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Martin J Stone - One of the best experts on this subject based on the ideXlab platform.
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semisynthesis of an evasin from tick saliva reveals a critical role of Tyrosine Sulfation for chemokine binding and inhibition
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Charlotte Franck, Martin J Stone, Simon R Foster, Jason Johansenleete, Sayeeda Tasneem Chowdhury, Michelle Cielesh, Ram Prasad Bhusal, Joel P Mackay, Mark Larance, Richard J PayneAbstract:Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the Amblyomma cajennense tick can be posttranslationally sulfated at two Tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that Tyrosine Sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.
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homogeneous sulfopeptides and sulfoproteins synthetic approaches and applications to characterize the effects of Tyrosine Sulfation on biochemical function
Accounts of Chemical Research, 2015Co-Authors: Martin J Stone, Richard J PayneAbstract:ConspectusPost-translational modification of proteins plays critical roles in regulating structure, stability, localization, and function. Sulfation of the phenolic side chain of Tyrosine residues to form sulfoTyrosine (sTyr) is a widespread modification of extracellular and integral membrane proteins, influencing the activities of these proteins in cellular adhesion, blood clotting, inflammatory responses, and pathogen infection. Tyrosine Sulfation commonly occurs in sequences containing clusters of Tyrosine residues and is incomplete at each site, resulting in heterogeneous mixtures of sulfoforms. Purification of individual sulfoforms is typically impractical. Therefore, the most promising approach to elucidate the influence of Sulfation at each site is to prepare homogeneously sulfated proteins (or peptides) synthetically. This Account describes our recent progress in both development of such synthetic approaches and application of the resulting sulfopeptides and sulfoproteins to characterize the funct...
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phosphate modulates receptor sulfoTyrosine recognition by the chemokine monocyte chemoattractant protein 1 mcp 1 ccl2
Organic and Biomolecular Chemistry, 2015Co-Authors: Justin P Ludeman, Richard J Payne, Brendan L Wilkinson, Mahdieh Nazarirobati, Cheng Huang, Martin J StoneAbstract:Tyrosine Sulfation is a widespread post-translational modification that mediates the interactions of secreted and membrane-associated proteins in such varied biological processes as peptide hormone action, adhesion, blood coagulation, complement activation and regulation of leukocyte trafficking. Due to the heterogeneous nature of Tyrosine Sulfation, detailed biochemical and biophysical studies of Tyrosine Sulfation rely on homogenous, synthetic sulfopeptides. Here we describe the synthesis of a fluorescent sulfopeptide (FL-R2D) derived from the chemokine receptor CCR2 and the application of FL-R2D in direct and competitive fluorescence anisotropy assays that enable the efficient measurement of binding affinities between sulfopeptides and their binding proteins. Using these assays, we have found that the binding of the chemokine monocyte chemoattractant protein-1 (MCP-1) to sulfated peptides derived from the chemokine receptor CCR2 is highly dependent on the assay buffer. In particular, phosphate buffer at close to physiological concentrations competes with the receptor sulfopeptide by binding to the sulfopeptide binding pocket on the chemokine surface. Thus, physiological phosphate may modulate the receptor binding selectivity of chemokines.
