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.     

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.     

Chemical modifications and amino acid substitutions in recombinant hirudin that increase hirudin-thrombin affinity

Recombinant hirudin (r-hirudin), unlike the naturally occurring leech protein, lacks a sulfate ester on Tyr-63 which reduces its binding affinity to thrombin by 3-10-fold. We demonstrate that nitration or iodination of Tyr-63 restores hirudin-thrombin affinity to levels similar to or exceeding that of the natural inhibitor. In contrast, nitration of Tyr-3 reduces the affinity of hirudin for thrombin. These chemical modifications results in multiple reaction products that are readily separated by reverse-phase HPLC. The mechanism of the observed changes in thrombin affinity may involve a reduction in the pK of the hydroxyl group of tyrosine due to substitution of the electrophilic iodo or nitro group on the phenyl ring, resulting in an increased negative charge at neutral pH. For Tyr-63, this effect mimics the sulfatotyrosine of natural hirudin, leading to an increased thrombin affinity at the anion-binding exosite. For Tyr-3, the increased polarity may destabilize its interaction within the apolar-binding site of thrombin. Substitution of the highly conserved Tyr-3 residue with Phe or Trp not only enables specific and quantitative chemical modification at Tyr-63 but also independently increases hirudin-thrombin affinity. Kinetic analysis of thrombin inhibition showed that enhanced binding by r-hirudin(nitro-Tyr-63) is due to an increase in the association rate between hirudin and thrombin whereas the reduced binding of r-hirudin(nitro-Tyr-3) results from a large increase in the dissociation rate. These observations indicate that specific segments within both the amino- and carboxy-terminal regions of hirudin interact with thrombin.     

Purification and characterization of tyrosylprotein sulfotransferase.

Tyrosylprotein sulfotransferase (TPST) is a Golgi membrane enzyme involved in the post-translational modification of secretory and membrane proteins. Here we describe the 140,000-fold purification of this enzyme from bovine adrenal medulla to apparent homogeneity and determine its substrate specificity. The key step in the purification was affinity chromatography on a substrate peptide to which the enzyme bound in the presence of nucleotide cosubstrate. TPST is a 54-50 kd integral membrane glycoprotein. The presence of sialic acid strongly suggests that within the Golgi complex, TPST is localized in the trans-most subcompartment. TPST was found to specifically sulfate tyrosine residues adjacent to acidic amino acids. These results define a major determinant for the specificity of protein sulfation in the trans Golgi.     

The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin.

Recombinant hirudin (hirudin), which lacks the sulphate group on Tyr-63, has a tenfold-reduced affinity for alpha-thrombin. Incubation of recombinant hirudin with [gamma-32P]ATP and protein tyrosine kinase III from spleen resulted in incorporation of radioactivity into the protein. Phosphatohirudin was purified to homogeneity (overall yield 5%) and shown to contain 1 mol phosphate/mol protein, as a phosphotyrosyl residue at position 63. The kinetics of the inhibition of human alpha-thrombin by phosphatohirudin were determined. It was found that the introduction of the negatively charged phosphate had fully restored the affinity of recombinant hirudin for alpha-thrombin to the level of the wild-type sulphatohirudin. The inhibition constant of phosphatohirudin was 18 fM compared with 20 fM for that of sulphatohirudin. Moreover, the values for the on- and off-rate constants of both forms of hirudin were indistinguishable.     

Recognition of substrates by tyrosylprotein sulfotransferase. Determination of affinity by acidic amino acids near the target sites.

The sulfation of proteins by tyrosylprotein sulfotransferase (TPST) is highly site-specific. In this study, we examined the sequence specificity of the target site for TPST by determining the kinetics of rat liver TPST with peptides related to the sequence of the C4 component of complement. The data obtained from this study demonstrate that selective elimination of negative charges from the -5 to +5 region of the acceptor tyrosine, either by removal or by isosteric substitution or the acidic amino acids in the region, produced a substantial change in the Km value, with relatively little effect on Vmax. Substitutions at -1 and +1 positions increase the Km value by 22- and 4-fold, respectively, whereas removal of the acidic amino acids from the -5, -4, or +4 positions increased the Km values by a factor of 2-4. The effect of elimination of an acidic amino acid on the Km value was constant and specific for its particular position in relation to tyrosine, and the effect of modification of more than one amino acid was multiplicative. This study provides evidence that: 1) acidic residues near tyrosines promote sulfation by increasing the affinity of enzyme-substrate binding and have little effect on catalytic rate; 2) the contribution of each acidic residue to affinity for TPST is independent and varies according to position relative to the acceptor tyrosine; and 3) the enzyme interacts with a segment of at least 4-5 residues on each side of the tyrosine, with the residues on the -1 and +1 positions being the most important determinants. In general, residues on the NH2-terminal side of the tyrosine have a greater effect on affinity for TPST.     

