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Kevin L Moore - One of the best experts on this subject based on the ideXlab platform.
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Tyrosine Sulfation of Native Mouse Psgl-1 Is Required for Optimal Leukocyte Rolling on P-Selectin In Vivo
2016Co-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 (WTRB6) or Tpst1;Tpst2 double knockout mice (Tpst DKORB6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKORB6 venules compared to WTRB6 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 o
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salivary gland hypofunction in Tyrosylprotein Sulfotransferase 2 knockout mice is due to primary hypothyroidism
PLOS ONE, 2013Co-Authors: Andrew D Westmuckett, Joseph C Siefert, Yasvir A Tesiram, David M Pinson, Kevin L MooreAbstract:Background Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi Tyrosylprotein Sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice.
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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 Tyrosylprotein Sulfotransferase 2 activity results in altered sperm egg interactions and loss of adam3 and adam6 in epididymal sperm
Journal of Biological Chemistry, 2011Co-Authors: Matthew R Marcello, Kevin L Moore, Julie A Leary, Weitao Jia, Janice Perry EvansAbstract: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.
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catalytic mechanism of golgi resident human Tyrosylprotein Sulfotransferase 2 a mass spectrometry approach
Journal of the American Society for Mass Spectrometry, 2010Co-Authors: Lieza M Danan, Kevin L Moore, Peter J Ludden, Weitao Jia, Julie A LearyAbstract:Human Tyrosylprotein Sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network, affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this study, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites, and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident Sulfotransferase.
Andrew D Westmuckett - One of the best experts on this subject based on the ideXlab platform.
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Tyrosine Sulfation of Native Mouse Psgl-1 Is Required for Optimal Leukocyte Rolling on P-Selectin In Vivo
2016Co-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 (WTRB6) or Tpst1;Tpst2 double knockout mice (Tpst DKORB6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKORB6 venules compared to WTRB6 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 o
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salivary gland hypofunction in Tyrosylprotein Sulfotransferase 2 knockout mice is due to primary hypothyroidism
PLOS ONE, 2013Co-Authors: Andrew D Westmuckett, Joseph C Siefert, Yasvir A Tesiram, David M Pinson, Kevin L MooreAbstract:Background Protein-tyrosine sulfation is a post-translational modification of an unknown number of secreted and membrane proteins mediated by two known Golgi Tyrosylprotein Sulfotransferases (TPST-1 and TPST-2). We reported that Tpst2-/- mice have mild-moderate primary hypothyroidism, whereas Tpst1-/- mice are euthyroid. While using magnetic resonance imaging (MRI) to look at the thyroid gland we noticed that the salivary glands in Tpst2-/- mice appeared smaller than in wild type mice. This prompted a detailed analysis to compare salivary gland structure and function in wild type, Tpst1-/-, and Tpst2 -/- mice.
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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 Tyrosylprotein Sulfotransferase activity in hematopoietic cells drastically attenuates atherosclerosis in ldlr mice
Arteriosclerosis Thrombosis and Vascular Biology, 2009Co-Authors: Andrew D Westmuckett, Kevin L MooreAbstract:Objective— Leukocyte recruitment is a major contributor in the development of atherosclerosis and requires a variety of proteins such as adhesion molecules, chemokines, and chemokine receptors. Several key molecular players implicated in this process are expressed on monocytes and require protein-tyrosine sulfation for optimal function in vitro, including human CCR2, CCR5, CX3CR1, and PSGL-1. We therefore hypothesized that protein-tyrosine sulfation in hematopoietic cells plays an important role in the development of atherosclerosis. Methods and Results— Lethally-irradiated Ldlr −/− mice were rescued with hematopoietic progenitors lacking Tyrosylprotein Sulfotransferase (TPST) activity attributable to deletion of the Tpst1 and Tpst2 genes. TPST deficient progenitors efficiently reconstituted hematopoiesis in Ldlr −/− recipients and transplantation had no effect on plasma lipids on a standard or atherogenic diet. However, we observed a substantial reduction in the size of atherosclerotic lesions and the number of macrophages in lesions from hyperlipidemic Ldlr −/− recipients transplanted with TPST deficient progenitors compared to wild-type progenitors. We also document for the first time that murine Psgl-1 and Cx3cr1 are tyrosine-sulfated. Conclusions— These data demonstrate that protein-tyrosine sulfation is an important contributor to monocytes/macrophage recruitment and/or retention in a mouse model of atherosclerosis.