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the structural role of receptor Tyrosine Sulfation in chemokine recognition
British Journal of Pharmacology, 2014Co-Authors: Justin P Ludeman, Martin J StoneAbstract:Tyrosine Sulfation is a post-translational modification of secreted and transmembrane proteins, including many GPCRs such as chemokine receptors. Most chemokine receptors contain several potentially sulfated Tyrosine residues in their extracellular N-terminal regions, the initial binding site for chemokine ligands. Sulfation of these receptors increases chemokine binding affinity and potency. Although receptor Sulfation is heterogeneous, insights into the molecular basis of sulfoTyrosine (sTyr) recognition have been obtained using purified, homogeneous sulfopeptides corresponding to the N-termini of chemokine receptors. Receptor sTyr residues bind to a shallow cleft defined by the N-loop and β3-strand elements of cognate chemokines. Tyrosine Sulfation enhances the affinity of receptor peptides for cognate chemokines in a manner dependent on the position of Sulfation. Moreover, Tyrosine Sulfation can alter the selectivity of receptor peptides among several cognate chemokines for the same receptor. Finally, binding to receptor sulfopeptides can modulate the oligomerization state of chemokines, thereby influencing the ability of a chemokine to activate its receptor. These results increase the motivation to investigate the structural basis by which Tyrosine Sulfation modulates chemokine receptor activity and the biological consequences of this functional modulation. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5
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Tyrosine Sulfation of chemokine receptor ccr2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein 1 mcp 1
Journal of Biological Chemistry, 2013Co-Authors: Justin P Ludeman, Richard J Payne, Deni Taleski, Jamie Wedderburn, Meritxell Canals, Pam Hall, Stephen J Butler, Arthur Christopoulos, Michael J Hickey, Martin J StoneAbstract:Abstract Chemokine receptors are commonly post-translationally sulfated on Tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of Tyrosine Sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 Sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1 (T10C) was less effective at inducing leukocyte recruitment in vivo. In 2D NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favour the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N-terminus of CCR2 destabilises the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.
Richard J Payne - One of the best experts on this subject based on the ideXlab platform.
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chemical synthesis of a haemathrin sulfoprotein library reveals enhanced thrombin inhibition following Tyrosine Sulfation
RSC Chemical Biology, 2020Co-Authors: Daniel Clayton, Jorge Ripollrozada, Pedro Pereira, Sameer S Kulkarni, Jessica Sayers, Luke J Dowman, Richard J PayneAbstract:The haemathrins are tick-derived thrombin-inhibiting proteins predicted to be post-translationally sulfated. This study reports the ligation-based assembly of eight homogeneously sulfated variants of haemathrin-1 and haemathrin-2. Functional assays revealed a two orders-of-magnitude enhancement in thrombin-inhibitory potency by Tyrosine Sulfation, thus reinforcing the crucial role of this post-translational modification for the activity of anticoagulant proteins.
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revealing the functional roles of Tyrosine Sulfation using synthetic sulfopeptides and sulfoproteins
Current Opinion in Chemical Biology, 2020Co-Authors: Joshua W C Maxwell, Richard J PayneAbstract:Abstract The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine Sulfation is a PTM that has been shown to be a key regulator of extracellular protein–protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of Tyrosine Sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation. In this account, we highlight recent advances in the prediction and detection of Tyrosine Sulfation and outline the need for continued innovation in this area. We also discuss the utility of emerging solid-phase synthesis and peptide ligation strategies for accessing libraries of homogeneously sulfated peptides and proteins to help reveal functional aspects of the sulfoproteome.
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semisynthesis of an evasin from tick saliva reveals a critical role of Tyrosine Sulfation for chemokine binding and inhibition
Proceedings of the National Academy of Sciences of the United States of America, 2020Co-Authors: Charlotte Franck, Martin J Stone, Simon R Foster, Jason Johansenleete, Sayeeda Tasneem Chowdhury, Michelle Cielesh, Ram Prasad Bhusal, Joel P Mackay, Mark Larance, Richard J PayneAbstract:Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the Amblyomma cajennense tick can be posttranslationally sulfated at two Tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that Tyrosine Sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.
