Carbohydrate modifications of epidermal growth factor-like modules and the O-linked fucose glycosylation pathway

Epidermal growth factor-like (EGF) modules are a common structural motif found within numerous extracellular proteins and are often involved in protein-protein interactions. Recently, two unusual forms of glycosylation were found on the EGF modules of a small number of proteins: O-linked fucose and O-linked glucose. In these modifications, the sugar, fucose or glucose, is directly attached to protein through the hydroxyl group of either serine or threonine. O-linked fucose was found on proteins containing the putative consensus sequence for O-linked fucose addition, CXXGGS/TC. Likewise, O-linked glucose was found on proteins containing the putative consensus sequence for O-linked glucose addition, CXSXPC. In order to study these modifications, we used Chinese hamster ovary culture-cells as our model system. Through our investigations, we discovered a previously unrecognized glycosylation system in mammals called "the O-linked fucose glycosylation pathway". O-linked fucose may be elongated with a beta-linked glucose residue to form a disaccharide or, alternatively, with a beta-linked N-acetylglycosamine resulting in the formation of a tetrasaccharide. These findings are unusual because fucose is commonly found as a terminal saccharide in mammals. We identified and characterized the enzymatic activity responsible for formation of the O-linked fucose disaccharide: a uridine diphosphoglucose: O-fucose beta1,3 glucosyltransferase. We showed that this enzyme activity is present in many species indicating that this is a widespread modification in biology. We also demonstrated that the EGF modules of mammalian Notch I, a biologically important receptor involved in numerous developmental pathways, are modified with O-linked fucose and O-linked glucose saccharides. Both modifications were found as monosaccharide and elongated species. Many of the predicted glycosylation sites fall within functionally important EGF modules and are conserved across species, suggesting that these modifications are biologically significant features of Notch 1 and may be crucial for Notch I receptor signalling events. These studies have enriched our understanding of the glycosylation of EGF modules with these novel sugar modifications.