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.

Keywords: EGF domain-specific O-GlcNAc transferase (EOGT); EOGT; N-linked glycosylation; Notch receptor; O-GlcNAc; O-linked N-acetylglucosamine (O-GlcNAc); endoplasmic reticulum; endoplasmic reticulum (ER); glycoprotein; oligomannose glycan; posttranslational modification; protein folding; protein maturation.

Figure 1.

EOGT is modified with N-glycans. A, schematic representation of mouse EOGT structure. Three N-glycosylation sequons, the N-terminal signal peptide, and the KDEL-like ER retention signal are indicated. B, multiple alignment of amino acid sequences from seven mammalian EOGT. Amino acid sequences and amino acid numbers surrounding the putative N-glycosylation sites are shown. The conserved N-glycosylation sites are highlighted by gray boxes. The amino acid residues identical to mouse EOGT are indicated by dashes. Three EOGT mutations found in the cancer database are also indicated. C, FLAG-EOGT was expressed in HEK293T cells and immunoprecipitated from the cell lysates by FLAG antibody. FLAG-EOGT was incubated with PNGase F to partially (left) or completely (right) remove N-glycans. EOGT-CT antibody was used for immunoblotting to detect the full-length form of EOGT. Partially or completely digested products are indicated by open or closed arrowheads, respectively. D, Eogt-transfected HEK293T cells were treated with or without tunicamycin for 24 h, and cell lysates were analyzed by immunoblotting using FLAG and GFP antibodies. Immunoblot bands corresponding to partially or completely deglycosylated products are indicated by open or closed arrowheads, respectively. GFP bands show similar transfection efficiency in the samples.

Figure 2.

Identification of two N-glycosylation sites on mouse EOGT. A, schematic representation showing the generation of FLAG-EOGT variants containing N-glycosylation site mutations. Asparagine (N) residues were replaced with glutamine (Q) residues. B, each FLAG-EOGT isoform was expressed in HEK293T cells and immunoprecipitated by FLAG antibody. FLAG-EOGT was incubated with PNGase F to partially remove N-glycans. EOGT-CT antibody was used to detect full-length EOGT. C, HEK293T cells were transfected to express each FLAG-EOGT isoform and treated with or without tunicamycin for 48 h. Cell lysates were analyzed by immunoblotting with EOGT-CT and α-tubulin antibodies. After immunoblotting, the PVDF membrane was stained with Coomassie Brilliant Blue (CBB) as the protein loading control. D, FLAG-EOGT harboring cancer-related mutations (S265A, S265L, and T356I) were expressed in HEK293T cells and treated with or without Endo H. WT and N263Q FLAG-EOGT were analyzed in parallel as controls. After immunoblotting with EOGT-CT and α-tubulin antibodies, the PVDF membrane was stained with Coomassie Brilliant Blue as the protein loading control.

Figure 3.

EOGT is modified with oligomannose N-glycans. A, lectin blot analysis of FLAG-EOGT isoforms. HEK293T cells were transfected to express each FLAG-EOGT isoform. The cell lysates were subjected to immunoprecipitation with FLAG-antibody followed by detection by biotinylated ConA lectin (ConA-biotin) or EOGT-CT antibody. An asterisk indicates nonspecific bands. B, LC–MS/MS spectra of glycopeptides modified with HexNAc2Hex9 N-glycan at N263 (top) and HexNAc2Hex7 N-glycan at Asn-354 (bottom) of FLAG-EOGT. Chymotryptic or tryptic glycopeptides prepared from recombinant FLAG-EOGT were analyzed by LC–MS/MS. Fragments ions corresponding to b and y ions, peptides with truncated glycans, and glycans are shown by arrows. Blue square, HexNAc (presumably GlcNAc); green circle, hexose (presumably mannose). C, bar graphs showing relative abundance of different N-glycan glycoforms at EOGT Asn-263 and Asn-354. D, endogenous EOGT sensitivity to Endo H digestion. HEK293T cell lysates were incubated in the absence or presence of Endo H and analyzed by immunoblotting with EOGT-specific AER61 antibody. Recombinant FLAG-EOGT and FLAG-EOGT^N263Q/N354Q were analyzed in parallel as controls.

Figure 4.

N-Glycans on EOGT are required for efficient O-GlcNAcylation. A, schematic representation of mutant alleles for EOGT newly generated by CRISPR/Cas9-mediated gene editing in HEK293T cells. B, detection of endogenous EOGT in cell lysates of HEK293T cells or EOGT-knockout (KO) HEK293T cells using EOGT-specific AER61 antibody. The immunoblot (IB) was re-probed using an α-tubulin antibody and stained with Coomassie Briliant Blue (CBB) as the protein loading control. C, immunoblotting with CTD110.6 antibody for detection of O-GlcNAc epitopes on FLAG-Notch1-TM. EOGT-knockout HEK293T cells were transfected to express FLAG-Notch1-TM together with WT or mutant Eogt. After purification with FLAG antibody-coated beads, FLAG-Notch1-TM was analyzed using the indicated antibodies. D, quantification of the relative O-GlcNAc level on FLAG-Notch1-TM. The band intensity, as shown in C, was measured using ImageJ software. The data were obtained from three independent transfection experiments and presented together with mean ± S.D. values. *, p < 0.01; the p values are from unpaired Welch's t test.

Figure 5.

