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Abstract
Dynamic activities of cell-cell signaling by the Notch receptor and its ligands are necessary to metazoan development. Activation of the Notch receptor is primarily mediated by interactions between the Extracellular Domain (ECD) of Notch and the ECD of its ligands Jagged1, 2 and Delta-like 1, 3 and 4. Biological and structural data have demonstrated that glycosylation of epidermal growth factor-like (EGF) repeats is a major component of pathway function. O-Fucosylation of Thr/Ser residues in Notch EGF repeats by the mammalian Protein O-Fucosyltransferase 1 (POFUT1) is necessary for efficient receptor trafficking, and addition of N-acetylglucosamine by Fringe glycosyltransferases enhances activation by the Delta family ligands while inhibiting activation by the Jagged family ligands. To investigate the specificity of glycosylation by POFUT1 and the three mammalian Fringe enzymes, we used mass spectrometry to detect EGF repeats carrying O-glycan modifications in pure protein from NOTCH1, NOTCH2, and Delta family proteins. To address the physiological relevance of significant NOTCH1 O-fucose sites identified in cell-based assays, we generated two mouse lines with point mutations in NOTCH1 and show opposing phenotypes in the retinal angiogenesis system.