Subcellular localization of DLL1 protein variants in CHO cells.

<p>(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.</p

Localization of endogenous DLL1 in PSM cells lacking POFUT1.

<p>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′).</p

Peptides from mouse DLL1 identified with O-fucose modifications.

<p>O-fucosylated peptides were identified by neutral loss of mass corresponding to fucose (146 daltons, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088571#pone.0088571.s001" target="_blank">Figure S1</a>) upon fragmentation. Spectra for each glycopeptide identified here are shown in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088571#pone.0088571.s001" target="_blank">Figure S1</a>. All masses were converted to the equivalent of singly charged (M+H⁺) for the table. For each glycopeptide, the mass of the parent ion, the deglycosylated product (lacking fucose), and the difference between these (corresponding to the mass of the modification) is shown. The predicted mass of the unglycosylated peptide is also shown. All peptide masses are adjusted for carbamidomethylation of cysteines. For peptides with a mass below 2000 Da, monoisotopic masses were used. For those above 2000 Da, average masses were used. Predicted O-fucose modification sites are bold underlined, and cysteines within the consensus sequence, C²XXXX(<u>S/T</u>)C³ are bold. *Note that this ion lost a water (16 Daltons).</p

The DLL3-S286A,T403V mutant does not complement the loss of endogenous DLL3 in somitogenesis.

<p>(A) Schematic representation of constructs introduced into HPRT deficient homozygous <i>Dll3</i> mutant ES cells. MSD2 is a dimer of the mesoderm-specific promoter element (MSD) from the <i>Dll1</i> locus [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref059" target="_blank">59</a>]. Locations of primers and restriction sites used for genotyping of embryos derived from tetraploid complementation are indicated below and above schemes b and c, respectively. (B) The MSD2 promoter element drives transgene expression in the PSM similar to endogenous <i>Dll3</i> expression. E8.5 MSD2::LacZ chimeric embryos were stained for β-galactosidase activity for one hour (a) and over night (b), or were analyzed by whole-mount in situ hybridization with a <i>lacZ</i> specific probe (c). (d) Expression pattern of endogenous <i>Dll3</i> transcripts in an E8.5 wt embryo for comparison. (C) Whole mount in situ hybridizations of E9.5 wt (a), homozygous <i>Dll3</i>^<i>pu</i> (b), and <i>Dll1</i>^{<i>Dll3ki</i>}, <i>Dll3</i>^<i>pu/pu</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#pone.0123776.ref038" target="_blank">38</a>] (c) embryos. In <i>Dll1</i>^{<i>Dll3ki</i>}, <i>Dll3</i>^<i>pu/pu</i> embryos, which lack endogenous DLL3 but express DLL3 from the <i>Dll1</i> locus, expression of the anterior-posterior (A-P) somite patterning marker <i>Uncx4.1</i> is restored except for minor irregularities (arrowhead in c). (d-h) Completely ES cell-derived embryos hybridized with <i>Uncx4.1</i>. Embryos generated from ES cells homozygous mutant for <i>Dll3</i> (<i>Dll3</i>^<i>pu/pu</i>) and carrying the HPRT deletion (ΔHPRT) display the same A-P somite patterning defects as homozygous <i>Dll3</i>^<i>pu</i> embryos (compare d with b). Expression of wild type DLL3 in ES cell-derived embryos almost completely rescues the <i>Dll3</i>^<i>pu</i> A-P somite patterning defect except for minor irregularities (arrowheads in e, g), whereas ES cell-derived embryos expressing mutant DLL3 display a <i>pudgy</i>-like somite phenotype (f, h) indicating that DLL3-S286A, T403V is not functional during somitogenesis. (D) Genotyping of tetraploid embryos shown in (C) using PCR and restriction digests as indicated in (A). The wild type <i>Dll3</i> PCR product is cut by <i>AleI</i> but not <i>NarI</i> (left panel), whereas the <i>Dll3</i>-S286A, T403V PCR product is cut by <i>NarI</i> (due to the presence of S286A) but not by <i>AleI</i> (due to the presence of T403V; middle and right panel; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123776#sec009" target="_blank">Material and Methods</a> for further details). Letters in parentheses refer to embryos shown in (C), arrowheads indicate cleavage products of the expected sizes. (E) Western blot analysis of lysates of wild type embryos (lane 1) or embryos obtained with ES cell clone B5 (lane 2) and lysates of CHO cells overexpressing Flag-tagged DLL3-S286A, T403V (lane 3) or wild type DLL3 (lane 4) using anti-Flag antibodies confirmed expression of DLL3-S286A, T403V-Flag in completely ES cell-derived embryos (red arrowhead). The equivalent of 4 trunks of E9 embryos was loaded in lanes 1 and 2. Asterisk between lanes 1 and 2 indicates a background band detected in embryo lysates.</p

