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• W1986322003 abstract "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 β1,3 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. 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 β1,3 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. Notch proteins are transmembrane receptors for a conserved signaling pathway that mediates a wide range of cell fate decisions during development (1Schweisguth F. Curr. Biol. 2004; 14: R129-R138Abstract Full Text Full Text PDF PubMed Google Scholar, 2Lai E.C. Development. 2004; 131: 965-973Crossref PubMed Scopus (868) Google Scholar). Notch receptors are activated by binding to transmembrane ligands expressed on adjacent cells. In a subset of Notch signaling events, such as occurs along the dorsal-ventral boundary of the wing disc in Drosophila or in developing somites in vertebrates, the interaction of ligands with the Notch receptor is modulated by differential glycosylation of Notch (3Haines N. Irvine K.D. Nat. Rev. Mol. Cell. Biol. 2003; 4: 786-797Crossref PubMed Scopus (338) Google Scholar, 4Haltiwanger R.S. Stanley P. Biochim. Biophys. Acta. 2002; 1573: 328-335Crossref PubMed Scopus (86) Google Scholar). This is effected by Fringes, β1,3-N-acetylglucosaminyltransferases that extend O-fucose glycans attached to epidermal growth factor-like (EGF) 3The abbreviations used are: EGF, epidermal growth factor-like;β4GalT,β1,4-galactosyltransferase; AP, alkaline phosphatase; β4GalNAcT, β1,4-N-acetylgalactosaminyltransferase; FNG, Fringe; SER, Serrate; MS/MS, tandem mass spectrometry; CID, collision-induced dissociation; LC, liquid chromatography; CHO, Chinese hamster ovary; HBSS, Hanks' balanced salt solution. domains (5Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar, 6Moloney 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). Drosophila has single fringe (fng) and Notch genes, and two Notch ligands, called Delta and Serrate (SER). Expression of fng potentiates the activation of Notch by Delta while inhibiting the activation of Notch by SER (7Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (508) Google Scholar, 8Fleming R.J. Gu Y. Hukriede N.A. Development. 1997; 124: 2973-2981Crossref PubMed Google Scholar). Mammals have four Notches, three Delta-related ligands, two Serrate-related ligands (called Jaggeds), and three Fringes. Although only some of the many possible Fringe-ligand-Notch combinations have been examined, mammalian Fringes can also modulate Notch signaling, both in vivo and in cell-based assays (6Moloney 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, 9Hicks 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, 10Shimizu 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, 11Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Mol. Biol. Cell. 2005; 16: 927-942Crossref PubMed Scopus (172) Google Scholar, 12Zhang N. Gridley T. Nature. 1998; 394: 374-377Crossref PubMed Scopus (363) Google Scholar, 13Evrard Y.A. Lun Y. Aulehla A. Gan L. Johnson R.L. Nature. 1998; 394: 377-381Crossref PubMed Google Scholar, 14Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar). Binding assays in which soluble forms of Notch or its ligands are bound to cells expressing a ligand or Notch have, in most cases, found that Fringes influence Notch-ligand binding, identifying this as a critical step modulated by Fringe (5Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar, 9Hicks 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, 11Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Mol. Biol. Cell. 2005; 16: 927-942Crossref PubMed Scopus (172) Google Scholar, 15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). However, because these studies all relied on in vivo glycosylation, they could not reveal whether glycosylation per se is sufficient to modulate Notch-ligand interactions, or instead whether it acts as a precursor to subsequent