human complement S-protein (vitronectin)

Human S-protein (vitronectin) and hemopexin, two structurally related plasma proteins of similar molecular mass and abundance, were analyzed for tyrosine sulfation. Both proteins were synthesized and secreted by the human hepatoma-derived cell line Hep G2, as shown by immunoprecipitation from the culture medium of [35S]methionine-labelled cells. When Hep G2 cells were labelled with [35S]sulfate, S-protein, but not hemopexin, was found to be sulfated. Half of the [35S]sulfate incorporated into S-protein was recovered as tyrosine sulfate. The stoichiometry of tyrosine sulfation was approximately two mol tyrosine sulfate/mol S-protein. Examination of the S-protein sequence for the presence of the known consensus features for tyrosine sulfation revealed three potential sulfation sites at positions 56, 59 and 401. Tyrosine 56 is the most probable site for stoichiometric sulfation, followed by tyrosine 59 which appears more likely to become sulfated than tyrosine 401. Tyrosines 56 and 59 are located in the anionic region of S-protein which has no homologous counterpart in hemopexin. We discuss the possibility that tyrosine sulfation of the anionic region of S-protein may stabilize the conformation of S-protein in the absence of thrombin-antithrombin III complexes and may play a role in its binding to thrombin-antithrombin III complexes during coagulation.     

CX3CR1 tyrosine sulfation enhances fractalkine-induced cell adhesion

Fractalkine is a unique CX(3)C chemokine/mucin hybrid molecule that functions like selectins in inducing the capture of receptor-expressing cells. Because of the importance of tyrosine sulfation for ligand binding of the selectin ligand PSGL1, we tested the role of tyrosine sulfation for CX(3)CR1 function in cell adhesion. Tyrosine residues 14 and 22 in the N terminus of CX(3)CR1 were mutated to phenylalanine and stably expressed on K562 cells. Cells expressing CX(3)CR1-Y14F were competent in signal transduction but defective in capture by and firm adhesion to immobilized fractalkine under physiologic flow conditions. In static binding assays, CX(3)CR1-Y14F mutants had a 2-4-fold decreased affinity to fractalkine compared with wild type CX(3)CR1. By surface plasmon resonance measurements of fractalkine binding to biosensor chip-immobilized cell membranes, CX(3)CR1-Y14F mutants had a 100-fold decreased affinity to fractalkine. CX(3)CR1-expressing cell membranes treated with arylsulfatase to desulfate tyrosine residues also showed a 100-fold decreased affinity for fractalkine. Finally, synthesized, sulfated N-terminal CX(3)CR1 peptides immobilized on biosensor chips showed a higher affinity for fractalkine than non-sulfated peptides. Thus, we conclude that sulfation of tyrosine 14 enhances the function of CX(3)CR1 in cell capture and firm adhesion. Further, tyrosine sulfation may represent a general mechanism utilized by molecules that function in the rapid capture of circulating leukocytes.     

Tyrosine modification enhances metal-ion binding

Tyrosine sulfation is a common modification of many proteins, and the ability to phosphorylate tyrosine residues is an intrinsic property of many growth-factor receptors. In the present study, we have utilized the peptide hormone CCK(8) (cholecystokinin), which occurs naturally in both sulfated and unsulfated forms, as a model to investigate the effect of tyrosine modification on metal-ion binding. The changes in absorbance and fluorescence emission on Fe(3+) binding indicated that tyrosine sulfation or phosphorylation increased the stoichiometry from 1 to 2, without greatly affecting the affinity (0.6-2.8 microM at pH 6.5). Measurement of Ca(2+) binding with a Ca(2+)-selective electrode revealed that phosphorylated CCK(8) bound two Ca(2+) ions. CCK(8) and sulfated CCK(8) each bound only one Ca(2+) ion with lower affinity. Binding of Ca(2+), Zn(2+) or Bi(3+) to phosphorylated CCK(8) did not cause any change in absorbance, but substantially increased the change in absorbance on subsequent addition of Fe(3+). The results of the present study demonstrate that tyrosine modification may increase the affinity of metal-ion binding to peptides, and imply that metal ions may directly regulate many signalling pathways.     

In vivo and in vitro tyrosine sulfation of a membrane glycoprotein

A431 cells incorporate 35SO4 into a protein of Mr 61,000 (P61). We examined sulfation of P61 by cells (in vivo) and by a cell-free system (in vitro) which requires only addition of A431 cell membranes and a 3'-phosphoadenosine 5'-phospho[35S]sulfate-generating system prepared from Krebs ascites cells. Sulfate is found exclusively in the form of tyrosine SO4 by two-dimensional high voltage electrophoresis following Pronase digestion. Endoglycosidase F digestion reduces the Mr by 2,000 but does not release the sulfate, indicating that P61 is a glycoprotein but that sulfate is not incorporated into the carbohydrate. Sulfated P61 is not found in the medium from cultured cells and remains associated with the membrane fraction following cell lysis. Treatment of membranes with 0.4 M NaCl, 0.3 M KCl, 15 mM EDTA, or pH 11.0 does not release sulfated P61. P61 is solubilized by Triton X-114 treatment of membranes and partitions into the detergent phase upon warming. Based on these characteristics, we conclude that P61 is an integral membrane protein. Trypsin digestion experiments with intact cells suggest that sulfated P61 is predominantly located in the plasma membrane. This is the first example of an integral membrane protein which is sulfated on tyrosine. The properties of the sulfation reaction are distinct from those reported for secreted proteins and are consistent with the possibility that this modification occurs at the plasma membrane rather than in the Golgi.     

The plasminogen-like molecule apically secreted by epithelial thyroid cells is sulfated

Plasminogen (Pl), a circulating protease synthesized in the liver, is also present in several tissues. In the thyroid gland a Pl-like protease was found in the apical lumen where it is involved, through its proteolytic activity, in luminal degradation of thyroglobulin (Tg). Here, we showed for the first time that the Pl-like protease apically secreted by epithelial thyroid cells is sulfated, both on tyrosine residue(s) and on oligosaccharide side chains. The Pl molecule is composed of a large N-terminal moiety made of five distinct Kringle domains (K1-K5) separated by small peptidic fragments, and of a C-terminal domain with serine protease activity. Using a software tool able to predict tyrosine sulfation sites in protein sequences we localized the potential tyrosine sulfation sites of Pl. Then, we became aware that, whatever the species considered, at least three of the four potential tyrosine sulfation sites of Pl were located on Kringle sites, and more precisely, for K1, on the highly conserved binding domain of K1. We determined with the same software tool which potential sulfation sites were the most likely to be really sulfated. We hypothesize that the sulfation of these sites modulates the binding properties of Pl.