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integrative physiology experimental medicine lack of Tyrosylprotein Sulfotransferase activity in hematopoietic cells drastically attenuates atherosclerosis in ldlr mice
2009Co-Authors: Andrew D Westmuckett, Kevin L MooreAbstract:Objective—Leukocyte recruitment is a major contributor in the development of atherosclerosis and requires a variety of proteins such as adhesion molecules, chemokines, and chemokine receptors. Several key molecular players implicated in this process are expressed on monocytes and require protein-tyrosine sulfation for optimal function in vitro, including human CCR2, CCR5, CX3CR1, and PSGL-1. We therefore hypothesized that protein-tyrosine sulfation in hematopoietic cells plays an important role in the development of atherosclerosis. Methods and Results—Lethally-irradiated Ldlr / mice were rescued with hematopoietic progenitors lacking Tyrosylprotein Sulfotransferase (TPST) activity attributable to deletion of the Tpst1 and Tpst2 genes. TPST deficient progenitors efficiently reconstituted hematopoiesis in Ldlr / recipients and transplantation had no effect on plasma lipids on a standard or atherogenic diet. However, we observed a substantial reduction in the size of atherosclerotic lesions and the number of macrophages in lesions from hyperlipidemic Ldlr / recipients transplanted with TPST deficient progenitors compared to wild-type progenitors. We also document for the first time that murine Psgl-1 and Cx3cr1 are tyrosine-sulfated. Conclusions—These data demonstrate that protein-tyrosine sulfation is an important contributor to monocytes/macrophage recruitment and/or retention in a mouse model of atherosclerosis. (Arterioscler Thromb Vasc Biol. 2009;29:1730-1736.)
Julie A Leary - One of the best experts on this subject based on the ideXlab platform.
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lack of Tyrosylprotein Sulfotransferase 2 activity results in altered sperm egg interactions and loss of adam3 and adam6 in epididymal sperm
Journal of Biological Chemistry, 2011Co-Authors: Matthew R Marcello, Kevin L Moore, Julie A Leary, Weitao Jia, Janice Perry EvansAbstract: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.
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catalytic mechanism of golgi resident human Tyrosylprotein Sulfotransferase 2 a mass spectrometry approach
Journal of the American Society for Mass Spectrometry, 2010Co-Authors: Lieza M Danan, Kevin L Moore, Peter J Ludden, Weitao Jia, Julie A LearyAbstract:Human Tyrosylprotein Sulfotransferases catalyze the transfer of a sulfuryl moiety from the universal sulfate donor PAPS to the hydroxyl substituent of tyrosine residues in proteins and peptides to yield tyrosine sulfated products and PAP. Tyrosine sulfation occurs in the trans-Golgi network, affecting an estimated 1% of the tyrosine residues in all secreted and membrane-bound proteins in higher order eukaryotes. In this study, an effective LC-MS-based TPST kinetics assay was developed and utilized to measure the kinetic properties of human TPST-2 and investigate its catalytic mechanism when G protein-coupled CC-chemokine receptor 8 (CCR8) peptides were used as acceptor substrates. Through initial rate kinetics, product inhibition studies, and radioactive-labeling experiments, our data strongly suggest a two-site ping-pong model for TPST-2 action. In this mechanistic model, the enzyme allows independent binding of substrates to two distinct sites, and involves the formation of a sulfated enzyme covalent intermediate. Some insights on the important amino acid residues at the catalytic site of TPST-2 and its covalent intermediate are also presented. To our knowledge, this is the first detailed study of the reaction kinetics and mechanism reported for human TPST-2 or any other Golgi-resident Sulfotransferase.
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pattern and temporal sequence of sulfation of ccr5 n terminal peptides by Tyrosylprotein Sulfotransferase 2 an assessment of the effects of n terminal residues
Biochemistry, 2009Co-Authors: Connie H Jen, Kevin L Moore, Julie A LearyAbstract:CC chemokine receptor 5 (CCR5) is the receptor for several inflammatory chemokines and is a coreceptor for HIV-1. Posttranslational sulfation of tyrosines in the N-terminal regions of chemokine receptors has been shown to be important in the binding affinity for chemokine ligands. In addition, sulfation of CCR5 is crucial for mediating interactions with HIV-1 envelope protein gp120. The major sulfation pathway for peptides derived from the N-terminal domains of CCR5 and CCR8 and variations of the peptides were determined by in vitro enzymatic sulfation by Tyrosylprotein sulfotranferase-2 (TPST-2), subsequent separation of products by RP-HPLC, and mass spectrometry analysis. It was found that the patterns of sulfation and the rates of sulfation for CCR5 and CCR8 depend on the number of amino acids N-terminal of Tyr-3. Results herein address previous seemingly contradictory studies and delineate the temporal sulfation of N-terminal chemokine receptor peptides.
