Differential O-Glc elongation on the specific EGF repeat within a ligand binding domain regulates NOTCH1 signaling

Three types of O-linked glycosylation - O-glucose, O-fucose, and O-N-acetylglucosamine - are crucial for the function of Notch receptors, which regulates critical cell fate determination processes in a wide variety of contexts. O-Glucose glycans are added to serine residues located between the first and second conserved cysteines within the epidermal growth factor (EGF) -like repeats in the Notch extracellular domain. Previously, O-glucose glycans were shown to be extended to a trisaccharide structure with two xyloses via an 1-3 linkage. Our recent studies, however, indicated that the O-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 NOTCH3 EGF9 (equivalent to NOTCH1 EGF10), with no detection in any other EGF domains in NOTCH1-3. Sequence comparison and mutagenesis experiments identified one amino acid at position -2 of the fourth cysteine in the EGF domain as crucial for the galactose elongation of O-glucose glycans. We further demonstrated that this site-specific elongation of O-glucose on NOTCH1 EGF10 significantly impact ligand binding and signal transduction of NOTCH1. Our findings contribute to the understanding of the intricate regulatory mechanisms of Notch receptor function mediated by distinct positions and structures of O-glycans. Significance StatementNotch, a signaling receptor critical for development, differentiation, and homeostasis in multicellular organisms, undergoes multiple post-translational glycan modifications to regulate its function. While O-glucose glycan elongation by xylose was previously known, we identified an O-glucose glycan extended by galactose and Neu5Ac on specific epidermal growth factor (EGF) -like repeats of certain Notch paralogs, NOTCH1 and NOTCH3. As far as we know, this structure has not been reported on any other proteins. We identify the enzymes involved in the biosynthesis of this glycan and demonstrate that modifications at a specific site on Notch direct receptor functions, including ligand binding and Notch signaling.