Notch ligands are substrates for protein O-fucosyltransferase-1 and Fringe

O-Fucose has been identified on epidermal growth factor-like (EGF) repeats of Notch, and elongation of O-fucose has been implicated in the modulation of Notch signaling by Fringe. O-Fucose modifications are also predicted to occur on Notch ligands based on the presence of the C(2)XXGG(S/T)C(3) consensus site (where S/T is the modified amino acid) in a number of the EGF repeats of these proteins. Here we establish that both mammalian and Drosophila Notch ligands are modified with O-fucose glycans, demonstrating that the consensus site was useful for making predictions. The presence of O-fucose on Notch ligands raised the question of whether Fringe, an O-fucose specific beta 1,3-N-acetylglucosaminyltransferase, was capable of modifying O-fucose on the ligands. Indeed, O-fucose on mammalian Delta 1 and Jagged1 can be elongated with Manic Fringe in vivo, and Drosophila Delta and Serrate are substrates for Drosophila Fringe in vitro. These results raise the interesting possibility that alteration of O-fucose glycans on Notch ligands could play a role in the mechanism of Fringe action on Notch signaling. As an initial step to begin addressing the role of the O-fucose glycans on Notch ligands in Notch signaling, a number of mutations in predicted O-fucose glycosylation sites on Drosophila Serrate have been generated. Interestingly, analysis of these mutants has revealed that O-fucose modifications occur on some EGF repeats not predicted by the C(2)XXGGS/TC(3) consensus site. A revised, broad consensus site, C(2)X(3-5)S/TC(3) (where X(3-5) are any 3-5 amino acid residues), is proposed.     

Modulation of receptor signaling by glycosylation: fringe is an O-fucose-beta1,3-N-acetylglucosaminyltransferase

The Notch family of signaling receptors plays key roles in determining cell fate and growth control. Recently, a number of laboratories have shown that O-fucose glycans on the epidermal growth factor (EGF)-like repeats of the Notch extracellular domain modulate Notch signaling. Fringe, a known modifier of Notch function, is an O-fucose specific beta1,3-N-acetylglucosaminyltransferase. The transfer of GlcNAc to O-fucose on Notch by fringe results in the potentiation of signaling by the Delta class of Notch ligands, but causes inhibition of signaling by the Serrate/Jagged class of Notch ligands. Interestingly, addition of a beta1,4 galactose by beta4GalT-1 to the GlcNAc added by fringe is required for Jagged1-induced Notch signaling to be inhibited in a co-culture assay. Thus, both fringe and beta4GalT-1 are modulators of Notch function. Several models have been proposed to explain how alterations in O-fucose glycans result in changes in Notch signaling, and these models are discussed.     

Notch signaling in normal and disease States: possible therapies related to glycosylation

The Notch signaling pathway is involved in a wide variety of highly conserved developmental processes in mammals. Importantly, mutations of the Notch protein and components of its signaling pathway have been implicated in an array of human diseases (T-cell leukemia and other cancers, Multiple Sclerosis, CADASIL, Alagille Syndrome, Spondylocostal Dysostosis). In mammals, Notch becomes activated upon binding of its extracellular domain to ligands (Delta and Jagged/Serrate) that are present on the surface of apposed cells. The extracellular domain of Notch contains up to 36 tandem Epidermal Growth Factor-like (EGF) repeats. Many of these EGF repeats are modified at evolutionarily-conserved consensus sites by an unusual form of O-glycosylation called O-fucose. Work from several groups indicates that O-fucosylation plays an important role in ligand mediated Notch signaling. Recent evidence also suggests that the enzyme responsible for addition of O-fucose to Notch, protein O-fucosyltransferase-1 (POFUT1), may serve a quality control function in the endoplasmic reticulum. Additionally, some of the O-fucose moieties are further elongated by the action of members of the Fringe family of beta-1,3-N-acetylglucosaminyltransferases. The alteration in O-fucose saccharide structure caused by Fringe modulates the response of Notch to its ligands. Thus, glycosylation serves an important role in regulating Notch activity. This review focuses on the role of glycosylation in the normal functioning of the Notch pathway. As well, potential roles for glycosylation in Notch-related human diseases, and possible roles for therapeutic targeting of POFUT1 and Fringe in Notch-related human diseases, are discussed.     

