Conversion of recombinant hirudin to the natural form by in vitro tyrosine sulfation. Differential substrate specificities of leech and bovine tyrosylprotein sulfotransferases

Hirudin, a tyrosine-sulfated protein secreted by the leech Hirudo medicinalis, is one of the most potent anticoagulants known. The hirudin cDNA has previously been cloned and has been expressed in yeast, but the resulting recombinant protein was found to be produced in the unsulfated form, which is known to have an at least 10 times lower affinity for thrombin than the naturally occurring tyrosine-sulfated hirudin. Here we describe the in vitro tyrosine sulfation of recombinant hirudin by leech and bovine tyrosylprotein sulfotransferase (TPST). With both enzymes, in vitro sulfation of recombinant hirudin occurred at the physiological site (Tyr-63) and rendered the protein biochemically and biologically indistinguishable from natural hirudin. However, leech TPST had an over 20-fold lower apparent Km value for recombinant hirudin than bovine TPST. Further differences in the catalytic properties of leech and bovine TPSTs were observed when synthetic peptides were tested as substrates. Moreover, a synthetic peptide corresponding to the 9 carboxyl-terminal residues of hirudin (which include Tyr-63) was sulfated by leech TPST with a similar apparent Km value as full length hirudin, indicating that structural determinants residing in the immediate vicinity of Tyr-63 are sufficient for sulfation to occur.     

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.     

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.     

酪氨酰蛋白磺基转移酶与植物蛋白质硫酸化修饰

蛋白质硫酸化是一种翻译后修饰,该修饰使分泌蛋白或膜蛋白具有成熟的生物学功能,在植物的生长发育中发挥重要的作用。催化这一修饰的酶是酪氨酰蛋白磺基转移酶（tyrosylprotein sulfotransferase, TPST）,它将底物3′-磷酸腺苷-5′磷酰硫酸（PAPS）的磺酸基团转移到蛋白质的酪氨酸残基上。近年来,随着植物中TPST的克隆,已有3个家族的植物多肽被发现存在硫酸化修饰。本文综述了植物TPST的生化特性与功能,介绍了植物TPST的3个底物多肽家族及其参与的分子信号途径。     

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.     

Identification and characterization of tyrosylprotein sulfotransferase from human saliva

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl2 for its activity. Maximum TPST activity was observed at 20 mM MnCl2. The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.     

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 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.     

Anticoagulant activity of synthetic hirudin peptides

Synthetic peptides based on the COOH-terminal 21 residues of hirudin were prepared in order to 1) evaluate the role of this segment in hirudin action toward thrombin, 2) define the shortest peptide derivative with anticoagulant activity, and 3) investigate the role of tyrosine sulfation in the peptides' inhibitory activities. A hirudin derivative of 20 amino acids, Hir45-64 (derived from residues 45-64 of the hirudin polypeptide), was found to effect a dose-dependent increase in the activated partial thromboplastin time (APTT) of normal human plasma but to have no measurable inhibitory activity toward thrombin cleavage of a tripeptidyl p-nitroanilide substrate. Anticoagulant activity in hirudin derivatives was comparable in peptides of 20, 16, and 12 residues truncated from the NH2 terminus. Additional truncated peptides prepared by synthesis and carboxypeptidase treatment reveal that the minimal sequence of a hirudin peptide fragment with maximal anticoagulant activity is contained within the sequence: NH2-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH. The 12-residue derivative thus identified was reacted with dicyclohexylcarbodiimide in the presence of sulfuric acid to yield a Tyr-sulfated peptide, S-Hir53-64. By comparison to unsulfated peptide, S-Hir53-64 was found to contain a specific inhibitory activity enhanced by one order of magnitude toward increase in APTT and to effect a dose-dependent increase in thrombin time of normal human plasma to yield a 4-fold increase in thrombin time with 2.5 micrograms/ml peptide using 0.8 units/ml alpha-thrombin. Comparison of S-Hir53-64 to hirudin in thrombin time and APTT assays reveals a 50-fold difference in molar specific activities toward inhibition of thrombin. Comparison of antithrombin activities of S-Hir53-64 using a variety of animal thrombins demonstrates greatest inhibitory activity toward murine, rat, and human enzymes and a 10-fold reduced activity toward bovine thrombin.     

