Amperometric determination of total phenolic content in wine by laccase immobilized onto silver nanoparticles/zinc oxide nanoparticles modified gold electrode

Tyrosine sulfation is a ubiquitous posttranslational modification that regulates extracellular protein-protein interactions, intracellular protein transportation modulation, and protein proteolytic process. However, identifying tyrosine sulfation sites remains a challenge due to the lability of sulfation sequences. In this study, we developed a method called PredSulSite that incorporates protein secondary structure, physicochemical properties of amino acids, and residue sequence order information based on support vector machine to predict sulfotyrosine sites. Three types of encoding algorithms-secondary structure, grouped weight, and autocorrelation function-were applied to mine features from tyrosine sulfation proteins. The prediction model with multiple features achieved an accuracy of 92.89% in 10-fold cross-validation. Feature analysis showed that the coil structure, acidic amino acids, and residue interactions around the tyrosine sulfation sites all contributed to the sulfation site determination. The detailed feature analysis in this work can help us to understand the sulfation mechanism and provide guidance for the related experimental validation. PredSulSite is available as a community resource at http://www.bioinfo.ncu.edu.cn/inquiries_PredSulSite.aspx.     

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.     

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.     

Recombinant expression of selectively sulfated proteins in Escherichia coli

Although tyrosine sulfation is a post-translational modification widespread across multicellular eukaryotes, its biological functions remain largely unknown. This is in part due to the difficulties of synthesizing selectively sulfated proteins. Here we report the selective incorporation of sulfotyrosine into proteins in bacteria by genetically encoding the modified amino acid in response to the amber nonsense codon TAG. Moreover, we show that this strategy enables direct expression in Escherichia coli of sulfo-hirudin, previously inaccessible through recombinant methods. The affinity of sulfo-hirudin toward human thrombin is enhanced more than tenfold over that of desulfo-hirudin, suggesting that sulfo-hirudin may offer clinical advantages for use as an anticoagulant. This general approach to the biosynthesis of sulfated proteins should facilitate further study and application of tyrosine sulfation.     

Is N-sulfation just a gateway modification during heparan sulfate biosynthesis?

Several biologically important growth factor-heparan sulfate (HS) interactions are regulated by HS sulfation patterns. However, the biogenesis of these combinatorial sulfation patterns is largely unknown. N-Deacetylase/N-sulfotrasferase (NDST) converts N-acetyl-d-glucosamine residues to N-sulfo-d-glucosamine residues. This enzyme is suggested to be a gateway enzyme because N-sulfation dictates the final HS sulfation pattern. It is known that O-sulfation blocks C5-epimerase, which acts immediately after NDST action. However, it is still unknown whether O-sulfation inhibits NDST action in a similar manner. In this article we radically change conventional assumptions regarding HS biosynthesis by providing in vitro evidence that N-sulfation is not necessarily just a gateway modification during HS biosynthesis.     

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.     

A mutation in Tpst2 encoding tyrosylprotein sulfotransferase causes dwarfism associated with hypothyroidism

The growth-retarded (grt) mouse has an autosomal recessive, fetal-onset, severe thyroid hypoplasia related to TSH hyporesponsiveness. Through genetic mapping and complementation experiments, we show that grt is a missense mutation of a highly conserved region of the tyrosylprotein sulfotransferase 2 (Tpst2) gene, encoding one of the two Tpst genes implicated in posttranslational tyrosine O-sulfation. We present evidence that the grt mutation leads to a loss of TPST2 activity, and TPST2 isoform has a high degree of substrate preference for TSH receptor (TSHR). The expression of TPST2 can restore TSH-TSHR-mediated cAMP production in fibroblasts derived from grt mice. Therefore, we propose that the tyrosine sulfation of TSHR by TPST2 is crucial for TSH signaling and resultant thyroid gland function.     