Biological functions of glycosyltransferase genes involved in O-fucose glycan synthesis

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. Well-known examples of such modification are O-linked fucose (O-fucose) and O-linked glucose (O-glucose) glycans on epidermal growth factor (EGF) domains. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as urinary-type plasminogen activator and Notch receptors. Two glycosyltransferases catalyze the initiation and elongation of O-fucose glycans. The initiation process is catalyzed by O-fucosyltransferase 1, which is essential for Notch signalling in both Drosophila and mice. O-fucosyltransferase 1 can affect the folding, ligand interaction and endocytosis of Notch receptors, and both the glycosyltransferase and non-catalytic activities of O-fucosyltransferase 1 have been reported. The elongation of O-fucose monosaccharide is catalyzed by Fringe-related genes, which differentially modulate the interaction between Notch and two classes of ligands, namely, Delta and Serrate/Jagged. In this article, we have reviewed the recent reports addressing the distinctive features of the glycosyltransferases and O-glycans present on the EGF domains.     

Highly conserved O-fucose sites have distinct effects on Notch1 function

The extracellular domain of mouse Notch1 contains 36 tandem epidermal growth factor-like (EGF) repeats, many of which are modified with O-fucose. Previous work from several laboratories has indicated that O-fucosylation plays an important role in ligand mediated Notch activation. Nonetheless, it is not clear whether all, or a subset, of the EGF repeats need to be O-fucosylated. Three O-fucose sites are invariantly conserved in all Notch homologues with 36 EGF repeats (within EGF repeats 12, 26, and 27). To investigate which O-fucose sites on Notch1 are important for ligand-mediated signaling, we mutated the three invariant O-fucose sites in mouse Notch1, along with several less highly conserved sites, and evaluated their ability to transduce Jagged1- and Delta1-mediated signaling in a cell-based assay. Our analysis revealed that mutation of any of the three invariant O-fucose sites resulted in significant changes in both Delta1 and Jagged1 mediated signaling, but mutations in less highly conserved sites had no detectable effect. Interestingly, mutation of each invariant site gave a distinct effect on Notch function. Mutation of the O-fucose site in EGF repeat 12 resulted in loss of Delta1 and Jagged1 signaling, while mutation of the O-fucose site in EGF repeat 26 resulted in hyperactivation of both Delta1 and Jagged1 signaling. Mutation of the O-fucose site in EGF repeat 27 resulted in faulty trafficking of the Notch receptor to the cell surface and a decreased S1 processing of the receptor. These results indicate that the most highly conserved O-fucose sites in Notch1 are important for both processing and ligand-mediated signaling in the context of a cell-based signaling assay.     

Regulation of Notch signaling by glycosylation

Notch receptors are approximately 300 kDa cell surface glycoproteins whose activation by Notch ligands regulates cell fate decisions in the metazoa. The extracellular domain of Notch receptors has many epidermal growth factor like repeats that are glycosylated with O-fucose and O-glucose glycans as well as N-glycans. Disruption of O-fucose glycan synthesis leads to severe Notch signaling defects in Drosophila and mammals. Removal or addition of O-fucose glycan consensus sites on Notch receptors also leads to Notch signaling defects. Ligand binding and ligand-induced Notch signaling assays have provided insights into how changes in the O-fucose glycans of Notch receptors alter Notch signaling.     

