O-fucosyltransferase 1/neurotic: Biological Overview | Regulation | Developmental Biology | Loss and Gain of Function | Evolutionary Homologs | References
Gene name - O-fucosyltransferase 1/neurotic
Synonyms - Ofut1
Cytological map position - 50E1
Function - enzyme
Keywords - Notch pathway
Symbol - O-fut1/ntc
FlyBase ID: FBgn0033901
Genetic map position - 2R
Classification - O-fucosyltransferase 1
Cellular location - golgi membrane
Notch and its ligands are modified by a protein O-fucosyltransferase (O-fut1, also known as Neurotic or Ofut1) that attaches fucose to a serine or threonine within EGF domains. By using RNAi to decrease O-fut1 expression in Drosophila, it has been demonstrated that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for O-fut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. Therefore, O-fut1 activity is required for the cell's ability to receive ligand signals, and would thus be consistent with the hypothesis that the key substrate of O-fut1 is Notch. The transcription of O-fut1 is developmentally regulated, and surprisingly, overexpression of O-fut1 inhibits Notch signaling. Together, these results indicate that O-fut1 is a core component of the Notch pathway, one that is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development (Okajima, 2002).
A mutation has been isolated in the gene encoding O-fucosyltransferase, and analysis of the mutant phenotype confirms the RNAi studies and reveals an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. A novel maternal neurogenic gene, neurotic, is essential for Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. neurotic has been shown to be required for Fringe activity. fringe encodes a fucose-specific ß1, 3 N-acetylglucosaminyltransferase that modulates Notch receptor activity. Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. These results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions (Sasamura, 2003).
Two types of Notch ligands, Delta and Serrate/Jagged, have been identified and shown to possess redundant and distinct functions. Specific functions of each of these two ligands are modified by Fringe (Fng), which adds N-acetylglucosamine (GlcNAc) to O-fucose residues on the EGF repeats of Notch. Fng is involved in establishing a boundary that segregates two cell groups, such as dorsal and ventral (D/V) compartments of the wing disc or leg segments of the leg disc in Drosophila. This GlcNAc modification had been thought to make Notch more sensitive to Delta and less sensitive to Serrate. An additional modification of the GlcNAc by a galactosyltransferase has been shown to be required for the inhibition of Jagged1. The O-fucose glycans are found at serine or threonine residues of consensus sequence CXXGGS/TC (X is any amino acid) that is between the second and third conserved cysteines of the EGF repeats. In mammals, the O-fucosyltransferase that catalyses this fucosylation has been purified and cloned. The functions of a Drosophila O-fucosyltransferase (O-fut1) have been examined using RNA interference (RNAi) and O-fut1 has been implicated in Notch signalling (Okajima, 2002). A knock out of the mouse Pofut1 gene that encodes mouse O-FucT-1 has been reported (Shi, 2003); Pofut1/ mice have a phenotype similar to those of the embryos lacking downstream effectors of all Notch signalling pathways. However, the molecular mechanism underlying the function of O-fucose was not determined (Sasamura, 2003).
Deficiency of essential genes for Notch signalling causes overproduction of neurons in the Drosophila embryo: the 'neurogenic' phenotype. Some essential components of Notch signalling are associated with the neurogenic phenotype when both the zygotic genes and maternally contributed mRNA are abolished. These genes are referred to as 'maternal neurogenic genes'. Several genes relatively recently identified as components of Notch signalling, such as Suppressor of Hairless, Kuzbanian, Presenilin and Nicastrin belong to this class of genes. Because of the difficulty to screen for maternal phenotypes, not all maternal neurogenic genes have yet been identified. A novel maternal neurogenic gene, neurotic (nti O-fut1 -- FlyBase), encodes the O-fucosyltransferase. The activity of nti is also needed in wing margin formation, indicating that nti is an essential component of Notch signalling. Epistatic analysis showed that nti is essential for full-length Notch and Fng function but not for NotchICD. Nti modification of Notch is essential for the physical interaction between Notch and Delta in Drosophila cultured cells. These results establish Neurotic/O-fut1 as a moderator of Notch-ligand interactions, this moderation has both Fng-dependent and Fng-independent functions (Sasamura, 2003).
Notch signalling is involved in two major classes of cell-cell signalling: lateral inhibition and inductive signalling. Neuroblast segregation in early embryogenesis is a well known example of lateral inhibition, and failure of the Notch signalling pathway during this process leads to an hypertrophy of neuroblasts, known as the neurogenic phenotype. However, induction of several genes, such as wg, along the dorsal/ventral compartmental boundary of the wing disc depends on inductive Notch signalling. In both classes of Notch signalling events, the Notch and nti mutant phenotypes are identical, indicating that Nti is essential for Notch signalling. This finding differs from the recent report using nti RNAi (Okajima, 2002), since apparent embryonic neurogenic phenotypes were not noted. This discrepancy is probably due to the partial knockdown effect generated by RNAi (Sasamura, 2003).
There are a considerable number of genes, including the ligands of the Notch receptor, that encode putative proteins that contain EGF motifs with consensus sequences for O-fucosylation in the Drosophila genome. However, mutant phenotypes of nti are strikingly similar to those of the Notch receptor. In fact, in addition to the wing phenotype, nti somatic clones exhibit mutant phenotypes that are characteristic of Notch loss-of-function mutations. These include defects on the notum bristles, rough eye and the leg segment fusion. These phenotypes were also observed in the knockdown study of nti (Okajima, 2002).
These results indicate that the primary target of O-fucosylation of Nti is Notch, while there may be unrevealed signalling pathways in which nti function is required. There is another putative O-fucosyltransferase in Drosophila and mammals (Roos, 2002). Thus, it is tempting to speculate that each O-fucosyltransferase has distinct target(s) that are involved in specific cell signalling pathways (Sasamura, 2003).
Since O-fucose on Notch has been shown to act as a molecular scaffold for GlcNAc that is elongated by Fng, one would expect that the phenotypes of O-fucosyltransferase mutant might be the same as those of fng. Unexpectedly, however, the nti and fng mutant phenotypes are quite different. Strikingly, the embryonic neurogenic phenotype that is evident in nti mutant, and is an indication of its essential role in Notch signalling, is not evident in fng mutants. Furthermore, it is thought that Fng does not have a significant role in lateral inhibition, while it is involved in the generation of the cell boundary between cells expressing Fng and cells not expressing Fng. Additionally, an in vitro binding assay revealed that Nti is essential for binding between Notch and Delta. Based on the previous findings and the present results, it is proposed that O-glycosylation of Notch EGF repeats has two distinct roles for binding to Delta. (1) O-fucosylation catalysed by Nti is an absolute requirement for binding between Notch and the ligand, and this binding is sufficient to accomplish lateral inhibition. For this function, no additional glycosylation to O-fucose residue is required. This idea is also supported by the observation that in the tissues and organisms that do not express fng, Delta is competent to activate the Notch receptor. In this respect, it is worth noting that in the C. elegans genome there is a highly conserved nti, while a fng homolog is not found. (2) Addition of GlcNAc to the O-fucose residue by Fng enhances the interactions between Notch and Delta, modulating the receptor-ligand interactions. In fact, the expression of fng shows a high degree of regional specificity, and the boundary of the cells expressing and not expressing Fng often defines the border of distinct tissue structures. Thus, the region-specific expression of fng allows modulation of Notch signalling, resulting in generation of complex structure of organs. As expected from the second function of Nti, its function is essential for Fng-dependent modulation of Notch signalling as well as Fng-independent function. In the wing disc, nti is epistatic to fng, and fng requires nti to induce Wg at the dorsal and ventral compartment boundary. Additionally, in the in vitro binding assay, Fng depends on Nti to enhance the binding between Notch and Delta. These lines of evidence indicate that Nti is involved in Fng-dependent modulation of Notch signalling, which is consistent with an O-glycan structure of the Notch EGF repeats (Sasamura, 2003).
