| Glypicans are major cell-surface proteoglycans that reliably carry heparan sulfate (HS) in vivo. Their core proteins consist of a globular domain, a single glycosaminoglycan (GAG) attachment domain with a cluster of 2--4 consecutive Ser-Gly dipeptides flanked by acidic residues, and a glycosylphosphatidylinositol membrane anchor. Using site-directed mutagenesis and a novel approach for measuring GAG substitution, we assessed how different core protein elements control glypican-1 glycanation by cultured cell lines. Deletion of the globular domain converted the proteoglycan from one that bore ∼90% HS to one that bore ∼90% chondroitin sulfate (CS). The effect required amino acids at least 70 residues from the GAG attachment site, and could be exerted on heterologous GAG attachment domains to which the globular domain had been fused. Preferential substitution with HS was also promoted by the multiplicity of Ser-Gly dipeptides (weakly) and by a short acidic cluster (strongly) in the GAG attachment domain. Interestingly, the globular domain and the acidic cluster had large, but opposite, effects on the fraction of molecules produced lacking any GAG. The results suggest a specific, sequential pathway for specifying GAG addition, in which local and distant core protein elements act at discrete steps. |
