Modulation of Notch-Ligand Binding by Protein O-Fucosyltransferase 1 and Fringe

Abstract

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. 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. Notch receptors are transmembrane proteins that regulate a wide range of cell fate decisions throughout the Metazoa (for review, see Ref. 1Artavanis-Tsakonas S. Rand M.D. Lake R.J. Science. 1999; 284: 770-776Crossref PubMed Scopus (4951) Google Scholar). The broad range of processes that require normal Notch signaling is reflected in the range of human diseases that result from mutations in components of the Notch signaling pathway. These include leukemia, Alagille syndrome, CADASIL, and spondylocostal dystoses (for review, see Refs. 2Joutel A. Tournier-Lasserve E. Semin. Cell Dev. Biol. 1998; 9: 619-625Crossref PubMed Scopus (165) Google Scholar and 3Gridley T. Hum. Mol. Genet. 2003; 12: 9-13Crossref PubMed Google Scholar). Notch receptors are activated by two types of ligands, Serrate ligands and Delta ligands. The core components of the Notch pathway are all highly conserved, but the Drosophila genome encodes a single Notch, a single Serrate (SER), 1The abbreviations used are: SER, Serrate; DL, Delta; EGF, epidermal growth factor-like; RNAi, RNA interference; dsRNA, double-stranded RNA; AP, alkaline phosphatase; OFUT1, protein O-fucosyltransferase 1; O-fucose, O-linked fucose. and a single Delta (DL), whereas mammalian genomes encode four Notches, three Deltas, and two Serrates (called Jagged in mammals). Interaction of ligand and receptor triggers proteolytic processing of Notch, resulting in the translocation of the intracellular domain of Notch from the plasma membrane to the nucleus (for review, see Ref. 4Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (850) Google Scholar). In the nucleus, Notch forms a complex with other proteins to regulate the transcription of downstream target genes. Recent studies have revealed that glycosylation is essential for normal Notch signaling. The extracellular domain of Notch includes 36 tandemly repeated epidermal growth factor-like (EGF) domains. Many of these EGF domains are subject to an unusual post-translational modification in which fucose is attached in an O-linkage to a serine or threonine residue that occurs right before the third cysteine of the EGF domain (5Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. J. Biol. Chem. 2000; 275: 9604-9611Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar). O-linked fucose (O-fucose) is essential for Notch signaling as down-regulation of the enzyme that mediates O-fucosylation by RNAi in Drosophila or by mutation in mouse impairs Notch signaling (6Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (326) Google Scholar, 7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). Subsequent to the action this enzyme, protein O-fucosyltransferase 1 (OFUT1 in Drosophila), O-fucose can be further elongated by the action of another glycosyltransferase, Fringe, which attaches N-acetylglucosamine (GlcNAc) in a β1,3 linkage to EGF-O-fucose (8Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar, 9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). In contrast to the general positive requirement for the O-fucose monosaccharide in Notch signaling, studies of Fringe indicate that elongated forms of O-fucose glycans differentially modulate the ability of the two types of Notch ligands to activate the Notch receptor (Refs. 10Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (339) Google Scholar, 11Klein T. Arias A.M. Development. 1998; 125: 2951-2962Crossref PubMed Google Scholar, 12Fleming R.J. Gu Y. Hukriede N.A. Development. 1997; 124: 2973-2981Crossref PubMed Google Scholar, 13Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar; for review, see Refs. 15Haines, N., and Irvine, K. D. (2003) Nat. Rev. Mol. Cell Biol., in pressGoogle Scholar). Genetic and cell culture studies further show that both OFUT1 and Fringe influence Notch signaling cell autonomously (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 10Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (339) Google Scholar, 13Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar), indicating that they act in the signal receiving cell, presumably by directly glycosylating Notch, which is a substrate for their glycosyltransferase activities (5Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. J. Biol. Chem. 2000; 275: 9604-9611Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 8Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar, 9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). Epistasis analyses indicate that both OFUT1 and Fringe act upstream of the activated (cleaved) form of the Notch receptor (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 10Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (339) Google Scholar, 13Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar), which positions the requirement for O-fucose glycans upstream of the proteolytic cleavages associated with Notch activation. In principle this leaves several possibilities for how O-fucose glycans