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Negative regulation of notch signaling by xylose.

Lee TV, Sethi MK, Leonardi J, Rana NA, Buettner FF, Haltiwanger RS, Bakker H, Jafar-Nejad H - PLoS Genet. (2013)

Bottom Line: A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases.Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor.Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14-20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.

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Shams functions as a glucoside xylosyltransferase on Notch.(A) Schematic of the xylose-xylose-glucose trisaccharide attached to the serine (S) residue in the consensus sequence on an EGF repeat and the glycosyltransferases involved in its generation. (B) Phylogenetic tree of human GXYLT1/2 and XXYLT1, and their Drosophila homologs CG9996 (Shams) and CG11388 based on the Clustal W algorithm. (C) Xylosyltransferase assays using UDP-[14C]xylose donor and synthetic lipophilic acceptors to determine acceptor specificity of Shams. R represents the drawn hydrophobic aglycon; pNP, para-nitrophenol. (D) Donor substrate specificity using Glc-R as substrate. (E) Mass spectrometric analysis of a glycosylated peptide of Drosophila Notch (d) EGF16–20 expressed in Sf9 insect cells shows an increase in the ratio of disaccharide- versus monosaccharide-modified form after incubation with Shams or human GXYLT1 and UDP-xylose in vitro. Extracted ion chromatograms on the right indicate that the ratio between xylosylated and non-xylosylated peptides (dashed and solid lines, respectively) is inverted by Shams and GXYLT1. (F) Mass spectrometry demonstrates the presence of O-glucose trisaccharide on a peptide from EGF16 (639QINECESNPCQFDGHCQDR657). Top and bottom panels show MS and MS/MS spectra, respectively. (G) Schematic representation of sites of dNotch xylosylation identified by mass spectrometry (spectra shown in Figure S2). The most elongated glycan structure detected on each EGF repeat is shown, but the shorter forms can also exist.
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pgen-1003547-g001: Shams functions as a glucoside xylosyltransferase on Notch.(A) Schematic of the xylose-xylose-glucose trisaccharide attached to the serine (S) residue in the consensus sequence on an EGF repeat and the glycosyltransferases involved in its generation. (B) Phylogenetic tree of human GXYLT1/2 and XXYLT1, and their Drosophila homologs CG9996 (Shams) and CG11388 based on the Clustal W algorithm. (C) Xylosyltransferase assays using UDP-[14C]xylose donor and synthetic lipophilic acceptors to determine acceptor specificity of Shams. R represents the drawn hydrophobic aglycon; pNP, para-nitrophenol. (D) Donor substrate specificity using Glc-R as substrate. (E) Mass spectrometric analysis of a glycosylated peptide of Drosophila Notch (d) EGF16–20 expressed in Sf9 insect cells shows an increase in the ratio of disaccharide- versus monosaccharide-modified form after incubation with Shams or human GXYLT1 and UDP-xylose in vitro. Extracted ion chromatograms on the right indicate that the ratio between xylosylated and non-xylosylated peptides (dashed and solid lines, respectively) is inverted by Shams and GXYLT1. (F) Mass spectrometry demonstrates the presence of O-glucose trisaccharide on a peptide from EGF16 (639QINECESNPCQFDGHCQDR657). Top and bottom panels show MS and MS/MS spectra, respectively. (G) Schematic representation of sites of dNotch xylosylation identified by mass spectrometry (spectra shown in Figure S2). The most elongated glycan structure detected on each EGF repeat is shown, but the shorter forms can also exist.

Mentions: O-linked glucose is attached to serine residues in the consensus sequence C1-X-S-X-(P/A)-C2 by the protein O-glucosyltransferase Rumi/POGLUT1 [8], [14], [19]. Rumi is required for both fly and mammalian Notch signaling at a step downstream of ligand-binding [14], [15], [20]. In vivo structure-function analyses indicate that all of the 18 Rumi target sequences in Drosophila Notch (dNotch) contribute to Notch activation, with O-glucose sites on EGF10-15 playing a more important role than others [20]. O-glucose can serve as a substrate for additional sugar modifications to generate xylose-xylose-glucose trisaccharides [8], [19], [21]. The human enzymes responsible for the addition of xylose to O-glucosylated Notch EGF repeats have recently been identified: glucoside xylosyltransferase (GXYLT)1 and GXYLT2 add the first xylose and xyloside xylosyltransferase (XXYLT1) adds the second (Figure 1A) [22], [23]. Thus far, no functional studies have been performed to analyze the role of xylosylation in Notch signaling.


Negative regulation of notch signaling by xylose.