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Tyrosine Sulfation modulates activity of tick derived thrombin inhibitors
Nature Chemistry, 2017Co-Authors: Robert E Thompson, Xuyu Liu, Jorge Ripollrozada, Noelia Alonsogarcia, Benjamin L Parker, Pedro Pereira, Richard J PayneAbstract:Madanin-1 and chimadanin are two small cysteine-free thrombin inhibitors that facilitate blood feeding in the tick Haemaphysalis longicornis. Here, we report a post-translational modification-Tyrosine Sulfation-of these two proteins that is critical for potent anti-thrombotic and anticoagulant activity. Inhibitors produced in baculovirus-infected insect cells displayed heterogeneous Sulfation of two Tyrosine residues within each of the proteins. One-pot ligation-desulfurization chemistry enabled access to homogeneous samples of all possible sulfated variants of the proteins. Tyrosine Sulfation of madanin-1 and chimadanin proved crucial for thrombin inhibitory activity, with the doubly sulfated variants three orders of magnitude more potent than the unmodified inhibitors. The three-dimensional structure of madanin-1 in complex with thrombin revealed a unique mode of inhibition, with the sulfated Tyrosine residues binding to the basic exosite II of the protease. The importance of Tyrosine Sulfation within this family of thrombin inhibitors, together with their unique binding mode, paves the way for the development of anti-thrombotic drug leads based on these privileged scaffolds.
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homogeneous sulfopeptides and sulfoproteins synthetic approaches and applications to characterize the effects of Tyrosine Sulfation on biochemical function
Accounts of Chemical Research, 2015Co-Authors: Martin J Stone, Richard J PayneAbstract:ConspectusPost-translational modification of proteins plays critical roles in regulating structure, stability, localization, and function. Sulfation of the phenolic side chain of Tyrosine residues to form sulfoTyrosine (sTyr) is a widespread modification of extracellular and integral membrane proteins, influencing the activities of these proteins in cellular adhesion, blood clotting, inflammatory responses, and pathogen infection. Tyrosine Sulfation commonly occurs in sequences containing clusters of Tyrosine residues and is incomplete at each site, resulting in heterogeneous mixtures of sulfoforms. Purification of individual sulfoforms is typically impractical. Therefore, the most promising approach to elucidate the influence of Sulfation at each site is to prepare homogeneously sulfated proteins (or peptides) synthetically. This Account describes our recent progress in both development of such synthetic approaches and application of the resulting sulfopeptides and sulfoproteins to characterize the funct...
Kevin L Moore - One of the best experts on this subject based on the ideXlab platform.
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direct identification of Tyrosine Sulfation by using ultraviolet photodissociation mass spectrometry
Journal of the American Society for Mass Spectrometry, 2014Co-Authors: Michelle R Robinson, Kevin L Moore, Jennifer S BrodbeltAbstract:Sulfation is a common post-translational modification of Tyrosine residues in eukaryotes; however, detection using traditional liquid chromatography-mass spectrometry (LC-MS) methods is challenging based on poor ionization efficiency in the positive ion mode and facile neutral loss upon collisional activation. In the present study, 193 nm ultraviolet photodissociation (UVPD) is applied to sulfopeptide anions to generate diagnostic sequence ions, which do not undergo appreciable neutral loss of sulfate even using higher energy photoirradiation parameters. At the same time, neutral loss of SO3 is observed from the precursor and charge-reduced precursor ions, a spectral feature that is useful for differentiating Tyrosine Sulfation from the nominally isobaric Tyrosine phosphorylation. LC-MS detection limits for UVPD analysis in the negative mode were determined to be around 100 fmol for three sulfated peptides, caerulein, cionin, and leu-enkephalin. The LC-UVPD-MS method was applied for analysis of bovine fibrinogen, and its key sulfated peptide was confidently identified.
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Tyrosine Sulfation of native mouse psgl 1 is required for optimal leukocyte rolling on p selectin in vivo
PLOS ONE, 2011Co-Authors: Andrew D Westmuckett, Kelly M Thacker, Kevin L MooreAbstract: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.
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lack of protein Tyrosine Sulfation disrupts photoreceptor outer segment morphogenesis retinal function and retinal anatomy
European Journal of Neuroscience, 2010Co-Authors: David M Sherry, Anne Murray, Yogita Kanan, Kelsey L Arbogast, Robert A Hamilton, Marie E Burns, Steven J. Fliesler, Kevin L Moore, Muayyad R AlubaidiAbstract:To investigate the role(s) of protein-Tyrosine Sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1 -/- /Tpst2 -/- ) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein-Tyrosine Sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.
