Mechanisms underlying preferential assembly of heparan sulfate on glypican-1

Glypicans are major cell surface heparan sulfate proteoglycans, the structu res of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylph osphatidylinositol membrane anchor. Despite strong evolutionary conservatio n of the globular domains of glypicans, no function has yet been attributed to them. By using a novel quantitative approach for assessing proteoglycan glycosylation, we show here that removal of the globular domain from rat g lypican-1 converts the proteoglycan from one that bears similar to 90% hepa ran sulfate (HS) to one that bears similar to 90% chondroitin sulfate. Mut ational analysis shows that sequences at least 70 amino acids away from the glypican-1 GAG attachment site are required for preferential HS assembly, although more nearby sequences also play a role. The effects of the glypica n-1 globular domain on HS assembly could also be demonstrated by fusing thi s domain to sequences representing the GAG attachment sites of other proteo glycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed glypican l GAG; attachment domain or on endogenous proteoglycans. Quantitative anal ysis of the effect of the globular domain on GAG addition to proteoglycan c ore proteins suggested that preferential HS assembly is achieved, at least in part, through the inhibition of chondroitin sulfate assembly. These data identify the glypican-1 globular domain as a structural motif that potentl y influences GAG class determination and suggest that an important role of glypican globular domains is to ensure a high level of HS substitution of t hese proteoglycans.