Limits...
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.

Show MeSH
Mutations in shams result in the loss of wing veins and head bristles.(A) Schematic representation of the genomic region containing shams (CG9996) and its neighboring genes, shams alleles, and the shams and CG11836 rescue transgene. Black boxes indicate the coding parts of exons. (B) Adult wing of a wild-type fly. (C,D) shamsPB/PB (e01256) mutants raised at 25°C exhibit a partially penetrant loss of the posterior cross-vein (C) and at 30°C lose the distal portion of the L5 wing vein (arrowheads) (D). (E) Precise excision of the piggyBac insertion results in animals with normal wing veins. (F,G) Adult wings of shamsΔ34/Df(3R)BSC494 flies raised at 25°C lose wing vein material at the distal end of L5 (F) and at 30°C exhibit substantial loss of L4, L5, and posterior cross-vein (G). (H,I) Overexpression of shams with nubbin-GAL4 does not cause any phenotypes in the wing (H), but rescues the wing vein loss in shamsΔ34/Df(3R)BSC494 animals (I). (J) shamsgt-wt rescues shamsΔ34/Df(3R)BSC494 wing defects. (K) A genomic rescue transgene harboring CG11836 does not rescue the wing vein phenotype of shamsΔ34/Df(3R)BSC494 animals. (L) Wild-type adult heads have two ocellar bristles and two post-vertical bristles (arrowheads). (M) In shamsΔ34/Df(3R)BSC494 mutants raised at 30°C, ocellar and post-vertical bristles are lost. (N) This bristle phenotype is rescued by shamsgt-wt (arrowheads).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3675014&req=5

pgen-1003547-g002: Mutations in shams result in the loss of wing veins and head bristles.(A) Schematic representation of the genomic region containing shams (CG9996) and its neighboring genes, shams alleles, and the shams and CG11836 rescue transgene. Black boxes indicate the coding parts of exons. (B) Adult wing of a wild-type fly. (C,D) shamsPB/PB (e01256) mutants raised at 25°C exhibit a partially penetrant loss of the posterior cross-vein (C) and at 30°C lose the distal portion of the L5 wing vein (arrowheads) (D). (E) Precise excision of the piggyBac insertion results in animals with normal wing veins. (F,G) Adult wings of shamsΔ34/Df(3R)BSC494 flies raised at 25°C lose wing vein material at the distal end of L5 (F) and at 30°C exhibit substantial loss of L4, L5, and posterior cross-vein (G). (H,I) Overexpression of shams with nubbin-GAL4 does not cause any phenotypes in the wing (H), but rescues the wing vein loss in shamsΔ34/Df(3R)BSC494 animals (I). (J) shamsgt-wt rescues shamsΔ34/Df(3R)BSC494 wing defects. (K) A genomic rescue transgene harboring CG11836 does not rescue the wing vein phenotype of shamsΔ34/Df(3R)BSC494 animals. (L) Wild-type adult heads have two ocellar bristles and two post-vertical bristles (arrowheads). (M) In shamsΔ34/Df(3R)BSC494 mutants raised at 30°C, ocellar and post-vertical bristles are lost. (N) This bristle phenotype is rescued by shamsgt-wt (arrowheads).

Mentions: To examine the role of xylosylation in Drosophila Notch signaling, we performed genetic experiments on two independent alleles of shams (Figure 2A). Flies homozygous for the piggyBac insertion shamse01256 (shamsPB/PB) are viable at 25°C and do not exhibit any adult phenotypes besides a loss of the posterior cross-vein in 20% of the flies (Figure 2C; compare to 2B). When raised at 30°C, 56% of shamsPB/PB flies lose the distal portion of the L5 wing vein (Figure 2D), similar to the phenotype observed in gain-of-function Abruptex alleles of Notch (NAx) [24]. Precise excision of this piggyBac insertion fully reverts the phenotype, indicating that the observed loss of the wing vein is due to the insertion (Figure 2E). We also generated a allele lacking 97% of the Shams coding region by using FLP/FRT-mediated recombination on two piggyBac insertions in the region (Figure 2A) [25]. Animals homozygous or hemizygous for the allele shamsΔ34 survive to adulthood and exhibit a 100% penetrant loss of the L5 wing vein at 25°C (Figure 2F). At 30°C, shamsΔ34/Df animals are semi-lethal and all the escapers exhibit partial loss of multiple wing veins (Figure 2G). The wing vein loss phenotype can be rescued by overexpression of shams cDNA (Figure 2H and 2I), by providing a shamsgt-wt genomic transgene that contains the shams locus (Figure 2J), or by an HA-tagged version of the shams genomic transgene (shamsgt-wt-HA, data not shown). However, even though the shamsΔ34 allele also affects CG11836 (Figure 2A), a genomic transgene containing this gene (CG11836gt-wt) does not rescue the wing vein phenotypes of the shamsΔ34/Df animals (Figure 2K). Of note, each of the shamsgt-wt and CG11836gt-wt genomic transgenes partially rescues the semi-lethality of these animals, indicating that both transgenes are functional. These observations indicate that loss of shams results in a wing vein loss phenotype. shamsΔ34/Df animals raised at 30°C also lose the ocellar and postvertical bristles in the head (Figure 2M; compare to 2L) similar to NAx alleles [26]. Again, this phenotype can be fully suppressed by a shams genomic transgene (Figure 2N). Together, these data indicate that loss of shams results in phenotypes reminiscent of Notch gain-of-function phenotypes.


