Famiglietti AL et al. (2017), Characterization and expression analysis of Gal...

ECB-ART-45447

PLoS One 2017 Apr 17;124:e0176479. doi: 10.1371/journal.pone.0176479.

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Characterization and expression analysis of Galnts in developing Strongylocentrotus purpuratus embryos.

Famiglietti AL , Wei Z , Beres TM , Milac AL , Tran DT , Patel D , Angerer RC , Angerer LM , Tabak LA .

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Mucin-type O-glycosylation is a ubiquitous posttranslational modification in which N-Acetylgalactosamine (GalNAc) is added to the hydroxyl group of select serine or threonine residues of a protein by the family of UDP-GalNAc:Polypeptide N-Acetylgalactosaminyltransferases (GalNAc-Ts; EC 2.4.1.41). Previous studies demonstrate that O-glycosylation plays essential roles in protein function, cell-cell interactions, cell polarity and differentiation in developing mouse and Drosophila embryos. Although this type of protein modification is highly conserved among higher eukaryotes, little is known about this family of enzymes in echinoderms, basal deuterostome relatives of the chordates. To investigate the potential role of GalNAc-Ts in echinoderms, we have begun the characterization of this enzyme family in the purple sea urchin, S. purpuratus. We have fully or partially cloned a total of 13 genes (SpGalnts) encoding putative sea urchin SpGalNAc-Ts, and have confirmed enzymatic activity of five recombinant proteins. Amino acid alignments revealed high sequence similarity among sea urchin and mammalian glycosyltransferases, suggesting the presence of putative orthologues. Structural models underscored these similarities and helped reconcile some of the substrate preferences observed. Temporal and spatial expression of SpGalnt transcripts, was studied by whole-mount in situ hybridization. We found that many of these genes are transcribed early in developing embryos, often with restricted expression to the endomesodermal region. Multicolor fluorescent in situ hybridization (FISH) demonstrated that transcripts encoding SpGalnt7-2 co-localized with both Endo16 (a gene expressed in the endoderm), and Gcm (a gene expressed in secondary mesenchyme cells) at the early blastula stage, 20 hours post fertilization (hpf). At late blastula stage (28 hpf), SpGalnt7-2 message co-expresses with Gcm, suggesting that it may play a role in secondary mesenchyme development. We also discovered that morpholino-mediated knockdown of SpGalnt13 transcripts, results in a deficiency of embryonic skeleton and neurons, suggesting that mucin-type O-glycans play essential roles during embryonic development in S. purpuratus.

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Species referenced: Echinodermata
Genes referenced: endo16 gcml gscl hnf6 LOC100887844 LOC100893907 LOC115917880 LOC115919910 LOC115926321 onecut2 pole
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	Fig 2. Structural representation incorporating variability data of human, mouse, and sea urchin GalNAc-T isoforms.Catalytic (left) and lectin (right) domains of human GalNAc-T2 (PDB code 2FFU), colored according to sequence conservation level, from bright red (perfectly conserved) to dark blue (highly variable). The active site of the catalytic domain contains the Mn ion (magenta sphere), a sugar donor fragment UDP (lines colored by atom type) and the acceptor peptide (green colored ribbon). Sequence variability is much higher within the lectin domain (right side), where the only conserved positions correspond to cysteine residues that form disulphide bonds which maintain structural integrity of the domain.
	Fig 3. Box and whisker plots of in vitro enzymatic activity assays of S. purpuratus GalNAc-Ts tested against a panel of peptide and glycopeptide substrates.Comparison of activity of (A) SpGalNAc-T1 and (B) SpGalNAc-T2 (C) SpGalNAc-T7 (D) SpGalNAc-T7-1 and (E) SpGalNAc-T7-2 using equivalent amounts of FLAG-purified protein. Each panel (A-E) shows the variability in activity (dpm/3hr) per enzyme from assays performed in triplicate from three separate transfections. Error bars indicate relative maximum and minimum activity of combined data points against each peptide tested.
	Fig 4. Structural mapping of enzyme sequence variability in the peptide-binding groove.Substrate peptide is depicted with dark-cyan spheres. Protein surface is colored gray, while substrate-binding groove (within 5 Å of the peptide) is colored as follows: yellow residues in the center of the groove are conserved, pink region is variable in terms of charge (sequence alignment shown in the “Pink loop” box), green region is variable in terms of flexibility (sequence alignment shown in the “Green loop” box), while the purple region indicates the residues interacting with the peptide only in the closed, compact conformation of the enzyme.
	Fig 5. Spatial and temporal expression of SpGalnts in developing sea urchin embryos.Whole-mount mRNA in situ hybridizations of SpGalnt1 (B), SpGalnt2 (C), SpGalnt7 (D), SpGalnt7-2 (E), SpGalnt10 (F) SpGalnt11 (G), SpGalnt13 (H) and SpGalnt13-2 (I) at 12, 18, 24, 36 and 48 hours post fertilization (hpf). In column (A), a sense SpGalnt7-1 probe was used as negative control. All representative embryos of three separate experiments are shown in the lateral view. Scale bar in (A) indicates 20 μm.
	Fig 6. SpGalnt7-2 is expressed in endomesoderm cells in mesenchyme blastula stage sea urchin embryos.Triple fluorescence in situ hybridization of SpGalnt7-2 (blue), Gcm (green), a secondary mesenchyme gene marker, and Endo16 (red) an endoderm marker in representative sea urchin embryos at 20 hpf and 28 hpf. All embryos are shown in vegetal view as depicted by DIC images (A) and (G). (B-E) triple and double fluorescence channel images of a 20 hpf embryo. (H-K) triple and double fluorescence channel images of a 28 hpf embryo. Arrows in (B), (D), and (E) indicate the co-localization of SpT7-2 with Gcm and Endo16 in the endomesoderm at 20 hpf. Arrows in (H), (J), (K) and (L) show a shift in SpGalnt7-2 expression to the aboral side in secondary mesenchyme cells at 28 hpf. Panels (F) and (L) show SpGalnt7-2 expression only. All representative embryos of three separate experiments are shown in the vegetal view. Scale bar in (A) indicates 20 μm.
	Fig 7. SpGalNAc-T13 is required for embryo skeleton and nerve cell development.(A-B) DIC images of 3-day embryos showing skeletal spicules in the control embryo (A, arrowheads) compared to a lack of spicules in the SpGalnt13 morphant (B). (C-H) fluorescent images of expression patterns of different markers. (C-D) Primary mesenchyme cell (PMC) antibody marker 6a9 (red) was detected in both control embryos (C) and SpGalnt13 morphants (D), indicating that PMC cells ingressed in the morphant but could not form spicules. (E-F) Oral ectoderm marker Goosecoid (GSC, red) was detected in both the control embryo (E) and the SpGalnt13 morphant (F), but ciliated band ectoderm marker Hnf6 (green) was not detected in the SpGalnt13 morphant. (G-H) Serotonergic neuron marker (green) and pan-neuronal marker Synaptotagmin B (SynB, red) were detected in the control embryo (G) but absent in the SpGalnt13 morphant (H). All embryos are shown in oral view, with the anterior pole to the top. Scale bar in (A) indicates 20 μm. Representative embryos are shown from three separate experiments.

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