Characterising the Notch-ligand binding interaction, and its modulation by glycosylation

The Notch signalling pathway is universally conserved in all metazoan species, and is involved in many aspects of cell fate determination and tissue homeostasis, during development and in adult organisms. Several developmental diseases are associated with defective Notch signalling, and the Notch pathway has been implicated in a growing number of cancers. The Notch signalling pathway requires direct cell-cell contact for ligand binding and receptor activation to occur. Specific domains within the Notch receptors and ligands have been identified as necessary for the interaction to take place, and a series of enzymes are known to regulate Notch signalling via glycosylation. Other domains beyond the minimal ligand binding region of the Notch receptor are also known to influence binding. The aim of this study was to characterise the molecular basis for ligand binding by the Notch receptor, and how this is regulated by glycosylation. The effects on ligand binding of specific amino acid substitutions and sugar modifications were tested using prokaryotically -expressed proteins, and a series of constructs containing additional domains N-terminal of the ligand binding region was produced prokaryotically and eukaryotically to test how additional domains might affect ligand binding. Binding was assessed by a flow cytometry-based binding assay and by SPR in order to investigate how particular modifications affected ligand binding. These assays indicated that an evolutionarily-conserved hydrophobic site exists within the central β-sheet of EGF12 in the Notch receptor that is directly adjacent to the O-fucosylation site within this domain. The GlcNAc-fucose disaccharide modification at this position was found to increase binding of hNotch1 to both Jagged1 and DLL4. Additional EGF domains N-terminal to the ligand binding region showed opposite effects on binding to these two ligand classes, suggesting that the precise mode of binding may vary slightly between different Notch ligands.EThOS - Electronic Theses Online ServiceGBUnited Kingdo