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protein Tyrosine Sulfation a critical posttranslation modification in plants and animals
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Kevin L MooreAbstract:Protein Tyrosine Sulfation is a posttranslational modification restricted to proteins that transit the secretory pathway that was first described by Bettelheim in bovine fibrinopeptide B in 1954 (1). Subsequent pioneering work by Wieland Huttner's group (2, 3) and others characterized the enzyme activity responsible for the reaction, called tyrosylprotein sulfotransferase (TPST), that catalyzes the transfer of sulfate from 3′-phosphoadenosine 5′-phosphosulfate to the hydroxyl group of peptidyl Tyrosine residues to form a Tyrosine O4-sulfate ester. The enzymes' subcellular localization in the trans-Golgi network and its widespread tissue and cellular distribution have been well documented in animals, and several dozen Tyrosine-sulfated proteins, mostly of animal origin, have been described, many of which play important roles in inflammation, hemostasis, immunity, and other processes (2–4). Finally, the general importance of protein Tyrosine Sulfation in protein–protein interactions has become widely accepted.
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detection and purification of Tyrosine sulfated proteins using a novel anti sulfoTyrosine monoclonal antibody
Journal of Biological Chemistry, 2006Co-Authors: Adam J Hoffhines, Eugen Damoc, Kristie Grove Bridges, Julie A Leary, Kevin L MooreAbstract:Abstract Protein Tyrosine O-Sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST1 and TPST2) that catalyze the transfer of sulfate to Tyrosine residues in secreted and transmembrane proteins. Tyrosine Sulfation plays a role in protein-protein interactions in several well defined systems. Although dozens of Tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of Tyrosine Sulfation in biological systems requires the development of new tools for the detection and study of Tyrosine-sulfated proteins. We have developed a novel anti-sulfoTyrosine monoclonal antibody (called PSG2) that binds with high affinity and exquisite specificity to sulfoTyrosine residues in peptides and proteins independently of sequence context. We show that it can detect Tyrosine-sulfated proteins in complex biological samples and can be used as a probe to assess the role of Tyrosine Sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of Tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 showed that certain sperm/epididymal proteins are undersulfated in Tpst2-/- mice. This indicates that TPST1 and TPST2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2-/- males. PSG2 should be widely applicable for identification of Tyrosine-sulfated proteins in other systems and organisms.
Justin P Ludeman - One of the best experts on this subject based on the ideXlab platform.
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phosphate modulates receptor sulfoTyrosine recognition by the chemokine monocyte chemoattractant protein 1 mcp 1 ccl2
Organic and Biomolecular Chemistry, 2015Co-Authors: Justin P Ludeman, Richard J Payne, Brendan L Wilkinson, Mahdieh Nazarirobati, Cheng Huang, Martin J StoneAbstract:Tyrosine Sulfation is a widespread post-translational modification that mediates the interactions of secreted and membrane-associated proteins in such varied biological processes as peptide hormone action, adhesion, blood coagulation, complement activation and regulation of leukocyte trafficking. Due to the heterogeneous nature of Tyrosine Sulfation, detailed biochemical and biophysical studies of Tyrosine Sulfation rely on homogenous, synthetic sulfopeptides. Here we describe the synthesis of a fluorescent sulfopeptide (FL-R2D) derived from the chemokine receptor CCR2 and the application of FL-R2D in direct and competitive fluorescence anisotropy assays that enable the efficient measurement of binding affinities between sulfopeptides and their binding proteins. Using these assays, we have found that the binding of the chemokine monocyte chemoattractant protein-1 (MCP-1) to sulfated peptides derived from the chemokine receptor CCR2 is highly dependent on the assay buffer. In particular, phosphate buffer at close to physiological concentrations competes with the receptor sulfopeptide by binding to the sulfopeptide binding pocket on the chemokine surface. Thus, physiological phosphate may modulate the receptor binding selectivity of chemokines.
