Negative regulation of notch signaling by xylose

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.

Figure 1. 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.

Figure 2. 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) shams^PB/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 shams^Δ34/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 shams^Δ34/Df(3R)BSC494 animals (I). (J) shams^gt-wt rescues shams^Δ34/Df(3R)BSC494 wing defects. (K) A genomic rescue transgene harboring CG11836 does not rescue the wing vein phenotype of shams^Δ34/Df(3R)BSC494 animals. (L) Wild-type adult heads have two ocellar bristles and two post-vertical bristles (arrowheads). (M) In shams^Δ34/Df(3R)BSC494 mutants raised at 30°C, ocellar and post-vertical bristles are lost. (N) This bristle phenotype is rescued by shams^gt-wt (arrowheads).

Figure 3. Xylosyltransferases inhibit Drosophila Notch signaling.

All animals were raised at 30°C. (A) N^55e11/+ animals show wing vein thickening (asterisk) and margin defects (arrows). (B) shams^PB/PB suppresses the N^55e11/+ phenotypes. (C) The Abruptex mutant N^Ax-E2/+ exhibits loss of wing vein at distal L5 (arrowhead) and occasionally L2. (D) shams^PB/PB enhances the N^Ax-E2/+ phenotype. (E) Wing-specific overexpression of HA-tagged human GXYLT1 by nubbin-GAL4 (nub>GXYLT1-HA) induces wing vein thickening (asterisk) and wing margin scalloping (arrow). (F) An additional copy of Notch suppresses the wing margin defect in nub>GXYLT1-HA. (G) The wing margin defect of nub>GXYLT1-HA flies is suppressed in a shams^PB/PB background. Note that GXYLT1-HA rescues the L5 wing vein loss phenotype of shams^PB/PB (compare to Figure 2D). (H) Co-overexpression of GXYLT1-HA and Shams results in the enhancement of the wing margin loss and wing vein expansion. (I) Overexpression of human XXYLT1-HA in the wing results in severe wing vein expansion and a complete loss of wing margin. (J) shams^PB/PB fully suppresses these phenotypes, but XXYLT1-HA overexpression does not suppress the L5 vein loss phenotype of shams ^PB/PB, indicating that the XXYLT1-HA phenotypes are strictly mediated by its enzymatic activity. (K) Overexpression of GXYLT1-HA by patched-GAL4 results in a mild wing margin loss in the patched domain (arrow). (L) Overexpression of XXYLT1-HA by patched-GAL4 results in wing vein thickening (bracket) and a more pronounced wing margin loss (arrow) in the patched domain. (M–N′) Double staining of wing imaginal discs by antibodies against HA (green) and the Notch downstream target Cut (red in M,N; gray in M′,N′). Note that the loss of Cut is less severe upon GXYLT1-HA overexpression (M,M′) compared to that resulting from XXYLT1-HA overexpression (N,N′).

Figure 4. 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; N^gt-wt/+, N⁻/Y; N^gt-10_15/+, and N⁻/Y; N^gt-24_35/+ males exhibit no wing vein loss, but N⁻/Y; N^gt-16_20/+ males (D) exhibit loss of L2, L4 and L5 veins (arrowheads). (F) At 25°C, N⁻/Y; N^gt-wt/+ males expressing GXYLT1-HA in the apterous-GAL4 domain show thickening of the distal wing veins. (G) In a N⁻/Y; N^gt-10_15/+ background, ap>GXYLT1-HA becomes lethal at 25°C, and is not suppressed at 22°C. (H) In a N⁻/Y; N^gt-16_20/+ background, the ap>GXYLT1-HA phenotype is fully suppressed. Note the presence of wing vein loss. (I) N^gt-24_35 does not suppress the ap>GXYLT1-HA phenotype. (J) At 25°C, N⁻/Y; N^gt-wt/+ males expressing nub>XXYLT1-HA show severe wing vein and margin defects. (K) The phenotypes are dramatically enhanced in N⁻/Y; N^gt-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; N^gt-16_20/+ males (L), but are enhanced in N⁻/Y; N^gt-24_35/+ males (M; compare to J). All wings, including the inset in M, are shown to scale.

Figure 5. 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 shams^Δ34 marked by nuclear GFP (GFP^NLS). 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 Notch^54l9 protein-null 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 Notch^gt-16_20 is increased compared to that in clones harboring Notch^gt-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; shams^gt-wt-HA-attVK22) or attVK22 control animals. Tubulin was used as loading control. Pupal extracts show relatively higher levels of HA-Shams.