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dc.contributor.authorTurnbull, Jeremy E
dc.contributor.authorGallagher, John T
dc.date.accessioned2010-06-21T14:02:26Z
dc.date.available2010-06-21T14:02:26Z
dc.date.issued1991-07-15
dc.identifier.citationSequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region. 1991, 277 ( Pt 2):297-303 Biochem. J.en
dc.identifier.issn0264-6021
dc.identifier.pmid1859357
dc.identifier.urihttp://hdl.handle.net/10541/106579
dc.description.abstractA strategy that we originally used to identify an N-acetylated domain adjacent to the protein-linkage sequence of heparan sulphate proteoglycan (HSPG) [Lyon, Steward, Hampson & Gallagher (1987) Biochem. J. 242, 493-498] has been adapted for analysis of the location of GlcNSO3-HexA and GlcNSO3(+/- 6S)-IdoA(2S) units most proximal to the core protein. [3H]Glucosamine-labelled HSPG from human skin fibroblasts was depolymerized by using HNO2 or heparinase under conditions that allowed cleavage of all susceptible linkages. The degraded PG was coupled to Sepharose beads through the protein component, enabling specific recovery of protein-linked resistant oligosaccharides. These were released by treatment with alkaline borohydride and analysed by gel filtration and gradient PAGE. This strategy allowed investigation of the sequence of sugar residues along the chain relative to a common reference point (i.e. the reducing end of the chain). HNO2 scission confirmed the presence of a well-defined N-acetylated sequence predominantly 9-12 disaccharide units in length proximal to the core protein. Heparinase scission produced two classes of oligosaccharides (Mr approx. 7000 and 15,000) with the general formula: IdoA(2S)-GlcNSO3-[HexA-GlcNR]n-HexA-GlcNSO3-[Hex A-GlcNAc]9 12-GlcA-Gal-Gal-Xyl in which the average value for n is 1-2 for the 7000-Mr species and approx. 22 for the 15,000-Mr species. The latter oligosaccharides extend to about one-third of the total length of the HS chains (Mr approx. 45,000). HNO2 scission of these oligosaccharides enabled hypothetical models for their sequence to be proposed. The general arrangement of N-sulphated and N-acetylated disaccharides between the proximal GlcNSO3 and terminal IdoA(2S) residues of the 15,000-Mr fragment was similar to that in the original polysaccharide, suggesting the possibility of a tandemly repeating pattern in the sequence of HS.
dc.language.isoenen
dc.subject.meshCarbohydrate Sequence
dc.subject.meshCells, Cultured
dc.subject.meshChromatography, Gel
dc.subject.meshFibroblasts
dc.subject.meshGlucosamine
dc.subject.meshHeparan Sulfate Proteoglycans
dc.subject.meshHeparin Lyase
dc.subject.meshHeparitin Sulfate
dc.subject.meshHumans
dc.subject.meshIduronic Acid
dc.subject.meshModels, Structural
dc.subject.meshMolecular Sequence Data
dc.subject.meshOligosaccharides
dc.subject.meshPolysaccharide-Lyases
dc.subject.meshProteochondroitin Sulfates
dc.subject.meshSkin
dc.subject.meshTritium
dc.titleSequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region.en
dc.typeArticleen
dc.contributor.departmentDepartment of Clinical Research, University of Manchester Christie Hospital and Holt Radium Institute, U.K.en
dc.identifier.journalBiochemical Journalen
html.description.abstractA strategy that we originally used to identify an N-acetylated domain adjacent to the protein-linkage sequence of heparan sulphate proteoglycan (HSPG) [Lyon, Steward, Hampson & Gallagher (1987) Biochem. J. 242, 493-498] has been adapted for analysis of the location of GlcNSO3-HexA and GlcNSO3(+/- 6S)-IdoA(2S) units most proximal to the core protein. [3H]Glucosamine-labelled HSPG from human skin fibroblasts was depolymerized by using HNO2 or heparinase under conditions that allowed cleavage of all susceptible linkages. The degraded PG was coupled to Sepharose beads through the protein component, enabling specific recovery of protein-linked resistant oligosaccharides. These were released by treatment with alkaline borohydride and analysed by gel filtration and gradient PAGE. This strategy allowed investigation of the sequence of sugar residues along the chain relative to a common reference point (i.e. the reducing end of the chain). HNO2 scission confirmed the presence of a well-defined N-acetylated sequence predominantly 9-12 disaccharide units in length proximal to the core protein. Heparinase scission produced two classes of oligosaccharides (Mr approx. 7000 and 15,000) with the general formula: IdoA(2S)-GlcNSO3-[HexA-GlcNR]n-HexA-GlcNSO3-[Hex A-GlcNAc]9 12-GlcA-Gal-Gal-Xyl in which the average value for n is 1-2 for the 7000-Mr species and approx. 22 for the 15,000-Mr species. The latter oligosaccharides extend to about one-third of the total length of the HS chains (Mr approx. 45,000). HNO2 scission of these oligosaccharides enabled hypothetical models for their sequence to be proposed. The general arrangement of N-sulphated and N-acetylated disaccharides between the proximal GlcNSO3 and terminal IdoA(2S) residues of the 15,000-Mr fragment was similar to that in the original polysaccharide, suggesting the possibility of a tandemly repeating pattern in the sequence of HS.


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