events. In Chinese hamster ovary (CHO) cells, the N-acetylglucosamine-fucose (GlcNAc-β1,3-Fuc) disaccharide that is the product of Fringes is further elongated by other glycosyltransferases to yield a tetrasaccharide, Sia-α2,3-Gal-β1,4-GlcNAc-β1,3-Fuc (6Moloney 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, 16Moloney 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). Investigations of the glycan structures that mediate Fringe-dependent modulation of Notch signaling have been carried out in CHO cells by using mutants deficient in specific steps of glycosylation. The ability of Manic fringe (Mfng) or Lunatic fringe (Lfng) to inhibit Jagged1 to Notch1 signaling in these cells requires the action of β1,4-galactosyltransferase 1 (β4GalT-1), which is also required for the elongation of the GlcNAc-β1,3-Fuc disaccharide to a Gal-β1,4-GlcNAc-β1,3-Fuc trisaccharide (14Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar). These observations, together with the lack of requirement for sialylation, suggested that the relevant glycan structure for Fringe-dependent modulation of Notch signaling in mammalian cells is the trisaccharide Gal-β1,4-GlcNAc-β1,3-Fuc (14Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar). Gene-targeted mutations in murine β4GalT-1 are viable and do not exhibit the defects in Notch signaling observed in Lfng mutants (17Asano M. Furukawa K. Kido M. Matsumoto S. Umesaki Y. Kochibe N. Iwakura Y. EMBO J. 1997; 16: 1850-1857Crossref PubMed Scopus (239) Google Scholar, 18Lu Q. Hasty P. Shur B.D. Dev. Biol. 1997; 181: 257-267Crossref PubMed Scopus (138) Google Scholar), although subtle defects in the expression of Notch pathway targets in developing somites have recently been identified (19Chen J. Lu L. Shi S. Stanley P. Gene Expr. Patterns. 2006; 6: 376-382Crossref PubMed Scopus (30) Google Scholar). The absence of a Lfng phenotype in β4GalT-1 mutant mice might be due to genetic redundancy, because there are six mammalian members of the β4GalT family with similar amino acid sequences that can catalyze the transfer of Gal in a β1,4 linkage to GlcNAc acceptors (20Amado M. Almeida R. Schwientek T. Clausen H. Biochim. Biophys. Acta. 1999; 1473: 35-53Crossref PubMed Scopus (263) Google Scholar, 21Guo S. Sato T. Shirane K. Furukawa K. Glycobiology. 2001; 11: 813-820Crossref PubMed Scopus (70) Google Scholar, 22Furukawa K. Sato T. Biochim. Biophys. Acta. 1999; 1473: 54-66Crossref PubMed Scopus (111) Google Scholar). However, Drosophila encode only two members of the β4GalT family, and animals that are doubly mutant for both genes are viable and do not exhibit fng-like phenotypes (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar). Although these genes actually encode GalNAcTs rather than GalTs (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar), they are highly similar at the amino acid sequence level to mammalian β4GalTs and thus the best candidates to encode any conserved biological functions of this gene family. The absence of fng phenotypes in β4GalNAcT mutants thus raised the possibility that elongation of the GlcNAc-Fuc disaccharide is not actually required for the influence of FNG on Notch signaling, at least in Drosophila. To determine whether any post-translational events subsequent to the addition of GlcNAc by FNG are required for its influence on Notch-ligand binding, we employed an in vitro glycosylation and ligand binding assay. The influence of FNG on Notch-SER and Notch-Delta binding can be reconstituted in vitro with purified components. Our results show that the simple addition of GlcNAc by FNG is sufficient to dramatically alter the interaction of Notch with its ligands, in a manner that suffices to explain the biological effects of fng on Notch signaling in vivo. Conversely, further elongation of the GlcNAc-β1,3-Fuc disaccharide by Gal has no noticeable effect on Notchligand binding in vitro. These observations