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.     

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.     

The role of tyrosine sulfation in the dimerization of the CXCR4:SDF‐1 complex

Oligomerization of G protein‐coupled receptors is a recognized mode of regulation of receptor activities, with alternate oligomeric states resulting in different signaling functions. The CXCR4 chemokine receptor is a G protein‐coupled receptor that is post‐translationally modified by tyrosine sulfation at three sites on its N‐terminus (Y7, Y12, Y21), leading to enhanced affinity for its ligand, stromal cell derived factor (SDF‐1, also called CXCL12). The complex has been implicated in cancer metastasis and is a therapeutic target in cancer treatment. Using molecular dynamics simulation of NMR‐derived structures of the CXCR4 N‐terminus in complex with SDF‐1, and calculations of electrostatic binding energies for these complexes, we address the role of tyrosine sulfation in this complex. Our results show that sulfation stabilizes the dimeric state of the CXCR4:SDF‐1 complex through hydrogen bonding across the dimer interface, conformational changes in residues at the dimer interface, and an enhancement in electrostatic binding energies associated with dimerization. These findings suggest a mechanism through which post‐translational modifications such as tyrosine sulfation might regulate downstream function through modulation of the oligomeric state of the modified system.     

Enzymatic sulfation of steroids. II. The sulfation of corticosterone by the glucocorticoid sulfotransferases of rat liver cytosol

A radioisotopic assay for the cytoplasmic corticosterone sulfotransferase activity of rat liver was developed. The steroid inhibits the enzyme reaction. For reliable results, a complex assay method, using three different corticosterone concentrations, each studied with several different amounts of enzyme, was necessary. This "mosaic" assay compensates for observed biological, gonadal and seasonal enzyme fluctuations. Cytosols from female rats contain 6--9-times the enzyme activity found in males. The sulfation product with both sexes is corticosterone-21-sulfate. The effects of castration and of androgen administration on hepatic cortisol and corticosterone sulfation were compared in female rats. Ovariectomy resulted in 20--32% and 25--35% decreases of hepatic corticosterone and cortisol sulfotransferase activity, respectively. Androgen administration caused 37--55% and 40--60% decreases of sulfation of the two steroids. The data suggest the equivalence of hepatic cortisol and corticosterone sulfotransferases. Fractionation of cytosols from female rats, on DEAE-Sephadex A-50 columns, resolved three peaks of corticosterone sulfotransferase activity which eluted concurrently with the hepatic cortisol sulfotransferases I, II and III. They appear to be the same enzymes. Cytosol from males contained cortisosterone sulfotransferase activity due mostly to sulfotransferase III. Sulfotransferases I and II appear to have higher turnover numbers for hepatic cortisol than for corticosterone. The reverse is true for sulfotransferase III.     

Detection of specific glycosaminoglycans and glycan epitopes by in vitro sulfation using recombinant sulfotransferases

Sulfated glycans play critical roles during the development, differentiation and growth of various organisms. The most well-studied sulfated molecules are sulfated glycosaminoglycans (GAGs). Recent incidents of heparin drug contamination convey the importance of having a convenient and sensitive method for detecting different GAGs. Here, we describe a molecular method to detect GAGs in biological and biomedical samples. Because the sulfation of GAGs is generally not saturated in vivo, it is possible to introduce the radioisotope (35)S in vitro using recombinant sulfotransferases, thereby allowing detection of minute quantities of these molecules. This strategy was also successfully applied in the detection of other glycans. As examples, we detected contaminant GAGs in commercial heparin, heparan sulfate and chondroitin samples. The identities of the contaminant GAGs were further confirmed by lyase digestion. Oversulfated chondroitin sulfate was detectable only following a simple desulfation step. Additionally, in vitro sulfation by sulfotransferases allowed us to map glycan epitopes in biological samples. This was illustrated using mouse embryo and rat organ tissue sections labeled with the following carbohydrate sulfotransferases: CHST3, CHST15, HS3ST1, CHST4 and CHST10.     