Two distinct pathways for O-fucosylation of epidermal growth factor-like or thrombospondin type 1 repeats

Epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) are both small cysteine-knot motifs known to be O-fucosylated. The enzyme responsible for the addition of O-fucose to EGF repeats, protein O-fucosyltransferase 1 (POFUT1), has been identified and shown to be essential in Notch signaling. Fringe, an O-fucose beta1,3-N-acetylglucosaminyltransferase, elongates O-fucose on specific EGF repeats from Notch to form a disaccharide that can be further elongated to a tetrasaccharide. TSRs are found in many extracellular matrix proteins and are involved in protein-protein interactions. The O-fucose moiety on TSRs can be further elongated with glucose to form a disaccharide. The discovery of O-fucose on TSRs raised the question of whether POFUT1, or a different enzyme, adds O-fucose to TSRs. Here we demonstrate the existence of a TSR-specific O-fucosyltransferase distinct from POFUT1. Similar to POFUT1, the novel TSR-specific O-fucosyltransferase is a soluble enzyme that requires a properly folded TSR as an acceptor substrate. In addition, we found that a previously identified fucose-specific beta1,3-glucosyltransferase adds glucose to O-fucose on TSRs, but it does not modify O-fucose on an EGF repeat. Similarly, Lunatic fringe, Manic fringe, and Radical fringe are all capable of modifying O-fucose on an EGF repeat, but not on a TSR. Taken together, these results suggest that two distinct O-fucosylation pathways exist in cells, one specific for EGF repeat and the other for TSRs.     

Fringe modifies O-fucose on mouse Notch1 at epidermal growth factor-like repeats within the ligand-binding site and the Abruptex region

Fringe plays a key role in the specification of boundaries during development by modulating the ability of Notch ligands to activate Notch receptors. Fringe is a fucose-specific beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose moieties on the epidermal growth factor-like (EGF) repeats of Notch. To investigate how the change in sugar structure caused by Fringe modulates Notch activity, we have analyzed the sites of O-fucose and Fringe modification on mouse Notch1. The extracellular domain of Notch1 has 36 tandem EGF repeats, many of which are predicted to be modified with O-fucose. We recently proposed a broadened consensus sequence for O-fucose, C(2)X(3-5)(S/T)C(3) (where C(2) and C(3) represent the second and third conserved cysteines), significantly expanding the potential number of modification sites on Notch. Here we demonstrate that sites predicted using this broader consensus sequence are modified with O-fucose on mouse Notch1, and we present evidence suggesting that the consensus can be further refined to C(2)X(4-5)(S/T)C(3). In particular, we demonstrate that EGF 12, a portion of the ligand-binding site, is modified with O-fucose and that this site is evolutionarily conserved. We also show that endogenous Fringe proteins in Chinese hamster ovary cells (Lunatic fringe and Radical fringe) as well as exogenous Manic fringe modify O-fucose on many but not all EGF repeats of mouse Notch1. These findings suggest that the Fringes show a preference for O-fucose on some EGF repeats relative to others. This specificity appears to be encoded within the amino acid sequence of the individual EGF repeats. Interestingly, our results reveal that Manic fringe modifies O-fucose both at the ligand-binding site (EGF 12) and in the Abruptex region. These findings provide insight into potential mechanisms by which Fringe action on Notch receptors may influence both the affinity of Notch-ligand binding and cell-autonomous inhibition of Notch signaling by ligand.     

O-linked N-acetylglucosamine is present on the extracellular domain of notch receptors

