Significance of EGF Repeat Glycosylation on Ligands Involved in Notch Signaling

The Notch signaling pathway is a fundamental cellular communication mechanism that is conserved across all metazoans. Notch activation occurs when a ligand on a signal-sending cell physically interacts with a receptor in a signal-receiving cell. In contrast, when Notch ligands and receptors are expressed in the same cell, their interactions inhibit the ability of the cell to send or receive signals. This process is called "cis-inhibition", and is a critical mechanism of pathway regulation. Because aberrant activation of Notch has been demonstrated in many diseases, understanding the mechanisms that regulate cis-inhibition is important. One such regulator of Notch activity is glycosylation of the EGF (epidermal growth factor-like) repeats of receptors. This mechanism begins with the addition of an O-linked fucose, which can then be extended by FRINGE family glycosyltransferases. This specific glycosylation modifies the strength of ligand/receptor interactions in cis and in trans. Ligands can also be glycosylated, but the functional relevance of this modification is not known. To examine the importance of ligand glycosylation, we utilized sequence analysis to identify ligand EGF repeats that contain consensus sequences for fucosylation. We identified two potential targets on the ligand DLK (delta-like) 1, on EGF repeats 3 and 6. DLK2 contains consensus sites on EGF repeats 2 and 5. We also identified several potential glycosylation motifs within JAGGED1 and are focusing on EGF repeats 4-6, which have been shown to be critical for cis-inhibition. After subcloning the relevant repeats, click chemistry utilizing a bioactive fucose demonstrated that fucosylation occurs on DLK1, DLK2, and JAGGED1. Furthermore, click chemistry assays using mutated constructs have revealed that EGF3 of DLK1 is not O-fucosylated. We are currently examining extension of the saccharide by FRINGE and the functional relevance of ligand glycosylation using mass spectroscopy and luciferase assays, respectively. Glycosylation is a relatively understudied modification in Notch signaling, this research could open the door for new insights into cell communication.Office of Undergraduate EducationUndergraduate Research OfficeA five-year embargo was granted for this item.Academic Major: Molecular Genetic