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)

Mutations in shams result in the loss of wing veins and head bristles.(A) Schematic representation of the genomic region containing shams (CG9996) and its neighboring genes, shams alleles, and the shams and CG11836 rescue transgene. Black boxes indicate the coding parts of exons. (B) Adult wing of a wild-type fly. (C,D) shamsPB/PB (e01256) mutants raised at 25°C exhibit a partially penetrant loss of the posterior cross-vein (C) and at 30°C lose the distal portion of the L5 wing vein (arrowheads) (D). (E) Precise excision of the piggyBac insertion results in animals with normal wing veins. (F,G) Adult wings of shamsΔ34/Df(3R)BSC494 flies raised at 25°C lose wing vein material at the distal end of L5 (F) and at 30°C exhibit substantial loss of L4, L5, and posterior cross-vein (G). (H,I) Overexpression of shams with nubbin-GAL4 does not cause any phenotypes in the wing (H), but rescues the wing vein loss in shamsΔ34/Df(3R)BSC494 animals (I). (J) shamsgt-wt rescues shamsΔ34/Df(3R)BSC494 wing defects. (K) A genomic rescue transgene harboring CG11836 does not rescue the wing vein phenotype of shamsΔ34/Df(3R)BSC494 animals. (L) Wild-type adult heads have two ocellar bristles and two post-vertical bristles (arrowheads). (M) In shamsΔ34/Df(3R)BSC494 mutants raised at 30°C, ocellar and post-vertical bristles are lost. (N) This bristle phenotype is rescued by shamsgt-wt (arrowheads).
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1003547-g002: Mutations in shams result in the loss of wing veins and head bristles.(A) Schematic representation of the genomic region containing shams (CG9996) and its neighboring genes, shams alleles, and the shams and CG11836 rescue transgene. Black boxes indicate the coding parts of exons. (B) Adult wing of a wild-type fly. (C,D) shamsPB/PB (e01256) mutants raised at 25°C exhibit a partially penetrant loss of the posterior cross-vein (C) and at 30°C lose the distal portion of the L5 wing vein (arrowheads) (D). (E) Precise excision of the piggyBac insertion results in animals with normal wing veins. (F,G) Adult wings of shamsΔ34/Df(3R)BSC494 flies raised at 25°C lose wing vein material at the distal end of L5 (F) and at 30°C exhibit substantial loss of L4, L5, and posterior cross-vein (G). (H,I) Overexpression of shams with nubbin-GAL4 does not cause any phenotypes in the wing (H), but rescues the wing vein loss in shamsΔ34/Df(3R)BSC494 animals (I). (J) shamsgt-wt rescues shamsΔ34/Df(3R)BSC494 wing defects. (K) A genomic rescue transgene harboring CG11836 does not rescue the wing vein phenotype of shamsΔ34/Df(3R)BSC494 animals. (L) Wild-type adult heads have two ocellar bristles and two post-vertical bristles (arrowheads). (M) In shamsΔ34/Df(3R)BSC494 mutants raised at 30°C, ocellar and post-vertical bristles are lost. (N) This bristle phenotype is rescued by shamsgt-wt (arrowheads).
Mentions: To examine the role of xylosylation in Drosophila Notch signaling, we performed genetic experiments on two independent alleles of shams (Figure 2A). Flies homozygous for the piggyBac insertion shamse01256 (shamsPB/PB) are viable at 25°C and do not exhibit any adult phenotypes besides a loss of the posterior cross-vein in 20% of the flies (Figure 2C; compare to 2B). When raised at 30°C, 56% of shamsPB/PB flies lose the distal portion of the L5 wing vein (Figure 2D), similar to the phenotype observed in gain-of-function Abruptex alleles of Notch (NAx) [24]. Precise excision of this piggyBac insertion fully reverts the phenotype, indicating that the observed loss of the wing vein is due to the insertion (Figure 2E). We also generated a allele lacking 97% of the Shams coding region by using FLP/FRT-mediated recombination on two piggyBac insertions in the region (Figure 2A) [25]. Animals homozygous or hemizygous for the allele shamsΔ34 survive to adulthood and exhibit a 100% penetrant loss of the L5 wing vein at 25°C (Figure 2F). At 30°C, shamsΔ34/Df animals are semi-lethal and all the escapers exhibit partial loss of multiple wing veins (Figure 2G). The wing vein loss phenotype can be rescued by overexpression of shams cDNA (Figure 2H and 2I), by providing a shamsgt-wt genomic transgene that contains the shams locus (Figure 2J), or by an HA-tagged version of the shams genomic transgene (shamsgt-wt-HA, data not shown). However, even though the shamsΔ34 allele also affects CG11836 (Figure 2A), a genomic transgene containing this gene (CG11836gt-wt) does not rescue the wing vein phenotypes of the shamsΔ34/Df animals (Figure 2K). Of note, each of the shamsgt-wt and CG11836gt-wt genomic transgenes partially rescues the semi-lethality of these animals, indicating that both transgenes are functional. These observations indicate that loss of shams results in a wing vein loss phenotype. shamsΔ34/Df animals raised at 30°C also lose the ocellar and postvertical bristles in the head (Figure 2M; compare to 2L) similar to NAx alleles [26]. Again, this phenotype can be fully suppressed by a shams genomic transgene (Figure 2N). Together, these data indicate that loss of shams results in phenotypes reminiscent of Notch gain-of-function phenotypes.

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