Protein O-fucosyltransferase Research Articles

Engineering of a mammalian O-glycosylation pathway in the yeast Saccharomyces cerevisiae: production of O-fucosylated epidermal growth factor domains

Development of a heterologous system for the production of homogeneous sugar structures has the potential to elucidate structure-function relationships of glycoproteins. In the current study, we used an artificial O-glycosylation pathway to produce an O-fucosylated epidermal growth factor (EGF) domain in Saccharomyces cerevisiae. The in vivo O-fucosylation system was constructed via expression of genes that encode protein O-fucosyltransferase 1 and the EGF domain, along with genes whose protein products convert cytoplasmic GDP-mannose to GDP-fucose. This system allowed identification of an endogenous ability of S. cerevisiae to transport GDP-fucose. Moreover, expression of EGF domain mutants in this system revealed the different contribution of three disulfide bonds to in vivo O-fucosylation. In addition, lectin blotting revealed differences in the ability of fucose-specific lectin to bind the O-fucosylated structure of EGF domains from human factors VII and IX. Further introduction of the human fringe gene into yeast equipped with the in vivo O-fucosylation system facilitated the addition of N-acetylglucosamine to the EGF domain from factor IX but not from factor VII. The results suggest that engineering of an O-fucosylation system in yeast provides a powerful tool for producing proteins with homogenous carbohydrate chains. Such proteins can be used for the analysis of substrate specificity and the production of antibodies that recognize O-glycosylated EGF domains.

DNA Micro-Array Gene Expression Profiling of Angiogenesis in Collagen Gel Culture

We examined angiogenesis-related gene expression profiles using collagen gel culture and a DNA chip. After isolation of total RNA from cultures before and after capillary tube formation, a mouse whole-genome array study was performed. Seventy-three out of over 35,000 transcripts were expressed after capillary tube formation. The majority of genes did not show any significant differences between before and after capillary tube formation. However, there were 7 upregulated genes; tumor necrosis factor alpha-induced protein (Tnfαip) 2, vascular endothelial growth factor b (Vegfb), corticotropin releasing hormone receptor (Crhr) 2, vascular endothelial growth factor C (Vegfc), fi broblast growth factor (Fgf) 6, protein O-fucosyltransferase (Pofut) 1, hypoxia inducible factor 1 alpha subunit (Hif-1a). Eleven down-regulated genes were identified; ras homolog gene family member B (RhoB), Fgf-2, Thrombospondin (Thbs) 1, Angiogenin (Ang) 3, guanine nucleotide binding protein alpha (Gna) 13, tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a), plasminogen (Plg), Elk3, upstream binding protein (Ubp) 1, integrin alpha V (Itgav), mitogen activated protein kinase (Mapk) 14. These up- and down-regulated genes identified in this study offer the strategies which improve the myocardial substrate during and following a myocardial infarction, such as the regrowth of functional blood vessels to the ischemic myocardium.

303 ORAL Intestinal inactivation of canonical Notch signaling by removal of protein O-fucosyltransferase 1 triggers secretory cell fate differentiation

303 ORAL Intestinal inactivation of canonical Notch signaling by removal of protein O-fucosyltransferase 1 triggers secretory cell fate differentiation

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.

O-Fucosylation Is Required for ADAMTS13 Secretion

ADAMTS13 is a plasma metalloproteinase that cleaves von Willebrand factor to smaller, less thrombogenic forms. Deficiency of ADAMTS13 activity in plasma leads to thrombotic thrombocytopenic purpura. ADAMTS13 contains eight thrombospondin type 1 repeats (TSR), seven of which contain a consensus sequence for the direct addition of fucose to the hydroxyl group of serine or threonine. Mass spectral analysis of tryptic peptides derived from human ADAMTS13 indicate that at least six of the TSRs are modified with an O-fucose disaccharide. Analysis of [(3)H]fucose metabolically incorporated into ADAMTS13 demonstrated that the disaccharide has the structure glucose-beta1,3-fucose. Mutation of the modified serine to alanine in TSR2, TSR5, TSR7, and TSR8 reduced the secretion of ADAMTS13. Mutation of more than one site dramatically reduced secretion regardless of the sites mutated. When the expression of protein O-fucosyltransferase 2 (POFUT2), the enzyme that transfers fucose to serines in TSRs, was reduced using siRNA, the secretion of ADAMTS13 decreased. A similar outcome was observed when ADAMTS13 was expressed in a cell line unable to synthesize the donor for fucose addition, GDP-fucose. Although overexpression of POFUT2 did not affect the secretion of wild-type ADAMTS13, it did increase the secretion of the ADAMTS13 TSR1,2 double mutant but not that of ADAMTS13 TSR1-8 mutant. Together these findings indicate that O-fucosylation is functionally significant for secretion of ADAMTS13.

