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Frank C Church - One of the best experts on this subject based on the ideXlab platform.
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molecular mapping of the thrombin heparin cofactor ii complex
Journal of Biological Chemistry, 2004Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Holly R Gentry, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:Abstract We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of Glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without Glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 × 108 m-1 min-1 for HCII-heparin when compared with 2.36 × 108 m-1 min-1 with wild-type thrombin and 0.03-0.53 × 108 m-1 min-1 for HCII-dermatan sulfate when compared with 5.23 × 108 m-1 min-1 with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of Glycosaminoglycans. However, our results are also consistent for HCII-Glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-Glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with Glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-Glycosaminoglycan inhibition reactions. Collectively, our results support a “double bridge” mechanism for HCII inhibition of thrombin in the presence of Glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the “hirudin-like” domain of HCII with thrombin exosite-1.
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aspartic acid residues 72 and 75 and tyrosine sulfate 73 of heparin cofactor ii promote intramolecular interactions during Glycosaminoglycan binding and thrombin inhibition
Journal of Biological Chemistry, 2002Co-Authors: Jennifer W Mitchell, Frank C ChurchAbstract:Abstract We used site-directed mutagenesis to investigate the role of Glu69, Asp70, Asp71, Asp72, Tyr-sulfate73, and Asp75 in the second acidic region (AR2) of the serpin heparin cofactor II (HCII) during formation of the thrombin·HCII complex with and without Glycosaminoglycans. E69Q/D70N/D71N recombinant (r)HCII, D72N/Y73F/D75N rHCII, and E69Q/D70N/D71N/D72N/Y73F/D75N rHCII were prepared to localize acidic residues important for thrombin inhibition. Interestingly, D72N/Y73F/D75N rHCII had significantly enhanced thrombin inhibition without Glycosaminoglycan (4-fold greater) and with heparin (6-fold greater), showing maximal activity at 2 μg/ml heparin compared with wild-type recombinant HCII (wt-rHCII) with maximal activity at 20 μg/ml heparin. The other rHCII mutants had lesser-enhanced activities, but they all eluted from heparin-Sepharose at significantly higher ionic strengths compared with wt-rHCII. Neutralizing and reversing the charge of Asp72, Tyr-sulfate73, and Asp75 were done to characterize their individual contribution to HCII activity. Only Y73K rHCII and D75K rHCII have significantly increased heparin cofactor activity compared with wt-rHCII; however, all of the individual rHCII mutants required substantially less Glycosaminoglycan at maximal inhibition than did wt-rHCII. Inhibition of either α-thrombin/hirugen or γT-thrombin (both with an altered anion-binding exosite-1) by the AR2 rHCII mutants was similar to wt-rHCII. D72N/Y73F/D75N rHCII and D75K rHCII were significantly more active than wt-rHCII in a plasma-based thrombin inhibition assay with Glycosaminoglycans. These results indicate that improved thrombin inhibition in the AR2 HCII mutants is mediated by enhanced interactions between the acidic domain and anion-binding exosite-1 of thrombin and that AR2 may be a “molecular rheostat” to promote thrombin inhibition in the presence of Glycosaminoglycans.