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the structural role of receptor Tyrosine Sulfation in chemokine recognition
British Journal of Pharmacology, 2014Co-Authors: Justin P Ludeman, Martin J StoneAbstract:Tyrosine Sulfation is a post-translational modification of secreted and transmembrane proteins, including many GPCRs such as chemokine receptors. Most chemokine receptors contain several potentially sulfated Tyrosine residues in their extracellular N-terminal regions, the initial binding site for chemokine ligands. Sulfation of these receptors increases chemokine binding affinity and potency. Although receptor Sulfation is heterogeneous, insights into the molecular basis of sulfoTyrosine (sTyr) recognition have been obtained using purified, homogeneous sulfopeptides corresponding to the N-termini of chemokine receptors. Receptor sTyr residues bind to a shallow cleft defined by the N-loop and β3-strand elements of cognate chemokines. Tyrosine Sulfation enhances the affinity of receptor peptides for cognate chemokines in a manner dependent on the position of Sulfation. Moreover, Tyrosine Sulfation can alter the selectivity of receptor peptides among several cognate chemokines for the same receptor. Finally, binding to receptor sulfopeptides can modulate the oligomerization state of chemokines, thereby influencing the ability of a chemokine to activate its receptor. These results increase the motivation to investigate the structural basis by which Tyrosine Sulfation modulates chemokine receptor activity and the biological consequences of this functional modulation. Linked ArticlesThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5
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Tyrosine Sulfation of chemokine receptor ccr2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein 1 mcp 1
Journal of Biological Chemistry, 2013Co-Authors: Joshua H Y Tan, Justin P Ludeman, Deni Taleski, Jamie Wedderburn, Meritxell Canals, Pam Hall, Stephen J Butler, Arthur Christopoulos, Michael J Hickey, Richard J PayneAbstract:Chemokine receptors are commonly post-translationally sulfated on Tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of Tyrosine Sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 Sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1(T10C) was less effective at inducing leukocyte recruitment in vivo. In two-dimensional NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favor the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N terminus of CCR2 destabilizes the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.
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Tyrosine Sulfation of chemokine receptor ccr2 enhances interactions with both monomeric and dimeric forms of the chemokine monocyte chemoattractant protein 1 mcp 1
Journal of Biological Chemistry, 2013Co-Authors: Justin P Ludeman, Richard J Payne, Deni Taleski, Jamie Wedderburn, Meritxell Canals, Pam Hall, Stephen J Butler, Arthur Christopoulos, Michael J Hickey, Martin J StoneAbstract:Abstract Chemokine receptors are commonly post-translationally sulfated on Tyrosine residues in their N-terminal regions, the initial site of binding to chemokine ligands. We have investigated the effect of Tyrosine Sulfation of the chemokine receptor CCR2 on its interactions with the chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2). Inhibition of CCR2 Sulfation, by growth of expressing cells in the presence of sodium chlorate, significantly reduced the potency for MCP-1 activation of CCR2. MCP-1 exists in equilibrium between monomeric and dimeric forms. The obligate monomeric mutant MCP-1(P8A) was similar to wild type MCP-1 in its ability to induce leukocyte recruitment in vivo, whereas the obligate dimeric mutant MCP-1 (T10C) was less effective at inducing leukocyte recruitment in vivo. In 2D NMR experiments, sulfated peptides derived from the N-terminal region of CCR2 bound to both the monomeric and dimeric forms of wild type MCP-1 and shifted the equilibrium to favour the monomeric form. Similarly, MCP-1(P8A) bound more tightly than MCP-1(T10C) to the CCR2-derived sulfopeptides. NMR chemical shift mapping using the MCP-1 mutants showed that the sulfated N-terminal region of CCR2 binds to the same region (N-loop and β3-strand) of both monomeric and dimeric MCP-1 but that binding to the dimeric form also influences the environment of chemokine N-terminal residues, which are involved in dimer formation. We conclude that interaction with the sulfated N-terminus of CCR2 destabilises the dimerization interface of inactive dimeric MCP-1, thus inducing dissociation to the active monomeric state.
