Modulation of notch-ligand binding by protein O-fucosyltransferase 1 and fringe

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 down-regulation of this enzyme by RNA interference or mutation of the Ofut1 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, we assayed the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Down-regulation of OFUT1 by RNA interference 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, overexpression of OFUT1 in cultured cells increases Serrate-Notch binding but inhibits Delta-Notch binding. These effects of OFUT1 are consistent with the consequences of OFUT1 overexpression on Notch signaling in vivo. Intriguingly, they are also opposite to, 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.     

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.     

敲除Pofut1不影响胚胎干细胞向神经元分化

蛋白O-连接岩藻糖基转移酶1 (Pofut1)基因缺失可导致Notch分子无法与配体结合并启动信号传递. 为研究Pofut1基因对哺乳动物胚胎干细胞（ESC）向神经分化的影响,利用Pofut1基因敲除的胚胎干细胞与野生型胚胎干细胞,经体外培养诱导拟胚体（EB）分化为神经细胞,计数分化为神经细胞的比例,采用细胞免疫组化染色和real-time PCR等方法,分析神经细胞特异性标志分子的表达. 结果显示,Pofut1基因缺失后,对EBC生长没有明显影响,分化过程中形成的拟胚体数量明显增多,分化的神经样细胞以及神经标志物分子的表达也明显多于对照组;Notch信号缺失对小鼠胚胎干细胞生长无明显影响,但可以促进ES细胞向神经细胞分化.     

Notch signaling in Sertoli cells regulates cyclical gene expression of Hes1 but is dispensable for mouse spermatogenesis

Mammalian spermatogenesis is a highly regulated system dedicated to the continuous production of spermatozoa from spermatogonial stem cells, and the process largely depends on microenvironments created by Sertoli cells, unique somatic cells that reside within a seminiferous tubule. Spermatogenesis progresses with a cyclical program known as the "seminiferous epithelial cycle," which is accompanied with cyclical gene expression changes in Sertoli cells. However, it is unclear how the cyclicity in Sertoli cells is regulated. Here, we report that Notch signaling, which is known to play an important role for germ cell development in Drosophila and Caenorhabditis elegans, is cyclically activated in Sertoli cells and regulates stage-dependent gene expression of Hes1. To elucidate the regulatory mechanism of stage-dependent Hes1 expression and the role of Notch signaling in mouse spermatogenesis, we inactivated Notch signaling in Sertoli cells by deleting protein O-fucosyltransferase 1 (Pofut1), using the cre-loxP system, and found that stage-dependent Hes1 expression was dependent on the activation of Notch signaling. Unexpectedly, however, spermatogenesis proceeded normally. Our results thus indicate that Notch signaling regulates cyclical gene expression in Sertoli cells but is dispensable for mouse spermatogenesis. This highlights the evolutionary divergences in regulation of germ cell development.     

The two N-glycans present on bovine Pofut1 are differently involved in its solubility and activity

O-Fucosylation is a post-translational glycosylation in which an O-fucose is covalently attached to the hydroxyl group of a specific serine or threonine residue. This modification occurs within the consensus sequence C2X(4-5)(S/T)C3 present on epidermal growth factor-like repeats of several proteins, including the Notch receptors and their ligands. The enzyme responsible for the addition of O-fucose to epidermal growth factor-like repeats is protein O-fucosyltransferase 1. Protein O-fucosyltransferase 1-mediated O-fucosylation is essential in Notch signaling, folding and targeting to the cell surface. Here, we studied the expression pattern of protein O-fucosyltransferase 1 in cattle and showed that the active enzyme is present in all tissues examined from embryo and adult as a glycoprotein with two N-glycans. By comparing protein O-fucosyltransferase 1 sequences available in databases, we observed that mammalian protein O-fucosyltransferase 1 enzymes possess two putative N-glycosylation sites, and that only the first is conserved among bilaterians. To gain more insight regarding the significance of N-glycans on protein O-fucosyltransferase 1, we substituted, by site-directed mutagenesis, bovine protein O-fucosyltransferase 1 N65, N163 or both, with L or Q. We demonstrated that the loss of N-glycan on N163 caused a slight decrease in protein O-fucosyltransferase 1 activity. In contrast, glycosylation of N65 was crucial for protein O-fucosyltransferase 1 functionality. Loss of glycosylation at N65 resulted in aggregation of protein O-fucosyltransferase 1, suggesting that N-glycosylation at this site is essential for proper folding of the enzyme.     

Notch signaling is required for the maintenance of enteric neural crest progenitors

Notch signaling is involved in neurogenesis, including that of the peripheral nervous system as derived from neural crest cells (NCCs). However, it remains unclear which step is regulated by this signaling. To address this question, we took advantage of the Cre-loxP system to specifically eliminate the protein O-fucosyltransferase 1 (Pofut1) gene, which is a core component of Notch signaling, in NCCs. NCC-specific Pofut1-knockout mice died within 1 day of birth, accompanied by a defect of enteric nervous system (ENS) development. These embryos showed a reduction in enteric neural crest cells (ENCCs) resulting from premature neurogenesis. We found that Sox10 expression, which is normally maintained in ENCC progenitors, was decreased in Pofut1-null ENCCs. By contrast, the number of ENCCs that expressed Mash1, a potent repressor of Sox10, was increased in the Pofut1-null mouse. Given that Mash1 is suppressed via the Notch signaling pathway, we propose a model in which ENCCs have a cell-autonomous differentiating program for neurons as reflected in the expression of Mash1, and in which Notch signaling is required for the maintenance of ENS progenitors by attenuating this cell-autonomous program via the suppression of Mash1.     