Given that nti has novel functions, what is the significance of Nti in Notch signalling at the level of cells and the organism? A model is proposed for nti function during development. This model involves a novel receptor on-off mechanism. In the early embryo, nti mRNA is detected uniformly. However, Okajima (2002) has reported that expression of nti/O-fut1 was spatially regulated during the late stages of embryogenesis and the imaginal discs. Therefore, O-fucosylation of Notch EGF repeats is probably regulated temporally and spatially, and may provide a novel mechanism to regulate Notch signalling. For example, in cells expressing nti at higher level than their neighbors, Notch may become more potent to receive a signal. Removal of O-fucose from the Notch EGF repeats may be part of this regulation (Sasamura, 2003).
In this work, no effect of nti on Notch-Serrate binding was demonstrated, because, as reported before, Notch-Serrate-AP binding can not be detected by this binding assay. However, phenotypes observed in the somatic clones of nti generated in the wing disc strongly suggests that Nti affects the Notch-Serrate binding. For example, in the nti clones near the dorsal and ventral boundary from the ventral side, induction of Wg is impaired, indicating that nti clones fail to receive the Serrate signal. Thus, it is proposed that the requirement of nti for Notch-ligand binding is not restricted to Delta. However, the neurogenic phenotype of nti can be wholly explained by the effect on Notch-Delta binding, since Delta is the only the ligand of Notch involved in neuroblast segregation during early embryogenesis (Sasamura, 2003).
In the Notch-Delta binding assay, co-expression of Nti and Fng with Notch increases the Notch-Delta binding over that of Fng with Notch, whereas co-expression of a wild-type Nti with Notch does not increase this binding. It is speculated that the amount of Nti endogenously expressed in S2 cells saturates the ability of fucose alone to promote the ligand binding. Indeed, it has been shown that S2 cells endogenously expressed Nti/O-fut1 (Okajima, 2002). However it does not imply that addition of O-fucose to Notch by Nti is also saturated under these conditions. Namely, the increase of O-fucosyltransferase activity, which does not lead to elevation of Delta binding per se, could increase the Notch-Delta binding after elongation of the O-fucose residue by Fng. These results suggest that the linear range by which fucose-GlcNAc moiety can elevate the ligand binding is wider than that by which fucose alone can. Multiple sites that are possibly O-fucosylated were found in the EGF repeats of Notch (Okajima, 2002). Thus, it is possible that some of these potential O-fucosylation sites may influence the Delta binding only after modification by Fng. It was also noticed that Nti-myc probably has a dominant-negative function. This dominant-negative activity of Nti-myc is apparently restored by co-expression with Fng; co-expression with Fng makes the level of ligand binding well over that by Fng expression alone. This is in contrast with the fact that the decreased Notch-Delta binding associated with RNAi of nti, which is supposed to merely reduce the endogenous Nti protein, is hardly restored by co-expression with Fng. Therefore, it is speculated that the Myc-tag does not simply disrupt the enzymatic activity of Nti-myc. Some kind of competition among Nti-myc, endogenous Nti, Fng and Notch may be involved in this dominant-negative effect. In support of this model, physical interaction between Notch and Fng has been reported. This dominant-negative effect may also account for the reduction of the Notch-Delta binding associated with Nti-G3-myc. It is speculated that Ntimyc and Nti-G3-myc, both of which carry the same Myc-tag, have similar dominant negative effects, which do not depend on the enzymatic activity of O-fucosyltransferase and which are restored by co-expressing Fng. The slight reduction of the Notch-Delta binding associated with Nti-G3-myc and Fng over that of Fng alone may also be caused by this dominant-negative effect of Nti-G3-myc (Sasamura, 2003).
The importance of glycoconjugates in cell adhesion and cell-cell communication has been affirmed for many years, since nearly all cells are covered with numerous carbohydrate-rich molecules. Gene disruption of a number of glycosyltransferases has been shown to resulted in embryonic lethality in mice, underscoring their importance in development of multicellular organisms. However, most of the previous reports are only descriptive of the phenotypes and the molecular mechanisms are still elusive. This study presents one of the very few examples of molecular explanation for such mechanisms of action. Furthermore, it is an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. The simplest interpretation of these phenomena would be that association of Delta to its receptor involves lectin-like protein-carbohydrate interaction. This hypothesis is consistent with the result that O-fucosylation induces little conformational change (Kao, 1999) in EGF repeat of factor VII (Sasamura, 2003).
The first protein shown to be O-fucosylated was urinary type plasminogen activator (uPA), followed by tissue-type plasminogen activators and several clotting factors. It has been shown that O-fucosylation of uPA is essential for activation of the uPA receptor that has diverse functions including plasminogen activation, although uPA receptor-deficient mice survive to adulthood with no overt phenotypic abnormalities and are fertile. Another O-fucosylated protein is Cripto, a member of the EGF-CFC family. Cripto is thought to act as an essential co-factor for Nodal signalling. Amino acid substitution of threonine to alanine, which prevents O-fucosylation reduces the signalling activity of Nodal, suggesting that O-fucosylation is important for Cripto function. Until now, more than ten proteins have been found to carry the consensus sequence of O-fucosylation site. In this paper, it has been shown that O-fucosylation is mandatory for Notch signalling. Although the O-fucosyltransferase presented in this study has remarkable functional specificity to the Notch receptor, there seems to be plurality of such enzymes. Therefore, the regulation of protein activity or function by O-fucosylation could be a general mechanism for many signal transduction systems (Sasamura, 2003 and references therein).
Proteins that bind to sugars without modifying them are classified as lectins. If the action of O-fucose glycans involves lectin-based recognition, it could be either that the Notch ligands themselves are lectins, or that there exist other Notch signaling cofactors that function as lectins. The hypothesis that lectin-based recognition is essential for Notch signaling suggests an explanation as to why the extracellular domain of Notch contains so many EGF repeats. Lectins typically have only low affinity for individual sugars, and high avidity lectin-sugar binding is achieved by multivalent interactions of lectins with sugars. Thus, it is proposed that an array of EGF repeats acts as a scaffold for the display of O-fucose glycans, and thus allows for high avidity binding to multivalent lectins. This hypothesis further implies that quantitative differences in the binding of lectins to EGF repeats may be achieved by varying the fucosylation status of the Notch receptor or its ligands (Okajima, 2002).
The lectin hypothesis also suggests explanations for how both the O-fucose monosaccharide and the more elongated O-fucose glycans that form in the presence of Fringe can influence Notch signaling. Because different lectins can recognize distinct glycans, the existence is postulated of O-fucose binding lectins that are required for all Notch signaling, as well as distinct lectins that bind Fringe-dependent glycans, such as the Gal-β1,4-GlcNAc-β1,3-Fuc trisaccharide that has been implicated in the inhibition of Jagged1 signaling (Chen, 2001). In this model, the general positive requirement for the O-fucose monosaccharide in Notch signaling and the localized positive effect of Fng-dependent glycans in certain tissues could be reconciled if only a subset of O-fucosylated EGF repeats are actually substrates for Fng. Alternatively, it could be that a positively required lectin binds O-fucose regardless of whether it is the terminal or an internal sugar, but its affinity is altered. In contrast, since the negative influence of O-fut1 overexpression on Notch signaling is suppressed by co-expression with Fng, it may involve a lectin that specifically requires a terminal O-fucose. The identification of lectins that bind to O-fucose glycans would make it possible to test and extend these hypotheses, and thus constitutes an important goal for future research (Okajima, 2002).