enhance our mechanistic understanding of the regulation of Notch signaling and confirm a striking example of the direct modulation of protein-protein interactions through glycosylation. Expression Constructs—Notch (N), Delta (DL), and Serrate (SER) constructs were all expressed under the control of the metallothionein promoter. The alkaline phosphatase (AP) fusion proteins N:AP, DL:AP, and SER:AP, cloned into the pRMHa-3 vector (N:AP/pRMHa-3, DL:AP/pRMHa-3, and SER:AP/pRMHa-3), were gifts from S. Cohen and have been described previously (5Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar). N:AP includes amino acids 1-1467 of Notch; DL:AP includes amino acids 1-592 of DL; and SER:AP includes amino acids 1-1213 of SER (see Fig. 1). N-EGF:FLAG includes amino acids 66-1452 of Notch and has been described previously (15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar) (see Fig. 1). Fc:AP includes the Fc domain from human IgG and has been described previously (15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). To facilitate production of larger quantities of DL:AP and SER:AP, these transgenes were cloned from pRMHa-3 into pMT(WB) (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar), which includes the blasticidin resistance gene as a selectable marker for making stable cell lines. The plasmids Delta:AP/pMT(WB) and Serrate:AP/pMT(WB) were constructed by ligating 3.5- or 5.3-kb, respectively, EcoRI-XbaI fragments from Delta-AP/pRmHa3 or Serrate-AP/pRmHa3, respectively (5Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar) into pMT(WB). To facilitate purification of ligand-AP fusion proteins, hexahistidine tags were inserted into DL:AP and SER:AP by amplifying across StuI-XbaI (for DL) or StuI-XhoI (for SER) fragments including the C terminus of AP from Delta:AP/pMT(WB) or Serrate:AP/pMT(WB) using as primers (forward) TGATGTGATCCTAGGTGGAGG and (reverse, Delta) GCTCTAGAGCATGGTGATGGTGATGATGACCCGGGTGCGCGGCGTCGGT or (reverse, Serrate) AACCGCTCGAGGCATGGTGATGGTGATGATGACCCGGGTGCGCGGCGTCGGT (underlined nucleotides encode His6 tag) and cloning into StuI-XbaI-cut Delta:AP/pMT(WB) or StuI-XhoI-cut Serrate:AP/pMT(WB) to create DL:AP:His6/pMT(WB) or SER:AP:His6/pMT(WB), respectively. Full-length, His-tagged FNG was expressed in S2 cells from a pMTHy vector construct that has been described previously (6Moloney 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). Full-length β4GalNAcTA and β4GalNAcTB were expressed from pMT(WB) constructs that have been described previously and are active on a pNp-GlcNAc substrate (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar). Protein Purification—For purification of hexahistidine-tagged proteins, stably transfected S2 cells were cultured to 40 ml in Schneider's Drosophila medium (Invitrogen), and then expression was induced by addition of 0.7 mm CuSO4 for 2 days. The cells were then pelleted by centrifugation, and the conditioned medium was mixed with 50 μl of His-Select nickel affinity gel (Sigma) with gentle agitation on an orbital shaker overnight at 4 °C. The beads were then pelleted by centrifugation at 5000 × g for 5 min and washed three times in 50 mm sodium phosphate, pH 8.0, 0.3 m sodium chloride, 10 mm imidazole. The ligands were eluted from beads in 100 μl of 50 mm sodium phosphate, pH 8.0, 0.3 m sodium chloride, 250 mm imidazole, and the eluate was dialyzed overnight at 4 °C in HBSS (1.26 mm CaCl2, 5.33 mm KCl, 0.44 mm KH2PO4, 0.5 mm MgCl2, 0.41 mm MgSO4, 138 mm NaCl, 4 mm NaHCO3, 0.3 mm Na2HPO4, 5.6 mm glucose). Cell-based Binding Assays—Cell-based binding assays were conducted as described previously (5Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar, 15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), using 4 μg of N:AP DNA, 2 μg each of glycosyltransferase DNA (pMTHy-FNG:His6, pMT(WB)-β4GalNAcTA, or pMT(WB)-β4GalNAcTB), and empty vector DNA (pRMHa-3) to bring the total transfected DNA