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

Abstract

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.

Keywords: X-ray; glycosylation; mass spectrometry.

Fig. 1.

Stoichiometric modification of T466 with O-fucose glycans. (A) Diagramatic representation of EGF domains 12 and 13 of hN1, with cysteines highlighted in yellow and residues responsible for coordination of calcium highlighted in red. O-fucose and -glucose glycans added in this study are indicated at the appropriate sites. EGF11 is not shown because it lacks O-glycosylation consensus sites. (B–F) Synthesis of O-fucose monosaccharide, disaccharide, trisaccharide, and tetrasaccharide forms of hN1_11–13. Representative HPLC profiles of hN1_11–13 incubated with the appropriate enzymes and donor substrates as indicated are shown. The masses of the species in each peak as determined by mass spectrometry are shown, indicating addition of the appropriate sugar. mAU, milli-absorbance units. * indicates oxidized species; # indicates an unmodified substrate. (B) No donor, Pofut1, and Lfng. (C) GDP–fucose, Pofut1, and Lfng. (D) GDP–fucose, UDP–GlcNAc, Pofut1, and Lfng. (E) GDP–fucose, UDP–GlcNAc, UDP–galactose, Pofut1, Lfng, and β4-galactosyltransferase. (F) After overnight incubation of E, CMP–sialic acid and α3-(N)sialyltransferase were added to an aliquot and incubated for an additional 6 h. (G) Extracted ion chromatograms (EICs) of the ions corresponding to unmodified (black line), O-fucose monosaccharide (red line), and disaccharide glycoforms (blue line) of the peptide containing the O-fucose consensus sequence from EGF12, ⁴⁵²DVNECVSNPCQNDATCL⁴⁶⁸, derived from Asp-N digests of hN1_11–13 modified with O-fucose monosaccharide (from C). See SI Appendix, Fig. S1A for description of the relevant ions. (H) EICs of the ions corresponding to the unmodified (black line), O-fucose monosaccharide (red line), and disaccharide (blue line) glycoforms of the same peptide from Asp-N digests of hN1_11–13 modified with O-fucose disaccharide (from D). See SI Appendix, Fig. S1B for description of the relevant ions.

Fig. 2.

Flow-cytometry analysis of the effect of the addition of sugars to T466 on binding to Notch ligands. (A–C) Flow cytometry of B16 cells expressing Jagged1 (A), B16 cells expressing DLL4 (B), or CHO cells expressing DLL1 (C) after interaction with biotinylated hN1_11–13 modified with various sugars at T466 (red line) bound to avidin-coated fluorescent beads. In each case, a representative trace is shown. Binding was compared with a negative control (fibrillin-1 cbEGF_12–14; gray shading) and positive control (hN1_11–13 WT; black line). A bimodal distribution is observed for the negative control in the presence of B16 cells due to fluorescence from unbound beads (Materials and Methods). (A) The effect on binding of each of the four separate sugar modifications is shown; the addition of the O-fucose monosaccharide (+mono) causes a small increase in binding compared with unmodified protein (indicated by the small rightward shift compared with the positive control), and the addition of the GlcNAc–fucose disaccharide (+di) causes a much larger increase in binding (indicated by a further shift to the right). Binding of hN1_11–13 modified with the trisaccharide (+tri) or tetrasaccharide (+tetra) is indistinguishable from the disaccharide. (B) None of the sugar modifications had any apparent effect on the interaction between hN1_11–13 and DLL4. (C) The addition of the O-fucose monosaccharide causes a slight increase in binding to DLL1, and the addition of the GlcNAc–fucose causes a much larger increase in binding. (D) Untransfected B16 and CHO cells do not bind unmodified or disaccharide (+di) hN1_11–13. (E) FACS binding data normalized to unmodified Notch1_11–13 with SD shown. For Jagged1, increased binding for the disaccharide, trisaccharide, and tetrasaccharide was found to be significant (*) by Tukey’s multiple comparison test (P = 0.0028, 0.0074, and 0.0220), as was increased binding of the disaccharide to DLL1 (P = 0.0001). None of the sugar modifications was found to significantly alter binding to DLL4.

Fig. 3.

SPR analysis of the effect of the addition of sugars to T466 on binding to Notch ligands. (A) Representative SPR traces for binding of 10 μM unmodified (black), monosaccharide (gray), disaccharide (cyan), and trisaccharide (brown) hN1_11–13 constructs over ∼3,000 response units (RU) Jagged1_NE3-Fc (Left), DLL1_NE3-Fc (Center), and DLL4_NE3-Fc (Right). Traces are shown corrected for refractive index changes seen in the control channel (Materials and Methods). (B) SPR binding of unmodified and glycosylated hN1_11–13 constructs to NE3-Fc fusion constructs of human Jagged1 (green), DLL1 (red), and DLL4 (blue) normalized to the monosaccharide construct, with SD shown. Increased binding to the disaccharide and trisaccharide for Jagged1_NE3-Fc and DLL1_NE3-Fc was found to be significant by Tukey’s multiple comparison test. *P < 0.0001.

Fig. 4.

Structure of O-fucosylated variants of hN1_11–13. (A) Superimposed X-ray structures of the unmodified hN1_11–13 (PDB ID code 2VJ3) and the monosaccharide and disaccharide structures with the subsequent additions to the T466 region highlighted. Ca²⁺ ions are shown in red. (B) hN1₁₂ disaccharide X-ray structure highlighting contacts between the C₆ methyl group of the O-fucose with Ile₄₇₇ (yellow) and Met₄₇₉ (orange), the C₆ methoxy group of GlcNAc with Asp₄₆₄ (pink), and the N-acetyl group of GlcNAc with Met₄₇₉ (orange). Thr₄₆₆ is highlighted in cyan. (C) hN1₁₂ unmodified and disaccharide X-ray structures highlighting residues shown to contribute to Jagged1 binding in a previous mutagenesis study (red); those residues that did not contribute to binding are also shown (blue) (32). T466 is highlighted in yellow, together with the disaccharide; the sugar may extend the known ligand-binding surface or act indirectly to promote complex formation.