Existence of a plant tyrosylprotein sulfotransferase: novel plant enzyme catalyzing tyrosine O-sulfation of preprophytosulfokine variants in vitro

An in vitro assay system to detect tyrosylprotein sulfotransferase (TPST) activity of higher plant cells was established, using synthetic oligopeptides based on the deduced amino acid sequence of a phytosulfokine-alpha (PSK-alpha) precursor. TPST activity was found in microsomal membrane fractions of rice, asparagus and carrot cells and it was confirmed that acidic amino acid residues adjacent to the tyrosine residues of acceptor peptides were essential to the sulfation reaction. The asparagus TPST exhibited a broad pH optimum of 7.0-8.5, required manganese ions for maximal activity and appeared to be a membrane-bound protein localized in the Golgi apparatus. These enzymes should be defined as a new class of plant sulfotransferases that catalyze tyrosine O-sulfation of a PSK-alpha precursor and other unknown proteins.     

Tyrosine sulfation of native mouse Psgl-1 is required for optimal leukocyte rolling on P-selectin in vivo

Background

We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.

Methodology/Principal Findings

Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT→B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO→B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO→B6 venules compared to WT→B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.

Conclusions/Significance

These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis.     

Two membrane-bound forms of tyrosylprotein sulfotransferase as revealed by phase partitioning in Triton X-114.

Tyrosylprotein sulfotransferase (TPST) is a membrane-associated enzyme of the trans Golgi network that catalyzes the posttranslational sulfation of a variety of secretory and membrane proteins. We have analyzed the membrane association of TPST in Golgi-enriched fractions from bovine adrenal medulla using carbonate treatment (pH 11) and Triton X-114 phase partitioning. TPST was not extracted by carbonate. Triton X-114 phase partitioning revealed that, unexpectedly, TPST from non-carbonate-treated membranes was present in both, a hydrophilic and a hydrophobic form with apparent sedimentation coefficients of approximately 13 and approximately 6, respectively. Extraction of membranes with carbonate converted the hydrophilic form TPST to the hydrophobic form. Addition of the carbonate extract to TPST solubilized from carbonate-treated membranes converted the hydrophobic form of the enzyme to the hydrophilic form. This conversion of TPST was specific in that it was not observed for the bulk of the proteins present in the carbonate-treated membranes. The factor in the carbonate extract responsible for this conversion, referred to as "phase-transfer factor", (i) was precipitable with ammonium sulfate and polyethylene glycol, (ii) was non-dialyzable, (iii) was not extracted from membranes by 0.5 M NaCl, and (iv) appeared to be more abundant than TPST itself. These results show that TPST is an integral membrane protein and suggested that the enzyme may exist in a complex with a peripheral membrane protein. Moreover, a phase-transfer factor was also observed in another system, PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine-sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.     

Sequential tyrosine sulfation of CXCR4 by tyrosylprotein sulfotransferases

CXC-chemokine receptor 4 (CXCR4) is a G protein-coupled receptor for stromal cell-derived factor-1 (SDF-1/CXCL12). SDF-1-induced CXCR4 signaling is indispensable for embryonic development and crucial for immune cell homing and has been implicated in metastasis of numerous types of cancer. CXCR4 also serves as the major coreceptor for cellular entry of T-cell line-tropic (X4) HIV-1 strains. Tyrosine residues in the N-terminal tail of CXCR4, which are post-translationally sulfated, are implicated in the high-affinity binding of SDF-1 to CXCR4. However, the specific roles of three potential tyrosine sulfation sites are not well understood. We investigated the pattern and sequence of CXCR4 sulfation by using recombinant human tyrosylprotein sulfotransferases TPST-1 and TPST-2 to modify a peptide that corresponds to amino acids 1-38 of the receptor (CXCR4 1-38). We analyzed the reaction products with a combination of reversed-phase HPLC, proteolytic cleavage, and mass spectrometry. We found that CXCR4 1-38 is sulfated efficiently by both TPST enzymes, leading to a final product with three sulfotyrosine residues. Sulfates were added stepwise to the peptide, producing specific intermediates with one or two sulfotyrosines. The pattern of sulfation in these intermediates indicates that with both enzymes Tyr-21 is sulfated first, followed by Tyr-12 or Tyr-7. Using heteronuclear NMR spectroscopy, we demonstrated that the SDF-1 binding affinity of CXCR4 1-38 increases with the number of sulfotyrosines present, which suggests a potential physiological role for sulfation of all three sites in the N-terminus of CXCR4. These results provide a structural basis for understanding the role of post-translational tyrosine sulfation in SDF-1-induced CXCR4 signaling.     

Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms.

The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-Phe-Pro-Arg-Pro-(Gly)4-hirudin (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower K(i) than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (P1-P1') seems to be cleaved.     

Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis

A DNA containing the coding sequence for the proteinase inhibitor protein hirudin from the leech Hirudo medicinalis has been obtained by enzymic ligation of chemically synthesized deoxyoligonucleotides. The 226 bp synthetic gene carries signals for the translation initiation and termination. Fragment synthesis was performed by the Khorana ligation method as well as by the fill-in method. Efficiencies of these two methods are compared. The synthetic gene was expressed in E. coli as a fusion protein with beta-galactosidase under the control of the lac-promoter as well as a non-hybrid protein under the control of the lambda PL-promoter. The non-hybrid expression product was shown to have similar biological properties as the authentic protein isolated from the leech.     

