Highly conserved O-fucose sites have distinct effects on Notch1 function

Area of Science:

• Cellular and Molecular Biology
• Glycobiology
• Signal Transduction

Background:

• Notch1 signaling is crucial for cell fate decisions and is regulated by O-fucosylation of its extracellular epidermal growth factor-like (EGF) repeats.
• The precise role of individual O-fucose sites in Notch1 activation and signaling remains incompletely understood.

Purpose of the Study:

• To investigate the functional importance of specific O-fucose sites within the Notch1 receptor for ligand-mediated signaling.
• To determine which O-fucose sites are essential for Notch1 processing and cell surface presentation.

Main Methods:

• Site-directed mutagenesis of invariant and less conserved O-fucose sites in mouse Notch1.
• Evaluation of mutant Notch1 receptor signaling in response to Jagged1 and Delta1 ligands using a cell-based assay.
• Assessment of Notch1 receptor trafficking and S1 processing.

Main Results:

• Mutations in the three invariant O-fucose sites (EGF repeats 12, 26, and 27) significantly altered Delta1 and Jagged1 signaling.
• Mutation of EGF repeat 12 O-fucose site abolished signaling; EGF repeat 26 O-fucose site mutation led to hyperactivation.
• Mutation of EGF repeat 27 O-fucose site impaired receptor trafficking and S1 processing, while less conserved sites had no effect.

Conclusions:

• The most conserved O-fucose sites in Notch1 are critical for proper receptor processing and ligand-induced signaling.
• Distinct invariant O-fucose sites differentially regulate Notch1 signaling outcomes, influencing both activation and receptor maturation.