ABSTRACT: Notch signaling is essential for cell-fate specification in metazoans, and dysregulation of the pathway leads to a variety of human diseases including heart and vascular defects as well as cancer. Glycosylation of the Notch extracellular domain has emerged as an elegant means for regulating Notch activity, especially since the discovery that Fringe is a glycosyltransferase that modifies O-fucose in 2000. Since then, several other O-glycans on the extracellular domain have been demonstrated to modulate Notch activity. Here we will describe recent results on the molecular mechanisms by which Fringe modulates Notch activity, summarize recent work on how O-glucose, O-GlcNAc, and O-GalNAc glycans affect Notch, and discuss several human genetic disorders resulting from defects in Notch glycosylation.
Project description:Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the ?3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity.
Project description:Fringe glycosyltransferases differentially modulate the binding of Notch receptors to Delta/DLL versus Serrate/Jagged ligands by adding GlcNAc to O-linked fucose on Notch epidermal growth factor-like (EGF) repeats. Although Notch has 22 O-fucosylation sites, the biologically relevant sites affecting Notch activity during animal development in vivo in the presence or absence of Fringe are not known. Using a variety of assays, we find important roles in Drosophila Notch signaling for GlcNAc-fucose-O glycans on three sites: EGF8, EGF9, and EGF12. O-Fucose monosaccharide on EGF12 (in the absence of Fringe) is essential for Delta-mediated lateral inhibition in embryos. However, wing vein development depends on the addition of GlcNAc to EGF8 and EGF12 by Fringe, with a minor contribution from EGF9. Fringe modifications of EGF8 and EGF12 together prevent Notch from cis-inhibiting Serrate, thereby promoting normal wing margin formation. Our work shows the combinatorial and context-dependent roles of GlcNAc-fucose-O glycans on these sites in Drosophila Notch-ligand interactions.
Project description:Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a beta 3N-acetylglucosaminyltransferase (beta 3GlcNAcT) that transfers GlcNAc to O-fucose in epidermal growth factor-like repeats of Notch. Here we use five different Chinese hamster ovary cell glycosylation mutants to identify a key aspect of the mechanism of fringe action. Although the beta 3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAc beta 3Fuc, is not elongated. The trisaccharide, Gal beta 4GlcNAc beta 3Fuc, is the minimal O-fucose glycan to support fringe modulation of Notch signaling. Of six beta 4galactosyltransferases (beta 4GalT) in Chinese hamster ovary cells, only beta 4GalT-1 is required to add Gal to GlcNAc beta 3Fuc, identifying beta 4GalT-1 as a new modulator of Notch signaling.
Project description:Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.
Project description:Fringe proteins are ?3-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 spectrometry 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 EGF12 enhanced Notch1 binding to and activation from Delta-like 1, while modifications at EGF6 and EGF36 (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.
Project description:The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.
Project description:Appropriate glycoprotein O-glycosylation is essential for normal development and tissue function in multicellular organisms. To comprehensively assess the developmental and functional impact of altered O-glycosylation, we have extensively analyzed the non-glycosaminoglycan, O-linked glycans expressed in Drosophila embryos. Through multidimensional mass spectrometric analysis of glycans released from glycoproteins by beta-elimination, we detected novel as well as previously reported O-glycans that exhibit developmentally modulated expression. The core 1 mucin-type disaccharide (Galbeta1-3GalNAc) is the predominant glycan in the total profile. HexNAcitol, hexitol, xylosylated hexitol, and branching extension of core 1 with HexNAc (to generate core 2 glycans) were also evident following release and reduction. After Galbeta1-3GalNAc, the next most prevalent glycans were a mixture of novel, isobaric, linear, and branched forms of a glucuronyl core 1 disaccharide. Other less prevalent structures were also extended with HexA, including an O-fucose glycan. Although the expected disaccharide product of the Fringe glycosyltransferase, (GlcNAcbeta1-3)fucitol, was not detectable in whole embryos, mass spectrometry fragmentation and exoglycosidase sensitivity defined a novel glucuronyl trisaccharide as GlcNAcbeta1-3(GlcAbeta1-4)fucitol. Consistent with the spatial distribution of the Fringe function, the GlcA-extended form of the Fringe product was enriched in the dorsal portion of the wing imaginal disc. Furthermore, loss of Fringe activity reduced the prevalence of the O-Fuc trisaccharide. Therefore, O-Fuc glycans necessary for the modulation of important signaling events in Drosophila are, as in vertebrates, substrates for extension beyond the addition of a single HexNAc.
Project description:Lunatic fringe belongs to a family of beta1-3 N-acetyltransferases that modulate the affinity of the Notch receptors for their ligands through the elongation of O-fucose moieties on their extracellular domain. A role for Notch signaling in vertebrate fertility has been predicted by the intricate expression of the Notch receptors and their ligands in the oocyte and granulosa cells of the ovary and the spermatozoa and Sertoli cells of the testis. It has been demonstrated that disruption of Notch signaling by inactivation of lunatic fringe led to infertility associated with pleiotropic defects in follicle development and meiotic maturation of oocytes. Lunatic fringe null males were found to be subfertile. Here, we report that gene expression data demonstrate that fringe and Notch signaling genes are expressed in the developing testis and the intratesticular ductal tract, predicting roles for this pathway during embryonic gonadogenesis and spermatogenesis. Spermatogenesis was not impaired in the majority of the lunatic fringe null males; however, spermatozoa were unilaterally absent in the epididymis of many mice. Histological and immunohistochemical analysis of these testes revealed the development of unilateral cystic dilation of the rete testis. Tracer dye experiments confirm a block in the connection between the rete testis and the efferent ducts. Further, the dye studies demonstrated that many lunatic fringe mutant males had partial blocks of the connection between the rete testis and the efferent ducts bilaterally.
Project description:<h4>Background</h4>O-fucosyltransferase1 (OFUT1) is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the N-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in Drosophila.<h4>Results</h4>We show that expression of an isoform lacking fucosyltransferase activity, Ofut1R245A, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development were evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose.<h4>Conclusion</h4>Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.
Project description:In the last two decades, our knowledge of the role of glycans in development and signal transduction has expanded enormously. While most work has focused on the importance of N-linked or mucin-type O-linked glycosylation, recent work has highlighted the importance of several more unusual forms of glycosylation that are the focus of this review. In particular, the ability of O-fucose glycans on the epidermal growth factor-like (EGF) repeats of Notch to modulate signaling places glycosylation alongside phosphorylation as a means to modulate protein-protein interactions and their resultant downstream signals. The recent discovery that O-glucose modification of Notch EGF repeats is also required for Notch function has further expanded the range of glycosylation events capable of modulating Notch signaling. The prominent role of Notch during development and in later cell-fate decisions underscores the importance of these modifications in human biology. The role of glycans in intercellular signaling events is only beginning to be understood and appears ready to expand into new areas with the discovery that thrombospondin type 1 repeats are also modified with O-fucose glycans. Finally, a rare form of glycosylation called C-mannosylation modifies tryptophans in some signaling competent molecules and may be a further layer of complexity in the field. We will review each of these areas focusing on the glycan structures produced, the consequence of their presence, and the enzymes responsible.