O-Fucosylation of Thrombospondin Type 1 Repeats in ADAMTS-like-1/Punctin-1 Regulates Secretion: IMPLICATIONS FOR THE ADAMTS SUPERFAMILY

The ADAMTS superfamily contains several metalloproteases (ADAMTS proteases) as well as ADAMTS-like molecules that lack proteolytic activity. Their common feature is the presence of one or more thrombospondin type-1 repeats (TSRs) within a characteristic modular organization. ADAMTS like-1/punctin-1 has four TSRs. Previously, O-fucosylation on Ser or Thr mediated by the endoplasmic reticulum-localized enzyme protein-O-fucosyltransferase 2 (POFUT2) was described for TSRs of thrombospondin-1, properdin, and F-spondin within the sequence Cys-Xaa(1)-Xaa(2)-(Ser/Thr)-Cys-Xaa-Xaa-Gly (where the fucosylated residue is underlined). On mass spectrometric analysis of tryptic peptides from recombinant secreted human punctin-1, the appropriate peptides from TSR2, TSR3, and TSR4 were found to bear either a fucose monosaccharide (TSR3, TSR4) or a fucose-glucose disaccharide (TSR2, TSR3, TSR4). Although mass spectral analysis did not unambiguously identify the relevant peptide from TSR1, metabolic labeling of cells expressing TSR1 and the cysteine-rich module led to incorporation of [(3)H]fucose into this construct. Mutation of the putative modified Ser/Thr residues in TSR2, TSR3, and TSR4 led to significantly decreased levels of secreted punctin-1. Similarly, expression of punctin-1 in Lec-13 cells that are deficient in conversion of GDP-mannose to GDP-fucose substantially decreased the levels of secreted protein, which were restored upon culture in the presence of exogenous l-fucose. In addition, mutation of the single N-linked oligosaccharide in punctin-1 led to decreased levels of secreted punctin-1. Taken together, the data define a critical role for N-glycosylation and O-fucosylation in the biosynthesis of punctin-1. From a broad perspective, these data suggest that O-fucosylation may be a widespread post-translational modification in members of the ADAMTS superfamily with possible regulatory consequences.

Notch Signaling in Normal and Disease States: Possible Therapies Related to Glycosylation

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

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.

Identification and Characterization of aβ1,3-Glucosyltransferase That Synthesizes the Glc-β1,3-Fuc Disaccharide on Thrombospondin Type 1 Repeats

Thrombospondin type 1 repeats (TSRs) are biologically important domains of extracellular proteins. They are modified with a unique Glcbeta1,3Fucalpha1-O-linked disaccharide on either serine or threonine residues. Here we identify the putative glycosyltransferase, B3GTL, as the beta1,3-glucosyltransferase involved in the biosynthesis of this disaccharide. This enzyme is conserved from Caenorhabditis elegans to man and shares 28% sequence identity with Fringe, the beta1,3-N-acetylglucosaminyltransferase that modifies O-linked fucosyl residues in proteins containing epidermal growth factor-like domains, such as Notch. beta1,3-Glucosyltransferase glucosylates properly folded TSR-fucose but not fucosylated epidermal growth factor-like domain or the non-fucosylated modules. Specifically, the glucose is added in a beta1,3-linkage to the fucose in TSR. The activity profiles of beta1,3-glucosyltransferase and protein O-fucosyltransferase 2, the enzyme that carries out the first step in TSR O-fucosylation, superimpose in endoplasmic reticulum subfractions obtained by density gradient centrifugation. Both enzymes are soluble proteins that efficiently modify properly folded TSR modules. The identification of the beta1,3-glucosyltransferase gene allows us to manipulate the formation of the rare Glcbeta1,3Fucalpha1 structure to investigate its biological function.

Molecular cloning and characterization of a novel human 1,3-glucosyltransferase, which is localized at the endoplasmic reticulum and glucosylates O-linked fucosylglycan on thrombospondin type 1 repeat domain