Notch receptors are glycoproteins that mediate a wide range of developmental processes. Notch is modified in its Epidermal growth factor-like domains by the addition of fucose to Serine or Threonine residues. O-fucosylation is mediated by Protein O-fucosyltransferase 1, and downregulation of this enzyme by RNAi or mutation of the O-fut1 gene in Drosophila, or by mutation of the Pofut1 gene in mouse, prevents Notch signaling. To investigate the molecular basis for the requirement for O-linked fucose on Notch, an assay was carried out of the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Downregulation of O-fut1 by RNAi in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. Conversely, over-expression of O-fut1 in cultured cells increases Serrate-Notch binding but inhibits DeltaNotch binding. These effects of O-fut1 are consistent with the consequences of O-fut1 overexpression on Notch signaling in vivo. Intriguingly, they are also the opposite of, and are suppressed by, expression of the glycosyltransferase Fringe, which specifically modifies O-linked fucose. Thus, Notch-ligand interactions are dependent upon both the presence and the type of O-fucose glycans (Okajima, 2003).
The requirement for O-Fut1 in Notch signaling has been demonstrated by RNAi in Drosophila (Okajima, 2002), and by a targeted mutation in the murine Pofut1 gene (Shi, 2003). One line from a large scale screen for lethal transposable element insertions in Drosophila has an insertion in the 3' end of O-fut1 (Oh, 2003), and is predicted to result in replacement of the seven C-terminal amino acids of O-fut1 with four different amino acids followed by a stop codon (Oh, 2003). To confirm that this insertion creates an O-fut1 mutation, animals in which patches of cells were made homozygous mutant for this allele were examined by mitotic recombination. These animals exhibit classic Notch mutant phenotypes, such as wing notching, thickened wing veins, and loss of sensory bristles on the notum, consistent with the phenotypes generated by RNAi of O-fut1 (Okajima, 2002). In developing wing imaginal discs, the expression of targets of Notch signaling, such as Wingless, is lost in cells mutant for O-fut1. This mutation (referred to hereafter as O-fut1SH) thus provides an independent demonstration of the requirement for O-fut1 for Notch signaling in Drosophila, and indicates that the seven C-terminal amino acids of O-fut1 are essential for function in vivo. The last four amino acids of O-fut1 conform to a consensus signal for retention in the endoplasmic reticulum, and experiments are in progress to determine whether the loss of function in O-fut1SH is due to loss of enzymatic activity or to mislocalization (Okajima, 2003).
The studies presented here indicate that O-fucosylation is required for the physical binding of Notch to its ligands Dl and Ser. These binding studies are consistent with prior genetic studies, which positioned a requirement for O-fucosylation in signal receiving cells, upstream of the cleavages associated with Notch activation (Okajima, 2002). Although the current results do not exclude the possibility that O-fucose glycans could also act at other steps, and indeed some influence of O-fut1 RNAi on secretion of Notch extracellular domain fusion proteins is detected, the requirement for O-fucose for Notch-ligand binding can in principle account for the requirement for O-fut1 in Notch signaling (Okajima, 2003).
Notably, O-fut1 is required for efficient binding of Notch to both Ser and Dl. This is consistent with the severe Notch phenotypes observed in vivo when O-fut1 is impaired by mutation or RNAi. By contrast, elongation of O-fucose by the GlcNAc transferase Fringe exerts opposing influences on the ability of Notch to bind to Ser and Dl. Fringe has clear and reproducible effects on both Dl-Notch and Ser-Notch binding. Importantly, these effects of Fringe on Notch-ligand binding recapitulate its effects on signaling by these two ligands in Drosophila. The ability of both the O-fucose monosaccharide and elongated forms of O-fucose to influence Notch-ligand binding, the influence of O-fucosylation on binding by both ligands, and the consistent correlations between the effects of O-fucosylation on binding in vitro and its effects on signaling in vivo all argue that O-fucose glycans act at the ligand binding step of Notch signaling (Okajima, 2003).
Beyond their importance to understanding regulation of Notch signaling, these observations thus provide a striking example of glycosylation as a mechanism for modulating protein-protein interactions. With the determination that O-fucosylation affects Notch-ligand binding, attention must now be turned to elucidating the mechanistic basis for this effect (Okajima, 2003). O-fut1 and Fringe always act in Notch-expressing cells to influence Notch signaling and Notch-ligand binding: this implicates Notch itself as the relevant substrate. However, the actual sites of glycosylation on Notch that mediate the effects of these glycosyltransferases remain to be identified. It is also not yet clear whether the importance of O-fucosylation reflects a role for lectin-like recognition of Notch by its ligands or other co-factors, or whether instead O-fucose glycans influence Notch-ligand binding indirectly, by altering the conformation or oligomerization of Notch (Okajima, 2003).
By contrast to the positive requirement for O-fut1 demonstrated by RNAi, over-expression of O-fut1 enhances Ser-Notch binding but inhibits Dl-Notch binding. It is intriguing that elevated O-fut1 expression provides a mechanism for differentially modulating the ability of different Notch ligands to interact with the Notch receptor. Previously, Fringe was the only factor known that could discriminate between the ability of Delta to activate Notch and that of Serrate to activate Notch. Indeed, elevated O-fut1 expression might be a mechanism for increasing the sensitivity of cells to the presence or absence of Fringe. In vivo, Fringe only affects a subset of Notch signaling events, and it remains unclear why certain processes are sensitive to Fringe whilst others are insensitive. Although O-fut1 action is the opposite of Fringe, its effects can be blocked by Fringe; therefore, the relative impact of Fringe on Dl-Notch or Ser-Notch interactions is expected to be greater in tissues where O-fut1 is expressed at higher levels. Indeed, even though expression of Fringe alone has no obvious effect on the patterning of notal bristles, it has a strong effect when O-fut1 is also overexpressed. Overexpression of O-fut1 inhibits Dl-Notch signaling, resulting in the formation of excess sensory bristles, but this effect is partially inhibited by co-expression with Fringe (Okajima, 2002). In addition to increasing the sensitivity of Notch signaling events to the presence or absence of Fringe, elevated O-fut1 expression presents a potential mechanism for modulating Notch signaling independently of Fringe. Although the in vivo relevance of Notch-ligand modulation by increased expression of O-fut1 at endogenous levels of expression remains uncertain, it is noted that certain tissues, such as the lymph gland, express much higher levels of O-fut1 than surrounding cells (Okajima, 2002). Intriguingly then, in most Drosophila tissues Dl is the sole or major Notch ligand. However, in the larval lymph gland, a role for Notch signaling in regulating cell fate decisions during hematopoeisis has recently been described, and Ser, rather than Dl, is the ligand that regulates Notch in this tissue. These observations provide some support for the possibility that transcriptional regulation of O-fut1 might provide a mechanism for Notch pathway regulation (Okajima, 2002), and suggest developmental contexts in which this issue may be investigated further (Okajima, 2003).
