title: Recycling of a glycosylphosphatidylinositol-anchored heparan sulphate proteoglycan (glypican) in skin fibroblasts.
abstract: We have used suramin and brefeldin A to investigate the nature of a heparan sulphate proteoglycan that appears to recycle from the cell surface to intracellular compartments which synthesize new heparan sulphate chains. Suramin, which would block internalization and deglycanation of a putative recycling cell surface proteoglycan, markedly increases the yield of a membrane-bound proteoglycan with a core protein of 60-70 kDa and unusually long heparan sulphate side chains. When transport of newly made core proteins to their Golgi sites for glycosaminoglycan assembly is blocked, by using brefeldin A, [3H]glucosamine and [35S]sulphate incorporation into cell surface-bound heparan sulphate proteoglycan can still take place. After chemical biotinylation of cell surface proteins in brefeldin A-treated cells, followed by metabolic [35S]sulphation in the presence of the same drug, biotin-tagged [35S]proteoglycan can be demonstrated, indicating the presence of recycling proteoglycan species. By pre-labelling cells with [3H]leucine or [3H]inositol in the presence of suramin, followed by chase labelling with [35S]sulphate in the presence of brefeldin A, a 3H- and 35S-labelled, hydrophobic heparan sulphate proteoglycan with a core protein of 60-65 kDa is obtained. The proteoglycan loses its hydrophobicity when glucosamine-inositol bonds are cleaved, indicating that it is membrane bound via a glycosylphosphatidylinositol anchor. However, treatment with phosphatidylinositol-specific phospholipase C has no effect, suggesting that the inositol moiety may be acylated. We propose that a portion of the lipid-anchored proteoglycan glypican is internalized, recycled via the Golgi, where heparan sulphate chains are added, and finally re-deposited at the cell surface.
authors.affiliation: Department of Cell and Molecular Biology, Lund University, Sweden.
authors.forename: L A
authors.initials: LA
authors.lastname: Fransson
authors.forename: G
authors.initials: G
authors.lastname: Edgren
authors.forename: B
authors.initials: B
authors.lastname: Havsmark
authors.forename: A
authors.initials: A
authors.lastname: Schmidtchen
mesh_terms: D001710:Biotin / Q000378:metabolism; D020126:Brefeldin A; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003517:Cyclopentanes / Q000494:pharmacology; D004622:Embryo, Mammalian; D005347:Fibroblasts / Q000378:metabolism*; D005944:Glucosamine / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D007294:Inositol / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011509:Proteoglycans / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D013498:Suramin / Q000494:pharmacology; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D001710:Biotin; D020126:Brefeldin A; D003517:Cyclopentanes; D005944:Glucosamine; D017261:Glycosylphosphatidylinositols; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D007294:Inositol; D011500:Protein Synthesis Inhibitors; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D013498:Suramin; D014316:Tritium; D010738:Type C Phospholipases