to 8 μg in all cases. Knockdown of β4GalNAcTA or β4GalNAcTB by RNA interference was performed as described previously (15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), using 40 μg of double-stranded RNA added 6 h after transfection of expression constructs. This procedure reduces but does not eliminate β4GalNAcT expression as monitored by reverse transcription-PCR. The cells were then cultured for 4 days, transgene expression was induced for 2 days using 0.7 mm CuSO4, and conditioned medium was collected, centrifuged 10 min at 14,000 × g to remove cell debris, and assayed for AP activity. In Vitro Binding Assays—In vitro binding assays were performed as described (15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), except that in some assays, affinity-purified ligands were used. Briefly, N-EGF:FLAG (∼1 μg) was loaded onto anti-FLAG beads (Sigma) and incubated with ligand-AP fusion proteins (affinity-purified or in conditioned medium). After washing, binding was quantified by assaying for alkaline phosphatase activity. N-EGF:FLAG was modified by FNG either in vivo (by coexpressing FNG in S2 cells as described above) or in vitro. In vitro glycosylation of N-EGF: FLAG by FNG was conducted as described previously (24Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). For in vitro glycosylation of N-EGF:FLAG by β4GalT-1, anti-FLAG beads (Sigma) loaded with Notch (N-EGF:FLAG or N-EGF:FLAG from cells co-transfected with FNG) or S2 conditioned medium were equilibrated with glycosylation buffer (50 mm Hepes, pH 7.7, 150 mm NaCl, 50 mm MnCl2), and 5 μl of beads were incubated with 100 milliunits of β4GalT-1 (Sigma) and 2.5 μm UDP-Gal (Sigma) for 4 h at 28°C. The beads were then washed four times in HBSS and used for in vitro binding (15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Sugars used in competition binding assays were purchased from Sigma, except for GlcNAc-β1,3-fucose, which was a gift from Dr. Kushi Matta (Roswell Park Memorial Institute, Buffalo, NY). The sugars were premixed with ligand:AP fusion proteins, and these solutions were then mixed with beads. For quantitation of labeling with FNG β4GalNAcTA, or β4GalNAcB, 1 μg of N-EGF:FLAG on anti-FLAG beads was incubated with purified FNG (6Moloney 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), β4GalNAcTA, or β4GalNAcTB (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar), and 3.6 μm UDP-[14C]GlcNAc (266 mCi/mmol, Amersham Biosciences), or 2.5 μm UDP-[14C]GalNAc (266 mCi/mmol, Amersham Biosciences) in 50 μl of glycosylation buffer for 4 h at 28°C.The beads were then washed four times in HBSS and subjected to scintillation counting. The counts on mock loaded (S2 conditioned medium) beads were taken as background. To normalize counts to the amount of labeled sugar, 2 μl of labeled UDP-sugar was counted directly in scintillation fluid. Quantitation of galactose added to GlcNAc on N-EGF:FLAG was performed by in vitro radiolabeling with UDP-[3H]galactose (60 Ci/mmol; American Radiolabeled Chemicals, Inc.) and saturating levels of bovine β4GalT-1 (Sigma) as described (25Haltiwanger R.S. Philipsberg G.A. J. Biol. Chem. 1997; 272: 8752-8758Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Removal of N-glycans by digestion with peptide N-glycosidase F, release of O-glycans by alkali-induced β-elimination, and characterization of the released glycans by gel filtration chromatography on a Superdex peptide column were all performed as described (26Moloney D.J. Lin A.I. Haltiwanger R.S. J. Biol. Chem. 1997; 272: 19046-19050Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Carbohydrate Analysis—Quantitative compositional analysis of carbohydrates was performed by acid hydrolysis and Dionex high pressure anion exchange chromatography as described (27Hardy M.R. Townsend R.R. Methods Enzymol. 