Since both Notch and its ligands are O-fucosylated, the effects of nti expression on the ligand-receptor interaction was investigated. S2 cultured cells were transfected with Notch and subsequently incubated with conditioned medium containing a Delta-alkaline phosphatase fusion protein (Delta-AP) prepared also by transfection to S2 cells. Because S2 cells are Notch-deficient, AP activity specifically bound to Notch-transfected cells will reflect the ligand binding activity of Notch. As previously reported, co-expression of Fng with Notch increases its ability to bind to Delta (Brückner, 2000). Nti co-expression alone does not significantly alter the binding ability of Notch. It is speculated that Nti, expressed endogenously, is saturated for the Notch-Delta binding under this condition. Endogenous activity of Nti/O-fut1 in S2 cells has been reported previously (Okajima, 2002). However, co-expression of Nti with Fng potentiates Notch binding activity. Importantly, co-expression of Nti or Fng does not significantly change the expression of Notch protein in the cells. It is noted that co-expression of Nti-Myc, but not wild-type Nti, with Notch inhibits the Notch-Delta binding, suggesting that Nti-Myc behaves as a dominant-negative protein (Sasamura, 2003).
The effect was examined of nti RNAi in this system. nti-IR reduces Notch binding activity to the level of vector-transfected cells, indicating Nti is required for Notch-Delta binding. Knock down of Nti activity by RNAi also results in the disruption of Fng function and an increase in Notch-Delta binding. This is consistent with the model that Fng attaches GlcNac to the O-fucose residue that is added by the Nti beforehand. Furthermore, these results suggest that the profound disruption of Notch signalling observed in vivo is due to the defective ligand-receptor interaction, because, during embryogenesis, only Delta is proposed to function as a ligand for Notch (Sasamura, 2003).
To test the possibility that nti might affect Notch presentation on the cell surface, expression of Notch was examined in live transfected cells using flow cytometry. In this experiment, S2 cells were co-transfected with GFP and Notch expression constructs, as well as either nti, nti-IR or control constructs, and stained with an antibody raised against the Notch extracellular domain. The ratio of double positive cells for GFP and the anti-Notch (cells expressing Notch on the cell surface) is not significantly affected by either the knockdown or the overexpression of Nti. Thus, Notch is expressed in a form accessible to the antibody and probably to the ligand irrespective of nti activity. These results suggest that Nti affects the physical interaction between Notch and Delta, rather than cellular distribution or transport of Notch. It has been reported that Delta and Serrate are also O-fucosylated. However, neither knockdown nor the overexpression of Nti affects the ability of Delta to bind Notch. These results are consistent with the in vivo result that nti functions cell autonomously (Sasamura, 2003).
Notch is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. Endocytic trafficking of Notch plays important roles in the activation and downregulation of this receptor. A Drosophila O-FucT-1 homolog, encoded by O-fut1, catalyzes the O-fucosylation of Notch, a modification essential for Notch signaling and ligand binding. It was recently proposed that O-fut1 acts as a chaperon for Notch in the endoplasmic reticulum and is required for Notch to exit the endoplasmic reticulum. O-fut1 has additional functions in the endocytic transportation of Notch. O-fut1 is indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which is independent of the O-fucosyltransferase activity of O-fut1. O-fut1 promotes the turnover of Notch, which consequently downregulates Notch signaling. O-fut1 formed a stable complex with the extracellular domain of Notch. In addition, O-fut1 protein added to conditioned medium and endocytosed is sufficient to rescue normal Notch transportation to the early endosome in O-fut1 knockdown cells. Thus, an extracellular interaction between Notch and O-fut1 is essential for the normal endocytic transportation of Notch. It is proposed that O-fut1 is the first example, except for ligands, of a molecule that is required extracellularly for receptor transportation by endocytosis (Sasamura, 2007).
Notch (N) is a transmembrane receptor that mediates the cell-cell interactions necessary for many cell-fate decisions. N has many epidermal growth factor-like repeats that are O-fucosylated by the protein O-fucosyltransferase 1 (O-fut1) (Neurotic), and the O-fut1 gene is essential for N signaling. However, the role of the monosaccharide O-fucose on N is unclear, because O-fut1 also appears to have O-fucosyltransferase activity-independent functions, including as an N-specific chaperon. Such an enzymatic activity-independent function could account for the essential role of O-fut1 in N signaling. To evaluate the role of the monosaccharide O-fucose modification in N signaling, this study generated a knock-in mutant of O-fut1 (O-fut1R245A knock-in), which expresses a mutant protein that lacks O-fucosyltransferase activity, but maintains the N-specific chaperon activity. Using O-fut1R245A knock-in and other gene mutations that abolish the O-fucosylation of N, it was found that the monosaccharide O-fucose modification of N has a temperature-sensitive function that is essential for N signaling. The O-fucose monosaccharide and O-glucose glycan modification, catalyzed by Rumi, function redundantly in the activation of N signaling. It was also showm that the redundant function of these two modifications is responsible for the presence of N at the cell surface. These findings elucidate how different forms of glycosylation on a protein can influence the protein's functions (Ushio, 2014: PubMed).
The demonstration that O-fut1 is broadly required for Notch signaling raises the question of whether it is simply a permissive factor that is expressed and required ubiquitously, or whether instead its expression might be patterned and contribute to the regulation of Notch activation. The human PO-fut1 gene has been described as being ubiquitously expressed, but its expression has only been analyzed at the level of whole tissue Northern blotting (Wang, 2001). To investigate the expression of Drosophila O-fut1, in situ hybridization was conducted to Drosophila tissues throughout development. O-fut1 mRNA is expressed ubiquitously and strongly in early embryos, from stages one through eight. Since zygotic transcription begins around stage four, the detection of high levels of expression prior to this indicates that O-fut1 mRNA is provided maternally. Indeed, high levels of O-fut1 are detected in germline cells during oogenesis. During germband extension, around stage 9, O-fut1 levels begin to decline, and there is a sharp drop in mRNA levels around stage 11. After stage 12 O-fut1 mRNA expression is greatly diminished in most cells, but it remains highly expressed in a few tissues including the lymph gland and the germ cells of the gonad (Okajima, 2002).
At larval stages, O-fut1 is uniformly expressed in some tissues, such as the wing, leg, and haltere imaginal discs, but in other tissues it is spatially regulated. In the eye disc, O-fut1 mRNA levels are upregulated behind the morphogenetic furrow, and in the larval brain and CNS O-fut1 is expressed in a complex pattern including the proliferation centers of the optic ganglia, and large cells that have been tentatively identified as neuroblasts in other regions (Okajima, 2002).
In order to examine loss-of-function phenotypes for O-fut1, dsRNA-mediated RNAi was employed in flies. dsRNA was generated by using the UAS-Gal4 system to express a portion of O-fut1 in an inverted repeat with an intervening loop (iO-fut1). This expressed dsRNA method was used rather than injecting dsRNA for several reasons, including more consistent phenotypes, greater temporal and spatial control, and the ability to downregulate O-fut1 during larval and adult development. Moreover, because UAS-Gal4 driven expression is temperature-sensitive in Drosophila, and different insertions of the same UAS construct can be expressed at different levels, this method allows for significant control over expression levels. As a test of the effectiveness of the RNAi construct, O-fut1 mRNA levels were monitored by quantitiative, real-time RT-PCR. O-fut1 mRNA levels are reduced to 7% of wild-type levels in whole larvae expressing iO-fut1 ubiquitously under da-Gal4 control (Okajima, 2002).