Lee TV, Sethi MK, Leonardi J, Rana NA, Buettner FF, Haltiwanger RS, Bakker H, Jafar-Nejad H - PLoS Genet. (2013)

Shams functions as a glucoside xylosyltransferase on Notch.(A) Schematic of the xylose-xylose-glucose trisaccharide attached to the serine (S) residue in the consensus sequence on an EGF repeat and the glycosyltransferases involved in its generation. (B) Phylogenetic tree of human GXYLT1/2 and XXYLT1, and their Drosophila homologs CG9996 (Shams) and CG11388 based on the Clustal W algorithm. (C) Xylosyltransferase assays using UDP-[14C]xylose donor and synthetic lipophilic acceptors to determine acceptor specificity of Shams. R represents the drawn hydrophobic aglycon; pNP, para-nitrophenol. (D) Donor substrate specificity using Glc-R as substrate. (E) Mass spectrometric analysis of a glycosylated peptide of Drosophila Notch (d) EGF16–20 expressed in Sf9 insect cells shows an increase in the ratio of disaccharide- versus monosaccharide-modified form after incubation with Shams or human GXYLT1 and UDP-xylose in vitro. Extracted ion chromatograms on the right indicate that the ratio between xylosylated and non-xylosylated peptides (dashed and solid lines, respectively) is inverted by Shams and GXYLT1. (F) Mass spectrometry demonstrates the presence of O-glucose trisaccharide on a peptide from EGF16 (639QINECESNPCQFDGHCQDR657). Top and bottom panels show MS and MS/MS spectra, respectively. (G) Schematic representation of sites of dNotch xylosylation identified by mass spectrometry (spectra shown in Figure S2). The most elongated glycan structure detected on each EGF repeat is shown, but the shorter forms can also exist.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3675014&req=5

pgen-1003547-g001: Shams functions as a glucoside xylosyltransferase on Notch.(A) Schematic of the xylose-xylose-glucose trisaccharide attached to the serine (S) residue in the consensus sequence on an EGF repeat and the glycosyltransferases involved in its generation. (B) Phylogenetic tree of human GXYLT1/2 and XXYLT1, and their Drosophila homologs CG9996 (Shams) and CG11388 based on the Clustal W algorithm. (C) Xylosyltransferase assays using UDP-[14C]xylose donor and synthetic lipophilic acceptors to determine acceptor specificity of Shams. R represents the drawn hydrophobic aglycon; pNP, para-nitrophenol. (D) Donor substrate specificity using Glc-R as substrate. (E) Mass spectrometric analysis of a glycosylated peptide of Drosophila Notch (d) EGF16–20 expressed in Sf9 insect cells shows an increase in the ratio of disaccharide- versus monosaccharide-modified form after incubation with Shams or human GXYLT1 and UDP-xylose in vitro. Extracted ion chromatograms on the right indicate that the ratio between xylosylated and non-xylosylated peptides (dashed and solid lines, respectively) is inverted by Shams and GXYLT1. (F) Mass spectrometry demonstrates the presence of O-glucose trisaccharide on a peptide from EGF16 (639QINECESNPCQFDGHCQDR657). Top and bottom panels show MS and MS/MS spectra, respectively. (G) Schematic representation of sites of dNotch xylosylation identified by mass spectrometry (spectra shown in Figure S2). The most elongated glycan structure detected on each EGF repeat is shown, but the shorter forms can also exist.
Mentions: O-linked glucose is attached to serine residues in the consensus sequence C1-X-S-X-(P/A)-C2 by the protein O-glucosyltransferase Rumi/POGLUT1 [8], [14], [19]. Rumi is required for both fly and mammalian Notch signaling at a step downstream of ligand-binding [14], [15], [20]. In vivo structure-function analyses indicate that all of the 18 Rumi target sequences in Drosophila Notch (dNotch) contribute to Notch activation, with O-glucose sites on EGF10-15 playing a more important role than others [20]. O-glucose can serve as a substrate for additional sugar modifications to generate xylose-xylose-glucose trisaccharides [8], [19], [21]. The human enzymes responsible for the addition of xylose to O-glucosylated Notch EGF repeats have recently been identified: glucoside xylosyltransferase (GXYLT)1 and GXYLT2 add the first xylose and xyloside xylosyltransferase (XXYLT1) adds the second (Figure 1A) [22], [23]. Thus far, no functional studies have been performed to analyze the role of xylosylation in Notch signaling.

Bottom Line: A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases.Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor.Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA.

ABSTRACT
The Notch signaling pathway controls a large number of processes during animal development and adult homeostasis. One of the conserved post-translational modifications of the Notch receptors is the addition of an O-linked glucose to epidermal growth factor-like (EGF) repeats with a C-X-S-X-(P/A)-C motif by Protein O-glucosyltransferase 1 (POGLUT1; Rumi in Drosophila). Genetic experiments in flies and mice, and in vivo structure-function analysis in flies indicate that O-glucose residues promote Notch signaling. The O-glucose residues on mammalian Notch1 and Notch2 proteins are efficiently extended by the addition of one or two xylose residues through the function of specific mammalian xylosyltransferases. However, the contribution of xylosylation to Notch signaling is not known. Here, we identify the Drosophila enzyme Shams responsible for the addition of xylose to O-glucose on EGF repeats. Surprisingly, loss- and gain-of-function experiments strongly suggest that xylose negatively regulates Notch signaling, opposite to the role played by glucose residues. Mass spectrometric analysis of Drosophila Notch indicates that addition of xylose to O-glucosylated Notch EGF repeats is limited to EGF14-20. A Notch transgene with mutations in the O-glucosylation sites of Notch EGF16-20 recapitulates the shams loss-of-function phenotypes, and suppresses the phenotypes caused by the overexpression of human xylosyltransferases. Antibody staining in animals with decreased Notch xylosylation indicates that xylose residues on EGF16-20 negatively regulate the surface expression of the Notch receptor. Our studies uncover a specific role for xylose in the regulation of the Drosophila Notch signaling, and suggest a previously unrecognized regulatory role for EGF16-20 of Notch.

Show MeSH