<i>O-fucosylation</i> of the Notch Ligand mDLL1 by POFUT1 Is Dispensable for Ligand Function

<div><p>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(<u>S/T</u>)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.</p></div

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

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088571#pone.0088571.s001" target="_blank">Figure S1</a>. (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).</p

Somite patterning and vertebral column defects in single and double <i>Dll3</i> and <i>Lfng</i> mutants.

<p>Whole-mount in situ hybridizations of E9.5 embryos with an <i>Uncx4.1</i> specific probe (a-d) and lateral (e-h) and dorsal (e‘-h‘) views of skeletal preparations of wild type (a, e,e‘), homozygous <i>Dll3</i>^<i>pu</i> (b, f, f‘), homozygous <i>Lfng</i>^<i>lacZ</i> (c, g, g‘) and double homozygous <i>Dll3</i>^<i>pu</i>; <i>Lfng</i>^<i>lacZ</i> E18.5 embryos (d, h, h‘). Absence of both proteins does not enhance somite A-P patterning or vertebral column defects present in single mutants.</p

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

<p>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).</p

Analysis of DLL1 cell surface presentation by biotinylation.

<p>(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.</p

Wild type DLL3 but not DLL3-S286A,T403V affects the subcellular localization of DLL1.

<p>Confocal images of CHO cells stably expressing Flag-tagged DLL1 (A), transfected either with HA-tagged wild type or fucosylation mutant DLL3 (B, C). (A) Costaining of DLL1-Flag (using MAb 1F9) with the cell surface marker Sodium potassium ATPase (a-c) detects DLL1 on the cell surface (arrowheads in a and c), costaining with GM130 (d-f) in the trans-Golgi (arrow heads in d and f). In contrast, staining of DLL1-Flag using anti-DLL1 antibody H-265 detects predominantly Golgi localized DLL1 (white arrowheads in g and i) and reacts only weakly with DLL1 on the cell surface (g and i, grey arrowhead). DLL1 staining with anti-Flag detects DLL1-Flag similar to staining with 1F9 (compare j with a or d) overlapping with H-265 staining in the Golgi (arrowhead in l). (B) Coexpression of wild type DLL3 in DLL1 CHO cells (a-f) leads to an intracellular accumulation of DLL1 protein (white arrowheads in a, c and d, f), whereas in cells coexpressing the fucosylation mutant of DLL3 (g-l) localization of DLL1 is similar to untransfected cells and readily detected at the cell surface (white arrowheads in g, i and j, l). (C) Coexpression of wild type DLL3 with DLL1 protein leads to colocalization of DLL3 and DLL1 in vesicular structures or punctae outside the Golgi (white arrowheads in a, c, e, f, h, j; 18 out of 21 cells). The asterisk in a-e marks a neighboring DLL1 expressing cell without DLL3 expression showing intracellular DLL1 protein confined to the trans-Golgi (grey arrowhead in a and c). Coexpression of the O-fucosylation-defective DLL3 mutant (k-t) had no obvious effect on the localization of intracellular DLL1 protein (k, m, o, p, r, t; 10 our of 14 cells) or resulted only in weak detection of DLL overlapping with DLL3 outside the Golgi (u-y). The asterisk in k-o marks a neighboring DLL3 non-expressing cell.</p