O-fucose serves as a substrate for the N-acetylglucosaminyltransferase activity of Fng. Hence loss of O-fucose by downregulation of O-fut1 should at least prevent Fng-dependent modulation of Notch signaling. The consequences of downregulation of O-fut1 in the wing were investigated by driving expression of iO-fut1 along the anterior-posterior (A-P) boundary under the control of the patched (ptc) promoter. In ptc-Gal4 UAS-iO-fut1 flies, wing tissue is lost from the distal edge, where the A-P boundary intersects the wing margin. The wing margin is specified by a Fng-dependent activation of the Notch pathway, and loss of tissue from the edge of the wing can also be induced by loss of fng or Notch. ptc-Gal4 UAS-iO-fut1 flies also have phenotypes in other adult tissues that are consistent with a loss of fng-dependent Notch signaling, including leg segment fusions, and at low frequency, small eyes (Okajima, 2002).
To confirm that the phenotypes of iO-fut1 expression result specifically from decreased levels of O-fut1, a UAS-O-fut1 transgene was created. Overexpression of O-fut1 under ptc-Gal4 control in wild-type flies can result in a mild wing vein phenotype. Importantly, however, when UAS-O-fut1 and UAS-iO-fut1 are co-expressed under ptc-Gal4 control, their phenotypes are mutually suppressed, resulting in phenotypically wild-type wings (Okajima, 2002).
To confirm that the loss of wing tissue in ptc-Gal4 UAS-iO-fut1 flies reflects a loss of Notch signaling, the expression of Notch-dependent target genes was examined in late third-instar wing imaginal discs. The expression of cut and wingless (wg), as well as expression driven by the boundary enhancer of vestigial (vg), is detected specifically along the D-V border of the wing imaginal disc, reflecting the peak of Notch activation in the wing. A fourth target, the E(spl)mβ gene, is expressed throughout the wing, presumably because it responds to much lower levels of Notch activation. The vg boundary enhancer and the E(spl)mβ enhancer contain binding sites for Su(H), and appear to be direct targets of Notch signaling. Importantly, the expression of all four Notch targets is repressed along the A-P border in wing discs from ptc-Gal4 UAS-iO-fut1 animals. Thus, reduction of O-fucose levels by downregulation of O-fut1 inhibits Notch signaling during Drosophila wing development (Okajima, 2002).
As a further test of the requirement for O-fut1 in fng-dependent Notch signaling, advantage was taken of the observation that ectopic Fng expression in ventral cells under ptc-Gal4 control induces ectopic Notch activation. The ability of Fng to induce Notch activation in ventral cells is completely suppressed by co-expression with iO-fut1. Since Fng requires an EGF-O-fucose substrate, this suppression also supports the conclusion that O-fut1 activity is significantly impaired in cells expression iO-fut1 (Okajima, 2002).
Because fng serves to position, rather than simply to promote, Notch activation in the wing, loss of fng also results in ectopic Notch activation, which is manifest as the induction of Notch target gene expression and ectopic wing margins. By contrast, ptc-Gal4 UAS-iO-fut1 flies never have ectopic Notch activation. This suggests that O-fucose is not simply a substrate for Fng, but rather that it may have some additional function that prevents ectopic Notch activation despite the absence of Fng-dependent glycans (Okajima, 2002).
In order to investigate other potential requirements for O-fucose during development, UAS-iO-fut1 was crossed to several different Gal4 lines that drive expression in a variety of different tissues and patterns. These resulted in a range of phenotypes including rough eyes, thickened wing veins, loss of notal and abdominal microchaete, induction of additional notal macrochaete, leg segment fusions, and loss of wing margin. When iO-fut1 was expressed broadly (e.g., da-Gal4), high levels of expression caused lethality. All of the phenotypes observed are consistent with a reduction in Notch signaling. Notably, however, many of these phenotypes, such as thickened wing veins or loss of notal microchaete are not associated with mutation of fng. This implies that O-fucose is not simply an acceptor substrate for Fng, but rather is required more generally for Notch signaling (Okajima, 2002).
Notch plays important roles in a wide variety of events, but its effects can be broken down into a few distinct modes. These modes are distinguished both by the way the Notch pathway is utilized and types of modulators that are employed to regulate Notch signaling. The two best studied and most widely utilized Fng-independent modes of Notch signaling are lateral inhibition and cell lineage decisions. During the development of the Drosophila nervous system, Notch-dependent lateral inhibition functions to limit the number of neural precursor cells. In the peripheral nervous system, the sensory organ precursor cells (SOPs) then go through asymmetric cell lineage decisions, each of which is regulated by Notch, to generate the distinct cell types of the sensory organ (Okajima, 2002).
Notal microchaete are mechanosensory organs, and their loss when iO-fut1 is expressed throughout the notum under ap-Gal4 control could be consistent either with increased Notch signaling during lateral inhibition, resulting in loss of SOPs, or decreased Notch signaling during lineage decisions, resulting in loss of the bristle-forming shaft cell. To investigate these possibilities, the influence of iO-fut1 expression on SOP development was examined using neuralized (neur) expression as a molecular marker of SOP fate. An enhancer trap insertion, neur-lacZA101, can be used to detect expression of neur in the SOP, and because of the perdurance of β-galactosidase, in its progeny. Notal microchaete SOPs are specified around 12 hr after puparium formation (APF), but for technical reasons pupal nota were stained at 15-18 hr APF, at which time well-spaced clusters of 2-3 cells were revealed by neur-lacZA101 staining in wild-type animals. By contrast, in ap-Gal4 UAS-iO-fut1 nota, a dramatic increase in neur-lacZA101 staining is observed, and neur-lacZA101 expression is not evenly spaced. Thus, ap-Gal4 UAS-iO-fut1 animals exhibit a failure in lateral inhibition (Okajima, 2002).
To directly examine the influence of O-fucose on cell lineage decisions, markers were used for two of the distinct cell types that are generated from each SOP. ELAV is a nuclear protein that is expressed in neurons, while Prospero is a transcription factor that is expressed in the sheath cell. While the neur-lacZA101 staining described above was conducted on animals raised at 29°C, by 24-30 hr APF, when the sheath and neuron cells have been specified, the nota were too fragile to be stained, presumably because of an extensive loss of epithelial cells. Thus, ap-Gal4 UAS-iO-fut1 animals were raised at 25°C, which generates a weaker phenotype but allowed the tissue to be stained. Strikingly, the number of neurons was significantly increased in these animals, while at the same time the number of sheath cells was significantly decreased. The simultaneous increase in neurons and decrease in sheath cells is diagnostic for a loss of Notch signaling in the presumptive sheath cell during the pIIIb cell division. Although the phenotype is somewhat variable, presumably due to the relatively weak expression of iO-fut1, it was noted that in many cases clusters of cells are observed that contain more than two neurons. This observation is consistent with the hypothesis that O-fut1 is in fact required for other asymmetric cell divisions in the SOP lineage. Similarly, the absence of bristle or socket cells in the adult notum, despite the substantial increase in SOPs, implies that O-fut1 is also required for Notch activation in the presumptive pIIa cell. Together, these results establish that O-fut1 is required for the Notch-dependent processes of lateral inhibition and asymmetric cell lineages during microchaete development, neither of which requires fng (Okajima, 2002).
When iO-fut1 is expressed under ptc-Gal4 control, a sharp boundary exists between iO-fut1 expressing and non-expressing cells. Expressing cells can be simultaneously marked by expression from a UAS-GFP transgene. Importantly, all four of the molecular markers of Notch signaling examined were repressed cell autonomously in ptc-Gal4 UAS-iO-fut1 wing imaginal discs. This cell-autonomous effect indicates that O-fut1 activity is required for the cell's ability to receive ligand signals, and would thus be consistent with the hypothesis that the key substrate of O-fut1 is Notch (Okajima, 2002).
