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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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Increased surface expression of Notch in shams and Notchgt-16_20 clones in the pupal wing.All animals were raised at 30°C. (A,A′) Shown are confocal images from a pupal wing at 22–24 hours after puparium formation (APF) with a MARCM clone of shamsΔ34 marked by nuclear GFP (GFPNLS). Surface expression of Notch is shown in red. Note, also in the xz section, that the Notch surface level at this stage is increased in shams mutant cells. (B–C′) Shown are confocal images of pupal wings around 22 hours APF from animals harboring MARCM clones of the Notch54l9 protein- allele (marked by CD8::GFP) with one copy of either a wild-type Notch transgene (B,B′) or a Notch transgene with O-glucose mutations in EGF16–20 (C,C′). The only source of Notch in the clones is the Notch transgene. Note, also in xz sections, that the level of surface Notch in clones harboring the Notchgt-16_20 is increased compared to that in clones harboring Notchgt-wt. (D) Anti-HA Western blot on larval and pupal protein extracts from animals harboring one copy of an HA-tagged Shams genomic transgene (HA-Shams; shamsgt-wt-HA-attVK22) or attVK22 control animals. Tubulin was used as loading control. Pupal extracts show relatively higher levels of HA-Shams.
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pgen-1003547-g005: Increased surface expression of Notch in shams and Notchgt-16_20 clones in the pupal wing.All animals were raised at 30°C. (A,A′) Shown are confocal images from a pupal wing at 22–24 hours after puparium formation (APF) with a MARCM clone of shamsΔ34 marked by nuclear GFP (GFPNLS). Surface expression of Notch is shown in red. Note, also in the xz section, that the Notch surface level at this stage is increased in shams mutant cells. (B–C′) Shown are confocal images of pupal wings around 22 hours APF from animals harboring MARCM clones of the Notch54l9 protein- allele (marked by CD8::GFP) with one copy of either a wild-type Notch transgene (B,B′) or a Notch transgene with O-glucose mutations in EGF16–20 (C,C′). The only source of Notch in the clones is the Notch transgene. Note, also in xz sections, that the level of surface Notch in clones harboring the Notchgt-16_20 is increased compared to that in clones harboring Notchgt-wt. (D) Anti-HA Western blot on larval and pupal protein extracts from animals harboring one copy of an HA-tagged Shams genomic transgene (HA-Shams; shamsgt-wt-HA-attVK22) or attVK22 control animals. Tubulin was used as loading control. Pupal extracts show relatively higher levels of HA-Shams.

Mentions: To examine the effects of loss of shams on Notch localization, we performed Notch surface staining on larval wing imaginal discs and pupal wings harboring shams mutant clones. Loss of shams does not affect the surface expression of Notch in third instar wing imaginal disc (Figure S7A–A″). However, more Notch protein is present in and at the surface of shams mutant cells in the pupal wing (Figure 5A and 5A′ and Figure S7B–B′). We also sought to determine whether mutating the O-glucose sites on EGF16–20 of Notch results in increased cell surface levels of Notch. To this end, we generated Mosaic Analysis with a Repressible Cell Marker (MARCM) clones [28] of a protein- allele of Notch in a background harboring one copy of the wild-type or an EGF16–20 mutant Notch, similar to what we have described before for other mutant Notch transgenes [20]. In these animals, heterozygous cells have both endogenous Notch and a copy of our transgene, but cells in the mutant clones only harbor a copy of the transgene. Accordingly, the level of Notch expressed from the wild-type transgene in the clones is less than that in the heterozygous cells (Figure 5B and 5B′), in agreement with our previous report [20]. Clones of Notchgt-16_20 in larval wing disc do not show an increase in Notch surface expression (data not shown). However, in the pupal wing, the level of surface Notch expressed from the Notchgt-16_20 transgene in the clones is significantly increased compared to that expressed from the wild-type Notchgt-wt transgene (Figure 5C and 5C′; compare to 5B′). One potential explanation for the difference between the effects of loss of Notch xylosylation in pupae versus larvae could be different levels of Shams expression at these stages. Indeed, Western blot confirmed higher Shams expression in pupae compared to third instar larvae (Figure 5D). In agreement with a role for xylose in surface expression of Notch, overexpression of human XXYLT1 results in a significant decrease and overexpression of human GXYLT1 results in a mild and partially penetrant decrease in Notch surface expression in the larval wing imaginal discs (Figure S8). Altogether, these observations suggest that addition of xylose residues to EGF16–20 of Notch decreases the availability of Notch at the cell surface.