Heterogeneity in the tyrosine sulfation of Chinese hamster ovary cell produced recombinant FVIII

By the use of recombinant technology, several stable Chinese hamster ovary (CHO) cell lines expressing human FVIII were established. Thrombin treatment and SDS-PAGE analysis of the purified recombinant FVIII (rFVIII) revealed a striking difference from plasma-derived FVIII (pFVIII). A 43-kDa fragment of the FVIII heavy chain appears as a double band from rFVIII, while a single band from pFVIII is observed. All other fragments from the two samples appeared similar by SDS-PAGE. The heterogeneity is caused by incomplete tyrosine sulfation of one or more of the three potential tyrosine sulfation sites (Tyr718, Tyr719, Tyr723). To investigate if there is a general limitation and heterogeneity in the tyrosine sulfation of rFVIII, two other potential tyrosine sulfation sites on the FVIII light chain (Tyr1664, Tyr1680) were analyzed. The results show that both sites on the pFVIII light chain and on the rFVIII light chain are completely sulfated. The limitation of CHO cells to tyrosine sulfate rFVIII is therefore only restricted to a few sites. The two sulfated forms of rFVIII can easily be separated by ion-exchange chromatography, indicating the importance of the sulfate groups on the charge and/or conformation of FVIII. Both forms of rFVIII possess identical in vitro coagulation activity, von Willebrand factor binding, and thrombin activation profile. However, the difference in tyrosine sulfation may change other biological properties of FVIII.     

Tyrosine polysulfation of human salivary histatin 1. A post-translational modification specific of the submandibular gland

Histatin 1 (His-1) derivatives showing serial mass increases of 80.0 +/- 0.1 Da were detected in human saliva by HPLC-ESI-MS. The same derivatives were also found in granules of submandibular glands and secretions of submandibular/sublingual glands, but not in granules and secretions of parotid glands. Only one phosphate group was present in His-1 and its derivatives, since treatment with alkaline phosphatase provided an 80.0 Da mass decrease. His-1 derivatives were almost completely transformed into His-1 by treatment with 1 M HCl at 100 degrees C, suggesting the presence of O-sulfotyrosine, which is more labile than phospho-Tyr to acidic hydrolysis. CE-MS analysis of pronase extensive digestion of derivatives confirmed the presence of sulfotyrosine. Derivatives were digested by trypsin, proteinase K, and protease V-8 and analyzed by different MS strategies. The results allowed locating sulfation on the last four tyrosines (Tyr 27, 30, 34, and 36). This study is the first report of the gland-specific sulfation of a salivary phosphopeptide in vivo.     

Recognition of a CXCR4 sulfotyrosine by the chemokine stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12)

Tyrosine sulfation of the chemokine receptor CXCR4 enhances its interaction with the chemokine SDF-1alpha. Given similar post-translational modification of other receptors, including CCR5, CX3CR1 and CCR2b, tyrosine sulfation may be of universal importance in chemokine signaling. N-terminal domains from seven transmembrane chemokine receptors have been employed for structural studies of chemokine-receptor interactions, but never in the context of proper post-translational modifications known to affect function. A CXCR4 peptide modified at position 21 by expressed tyrosylprotein sulfotransferase-1 and unmodified peptide are both disordered in solution, but bind SDF-1alpha with low micromolar affinities. NMR and fluorescence polarization measurements showed that the CXCR4 peptide stabilizes dimeric SDF-1alpha, and that sulfotyrosine 21 binds a specific site on the chemokine that includes arginine 47. We conclude that the SDF-1alpha dimer preferentially interacts with receptor peptide, and residues beyond the extreme N-terminal region of CXCR4, including sulfotyrosine 21, make specific contacts with the chemokine ligand.     

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.     

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.     

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.     

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.     

