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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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Xylosylation of EGF16–20 negatively regulates Drosophila Notch signaling in vivo.(A) Schematic of the EGF repeats of wild-type and mutant Notch genomic transgenes. Blue boxes show EGF repeats with a consensus O-glucosylation site; orange boxes denote EGF repeats with a serine-to-alanine mutation in the O-glucosylation site, which prevents the addition of O-glucose and therefore xylose. (B–E) N−/Y; Ngt-wt/+, N−/Y; Ngt-10_15/+, and N−/Y; Ngt-24_35/+ males exhibit no wing vein loss, but N−/Y; Ngt-16_20/+ males (D) exhibit loss of L2, L4 and L5 veins (arrowheads). (F) At 25°C, N−/Y; Ngt-wt/+ males expressing GXYLT1-HA in the apterous-GAL4 domain show thickening of the distal wing veins. (G) In a N−/Y; Ngt-10_15/+ background, ap>GXYLT1-HA becomes lethal at 25°C, and is not suppressed at 22°C. (H) In a N−/Y; Ngt-16_20/+ background, the ap>GXYLT1-HA phenotype is fully suppressed. Note the presence of wing vein loss. (I) Ngt-24_35 does not suppress the ap>GXYLT1-HA phenotype. (J) At 25°C, N−/Y; Ngt-wt/+ males expressing nub>XXYLT1-HA show severe wing vein and margin defects. (K) The phenotypes are dramatically enhanced in N−/Y; Ngt-10_15/+ males raised at 25°C (inset) and are comparable to (J) when raised at 18°C. (L,M) The nub>XXYLT1-HA phenotypes are fully suppressed in N−/Y; Ngt-16_20/+ males (L), but are enhanced in N−/Y; Ngt-24_35/+ males (M; compare to J). All wings, including the inset in M, are shown to scale.
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pgen-1003547-g004: Xylosylation of EGF16–20 negatively regulates Drosophila Notch signaling in vivo.(A) Schematic of the EGF repeats of wild-type and mutant Notch genomic transgenes. Blue boxes show EGF repeats with a consensus O-glucosylation site; orange boxes denote EGF repeats with a serine-to-alanine mutation in the O-glucosylation site, which prevents the addition of O-glucose and therefore xylose. (B–E) N−/Y; Ngt-wt/+, N−/Y; Ngt-10_15/+, and N−/Y; Ngt-24_35/+ males exhibit no wing vein loss, but N−/Y; Ngt-16_20/+ males (D) exhibit loss of L2, L4 and L5 veins (arrowheads). (F) At 25°C, N−/Y; Ngt-wt/+ males expressing GXYLT1-HA in the apterous-GAL4 domain show thickening of the distal wing veins. (G) In a N−/Y; Ngt-10_15/+ background, ap>GXYLT1-HA becomes lethal at 25°C, and is not suppressed at 22°C. (H) In a N−/Y; Ngt-16_20/+ background, the ap>GXYLT1-HA phenotype is fully suppressed. Note the presence of wing vein loss. (I) Ngt-24_35 does not suppress the ap>GXYLT1-HA phenotype. (J) At 25°C, N−/Y; Ngt-wt/+ males expressing nub>XXYLT1-HA show severe wing vein and margin defects. (K) The phenotypes are dramatically enhanced in N−/Y; Ngt-10_15/+ males raised at 25°C (inset) and are comparable to (J) when raised at 18°C. (L,M) The nub>XXYLT1-HA phenotypes are fully suppressed in N−/Y; Ngt-16_20/+ males (L), but are enhanced in N−/Y; Ngt-24_35/+ males (M; compare to J). All wings, including the inset in M, are shown to scale.

Mentions: Notch transgenes harboring serine-to-alanine mutations in all or most O-glucosylation sites show a temperature-sensitive loss of Notch signaling, similar to rumi animals [14], [20]. However, when smaller subsets of the O-glucosylation sites are mutated and the animals are raised at 25°C or lower, the negative effects of loss of O-glucose on Notch is significantly decreased [20]. If loss of xylose on specific EGF repeats results in increased Notch signaling, these mutations should recapitulate the shams mutant phenotypes, as loss of O-glucose precludes the addition of xylose. To test this, we generated animals that lack endogenous Notch but are rescued by one copy of Notch genomic transgenes carrying mutations in various subsets of O-glucosylation sites (Figure 4A) [20]. Serine-to-alanine mutations in EGF10–15 or EGF24–35 did not result in loss of wing vein, similar to a wild-type Notch transgene (Figure 4B, 4C and 4E). However, O-glucose mutations in EGF16–20 resulted in a partial loss of wing veins L2, L4, and L5 (Figure 4D), similar to but somewhat stronger than the shams phenotypes (Figure 2F and 2G). N54l9/Y; Ngt-16_20/+ animals also exhibited head bristle defects similar to shams mutants (Figure S6). These observations nicely match our mass spectrometry data (Figure 1G) and indicate that xylosylation of EGF16–20 plays a negative regulatory role in Drosophila 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)

