title: O-fucosylation of the notch ligand mDLL1 by POFUT1 is dispensable for ligand function.
abstract: Fucosylation of Epidermal Growth Factor-like (EGF) repeats by protein O-fucosyltransferase 1 (POFUT1 in vertebrates, OFUT1 in Drosophila) is pivotal for NOTCH function. In Drosophila OFUT1 also acts as chaperone for Notch independent from its enzymatic activity. NOTCH ligands are also substrates for POFUT1, but in Drosophila OFUT1 is not essential for ligand function. In vertebrates the significance of POFUT1 for ligand function and subcellular localization is unclear. Here, we analyze the importance of O-fucosylation and POFUT1 for the mouse NOTCH ligand Delta-like 1 (DLL1). We show by mass spectral glycoproteomic analyses that DLL1 is O-fucosylated at the consensus motif C²XXXX(S/T)C³ (where C² and C³ are the second and third conserved cysteines within the EGF repeats) found in EGF repeats 3, 4, 7 and 8. A putative site with only three amino acids between the second cysteine and the hydroxy amino acid within EGF repeat 2 is not modified. DLL1 proteins with mutated O-fucosylation sites reach the cell surface and accumulate intracellularly. Likewise, in presomitic mesoderm cells of POFUT1 deficient embryos DLL1 is present on the cell surface, and in mouse embryonic fibroblasts lacking POFUT1 the same relative amount of overexpressed wild type DLL1 reaches the cell surface as in wild type embryonic fibroblasts. DLL1 expressed in POFUT1 mutant cells can activate NOTCH, indicating that POFUT1 is not required for DLL1 function as a Notch ligand.
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Müller
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States of America.
authors.forename: Nadia A
authors.initials: NA
authors.lastname: Rana
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Katrin
authors.initials: K
authors.lastname: Serth
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States of America.
authors.forename: Shinako
authors.initials: S
authors.lastname: Kakuda
authors.affiliation: Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, United States of America.
authors.forename: Robert S
authors.initials: RS
authors.lastname: Haltiwanger
authors.affiliation: Institut für Molekularbiologie, Medizinische Hochschule Hannover, Hannover, Germany.
authors.forename: Achim
authors.initials: A
authors.lastname: Gossler
mesh_terms: D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002135:Calcium-Binding Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid; D003412:Cricetulus; D004815:Epidermal Growth Factor / Q000737:chemistry; D005347:Fibroblasts / Q000378:metabolism; D005643:Fucose / Q000737:chemistry*; D005647:Fucosyltransferases / Q000235:genetics / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D040901:Proteomics / Q000379:methods; D051880:Receptors, Notch / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D053719:Tandem Mass Spectrometry; D015533:Transcriptional Activation
chemical_list: D002135:Calcium-Binding Proteins; C512925:Dlk1 protein, mouse; D036341:Intercellular Signaling Peptides and Proteins; D008024:Ligands; D051880:Receptors, Notch; D005643:Fucose; D004815:Epidermal Growth Factor; D005647:Fucosyltransferases; C539426:Pofut1 protein, mouse