Human mononuclear cells contain an endoglycosidase specific for heparan sulphate glycosaminoglycan demonstrable with the use of a specific solid-phase metabolically radiolabelled substrate.
Ontology highlight
ABSTRACT:
SUBMITTER: Sewell RF
PROVIDER: S-EPMC1133653 | biostudies-other | 1989 Dec
REPOSITORIES: biostudies-other
Similar Datasets
Project description:An endoglycosidase is described in isolated liver plasma membranes that brings about a rapid and selective degradation of membrane-associated heparan sulphate, pre-labelled biosynthetically with Na2(35)SO4. The enzyme attacked mainly the polysaccharide chains of a hydrophobic membrane proteoglycan and it had little effect on a proteoglycan that could be displaced from the membranes with 1.0 M-NaCl. The highest activity was measured in the pH range 7.5-8.0, and the enzyme was almost completely inhibited below pH 5.5. Breakdown of susceptible polysaccharide chains was fast, being complete in 20-30 min. The major oligosaccharide fraction (Mr approx. 6000) produced by the enzyme was considerably smaller than the intact heparan sulphate chains. Enzyme activity was retained in membranes solubilized in 1% (v/v) Triton X-100. The high pH optimum and plasma-membrane association distinguish this enzyme from other heparan sulphate-degrading endoglycosidases that have acid pH optima and may be of lysosomal origin. A plasma-membrane endoglycosidase could modulate cellular interactions mediated by heparan sulphate, and/or release biologically active fragments of the polysaccharide from the cell periphery.
| S-EPMC1148917 | biostudies-other
Project description:HS (heparan sulphate) proteoglycans bind secreted signalling proteins, including FGFs (fibroblast growth factors) through their HS side chains. Such chains contain a wealth of differentially sulphated saccharide epitopes. Whereas specific HS structures are commonly believed to modulate FGF-binding and activity, selective binding of defined HS epitopes to FGFs has generally not been demonstrated. In the present paper, we have identified a series of sulphated HS octasaccharide epitopes, derived from authentic HS or from biosynthetic libraries that bind with graded affinities to FGF4, FGF7 and FGF8b. These HS species, along with previously identified oligosaccharides that interact with FGF1 and FGF2, constitute the first comprehensive survey of FGF-binding HS epitopes based on carbohydrate sequence analysis. Unexpectedly, our results demonstrate that selective modulation of FGF activity cannot be explained in terms of binding of individual FGFs to specific HS target epitopes. Instead, different FGFs bind to identical HS epitopes with similar relative affinities and low selectivity, such that the strength of these interactions increases with increasing saccharide charge density. We conclude that FGFs show extensive sharing of binding sites in HS. This conclusion challenges the current notion of specificity in HS-FGF interactions, and instead suggests that a set of common HS motifs mediates cellular targeting of different FGFs.
| S-EPMC1188264 | biostudies-literature
Project description:A targeted heptasaccharide library was synthesised to prepare a heparan sulphate (HS) microarray. The array was probed with two glycan-binding proteins, HS 3-O-sulphotransferase 1 and antithrombin, demonstrating the binding selectivity between HS and proteins. The HS microarray technique will accelerate the understanding of the structure and function relationships of HS.
| S-EPMC6516466 | biostudies-literature
Project description:The glycosaminoglycan, heparan sulphate (HS), orchestrates many developmental processes. Yet its biological role has not yet fully been elucidated. Small molecule chemical inhibitors can be used to perturb HS function and these compounds provide cheap alternatives to genetic manipulation methods. However, existing chemical inhibition methods for HS also interfere with chondroitin sulphate (CS), complicating data interpretation of HS function. Herein, a simple method for the selective inhibition of HS biosynthesis is described. Using endogenous metabolic sugar pathways, Ac4GalNAz produces UDP-GlcNAz, which can target HS synthesis. Cell treatment with Ac4GalNAz resulted in defective chain elongation of the polymer and decreased HS expression. Conversely, no adverse effect on CS production was observed. The inhibition was transient and dose-dependent, affording rescue of HS expression after removal of the unnatural azido sugar. The utility of inhibition is demonstrated in cell culture and in whole organisms, demonstrating that this small molecule can be used as a tool for HS inhibition in biological systems.
