Notch receptors play an essential role in numerous stages of development. Deregulation of Notch signaling leads to a variety of human pathologies, including cancer, vascular disorders, and numerous developmental disorders. Notch activation is regulated at numerous levels. The Notch extracellular domain contains up to 36 tandem epidermal growth factor-like (EGF) repeats, many of which are predicted to be modified by two unusual forms of glycosylation: O-fucose and O-glucose. This observation has led to the hypothesis that Notch can be regulated by differential glycosylation of its extracellular domain. Recent studies from several laboratories have provided significant support for this hypothesis. For instance, genetic alteration of the gene encoding the enzyme responsible for addition of O-fucose to Notch, protein O-fucosyltransferase 1 (O-FucT-1), leads to severe embryonic lethal phenotypes closely resembling those associated with loss of Notch function. Similarly, mutations in the Fringe family of proteins (O-fucose specific b1, 3-N-acetylglucosaminyltransferases responsible for modification of O-fucose residues on Notch) show less severe, but significant Notch-like phenotypes. The proposed experiments are designed to provide further support for this hypothesis and to address the mechanism by which these sugars affect Notch function. To understand the mechanism by which O-fucose saccharides modulate Notch function, one must know where the modifications occur and what structures exist at each site.
In Aim 1 mass spectral methodologies will be used to map O-fucose and O-glucose modifications on Notch. Identification of enzymes involved in O-fucose glycan biosynthesis has been critical for evaluating the function of these modifications.
In Aim 2, the specificity and localization of Fringe and O-FucT-1 will be examined, and the enzyme responsible for adding O-glucose to Notch, the protein O-glucosyltransferase, will be identified.
In Aim 3, mutations in specific glycosylation sites on Notch, as well as mutations that prevent the action of Fringe, will be generated and analyzed for their effects on Notch function. The studies proposed in these three aims will provide fundamental information regarding the role of these unusual carbohydrate modifications in Notch activation.
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