Site‐Specific Analysis of O ‐Fucose and O ‐Glucose Glycans on Notch

The extracellular domain (ECD) of the Notch receptor contains up to 36 tandem Epidermal Growth Factor‐like (EGF) repeats, many of which are modified at consensus sites with one or both of two unusual forms of O ‐glycosylation: O ‐fucose and O ‐glucose. O ‐Fucose can be elongated to a tetrasaccharide, Siaα2–3Galβ1–4GlcNAcβ1–3Fucα‐ O ‐Ser/Thr, and O ‐glucose to the trisaccharide Xylα1–3Xylα1–3Glcβ‐ O ‐Ser. Genetic, cell biological, and biochemical studies reveal that both O ‐fucose and O ‐glucose glycans are essential for Notch function, and elongation past the monosaccharide modulates Notch function. In order to understand the molecular mechanism by which these glycans affect Notch activity, the presence and structure of the glycans at specific sites must be determined. We have used nano‐LC‐MS/MS glycoproteomic methods to map glycans to predicted sites on mouse Notch1 expressed in cells in the presence or absence of β3GnT's of the Fringe family (Lunatic, Manic, or Radical Fringe). We have also mapped glycans on Drosophila Notch expressed in cells in the presence or absence of Fringe. Using semi‐quantitative mass spectral methods, we have analyzed site occupancy and extent of elongation at each site. Our results suggest that individual O ‐fucose or O ‐glucose sites are modified at high but variable stoichiometries, and elongation beyond the monosaccharide is site specific. Supported by NIH grant GM061126.