Updated perspectives on the cytosolic sulfotransferases (SULTs) and SULT-mediated sulfation

The cytosolic sulfotransferases (SULTs) are Phase II detoxifying enzymes that mediate the sulfate conjugation of numerous xenobiotic molecules. While the research on the SULTs has lagged behind the research on Phase I cytochrome P-450 enzymes and other Phase II conjugating enzymes, it has gained more momentum in recent years. This review aims to summarize information obtained in several fronts of the research on the SULTs, including the range of the SULTs in different life forms, concerted actions of the SULTs and other Phase II enzymes, insights into the structure–function relationships of the SULTs, regulation of SULT expression and activity, developmental expression of SULTs, as well as the use of a zebrafish model for studying the developmental pharmacology/toxicology.     

CXCR3 requires tyrosine sulfation for ligand binding and a second extracellular loop arginine residue for ligand-induced chemotaxis

CXCR3 is a G-protein-coupled seven-transmembrane domain chemokine receptor that plays an important role in effector T-cell and NK cell trafficking. Three gamma interferon-inducible chemokines activate CXCR3: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11 (I-TAC). Here, we identify extracellular domains of CXCR3 that are required for ligand binding and activation. We found that CXCR3 is sulfated on its N terminus and that sulfation is required for binding and activation by all three ligands. We also found that the proximal 16 amino acid residues of the N terminus are required for CXCL10 and CXCL11 binding and activation but not CXCL9 activation. In addition, we found that residue R216 in the second extracellular loop is required for CXCR3-mediated chemotaxis and calcium mobilization but is not required for ligand binding or ligand-induced CXCR3 internalization. Finally, charged residues in the extracellular loops contribute to the receptor-ligand interaction. These findings demonstrate that chemokine activation of CXCR3 involves both high-affinity ligand-binding interactions with negatively charged residues in the extracellular domains of CXCR3 and a lower-affinity receptor-activating interaction in the second extracellular loop. This lower-affinity interaction is necessary to induce chemotaxis but not ligand-induced CXCR3 internalization, further suggesting that different domains of CXCR3 mediate distinct functions.     

肝素硫酸转移酶优化表达及其在动物源肝素硫酸化中的应用

肝素是一种重要的凝血药物,目前主要依赖于动物小肠粘膜的提取。动物源肝素含有的抗凝血活性五糖单位GlcNS6S-GlcA-GlcNS6S3S-Ido2S-GlcNS6S少,抗凝血活性低下。文中提出并验证了一种基于酶法催化动物源肝素,提高其硫酸化程度和抗凝血活性的方法。通过比较3种硫酸转移酶肝素-2-硫酸转移酶(Heparan sulfate-2-O-sulfotransferase,HS2ST)、肝素-6-硫酸转移酶(Heparan sulfate-6-O-sulfotransferase,HS6ST)、肝素-3-硫酸转移酶(Heparan sulfate-3-O-sulfotransferase,HS3ST)在重组大肠杆菌及重组毕赤酵母中表达,确定了毕赤酵母作为3种硫酸转移酶的表达宿主;进一步通过N端融合麦芽糖融合蛋白MBP和硫氧还蛋白Trx A,HS2ST和HS6ST的酶表达水平分别提高至(839?14) U/L和(792?23) U/L。通过3种硫酸转移酶HS2ST、HS6ST和HS3ST共同催化动物源肝素,其抗凝血活性由(76?2) IU/mg提高至(189?17) IU/mg。     

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.     

Intracellular transport and tyrosine sulfation of procollagens V

Several tyrosine residues of the extracellular p-collagens V and collagens V are sulfated [Fessler, L. I., Brosh, S., Chapin, S. and Fessler, J. H. (1986) J. Biol. Chem. 261, 5034-5040]. Here, the sulfation of their intracellular precursors, the procollagens V, was studied. A Golgi-enriched subcellular fraction of chick embryo tendon catalyzed the sulfation of tyrosine residues in both endogenous and added, unsulfated procollagens V with the sulfate donor 3'-phosphoadenosine 5'-[35S]phosphosulfate. Intracellular tyrosine sulfation of procollagen V occurred at a point distal to the cis Golgi compartment as judged by change of the N-linked carbohydrate of procollagen V from being endoglycosidase-H-sensitive to being resistant. The time course of the intracellular modifications of procollagen V was determined by incubating tendons with 3H-labeled amino acids and with [35S]sulfate. The pro alpha(V) chains were synthesised in about 10 min and then assembled into unsulfated procollagen V molecules. Tyrosine sulfation occurred 50 min after completion of polypeptide synthesis and the molecules were successively sulfated in the order in which they had been synthesized. The antimicrotubular drug Nocodazole, which disrupts the spatial organization of the Golgi, decreased the time interval between synthesis of procollagens V and sulfation. The sulfated procollagens V were soon secreted and cut to sulfated p-collagens V. Sulfated pro alpha 1(V) chains were cleaved faster than sulfated pro alpha 1'(V) chains. The relationship of sequential protein modification to spatial cellular organization is discussed.     

