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

The membrane lipid-anchored glypicans (Gpcs) [heparan sulfate (HS) proteoglycans (PGs)] 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 HS side chains and the lipid anchor. Cysteines in Gpc-1 can be S-nitrosylated but their positions have not been identified. The recently determined crystal structure of the N-terminal domain of Gpc-1 has revealed that all the evolutionary conserved cysteines form intramolecular disulfide bonds. However, Gpc-1 contains two more, non-conserved cysteines in the C-terminal stalk, located near the HS attachment sites. We show here that the non-conserved cysteines are free thiols as a Gpc-1 core protein containing the C-terminal stalk could be biotinylated by 1-biotinamido-4-(4'-[maleimidomethyl-cyclohexane]-carboxyamido)butane. After S-nitrosylation by using a nitric oxide (NO) donor and copper ions, the Gpc-1 core protein was retained on an affinity matrix substituted with HS oligosaccharides containing N-unsubstituted glucosamines (GlcNH(2)/NH(3)(+)). The protein was displaced with 0.2 M glucosamine but also by 2 mM ascorbate. In the latter case, the HS of the affinity matrix was simultaneously cleaved into fragments containing anhydromannose (anMan). We propose that the S-nitrosocysteine residues interact with closely located GlcNH(2)/NH(3)(+) in the HS side chains of the Gpc-1 PG. Addition of ascorbate induces a series of reactions that eventually releases HS fragments with reducing terminal anMan, presumably without the formation of free NO.