| الوصف: |
The Notch signalling pathway is conserved in all metazoan species and plays a crucial role in development and tissue maintenance. Canonical Notch signalling requires cell-cell contact to allow the interaction between Drosophila Notch receptor and its ligands, Serrate and Delta. The Notch Abruptex (Ax) region comprises 24-29 of the 36 epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Mutations in the Ax region give rise to three distinct phenotypes in Drosophila. Notch EGF repeats 11-12 form the ligand binding region (LBR). The recently solved structure of the module at the N-terminal of Notch ligands (MNNL) of a human Notch ligand, Jagged1, revealed that the domain was a calcium-binding C2 domain with Ca2+-dependent lipid binding. This study aimed to investigate the intra- and intermolecular properties of Drosophila Notch Ax region, LBR, and the MNNL of Notch ligand Serrate. In WT Drosophila Notch EGF23-25, all three EGF domains were found to be Ca2+-binding, and a previously unknown Ca2+ binding consensus sequence was identified. AxN-suppressor mutations D948V and N986I were shown to impair the Ca2+-binding properties of the mutant EGF domain without affecting the neighbouring domains, suggesting a mechanism to explain the signalling phenotype associated with this mutation type. Notch EGF11-13 showed Ca2+-binding in each EGF domain and binding to ligand-expressing cells. Its C-terminal tag was found to influence the Ca2+-dependent fold of EGF13, suggesting a future strategy for protein expression. A Serrate fragment MNNL-EGF3 showed Ca2+-dependent lipid binding, which was not observed in a construct lacking MNNL. The lipid binding could be reduced by a substitution D197A in MNNL, suggesting this mutant could be used to probe functional importance of MNNL in model organism studies. Binding between Serrate and Notch was assessed with a new cell aggregation assay method using flow cytometry, and agreed with previously published studies. Binding to Delta was subsequently measured, which suggested ligand specific differences although Notch residue L504 was important for both Serrate- and Delta-binding. Collectively these studies establish that Drosophila Notch and its ligand Serrate has similar properties to mammalian homologues, which will facilitate future structural and functional studies. |