| S-EPMC8269443 | biostudies-literature
Project description:Transglutaminase-2 (TG2) is a new anti-fibrotic target for chronic kidney disease, for its role in altering the extracellular homeostatic balance leading to excessive build-up of matrix in kidney. However, there is no confirmation that TG2 is the only transglutaminase involved, neither there are strategies to control its action specifically over that of the conserved family-members. In this study, we have profiled transglutaminase isozymes in the rat subtotal nephrectomy (SNx) model of progressive renal scarring. All transglutaminases increased post-SNx peaking at loss of renal function but TG2 was the predominant enzyme. Upon SNx, extracellular TG2 deposited in the tubulointerstitium and peri-glomerulus via binding to heparan sulphate (HS) chains of proteoglycans and co-associated with syndecan-4. Extracellular TG2 was sufficient to activate transforming growth factor-?1 in tubular epithelial cells, and this process occurred in a HS-dependent way, in keeping with TG2-affinity for HS. Analysis of heparin binding of the main transglutaminases revealed that although the interaction between TG1 and HS is strong, the conformational heparin binding site of TG2 is not conserved, suggesting that TG2 has a unique interaction with HS within the family. Our data provides a rationale for a novel anti-fibrotic strategy specifically targeting the conformation-dependent TG2-epitope interacting with HS.
| S-EPMC5046136 | biostudies-literature
Project description:Heparan sulphate proteoglycans (HSPGs) consist of a core protein decorated with sulphated HS-glycosaminoglycan (GAG) chains. These negatively charged HS-GAG chains rely on the activity of PAPSS synthesising enzymes for their sulfation, which allows them to bind to and regulate the activity of many positively charged HS-binding proteins. HSPGs are found on the surfaces of cells and in the pericellular matrix, where they interact with various components of the cell microenvironment, including growth factors. By binding to and regulating ocular morphogens and growth factors, HSPGs are positioned to orchestrate growth factor-mediated signalling events that are essential for lens epithelial cell proliferation, migration, and lens fibre differentiation. Previous studies have shown that HS sulfation is essential for lens development. Moreover, each of the full-time HSPGs, differentiated by thirteen different core proteins, are differentially localised in a cell-type specific manner with regional differences in the postnatal rat lens. Here, the same thirteen HSPG-associated GAGs and core proteins as well as PAPSS2, are shown to be differentially regulated throughout murine lens development in a spatiotemporal manner. These findings suggest that HS-GAG sulfation is essential for growth factor-induced cellular processes during embryogenesis, and the unique and divergent localisation of different lens HSPG core proteins indicates that different HSPGs likely play specialized roles during lens induction and morphogenesis.
| S-EPMC10216587 | biostudies-literature
Project description:Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.
| S-EPMC1144099 | biostudies-other
Project description:Glycosaminoglycans (GAGs) affect human physiology and pathology by modulating more than 500 proteins. GAG-protein interactions are generally assumed to be ionic and nonspecific, but specific interactions do exist. Here, we present a simple method to identify the GAG-binding site (GBS) on proteins that in turn helps predict high specific GAG-protein systems. Contrary to contemporary thinking, we found that the electrostatic potential at basic arginine and lysine residues neither identifies the GBS consistently, nor its specificity. GBSs are better identified by considering the potential at neutral hydrogen bond donors such as asparagine or glutamine sidechains. Our studies also reveal that an unusual constellation of ionic and non-ionic residues in the binding site leads to specificity. Nature engineers the local environment of Asn45 of antithrombin, Gln255 of 3-O-sulfotransferase 3, Gln163 and Asn167 of 3-O-sulfotransferase 1 and Asn27 of basic fibroblast growth factor in the respective GBSs to induce specificity. Such residues are distinct from other uncharged residues on the same protein structure in possessing a significantly higher electrostatic potential, resultant from the local topology. In contrast, uncharged residues on nonspecific GBSs such as thrombin and serum albumin possess a diffuse spread of electrostatic potential. Our findings also contradict the paradigm that GAG-binding sites are simply a collection of contiguous Arg/Lys residues. Our work demonstrates the basis for discovering specifically interacting and druggable GAG-protein systems based on the structure of protein alone, without requiring access to any structure-function relationship data.
