Structure of heparan sulphate oligosaccharides and their degradation by exo-enzymes.
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ABSTRACT:
SUBMITTER: Linker A
PROVIDER: S-EPMC1161653 | biostudies-other | 1979 Dec
REPOSITORIES: biostudies-other
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Project description:The biological activity of heparan sulphate (HS) and heparin largely depends on internal oligosaccharide sequences that provide specific binding sites for an extensive range of proteins. Identification of such structures is crucial for the complete understanding of glycosaminoglycan (GAG)-protein interactions. We describe here a simple method of sequence analysis relying on the specific tagging of the sugar reducing end by 3H radiolabelling, the combination of chemical scission and specific enzymic digestion to generate intermediate fragments, and the analysis of the generated products by strong-anion-exchange HPLC. We present full sequence data on microgram quantities of four unknown oligosaccharides (three HS-derived hexasaccharides and one heparin-derived octasaccharide) which illustrate the utility and relative simplicity of the technique. The results clearly show that it is also possible to read sequences of inhomogeneous preparations. Application of this technique to biologically active oligosaccharides should accelerate progress in the understanding of HS and heparin structure-function relationships and provide new insights into the primary structure of these polysaccharides.
| S-EPMC1220215 | biostudies-other
Project description:Heparan sulfate (HS) has a domain structure in which regions that are modified by epimerization and sulfonation (NS domains) are interspersed by unmodified fragments (NA domains). There is data to support that domain organization of HS can regulate binding of proteins, however, such model has been difficult to probe. Here, we report a chemoenzymatic methodology that can provide HS oligosaccharides composed of two or more NS domains separated by NA domains of different length. It is based on the chemical synthesis of a HS oligosaccharide that enzymatically was extended by various GlcA-GlcNAc units and terminated in GlcNAc having an azido moiety at C-6 position. HS oligosaccharides having an azide and alkyne moiety could be assembled by copper catalyzed alkyne-azide cycloaddition to give compounds having various NS domains separated by unsulfonated regions. Competition binding studies showed that the length of an NA domain modulates the binding of the chemokines CCL5 and CXCL8.
| S-EPMC9828060 | biostudies-literature
Project description:NO is a bioactive free radical produced by NO synthase in various tissues including vascular endothelium. One of the degradation products of NO is HNO2, an agent known to degrade heparin and heparan sulphate. This report documents degradation of heparin by cultured endothelial-cell-derived as well as exogenous NO. An exogenous narrow molecular-mass preparation of heparin was recovered from the medium of cultured endothelial cells using strong-anion exchange. In addition, another narrow molecular-mass preparation of heparin was gassed with exogenous NO under argon. Degradation was evaluated by gel-filtration chromatography. Since HNO2 degrades heparin under acidic conditions, the reaction with NO gas was studied under various pH conditions. The results show that the degradation of exogenous heparin by endothelial cells is inhibited by NO synthase inhibitors. Exogenous NO gas at concentrations as low as 400 p.p.m. degrades heparin and heparan sulphate. Exogenous NO degrades heparin at neutral as well as acidic pH. Endothelial-cell-derived NO, as well as exogenous NO gas, did not degrade hyaluronan, an unrelated glycosaminoglycan that resists HNO2 degradation. Peroxynitrite, a metabolic product of the reaction of NO with superoxide, is an agent that degrades hyaluronan; however, peroxynitrite did not degrade heparin. Thus endothelial-cell-derived NO is capable of degrading heparin and heparan sulphate via HNO2 rather than peroxynitrite. These observations may be relevant to various pathophysiological processes in which extracellular matrix is degraded, such as bone development, apoptosis, tissue damage from inflammatory responses and possible release of growth factors and cytokines.
| S-EPMC1218454 | biostudies-other
Project description:The domain structure of heparan sulphate chains from an endothelial low-density proteoglycan was examined using specific degradations of the chains while attached to the intact proteoglycan. 'Inner' chain fragments, remaining on the protein core, were separated from 'outer' fragments by gel chromatography, and were subsequently released from the protein core by alkaline cleavage. The structure of 'inner' and 'outer' chain fragments was then examined and compared. Using deaminative cleavage we obtained evidence that the first N-sulphated glucosamine residue is variably positioned some 10-17 disaccharides from the xylose-serine linkage of the proteoglycan. Digestion with heparinase yielded 'inner' and 'outer' fragments covering a broad range of different sizes, indicating a scarce and variable distribution of sulphated iduronic acid in the native chains. N-sulphated glucosamine occurred more frequently in the 'outer' fragments. We also studied the affinity of the endothelial heparan sulphate chains towards two presumptive biological ligands, namely antithrombin III and lipoprotein lipase. A major part of the endothelial heparan sulphate chains showed a weak affinity for antithrombin III and the affinity was essentially lost on heparinase digestion. On lipoprotein lipase-agarose the endothelial heparan sulphate chains were eluted at the same salt concentration as heparin, and the binding persisted, although with decreased strength, after digestion with heparinase.
