Embryonic expression of zebrafish <i>b3glct</i> genes.

<p>(A) RT-PCR analysis of <i>b3glct</i> expression demonstrates robust expression of both <i>b3glcta</i> (left panel) and <i>b3glctb</i> (middle panel) at different stages of development in whole embryos as well as various embryonic tissues at 48-hpf (right panel). Controls included <i>pitx2c</i> as negative control for 0-hpf, <i>rhodopsin</i> as negative control for the lens, <i>beta-actin</i> as positive control for all tissues and H₂O as negative contamination control for all reactions. (B) In-situ hybridization analysis of <i>b3glcta</i> and <i>b3glctb</i> expression demonstrates broad expression in 24-120-hpf embryos with enrichment in the developing eyes, fins, brain, craniofacial region and somites. aer–apical ectodermal ridge, ase–anterior segment of the eye, b–brain, cmz–ciliary marginal zone, crc–craniofacial cartilage, e–eye, f–fins, h–heart, le–lens, sm–skeletal muscles.</p

Summary of differentially regulated genes implicated in ER quality control, unfolded protein response or cell survival.

<p>Summary of differentially regulated genes implicated in ER quality control, unfolded protein response or cell survival.</p

Functional characterization of zebrafish orthologs of the human Beta 3-Glucosyltransferase <i>B3GLCT</i> gene mutated in Peters Plus Syndrome

<div><p>Peters Plus Syndrome (PPS) is a rare autosomal recessive disease characterized by ocular defects, short stature, brachydactyly, characteristic facial features, developmental delay and other highly variable systemic defects. Classic PPS is caused by loss-of-function mutations in the <i>B3GLCT</i> gene encoding for a β3-glucosyltransferase that catalyzes the attachment of glucose via a β1–3 glycosidic linkage to <i>O</i>-linked fucose on thrombospondin type 1 repeats (TSRs). B3GLCT was shown to participate in a non-canonical ER quality control mechanism; however, the exact molecular processes affected in PPS are not well understood. Here we report the identification and characterization of two zebrafish orthologs of the human <i>B3GLCT</i> gene, <i>b3glcta</i> and <i>b3glctb</i>. The <i>b3glcta</i> and <i>b3glctb</i> genes encode for 496-aa and 493-aa proteins with 65% and 57% identity to human B3GLCT, respectively. Expression studies demonstrate that both orthologs are widely expressed with strong presence in embryonic tissues affected in PPS. <i>In vitro</i> glucosylation assays demonstrated that extracts from wildtype embryos contain active b3glct enzyme capable of transferring glucose from UDP-glucose to an <i>O</i>-fucosylated TSR, indicating functional conservation with human B3GLCT. To determine the developmental role of the zebrafish genes, single and double <i>b3glct</i> knockouts were generated using TALEN-induced genome editing. Extracts from double homozygous <i>b3glct</i>^<i>-/-</i> embryos demonstrated complete loss of <i>in vitro</i> b3glct activity. Surprisingly, <i>b3glct</i>^<i>-/-</i> homozygous fish developed normally. Transcriptome analyses of head and trunk tissues of <i>b3glct</i>^<i>-/-</i> 24-hpf embryos identified 483 shared differentially regulated transcripts that may be involved in compensation for b3glct function in these embryos. The presented data show that both sequence and function of <i>B3GLCT/b3glct</i> genes is conserved in vertebrates. At the same time, complete <i>b3glct</i> deficiency in zebrafish appears to be inconsequential and possibly compensated for by a yet unknown mechanism.</p></div

Exonic structure, genomic context and multiple species alignment of B3GLCT/b3glct.

<p>(A) The two zebrafish orthologs of B3GLCT show overall similar exonic arrangement. The number of each exon is located within each box and the size of the exon (in base pairs) is shown above each exon. The 5’ and 3’ UTRs are indicated preceding the first ATG and following the stop codon (TAA/TAG). White indicates the N-terminal signal sequence, light grey indicates the stem region and dark grey indicates the catalytic domain. The vertical black bar in exon 12 of each gene indicates the location of nucleotides encoding for the catalytic tri-aspartic acid residues. Horizontal lines underneath the zebrafish genes indicate previously annotated sequence and sequence identified in this study. (B) Schematic of genomic context for B3GLCT/b3glct. (C) Multiple species alignment of B3GLCT orthologs from human (NP_919299), mouse (NP_001074673), Xenopus (NP_001072551), and zebrafish. Blue bar indicates signal peptide, green indicates stem region and orange indicates catalytic core. Grey shading of amino acids indicates conservation. The DxD motif is boxed in red.</p

Notch activation by DLL1 in POFUT1 deficient fibroblasts.

<p>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.</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

<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

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

<p>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.</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

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