Identification of two sites of sulfation of human heparin cofactor II

Tyrosine sulfate was identified as a constituent of human heparin cofactor II by analysis of sulfate-labeled protein secreted by a human hepatoma-derived cell line and of purified protein from human plasma. Alkaline hydrolysis of heparin cofactor II released tyrosine sulfate as demonstrated by anion-exchange high performance liquid chromatography of hydrolysates. Two sites of sulfation were identified, and the amino acid sequences of the sites were established by sequential Edman degradation of sulfate-containing tryptic peptides that were isolated by reverse-phase high performance liquid chromatography. Each peptide contains only a single tyrosine residue so that the sites of sulfation can be assigned unambiguously. The two sites of sulfation are separated by 13 residues and represent an internal sequence repeat in the heparin cofactor II molecule. The two sites have the following sequences. Glu56-Asp-Asp-Asp-Tyr(SO4)-Leu-Asp62 Glu69-Asp-Asp-Asp-Tyr(SO4)-Ile-Asp75 Sulfate-labeled heparin cofactor II formed a covalent complex with thrombin in a heparin-dependent manner. Thus, the sulfate-containing form of the protein was shown to be biologically active. The characteristic sulfate-containing segment of heparin cofactor II, which contains 17 acidic amino acid residues over a span of 30 residues, may contribute to the unique properties of this thrombin inhibitor.     

Preparation of tyrosine-O-[35S]sulfated cholecystokinin octapeptide from a nonsulfated precursor peptide

A rapid and simple one-pot method for O-sulfation of nonsulfated cholecystokinin octapeptide (CCK-8) was developed using sulfuric acid and dicyclohexylcarbodiimide (DCC) without protection of the amino acid side chains. The extent of sulfation was increased with increasing the amount of reactants, sulfuric acid, and DCC, and reached maximum (40%) with fourfold molar excess of sulfuric acid and 40-fold molar excess of DCC. The excess of nonsulfated peptide inhibited the sulfation. The sulfation product was purified by HPLC or TLC to give a pure sulfated substance which showed exactly the same behavior as that of an authentic O-sulfated CCK-8 on HPLC or TLC. The purified sulfated peptide was active in stimulating amylase secretion from rat pancreatic fragments, and amino acid analysis showed that the tyrosine residue in the peptide existed in O-sulfated form. Sulfation with [35S]sulfuric acid-DCC produced a radioactive substance, from which O-[35S]sulfated CCK-8 could be easily purified by two-dimensional TLC.     

Lack of tyrosylprotein sulfotransferase-2 activity results in altered sperm-egg interactions and loss of ADAM3 and ADAM6 in epididymal sperm

Tyrosine O-sulfation is a post-translational modification catalyzed by two tyrosylprotein sulfotransferases (TPST-1 and TPST-2) in the trans-Golgi network. Tpst2-deficient mice have male infertility, sperm motility defects, and possible abnormalities in sperm-egg membrane interactions. Studies here show that compared with wild-type sperm, fewer Tpst2-null sperm bind to the egg membrane, but more of these bound sperm progress to membrane fusion. Similar outcomes were observed with wild-type sperm treated with the anti-sulfotyrosine antibody PSG2. The increased extent of sperm-egg fusion is not due to a failure of Tpst2-null sperm to trigger establishment of the egg membrane block to polyspermy. Anti-sulfotyrosine staining of sperm showed localization similar to that of IZUMO1, a sperm protein that is essential for gamete fusion, but we detected little to no tyrosine sulfation of IZUMO1 and found that IZUMO1 expression and localization were normal in Tpst2-null sperm. Turning to a discovery-driven approach, we used mass spectrometry to characterize sperm proteins that associated with PSG2. This identified ADAM6, a member of the A disintegrin and A metalloprotease (ADAM) family; members of this protein family are associated with multiple sperm functions. Subsequent studies revealed that Tpst2-null sperm lack ADAM6 and ADAM3. Loss of ADAM3 is strongly associated with male infertility and is observed in knockouts of male germ line-specific endoplasmic reticulum-resident chaperones, raising the possibility that TPST-2 may function in quality control in the secretory pathway. These data suggest that TPST-2-mediated tyrosine O-sulfation participates in regulating the sperm surface proteome or membrane order, ultimately affecting male fertility.     

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.