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Tyrosylprotein Sulfotransferase 2 expression is required for sulfation of rnase 9 and mfge8 in vivo
Journal of Biological Chemistry, 2009Co-Authors: Adam J Hoffhines, Julie A Leary, Constance H Jen, Kevin L MooreAbstract:Protein-tyrosine sulfation is mediated by two Golgi tyrosyl-protein Sulfotransferases (TPST-1 and TPST-2) that are widely expressed in vivo. However, the full substrate repertoire of this enzyme system is unknown and thus, our understanding of the biological role(s) of tyrosine sulfation is limited. We reported that whereas Tpst1-/- male mice have normal fertility, Tpst2-/- males are infertile despite normal spermatogenesis. However, Tpst2-/- sperm are severely defective in their motility in viscous media and in their ability to fertilize eggs. These findings suggest that sulfation of unidentified substrate(s) is crucial for normal sperm function. We therefore sought to identify tyrosine-sulfated proteins in the male genital tract using affinity chromatography on PSG2, an anti-sulfotyrosine monoclonal antibody, followed by mass spectrometry. Among the several candidate tyrosine-sulfated proteins identified, RNase 9 and Mfge8 were examined in detail. RNase 9, a catalytically inactive RNase A family member of unknown function, is expressed only in the epididymis after onset of sexual maturity. Mfge8 is expressed on mouse sperm and Mfge8-/- male mice are subfertile. Metabolic labeling coupled with sulfoamino acid analysis confirmed that both proteins are tyrosine-sulfated and both proteins are expressed at comparable levels in wild type, Tpst1-/-, and Tpst2-/- epididymides. However, we demonstrate that RNase 9 and Mfge8 are tyrosine-sulfated in wild type and Tpst1-/-, but not in Tpst2-/- mice. These findings suggest that lack of sulfation of one or both of these proteins may contribute mechanistically to the infertility of Tpst2-/- males.
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mass spectrometric kinetic analysis of human Tyrosylprotein Sulfotransferase 1 and 2
Journal of the American Society for Mass Spectrometry, 2008Co-Authors: Lieza M Danan, Kevin L Moore, Adam J Hoffhines, Julie A LearyAbstract:Protein tyrosine O-sulfation, a widespread post-translational modification, is mediated by two Golgi enzymes, Tyrosylprotein Sulfotransferase-1 and-2. These enzymes catalyze the transfer of sulfate from the universal sulfate donor 3′-phosphoadenosine-5′-phosphosulfate (PAPS) to the hydroxyl group of tyrosine residues to form tyrosine O-sulfate ester and PAP. More than 60 proteins have been identified to be tyrosine sulfated including several G protein-coupled receptors, such as CC-chemokine receptor 8 (CCR8) that is implicated in allergic inflammation, asthma, and atherogenesis. However, the kinetic properties of purified Tyrosylprotein Sulfotransferase (TPST)-1 and −2 have not been previously reported. Moreover, currently there is no available quantitative TPST assay that can directly monitor individual sulfation of a series of tyrosine residues, which is present in most known substrates. We chose an MS-approach to address this limitation. In this study, a liquid chromatography electrospray ionisation mass spectrometry (LC/ESI-MS)-based TPST assay was developed to determine the kinetic parameters of individual TPSTs and a mixture of both isozymes using CCR8 peptides as substrates that have three tyrosine residues in series. Our method can differentiate between mono-and disulfated products, and our results show that the Km,app for the monosulfated substrate was 5-fold less than the nonsulfated substrate. The development of this method is the initial step in the investigation of kinetic parameters of the sequential tyrosine sulfation of chemokine receptors by TPSTs and in determining its catalytic mechanism.
Chinnaswamy Kasinathan - One of the best experts on this subject based on the ideXlab platform.