Rare types of glycosylation often occur in a domain-specific manner and are involved in specific biological processes. In particular, O-fucose glycans are reported to regulate the functions of EGF domain-containing proteins such as Notch receptors. In the course of mass spectrometric analysis of O-glycans displayed on Drosophila Notch receptors expressed in S2 cells, we found an unusual O-linked N-acetylhexosamine (HexNAc) modification which occurs at a site distinct from those of O-fucose and O-glucose glycosylations. Modification site mapping by mass spectrometry and amino acid substitution studies revealed that O-HexNAc modification occurs on a serine or threonine located between the fifth and sixth cysteines within the EGF domain. This modification occurs simultaneously along with other closely positioned O-glycosylations. This modification was determined to be O-beta-GlcNAc by galactosyltransferase labeling and beta-N-acetyl-hexosaminidase digestion experiments and by immunoblotting with a specific antibody. O-GlcNAc modification occurs at multiple sites on Notch epidermal growth factor repeats. O-GlcNAc modification was also found on the extracellular domain of Delta, a ligand for Notch receptors. Although the O-GlcNAc modification is known to regulate a wide range of cellular processes, the list of known modified proteins has previously been limited to intracellular proteins in animals. Thus, the finding of O-GlcNAc modification in extracellular environments predicts a distinct glycosylation process that might be associated with a novel regulatory mechanism for Notch receptor activity.     

糖基化对Notch信号传递系统的影响

Notch信号分子是多细胞生物发育过程中高度保守的一类十分重要的跨膜信 号受体糖蛋白家族.这一信号途径通过局部细胞间的相互作用而产生对多种不成熟细胞分化 的抑制信号, 精确调控细胞的分化潜能,在细胞发育、增殖、分化中起关键作用,参与造血 、T细胞发育、血管生成等重要生理过程.Notch受体分子上具有多种寡糖链,包括N-聚糖、O-岩藻糖聚糖、O-葡萄糖聚糖等,这些寡糖以及相关糖基转移酶对Notch受体-配体结合以及Notch信号传递功能有重要影响.本文就近年来有关Notch受体糖基化及其对Notch信号传递过程的研究进行综述.     

Regulation of signal transduction pathways in development by glycosylation

Recent studies from several laboratories have provided evidence that cell surface complex carbohydrates play key roles in the regulation of developmentally relevant signal transduction events. The demonstration that Fringe, a known modifier of Notch function, is a fucose-specific N-acetylglucosaminyltransferase provided strong evidence that the Notch signaling pathway could be regulated by alterations of O-fucose structures. More recently, the demonstration that O-fucose modification of Cripto is essential for Nodal-dependent signaling provides further evidence of a role for glycosylation in signal transduction. These and other examples provide a new paradigm for the regulation of signal transduction events by glycosylation.     

Regions of Drosophila Notch that contribute to ligand binding and the modulatory influence of Fringe

Two glycosyltransferases that transfer sugars to epidermal growth factor (EGF) domains, OFUT1 and Fringe, regulate Notch signaling. To characterize the impact of glycosylation at the 23 consensus O-fucose sites in Drosophila Notch, we conducted deletion mapping and site-specific mutagenesis and then assayed the binding of soluble forms of Notch to cell-surface ligands. Our results support the conclusion that EGF11 and EGF12 are essential for ligand binding, but indicate that other EGF domains also make substantial contributions to ligand binding. Characterization of Notch deletion constructs and O-fucose site mutants further revealed that no single site or region can account for the influence of Fringe on Notch-ligand binding. Additionally, we observed an influence of Fringe on a Notch fragment including only 4 of its 36 EGF domains (EGF10-13). Together, our observations imply that glycosylation influences Notch-ligand interactions through a distributive mechanism that involves local interactions with multiple EGF domains and led us to suggest a structural model for how Notch interacts with its ligands.     

Lunatic fringe, manic fringe, and radical fringe recognize similar specificity determinants in O-fucosylated epidermal growth factor-like repeats

