Abstract
O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.
Highlights
O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe
Expansion of the Number of Substrates for O-FucT-1 and Fringe—In this study, we have experimentally demonstrated that both Drosophila Notch ligands, Serrate and Delta, as well as their mammalian counterparts, Delta1 and Jagged1, are modified with O-fucose and can be further elongated with GlcNAc by Fringe
The Notch ligands define a new class of substrates for O-fucose modification and are only the third class of proteins identified in which O-fucose is further elongated by GlcNAc
Summary
Notch ligands Drosophila Deltaa Drosophila Serratea Human Jagged1a Rat Delta-like 1a a O-Fucose further elongated by Fringe. The C2XXGG(S/T)C3 consensus sequence exists in many other EGF repeat-containing proteins [16, 24], including all of the known Notch ligands. This raises the possibility that O-fucosylation occurs on both the Notch receptor and its ligands, and that modification of O-fucose saccharides on Notch ligands may contribute to the modulatory effects of Fringe on Notch signaling. We found that O-fucosylation can occur on EGF repeats that do not match the C2XXGG(S/T)C3 consensus sequence for O-fucosylation These observations suggest that the role of O-fucosylation in the modulation of Notch signaling and other processes is likely to be broader and more complex than suggested previously
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