Two novel protein O -glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling

Significance The Notch-signaling pathway is normally activated by receptor–ligand interactions. Extracellular domains (ECDs) of Notch receptors are heavily modified withO -linked glycans, such asO -glucose (O -Glc),O -fucose (O -Fuc), andO -GlcNAc. The significance of multiple types ofO -glycans on Notch is not understood. NOTCH1 ECD interacts with ligands at multiple points, including anO -Glc monosaccharide on the 11th Epidermal Growth Factor (EGF) repeat (EGF11). Here, we identify two novel proteinO -glucosyltransferases that modify NOTCH1 EGF11 withO -Glc. Combined deletion of theO -Glc site on EGF11 withO -Fuc modification sites on EGF8 or EGF12 markedly reduced NOTCH1 cell-surface expression or activation of NOTCH1 by Delta-like ligand 1, respectively. This study identifies a cooperative mechanism for fine-tuning the Notch-signaling pathway by different types ofO -glycans.