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.

Figure 1. Schematic overview of DLL1 wild type and mutant proteins and mass spectrometry results.

(A) Schematic representation of the DLL1 protein. Boxes indicate DSL, EGF1-EGF8 (E1–E8) repeats, and the transmembrane (TM) domain. The presence of different classes of O-fucosylation consensus sites is indicated by light grey, dark grey and black shading. (B) Extracted Ion Chromatograms (EIC) show that peptides derived from EGF repeats 3, 4, 7, and 8 are modified at high stoichiometry, but the peptide derived from EGF repeat 2 is not O-fucosylated. Masses corresponding to the glycopeptides (red lines) or unmodified peptide (black lines) used to generate the EIC are shown in Figure S1. (C) Schematic overview over single and multiple mutations (indicated by black dots aligned to EGF domains) introduced into EGF repeats of DLL1. (D) Western Blot analysis of cell lysates of clonal CHO cell lines stably expressing DLL1 and mutant variants at comparable levels. Beside CHO control cells (CHO), and CHO cells expressing DLL1wt (DLL1), roman letters refer to DLL1mEGF7 (I), DLL1mEGF3/4/7/8 (II), DLL1mEGF3/4/7 (III) and DLL1mEGF3/4/8 (IV).

Figure 2. Subcellular localization of DLL1 protein variants in CHO cells.

(A) Confocal images stained with antibodies against DLL1 and a marker for the cell surface (Sodium potassium ATPase). (a-r) CHO cells stably transfected with control (a-c), wild type DLL1 (d-f), or mutant DLL1 (g-r) show wild type DLL1 on the surface (d-f′) and the protein variants DLL1mEGF7, DLL1mEGF3/4/7/8, DLL1mEGF3/4/7 and DLL1mEGF3/4/8 were detected on the cell surface and intracellularly (g-i′, j-l′, m-o′, p-r′). (B) Western Blot analysis of DLL1 protein variants after cell surface biotinylation, cell lysis and immunoprecipitation with Flag-Agarose beads (Flag Input) and Streptavidin-Sepharose to detect the cell surface biotinylated fraction (Streptavidin). Cells and constructs are indicated above the lanes.

Figure 3. Colocalization of DLL1mEGF7 and DLL1mEGF3/4/7/8 protein with intracellular compartment markers.

Confocal images of cells stably expressing DLL1mEGF7 or DLL1mEGF3/4/7/8 co-stained with antibodies against DLL1 and the ER markers KDEL (a-f′) and Rab6A (g-l′), the trans-Golgi markers GM130 (m-r′) and Rab11 (s-x′), the endocytotic vesicle marker Caveolin (y-Ad′), and the early endosome markers EEA1 (Ae-Aj′) and Rab5 (Ak-As′). DLL1mEGF7 is present on the cell surface and intracellulary partially overlaping with KDEL, Rab6A and GM130, and to a lesser extent with Rab11, Caveolin, EEA1 and Rab5. The DLL1mEGF3/4/7/8 variant shows a similar distribution.

Figure 4. Localization of endogenous DLL1 in PSM cells lacking POFUT1.

Confocal images of flat mounted PSMs from wild type and POFUT1 mutant E9.5 embryos (a-f). In wild type PSM cells, DLL1 was present almost exclusively on the cell surface and colocalized with Pan-Cadherin staining (c-c′). In POFUT1 mutant PSM cells most of the DLL1 protein was detected intracellularly in punctae or dots, reflecting significantly reduced colocalization with Pan-Cadherin (f-f′).

Figure 5. Localization of endogenous DLL1 in wild type and POFUT1 mutant MEFs.

Confocal images of wt and Pofut deficient cells stably overexpressing flag-tagged DLL1. Costaining were performed with antibodies against DLL1 (green) and marker for intracellular compartments (red). Both in wt and POFUT1 deficient cells DLL1 is located on the cell surface and colocalizes with the cell surface marker ATPase (a-f′). In addition, DLL1 protein was also detected intracellulary in both cell lines showing partially colocalization with the ER markers KDEL (g-l′), the trans-Golgi markers GM130 (m-r′) and Rab11 (s-x′), the endocytotic marker Caveolin (y-ad), the early endosome markers EEA1 (ae-aj′), Rab5 (ak-ap′) and the transferrin receptor (TfR, aq-av).

Figure 6. Analysis of DLL1 cell surface presentation by biotinylation.

(A) Representative examples of Western blots used for quantification of DLL1 cell surface presentation in wt and POFUT1 deficient cells stably expressing flag-tagged DLL1. Input lane represents total DLL1 protein from whole cell lysates. IP lane represents DLL1 surface protein, which was biotinylated and immunoprecipitated with Neutravidin Agarose Resin (IP). As an additional control blots were reprobed with β-Actin antibody to exclude cell lysis during the biotinylation step. (B) Calculated relative cell surface levels of DLL1. 17.3% of total DLL1 protein was detected on the cell surface in POFUT wt cells, whereas in POFUT deficient cells 21.5% (p = 0.39) of total DLL1 reached the cell surface.

Figure 7. Notch activation by DLL1 in POFUT1 deficient fibroblasts.

Western Blot analysis of cell lysates of NOTCH1 expressing HeLa cells (HeLaN1) co-cultured with POFUT1+/+ or POFUT1−/− fibroblasts or with CHO cells over-expressing DLL1 using the anti-Cleaved Notch1 (Val1744) antibody, which specifically detects the Notch1 intracellular domain (NICD) after S3 cleavage. Non-fucosylated DLL1 can efficiently activate Notch1.