Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules

Notch is a large cell-surface receptor known to be an essential player in a wide variety of developmental cascades, Here we show that Notch1 endogenou sly expressed in Chinese hamster ovary cells is modified with O-linked fuco se and O-linked glucose saccharides, two unusual forms of O-linked glycosyl ation found on epidermal growth factor-like (EGF) modules. Interestingly, b oth modifications occur as monosaccharide and oligosaccharide species, Thro ugh exoglycosidase digestions we determined that the O-linked fucose oligos accharide is a tetrasaccharide with a structure identical to that found on human clotting factor IX: Sia-alpha 2,3-Gal-beta 1,4-GlcNAc-beta 1,3-Fuc-al pha 1-O-Ser/Thr. The elongated form of O-linked glucose appears to be a tri saccharide. Notch1 is the first membrane-associated protein identified with either O-linked fucose or O-linked glucose modifications. It also represen ts the second protein discovered with an elongated form of O-linked fucose, The sites of glycosylation, which fall within the multiple EGF modules of Notch, are highly conserved across species and within Notch homologs, Since Notch is known to interact with its ligands through subsets of EGF modules , these results suggest that the O-linked carbohydrate modifications of the se modules may influence receptor-ligand interactions.