Fringe-mediated extension of
            <i>O</i>
            -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

Taylor, Paul; Takeuchi, Hideyuki; Sheppard, Devon; Chillakuri, Chandramouli; Lea, Susan M.; Haltiwanger, Robert S.; Handford, Penny A.

doi:10.1073/pnas.1319683111

Cited by 99 publications

(149 citation statements)

References 49 publications

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“…Significantly, addition of the O-glucose trisaccharide did not alter the conformation of the EGF repeat. We had previously shown that addition of O-fucose monosaccharide or disaccharide to EGF12 of Notch1 also does not induce conformational change of the EGF repeat, but similar to what we observed here, the O-fucose glycan has multiple interactions with underlying amino acids (40). Similar results have been reported by other investigators (37,39).…”

Section: O-glycans Modulate Egf Protein Stabilitysupporting

confidence: 92%

“…We further compared the above-described structures with the structure of EGF12 either unmodified or modified with an O-fucose monosaccharide or an O-fucose disaccharide (Fig. 5, A and E-G) (40), and we found that O-fucose glycans are on the opposite side of the protein from where the O-glucose glycans attach. This result supports our cell biological and biochemical observation that the effects of O-fucose and O-glucose on EGF repeats are independent and therefore additive.…”

Section: Structural Analysis Of Hfa9 Egf Repeat Modified With Oglucosmentioning

confidence: 99%

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O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

Takeuchi

Hao

et al. 2017

Journal of Biological Chemistry

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Glycosylation in the endoplasmic reticulum (ER) is closely associated with protein folding and quality control. We recently described a non-canonical ER quality control mechanism for folding of thrombospondin type 1 repeats by protein O-fucosyltransferase 2 (POFUT2). Epidermal growth factor-like (EGF) repeats are also small cysteine-rich protein motifs that can be O-glycosylated by several ER-localized enzymes, including protein O-glucosyltransferase 1 (POGLUT1) and POFUT1. Both POGLUT1 and POFUT1 modify the Notch receptor on multiple EGF repeats and are essential for full Notch function. The fact that POGLUT1 and POFUT1 can distinguish between folded and unfolded EGF repeats raised the possibility that they participate in a quality control pathway for folding of EGF repeats in proteins such as Notch. Here, we demonstrate that cell-surface expression of endogenous Notch1 in HEK293T cells is dependent on the presence of POGLUT1 and POFUT1 in an additive manner. In vitro unfolding assays reveal that addition of O-glucose or O-fucose stabilizes a single EGF repeat and that addition of both O-glucose and O-fucose enhances stability in an additive manner. Finally, we solved the crystal structure of a single EGF repeat covalently modified by a full O-glucose trisaccharide at 2.2 Å resolution. The structure reveals that the glycan fills up a surface groove of the EGF with multiple contacts with the protein, providing a chemical basis for the stabilizing effects of the glycans. Taken together, this work suggests that O-fucose and O-glucose glycans cooperatively stabilize individual EGF repeats through intramolecular interactions, thereby regulating Notch trafficking in cells.

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Section: O-glycans Modulate Egf Protein Stabilitysupporting

confidence: 92%

Section: Structural Analysis Of Hfa9 Egf Repeat Modified With Oglucosmentioning

confidence: 99%

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

Takeuchi

Hao

et al. 2017

Journal of Biological Chemistry

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“…In our mass spectral analyses, we were particularly interested in determining which O-fucose sites were modified by Fringe because those sites should play important roles in the effects Fringe has on Notch. Recent structural studies on the ligand binding domain of mammalian Notch1 show that the O-fucose glycans on EGF12 directly interact with ligands upon binding (22,50). From the EIC analysis, we observed that Fringe elongates some sites more efficiently than others.…”

Section: Xxxx(s/t)cmentioning

confidence: 73%

“…Loss of Ofut1/Pofut1 in flies or mice results in Notch-null phenotypes, indicating that O-fucosylation is essential for Notch function (18 -20). The ␤3GlcNAc-transferases of the Fringe family can extend O-fucose in flies and mammals (21) and most notably play important roles in modulating Notch-ligand binding (22)(23)(24)(25). In mammals, the GlcNAc␤1-3Fuc-O-Ser/Thr disaccharide can be further elongated to a tetrasaccharide: Sia␣2-6Gal␤1-4GlcNAc␤1-3Fuc-O-Ser/Thr (26).…”

mentioning

confidence: 99%

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch

Harvey

Rana

Moss

et al. 2016

Journal of Biological Chemistry

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Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the ␤3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.

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“…The analysis by Luca and colleagues also revealed how the addition of O-fucose and O-glucose to threonine and serine residues on Notch1 functionalizes the receptor by making specific and essential contacts with residues on DLL4. Collaborative work from Robert Haltiwanger (Stony Brook University, USA) and Handford further showed how Fringemediated addition of GlcNAc to O-fucose increases binding of Notch1 to Notch ligands (Taylor et al, 2014). These observations illustrate how post-translational modifications of ligand-binding sites on Notch proteins that are regulated by specific biosynthetic pathways provide a strategy for regulating Notch function during development.…”

Section: Notch-ligand Interactions and The Mechanics Of Notch Activationmentioning

confidence: 90%

The Notch meeting: an odyssey from structure to function

Chitnis

Bally‐Cuif

2016

Development

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There was an error published in Development 143, 547-553.The name of the second author was spelt incorrectly. This error has now been corrected in the print, online and PDF versions of the paper.We apologise to the authors for this mistake. 1229 ABSTRACTThe Notch signaling pathway plays fundamental roles in diverse developmental processes. Studies of the basic biology of Notch function have provided insights into how its dysfunction contributes to multi-systemic diseases and cancer. In addition, our understanding of Notch signaling in maintaining stem/progenitor cell populations is revealing new avenues for rekindling regeneration. The Notch IX meeting, which was held in Athens, Greece in October 2015, brought together scientists working on different model systems and studying Notch signaling in various contexts. Here, we provide a summary of the key points that were presented at the meeting. Although we focus on the molecular mechanisms that determine Notch signaling and its role in development, we also cover talks describing roles for Notch in adulthood. Together, the talks revealed how interactions between adjacent cells mediated by Notch regulate development and physiology at multiple levels.

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Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

Cited by 99 publications

References 49 publications

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

O-Glycosylation modulates the stability of epidermal growth factor-like repeats and thereby regulates Notch trafficking

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch

The Notch meeting: an odyssey from structure to function

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