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heparin promotes proteolytic inactivation by thrombin of a reactive site mutant l444r of recombinant heparin cofactor ii
Journal of Biological Chemistry, 1997Co-Authors: Angelina V Ciaccia, Annemieke J Willemze, Frank C ChurchAbstract:Abstract A heparin cofactor II (HCII) mutant with an Arg substituted for Leu444 at the P1 position (L444R-rHCII) was previously found to have altered proteinase specificity (Derechin, V. M., Blinder, M. A., and Tollefsen, D. M. (1990) J. Biol. Chem. 265, 5623-5628). The present study characterizes the effect of Glycosaminoglycans on the substrate versus inhibitor activity of L444R-rHCII. Heparin increased the stoichiometry of inhibition of L444R-rHCII with α-thrombin (compared with minus Glycosaminoglycan) but decreased it with R93A,R97A,R101A-thrombin, a mutant thrombin that does not bind Glycosaminoglycans. Dermatan sulfate decreased the stoichiometry of inhibition of L444R-rHCII with both proteinases. SDS-polyacrylamide gel electrophoresis showed no proteolysis of L444R-rHCII when incubated with R93A,R97A,R101A-thrombin in the absence or the presence of Glycosaminoglycan or with α-thrombin and dermatan sulfate. In contrast, greater than 75% of the L444R-rHCII was converted to a lower molecular weight form when incubated with α-thrombin/heparin. A time course of α-thrombin inhibition by L444R-rHCII/heparin showed a rapid but transient inhibition with approximately 80% of the α-thrombin activity being regained after 6 h of incubation. In contrast, all other combinations of inhibitor, proteinase, and Glycosaminoglycan resulted in complete and sustained inhibition of the proteinase. Heparin fragments of 8-20 polysaccharides in length rapidly accelerated L444R-rHCII inhibition of both α-thrombin and R93A,R97A,R101A-thrombin. After extended incubations, R93A,R97A,R101A-thrombin was completely inhibited by L444R-rHCII with all the heparin fragments, but approximately 30-50% of α-thrombin activity remained with fragments long enough to bridge HCII-thrombin. These results collectively indicate that ternary complex formation, mediated by heparin, increases L444R-rHCII inactivation by α-thrombin.
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inhibition of dysthrombins quick i and ii by heparin cofactor ii and antithrombin
Journal of Biological Chemistry, 1993Co-Authors: Jeanne E Phillips, Ruth Ann Henriksen, Herbert C. Whinna, Rebecca A. Shirk, Frank C ChurchAbstract:Abstract Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by Glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of Glycosaminoglycan. In contrast, Glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of Glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with Glycosaminoglycans and the functional availability of thrombin exosites.
Robert J Linhardt - One of the best experts on this subject based on the ideXlab platform.
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analysis of total human urinary Glycosaminoglycan disaccharides by liquid chromatography tandem mass spectrometry
Analytical Chemistry, 2015Co-Authors: Lingyun Li, Katherine H Overdier, Lee Anne Ammons, Ivor S Douglas, Eric P Schmidt, Fuming Zhang, Clay Cothren Burlew, Robert J LinhardtAbstract:The determination of complex analytes, present at low concentrations, in biological fluids poses a difficult challenge. This study relies on an optimized method of recovery, enzymatic treatment, and disaccharide analysis by liquid chromatography–tandem mass spectrometry to rapidly determine low concentrations of Glycosaminoglycans in human urine. The approach utilizes multiple reaction monitoring (MRM) of Glycosaminoglycan disaccharides obtained from treating urine samples with recombinant heparin lyases and chondroitin lyase. This rapid and sensitive method allows the analysis of Glycosaminoglycan content and disaccharide composition in urine samples having concentrations 10- to 100-fold lower than those typically analyzed from patients with metabolic diseases, such as mucopolysaccharidosis. The current method facilitates the analysis low (ng/mL) levels of urinary Glycosaminoglycans present in healthy individuals and in patients with pathological conditions, such as inflammation and cancers, that can sub...
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disaccharide analysis of Glycosaminoglycan mixtures by ultra high performance liquid chromatography mass spectrometry
Journal of Chromatography A, 2012Co-Authors: Bo Yang, Yuqing Chang, Amanda Weyers, Eric Sterner, Robert J LinhardtAbstract:Glycosaminoglycans are a family of polysaccharides widely distributed in all eukaryotic cells. These polyanionic, linear chain polysaccharides are composed of repeating disaccharide units that are often differentially substituted with sulfo groups. The diversity of Glycosaminoglycan structures in cells, tissues and among different organisms reflect their functional an evolutionary importance. Glycosaminoglycan composition and structure also changes in development, aging and in disease progression, making their accurate and reliable analysis a critical, albeit, challenging endeavor. Quantitative disaccharide compositional analysis is one of the primary ways to characterize Glycosaminoglycan composition and structure and has a direct relationship with Glycosaminoglycan biological functions. In this study, Glycosaminoglycan disaccharides, prepared from heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate and neutral hyaluronic acid using multiple polysaccharide lyases, were fluorescently labeled with 2-aminoacridone, fractionated into 17 well-resolved components by reverse-phase ultra-performance liquid chromatography, and analyzed by electrospray ionization mass spectrometry. This analysis was successfully applied to cell, tissue, and biological fluid samples for the picomole level detection of Glycosaminoglycan composition and structure.
