O-fucosylation of the Notch Ligand mDLL1 by POFUT1 Is Dispensable for Ligand Function

Julia Müller,Shinako Kakuda,Achim Gossler,Nadia A Rana,Katrin Serth,Robert S Haltiwanger

doi:10.1371/journal.pone.0088571

Julia Müller, Shinako Kakuda + Show 4 more

Open Access

https://doi.org/10.1371/journal.pone.0088571

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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.

Highlights

The evolutionarily conserved Notch signaling pathway mediates direct cell-to-cell communication and regulates numerous developmental processes [1,2,3,4,5]
In an attempt to identify the intracellular compartment in which Delta-like 1 (DLL1) might accumulate in the absence of Protein O-fucosyltransferase 1 (POFUT1) we analyzed wild type and POFUT1 mutant mouse fibroblasts (MEFs) expressing wild type DLL1 with endoplasmatic reticulum (ER), Golgi and endosome markers used for the analysis of CHO cells expressing mutant DLL1 proteins (Figure 5)
To assess the portion of total DLL1 that is present on the cell surface in wild type and POFUT1 mutant cells quantitatively, we analyzed total and cell-surface DLL1 after surface biotinylation in six independent experiments. 17.3 and 21.5% of DLL1 was present on the surface of wild type and POFUT1 mutant MEFs, respectively (Figure 6 A, B), the difference being statistically not significant (p = 0.39), indicating that DLL1 expressed in MEFs reaches the cell surface efficiently even in the absence of POFUT1 and O-fucosylation

Summary

Introduction

The evolutionarily conserved Notch signaling pathway mediates direct cell-to-cell communication and regulates numerous developmental processes [1,2,3,4,5]. Mutations disrupting O-fucosylation sites cause intracellular accumulation of DLL1 To address the significance of these potential O-fucosylation sites for DLL1 function we generated DLL1 variants (Figure 1 C), in which the Ser or Thr residues in the consensus sites were replaced with Ala and Val residues, respectively, to prevent Ofucosylation, and established CHO cell lines expressing these variants at similar levels (Figure 1 D).

Results

Conclusion