1994; 230: 208-225Crossref PubMed Scopus (157) Google Scholar). N-EGF:FLAG was eluted from anti-FLAG beads using 3× FLAG peptide (Sigma) by mixing 0.5-ml beads with 0.5 ml of 3× FLAG peptide (150 ng/ml in Tris-buffered saline) and incubating 30 min at 4 °C. The beads were then pelleted for 30 s at 8200 × g, and the supernatant was dialyzed in HBSS to remove 3× FLAG peptide. Coomassie staining of SDS-PAGE gels (not shown) confirmed that N-EGF:FLAG was purified to apparent homogeneity. Approximately 10 μg of N-EGF:FLAG purified from S2 cells with or without exogenous FNG was concentrated by acetone precipitation and hydrolyzed in 2 m trifluoroacetic acid (Pierce) for 4 h at 100°C. The samples were dried in a SpeedVac, resuspended in water, passed through a C18 ZipTip (Millipore), and dried again. The pellets were dissolved in 100 μl of water and analyzed by high pH anion exchange chromatography on a CarboPac PA-1 column (20 μl/run) on a Dionex DX-300 system with pulsed amperometric detection. All of the experiments were performed in duplicate. Mass spectral analysis of O-fucose glycosylation sites was performed essentially as described (28Nita-Lazar A. Haltiwanger R.S. Methods Enzymol. 2006; 417: 93-111Crossref PubMed Scopus (18) Google Scholar). Briefly, ∼1 μg of N-EGF:FLAG expressed in S2 cells with exogenous FNG was reduced and alkylated, separated by SDS-PAGE, and subjected to in-gel tryptic digestion. The resulting peptides were analyzed by LC-MS/MS on an Agilent XCT Ion Trap mass spectrometer. Glycosylated peptides were identified by searching MS/MS data for neutral losses of the GlcNAc-fucose disaccharide (349.1 Da). Loss of the disaccharide gave a characteristic fragmentation pattern allowing rapid identification of glycopeptides (see Fig. 6). The mass of the unglycosylated peptide was then matched to predicted masses of tryptic peptides from Notch containing the O-fucose consensus sequence:C2XXXX(S/T)C3, where C2 and C3 are the second and third conserved cysteines of an EGF domain (29Shao L. Moloney D.J. Haltiwanger R. J. Biol. Chem. 2003; 278: 7775-7782Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) (see Table 1). Once glycopeptides were identified, additional searches of the MS/MS data for the unmodified peptides were performed (Extracted Ion Searches). The extracted ion searches take advantage of the fact that glycosidic linkages are more labile than peptide bonds upon collision-induced dissociation (CID), and hence the major product ion from fragmentation of a glycopeptide is the unglycosylated peptide. Using this method, peptides bearing different forms of the O-fucose glycans (e.g. mono- or disaccharide) were found (see Fig. 6, Table 1, and supplemental Fig. S3). No glycopeptides with tri- or tetrasaccharide forms of O-fucose were identified, nor, aside from O-glucose glycans (to be reported elsewhere) were any other modifications of O-fucose bearing peptides identified.TABLE 1O-Fucosylated peptides from N-EGF:FLAG produced in S2 cells with FNGEGFSequenceParent ion (M+H+)Deglycosylated product (M+H+)Mass ΔPredicted mass (M+H+)373CPLGFDESLCEIAVPNACDHVTCLNGGTCQLK1043986.23636.7349.53638.1373CPLGFDESLCEIAVPNACDHVTCLNGGTCQLK1043782.23636.7145.53638.15175YGGTCVNTHGSYQCMCPTGYTGK1972949.12600.5348.62600.97236NCEQNYDDCLGHLCQNGGTCIDGISDYTCR2653945.43597.7347.73597.823866NGASCLNVPGSYR8781744.81396.0348.81395.523866NGASCLNVPGSYR8781541.81396.0145.81395.5 Open table in a new tab fng null mutant flies die as first instar larvae, whereas hypomorphic alleles are viable but exhibit defects in wing and eye development (30Irvine K.D. Wieschaus E. Cell. 1994; 79: 595-606Abstract Full Text PDF PubMed Scopus (306) Google Scholar, 31Correia T. Papayannopoulos V. Panin V. Woronoff P. Jiang J. Vogt T.F. Irvine K.D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6404-6409Crossref PubMed Scopus (42) Google Scholar, 32Papayannopoulos V. Tomlinson A. Panin V.M. Rauskolb C. Irvine K.D. Science. 