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Capillary electrophoresis for the analysis of Glycosaminoglycans and Glycosaminoglycan-derived oligosaccharides.
Biomedical Chromatography, 2002Co-Authors: Charuwan Thanawiroon, Robert J LinhardtAbstract:Glycosaminoglycans are a family of polydisperse, highly sulfated complex mixtures of linear polysaccharides that are involved in many life processes. Defining the structure of Glycosaminoglycans is an important factor in elucidating their structure–activity relationship. Capillary electrophoresis has emerged as a highly promising technique consuming an extremely small amount of sample and capable of rapid, high-resolution separation, characterization and quantitation of analytes. Numerous capillary electrophoresis methods for analysis of intact Glycosaminoglycans and Glycosaminoglycan-derived oligosaccharides have been developed. These methods allow for both qualitative and quantitative analysis with a high level of sensitivity. This review is concerned with separation methods of capillary electrophoresis, detection methods and applications to several aspects of research into Glycosaminoglycans and Glycosaminoglycan-derived oligosaccharides. The importance of capillary electrophoresis in biological and pharmaceutical samples in glycobiology and carbohydrate biochemistry and its possible applications in disease diagnosis and monitoring chemical synthesis are described. Copyright © 2002 John Wiley & Sons, Ltd.
Bo Yang - One of the best experts on this subject based on the ideXlab platform.
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disaccharide analysis of Glycosaminoglycan mixtures by ultra high performance liquid chromatography mass spectrometry
Journal of Chromatography A, 2012Co-Authors: Bo Yang, Yuqing Chang, Amanda Weyers, Eric Sterner, Robert J LinhardtAbstract:Glycosaminoglycans are a family of polysaccharides widely distributed in all eukaryotic cells. These polyanionic, linear chain polysaccharides are composed of repeating disaccharide units that are often differentially substituted with sulfo groups. The diversity of Glycosaminoglycan structures in cells, tissues and among different organisms reflect their functional an evolutionary importance. Glycosaminoglycan composition and structure also changes in development, aging and in disease progression, making their accurate and reliable analysis a critical, albeit, challenging endeavor. Quantitative disaccharide compositional analysis is one of the primary ways to characterize Glycosaminoglycan composition and structure and has a direct relationship with Glycosaminoglycan biological functions. In this study, Glycosaminoglycan disaccharides, prepared from heparan sulfate/heparin, chondroitin sulfate/dermatan sulfate and neutral hyaluronic acid using multiple polysaccharide lyases, were fluorescently labeled with 2-aminoacridone, fractionated into 17 well-resolved components by reverse-phase ultra-performance liquid chromatography, and analyzed by electrospray ionization mass spectrometry. This analysis was successfully applied to cell, tissue, and biological fluid samples for the picomole level detection of Glycosaminoglycan composition and structure.
John T Gallagher - One of the best experts on this subject based on the ideXlab platform.
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the mode of action of heparan and dermatan sulfates in the regulation of hepatocyte growth factor scatter factor
Journal of Biological Chemistry, 2002Co-Authors: Malcolm Lyon, Jon A Deakin, John T GallagherAbstract:Abstract Hepatocyte growth factor/scatter factor, in addition to binding to its specific signal-transducing receptor, Met, also interacts with both heparan and dermatan sulfates with high affinity. We have investigated the comparative role of these two Glycosaminoglycans in the activation of Met by hepatocyte growth factor/scatter factor. Using Glycosaminoglycan-deficient CHOpgsA-745 cells we have shown that growth factor activity is critically dependent upon Glycosaminoglycans, and that heparan sulfate and dermatan sulfate are equally potent as co-receptors. Cross-linked 1:1 conjugates of growth factor and either heparan or dermatan sulfate do not dimerize under physiological conditions and are biologically active. This implies that a ternary signaling complex with Met formsin vivo. Native Met isolated from CHO pgsA-745 cells shows only very weak intrinsic affinity for heparin in vitro. Also, a heparin-derived hexasaccharide, which is the minimal size for high affinity binding to the growth factor alone, is sufficient to induce biological activity. Together these observations imply that the role of these Glycosaminoglycan may be primarily to effect a conformational change in hepatocyte growth factor/scatter factor, rather than to induce a necessary growth factor dimerization, or to stabilize a ternary complex by additionally interacting with Met.
