ReviewRole of glycosylation of Notch in development
Introduction
Notch signaling is essential for proper development in metazoans, and defects in this pathway result in a number of human diseases [1], [2]. Notch is regulated at numerous overlapping levels, including endocytosis, ubiquitination, intracellular trafficking, degradation, and glycosylation [2], [3], [4], [5], [6]. Many genes impinge on this pathway, and the number of these genes continues to increase with the improved techniques for genome-wide analysis [7]. This review focuses on regulation of the Notch pathway by glycosylation.
The Notch phenotype was originally described in Drosophila nearly 100 years ago as an X-linked, dominant mutation which showed irregular “notches” at the tips of the wings [8]. Subsequent work demonstrated that Notch plays key roles in development of many tissues in flies, including formation of neurons and glial cells, leg segments, eyes, heart, muscles, and blood lineages [2], [9], [10]. Drosophila has a single Notch receptor, while mammals have four [1]. Targeted disruption of the four mouse Notch genes demonstrated that these genes play important roles in development of many tissues. Loss of mouse Notch1 results in an embryonic lethal phenotype with severe defects in somitogenesis [11], [12]. Subsequent studies showed that Notch1 is also involved in neurogenesis and vasculogenesis [13], [14]. Deletion of mouse Notch2 also results in an embryonic lethal phenotype with apoptotic cell death in a wide variety of tissues, especially neural tissues, from embryonic day 9.5 [15]. Notch3−/− mice are viable and fertile, but have defects in arterial differentiation and maturation of vascular smooth muscle [16]. Although Notch4−/− mice are viable and fertile [14], loss of Notch4 exacerbates the vascular remodeling defects observed in Notch1−/− embryo [14], suggesting partially overlapping function of Notch1 and 4 during embryogenesis. Aberrant Notch signaling leads to multiple human disorders [1], [17]. Mutations of Notch and the components of this pathway are implicated in human developmental disorders such as Alagille Syndrome and Spondylocostal Dysostosis, adult onset diseases such as CADASIL and Multiple Scleorosis, and cancers such as T cell acute lymphoblastic leukemia (T-ALL) and colon cancer.
Notch receptors are large type I transmembrane proteins [2]. Their basic molecular structure is evolutionarily conserved and consists of three domains: an extracellular domain (ECD) with 29–36 tandem epidermal growth factor-like (EGF) repeats and a unique negative regulatory region (NRR) which consists of three Lin-12/Notch repeats and a heterodimerization domain; a single transmembrane domain; and an intracellular domain with an RBP-Jκ (recombination signal sequence-binding protein-Jκ) association module domain, several nuclear localization sequences, seven ankyrin repeats, and a transactivation domain that harbors proline/glutamic acid/serine/threonine-rich motifs responsible for rapid degradation. The mature receptor is a heterodimer with the ECD tethered to the transmembrane/intracellular domain (T/ICD) through non-covalent, calcium dependent interactions. The heterodimer is formed by cleavage of the nascent polypeptide at site 1 by a furin-like protease in the Golgi [18], [19].
Notch ligands are also type I transmembrane proteins with a similar overall architecture: an ECD containing an N-terminal DSL (Delta/Serrate/LAG-2) motif, specialized tandem EGF repeats termed the DOS (Delta and OSM-11-like proteins) domain, and several tandem EGF repeats; a single transmembrane domain; and a small intracellular domain [20]. Drosophila has two ligands, Delta and Serrate, while mammals have three Delta-like ligands (Dll1, 3, and 4) and two Serrate homologues (Jagged1 and 2).
Notch activation is initiated by ligand binding, and accomplished through a proteolytic mechanism [21]. The first cleavage occurs at site 2 (S2), just outside the membrane on the T/ICD, and is catalyzed by a metalloprotease of the ADAM family. In the absence of ligand, S2 appears to be covered by the NRR, sterically blocking access of the ADAM protease to the site. Ligand binding results in a conformational change in the NRR, exposing the site and allowing cleavage [22], [23], [24]. Subsequently, cleavage at site 3 (S3) in the Notch transmembrane domain by the γ-secretase complex results in the release of the Notch intracellular domain (NICD), and translocation of the NICD into the nucleus [25]. Interaction between NICD and DNA binding proteins such as RBP-Jκ, activate target gene transcription [26].
Section snippets
Regulation of Notch function with glycosylation
The discovery that Fringe, a known modulator of Notch activity, is a glycosyltransferase modifying O-fucose glycans on Notch EGF repeats [27], [28], brought the study of Notch into the field of Glycobiology [29]. The EGF repeats of Notch are modified with three different types of O-linked glycosylation: O-fucosylation, O-glucosylation, and O-GlcNAc’ylation (Fig. 1) [30], [31], [32]. Addition of O-fucose to Ser/Thr occurs within the consensus sequence C2-X-X-X-X-(S/T)-C3 (C, cysteine; X, any
Conclusions
Evidence for the importance of carbohydrate modifications on Notch for signaling is largely based on genetic studies. While unidentified sugars may yet exist on Notch, most of the genes encoding the enzymes responsible for the synthesis of the known structures have been identified. The potential sites for O-fucosylation and O-glucosylation on the ECD of Notch are well conserved among species (Fig. 2), suggesting a distinct pattern of each modification on the entire ECD of Notch. Such
Acknowledgements
We would like to thank Dr. Kelly Ten Hagen for giving us an opportunity to write this manuscript, and Drs. Bernadette C. Holdener, Hamed Jafar-Nejad and Haltiwanger lab members for helpful comments. Primary work was supported by NIH grant GM061126 (to RSH) and the research grant from Mizutani Foundation for Glycoscience (to HT).