Protein O-linked fucosylation is an unusual glycosylation associated with many important biological functions such as Notch signaling. Two fucosylation pathways synthesizing O-fucosylglycans have been reported on cystein-knotted proteins, that is, on epidermal growth factor-like (EGF-like) domains and on thrombospondin Type 1 repeat (TSR) domains. We report here the molecular cloning and characterization of a novel beta1,3-glucosyltransferase (beta3Glc-T) that synthesizes a Glcbeta1,3Fucalpha- structure on the TSR domain. We found a novel glycosyltransferase gene with beta1,3-glycosyltransferase (beta3GT) motifs in databases. The recombinant enzyme expressed in human embryonic kidney 293T (HEK293T) cells exhibited glucosyltransferase activity toward fucose-alpha-para-nitrophenyl (Fucalpha-pNp). Thin-layer chromatography (TLC) analysis revealed that the product of the recombinant enzyme migrated to the same position as did the product of endogenous beta3Glc-T of Chinese hamster ovary (CHO) cells. The two products could be digested by beta-glucosidase from almond and by exo-1,3-beta-glucanase from Trichoderma sp. These results strongly suggested that the product has the structure of Glcbeta1-3Fuc. Therefore, we named this novel enzyme beta3Glc-T. Immunostaining revealed that FLAG-tagged beta3Glc-T is an enzyme residing in the endoplasmic reticulum (ER) via retention signal, "REEL," which is a KDEL-like sequence, at the C-terminus. The TSR domain expressed in Escherichia coli was first fucosylated by the recombinant protein O-fucosyltransferase 2 (POFUT2), after which it became an acceptor substrate for the recombinant beta3Glc-T, which could apparently transfer Glc to the fucosylated TSR domain. Our results suggest that a novel glycosyltransferase, beta3Glc-T, contributes to the elongation of O-fucosylglycan and that this occurs specifically on TSR domains.

Molecular evolution of protein O-fucosyltransferase genes and splice variants

O-Fucose has been described on both epidermal growth factor-like (EGF-like) repeats and Thrombospondin type 1 repeats (TSRs). The enzyme adding fucose to EGF-like repeats, protein O-fucosyltransferase 1 (Pofut1), is a soluble protein located in the lumen of endoplasmic reticulum (ER). A second protein O-fucosyltransferase, Pofut2, quite divergent from its homolog Pofut1, has recently been shown to O-fucosylate TSRs but not EGF-like repeats. To date, Pofut1 genes have only been characterized in human, mouse, and fly, and Pofut2 in mouse, fly, and partially in the nematode Caenorhabditis elegans. Here, we report cDNA sequences and genomic structures of bovine Pofut1 and Pofut2 genes and describe for the first time five alternative spliced transcripts for each gene. Only one transcript for both Pofut1 and Pofut2 encodes an active bovine O-fucosyltransferase. Variant transcript distribution was examined in 13 bovine tissues. Transcripts encoding active forms are ubiquitous, whereas other forms possess a more restricted tissue-expression profile. Sequence comparison and phylogenetic analyses revealed that both Pofut genes are present as a single copy in animal genomes, and their exon-intron organizations are conserved among vertebrates. The last common ancestor of all analyzed bilaterian species would be predicted to possess polyexonic Pofut genes in their genome.

Two Distinct Pathways for O-Fucosylation of Epidermal Growth Factor-like or Thrombospondin Type 1 Repeats

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

Protein O-Fucosyltransferase 2 Adds O-Fucose to Thrombospondin Type 1 Repeats

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

Canonical Notch Signaling Is Dispensable for Early Cell Fate Specifications in Mammals

The canonical Notch signaling pathway mediated by Delta- and Jagged-like Notch ligands determines a variety of cell fates in metazoa. In Caenorhabditis elegans and sea urchins, canonical Notch signaling is essential for different cell fate specifications during early embryogenesis or the formation of endoderm, mesoderm, or ectoderm germ layers. Transcripts of Notch signaling pathway genes are present during mouse blastogenesis, suggesting that the canonical Notch signaling pathway may also function in early mammalian development. To test this directly, we used conditional deletion in oocytes carrying a ZP3Cre recombinase transgene to generate mouse embryos lacking both maternal and zygotic protein O-fucosyltransferase 1, a cell-autonomous and essential component of canonical Notch receptor signaling. Homozygous mutant embryos derived from eggs lacking Pofut1 gene transcripts developed indistinguishably from the wild type until approximately embryonic day 8.0, a postgastrulation stage after the formation of the three germ layers. Thus, in contrast to the case with C. elegans and sea urchins, canonical Notch signaling is not required in mammals for earliest cell fate specifications or for formation of the three germ layers. The use of canonical Notch signaling for early cell fate specifications by lower organisms may represent co-option of a regulatory pathway originally used later in development by all metazoa.

CADASIL mutations impair Notch3 glycosylation by Fringe

Mutations in the NOTCH3 gene trigger adult-onset stroke and vascular dementia in patients with CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy). All CADASIL mutations described to date affect the epidermal growth factor-like (EGF-like) repeats located in the extracellular domain of the Notch3 receptor. These domains are also the target of sequential complex O-linked glycosylation mediated by protein O-fucosyltransferase 1 and Fringe. We investigated whether O-fucosylation or Fringe-mediated elongation of O-fucose on Notch3 is impaired by CADASIL mutations. Biochemical studies of a Notch3 fragment containing the first five EGF-like repeats of Notch3, including the mutational hot spot, showed that CADASIL mutations do not affect the addition of O-fucose but do impair carbohydrate chain elongation by Fringe. CADASIL changes also induced aberrant homodimerization of mutant Notch3 fragments and heterodimerization of mutant Notch3 with Lunatic Fringe itself. Together, these data suggest that Fringe plays a role in CADASIL pathophysiology.