However, the Notch ligands Dl and Ser are also modified by O-fucose. Expression of Dl and Ser in the wing is maintained by Notch activation, hence their transcription decreases when Notch signaling is impaired. Thus, in order to examine the ability of Notch ligands to signal in the presence of iO-fut1, the ligands were expressed under the control of the ptc promoter. This results in strong Notch activation in cells outside the ptc stripe. Inside the ptc stripe, Notch activation is weaker and incomplete because high level expression of Notch ligands inhibits Notch signaling. When Dl or Ser is co-expressed with iO-fut1, Notch activation, as monitored by Wg expression, is suppressed inside the ptc stripe, but not outside of the stripe. Although these experiments are subject to the caveat that O-fucosylation may not be completely inhibited, these results indicate that a reduction in O-fut1 levels that impairs the ability of cells to receive Notch signals does not detectably diminish their ability to send them. They also show that the block in Notch activation associated with reduction of O-fucose is not overcome by elevating expression of Notch ligands (Okajima, 2002).
Activation of Notch involves proteolytic cleavages, and expression of the intracellular domain of Notch alone results in a constitutive, ligand-independent activation of the Notch pathway. If O-fut1 influences Notch signaling by glycosylating its extracellular EGF domains, then the expression of the intracellular domain of Notch should be epistatic to expression of iO-fut1. To test this, both constructs were expressed under ptc-Gal4 control. Indeed, the ability of the intracellular domain of Notch to activate downstream targets is unaffected by co-expression with iO-fut1 (Okajima, 2002).
Whether O-fucose influences the stability or subcellular localization of Notch was also investigated. In wild-type, Notch is expressed ubiquitously throughout third instar wing imaginal disc, with the highest protein levels concentrated apically. In wing cells expressing iO-fut1, Notch is readily detected and the subcellular distribution of Notch is similar to wild-type, with the highest levels of Notch protein observed at the apical and basal edges. This distribution indicates that Notch is not unstable when O-fut1 levels are decreased, nor is it stuck in the ER or Golgi. Rather, it appears to traffic through the secretory pathway to the cell surface. Strikingly however, the levels of Notch protein were substantially increased. The increased level of Notch protein detected is consistent with the hypothesis that O-fucose is required for the activation of Notch -- because Notch is cleaved upon activation, failure to signal to Notch can be expected to result in increased levels of Notch. Indeed, loss of Dl has been reported to cause just such an increase in the Drosophila ovary (Okajima, 2002).
The patterned expression of O-fut1 mRNA suggests that O-fut1 expression levels might be regulated during normal development to influence Notch signaling. In order to begin to examine this possibility, the consequence of ectopic or overexpression of O-fut1 was examined. While lower level expression of O-fut1 generates only the mild wing vein phenotype, higher level expression results in a wide range of phenotypes that are characteristic of a loss of Notch signaling. Thus, depending on the Gal4 line used to drive expression of UAS-O-fut1, and the strength of expression driven by a particular insertion, rough eyes, thickened wing veins, loss of tissue from the wing margin, or extra bristles on the notum or abdomen are observed (Okajima, 2002).
While many of the O-fut1 overexpression phenotypes are similar to those observed when iO-fut1 is expressed, overexpression of O-fut1 results in increased bristle density, instead of bald cuticle. This suggests that increased O-fucosylation impairs lateral inhibition, but not SOP lineage decisions. Consistent with this, staining for expression of ELAV and Prospero at pupal stages reveals an increased density of sensory organs, yet each ELAV-expressing cell remains closely associated with a Prospero-expressing cell. The difference in phenotypes is not simply quantitative, because reduced expression of iO-fut1 never yields a situation in which lateral inhibition is impaired but SOP lineage decisions are not. Instead, as the levels of iO-fut1 expression are lowered, sensory organs with bristle forming shaft cells are recovered in a well-spaced pattern (Okajima, 2002).
Intriguingly, it was also noticed that in flies expressing O-fut1 under ap-Gal4 control, a particularly strong increase in bristle density occurs in reproducible stripes on the notum. This implies that some other factor exists that can modulate the consequences of O-fut1 overexpression. Since Fng is expressed in stripes in the pupal notum, the influence of Fng on the O-fut1 overexpression phenotype was examined by co-expressing Fng and O-fut1. Indeed, the induction of additional microchaete is partially suppressed in the notums of ap-Gal4 UAS-fng UAS-O-fut1 flies (Okajima, 2002).
Therefore, overexpression of O-fut1 suggests that O-fucose, besides being required for activation of Notch, has a second, negative role in Notch signaling. This negative role appears to be qualitatively distinct, as it impairs inductive signaling and lateral inhibition, but not cell lineage decisions. It is speculated that these opposing influences of O-fucosylation could involve positive and negative roles for distinct EGF domains on Notch or its ligands, coupled to different affinities of these EGF domains for O-fut1. As enzyme levels vary, so too would the profile of O-fucosylation on EGF domains. Intriguingly, O-fut1 mRNA is downregulated in most but not all cells during embryonic development, and its levels are also modulated in certain tissues including the eye imaginal disc, larval brain, and larval CNS. The transition between high and low levels of O-fut1 during embryogenesis corresponds to a period of lateral inhibition, when the neural and ectodermal precursors are specified by Notch signaling. Thus, this process may require moderate levels of O-fut1 for effective Notch regulation. Although there are many regulatory inputs into the Notch pathway, the present data suggest that O-fucose may have multiple, distinct roles in Notch signaling, and that regulation of O-fut1 expression may contribute to the pattern of Notch activation during development (Okajima, 2002).
To identify novel genes involved in the Notch pathway, a collection of zygotic lethal mutation with specific maternal effects was screened for mutations associated with a neurogenic phenotype. Such a mutation, 4R6, was identified in a novel gene that has been named neurotic (nti). While nti zygotic mutant embryos have normal nervous systems, embryos derived from nti homozygous germline clones show a strong neurogenic phenotype that is not significantly paternally rescued. Thus, the maternal effect phenotype of nti is similar to the neurogenic phenotypes associated with mutations that are defective in Notch signalling. Next, tests were performed to see whether nti is required for other Notch-dependent developmental events, such as adult wing patterning. In the adult wing, nti mutant clones result in loss of wing margin and vein thickening, phenotypes that are reminiscent of loss of Notch activity. Notch regulates the expression of wingless (wg) at the dorsal and ventral (D/V) compartmental boundary. Like Notch mutant clones, nti mutant clones across the D/V boundary are associated with loss of Wg expression. These phenotypes suggest that nti encodes an essential component of the Notch pathway (Sasamura, 2003).