| S-EPMC4619353 | biostudies-literature
Project description:Enterovirus 71 (EV-71) infections are usually associated with mild hand, foot, and mouth disease in young children but have been reported to cause severe neurological complications with high mortality rates. To date, four EV-71 receptors have been identified, but inhibition of these receptors by antagonists did not completely abolish EV-71 infection, implying that there is an as yet undiscovered receptor(s). Since EV-71 has a wide range of tissue tropisms, we hypothesize that EV-71 infections may be facilitated by using receptors that are widely expressed in all cell types, such as heparan sulfate. In this study, heparin, polysulfated dextran sulfate, and suramin were found to significantly prevent EV-71 infection. Heparin inhibited infection by all the EV-71 strains tested, including those with a single-passage history. Neutralization of the cell surface anionic charge by polycationic poly-d-lysine and blockage of heparan sulfate by an anti-heparan sulfate peptide also inhibited EV-71 infection. Interference with heparan sulfate biosynthesis either by sodium chlorate treatment or through transient knockdown of N-deacetylase/N-sulfotransferase-1 and exostosin-1 expression reduced EV-71 infection in RD cells. Enzymatic removal of cell surface heparan sulfate by heparinase I/II/III inhibited EV-71 infection. Furthermore, the level of EV-71 attachment to CHO cell lines that are variably deficient in cell surface glycosaminoglycans was significantly lower than that to wild-type CHO cells. Direct binding of EV-71 particles to heparin-Sepharose columns under physiological salt conditions was demonstrated. We conclude that EV-71 infection requires initial binding to heparan sulfate as an attachment receptor.
| S-EPMC3536405 | biostudies-literature
Project description:Heparin and heparan sulfate are very large linear polysaccharides that undergo a complex variety of modifications and are known to play important roles in human development, cell-cell communication and disease. Sequencing of highly sulfated glycosaminoglycan oligosaccharides like heparin and heparan sulfate by liquid chromatography-tandem mass spectrometry (LC-MS/MS) remains challenging because of the presence of multiple isomeric sequences in a complex mixture of oligosaccharides, the difficulties in separation of these isomers, and the facile loss of sulfates in MS/MS. We have previously introduced a method for structural sequencing of heparin/heparan sulfate oligosaccharides involving chemical derivatizations that replace labile sulfates with stable acetyl groups. This chemical derivatization scheme allows the use of reversed phase LC for high-resolution separation and MS/MS for sequencing of isomeric heparan sulfate oligosaccharides. However, because of the large number of analytes present in complex mixtures of heparin/HS oligosaccharides, the resulting LC-MS/MS data sets are large and cannot be annotated with existing glycomics software because of the specifically designed chemical derivatization strategy. We have developed a tool, called GAG-ID, to automate the interpretation of derivatized heparin/heparan sulfate LC-MS/MS data based on a modified multivariate hypergeometric distribution to weight the annotation of more intense peaks. The software is tested on a LC-MS/MS data set collected from a mixture of 21 synthesized heparan sulfate tetrasaccharides. By testing the discrimination of scoring with this system, we show that stratifying peaks into different intensity classes benefits the discrimination of scoring, and GAG-ID is able to properly assign all 21 synthetic tetrasaccharides in a defined mixture from a single LC-MS/MS run.
| S-EPMC4458732 | biostudies-literature