References (104)
- et al.
The canonical Notch signaling pathway: unfolding the activation mechanism
Cell
(2009) Notch signaling: the core pathway and its posttranslational regulation
Dev Cell
(2009)- et al.
Arrestin development: emerging roles for beta-arrestins in developmental signaling pathways
Dev Cell
(2009) - et al.
Role of unusual O-glycans in intercellular signaling
Int J Biochem Cell Biol
(2009) Regulation of Notch signaling by glycosylation
Curr Opin Struct Biol
(2007)- et al.
Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL
Blood
(2009) - et al.
Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules
J Biol Chem
(2000) - et al.
O-linked N-acetylglucosamine is present on the extracellular domain of Notch receptors
J Biol Chem
(2008) - et al.
Lunatic Fringe, Manic Fringe, and Radical Fringe recognize similar specificity determinants in O-fucosylated epidermal growth factor-like repeats
J Biol Chem
(2005) - et al.
In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe
J Biol Chem
(2007)
Notch ligands are substrates for EGF protein O-fucosyltransferase and Fringe
J Biol Chem
Notch-dependent control of myelopoiesis is regulated by fucosylation
Blood
Fucose is on the TRAIL of colon cancer
Gastroenterology
Modification of epidermal growth factor-like repeats with O-fucose: molecular cloning of a Novel GDP-Fucose: protein O-fucosyltransferase
J Biol Chem
O-fucosylation of Notch occurs in the endoplasmic reticulum
J Biol Chem
Purification and characterization of a GDP-fucose: polypeptide fucosyltransferase from Chinese hamster ovary cells
J Biol Chem
Interaction between Notch signalling and Lunatic Fringe during somite boundary formation in the mouse
Curr Biol
Regulation of Notch signaling by O-linked fucose
Cell
Fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development
Cell
Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype
Am J Hum Genet
Oscillating signaling pathways during embryonic development
Curr Opin Cell Biol
Lunatic and Manic Fringe cooperatively enhance marginal zone B cell precursor competition for delta-like 1 in splenic endothelial niches
Immunity
The Notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis
Cell
The diversity of O-linked glycans expressed during Drosophila melanogaster development reflects stage- and tissue-specific requirements for cell signaling
J Biol Chem
Expression of Notch signaling pathway genes in mouse embryos lacking beta4galactosyltransferase-1
Gene Expr Patterns
Human factor IX has a tetrasaccharide O-glycosidically linked to serine 61 through the fucose residue
J Biol Chem
Pofut1 is required for the proper localization of the Notch receptor during mouse development
Mech Dev
Roles of Pofut1 and O-fucose in mammalian Notch signaling
J Biol Chem
Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the abruptex region
J Biol Chem
Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor
Cell
Structural and functional properties of the human Notch-1 ligand binding region
Structure (Camb)
Localisation of the Delta-like-1 binding site in human Notch-1 and its modulation by calcium affinity
J Biol Chem
Regions of Drosophila Notch that contribute to ligand binding and the modulatory influence of Fringe
J Biol Chem
Highly conserved O-fucose sites have distinct effects on Notch1 function
J Biol Chem
Identification of a disaccharide (Xyl-Glc) and a trisaccharide (Xyl2-Glc) O-glycosidically linked to a serine residue in the first epidermal growth factor-like domain of human factors VII and IX and protein Z and bovine protein Z
J Biol Chem
The structure of (xylose)2glucose-O-serine 53 found in the first epidermal growth factor-like domain of bovine blood clotting factor IX
J Biol Chem
Rumi is a CAP10 domain glycosyltransferase that modifies Notch and is required for Notch signaling
Cell
Glycosylation regulates Notch signaling
Nat Rev Mol Cell Biol
Genome-wide analysis of Notch signalling in Drosophila by transgenic RNAi
Nature
Character changes caused by mutation of an entire region of a chromosome in Drosophila
Genetics
Notch signaling: cell fate control and signal integration in development
Science
Notch signaling: control of cell communication and cell fate
Development
Notch1 is essential for postimplantation development in mice
Genes Dev
Notch1 is required for the coordinate segmentation of somites
Development
Conservation of the Notch signalling pathway in mammalian neurogenesis
Development
Notch signaling is essential for vascular morphogenesis in mice
Genes Dev
Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality
Development
Characterization of Notch3-deficient mice: normal embryonic development and absence of genetic interactions with a Notch1 mutation
Genesis
Notch signaling in normal and disease states: possible therapies related to glycosylation
Curr Mol Med
In vivo analysis of the Notch receptor S1 cleavage
PLoS One
Cited by (74)
Identifications of three novel alleles of Serrate in Drosophila
2024, Cells and DevelopmentProtein modifications | Glycoprotein-mediated cell interactions, nonmucin-type O-linked
2021, Encyclopedia of Biological Chemistry: Third EditionSeparation and Purification of Sialylglycopeptide from Egg Yolk Based on Cotton Hydrophilic Chromatography
2020, Chinese Journal of Analytical Chemistry