Xylosylation of EGF16–20 negatively regulates Drosophila Notch signaling in vivo.(A) Schematic of the EGF repeats of wild-type and mutant Notch genomic transgenes. Blue boxes show EGF repeats with a consensus O-glucosylation site; orange boxes denote EGF repeats with a serine-to-alanine mutation in the O-glucosylation site, which prevents the addition of O-glucose and therefore xylose. (B–E) N−/Y; Ngt-wt/+, N−/Y; Ngt-10_15/+, and N−/Y; Ngt-24_35/+ males exhibit no wing vein loss, but N−/Y; Ngt-16_20/+ males (D) exhibit loss of L2, L4 and L5 veins (arrowheads). (F) At 25°C, N−/Y; Ngt-wt/+ males expressing GXYLT1-HA in the apterous-GAL4 domain show thickening of the distal wing veins. (G) In a N−/Y; Ngt-10_15/+ background, ap>GXYLT1-HA becomes lethal at 25°C, and is not suppressed at 22°C. (H) In a N−/Y; Ngt-16_20/+ background, the ap>GXYLT1-HA phenotype is fully suppressed. Note the presence of wing vein loss. (I) Ngt-24_35 does not suppress the ap>GXYLT1-HA phenotype. (J) At 25°C, N−/Y; Ngt-wt/+ males expressing nub>XXYLT1-HA show severe wing vein and margin defects. (K) The phenotypes are dramatically enhanced in N−/Y; Ngt-10_15/+ males raised at 25°C (inset) and are comparable to (J) when raised at 18°C. (L,M) The nub>XXYLT1-HA phenotypes are fully suppressed in N−/Y; Ngt-16_20/+ males (L), but are enhanced in N−/Y; Ngt-24_35/+ males (M; compare to J). All wings, including the inset in M, are shown to scale.
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pgen-1003547-g004: Xylosylation of EGF16–20 negatively regulates Drosophila Notch signaling in vivo.(A) Schematic of the EGF repeats of wild-type and mutant Notch genomic transgenes. Blue boxes show EGF repeats with a consensus O-glucosylation site; orange boxes denote EGF repeats with a serine-to-alanine mutation in the O-glucosylation site, which prevents the addition of O-glucose and therefore xylose. (B–E) N−/Y; Ngt-wt/+, N−/Y; Ngt-10_15/+, and N−/Y; Ngt-24_35/+ males exhibit no wing vein loss, but N−/Y; Ngt-16_20/+ males (D) exhibit loss of L2, L4 and L5 veins (arrowheads). (F) At 25°C, N−/Y; Ngt-wt/+ males expressing GXYLT1-HA in the apterous-GAL4 domain show thickening of the distal wing veins. (G) In a N−/Y; Ngt-10_15/+ background, ap>GXYLT1-HA becomes lethal at 25°C, and is not suppressed at 22°C. (H) In a N−/Y; Ngt-16_20/+ background, the ap>GXYLT1-HA phenotype is fully suppressed. Note the presence of wing vein loss. (I) Ngt-24_35 does not suppress the ap>GXYLT1-HA phenotype. (J) At 25°C, N−/Y; Ngt-wt/+ males expressing nub>XXYLT1-HA show severe wing vein and margin defects. (K) The phenotypes are dramatically enhanced in N−/Y; Ngt-10_15/+ males raised at 25°C (inset) and are comparable to (J) when raised at 18°C. (L,M) The nub>XXYLT1-HA phenotypes are fully suppressed in N−/Y; Ngt-16_20/+ males (L), but are enhanced in N−/Y; Ngt-24_35/+ males (M; compare to J). All wings, including the inset in M, are shown to scale.
Mentions: Notch transgenes harboring serine-to-alanine mutations in all or most O-glucosylation sites show a temperature-sensitive loss of Notch signaling, similar to rumi animals [14], [20]. However, when smaller subsets of the O-glucosylation sites are mutated and the animals are raised at 25°C or lower, the negative effects of loss of O-glucose on Notch is significantly decreased [20]. If loss of xylose on specific EGF repeats results in increased Notch signaling, these mutations should recapitulate the shams mutant phenotypes, as loss of O-glucose precludes the addition of xylose. To test this, we generated animals that lack endogenous Notch but are rescued by one copy of Notch genomic transgenes carrying mutations in various subsets of O-glucosylation sites (Figure 4A) [20]. Serine-to-alanine mutations in EGF10–15 or EGF24–35 did not result in loss of wing vein, similar to a wild-type Notch transgene (Figure 4B, 4C and 4E). However, O-glucose mutations in EGF16–20 resulted in a partial loss of wing veins L2, L4, and L5 (Figure 4D), similar to but somewhat stronger than the shams phenotypes (Figure 2F and 2G). N54l9/Y; Ngt-16_20/+ animals also exhibited head bristle defects similar to shams mutants (Figure S6). These observations nicely match our mass spectrometry data (Figure 1G) and indicate that xylosylation of EGF16–20 plays a negative regulatory role in Drosophila 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.

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