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

In the endoplasmic reticulum (ER), O-glycosylation by O-fucose, O-glucose, and O-N-acetylglucosamine (O-GlcNAc) occur in the epidermal growth factor-like (EGF) domains of secreted or transmembrane glycoproteins. Previous studies on Notch receptors have revealed the pivotal role of these O-glycans in the cell surface expression of Notch and 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 following the completion of the folding process remains unexplored. In this study, we used delta-like homolog 1 (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, along with an unusual EGF-domain-specific O-linked N-acetylglucosamine transferase (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, as assessed using the selective hooks (RUSH) system. Importantly, the absence of O-fucose did not affect O-glucose modification within the same EGF domain, and vice versa. Since 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.