title: In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe.
abstract: Notch signaling plays critical roles in animal development and physiology. The activation of Notch receptors by their ligands is modulated by Fringe-dependent glycosylation. Fringe catalyzes the addition of N-acetylglucosamine in a beta1,3 linkage onto O-fucose on epidermal growth factor-like domains. This modification of Notch by Fringe influences the binding of Notch ligands to Notch receptors. However, prior studies have relied on in vivo glycosylation, leaving unresolved the question of whether addition of N-acetylglucosamine is sufficient to modulate Notch-ligand interactions on its own, or whether instead it serves as a precursor to subsequent post-translational modifications. Here, we describe the results of in vitro assays using purified components of the Drosophila Notch signaling pathway. In vitro glycosylation and ligand binding studies establish that the addition of N-acetylglucosamine onto O-fucose in vitro is sufficient both to enhance Notch binding to the Delta ligand and to inhibit Notch binding to the Serrate ligand. Further elongation by galactose does not detectably influence Notch-ligand binding in vitro. Consistent with these observations, carbohydrate compositional analysis and mass spectrometry on Notch isolated from cells identified only N-acetylglucosamine added onto Notch in the presence of Fringe. These observations argue against models in which Fringe-dependent glycosylation modulates Notch signaling by acting as a precursor to subsequent modifications and instead establish the simple addition of N-acetylglucosamine as a basis for the effects of Fringe on Drosophila Notch-ligand binding.
authors.affiliation: Howard Hughes Medical Institute, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
authors.forename: Aiguo
authors.initials: A
authors.lastname: Xu
authors.forename: Nicola
authors.initials: N
authors.lastname: Haines
authors.forename: Malgosia
authors.initials: M
authors.lastname: Dlugosz
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Rana
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haltiwanger
authors.forename: Kenneth D
authors.initials: KD
authors.lastname: Irvine
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002241:Carbohydrates / Q000737:chemistry; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000378:metabolism*; D005643:Fucose / Q000737:chemistry; D005786:Gene Expression Regulation; D006031:Glycosylation; D066298:In Vitro Techniques; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D011485:Protein Binding; D051880:Receptors, Notch; D000072099:Serrate-Jagged Proteins; D019076:Transgenes
chemical_list: D002135:Calcium-Binding Proteins; D002241:Carbohydrates; D029721:Drosophila Proteins; D036341:Intercellular Signaling Peptides and Proteins; D047908:Intracellular Signaling Peptides and Proteins; D000072100:Jagged-1 Protein; D008024:Ligands; D008565:Membrane Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch; C000606278:Ser protein, Drosophila; D000072099:Serrate-Jagged Proteins; C064045:delta protein; D005643:Fucose; D017351:N-Acetylglucosaminyltransferases; C090576:fng protein, Drosophila