O-Fucosylation of Notch Occurs in the Endoplasmic Reticulum

LADII (leukocyte adhesion deficiency type II)/CDGIIc (congenital disorder of glycosylation type IIc) is a rare autosomal recessive disease characterized by leukocyte adhesion deficiency as well as severe neurological and developmental abnormalities. It is caused by mutations in the Golgi GDP-fucose transporter, resulting in a reduction of fucosylated antigens on the cell surface. A recent study using fibroblasts from LADII/CDGIIc patients suggested that although terminal fucosylation of N-glycans is reduced severely, protein O-fucosylation is generally unaffected (Sturla, L., Rampal, R., Haltiwanger, R. S., Fruscione, F., Etzioni, A., and Tonetti, M. (2003) J. Biol. Chem. 278, 26727-26733). A potential explanation for this phenomenon is that enzymes adding O-fucose to proteins localize to cell organelles other than the Golgi apparatus. In this study, we investigated the subcellular localization of protein O-fucosyltransferase 1 (O-FucT-1), which is responsible for adding O-fucose to epidermal growth factor-like repeats. Our analysis reveals that, unlike all other known fucosyltransferases, O-FucT-1 is a soluble protein that localizes to the endoplasmic reticulum (ER). In addition, it appears that O-FucT-1 is retained in the ER by a KDEL-like sequence at its C terminus. Our results also suggest that enzymatic addition of O-fucose to proteins occurs in the ER, suggesting that a novel, ER-localized GDP-fucose transporter may exist. The fact that O-FucT-1 recognizes properly folded epidermal growth factor-like repeats, together with this unique localization, suggests that it may play a role in quality control.

The Caenorhabditis elegans ortholog of C21orf80, a potential new protein O-fucosyltransferase, is required for normal development

Down syndrome (DS), as a phenotypic result of trisomy 21, is the most frequent aneuploidy at birth and the most common known genetic cause of mental retardation. DS is also characterized by other phenotypes affecting many organs, including brain, muscle, heart, limbs, gastrointestinal tract, skeleton, and blood. Any of the human chromosome 21 (Hsa21) genes may contribute to some of the DS phenotypes. To determine which of the Hsa21 genes are involved in DS, the effects of disrupting and overexpressing individual human gene orthologs in model organisms, such as the nematode Caenorhabditis elegans, can be analyzed. Here, we isolated and characterized C21orf80 (human chromosome 21 open reading frame 80), a potential novel protein O-fucosyltransferase gene that encodes three alternatively spliced transcripts. Transient expression of tagged C21orf80 proteins suggests a primary intracellular localization in the Golgi apparatus. To gain insight into the biological role of C21orf80 and its potential role in DS, we isolated its C. elegans ortholog, pad-2, and performed RNA interference (RNAi) and overexpression experiments. pad-2(RNAi) embryos showed failure to undergo normal morphogenesis. Transgenic worms with elevated dosage of pad-2 displayed severe body malformations and abnormal neuronal development. These results show that pad-2 is required for normal development and suggest potential roles for C21orf80 in the pathogenesis of DS.

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.

Neurotic, a novel maternal neurogenic gene, encodes an O-fucosyltransferase that is essential for Notch-Delta interactions.

Notch signalling, which is highly conserved from nematodes to mammals, plays crucial roles in many developmental processes. In the Drosophila embryo, deficiency in Notch signalling results in neural hyperplasia, commonly referred to as the neurogenic phenotype. We identify a novel maternal neurogenic gene, neurotic, and show that it is essential for Notch signalling. neurotic encodes a Drosophila homolog of mammalian GDP-fucose protein O-fucosyltransferase, which adds fucose sugar to epidermal growth factor-like repeats and is known to play a crucial role in 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. Further, we show that neurotic is required for Fringe activity, which encodes a fucose-specific beta1, 3 N-acetylglucosaminyltransferase, previously shown to modulate Notch receptor activity. Finally, 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. We present an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. Our 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.

Regulation of Notch Signaling by O-Linked Fucose

Notch and its ligands are modified by a protein O-fucosyltransferase (OFUT1) that attaches fucose to a Serine or Threonine within EGF domains. By using RNAi to decrease Ofut1 expression in Drosophila, we demonstrate that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for Ofut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. The transcription of Ofut1 is developmentally regulated, and surprisingly, overexpression of Ofut1 inhibits Notch signaling. Together, these results indicate that OFUT1 is a core component of the Notch pathway, which is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development.