| S-EPMC1151520 | biostudies-other
Project description:Although hundreds of heparan sulfate binding proteins have been identified and implicated in a myriad of physiological and pathological processes, very little information is known about the ligand requirements for binding and mediating biological activities by these proteins. This difficulty results from a lack of technology for establishing structure-activity relationships, which in turn is due to the structural complexity of natural heparan sulfate (HS) and difficulties of preparing well-defined HS oligosaccharides. To address this deficiency, we developed a modular approach for the parallel combinatorial synthesis of HS oligosaccharides that utilizes a relatively small number of selectively protected disaccharide building blocks, which can easily be converted into glycosyl donors and acceptors. The utility of the modular building blocks has been demonstrated by the preparation of a library of 12 oligosaccharides, which has been employed to probe the structural features of HS for inhibiting the protease, BACE-1. The complex variations in activity with structural changes support the view that important functional information is embedded in HS sequences. Furthermore, the most active derivative provides an attractive lead compound for the preparation of more potent compounds, which may find use as a therapeutic agent for Alzheimer's disease.
| S-EPMC2820250 | biostudies-literature
Project description:Heparan sulfates are implicated in a wide range of biological processes. A major challenge in deciphering their structure and activity relationship is the synthetic difficulties to access diverse heparan sulfate oligosaccharides with well-defined sulfation patterns. In order to expedite the synthesis, a divergent synthetic strategy was developed. By integrating chemical synthesis and two types of O-sulfo transferases, seven different hexasaccharides were obtained from a single hexasaccharide precursor. This approach combined the flexibility of chemical synthesis with the selectivity of enzyme-catalyzed sulfations, thus simplifying the overall synthetic operations. In an attempt to establish structure activity relationships of heparan sulfate binding with its receptor, the synthesized oligosaccharides were incorporated onto a glycan microarray, and their bindings with a growth factor FGF-2 were examined. The unique combination of chemical and enzymatic approaches expanded the capability of oligosaccharide synthesis. In addition, the well-defined heparan sulfate structures helped shine light on the fine substrate specificities of biosynthetic enzymes and confirm the potential sequence of enzymatic reactions in biosynthesis.
| S-EPMC4685427 | biostudies-literature
Project description:Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.
| S-EPMC2962522 | biostudies-literature
Project description:Rat liver cells grown in primary cultures in the presence of [(35)S]sulphate synthesize a labelled heparan sulphate-like glycosaminoglycan. The characterization of the polysaccharide as heparan sulphate is based on its resistance to digestion with chondroitinase ABC or hyaluronidase and its susceptibility to HNO(2) treatment. The sulphate groups (including sulphamino and ester sulphate groups) are distributed along the polymer in the characteristic block fashion. In (3)H-labelled heparan sulphate, isolated after incubation of the cells with [(3)H]galactose, 40% of the radioactive uronic acid units are l-iduronic acid, the remainder being d-glucuronic acid. The location of heparan sulphate at the rat liver cell surface is demonstrated; part of the labelled polysaccharide can be removed from the cells by mild treatment with trypsin or heparitinase. Further, a purified plasma-membrane fraction isolated from rats previously injected with [(35)S]sulphate contains radioactively labelled heparan sulphate. A proteoglycan macromolecule composed of heparan sulphate chains attached to a protein core can be solubilized from the membrane fraction by extraction with 6m-guanidinium chloride. The proteoglycan structure is degraded by treatment with papain, Pronase or alkali. The production of heparan [(35)S]sulphate by rat liver cells incubated in the presence of [(35)S]sulphate was followed. Initially the amount of labelled polysaccharide increased with increasing incubation time. However, after 10h of incubation a steady state was reached where biosynthetic and degradative processes were in balance.
| S-EPMC1164759 | 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