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

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.

SUBMITTER: Turnbull JE

PROVIDER: S-EPMC1151232 | biostudies-other | 1991 Jul

REPOSITORIES: biostudies-other

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Project description:The structure of human skin fibroblast heparan sulphate has been examined by depolymerization with heparinase, which specifically cleaves highly sulphated disaccharides of structure GlcNSO3 (+/-6S)-alpha 1,4IdoA(2S) [N-sulphated glucosamine (6-sulphate)-alpha 1,4-iduronic acid 2-sulphate]. Heparan sulphate contained only a small proportion (approximately 10%) of linkages susceptible to this enzyme. The major products of depolymerization with heparinase were large oligosaccharides with an average molecular mass of 10 kDa (dp approximately 40, where dp is degree of polymerization; for disaccharides, dp = 2 etc.) as assessed by gel filtration on Sepharose CL-6B, compared with a molecular mass of 45 kDa (dp approximately 200) for the intact chains. The large heparinase-resistant oligosaccharides were highly susceptible to depolymerization with the enzyme heparitinase, which cleaves heparan sulphate in areas of low sulphation, where N-acetylated disaccharides [GlcNAc-alpha 1,4GlcA (N-acetylglucosaminyl-alpha 1,4-glucuronic acid)] are the predominant structural unit. Further analysis of the location of the heparinase cleavage sites indicated that they were predominantly found in a central position in GlcNSO3-alpha 1,4IdoA repeat sequences of average length four to seven disaccharides (dp 8-14). These results indicate that heparinase cleaves heparan sulphate in approximately four or five N-sulphated domains, each domain containing a cluster of two or three susceptible disaccharides; the domains are separated by long N-acetyl-rich sequences that are markedly deficient in sulphate groups. On the basis of these findings a model is proposed which depicts heparan sulphate as an ordered polymeric structure composed of an alternate arrangement of sulphate-rich and sulphate-poor regions. The sulphate-rich regions are likely to be flexible areas of the chain because of their high content of the conformationally versatile IdoA and IdoA(2S) residues. The model has important implications for the biosynthesis and functions of heparan sulphate.

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

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