PMID: 21757702

 

    Legend: Sugar

Title : O-glucose trisaccharide is present at high but variable stoichiometry at multiple sites on mouse Notch1

Abstract :

1. Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both
2. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 ( mN1 ) using the consensus sequence C(1) XSXPC(2)
3. We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent
4. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type
5. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9
6. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2 : C(1) XSX(A/P) C(2)
7. We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats.
8. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc.
9. To establish the functional importance of individual O-glucose sites in mN1 , we used a cell-based signaling assay
10. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites
11. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch , does significantly reduce activity
12. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1 , and they play important roles in Notch function