Notch receptors are cell surface glycoproteins that transduce signals to control cell fate and cell division in metazoans. Constitutive or disregulated signaling leads to developmental defects and cancer. The ligands that bind to Notch and stimulate signal transduction are Delta and Serrate/Jagged. We have shown in collaboration with others that Drosophila and mammalian fringe proteins have a beta3N-acetylglucosaminyltransferase activity and act directly on Notch to add GIcNAc to O-fucose on Notch EGF repeats, thereby modulating ligand-induced Notch signaling. In Drosophila and mammalian cells, fringe acts cell autonomously to inhibit the Notch response to Serrate(Jagged) while potentiating the Notch response to Delta. We have used a co-culture reporter assay to show that manic or lunatic fringe in Notch expressing CHO cells inhibit the response to Jagged1. We have used a panel of CHO glycosylation mutants to show that inhibition of Notch signaling by fringe does not require complex or hybrid N-glycans or sialic acid but does require the action of fringe on O-fucose attached to Notch EGF repeats, and the subsequent action of beta4GalT- 1. Thus O. fucosyltransferase and beta4GalT-1 are novel modulators of Notch signaling. Using CHO glycosylation mutants, co-culture Notch signaling and soluble ligand binding assays, and mouse mutants, we propose 1 )To identify the different 0-fucose glycans required for Jagged- and Delta-induced signaling by Notch receptors 1, 2, 3 and 4 and for the modulation of Notch signaling by mammalian fringes (manic, lunatic and radical); 2) To determine the biochemical mechanism by which fringe action perturbs Notch signaling; and 3) To identify in vivo consequences to Notch signaling in the mouse of inactivating the genes encoding new modulators of Notch signaling.
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