Differential <i>O</i> -glucose elongation on a specific EGF repeat within the canonical ligand–binding domain regulates DLL1/4-NOTCH1 signaling

Three types of <i>O</i>-linked glycosylation-<i>O</i>-glucose, <i>O</i>-fucose, and <i>O</i>-<i>N</i>-acetylglucosamine- are crucial for the function of Notch receptors, which regulate critical cell fate determination processes in a wide variety of contexts. <i>O</i>-Glucose glycans are transferred to serine residues located between the first and second conserved cysteines within the epidermal growth factor-like (EGF) repeats in the Notch extracellular domain. Previously, <i>O</i>-glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via α1-3 linkages. Our recent studies, however, indicated that the <i>O</i>-glucose glycan on NOTCH1 EGF10 can be extended by hexose and Neu5Ac. Here, we demonstrated that this hexose- and Neu5Ac-extended glycan has a 3'-sialyllactose-like structure synthesized by specific members of two isoenzyme families, B4GALT1 and ST3GAL4. Using mass spectrometry, we identified this modification exclusively on NOTCH1 EGF10 and the analogous NOTCH3 EGF9 domain, with no detection in any other EGF domains in NOTCH1, NOTCH2, and NOTCH3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine (C⁴-2) in the EGF domain as crucial for the galactose elongation of <i>O</i>-glucose glycans. We further demonstrated that this site-specific elongation of <i>O</i>-glucose on NOTCH1 EGF10 significantly impacts ligand binding and signal transduction of NOTCH1. In the context of early T cell development, the C⁴-2 mutants NOTCH1 A396Y and A396F enhance T cell differentiation through DLL1- and DLL4-dependent NOTCH1 signaling. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of <i>O</i>-glycans.