affect Notch signaling, such as an influence on intracellular trafficking, an influence on Notch-ligand binding, or an influence on Notch proteolysis. The possibility that OFUT1 affects Notch-ligand binding has not been investigated previously, and evidence both for and against the possibility that Fringe influences Notch signaling at the level of Notch-ligand binding has been presented in prior studies (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar, 10Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (339) Google Scholar, 16Shimizu K. Chiba S. Saito T. Kumano K. Takahashi T. Hirai H. J. Biol. Chem. 2001; 276: 25753-25758Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 17Klueg K.M. Muskavitch M.A. J. Cell Sci. 1999; 112: 3289-3297Crossref PubMed Google Scholar). Here, we demonstrate that the O-fucose monosaccharide is essential for the normal binding of Notch to its ligands, DL and SER. We also extend prior studies of the influence of O-fucose elongation by demonstrating that Fringe not only enhances DL-Notch binding (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar) but also inhibits SER-Notch binding, and we show that OFUT1 overexpression and Fringe expression exert opposing influences on Notch-ligand binding. Our results underscore the central importance of O-fucosylation to Notch signaling and identify a mechanism by which O-fucose glycans exert their influence. Expression Constructs—All proteins were expressed under control of the metallothionein promoter. N:AP and expression were from S. Cohen (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar), and and OFUT1 expression have been (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 8Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar, 13Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar). We also used an OFUT1 expression in which and were to the These have influence on its To the by of human alkaline from the N:AP (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar) the and with and and with the domain of human from The for were by from S.H. C. Wilson R. Irvine K.D. Vogt T.F. Development. 1997; 124: PubMed Google Scholar). by of Notch and a from and with and and binding from the of (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar) and N:AP or with cells a Notch ligand or with control of were cultured in in to a of with expression of in Drosophila for were cultured for in and expression in by the addition of to the at for at to cell and assayed for that to of the in the N:AP in the by molecular The of in the presented in the by the addition of from N:AP with or the of by as RNA as (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). RNAi for Ofut1 by of 1 of and the cells were cultured for before of expression in of for cells were a and cells were R.J. L. P. S. Cell. Full Text PDF PubMed Scopus Google Scholar). cells for were with In the of not by of control were cultured to a of in of and with in for were in and in with the of and of cells were and for at with were at with with and and in at for 1 cell by for for and assayed as a substrate in 1 at for The by the addition of 1 and by the of a a and expressed as of in which with cell type used as the In binding or with the extracellular domain of Notch or with control proteins as of of from cells or control cells with with of at and with of with of from normal and cells were and the to by a mouse a and substrate and assayed a were by with with of or control were for 1 at with 1 of with were in of for 1 at and in 1 of assayed as In Fringe or from or The of Notch on by for N:AP and by as three with were with Fringe of were with (8Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar) and for at were four with and to The are for and are as the with as a used were mouse mouse mouse V. A. Panin V.M. C. Irvine K.D. Science. 1998; PubMed Scopus Google Scholar), mouse and were as V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar). at by with a to the of OFUT1, to Drosophila and were from S. T. Chen Chen Wang H. P. 2003; Google Scholar). mutation by and were by to and to T. Development. PubMed Google Scholar). in the Drosophila Ofut1 requirement for OFUT1 in Notch signaling has been by RNAi in Drosophila (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) and by a mutation in the Pofut1 gene (6Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (326) Google Scholar). T. Chen Chen Wang H. P. 2003; Google Scholar) the results of a for in to have an in the of Ofut1 T. Chen Chen Wang H. P. 2003; Google Scholar) and is to result in of the of OFUT1 with four by a T. Chen Chen Wang H. P. 2003; Google Scholar). To that this an Ofut1 we in which of cells were for this by These Notch such as and of on the 1, consistent with the by RNAi of Ofut1 (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). In the expression of of Notch signaling, such as is in cells for Ofut1 mutation to as an of the requirement for OFUT1 for Notch signaling in Drosophila and that the of OFUT1 are essential for in vivo. The four of OFUT1 to a signal for in the Y. Shao L. S. R.J. Stanley P. Haltiwanger R.S. