Mechanisms Underlying Preferential Assembly of Heparan Sulfate on Glypican-1

Abstract

Glypicans are major cell surface heparan sulfate proteoglycans, the structures of which are characterized by the presence of a cysteine-rich globular domain, a short glycosaminoglycan (GAG) attachment region, and a glycosylphosphatidylinositol membrane anchor. Despite strong evolutionary conservation 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 glypican-1 converts the proteoglycan from one that bears ϳ90% heparan sulfate (HS) to one that bears ϳ90% chondroitin sulfate. Mutational 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 glypican-1 globular domain on HS assembly could also be demonstrated by fusing this domain to sequences representing the GAG attachment sites of other proteoglycans or, surprisingly, simply by expressing the isolated globular domain in cells and analyzing effects either on an exogenously expressed glypican-1 GAG attachment domain or on endogenous proteoglycans. Quantitative analysis of the effect of the globular domain on GAG addition to proteoglycan core 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 potently influences GAG class determination and suggest that an important role of glypican globular domains is to ensure a high level of HS substitution of these proteoglycans.

Expression of Collagen XVIII and Localization of Its Glycosaminoglycan Attachment Sites

“…Recent studies, however, showed that the clear-cut identification of an SG consensus sequence as a glycosylation site or the prediction of the type of glycosylation at a specific site is impossible. Some peptide domains distant to the SG sites were shown also to be important in the glycosylation (23,24). Furthermore, the fact that endogenous and recombinant chick collagen XVIII is different in its GAG glycosylation suggests that additional regulatory factors such as the cell type in which the protein is expressed and cell culture conditions are also important for glycosylation.…”

Heparin and Heparan Sulfate Biosynthesis

“…Hydrophobic residues (aromatic and aliphatic) in close proximity to the Ser-Gly attachment site have been proposed to be an enhancer element for HS assembly. In addition, the sequence distant from the GAG attachment site, for example, on the glypican core protein, has a profound influence for selectively assembling an HS chain (34). In this context, it has been hypothesized that modifications of the linkage region tetrasaccharide may be involved in the selective assembly of CS and HS chains on the linkage region (14,35,36).…”

Human tumor suppressor EXT gene family members EXTL1 and EXTL3 encode α1,4- N -acetylglucosaminyltransferases that likely are involved in heparan sulfate/ heparin biosynthesis

“…Our observations that EXTL2 and EXTL3 transferred a GlcNAc residue to chemically synthesized disaccharides with a hydrophobic aglycon, but not to GlcA␤1-3Gal␤1-3Gal␤1-4Xyl␤1-O-Ser, are consistent with this concept. In addition, the importance of distant regions from the GAG attachment sites also has been emphasized recently in the preferential addition of HS chains (50): the cysteine-rich globular domain of glypican-1 is as much indispensable as the GAG attachment region to direct HS synthesis. If either EXTL3 or EXTL2 becomes associated with either EXT1 or EXT2, HS and͞or Hep chains possibly might be initiated and polymerized efficiently on the tetrasaccharide core.…”