Post-translational modification of bovine pro-opiomelanocortin. Tyrosine sulfation and pyroglutamate formation, a mass spectrometric study

The amino-terminal fragment of beta-lipotropin (i.e. beta-lipotropin (1-40)) and joining peptide portions of pro-opiomelanocortin have been purified from extracts of bovine posterior pituitaries. Peptides were purified using a combination of reversed-phase and ion-exchange batch extraction procedures followed by reversed-phase high performance liquid chromatography. beta-Lipotropin (1-40) was found to consist of four major components while joining peptide was found to consist of two major components. Fast atom bombardment-mass spectrometric analysis of the tryptic fragments of both peptides revealed that the observed heterogeneity could be explained in terms of post-translational modifications. beta-Lipotropin (1-40) was found to be sulfated at tyrosine residue 28 to an extent of about 50%. The tyrosine residue in beta-lipotropin (1-40) is situated within an amino acid sequence with a preponderance of glutamate residues. Sulfation of this amino acid residue is entirely compatible with the known primary structure requirements of the sulfotransferase enzyme located in the trans-Golgi fraction. Both beta-lipotropin (1-40) and joining peptide were found to have pyroglutamate at their amino termini to an extent of about 50%. The cDNA sequence for bovine pro-opiomelanocortin predicts the presence of glutamic acid at position 1 of both peptides. Pyroglutamate is normally formed through the cyclization of glutamine. This reaction is thought to be catalyzed by a pyroglutamate forming enzyme located within the secretory granule fraction. Under certain circumstances peptides with glutamate at their amino termini may act as substrates for this enzyme.     

Differential enzymatic characteristics and tissue-specific expression of human TPST-1 and TPST-2

Protein tyrosine sulfation is emerging as a widespread post-translational modification in multicellular eukaryotes. The responsible enzyme, named tyrosylprotein sulfotransferase (TPST), catalyzes the sulfate transfer from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues of proteins. Two distinct TPSTs, designated TPST-1 and TPST-2, had previously been identified. In the present study, we cloned human TPST-1 and TPST-2 expressed and characterized the recombinant enzymes using peptide substrates. These enzymes displayed distinct acidic pH optima and stimulatory effects of Mn(2+). Additionally, the activity of TPST-2, but not TPST-1, was stimulated in the presence of Mg(2+). Compared with TPST-2, TPST-1 displayed considerably lower K(m) and V(max) for the majority of the tested peptide substrates, implying their differential substrate specificity. Quantitative real-time PCR analysis showed that although the two TPSTs were co-expressed in all 20 human tissues examined, the levels of expression of TPST-1 and TPST-2 varied significantly among different tissues. These latter findings may imply distinct physiological functions of TPST-1 and TPST-2.     

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.     

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.     

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.     

Requirement of tyrosylprotein sulfotransferase-A for proper cuticle formation in the nematode C. elegans

Tyrosine O-sulfation is one of the post-translational modification processes that occur to membrane proteins and secreted proteins in eukaryotes. Tyrosylprotein sulfotransferase (TPST) is responsible for this modification, and in this report, we describe the expression pattern and the biological role of TPST-A in the nematode Caenorhabditis elegans. We found that TPST-A was mainly expressed in the hypodermis, especially in the seam cells. Reduction of TPST-A activity by RNAi caused severe defects in cuticle formation, indicating that TPST-A is involved in the cuticle formation in the nematode. We found that RNAi of TPST-A suppressed the roller phenotype caused by mutations in the rol-6 collagen gene, suggesting that sulfation of collagen proteins may be important for proper organization of the extracellular cuticle matrix. The TPST-A RNAi significantly decreased the dityrosine level in the worms, raising the possibility that the sulfation process may be a pre-requisite for the collagen tyrosine cross-linking.     

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.     

Identification and functional importance of tyrosine sulfate residues within recombinant factor VIII.

Sulfated tyrosine residues within recombinant human factor VIII were identified by [35S]sulfate biosynthetic labeling of Chinese hamster ovary cells which express human recombinant factor VIII. Alkaline hydrolysis of purified [35S]sulfate-labeled factor VIII showed that greater than 95% of the [35S]sulfate was incorporated into tyrosine. [3H]Tyrosine and [35S]sulfate double labeling was used to quantify the presence of 6 mol of tyrosine sulfate per mole of factor VIII. Amino acid sequence analysis of thrombin and tryptic peptides isolated from [35S]sulfate-labeled factor VIII demonstrated tyrosine sulfate at residue 346 in the factor VIII heavy chain and at residues 1664 and 1680 in the factor VIII light chain. In addition, the carboxyl-terminal half of the A2 domain contained three tyrosine sulfate residues, likely at positions 718, 719, and 723. Interestingly, all sites of tyrosine sulfation border thrombin cleavage sites. The functional importance of tyrosine sulfation was examined by treatment of cells expressing factor VIII with sodium chlorate, a potent inhibitor of tyrosine sulfation. Increasing concentrations of sodium chlorate inhibited sulfate incorporation into factor VIII without affecting its synthesis and/or secretion. However, factor VIII secreted in the presence of sodium chlorate exhibited a 5-fold reduction in procoagulant activity, although the protein was susceptible to thrombin cleavage. These results suggest that tyrosine sulfation is required for full factor VIII activity and may affect the interaction of factor VIII with other components of the coagulation cascade.