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. Full Text PDF PubMed Google Scholar), and are in to the of in is to of or to studies that O-fucose is on Notch in for to be activated by its ligands (6Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (326) Google Scholar, 7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). To investigate this requirement an influence on the binding of Notch to its ligands, we assayed the ability of an extracellular of Notch to bind to N:AP the culture and the used for binding has been to demonstrate that elongation of O-fucose by the Fringe can DL-Notch binding (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). is to both N:AP binding to cells and binding to we that in OFUT1 the of N:AP from cells of a that the of Notch on the cell with of O-fucosylation and the of for such we on N:AP binding to of N:AP were before with cells and by with from cultured Drosophila binding and the of N:AP by cells by cells OFUT1, but not Fringe (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, 8Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar). N:AP from cells to cells (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). we have not been to the binding of N:AP to the is to the of N:AP with cells at a range and is to the of cells at a range which the of the is a control protein of the domain of human is with cells or N:AP is with cells that not a Notch ligand we have also been to SER-Notch binding with this which not (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). for the of N:AP to cells is to the of N:AP with cells O-Fucosylation for O-fucosylation influences Notch-ligand binding, N:AP from cells with a N:AP expression and with to In the addition of to the culture is to RNAi T. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). that of OFUT1 expression by RNAi highly N:AP is from cells with Ofut1 RNAi the binding of N:AP to cells is to of binding is which be to of by RNAi or indicate that O-fucose binding but is not for binding. these results that O-fucose is on Notch for normal binding to down-regulation of OFUT1 in Notch-secreting cells by RNAi also inhibits binding The requirement for OFUT1 for both DL-Notch and SER-Notch binding an for the Notch by of Ofut1 in (6Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (326) Google Scholar, 7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). of OFUT1 DL-Notch and SER-Notch overexpression of OFUT1 inhibits of Notch signaling in Drosophila (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). To investigate this of Notch signaling also be at the level of Notch-ligand binding, we assayed the consequences of OFUT1 expression on the binding of N:AP to The ability of N:AP from cells that were with an OFUT1 expression to bind to cells in to N:AP from control cells of OFUT1 and N:AP in binding of N:AP to cells The effects of OFUT1 overexpression on DL-Notch and SER-Notch binding an for the of Notch signaling OFUT1 is in the Drosophila (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). of OFUT1 impairs which on DL but not affect the decisions of the which Notch can be by or DL C. S. Dev. 1998; 12: PubMed Scopus Google Scholar). The of OFUT1 by the influence of OFUT1 expression is opposite to that of Fringe in the of Notch signaling (Refs. 10Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (339) Google Scholar, 11Klein T. Arias A.M. Development. 1998; 125: 2951-2962Crossref PubMed Google Scholar, 12Fleming R.J. Gu Y. Hukriede N.A. Development. 1997; 124: 2973-2981Crossref PubMed Google Scholar, 13Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar; for review, see Ref. R.S. Stanley P. 2002; PubMed Scopus Google Scholar and 15Haines, N., and Irvine, K. D. (2003) Nat. Rev. Mol. Cell Biol., in pressGoogle Scholar). we used the N:AP binding to investigate the the influences of OFUT1 and Fringe on Notch-ligand binding. Expression of Fringe enhances binding of N:AP to cells (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). Fringe and OFUT1 are the influence of OFUT1 on binding is and binding is as as in the of OFUT1 The to N:AP binding to in a prior of the influence of Fringe on SER-Notch binding in Drosophila (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar). We that Fringe can binding of N:AP to cells Fringe and OFUT1 are the binding of N:AP to is by with Fringe in with Expression of Fringe and that in OFUT1 and Fringe expression are with glycosylation of Notch and to an of the to which Notch glycosylation is we in glycosylation glycosylation by Fringe is to O-fucose on EGF domains (8Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Nature. 