Reduced O-GlcNAc stoichiometry on Notch1 in HEK293T cells expressing N-glycan-deficient EOGT. A, MS analysis of tryptic glycopeptides prepared from FLAG-Notch1-TM harboring O-GlcNAcylation sites. FLAG-Notch1-TM was expressed in EOGT-deficient HEK293T cells exogenously expressing WT or the N263Q/N354Q EOGT. EICs show the relative signal intensity corresponding to the peptides without modification (orange) or with O-GlcNAc (blue) on EGF2, EGF10, EGF11, EGF21, and EGF23 of Notch1. Note that no elongated O-GlcNAc glycoforms were detected. Data are presented as mean ± S.D. (n = 2). LC–MS/MS spectra of tryptic glycopeptides are shown in Fig. S3. B, quantification of O-GlcNAc glycans on representative EGF domains. EIC peak heights were measured and expressed as percent area. The color code is same as described in A.

Figure 6.

N-Glycans are dispensable for the enzymatic activity of EOGT. A, bacterially expressed dEGF20 was analyzed by HPLC with a 30-min linear gradient (10–80% ACN in 0.1% TFA). Absorbance at 280 nm was monitored. The fraction containing folded dEGF20 is highlighted in red. B, HPLC analysis of enzymatic products. In vitro O-GlcNAc transferase assay was performed using dEGF20 as an acceptor substrate, UDP-GlcNAc as a donor substrate, and WT or mutated FLAG-EOGT as an enzyme or buffer as a negative control. C, Coomassie staining showing the purified WT and mutant FLAG- EOGT. An empty well is marked by #. D, the enzymatic activities of the respective FLAG-EOGT isoforms measured by UDP-Glo assay using an equal amount of purified enzymes. The results are expressed as mean ± S.D. from three assays performed independently.

Figure 7.

N-Glycans affect the protein expression of EOGT. A, immunoblot (IB) analysis for protein expression of WT and mutant EOGT. Each FLAG-EOGT isoform was co-expressed together with GFP that served as an internal control of protein expression in HEK293T cells. Cell lysates were analyzed with an EOGT-CT or a GFP antibody. B, quantification of EOGT protein levels normalized to GFP. The data were obtained from three independent transfection experiments and presented together with mean ± S.D. values. **, p < 0.05; the p values are from unpaired Welch's t test. C, detection of FLAG-EOGT isoforms in the soluble and insoluble fractions. Cells stably expressing WT or mutant EOGT were lysed in TBS buffer containing 0.1% digitonin. Soluble/insoluble fractions were separated and detected with an EOGT-CT antibody. After immunoblotting, the PVDF membrane was stained with Coomassie Brilliant Blue (CBB) as the protein loading control. The data are representative of two independent transfection experiments. D, purified FLAG-EOGT was incubated with or without PNGase F. After 3 h, soluble/insoluble fractions of reaction mixtures were separated and detected with EOGT-CT antibody. n.s., not significant.

Figure 8.

Loss of N-glycans on EOGT diminished its peripheral ER localization. A, subcellular localization of FLAG-EOGT and EOGT mutants. Confocal microscopy was performed on HEK293T cells transiently transfected with WT FLAG-Eogt or one of its N-glycosylation mutants. Staining was performed with anti-FLAG (for EOGT, green), anti-calnexin (ER marker, red), and phalloidin (white). It is noted that EOGT staining at cell periphery is diminished, whereas the perinuclear staining remained in the N263Q/N354Q mutant (arrowheads). Scale bar, 5 μm. Additional data are shown in Fig. S5A. B, staining intensity ratio between peripheral and perinuclear regions in the images shown in A. The results were obtained from eight transfected cells in a single experiment. Similar results were obtained in three independent transfection experiments. Data are presented as mean ± S.D. ***, p < 0.001; the p values are from unpaired Welch's t test. C, subcellular localization of Notch1EGF1-36:MycHis. Confocal microscopy imaging was performed on HEK293T cells transiently transfected with Notch1EGF1-36:MycHis alone or together with FLAG-Eogt or its mutant. Staining was performed with anti-Myc (for Notch1EGF1-36, green) and anti-calnexin. Scale bar, 5 μm. D, staining intensity ratio between peripheral and perinuclear regions in the images shown in C. The results were obtained from eight transfected cells in a single experiment. Similar results were obtained in three independent transfection experiments. Data are presented as mean ± S.D.

Figure 9.

Decreased peripheral ER localization of endogenous EOGT in cells treated with tunicamycin. A, detection of endogenous EOGT by the specific AER61 antibody. B, subcellular localization of endogenous EOGT in HEK293T or HeLa cells. Cells were treated with or without tunicamycin for 48 h and stained with the EOGT-specific AER61 (green) antibody, calnexin (red) antibody, and phalloidin (white). Scale bar, 5 μm. C, staining intensity ratio between peripheral and perinuclear regions in the images shown in B. The results were obtained from eight transfected cells in a single experiment. Similar results were obtained in three independent transfection experiments. Data are presented as mean ± S.D. ***, p < 0.001; the p values are from unpaired Welch's t test. D, subcellular localization of endogenous EOGT in endothelial cells. HUVEC cells were treated with or without tunicamycin for 48 h and stained with the EOGT-specific AER61 (green) antibody and phalloidin (white). Scale bar, 5 μm.