Deciphering the Fringe-Mediated Notch Code: Identification of Activating and Inhibiting Sites Allowing Discrimination between Ligands

Fringe proteins are β3-N-acetylglucosaminyltransferases that modulate Notch activity by modifying O-fucose residues on epidermal growth factor-like (EGF) repeats of Notch. Mammals have three Fringes: Lunatic, Manic, and Radical. While Lunatic and Manic Fringe inhibit Notch1 activation from Jagged1 and enhance activation from Delta-like 1, Radical Fringe enhances signaling from both. We used a mass spectrometry approach to determine whether the variable effects of Fringes on Notch1 result from generation of unique glycosylation patterns on Notch1. We found that Lunatic and Manic Fringe modified similar sites on Notch1, while Radical Fringe modified a subset. Fringe modifications at EGF8 and EGF12 enhanced Notch1 binding to and activation from Delta-like 1, while modifications at EGF6 and EGF36 (added by Manic and Lunatic but not Radical) inhibited Notch1 activation from Jagged1. Combined, these results suggest that Fringe modifications “mark” different regions in the Notch1 extracellular domain for activation or inhibition. [Display omitted] •Fringe modulates Notch activity by addition of GlcNAc to O-fucose on EGF repeats•O-fucose was found on 17 EGF repeats, but only a few are modified by any Fringe•Fringe modifications at EGF8 and 12 enhance activation of Notch1 by Delta-like 1•Fringe modifications at EGF6 and 36 inhibit Notch1 activation by Jagged1 Kakuda and Haltiwanger examine how Fringes regulate Notch1. They show that Radical Fringe modifies a subset of EGF repeats modified by Lunatic and Manic Fringe. Modifications at distinct EGF repeats “mark” the Notch1 extracellular domain, either enhancing or inhibiting activity.