Xylosylation of the Notch receptor preserves the balance between its activation by <i>trans</i>-Delta and inhibition by <i>cis</i>-ligands in <i>Drosophila</i>

<div><p>The <i>Drosophila</i> glucoside xylosyltransferase Shams xylosylates Notch and inhibits Notch signaling in specific contexts including wing vein development. However, the molecular mechanisms underlying context-specificity of the <i>shams</i> phenotype is not known. Considering the role of Delta-Notch signaling in wing vein formation, we hypothesized that Shams might affect Delta-mediated Notch signaling in <i>Drosophila</i>. Using genetic interaction studies, we find that altering the gene dosage of <i>Delta</i> affects the wing vein and head bristle phenotypes caused by loss of Shams or by mutations in the Notch xylosylation sites. Clonal analysis suggests that loss of <i>shams</i> promotes Delta-mediated Notch activation. Further, Notch <i>trans</i>-activation by ectopically overexpressed Delta shows a dramatic increase upon loss of <i>shams</i>. In agreement with the above <i>in vivo</i> observations, cell aggregation and ligand-receptor binding assays show that <i>shams</i> knock-down in Notch-expressing cells enhances the binding between Notch and <i>trans</i>-Delta without affecting the binding between Notch and <i>trans</i>-Serrate and cell surface levels of Notch. Loss of Shams does not impair the <i>cis</i>-inhibition of Notch by ectopic overexpression of ligands <i>in vivo</i> or the interaction of Notch and <i>cis</i>-ligands in S2 cells. Nevertheless, removing one copy of endogenous ligands mimics the effects of loss <i>shams</i> on Notch <i>trans</i>-activation by ectopic Delta. This favors the notion that <i>trans</i>-activation of Notch by Delta overcomes the <i>cis</i>-inhibition of Notch by endogenous ligands upon loss of <i>shams</i>. Taken together, our data suggest that xylosylation selectively impedes the binding of Notch with <i>trans</i>-Delta without affecting its binding with <i>cis</i>-ligands and thereby assists in determining the balance of Notch receptor’s response to <i>cis-</i>ligands vs. <i>trans-</i>Delta during <i>Drosophila</i> development.</p>