Sequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region.

2.50
Hdl Handle:
http://hdl.handle.net/10541/106579
Title:
Sequence analysis of heparan sulphate indicates defined location of N-sulphated glucosamine and iduronate 2-sulphate residues proximal to the protein-linkage region.
Authors:
Turnbull, Jeremy E; Gallagher, John T
Abstract:
A 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.
Affiliation:
Department of Clinical Research, University of Manchester Christie Hospital and Holt Radium Institute, U.K.
Citation:
Sequence 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.
Journal:
Biochemical Journal
Issue Date:
15-Jul-1991
URI:
http://hdl.handle.net/10541/106579
PubMed ID:
1859357
Type:
Article
Language:
en
ISSN:
0264-6021
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorTurnbull, Jeremy Een
dc.contributor.authorGallagher, John Ten
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.en
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

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