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.     

Pofut1 is required for the proper localization of the Notch receptor during mouse development

Protein O-fucosyltransferase 1 (Pofut1), which catalyzes the addition of O-linked fucose to the EGF domains of the Notch receptor, is indispensable for Notch signaling activation. However, the mechanism of action of Pofut1 in mice is still unclear. Mouse embryos lacking Pofut1 shows defects in valve formation and trabeculation in the cardiovascular system, which are almost identical abnormalities to those of the RBP-Jk mutants. In our current study, we have examined the epistatic relationship between the functions of Pofut1 and activated-Notch1 (NICD1) by taking advantage of the fact that forced expression of NICD1 results in myocardial defects. These defects were still evident in NICD1-expressing embryos irrespective of the presence or absence of Pofut1, which indicates that Pofut1 is required for Notch signaling upstream of NICD1. We further found that Pofut1-null cells do not possess normally localized Notch1 receptors, which may results in their lack of interaction with the Dll1 ligand in the presomitic mesoderm where Notch signaling plays a pivotal role. We propose that altered trafficking pathways may account for the abnormal accumulation of the Notch1 receptor in the endoplasmic reticulum in Pofut1-null mouse embryos.     

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.     

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.     

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.     

The O-fucosyltransferase O-fut1 is an extracellular component that is essential for the constitutive endocytic trafficking of Notch in Drosophila

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. Here, we report that O-fut1 has additional functions in the endocytic transportation of Notch. O-fut1 was indispensable for the constitutive transportation of Notch from the plasma membrane to the early endosome, which we show was independent of the O-fucosyltransferase activity of O-fut1. We also found that O-fut1 promoted the turnover of Notch, which consequently downregulated 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 was 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. We propose that O-fut1 is the first example, except for ligands, of a molecule that is required extracellularly for receptor transportation by endocytosis.     

An Evolutionary-Conserved Function of Mammalian Notch Family Members as Cell Adhesion Molecules

Notch family members were first identified as cell adhesion molecules by cell aggregation assays in Drosophila studies. However, they are generally recognized as signaling molecules, and it was unclear if their adhesion function was restricted to Drosophila. We previously demonstrated that a mouse Notch ligand, Delta-like 1 (Dll1) functioned as a cell adhesion molecule. We here investigated whether this adhesion function was conserved in the diversified mammalian Notch ligands consisted of two families, Delta-like (Dll1, Dll3 and Dll4) and Jagged (Jag1 and Jag2). The forced expression of mouse Dll1, Dll4, Jag1, and Jag2, but not Dll3, on stromal cells induced the rapid and enhanced adhesion of cultured mast cells (MCs). This was attributed to the binding of Notch1 and Notch2 on MCs to each Notch ligand on the stromal cells themselves, and not the activation of Notch signaling. Notch receptor-ligand binding strongly supported the tethering of MCs to stromal cells, the first step of cell adhesion. However, the Jag2-mediated adhesion of MCs was weaker and unlike other ligands appeared to require additional factor(s) in addition to the receptor-ligand binding. Taken together, these results demonstrated that the function of cell adhesion was conserved in mammalian as well as Drosophila Notch family members. Since Notch receptor-ligand interaction plays important roles in a broad spectrum of biological processes ranging from embryogenesis to disorders, our finding will provide a new perspective on these issues from the aspect of cell adhesion.     

Protein O-Fucosyltransferase 1 Expression Impacts Myogenic C2C12 Cell Commitment via the Notch Signaling Pathway

The Notch signaling pathway plays a crucial role in skeletal muscle regeneration in mammals by controlling the transition of satellite cells from quiescence to an activated state, their proliferation, and their commitment toward myotubes or self-renewal. O-fucosylation on Notch receptor epidermal growth factor (EGF)-like repeats is catalyzed by the protein O-fucosyltransferase 1 (Pofut1) and primarily controls Notch interaction with its ligands. To approach the role of O-fucosylation in myogenesis, we analyzed a murine myoblastic C2C12 cell line downregulated for Pofut1 expression by short hairpin RNA (shRNA) inhibition during the time course of differentiation. Knockdown of Pofut1 affected the signaling pathway activation by a reduction of the amount of cleaved Notch intracellular domain and a decrease in downstream Notch target gene expression. Depletion in Pax7⁺/MyoD⁻ cells and earlier myogenic program entrance were observed, leading to an increase in myotube quantity with a small number of nuclei, reflecting fusion defects. The rescue of Pofut1 expression in knockdown cells restored Notch signaling activation and a normal course in C2C12 differentiation. Our results establish the critical role of Pofut1 on Notch pathway activation during myogenic differentiation.