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.
Highlights
Lular O-GlcNAc modifications of transmembrane or secreted proteins [4]
The biological significance of extracellular OGlcNAc is further suggested by O-GlcNAc transferase (EOGT) mutations found in patients with Adams-Oliver syndrome, a rare disorder characterized by congenital limb abnormalities and scalp defects [5, 6]
The predicted N-glycosylation sites on EOGT are located in highly conserved regions in various mammalian species including Mus musculus, Rattus norvegicus (Norway rat), Oryctolagus cuniculus, Bos taurus, Sus scrofa, Homo sapiens, and Canis lupus familiaris (Fig. 1B)
Summary
The biological significance of extracellular OGlcNAc is further suggested by EOGT mutations found in patients with Adams-Oliver syndrome, a rare disorder characterized by congenital limb abnormalities and scalp defects [5, 6]. Genomic analysis of EOGT in Adams-Oliver syndrome patients revealed several missense and nonsense mutations, including W206S, R377Q, and 359Dfs*28, all of which result in the loss of enzyme activity [7]. We characterized mouse EOGT to determine all N-glycosylation sites, detailed N-glycan structures, and their effect on EOGT expression, localization, and enzymatic function. Epidermal growth factor-like (EGF) domain-specific OGlcNAc transferase (EOGT) is a luminal endoplasmic reticulum (ER) protein that modifies Ser/Thr residues conserved on a subset of EGF domains of secreted and transmembrane proteins [1,2,3]. Unlike OGT-mediated regulation of O-GlcNAcylated cytoplasmic, mitochondrial, and nuclear proteins, EOGT can modulate pericellular protein function directly by extracel-
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