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Non-conserved, S-nitrosylated cysteines in glypican-1 react with N-unsubstituted glucosamines in heparan sulfate and catalyze deaminative cleavage.

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Author

Summary, in English

The membrane lipid-anchored glypicans (heparan sulfate proteoglycans) are present in both vertebrates and invertebrates and serve as important modulators of growth factors and morphogens during development. Their core proteins are similar and consist of a large N-terminal domain comprising 14 evolutionary conserved cysteines and a C-terminal stalk carrying the heparan sulfate side-chains and the lipid anchor. Cysteines in glypican-1 can be S-nitrosylated but their positions have not been identified. The recently determined crystal structure of the N-terminal domain of glypican-1 has revealed that all the evolutionary conserved cysteines form intramolecular disulfide bonds. However, glypican-1 contains two more, non-conserved cysteines in the C-terminal stalk, located near the heparan sulfate attachment sites. We show here that the non-conserved cysteines are free thiols as a glypican-1 core protein containing the C-terminal stalk could be biotinylated by biotin-BMCC. After S-nitrosylation by using an NO-donor and copper ions, the glypican-1 core protein was retained on an affinity matrix substituted with heparan sulfate oligosaccharides containing N-unsubstituted glucosamines. The protein was displaced with 0.2 M glucosamine but also by 2 mM ascorbate. In the latter case, the heparan sulfate of the affinity matrix was simultaneously cleaved into fragments containing anhydromannose. We propose that the S-nitrosocysteine residues interact with closely located N-unsubstituted glucosamines in the heparan sulfate side-chains of the glypican-1 proteoglycan. Addition of ascorbate induces a series of reactions that eventually releases heparan sulfate fragments with reducing terminal anhydromannose, presumably without the formation of free nitric oxide.

Department/s

Publishing year

2012

Language

English

Pages

1480-1485

Publication/Series

Glycobiology

Volume

22

Issue

11

Document type

Journal article

Publisher

Oxford University Press

Topic

  • Biochemistry and Molecular Biology

Status

Published

Research group

  • Glycobiology

ISBN/ISSN/Other

  • ISSN: 1460-2423