Notch signaling is a component of a wide variety of developmental processes in many organisms. Notch activity can be modulated by O-fucosylation (mediated by protein O-fucosyltransferase-1) and Fringe, a beta1,3-N-acetylglucosaminyltransferase that modifies O-fucose in the context of epidermal growth factor-like (EGF) repeats. Fringe was initially described in Drosophila, and three mammalian homologues have been identified, Manic fringe, Lunatic fringe, and Radical fringe. Here for the first time we have demonstrated that, similar to Manic and Lunatic, Radical fringe is also a fucose-specific beta1,3-N-acetylglucosaminyltransferase. The fact that three Fringe homologues exist in mammals raises the question of whether and how these enzymes differ. Although Notch contains numerous EGF repeats that are predicted to be modified by O-fucose, previous studies in our laboratory have demonstrated that not all O-fucosylated EGF repeats of Notch are further modified by Fringe, suggesting that the Fringe enzymes can differentiate between them. In this work, we have sought to identify specificity determinants for the recognition of an individual O-fucosylated EGF repeat by the Fringe enzymes. We have also sought to determine differences in the biochemical behavior of the Fringes with regard to their in vitro enzymatic activities. Using both in vivo and in vitro experiments, we have found two amino acids that appear to be important for the recognition of an O-fucosylated EGF repeat by all three mammalian Fringes. These amino acids provide an initial step toward defining sequences that will allow us to predict which O-fucosylated EGF repeats are modified by the Fringes.     

Significance of glycosylation in Notch signaling

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.     

Protein O-fucosyltransferase 2 adds O-fucose to thrombospondin type 1 repeats

O-Fucose is an unusual form of glycosylation found on epidermal growth factor-like (EGF) repeats and thrombospondin type 1 repeats (TSRs) in many secreted and transmembrane proteins. Recently O-fucose on EGF repeats was shown to play important roles in Notch signaling. In contrast, physiological roles for O-fucose on TSRs are unknown. In the accompanying paper (Luo, Y., Nita-Lazar, A., and Haltiwanger, R. S. (2006) J. Biol. Chem. 281, 9385-9392), we demonstrated that an enzyme distinct from protein O-fucosyltransferase 1 adds O-fucose to TSRs. A known homologue of O-fucosyltransferase 1 is putative protein O-fucosyltransferase 2. The cDNA sequence encoding O-fucosyltransferase 2 was originally identified during a data base search for fucosyltransferases in Drosophila. Like O-fucosyltransferase 1, O-fucosyltransferase 2 is conserved from Caenorhabditis elegans to humans. Although O-fucosyltransferase 2 was assumed to be another protein O-fucosyltransferase, no biochemical characterization existed supporting this contention. Here we show that RNAi-mediated reduction of the O-fucosyltransferase 2 message significantly decreased TSR-specific O-fucosyltransferase activity in Drosophila S2 cells. We also found that O-fucosyltransferase 2 is predominantly localized in the endoplasmic reticulum compartment of these cells. Furthermore, we expressed recombinant Drosophila O-fucosyltransferase 2 and showed that it O-fucosylates TSRs but not EGF repeats in vitro. These results demonstrate that O-fucosyltransferase 2 is in fact a TSR-specific O-fucosyltransferase.     

The threonine that carries fucose, but not fucose, is required for Cripto to facilitate Nodal signaling

Cripto is a membrane-bound co-receptor for Nodal, a member of the transforming growth factor-beta superfamily. Mouse embryos lacking either Cripto or Nodal have the same lethal phenotype at embryonic day 7.5. Previous studies suggest that O-fucosylation of the epidermal growth factor-like (EGF) repeat in Cripto is essential for the facilitation of Nodal signaling. Substitution of Ala for the Thr to which O-fucose is attached led to functional inactivation of both human and mouse Cripto. However, embryos null for protein O-fucosyltransferase 1, the enzyme that adds O-fucose to EGF repeats, do not exhibit a Cripto null phenotype and die at about embryonic day 9.5. This suggested that the loss of O-fucose from the EGF repeat may not have led to the inactivation of Cripto in previous studies. Here we investigate this hypothesis and show the following: 1) protein O-fucosyltransferase 1 is indeed the enzyme that adds O-fucose to Cripto; 2) Pofut1(-/-) embryonic stem cells behave the same as Pofut1(+/+) embryonic stem cells in a Nodal signaling assay; 3) Pofut1(-/-) and Pofut1(+/+) embryoid bodies are indistinguishable in their ability to differentiate into cardiomyocytes; and 4) none of 10 amino acid substitutions at Thr(72), including Ser which acquires O-fucose, rescues the activity of mouse Cripto in Nodal signaling assays. Therefore, the Thr to which O-fucose is linked in Cripto plays a key functional role, but O-fucose at Thr(72) is not required for Cripto to function in cell-based signaling assays or in vivo. By contrast, we show that O-fucose, and not the Thr to which it is attached, is required in the ligand-binding domain of Notch1 for Notch1 signaling.     