Human anti-CXCR4 antibodies undergo VH replacement, exhibit functional V-region sulfation, and define CXCR4 antigenic heterogeneity

The chemokine receptor CXCR4 and its ligand stromal-derived factor-1 (SDF-1/CXCL12) are essential for many biological processes and various pathological conditions. However, the relationship between CXCR4 antigenic structure and SDF-1-mediated biological responses is poorly understood. In this report, a panel of human anti-CXCR4 Abs were isolated and used to explore CXCR4 antigenic heterogeneity and function. Multiple fixed CXCR4 antigenic isoforms were detected on the surface of hemopoietic cells. Epitope mapping studies demonstrated the complex nature of the surface-exposed CXCR4 epitopes. Ab-mediated inhibition of chemotaxis correlated strongly with binding affinity, epitope recognition, as well as the level of CXCR4 isoform expression. In addition, detailed genetic analyses of these Abs showed evidence of V(H) replacement. Importantly, structural and biochemical studies demonstrated tyrosine sulfation in novel regions of the V genes that contributed bidirectionally to the binding activity of the Abs. These data provide the first evidence that functional tyrosine sulfation occurs in self-reactive Abs and suggest a potential new mechanism that may contribute to the pathogenesis of Ab-mediated autoimmune disease. These Abs also provide valuable tools to explore the selective in vivo targeting of CXCR4 isoforms that may be preferentially expressed in certain disease states and involved in steady-state CXCR4-SDF-1 homeostasis.     

A quantitative assay for tyrosine sulfation and tyrosine phosphorylation in peptides.

A method was developed to measure sulfation and phosphorylation of tyrosine in proteins after alkaline hydrolysis, ion-exchange chromatography, reaction with [3H]dinitrofluorobenzene and subsequent thin-layer chromatography. The method allows the detection of 10-20 pmol of modified tyrosine and was applied to determine the content of tyrosine-phosphate and -sulfate in fibrinogens, thyroglobulin, alpha-casein, cytochrome c and glyceraldehyde dehydrogenase.     

Analysis of sequence requirements for protein tyrosine sulfation.

We analyzed sequences surrounding known tyrosine sulfation sites to determine the characteristics that distinguish these sites from those that do not undergo sulfation. Tests evaluated the number and position of acidic, basic, hydrophobic, and small amino acids, as well as disulfide and N-glycosylation (sugar) sites. We determined that composition-based tests that select close to 100% of known tyrosine sulfation sites reject 97% of the non-sulfated tyrosines. The acidic test, by far the most selective, eliminated 95% of the non-sulfated tyrosine residues and none of the sulfated tyrosines. Including the basic, hydrophobic, and disulfide tests increased the elimination rate to 97%. Whereas no position flanking the tyrosine residues had the same amino acid always present, imperfectly conserved amino acids found in some positions will improve the specificity of the tests.     

4-Hydroxytamoxifen sulfation metabolism

Tamoxifen (TAM) is an important chemotherapeutic agent for the treatment of breast cancer. It has also been shown to decrease breast cancer incidence in healthy women at high risk for the disease. The increased risk of endometrial cancer in women has raised concerns in the use of the drug. Tamoxifen has also been shown to be a potent hepatocarcinogen in rats. The oxidative metabolites of TAM include alpha-hydroxytamoxifen (alpha-OH-TAM) and 4-hydroxytamoxifen (4-OH-TAM). The studies on the sulfation of these metabolites are very limited. It has been reported that alpha-OH-TAM is a substrate for rat hydroxysteroid sulfotransferase a (STa). Our studies on the sulfation of 4-OH-TAM demonstrated that 4-hydroxytamoxifen can be sulfated by human liver and human intestinal cytosols. Human phenol-sulfating sulfotransferase and human estrogen sulfotransferase are the major enzymes for the sulfation of 4-OH-TAM. Human dopamine-sulfating sulfotransferase also has sulfation activity for 4-OH-TAM. In contrast, rat liver and intestine cytosols have no detectable sulfation activity for 4-OH-TAM. The results suggest that the alpha-OH-TAM sulfation pathway leads to bioactivation of TAM, and the 4-OH-TAM sulfation pathway leads to detoxification of TAM. This agrees with the fact that TAM is more toxic for rats than for human beings.     