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in vivo induction of Tyrosylprotein Sulfotransferase by ethanol role of increased enzyme synthesis
Alcoholism: Clinical and Experimental Research, 1998Co-Authors: Patalapati Ramaprasad, Chinnaswamy KasinathanAbstract:Tyrosine sulfation is a posttranslational modification involved in the synthesis, secretion, and biological activity of proteins and peptides. Our previous studies have demonstrated that the enzyme activity was induced by ethanol. In the present work, the induction was studied in detail. Initial experiments were conducted to examine the time course of Tyrosylprotein Sulfotransferase (TPST) induction in rats pair-fed liquid diets containing either ethanol or carbohydrate substitute (controls). Marked elevation of TPST activity (3-fold) was measured on day 10 in the liver and gastric mucosa of ethanol-fed rats. Ethanol-mediated enhancement was also noticed by Western-blot analysis with anti-TPST antibody in both the liver and gastric mucosa on days 5 and 10. We then determined the steady-state TPST protein turnover in ethanol-fed and control animals that were given 35S-methionine after 10 days of pair-feeding with liquid diet. The rates of TPST synthesis assessed by measuring initial rates of incorporation of 35S-methionine into TPST was increased in the liver and gastric mucosa of animals fed with ethanol. Monophasic exponential decay curves showed that TPST protein half-lives for liver (control: 34 hr, ethanol: 32 hr) and gastric mucosa (control: 52 hr, ethanol: 48 hr) did not differ between control and ethanol groups. Our overall results indicate that the in vivo induction of TPST by ethanol involves increased enzyme synthesis rather than decreased enzyme degradation.
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stimulation of Tyrosylprotein Sulfotransferase activity by ethanol role of increased enzyme level
Alcohol, 1998Co-Authors: Chinnaswamy Kasinathan, Patalapati Ramaprasad, Samuel William, Noel EspinaAbstract:Tyrosylprotein Sulfotransferase (TPST), an enzyme involved in the posttranslational modification of proteins, plays important role in the biological activity and secretion of proteins. Previously we have shown an increased activity of this enzyme in gastric mucosa of alcoholics. In the present study, effect of ethanol on TPST was examined in rat liver and gastric mucosa utilizing enzyme assays and Western blot analyses for TPST levels. Male Sprague-Dawley rats were pair-fed Leiber-DeCarli liquid diets for 10 days and controls received a liquid diet in which dextrose was isocalorically substituted for ethanol. After ethanol feeding, rats were sacrificed and liver and gastric mucosa were processed for Golgi membrane preparation. The TPST activity was measured using poly(Glu6, Ala3, Tyr1) as the sulfate acceptor and PAPS as sulfate donor. There was a threefold increase in TPST activity of gastric mucosa of animals subjected to chronic alcohol feeding. In the liver, the increase in tyrosine sulfating activity was also around threefold. The kinetic studies performed to understand the mechanism involved in ethanol stimulation of TPST activity showed no change in the Km values of the enzyme by ethanol. In control and ethanol-treated animals, the Km for EAY was 0.41-0.53 and 0.43-0.53 microM, and the Km for PAPS was 10-12.5 and 9-17 microM, respectively. The Vmax in ethanol-fed animals was increased by 1.5- to 2-fold. The increase in TPST activity in experimental rats was further tested by analyzing the Western blots by Imaging Densitometer for TPST levels. Analysis of TPST levels also showed over threefold increase in the stomach and liver of ethanol-fed rats. Our results indicate that stimulation of TPST by ethanol involves increased TPST level rather than change in affinity for its substrates.
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isolation of Tyrosylprotein Sulfotransferase from rat liver
General Pharmacology-the Vascular System, 1998Co-Authors: Patalapati Ramaprasad, Chinnaswamy KasinathanAbstract:Abstract 1. Tyrosylprotein Sulfotransferase (TPST) is involved in the posttranslational modification of proteins and plays a critical role in the biological activity and secretion of proteins. A simple method has been developed to isolate the TPST (28% yield) from rat liver, using polyclonal anti-TPST antibodies. 2. The protein fractions eluted from antibody affinity column showed TPST activity and revealed a 50–54 kDa protein band in the silver stained SDS-polyacrylamide gels. 3. The enzyme exhibited optimum activity at pH 5.5 with 20 mM MnCl2. Unlike the TPST activity of the Golgi membrane, the activity of the purified enzyme was not stimulated by NaF, 5′-AMP, and Triton X-100. 4. The antibody was also used to study the TPST protein turnover in rat liver of animals that were given [35S]methionine. The TPST protein synthesis assessed by measuring initial rates of incorporation of [35S]methionine into TPST protein showed enzyme synthesis for up to 60 min. 35S-labeled TPST protein of rat liver was degraded with a half-life of 30 hr. 5. The immunoaffinity purification method using rat liver as an enzyme source appeared to be very simple, rapid, and easy to perform with significant enzyme recovery. Further, the antibody was also found to be useful in the study involving TPST protein metabolism.
