Notch receptor activation initiates cell fate decisions, and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5 angstrom crystal structure of the extracellular interacting regions of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ∼120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 EGF domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch domains are favored in binding to Jag1 compared to those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate amongst Notch ligands and potentiate Notch signaling.

Summary Fringe proteins are β3-N-acetylglucosaminyltransferases that modulate Notch activity by modifying O-fucose residues on Epidermal Growth Factor-like (EGF) repeats of Notch. Mammals have three Fringes: Lunatic, Manic, and Radical. While Lunatic and Manic Fringe inhibit Notch1 activation from Jagged1 and enhance activation from Delta-like 1, Radical Fringe enhances signaling from both. We used a mass spectral approach to determine whether the variable effects of Fringes on Notch1 result from generation of unique glycosylation patterns on Notch1. We found that Lunatic and Manic Fringe modified similar sites on Notch1, while Radical Fringe modified a subset. Fringe modifications at EGF8 and 12 enhanced Notch1 binding to and activation from Delta-like 1, while modifications at EGF6 and 36 (added by Manic and Lunatic but not Radical) inhibited Notch1 activation from Jagged1. Combined, these results suggest that Fringe modifications “mark” different regions in the Notch1 extracellular domain for activation or inhibition.

Haploinsufficiency for the Notch ligand JAG1 in humans results in an autosomal dominant, multisystem disorder known as Alagille syndrome, which is characterized by a congenital cholangiopathy of variable severity. Here we show that on a C57BL/6 background, jagged1 heterozygous mice (Jag1+/−) exhibit impaired intrahepatic bile duct (IHBD) development, decreased SOX9 expression and thinning of the peri-portal vascular smooth muscle cell (VSMC) layer, which are apparent at embryonic day 18 and the first postnatal week. In contrast, mice double-heterozygous for Jag1 and the glycosyltransferase Poglut1 (Rumi) start showing a significant improvement in IHBD development and VSMC differentiation during the first week. At P30, Jag1+/− mice show widespread ductular reactions and ductopenia in their livers and a mild but statistically significant bilirubinemia. In contrast, P30 Jag1/Rumi double-heterozygous mice show well-developed portal triads around most portal veins, with no elevation of serum bilirubin. Conditional deletion of Rumi in VSMCs results in progressive arborization of the IHBD tree, whereas deletion of Rumi in hepatoblasts frequently results in an increase in the number of hepatic arteries without affecting bile duct formation. Nevertheless, removing one copy of Rumi from either VSMCs or hepatoblasts is sufficient to partially suppress the Jag1+/− bile duct defects. Finally, all Rumi target sites of the human JAG1 are efficiently glucosylated, and loss of Rumi in VSMCs results in increased levels of full-length JAG1 and a shorter fragment of JAG1 without affecting Jag1 mRNA levels. Conclusions On a C57BL/6 background, Jag1 haploinsufficiency results in bile duct paucity in mice. Removing one copy of Rumi suppresses the Jag1+/− bile duct phenotype, indicating that Rumi opposes the JAG1 function in the liver.

Notch activity is regulated by both O-fucosylation and O-glucosylation, and Notch receptors contain multiple predicted sites for both. Here we examine the occupancy of the predicted O-glucose sites on mouse Notch1 (mN1) using the consensus sequence C 1 XSXPC 2 . We show that all of the predicted sites are modified, although the efficiency of modifying O-glucose sites is site-and cell type-dependent. For instance, although most sites are modified at high stoichiometries, the site at EGF 27 is only partially glucosylated, and the occupancy of the site at EGF 4 varies with cell type. O-Glucose is also found at a novel, nontraditional consensus site at EGF 9. Based on this finding, we propose a revision of the consensus sequence for O-glucosylation to allow alanine N-terminal to cysteine 2: C 1 XSX(A/P)C 2 . We also show through biochemical and mass spectral analyses that serine is the only hydroxyamino acid that is modified with O-glucose on EGF repeats. The O-glucose at all sites is efficiently elongated to the trisaccharide Xyl-Xyl-Glc. To establish the functional importance of individual O-glucose sites in mN1, we used a cell-based signaling assay. Elimination of most individual sites shows little or no effect on mN1 activation, suggesting that the major effects of O-glucose are mediated by modification of multiple sites. Interestingly, elimination of the site in EGF 28, found in the Abruptex region of Notch, does significantly reduce activity. These results demonstrate that, like O-fucose, the O-glucose modifications of EGF repeats occur extensively on mN1, and they play important roles in Notch function.The Notch family of single-pass transmembrane receptors is essential for early metazoan development, activating the expression of many genes involved in cell differentiation and tissue morphogenesis (1-3). Defects in Notch signaling have been implicated in a number of human diseases, including several forms of cancer, vascular defects such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (4 -6), multiple sclerosis (6), and a number of developmental syndromes (4, 7-10). The canonical Notch signaling pathway is initiated by the interaction of Notch with its ligand on an apposed cell. Upon ligand binding, Notch undergoes presenilin-1-dependent proteolysis, releasing a soluble Notch intracellular domain, which enters the cell nucleus to interact directly with the transcription factors from the CSL family and regulate Notch target genes (2). There are four members of the Notch family in vertebrates (Notch1 to Notch4) interacting with several classes of Notch ligands: ligands of the DSL family (Delta-like 1, 3, and 4 and Jagged 1 and 2) (1) and newly characterized ligands without the DSL domain, such as DNER and MAGP-1 and -2 (11-13).The Notch proteins consist of a large extracellular domain (ECD), 5 a transmembrane region, and a large intracellular domain (1, 2, 14). The majority of the ECD consists of tandem epidermal growth factor-like (EGF) repeats (36 EGF repeats are ...

Notch signaling affects many developmental and cellular processes and has been implicated in congenital disorders, stroke, and numerous cancers. The Notch receptor binds its ligands Delta and Serrate and is able to discriminate between them in different contexts. However, the specific domains in Notch responsible for this selectivity are poorly defined. Through genetic screens in Drosophila, we isolated a mutation, Notchjigsaw, that affects Serrate- but not Delta-dependent signaling. Notchjigsaw carries a missense mutation in epidermal growth factor repeat-8 (EGFr-8) and is defective in Serrate binding. A homologous point mutation in mammalian Notch2 also exhibits defects in signaling of a mammalian Serrate homolog, Jagged1. Hence, an evolutionarily conserved valine in EGFr-8 is essential for ligand selectivity and provides a molecular handle to study numerous Notch-dependent signaling events.