Notch receptor signaling is regulated by glycosylation

Abstract

Notch receptors are cell surface glycoproteins that are stimulated by glycoprotein ligands Jagged or Delta on adjacent cells to induce target gene expression, thereby altering cell fate determination. Disregulated Notch signal transduction leads to developmental defects and cancer. Fringe is a beta3 N-acetylglucosaminyltransferase that adds GlcNAc to O-fucose on the epidermal growth factor (EGF)-like repeats of Notch receptors. In Drosophila, Fringe action inhibits Serrate/Jagged-induced Notch signaling, but potentiates Delta-dependent Notch activation. Experiments with a co-culture Notch signaling assay and Chinese hamster ovary (CHO) glycosylation mutants indicated that O-fucose on Notch is not only the substrate for Fringe, but is also important for optimal Notch signaling itself. In CHO cells, it was found that elongation of Notch-O-fucosebeta3GlcNAc glycans with a galactose residue added by beta4GalT-1 is necessary for the modulation of Jaggd1-induced Notch signaling by Fringe. By contrast, neither sialic acid on O-fucose glycans, nor complex or hybrid N-glycans were found to be required. Inactivation of Lunatic fringe in mice leads to perinatal lethality due to altered Notch signaling during somitogenesis. Therefore, somitogenesis was investigated in beta4GalT-1 null mice. While there was no defect in the skeletal preparation of E15 and E18 beta4GalT-1 mutant embryos and no significant difference in size or morphology were observed in E9.5 mutant embryos, in situ hybridization with a subset of somitogenic and Notch pathway gene probes showed that the expression of some Notch pathway genes, in particular Hes5, Mesp2, Dll1 and Dll3 are reduced and altered in at least 50% of beta 4GalT-1 mutant embryos. These studies provided evidence that Gal is involved in Notch signaling during somitogenesis in mice. The phenotype is subtle and suggests that more than one of the six mammalian beta4galactosyltransferases may need to be inactivated to obtain severe somitogenic phenotypes.