2000; 406: 369-375Crossref PubMed Scopus (723) Google Scholar, V.M. Shao L. L. D.J. Irvine K.D. Haltiwanger R.S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, L. D.J. Haltiwanger R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), in glycosylation by Fringe is to O-fucose N:AP from cells from as a Fringe substrate in the N:AP from cells a substrate for Fringe N:AP from cells that not Fringe Conversely, overexpression of OFUT1 Notch-ligand binding by O-fucosylation of Notch, N:AP from cells OFUT1 be to be a substrate for Fringe and a substrate for this not the as in in glycosylation of N:AP and not the possibility that the of Notch-ligand binding by OFUT1 expression is not to is also that O-fucosylation of only or a EGF domains is for the influence of OFUT1 are O-fucose on Notch, such a be to be in glycosylation in also to that expression of OFUT1 by RNAi glycosylation of the of N:AP from cells with this we an Notch-ligand binding in which the EGF domains of Notch were to a RNAi for Ofut1 of N:AP from cells to that from but of is only to that from cells To Notch-ligand binding, we to and these with from cells or as a and bind specifically to and not to control are from of assayed by from cells with for the binding of and is in binding an of the requirement for O-fucose for Notch-ligand binding. in glycosylation that from cells is a substrate for Fringe from cells consistent with the that O-fucosylation of is by Ofut1 RNAi (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). for studies presented indicate that O-fucosylation is for the binding of Notch to its ligands DL and SER. These binding studies are consistent prior which a requirement for O-fucosylation in upstream of the cleavages associated with Notch (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). results not the possibility that O-fucose glycans also act at other and we influence of Ofut1 RNAi on of Notch extracellular domain the requirement for O-fucose for Notch-ligand binding can in principle for the requirement for OFUT1 in Notch signaling. OFUT1 is for efficient binding of Notch to both and is consistent with the Notch in Ofut1 is by mutation or RNAi (6Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (326) Google Scholar, 7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). elongation of O-fucose by the Fringe opposing influences on the ability of Notch to bind to and that Fringe influences Notch-ligand binding has been (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (596) Google Scholar, 16Shimizu K. Chiba S. Saito T. Kumano K. Takahashi T. Hirai H. J. Biol. Chem. 2001; 276: 25753-25758Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), but other studies have results C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (339) Google Scholar, 17Klueg K.M. Muskavitch M.A. J. Cell Sci. 1999; 112: 3289-3297Crossref PubMed Google Scholar). In Fringe has and effects on both DL-Notch and SER-Notch binding. these effects of Fringe on Notch-ligand binding its effects on signaling by these two ligands in 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 the effects of O-fucosylation on binding in and its effects on signaling in all that O-fucose glycans act at the ligand binding of Notch signaling. their importance to of Notch signaling, these a of glycosylation as a mechanism for the that O-fucosylation affects Notch-ligand binding, be to the basis for this OFUT1 and Fringe act in cells to influence Notch signaling and Notch-ligand binding, which Notch as the the of glycosylation on Notch that mediate the effects of these to be is also not the importance of O-fucosylation a for of Notch by its ligands or other or O-fucose glycans influence Notch-ligand binding by the or of of contrast to the positive requirement for OFUT1 by RNAi, overexpression of OFUT1 enhances SER-Notch binding but inhibits DL-Notch binding. is that OFUT1 expression a mechanism for differentially the ability of Notch ligands to with the Notch the only that the ability of Delta to activate Notch and that of Serrate to activate Notch OFUT1 expression be a mechanism for the of cells to the presence or of In Fringe only affects a of Notch signaling and processes are to Fringe, whereas are (for review, see Refs. 15Haines, N., and Irvine, K. D. (2003) Nat. Rev. Mol. Cell Biol., in pressGoogle Scholar and V.M. Irvine K.D. Semin. Cell Dev. Biol. 1998; 9: PubMed Scopus Google Scholar). OFUT1 to Fringe, its effects can be by Fringe, the of Fringe on DL-Notch or SER-Notch interactions is to be in OFUT1 is expressed at expression of Fringe has on the of has a OFUT1 is also of OFUT1 inhibits DL-Notch signaling, resulting in the of but this is by with Fringe (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). In addition to the of Notch signaling to the presence or of Fringe, OFUT1 expression a mechanism for Notch signaling of the in of Notch-ligand by expression of OFUT1 at of expression we that such as the of Ofut1 cells (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). in Drosophila DL is the or Notch in the a for Notch signaling in cell fate decisions has been and SER, DL, is the ligand that Notch in this J. Biol. 2002; 12: Full Text Full Text PDF PubMed Scopus Google Scholar, T. S.H. U. Dev. 2003; PubMed Scopus Google Scholar). These for the possibility that of Ofut1 a mechanism for Notch pathway (7Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) and developmental in which this be investigated We T. for with Drosophila and L. for of Fringe, S. S. R. and R. Haltiwanger for cell and expression the for Drosophila the at the of for and P. C. and R. Haltiwanger for and on the