Structural and Functional Properties of the Human Notch-1 Ligand Binding Region

Hambleton, Sophie; Valeyev, Najl V.; Muranyi, Andreas; Knott, Vroni; Werner, Jörn M.; McMichael, Andrew J.; Handford, Penny A.; Downing, A. Kristina

doi:10.1016/j.str.2004.09.012

Cited by 106 publications

(124 citation statements)

References 56 publications

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“…S11). By contrast, another NMR study (41), performed on hN1 [11][12][13] at physiologically relevant Ca 2+ concentrations, demonstrated that EGF12 residues are characterized by order parameters >0.8, suggesting that this domain does not contain backbone segments that are dynamic on a fast timescale (42,43). Our crystal structures, obtained in the presence of Ca 2+ , demonstrate that the addition of sugars to T466 does not appear to induce a conformational change either in the orientation of the Thr side chain or more globally in the overall structure of the EGF12 domain, or alter the packing interactions it makes with adjacent cbEGF domains.…”

Section: Discussionmentioning

confidence: 90%

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

Taylor

Takeuchi

Sheppard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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123

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The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.glycosylation | mass spectrometry | X-ray

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Section: Discussionmentioning

confidence: 90%

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

Taylor

Takeuchi

Sheppard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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123

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“…EGF 28 is in the Abruptex region of Notch, named for a series of mutations in Drosophila Notch that result in a hyperactive Notch refractory to Fringe (17,62). Recent structural studies have suggested that the presence of calcium-binding motifs reduces the flexibility of the linker between adjacent EGF repeats (63). Most of the EGF repeats in Notch1 contain the calcium-binding motif, and as a result, the linkers are predicted to be rigid (Fig.…”

Section: Discussionmentioning

confidence: 99%

O-Glucose Trisaccharide Is Present at High but Variable Stoichiometry at Multiple Sites on Mouse Notch1

Rana¹,

Nita‐Lazar²,

Takeuchi

et al. 2011

Journal of Biological Chemistry

120

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Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C 1 XSXPC 2 . We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site-and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, nontraditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C 1 XSX(A/P)C 2 . We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.The Notch family of single-pass transmembrane receptors is essential for early metazoan development, activating the expression of many genes involved in cell differentiation and tissue morphogenesis (1-3). Defects in Notch signaling have been implicated in a number of human diseases, including several forms of cancer, vascular defects such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (4 -6), multiple sclerosis (6), and a number of developmental syndromes (4, 7-10). The canonical Notch signaling pathway is initiated by the interaction of Notch with its ligand on an apposed cell. Upon ligand binding, Notch undergoes presenilin-1-dependent proteolysis, releasing a soluble Notch intracellular domain, which enters the cell nucleus to interact directly with the transcription factors from the CSL family and regulate Notch target genes (2). There are four members of the Notch family in vertebrates (Notch1 to Notch4) interacting with several classes of Notch ligands: ligands of the DSL family (Delta-like 1, 3, and 4 and Jagged 1 and 2) (1) and newly characterized ligands without the DSL domain, such as DNER and MAGP-1 and -2 (11-13).The Notch proteins consist of a large extracellular domain (ECD), 5 a transmembrane region, and a large intracellular domain (1, 2, 14). The majority of the ECD consists of tandem epidermal growth factor-like (EGF) repeats (36 EGF repeats are ...

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“…Deletion experiments have confirmed that EGF 11 and 12 constitute the Notch ligand binding domain [28]. While Notch1 EGF repeats 11-13 made in E.coli, and therefore not O-fucosylated, bind to Delta-expressing cells, this was only observed after tetramerization of the Notch fragment [29]. In fact, elimination of O-fucose from EGF 12 has profound conequences for Notch signaling.…”

Section: Elimination or Acquisition Of O-fucose Sites In Notchmentioning

confidence: 97%