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purification of Tyrosylprotein Sulfotransferase from rat submandibular salivary glands
Archives of Biochemistry and Biophysics, 1997Co-Authors: Samuel William, Patalapati Ramaprasad, Chinnaswamy KasinathanAbstract:Tyrosylprotein Sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands. In the present study, this enzyme was purified from the Golgi membranes of rat submandibular salivary glands using a Cibacron blue F3GA affinity column chromatography. Antibodies raised in rabbit against TPST detected the purified enzyme (50-54 kDa) and proteins consisting of molecular mass 50-54 kDa in the Golgi membranes of liver, submandibular salivary glands, stomach, cerebellum, thalamus, and pituitary. The protein levels in liver and salivary glands were higher compared to those found in the stomach, cerebellum, thalamus, and pituitary. The levels of immunoreactivity in cytosol and endoplasmic reticulum fractions of salivary glands were either undetectable or very low. The antibody was also used to immunoprecipitate the TPST activity and to isolate protein by immunoaffinity column. MnCl2 was required for the purified TPST. The enzyme exhibited optimum activity between pH 6.2 and 6.8 at 20 mM MnCl2. The apparent K(m) values of the purified enzyme for poly-(Glu6, Ala3, Tyr1) (EAY: M(r) 47,000) and 3'-phosphoadenosine 5'-phosphosulfate were 3 and 20 microM, respectively. The results presented here collectively demonstrate the purification of TPST and, for the first time, development of polyclonal antibody that recognizes this enzyme.
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effect of prostaglandins on Tyrosylprotein Sulfotransferase activity in rat submandibular salivary glands
General Pharmacology-the Vascular System, 1995Co-Authors: Chinnaswamy Kasinathan, R Sundaram, Samuel WilliamAbstract:Abstract 1. 1. Tyrosylprotein Sulfotransferase (TPST) is a key enzyme in the processing of several secretory proteins, including those found in saliva. In this report, the effect of prostaglandins (PG) on TPST activity in submandibular salivary gland was investigated. 2. 2. The results revealed that PGE2 exhibited TPST stimulatory activity with a 1.5-fold stimulation at 100 μM concentration and a half maximal stimulation at 50 μM. The PGE2 stimulation was accompanied by an increase in the affinity of TPST towards sulfate acceptor (Km 1.4 μM → 0.12 μM) with little change in Vmax. 3. 3. The TPST activity was also stimulated by two other major prostaglandins of salivary glands, PGF2α and 6-Keto-PGF1α, however to lesser extent, 22 and 23%, respectively. Arachidonic acid, an intermediate prostaglandin precursor, had no effect on TPST activity. 4. 4. The results suggest that prostaglandins and in particular PGE2 may play a role in the regulation of TPST catalyzed secretory protein tyrosine sulfation in salivary glands.
Miklós Sahin-tóth - One of the best experts on this subject based on the ideXlab platform.
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A common African polymorphism abolishes tyrosine sulfation of human anionic trypsinogen (PRSS2).
The Biochemical journal, 2009Co-Authors: Zsolt Rónai, Heiko Witt, Olga Rickards, Giovanni Destro-bisol, Andrew Bradbury, Miklós Sahin-tóthAbstract:Human pancreatic trypsinogens undergo post-translational sulfation on Tyr154, catalyzed by the Golgi-resident enzyme Tyrosylprotein Sulfotransferase 2. Sequence alignments suggest that sulfation of Tyr154 is facilitated by a unique sequence context characteristically found in primate trypsinogens. In search for genetic variants that might alter this sulfation motif, we identified a single nucleotide polymorphism (c.457G>C) in the human anionic trypsinogen gene (PRSS2), which changed Asp153 to His (p.D153H). The p.D153H variant is common in subjects of African origin, with a minor allele frequency of 9.2%, whereas it is absent in subjects of European descent. We demonstrate that Asp153 is the main determinant of tyrosine sulfation in anionic trypsinogen, as both the natural p.D153H variation and the p.D153N mutation result in complete loss of trypsinogen sulfation. In contrast, mutation of Asp156 and Glu157 only slightly decrease tyrosine sulfation, whereas mutation of Gly151 and Pro155 are without consequence. With respect to the biological relevance of the p.D153H variant, we found that tyrosine sulfation had no significant effect on the activation of anionic trypsinogen or the catalytic activity and inhibitor sensitivity of anionic trypsin. Taken together with previous studies, the observations suggest that the primary role of trypsinogen sulfation in humans is to stimulate autoactivation of cationic trypsinogen (PRSS1), whereas sulfation of anionic trypsinogen is unimportant for normal digestive physiology. As a result, the p.D153H polymorphism which eliminates this modification could become widespread in a healthy population.