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a general method for the detection and mapping of submicrogram quantities of Glycosaminoglycan oligosaccharides on polyacrylamide gels by sequential staining with azure a and ammoniacal silver
Analytical Biochemistry, 1990Co-Authors: Malcolm Lyon, John T GallagherAbstract:Abstract A sensitive method has been developed for the visualization of nonradiolabeled Glycosaminoglycan oligosaccharides resolved by polyacrylamide gel electrophoresis using fixation with azure A followed by staining with ammoniacal silver. This method, which can detect as little as 1–2 ng of a single oligosaccharide species, can be used to stain a few micrograms of a complex oligosaccharide mixture. The combination of gradient polyacrylamide gel electrophoresis and sequential azure A/silver staining can be applied to the analysis of all the complex Glycosaminoglycans (i.e., heparin, heparan sulfate, chondroitin/dermatan sulfate, keratan sulfate) and hyaluronate, as well as to comparisons of specificities of the Glycosaminoglycan-degrading enzymes. This procedure may be particularly valuable in situations where the availability of Glycosaminoglycan is very limited and/or where radiolabeling is impractical or undesirable.
Herbert C. Whinna - One of the best experts on this subject based on the ideXlab platform.
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molecular mapping of the thrombin heparin cofactor ii complex
Journal of Biological Chemistry, 2004Co-Authors: Yolanda M Fortenberry, Herbert C. Whinna, Holly R Gentry, Timothy Myles, Lawrence L K Leung, Frank C ChurchAbstract:Abstract We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of Glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without Glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 × 108 m-1 min-1 for HCII-heparin when compared with 2.36 × 108 m-1 min-1 with wild-type thrombin and 0.03-0.53 × 108 m-1 min-1 for HCII-dermatan sulfate when compared with 5.23 × 108 m-1 min-1 with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of Glycosaminoglycans. However, our results are also consistent for HCII-Glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-Glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with Glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-Glycosaminoglycan inhibition reactions. Collectively, our results support a “double bridge” mechanism for HCII inhibition of thrombin in the presence of Glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the “hirudin-like” domain of HCII with thrombin exosite-1.
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inhibition of dysthrombins quick i and ii by heparin cofactor ii and antithrombin
Journal of Biological Chemistry, 1993Co-Authors: Jeanne E Phillips, Ruth Ann Henriksen, Herbert C. Whinna, Rebecca A. Shirk, Frank C ChurchAbstract:Abstract Heparin cofactor II and antithrombin are plasma serine proteinase inhibitors whose ability to inhibit alpha-thrombin is accelerated by Glycosaminoglycans. Dysfunctional thrombin mutants Quick I (Arg67-->Cys) and Quick II (Gly226-->Val) were used to further compare heparin cofactor II and antithrombin interactions. Quick I, Quick II, and alpha-thrombin were eluted at the same salt concentration from heparin-Sepharose suggesting that the putative heparin-binding site (also termed anion binding exosite-II) is functional. Antithrombin yielded similar inhibition rates for Quick I and alpha-thrombin in the absence or presence of various amounts of heparin. Also, Quick I was inhibited similarly to alpha-thrombin by heparin cofactor II in the absence of Glycosaminoglycan. In contrast, Glycosaminoglycan-accelerated Quick I inhibition by heparin cofactor II was greatly reduced indicating that anion binding exosite-I (where the mutation occurs in Quick I) is critical for increased inhibition by heparin cofactor II. We also found that heparin cofactor II formed a SDS-resistant bimolecular complex with Quick II and alpha-thrombin at similar rates and the rate of complex formation was accelerated in the presence of Glycosaminoglycans. A three-dimensional molecular model of the Quick II active site compared to alpha-thrombin suggested that the heparin cofactor II Leu-Ser-reactive site sequence (P1-P1') is a compatible "pseudosubstrate" in contrast to the Arg-Ser sequence found in antithrombin. The importance of heparin cofactor II as a thrombin regulator will depend upon its ability to interact with Glycosaminoglycans and the functional availability of thrombin exosites.