The nti mutation 4R6 was mapped to 50C-D by complementation tests to deficiency lines, and nucleotide sequence analysis of the mutant chromosome revealed a nonsense mutation in the annotated gene CG12366. The single mutation changes the Lysine at position 133 in the putative ORF of 402 amino acids to a premature stop codon (AAG to TAG), strongly suggesting that 4R6 causes complete loss of CG12366 function. As expected from the nti maternal effect, CG12366 mRNA is uniformly distributed in early embryos but decreases during later stages. To confirm that the nti mutant phenotype is caused by a disruption in CG12366, whether inducible expression of CG12366 could rescue nti mutant cells was tested. Wg expression in the nti clones is rescued in wing imaginal discs by ubiquitous expression of CG12366 (hs-nti). The maternal neurogenic phenotype can also be partially rescued. A hairpin construct (nti-IR) was constructed to express in vivo a dsRNA that would inhibit CG12366 function. Expression of nti-IR using ptc-Gal4 causes wing nicks reminiscent of loss of Notch activity and nti mutant clones, consistent with RNAi-mediated inactivation of nti. This nti-IR expression construct was used to knock down nti function in cultured cells. These results demonstrate that CG12366 encodes nti function (Sasamura, 2003).
Database searches reveal that nti encodes a putative protein with significant homology to human GDP-fucose O-fucosyltransferase 1 (Wang, 2001). O-FucT-1 is an enzyme that adds a carbohydrate, fucose, to serine or threonine residues in the consensus sequence CXXGGS/TC (X is any amino acids) between the second and third conserved cysteines of EGF repeats. Notch and its ligands, Delta and Serrate, have such sequences in their extracellular EGF repeats, and all of them are O-fucosylated (Moloney, 2000; Moloney, 2000b; Panin, 2002). Drosophila O-FucT-1 has been shown to be required for the activation of Notch by its ligands using a RNAi knockdown approach (Okajima, 2002) and O-FucT-1 mutant mice have been shown to have a phenotype similar to that of Presenilins or RBP-Jkappa (Shi, 2003). Thus the current results confirm these previous observations and support the model that O-FucT-1 is an essential component of Notch signalling (Sasamura, 2003).
Fringe (Fng), a modulator of Notch signalling, adds N-acetylglucosamine (GlcNAc) onto the O-fucose moieties of Notch and its ligands. Modification of Notch by Fng, and subsequent glycosylation of Notch, modulates the binding of Delta and Serrate to Notch. This result suggests that nti enables Notch to respond to its ligands by adding O-linked fucose to the Notch EGF repeats. However, this finding appears unexpectedly because no report has ever associated fng mutations with the neurogenic phenotype. Therefore, nti, like Notch, is essential for lateral inhibition during neuroblast segregation, a process for which fng is probably dispensable. Furthermore, the nti mutant phenotype in adult wings is also distinct from that of fng. These results indicate that nti has a function that is independent of fng. Such a fng-independent function was also proposed from the analysis of the neurogenic phenotype induced by RNAi of Nti/O-fut1 in Drosophila bristles (Okajima, 2002; Sasamura, 2003 and references therein).
To define precisely the function of nti in the Notch signalling cascade, the epistatic relationship between nti and other genes involved in Notch signalling was investigated. It was first asked whether the activity of the Notch intracellular domain (NotchICD), which acts as a constitutively active Notch receptor, requires nti. Expression of Wg is absent in nti mutant clones. However, NotchICD can induce Wg expression in nti mutant clones, just as it can in wild-type cells. A similar experiment was carried out with an overexpressed full-length Notch receptor, which in wild type also induces ectopic Wg. Interestingly, in contrast to NotchICD, the full-length Notch cannot induce ectopic Wg expression in nti mutant clones, indicating that nti is required for the activity of full-length Notch but not for its intracellular domain. These interactions are consistent with the idea that Nti modifies the extracellular domain of Notch or its ligands (Sasamura, 2003).
Next, the epistatic relationship between nti and fng was examined. From the structure of the O-linked tetrasaccharide attached to the EGF repeats of Notch and from enzymatic functions of Nti and Fng, it was speculated that Nti activity is a prerequisite for the function of Fng. Ectopic expression of Fng in the ventral compartment of the wing imaginal disc is known to induce Wg ectopically. By contrast, ectopic expression of Fng in nti mutant clones does not induce ectopic expression of Wg, and endogeneous expression of Wg is absent in these clones. These results indicate that Nti is essential for Fng function. These findings are consistent with the RNAi experiments against Nti/O-fut1 (Okajima, 2002; Sasamura, 2003).
The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex
Cell-cell signaling mediated by the Notch (N) receptor is implicated in a wide variety of developmental processes in multicellular organisms, across phyla. In humans, N-signaling abnormalities cause diseases that include leukemia, other cancers, and pulmonary arterial hypertension. Drosophila N encodes a transmembrane receptor with 36 epidermal growth factor (EGF)- like repeats in its extracellular domain. During maturation of N, its extracellular domain is cleaved by Furin protease (S1 cleavage) in the Golgi. After reaching the cell surface, the binding of N to its transmembrane ligand, Delta or Serrate, leads to a second cleavage in the extracellular domain of N by Kuzbanian (Kuz)/ADAM10 or ADAM17 (S2 cleavage). This cleavage removes most of the N extracellular domain and produces a membrane-tethered form of the N intracellular domain (NEXT). Subsequently, NEXT is cleaved within its transmembrane domain by γ-secretase (S3 cleavage), which liberates the intracellular domain, termed NICD. NICD then translocates to the nucleus and regulates the transcription of downstream genes (Yamakawa, 2012).
N requires various post-translational modifications to its extracellular domain to be activated. For example, O-glycosylation of the N extracellular domain by O-fucosyltransferase 1 (O-fut1) and Fringe regulates the binding between N and its ligands. O-fut1 is also known to act as an N-specific chaperone in Drosophila. In addition, analysis of a Drosophila thiol oxidase, endoplasmic reticulum (ER) oxidoreductin 1-like (Ero1L), showed that disulfide-bond formation in the extracellular domain of N is indispensable for the activation of the N signal (Yamakawa, 2012).
Many roles played by N signaling in Drosophila development are crucial and have been studied extensively. Its best-known role during the early development of the central nervous system, is to prevent cells that neighbor a neuroblast from choosing the neuroblast fate, a phenomenon called 'lateral inhibition'. This is achieved when the neuroblast-fated cell activates N signaling in its neighbors; these cells become epidermoblasts. Thus, disruption of N signaling in Drosophila embryos results in the failure of lateral inhibition and the consequent hyperplasia of neuroblasts at the expense of epidermoblasts, which is referred to as the ‘neurogenic’ phenotype. Because most of the genes that encode N-signaling components are essential for lateral inhibition, these genes were first identified by the neurogenic phenotype resulting from their disruption (Yamakawa, 2012).
pecanex was originally identified as a mutant showing recessive female sterility (Perrimon, 1984). Thus, pcx homozygous or hemizygous embryos obtained from pcx heterozygous females survive until adulthood. However, embryos obtained from pcx homozygous females mated with pcx hemizygous males, which are fertile, show neuronal hyperplasia, i.e. the neurogenic phenotype, suggesting that the maternally supplied pcx function rescues this phenotype. Therefore, pcx is considered to be a maternal neurogenic gene. pcx encodes a multi-pass transmembrane protein consisting of 3433 amino acids that is highly conserved from Drosophila to humans. A rat homolog of pcx, pecanex1, is expressed in spermatocytes and probably functions in the testes. However, no molecular function of the pcx protein has been identified in any species. This study has established that pcx is an N-signaling component in Drosophila. Evidence is also provided that pcx might be involved in ER functioning (Yamakawa, 2012)
No motifs that might suggest pcx's biochemical function have been found in its amino acid sequence. Although pcx was previously suggested to be involved in N signaling, based on the neurogenic phenotype associated with its mutant in Drosophila, this possibility had not been explored. This study provides evidence that pcx is a component of the N-signaling pathway (Yamakawa, 2012).
