S-Nitrosylation of secreted recombinant human glypican-1.
Glypican-1 is a glycosylphosphatidylinositol anchored cell surface S-nitrosylated heparan sulfate proteoglycan that is processed by nitric oxide dependent degradation of its side chains. Cell surface-bound glypican-1 becomes internalized and recycles via endosomes, where the heparan sulphate chains undergo nitric oxide and copper dependent autocleavage at N-unsubstituted glucosamines, back to the Golgi. It is not known if the S-nitrosylation occurs during biosynthesis or recycling of the protein. Here we have generated a recombinant human glypican-1 lacking the glycosylphosphatidylinositol-anchor. We find that this protein is directly secreted into the culture medium both as core protein and proteoglycan form and is not subjected to internalization and further modifications during recycling. By using SDS-PAGE, Western blotting and radiolabeling experiments we show that the glypican-1 can be S-nitrosylated. We have measured the level of S-nitrosylation in the glypican-1 core protein by biotin switch assay and find that the core protein can be S-nitrosylated in the presence of copper II ions and NO donor. Furthermore the glypican-1 proteoglycan produced in the presence of polyamine synthesis inhibitor, alpha-difluoromethylornithine, was endogenously S-nitrosylated and release of nitric oxide induced deaminative autocleavage of the HS side chains of glypican-1. We also show that the N-unsubstituted glucosamine residues are formed during biosynthesis of glypican-1 and that the content increased upon inhibition of polyamine synthesis. It cannot be excluded that endogenous glypican-1 can become further S-nitrosylated during recycling.
Svensson
Gabriel
G
Department of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University, Biomedical Center A13, SE-221 84, Lund, Sweden.
Mani
Katrin
K
D002460:Cell Line; D003641:Deamination / Q000187:drug effects; D000518:Eflornithine / Q000494:pharmacology; D005822:Genetic Vectors; D005944:Glucosamine / Q000378:metabolism; D053673:Glypicans / Q000302:isolation & purification / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism*
D053673:Glypicans; D019812:Heparan Sulfate Proteoglycans; D011073:Polyamines; D011994:Recombinant Proteins; D009569:Nitric Oxide; D005944:Glucosamine; D000518:Eflornithine