RECYCLING OF A GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED HEPARAN-SULFATE PROTEOGLYCAN (GLYPICAN) IN SKIN FIBROBLASTS

We have used suramin and brefeldin A to investigate the nature of a he paran sulphate proteoglycan that appears to recycle from the cell surf ace to intracellular compartments which synthesize new heparan sulphat e 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 glycosaminoglyca n assembly is blocked, by using brefeldin A, [H-3]glucosamine and [S-3 5]sulphate incorporation into cell surface-bound heparan sulphate prot eoglycan can still take place, After chemical biotinylation of cell su rface proteins in brefeldin A-treated cells, followed by metabolic [S- 35]sulphation in the presence of the same drug, biotin-tagged [S-35]pr oteoglycan can be demonstrated, indicating the presence of recycling p roteoglycan species, By pre-labelling cells with [H-3]leucine or [H-3] inositol in the presence of suramin, followed by chase labelling with [S-35]sulphate in the presence of brefeldin A, a H-3- and S-35-labelle d, hydrophobic heparan sulphate proteoglycan with a core protein of 60 -65 kDa is obtained, The proteoglycan loses its hydrophobicity when gl ucosamine-inositol bonds are cleaved, indicating that it is membrane b ound via a glycosylphosphatidylinositol anchor. However, treatment wit h phosphatidylinositol-specific phospholipase C has no effect, suggest ing that the inositol moiety may be acylated, We propose that a portio n of the lipid-anchored proteoglycan glypican is internalized, recycle d via the Golgi, where heparan sulphate chains are added, and finally re-deposited at the cell surface.