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)

Increased surface expression of Notch in shams and Notchgt-16_20 clones in the pupal wing.All animals were raised at 30°C. (A,A′) Shown are confocal images from a pupal wing at 22–24 hours after puparium formation (APF) with a MARCM clone of shamsΔ34 marked by nuclear GFP (GFPNLS). Surface expression of Notch is shown in red. Note, also in the xz section, that the Notch surface level at this stage is increased in shams mutant cells. (B–C′) Shown are confocal images of pupal wings around 22 hours APF from animals harboring MARCM clones of the Notch54l9 protein- allele (marked by CD8::GFP) with one copy of either a wild-type Notch transgene (B,B′) or a Notch transgene with O-glucose mutations in EGF16–20 (C,C′). The only source of Notch in the clones is the Notch transgene. Note, also in xz sections, that the level of surface Notch in clones harboring the Notchgt-16_20 is increased compared to that in clones harboring Notchgt-wt. (D) Anti-HA Western blot on larval and pupal protein extracts from animals harboring one copy of an HA-tagged Shams genomic transgene (HA-Shams; shamsgt-wt-HA-attVK22) or attVK22 control animals. Tubulin was used as loading control. Pupal extracts show relatively higher levels of HA-Shams.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003547-g005: Increased surface expression of Notch in shams and Notchgt-16_20 clones in the pupal wing.All animals were raised at 30°C. (A,A′) Shown are confocal images from a pupal wing at 22–24 hours after puparium formation (APF) with a MARCM clone of shamsΔ34 marked by nuclear GFP (GFPNLS). Surface expression of Notch is shown in red. Note, also in the xz section, that the Notch surface level at this stage is increased in shams mutant cells. (B–C′) Shown are confocal images of pupal wings around 22 hours APF from animals harboring MARCM clones of the Notch54l9 protein- allele (marked by CD8::GFP) with one copy of either a wild-type Notch transgene (B,B′) or a Notch transgene with O-glucose mutations in EGF16–20 (C,C′). The only source of Notch in the clones is the Notch transgene. Note, also in xz sections, that the level of surface Notch in clones harboring the Notchgt-16_20 is increased compared to that in clones harboring Notchgt-wt. (D) Anti-HA Western blot on larval and pupal protein extracts from animals harboring one copy of an HA-tagged Shams genomic transgene (HA-Shams; shamsgt-wt-HA-attVK22) or attVK22 control animals. Tubulin was used as loading control. Pupal extracts show relatively higher levels of HA-Shams.
Mentions: To examine the effects of loss of shams on Notch localization, we performed Notch surface staining on larval wing imaginal discs and pupal wings harboring shams mutant clones. Loss of shams does not affect the surface expression of Notch in third instar wing imaginal disc (Figure S7A–A″). However, more Notch protein is present in and at the surface of shams mutant cells in the pupal wing (Figure 5A and 5A′ and Figure S7B–B′). We also sought to determine whether mutating the O-glucose sites on EGF16–20 of Notch results in increased cell surface levels of Notch. To this end, we generated Mosaic Analysis with a Repressible Cell Marker (MARCM) clones [28] of a protein- allele of Notch in a background harboring one copy of the wild-type or an EGF16–20 mutant Notch, similar to what we have described before for other mutant Notch transgenes [20]. In these animals, heterozygous cells have both endogenous Notch and a copy of our transgene, but cells in the mutant clones only harbor a copy of the transgene. Accordingly, the level of Notch expressed from the wild-type transgene in the clones is less than that in the heterozygous cells (Figure 5B and 5B′), in agreement with our previous report [20]. Clones of Notchgt-16_20 in larval wing disc do not show an increase in Notch surface expression (data not shown). However, in the pupal wing, the level of surface Notch expressed from the Notchgt-16_20 transgene in the clones is significantly increased compared to that expressed from the wild-type Notchgt-wt transgene (Figure 5C and 5C′; compare to 5B′). One potential explanation for the difference between the effects of loss of Notch xylosylation in pupae versus larvae could be different levels of Shams expression at these stages. Indeed, Western blot confirmed higher Shams expression in pupae compared to third instar larvae (Figure 5D). In agreement with a role for xylose in surface expression of Notch, overexpression of human XXYLT1 results in a significant decrease and overexpression of human GXYLT1 results in a mild and partially penetrant decrease in Notch surface expression in the larval wing imaginal discs (Figure S8). Altogether, these observations suggest that addition of xylose residues to EGF16–20 of Notch decreases the availability of Notch at the cell surface.

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