O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

Department of Biochemistry and Cell Biology, Institute of Cell and Developmental Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 07/2011; 286(36):31623-37. DOI: 10.1074/jbc.M111.268243
Source: PubMed


Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C(1)XSXPC(2). We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site- and cell type-dependent. 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. O-Glucose is also found at a novel, non-traditional consensus site at EGF 9. 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). 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. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. 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. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. 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.

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    • "We recently revised this to C 1 -X-S-X(P/A )-C 2 based on our biochemical and mass spectral analyses of O-glucose glycan modification sites on mouse Notch1 and Drosophila Notch [67]. Database searches for this consensus sequence identify over 40 proteins predicted to be O-glucosylated, although like O-fucose, the Notch family of receptors has more consensus sites than any other protein (Fig. 1) [67]. O-Glucose glycans are also essential for Notch activity in both mice and flies [66] [68]. "
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