In pcxm/z embryos, the ER was abnormally enlarged. Various factors regulating the architecture of the ER have been identified. In Drosophila, Atlastin, a dynamin-like GTPase, is required for fusion of the ER membrane. Thus, the overexpression of Atlastin induces an enlarged ER (Orso, 2009). In addition, the peripheral ER shows two distinct structures: tubules and sheets. Several factors organizing the shape of the ER membrane into tubules or sheets have been identified. Therefore, pcx might contribute to the regulatory machinery that accomplishes the normal organization of the ER (Yamakawa, 2012).
In pcxm/z embryos, the enlarged ER was observed predominantly in the region corresponding to the dorsal epidermis of wild-type embryos. Therefore, sensitivity to the absence of pcx function might differ among groups of cells. This distinct behavior could reflect differences in the cell-cycle phase or level of UPR activity (Yamakawa, 2012).
Although the results showed that the reduction of N signaling was not responsible for the enlargement of the ER in pcxm/z embryos, the ectopic activation of N signaling by overexpression of NICD also suppressed this ER defect. It is speculated that the ectopic activation of N signaling might affect the progression of the cell-cycle or the level of UPR, which could in turn affect the regulation of the ER architecture. It has been shown that N signaling directly or indirectly affects the cell cycle. However, the biological significance and mechanisms of this phenomenon remain elusive (Yamakawa, 2012).
Induction of the UPR was found to suppress the ER enlargement in pcxm/z embryos. The suppression of the ER enlargement by the expression of genes that induce the UPR coincided with the rescue of N signaling activity in these embryos. Therefore, the reduced N signaling in pcxm/z embryos might be attributable to the enlargement of the ER. However, the possibility cannot be excluded that pcx is independently involved in the activation of N signaling and the regulation of the ER architecture. Nevertheless, the results suggest that some downstream events induced by the UPR compensate for the defect of N signaling associated with the absence of pcx function. It was found that overexpression of O-fut1, an N-specific chaperone, partially compensated for the loss of pcx function. Thus, a disruption of N signaling in the absence of pcx function might be partly due to the mis-folding of N, which is consistent with the hypothesis that pcx acts upstream of the activated forms of N and probably functions in signal-receiving cells (Yamakawa, 2012).
The UPR induces various downstream events, including the attenuation of protein synthesis, the enhancement of misfolded ER protein degradation, and the induction of genes encoding various chaperones. Therefore, in future experiments, it will be important to determine the specific defects that are compensated for by the UPR in the absence of pcx function (Yamakawa, 2012).
A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using as ligands the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The Km values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 microM, respectively. The Vmax is 2.5 micromol.min-1.mg-1. The presence of 50 mM Mn2+ increases the enzyme activity 17-fold, but Mn2+ is not absolutely required, since the enzyme exhibits some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity (Wang, 1998).
The O-fucose modification is found on epidermal growth factor-like repeats of a number of cell surface and secreted proteins. O-Fucose glycans play important roles in ligand-induced receptor signaling. For example, elongation of O-fucose on Notch by the beta1,3-N-acetylglucosaminyltransferase Fringe modulates the ability of Notch to respond to its ligands. The enzyme that adds O-fucose to epidermal growth factor-like repeats, GDP-fucose protein O-fucosyltransferase (O-FucT-1), was purified previously from Chinese hamster ovary (CHO) cells. A cDNA that encodes human O-FucT-1 has been isolated. A probe deduced from N-terminal sequence analysis of purified CHO O-FucT-1 was used to screen a human heart cDNA library and expressed sequence tag and genomic data bases. The cDNA contains an open reading frame encoding a protein of 388 amino acids with a predicted N-terminal transmembrane sequence typical of a type II membrane orientation. Likewise, the mouse homolog obtained from an expressed sequence tag and 5'-rapid amplification of cDNA ends of a mouse liver cDNA library encodes a type II transmembrane protein of 393 amino acids with 90.4% identity to human O-FucT-1. Homologs were also found in Drosophila and Caenorhabditis elegans with 41.2% and 29.4% identity to human O-FucT-1, respectively. The human gene (PO-fut1) is on chromosome 20 between PLAGL2 and KIF3B, near the centromere at 20p11. The mouse gene (Pofut1) maps near Plagl2 on a homologous region of mouse chromosome 2. PO-fut1 gene transcripts were expressed in all tissues examined, consistent with the widespread localization of the modification. Expression of a soluble form of human O-FucT-1 in insect cells yields a protein of the predicted molecular weight with O-FucT-1 kinetic and enzymatic properties similar to those of O-FucT-1 purified from CHO cells. The identification of the gene encoding protein O-fucosyltransferase I now makes possible mutational strategies to examine the functions of the unusual O-fucose post-translational modification (Wang, 2001).
Notch receptor signaling regulates cell growth and differentiation, and core components of Notch signaling pathways are conserved from Drosophila to humans. Fringe glycosyltransferases are crucial modulators of Notch signaling that act on epidermal growth factor (EGF)-like repeats in the Notch receptor extracellular domain. The substrate of Fringe is EGF-O-fucose and the transfer of fucose to Notch by protein O-fucosyltransferase 1 is necessary for Fringe to function. O-fucose also occurs on Cripto and on Notch ligands. Mouse embryos lacking protein O-fucosyltransferase 1 die at midgestation with severe defects in somitogenesis, vasculogenesis, cardiogenesis, and neurogenesis. The phenotype is similar to that of embryos lacking downstream effectors of all Notch signaling pathways such as presenilins or RBP-J kappa, and is different from Cripto, Notch receptor, Notch ligand, or Fringe null phenotypes. Protein O-fucosyltransferase 1 is therefore an essential core member of Notch signaling pathways in mammals (Shi, 2003).
Search PubMed for articles about Drosophila O-fucosyltransferase 1/neurotic
Brückner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406: 411-415. 10935637
Chen, J., Moloney, D. J., and Stanley, P. (2001). Fringe modulation of Jagged1-induced Notch signaling requires the action of ß4galactosyltransferase-1. Proc. Natl. Acad. Sci. 98: 13716-13721. 11707585
Ishio, A., Sasamura, T., Ayukawa, T., Kuroda, J., Ishikawa, H. O., Aoyama, N., Matsumoto, K., Gushiken, T., Okajima, T., Yamakawa, T. and Matsuno, K. (2014). O-fucose monosaccharide of Drosophila Notch has a temperature-sensitive function and cooperates with O-glucose glycan in Notch transport and Notch signaling activation. J Biol Chem 290(1):505-19. PubMed ID: 25378397
Kao, Y. H., Lee, G. F., Wang, Y., Starovasnik, M. A., Kelley, R. F., Spellman, M. W. and Lerner, L. (1999). The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII. Biochemistry 38: 7097-7110. 10353820
Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. and Vogt, T. F. (2000a). Fringe is a glycosyltransferase that modifies Notch. Nature 406: 369-375. 10935626
Moloney, D. J., Shair, L. H., Lu, F. M., Xia, J., Locke, R., Matta, K. L. and Haltiwanger, R. S. (2000b). Mammalian Notch1 is modified with two unusual forms of O-linked glycosylation found on epidermal growth factor-like modules. J. Biol. Chem. 275: 9604-9611. 10734111
Oh, S. W., Kingsley, T., Shin, H. H., Zheng, Z., Chen, H. W., Chen, X., Wang, H., Ruan, P., Moody, M., and Hou, S. X. (2003) Genetics 163: 195-201. 12586707
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date revised: 20 November 2012
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