Contribution of extracellular O-GlcNAc to the stability of folded epidermal growth factor-like domains and Notch1 trafficking

Ogawa, Masamichi; Tashima, Yohtalou; Sakaguchi, Yamato; Takeuchi, Hideyuki; Okajima, Tetsuya

doi:10.1016/j.bbrc.2020.03.066

Cited by 20 publications

(13 citation statements)

References 35 publications

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“…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).…”

Section: Hybrid Quadrupole Ft Linear Ion Trap Ms Analysis -mentioning

confidence: 74%

“…Furthermore, extracellular O-GlcNAc is reported to exert its function in other biological contexts, including regulatory T cell differentiation in autoimmune hepatitis (19) and the regulation of Adropin expression in the decidualizing human endometrium (20). The molecular function of O-GlcNAc has been analyzed using Notch receptors as model proteins (21); however, many other proteins are expected to be modified by EOGT (13,22). O-GlcNAcylation of EGF domains occurs on Thr/Ser residues located between the 5th and 6th conserved cysteines (8).…”

Section: Introductionmentioning

confidence: 99%

“…Comprehensive O-GlcNAc glycoproteomics on NOTCH1 EGF repeats O-GlcNAc is reported to exert its function in other biological contexts, including regulatory T cell differentiation in autoimmune hepatitis (Hao, X., Li, Y., et al 2018) and the regulation of Adropin expression in the decidualizing human endometrium (Muter, J., Alam, M.T., et al 2018). The molecular function of O-GlcNAc has been analyzed using Notch receptors as model proteins (Ogawa, M., Tashima, Y., et al 2020); however, many other proteins are expected to be modified by EOGT (Muller, R., Jenny, A., et al 2013, Pennarubia, F., Germot, A., et al 2021.…”

Section: Introductionmentioning

confidence: 99%

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

Tsukamoto

Ogawa

Yogi

et al. 2021

Preprint

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The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Notably, the Lewis X structure on O-glycans was identified in endogenous NOTCH1 isolated from MCF7 cells. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.HighlightsComprehensive analysis of O-GlcNAc glycan on NOTCH1 refined O-GlcNAcylation sequonsFUT1, FUT2, and FUT9 modify O-GlcNAc glycans in the selective EGF domains of NOTCH1The Lewis X epitope can be formed on both O-GlcNAc and O-fucose glycans on NOTCH1In BriefThis study performed comprehensive mass spectrometric analyses of O-GlcNAc glycans in the NOTCH1 extracellular region in cultured mammalian cells. The results indicated that EOGT-dependent O-GlcNAcylation occurs on a limited number of EGF repeats harboring putative O-GlcNAcylation sites, refining the sequons for extracellular O-GlcNAc modification in mammals. Moreover, novel structures with fucosylated O-GlcNAc glycans were identified, and a similar structure was detectable on O-fucose glycans, extending the view of structural diversity of functional Notch O-glycans.

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Section: Hybrid Quadrupole Ft Linear Ion Trap Ms Analysis -mentioning

confidence: 74%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Tsukamoto

Ogawa

Yogi

et al. 2021

Preprint

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“…Hence, the authors proposed that O-GlcNAc as well as calcium ions regulate the flexibility and rigidity of the connection of NOTCH1 EGF repeats [81]. Very recently, we demonstrated that similar to O-fucose and O-glucose [82], O-GlcNAc enhances the stability of a single EGF repeat, Drosophila NOTCH EGF20 [83]. Further insights are required into how O-GlcNAc alters the rigidity of sequential EGF repeats, whether O-GlcNAcylation and calcium ions affect each other, and if either of these affects the activation of Notch.…”

Section: O-glcnac Glycans In the Lbd Of Notchmentioning

confidence: 88%

Current Views on the Roles of O-Glycosylation in Controlling Notch-Ligand Interactions

Saiki

Okajima

et al. 2021

Biomolecules

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The 100th anniversary of Notch discovery in Drosophila has recently passed. The Notch is evolutionarily conserved from Drosophila to humans. The discovery of human-specific Notch genes has led to a better understanding of Notch signaling in development and diseases and will continue to stimulate further research in the future. Notch receptors are responsible for cell-to-cell signaling. They are activated by cell-surface ligands located on adjacent cells. Notch activation plays an important role in determining the fate of cells, and dysregulation of Notch signaling results in numerous human diseases. Notch receptors are primarily activated by ligand binding. Many studies in various fields including genetics, developmental biology, biochemistry, and structural biology conducted over the past two decades have revealed that the activation of the Notch receptor is regulated by unique glycan modifications. Such modifications include O-fucose, O-glucose, and O-N-acetylglucosamine (GlcNAc) on epidermal growth factor-like (EGF) repeats located consecutively in the extracellular domain of Notch receptors. Being fine-tuned by glycans is an important property of Notch receptors. In this review article, we summarize the latest findings on the regulation of Notch activation by glycosylation and discuss future challenges.

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“…The reaction was stopped by the addition of 900 ml of 100 mM EDTA at pH 8.2. The samples were analyzed by an HPLC system (Shimazu) equipped with a C18 column as described previously (43).…”

Section: In Vitro Eogt Activity Assaymentioning

confidence: 99%

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

Alam

Tsukamoto

Ogawa

et al. 2020

Journal of Biological Chemistry

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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.

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Contribution of extracellular O-GlcNAc to the stability of folded epidermal growth factor-like domains and Notch1 trafficking

Cited by 20 publications

References 35 publications

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

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

Current Views on the Roles of O-Glycosylation in Controlling Notch-Ligand Interactions

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

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