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.
Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Centre, Lund, Sweden.
Fang
F
Cheng
Gabriel
G
Svensson
Lars-Åke
LÅ
Fransson
Katrin
K
Mani
D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry; D005944:Glucosamine / Q000737:chemistry*; D053673:Glypicans / Q000737:chemistry*; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D026403:S-Nitrosothiols / Q000737:chemistry*
D003545:Cysteine; D005944:Glucosamine; D053673:Glypicans; D006497:Heparan Sulfate; D026403:S-Nitrosothiols; C021315:S-nitrosocysteine