Secretion of biologically active leech hirudin from baculovirus-infected insect cells.

The thrombin inhibitor, hirudin, from the leech Hirudo Medicinalis, is the most powerful natural anticoagulant known. It has been characterized as a polypeptide of 65 amino acids which exhibits its anticoagulant properties by binding tightly and specifically to alpha-thrombin. The potency and specificity of hirudin have generated interest on its possible use in the treatment or prophylaxis of various thrombotic diseases. We have used the baculovirus expression system to efficiently produce active hirudins in insect cells. The Autographa californica nuclear polyhedrosis virus has proved useful as a helper-independent viral expression vector for high-level production of recombinant proteins in cultured insect cells. Hirudin variants (HV1 and HV2) were produced in infected insect cells as secreted proteins by joining their coding sequences to the leader peptide sequence of the vescicular stomatitis virus G protein. The recombinant products were biologically active and, interestingly, N-terminal sequencing of HV1 revealed that the heterologous leader peptide is correctly removed.     

Tyrosine sulfation of statherin

Tyrosylprotein sulfotransferase (TPST), responsible for the sulfation of a variety of secretory and membrane proteins, has been identified and characterized in submandibular salivary glands (William et al. Arch Biochem Biophys 1997; 338: 90-96). In the present study we demonstrate the sulfation of a salivary secretory protein, statherin, by the tyrosylprotein sulfotransferase present in human saliva. Optimum statherin sulfation was observed at pH 6.5 and at 20 mm MnCl(2). Increase in the level of total sulfation was observed with increasing statherin concentration. The K(m)value of tyrosylprotein sulfotransferase for statherin was 40 microM. Analysis of the sulfated statherin product on SDS-polyacrylamide gel electrophoresis followed by autoradiography revealed (35)S-labelling of a 5 kDa statherin. Further analysis of the sulfated statherin revealed the sulfation on tyrosyl residue. This study is the first report demonstrating tyrosine sulfation of a salivary secretory protein. The implications of this sulfation of statherin in hydroxyapatite binding and Actinomyces viscosus interactions are discussed.     

Tyrosine sulfation of yolk proteins 1, 2, and 3 in Drosophila melanogaster

Protein sulfation was studied in Drosophila melanogaster after in vivo labeling of flies with inorganic [35S]sulfate. After separation of total fly protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins with sulfated carbohydrates and proteins containing tyrosine sulfate were found in all the molecular weight ranges analyzed. When female and male fly proteins were compared with each other, the electrophoretic patterns of protein-bound carbohydrate sulfate were found to be similar, whereas those of protein-bound tyrosine sulfate were distinct. The most prominent difference was the exclusive presence in female flies of three major tyrosine-sulfated proteins with apparent molecular masses between 48 and 45 kDa. Radioimmunolabeling after two-dimensional polyacrylamide gel electrophoresis was used to identify these proteins as yolk proteins 1, 2, and 3. Each of the three yolk proteins existed in several isoelectric forms, all of which were sulfated. Since the number of tyrosine residues in the yolk proteins is known, the stoichiometry of tyrosine sulfation could be determined by a novel method and was found to be 2.2, 0.9, and 1.2 mol of tyrosine sulfate per mol of yolk protein 1, 2, and 3, respectively. The present results, together with the recently reported molecular cloning of the yolk protein genes, make the yolk proteins suitable objects for genetic approaches to investigate the biological role(s) of tyrosine sulfation of secretory proteins.     

Rapid purification and revised amino-terminal sequence of hirudin: a specific thrombin inhibitor of the bloodsucking leech

Hirudin is a specific polypeptide thrombin inhibitor consisting of 65 amino acids that is produced by the leech, Hirudo medicinalis. We describe a rapid method for the purification of hirudin from a leech extract. Crude hirudin, purchased from a commercial source, was first fractionated on a DEAE-HPLC column using a salt gradient. Hirudin activity was monitored by inhibition of the thrombin-mediated hydrolysis of a synthetic substrate H-D-Phenylalanyl-Pipecolyl-Arginine-p-Nitroanilide. The fractions containing antithrombin activity were pooled and further purified by reverse-phase HPLC. The homogeneity of purified hirudin was confirmed by a single amino-terminal sequence for 43 residues with Val-Val as the first two amino acids. Residue 33 was Asn rather than Asp as reported previously.     

Tyrosylprotein sulfotransferase suppresses ABA signaling via sulfation of SnRK2.2/2.3/2.6

Phytohormone abscisic acid (ABA) plays vital roles in stress tolerance, while long-term overactivation of ABA signaling suppresses plant growth and development. However, the braking mechanism of ABA responses is not clear. Protein tyrosine sulfation catalyzed by tyrosylprotein sulfotransferase (TPST) is a critical post-translational modification. Through genetic screening, we identified a tpst mutant in Arabidopsis that was hypersensitive to ABA. In-depth analysis revealed that TPST could interact with and sulfate SnRK2.2/2.3/2.6, which accelerated their degradation and weakened the ABA signaling. Taken together, these findings uncovered a novel mechanism of desensitizing ABA responses via protein sulfation.