O-fucosyltransferase 1/neurotic
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).
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