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

Abstract

Epidermal growth factor (EGF) domain-specific O -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 EOGT mutations that may affect putative N -glycosylation consensus sites are recorded in the cancer database, but the presence and function of N -glycans in EOGT have not yet been characterized. Here, we identified N -glycosylation sites in mouse EOGT and elucidated their molecular functions. Three predicted N -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 N -glycans. Lectin blotting, endoglycosidase H digestion, and MS analysis revealed that both residues are modified with oligomannose N -glycans. Loss of an individual N -glycan on EOGT did not affect its endoplasmic reticulum (ER) localization, enzyme activity, and ability to O -GlcNAcylate Notch1 in HEK293T cells. However, simultaneous substitution of both N -glycosylation sites affected both EOGT maturation and expression levels without an apparent change in enzymatic activity, suggesting that N -glycosylation at a single site is sufficient for EOGT maturation and expression. Accordingly, a decrease in O -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 N -glycosylation of EOGT is required for its ER localization at the cell periphery. These results suggest critical roles of N -glycans in sustaining O -GlcNAc transferase function both by maintaining EOGT levels and by ensuring its proper subcellular localization in the ER.