Prediction of tyrosine sulfation with mRMR feature selection and analysis

Protein tyrosine sulfation is a ubiquitous post-translational modification (PTM) of secreted and transmembrane proteins that pass through the Golgi apparatus. In this study, we developed a new method for protein tyrosine sulfation prediction based on a nearest neighbor algorithm with the maximum relevance minimum redundancy (mRMR) method followed by incremental feature selection (IFS). We incorporated features of sequence conservation, residual disorder, and amino acid factor, 229 features in total, to predict tyrosine sulfation sites. From these 229 features, 145 features were selected and deemed as the optimized features for the prediction. The prediction model achieved a prediction accuracy of 90.01% using the optimal 145-feature set. Feature analysis showed that conservation, disorder, and physicochemical/biochemical properties of amino acids all contributed to the sulfation process. Site-specific feature analysis showed that the features derived from its surrounding sites contributed profoundly to sulfation site determination in addition to features derived from the sulfation site itself. The detailed feature analysis in this paper might help understand more of the sulfation mechanism and guide the related experimental validation.     

Emerging patterns of tyrosine sulfation and O-glycosylation cross-talk and co-localization

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Role of tyrosine phosphorylation in ligand-independent sequestration of CXCR4 in human primary monocytes-macrophages

The chemokine stromal cell-derived factor (SDF)-1 and its receptor, CXCR4, play important roles in human immunodeficiency virus type 1 (HIV-1) pathophysiology, leukocyte trafficking, inflammation, hematopoiesis, embryogenesis, angiogenesis, and cancer metastasis. The effects of cytokines on the regulation of CXCR4 function were investigated in human primary monocytes-macrophages. The expression of functional CXCR4 on the cell surface was demonstrated by the detection of ligand-induced Ca(2+) mobilization, chemotaxis, and ligand-induced receptor endocytosis. Surface CXCR4 expression was down-regulated by cytokines interleukin-4 (IL-4), IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF) and up-regulated by IL-10 and transforming growth factor-beta 1. Down-regulation was mediated post-translationally, in the absence of protein degradation, through an endocytotic mechanism. In contrast to SDF-1 alpha-induced CXCR4 endocytosis, cytokine-induced endocytosis of this receptor was independent of actin filament polymerization. GM-CSF increased the expression of G protein-coupled receptor kinase 3 (GRK3), beta-arrestin-1, Pyk2, and focal adhesion kinase (FAK). Cytokine treatment also increased the total and tyrosine-specific phosphorylation of CXCR4 as well as the phosphorylation of FAK on tyrosine 397. It also induced the formation of GRK3.CXCR4 or FAK.CXCR4 complexes. Infection of macrophages by primary R5X4 and X4 isolates of HIV-1 was inhibited by IL-4, IL-13, and GM-CSF, an effect that was associated with down-regulation of surface CXCR4 expression. These data indicate that ligand-dependent and ligand-independent endocytoses of CXCR4 are mediated by different mechanisms. Cytokine-induced endocytosis of chemokine receptors may be of therapeutic value in HIV-1 infection, inflammation, tumor metastasis, and defective hematopoiesis.     

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.     

The Sulfinator: predicting tyrosine sulfation sites in protein sequences

Protein tyrosine sulfation is an important post-translational modification of proteins that go through the secretory pathway. No clear-cut acceptor motif can be defined that allows the prediction of tyrosine sulfation sites in polypeptide chains. The Sulfinator is a software tool that can be used to predict tyrosine sulfation sites in protein sequences with an overall accuracy of 98%. Four different Hidden Markov Models were constructed, each of them specialized to recognize sulfated tyrosine residues depending on their location within the sequence: near the N-terminus, near the C-terminus, in the center of a window with a size of at least 25 amino acids, as well as in windows containing several tyrosine residues. AVAILABILITY: The Sulfinator is accessible at (http://www.expasy.org/tools/sulfinator/). Supplementary information: Sulfinator documentation is accessible at (http://www.expasy.org/tools/sulfinator/sulfinator-doc.html).     

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.