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Tyrosine Sulfation influences the chemokine binding selectivity of peptides derived from chemokine receptor CCR3.
Biochemistry, 2011Co-Authors: John Z. Zhu, Justin P Ludeman, Christopher J Millard, Richard J Payne, Levi S. Simpson, Daniel Clayton, Theodore S. Widlanski, Martin J StoneAbstract:The interactions of chemokines with their G protein-coupled receptors play critical roles in the control of leukocyte trafficking in normal homeostasis and in inflammatory responses. Tyrosine Sulfation is a common post-translational modification in the amino-terminal regions of chemokine receptors. However, Tyrosine Sulfation of chemokine receptors is commonly incomplete or heterogeneous. To investigate the possibility that differential Sulfation of two adjacent Tyrosine residues could bias the responses of chemokine receptor CCR3 to different chemokines, we have studied the binding of three chemokines (eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26) to an N-terminal CCR3-derived peptide in each of its four possible Sulfation states. Whereas the nonsulfated peptide binds to the three chemokines with approximately equal affinity, Sulfation of Tyr-16 gives rise to 9-16-fold selectivity for eotaxin-1 over the other two chemokines. Subsequent Sulfation of Tyr-17 contributes additively to the affinity for eotaxin-1 and eotaxin-2 but cooperatively to the affinity for eotaxin-3. The doubly sulfated peptide selectively binds to both eotaxin-1 and eotaxin-3 approximately 10-fold more tightly than to eotaxin-2. Nuclear magnetic resonance chemical shift mapping indicates that these variations in affinity probably result from only subtle differences in the chemokine surfaces interacting with these receptor peptides. These data support the proposal that variations in Sulfation states or levels may regulate the responsiveness of chemokine receptors to their cognate chemokines.
Wieland B. Huttner - One of the best experts on this subject based on the ideXlab platform.
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Tyrosine o Sulfation
Current protocols in protein science, 2005Co-Authors: Denis Corbeil, Christoph Thiele, Wieland B. HuttnerAbstract:The O-Sulfation of Tyrosine residues of plasma membrane and secretory proteins that transit through the secretory pathway of eukaryotic cells is a widespread post-translational modification. This enzymatic reaction is catalyzed by trans-Golgi-associated tyrosylprotein sulfotransferases, which recognize Tyrosine residues located in a specific acidic amino acid sequence. Tyrosine Sulfation promotes extracellular protein-protein interactions involved in diverse biological processes, ranging from the receptor binding of regulatory peptides to the interaction of viral envelope proteins with the cell surface. This unit outlines procedures to determine whether a protein of interest contains sulfated Tyrosine residues, using methods based on labeling proteins with inorganic [35S]-sulfate, alkaline hydrolysis, and one-dimensional thin-layer electrophoresis.
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protein Tyrosine Sulfation 1993 an update
Chemico-Biological Interactions, 1994Co-Authors: Christof Niehrs, Roland Beiswanger, Wieland B. HuttnerAbstract:Sulfation is the most abundant post-translational modification of Tyrosine residues and occurs in many soluble and membrane proteins passing through the secretory pathway of metazoan cells. The Sulfation reaction is catalysed by tyrosylprotein sulfotransferase, a membrane-bound enzyme of the trans-Golgi-network. Tyrosylprotein sulfotransferase has been purified and its substrate specificity characterized. Tyrosine Sulfation has been shown to be important for protein-protein interactions occurring during the intracellular transport of proteins and upon their secretion.
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in vivo expression and stoichiometric Sulfation of the artificial protein sulfophilin a polymer of Tyrosine Sulfation sites
Journal of Biological Chemistry, 1992Co-Authors: Christof Niehrs, Wieland B. Huttner, U RutherAbstract: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.
