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.

Bioinformatics and Functional Analyses Implicate Potential Roles for EOGT and L-fringe in Pancreatic Cancers

“…Then, cells were counter-stained with DAPI (Southern Biotech, Birmingham, AL, USA). All the slides were examined by TiEA1R Confocal Microscopy with NIS Elements (Nikon, Japan) [ 52 ].…”

Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats implicated unique Lewis X epitopes in mammals

“…The antibodies used were anti-Lewis X antibody Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats reported a comprehensive analysis of whole O-Glc and O-Fuc sites in the same mouse NOTCH1 expressed in HEK293T cells (28,29). In particular, our study showed that the 16 EGF domains (EGF2, 4,10,12,13,14,16,19,20,21,25,27,28,31,33) with putative O-Glc sites were O-glucosylated, except for EGF9 (28).…”

“…Plasmid constructs -Expression vectors for NOTCH1 EGF repeats (pSectag2C/mNotch1-EGF1-36:MycHis-IRES-EGFP), EOGT (pSectag2C/EOGT), and dEGF20 ΔGlcΔFuc (pSectag2C/dEGF20 ΔGlcΔFuc :MycHis-IRES-EGFP) have been previously reported (18,23). Expression vectors for NOTCH1 EGF24-28 repeats (pSectag2C/mNotch1-EGF [24][25][26][27][28]:MycHis) have been reported…”

“…Cell culture and stable cell line -HEK293T cells, EOGT-deficient HEK293T cells (25), MCF7 cells, and H1299 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen). To express full-length or partial NOTCH1 EGF repeats, the expression vectors were transfected into HEK293T cells with or without those encoding the indicated glycosyltransferases using PEI-MAX, as described previously (23).…”

“…The glycosyltransferase responsible for this modification is EOGT, the endoplasmic reticulum (ER)localized O-GlcNAc transferase (5)(6)(7)(8)(9). More than ten proteins, including Notch1 and thrombospondin-1 (TSP-1), are substrates of EOGT in mammals (10)(11)(12)(13)(14).…”