In vitro reconstitution of the modulation of Drosophila Notch-ligand binding by Fringe

Notch signaling plays critical roles in animal development and physiology. The activation of Notch receptors by their ligands is modulated by Fringe-dependent glycosylation. Fringe catalyzes the addition of N-acetylglucosamine in a beta1,3 linkage onto O-fucose on epidermal growth factor-like domains. This modification of Notch by Fringe influences the binding of Notch ligands to Notch receptors. However, prior studies have relied on in vivo glycosylation, leaving unresolved the question of whether addition of N-acetylglucosamine is sufficient to modulate Notch-ligand interactions on its own, or whether instead it serves as a precursor to subsequent post-translational modifications. Here, we describe the results of in vitro assays using purified components of the Drosophila Notch signaling pathway. In vitro glycosylation and ligand binding studies establish that the addition of N-acetylglucosamine onto O-fucose in vitro is sufficient both to enhance Notch binding to the Delta ligand and to inhibit Notch binding to the Serrate ligand. Further elongation by galactose does not detectably influence Notch-ligand binding in vitro. Consistent with these observations, carbohydrate compositional analysis and mass spectrometry on Notch isolated from cells identified only N-acetylglucosamine added onto Notch in the presence of Fringe. These observations argue against models in which Fringe-dependent glycosylation modulates Notch signaling by acting as a precursor to subsequent modifications and instead establish the simple addition of N-acetylglucosamine as a basis for the effects of Fringe on Drosophila Notch-ligand binding.     

Effects of Notch glycosylation on health and diseases

Notch signaling is an evolutionarily conserved signaling pathway and is essential for cell-fate specification in metazoans. Dysregulation of Notch signaling results in various human diseases, including cardiovascular defects and cancer. In 2000, Fringe, a known regulator of Notch signaling, was discovered as a Notch-modifying glycosyltransferase. Since then, glycosylation—a post-translational modification involving literal sugars—on the Notch extracellular domain has been noted as a critical mechanism for the regulation of Notch signaling. Additionally, the presence of diverse O-glycans decorating Notch receptors has been revealed in the extracellular domain epidermal growth factor-like (EGF) repeats. Here, we concisely summarize the recent studies in the human diseases associated with aberrant Notch glycosylation.     

An O-fucose site in the ligand binding domain inhibits Notch activation

Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. Here, we describe the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. Our results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicate that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling.     

Modulation of receptor signaling by glycosylation: fringe is an O-fucose-β1,3-N-acetylglucosaminyltransferase

The Notch family of signaling receptors plays key roles in determining cell fate and growth control. Recently, a number of laboratories have shown that O-fucose glycans on the epidermal growth factor (EGF)-like repeats of the Notch extracellular domain modulate Notch signaling. Fringe, a known modifier of Notch function, is an O-fucose specific β1,3-N-acetylglucosaminyltransferase. The transfer of GlcNAc to O-fucose on Notch by fringe results in the potentiation of signaling by the Delta class of Notch ligands, but causes inhibition of signaling by the Serrate/Jagged class of Notch ligands. Interestingly, addition of a β1,4 galactose by β4GalT-1 to the GlcNAc added by fringe is required for Jagged1-induced Notch signaling to be inhibited in a co-culture assay. Thus, both fringe and β4GalT-1 are modulators of Notch function. Several models have been proposed to explain how alterations in O-fucose glycans result in changes in Notch signaling, and these models are discussed.