The Experts below are selected from a list of 26115 Experts worldwide ranked by ideXlab platform
Robert J. Linhardt - One of the best experts on this subject based on the ideXlab platform.
-
analysis of total human urinary glycosaminoglycan disaccharides by liquid chromatography tandem mass spectrometry
Analytical Chemistry, 2015Co-Authors: Lingyun Li, Lee Anne Ammons, Clay Cothren Burlew, Eric P Schmidt, Katherine H Overdier, Ivor S Douglas, Fuming Zhang, 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...
-
disaccharide analysis of glycosaminoglycan mixtures by ultra high performance liquid chromatography mass spectrometry
Journal of Chromatography A, 2012Co-Authors: Bo Yang, Amanda Weyers, Eric Sterner, Yuqing Chang, 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.
-
a new glycosaminoglycan from the giant african snail achatina fulica
Journal of Biological Chemistry, 1996Co-Authors: Y S Kim, Il Moo Chang, Toshihiko Toida, Youmie Park, Robert J. LinhardtAbstract:Abstract A new glycosaminoglycan has been isolated from the giant African snail Achatina fulica. This polysaccharide had a molecular weight of 29,000, calculated based on the viscometry, and a uniform repeating disaccharide structure of 4)-2-acetyl,2-deoxy-α-D-glucopyranose (14)-2-sulfo-α-L-idopyranosyluronic acid (1. This polysaccharide represents a new, previously undescribed glycosaminoglycan. It is related to the heparin and heparan sulfate families of Glycosaminoglycans but is distinctly different from all known members of these classes of Glycosaminoglycans. The structure of this polysaccharide, with adjacent N-acetylglucosamine and 2-sulfo-iduronic acid residues, also poses interesting questions about how it is made in light of our current understanding of the biosynthesis of heparin and heparan sulfate. This glycosaminoglycan represents 3-5% of the dry weight of this snail's soft body tissues, suggesting important biological roles for the survival of this organism, and may offer new means to control this pest. Snail glycosaminoglycan tightly binds divalent cations, such as copper(II), suggesting a primary role in metal uptake in the snail. Finally, this new polysaccharide might be applied, like the Escherichia coli K5 capsular polysaccharide, to the study of glycosaminoglycan biosynthesis and to the semisynthesis of new glycosaminoglycan analogs having important biological activities.
Alan Cartmell - One of the best experts on this subject based on the ideXlab platform.
-
Metabolism of multiple Glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus
Nature Communications, 2020Co-Authors: Didier Ndeh, Arnaud Baslé, Henrik Strahl, Urszula L. Mcclurgg, Bernard Henrissat, Nicolas Terrapon, Alan CartmellAbstract:Glycosaminoglycans (GAGs) are an important nutrient source for the gut microbiome. Here, the authors characterize the genetic loci that underpins glycosaminoglycan utilization in Bacteroides thetaiotaomicron; providing insights into the metabolism of GAGs by a predominant member of the gut microbiota. The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the 'sulfation problem' is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
-
Metabolism of multiple Glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus.
Nature Communications, 2020Co-Authors: Didier Ndeh, Arnaud Baslé, Henrik Strahl, Edwin A. Yates, Urszula L. Mcclurgg, Bernard Henrissat, Nicolas Terrapon, Alan CartmellAbstract:The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the ‘sulfation problem’ is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
Frank C Church - One of the best experts on this subject based on the ideXlab platform.
-
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.
-
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.
-
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.
-
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.
Steven M Albelda - One of the best experts on this subject based on the ideXlab platform.
-
platelet endothelial cell adhesion molecule 1 cd31 mediated cellular aggregation involves cell surface Glycosaminoglycans
Journal of Biological Chemistry, 1993Co-Authors: Horace M Delisser, Horng Chin Yan, Peter J Newman, William A Muller, Clayton A Buck, Steven M AlbeldaAbstract:Platelet/endothelial cell adhesion molecule-1 (PECAM-1, CD31) is a 130-kDa integral membrane glycoprotein expressed on endothelial cells, platelets, and leukocytes. Experiments analyzing the aggregation of mouse L-cells stably transfected with full-length PECAM-1 cDNA have demonstrated that PECAM-1 is capable of mediating calcium-dependent heterophilic aggregation. In this report the ligand interactions involved in the aggregation process were studied. This aggregation was inhibited by heparin and chondroitin sulfate, but not by other Glycosaminoglycans. Enzymatic removal of cell surface Glycosaminoglycans confirmed a PECAM-1-glycosaminoglycan interaction and suggested that this interaction involved Glycosaminoglycans on adjacent cells. PECAM-1 contains a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like domain of the molecule's extracellular domain. A comparable region in the related adhesion protein N-CAM has been shown to mediate the adhesive properties of N-CAM. Cells expressing mutant PECAM-1 protein missing the second domain failed to aggregate. Synthetic peptides mimicking the consensus glycosaminoglycan binding sequence, L-K-R-E-K-N, inhibited aggregation. These results demonstrate that PECAM-1-mediated aggregation is dependent on the binding of PECAM-1 to specific Glycosaminoglycans on adjacent cells via a glycosaminoglycan consensus binding sequence in the second immunoglobulin-like homology domain.
Didier Ndeh - One of the best experts on this subject based on the ideXlab platform.
-
Metabolism of multiple Glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus
Nature Communications, 2020Co-Authors: Didier Ndeh, Arnaud Baslé, Henrik Strahl, Urszula L. Mcclurgg, Bernard Henrissat, Nicolas Terrapon, Alan CartmellAbstract:Glycosaminoglycans (GAGs) are an important nutrient source for the gut microbiome. Here, the authors characterize the genetic loci that underpins glycosaminoglycan utilization in Bacteroides thetaiotaomicron; providing insights into the metabolism of GAGs by a predominant member of the gut microbiota. The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the 'sulfation problem' is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.
-
Metabolism of multiple Glycosaminoglycans by Bacteroides thetaiotaomicron is orchestrated by a versatile core genetic locus.
Nature Communications, 2020Co-Authors: Didier Ndeh, Arnaud Baslé, Henrik Strahl, Edwin A. Yates, Urszula L. Mcclurgg, Bernard Henrissat, Nicolas Terrapon, Alan CartmellAbstract:The human gut microbiota (HGM), which is critical to human health, utilises complex glycans as its major carbon source. Glycosaminoglycans represent an important, high priority, nutrient source for the HGM. Pathways for the metabolism of various glycosaminoglycan substrates remain ill-defined. Here we perform a biochemical, genetic and structural dissection of the genetic loci that orchestrates glycosaminoglycan metabolism in the organism Bacteroides thetaiotaomicron. Here, we report: the discovery of two previously unknown surface glycan binding proteins which facilitate glycosaminoglycan import into the periplasm; distinct kinetic and genetic specificities of various periplasmic lyases which dictate glycosaminoglycan metabolic pathways; understanding of endo sulfatase activity questioning the paradigm of how the ‘sulfation problem’ is handled by the HGM; and 3D crystal structures of the polysaccharide utilisation loci encoded sulfatases. Together with comparative genomic studies, our study fills major gaps in our knowledge of glycosaminoglycan metabolism by the HGM.