Tsukamoto, N. | bioRxiv Science

bioRxiv2025

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.

biochemistry↗

bioRxiv2024

Retention of DLK1 in the endoplasmic reticulum identifies roles for EGF domain-specific O-glycans in the secretory pathway

In the endoplasmic reticulum (ER), O-glycosylation by O-fucose, O-glucose, and O-GlcNAc occurs in the epidermal growth factor-like (EGF) domains of secreted or transmembrane glycoproteins. Previous studies focusing on Notch receptors have revealed the pivotal role of these O-glycans in the cell surface expression of Notch or secretion of truncated Notch fragments. Although it has been demonstrated that O-fucose, O-glucose, and O-GlcNAc stabilize individual EGF domains, their role in the secretory pathway after the completion of the folding process remains unexplored. In this study, we used delta-like 1 homolog (DLK1) containing six consecutive EGF domains as a model glycoprotein to investigate the role of EGF domain-specific O-glycans in the secretory pathway. Semi-quantitative site-specific glycoproteomics of recombinantly expressed DLK1 revealed multiple O-fucose and O-glucose modifications in addition to an unusual EOGT-dependent O-hexose modification. Consistent with the results of the secretion assay, inactivation of the glycosyltransferases modifying O-fucose and O-glucose, but not the newly identified O-hexose, perturbed the transport of DLK1 from the ER during retention using the selective hooks (RUSH) system. Importantly, the absence of O-fucose did not result in an apparent loss of O-glucose modification within the same EGF domain, and vice versa. Given that protein O-fucosyltransferase 1 and protein O-glucosyltransferase 1 activities depend on the folded state of the EGF domains, O-glycans affected DLK1 transport independently of the folding process required for O-glycosylation in the ER. These findings highlight the distinct roles of O-glycans in facilitating the transport of DLK1 from the ER to the cell surface.

biochemistry↗