-Glycans on EGF domain-specific -GlcNAc transferase (EOGT) facilitate EOGT maturation and peripheral endoplasmic reticulum localization.

Epidermal growth factor (EGF) domain-specific -GlcNAc transferase (EOGT) is an endoplasmic reticulum (ER)-resident protein that modifies EGF repeats of Notch receptors and thereby regulates Delta-like ligand-mediated Notch signaling. Several mutations that may affect putative -glycosylation consensus sites are recorded in the cancer database, but the presence and function of -glycans in EOGT have not yet been characterized. Here, we identified -glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted -glycosylation consensus sequences on EOGT are highly conserved among mammalian species. Within these sites, we found that Asn-263 and Asn-354, but not Asn-493, are modified with -glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose -glycans. Loss of an individual -glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to -GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both -glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that -glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in -GlcNAc stoichiometry was observed in Notch1 co-expressed with an N263Q/N354Q variant compared with WT EOGT. Moreover, the N263Q/N354Q variant exhibited altered subcellular distribution within the ER in HEK293T cells, indicating that -glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of -glycans in sustaining -GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.