1998; 281: 2031-2034Crossref PubMed Scopus (194) Google Scholar). By contrast, flies doubly mutant for β4GalNAcTA and β4GalNAcTB are viable and appear morphologically normal (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar). Because these are the only Drosophila homologues of mammalian β4GalTs that act on GlcNAc acceptors, the absence of developmental phenotypes indicated that this gene family does not influence Notch signaling during Drosophila development and suggested that further elongation of the GlcNAcβ1,3-Fuc disaccharide might not be required for modulation of Notch signaling in Drosophila. Nonetheless, we examined the influence of these glycosyltransferases in Notchligand binding assays. A secreted fragment of Notch including most of the extracellular domain fused to alkaline phosphatase (N:AP, Fig. 1) can bind specifically to ligand-expressing cells. This assay has been used previously to demonstrate that FNG can influence Notch-ligand binding (5Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (597) Google Scholar, 15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). To examine their potential influence, we overexpressed β4GalNAcTA or β4GalNAcTB in cells producing N:AP and FNG or down-regulated their expression by RNA interference. However, neither elevated nor decreased expression of these glycosyltransferases detectably influenced Notch-ligand binding (Fig. 2), consistent with the absence of Notch or fng phenotypes in β4GalNAcTA and β4GalNAcTB null mutant animals (23Haines N. Irvine K.D. Glycobiology. 2005; 15: 335-346Crossref PubMed Scopus (61) Google Scholar). The Influence of Fringe Is Detectable in an in Vitro Binding System—The above observations argued against the possibility that β4GalNAcTA or β4GalNAcTB are required for the modulation of Notch signaling by FNG but left open the possibility that some other as yet unidentified glycosyltransferases effect a functionally important modification of GlcNAc on Notch, be it addition of Gal or some other sugar. Moreover, prior studies investigating the influence of FNG on Notch-ligand binding have all relied on in vivo glycosylation by FNG and have been conducted with conditioned medium rather than purified proteins. Thus, they could not address the potential importance of modifications subsequent to glycosylation or of additional, accessory factors in FNG-dependent modulation of Notch signaling. We established an in vitro binding assay using a Notch construct (N-EGF:FLAG; Fig. 1) comprising all 36 EGF domains of Drosophila Notch fused to a triple FLAG epitope tag (15Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). This Notch polypeptide was then purified on anti-FLAG agarose beads, and the beads were mixed with ligands fused to AP (Fig. 1). This in vitro binding system provides a means for determining whether or not additional factors or modifications are required for the influence of FNG on Notch. Importantly, both the positive modulation of Notch-Delta binding and the negative modulation of Notch-Serrate can be detected with this assay when N-EGF:FLAG expressed in the presence of FNG is compared with N:EGF-FLAG expressed in the absence of FNG (Fig. 3A). Thus, the influence of FNG can be detected using an in vitro binding assay. The GlcNAc-Fuc Disaccharide Is Sufficient to Modulate Notch-Ligand Binding—To evaluate the potential significance of post-translational modifications subsequent to FNG, we modified this in vitro assay by first purifying N-EGF:FLAG expressed from S2 cells without FNG and then glycosylating it in vitro with FNG. When N-EGF:FLAG is purified on anti-FLAG beads, only a single prominent band is detected on Coomassie-stained gels (supplemental Fig. S1A). Similarly, when Fringe:His6 was purified using agarose-Ni2+ beads, only a single prominent band was detected by Coomassie staining (supplemental Fig. S1B). We then conducted in vitro glycosylation experiments, using N-EGF:FLAG attached to beads, soluble Fringe:His6, and UDP-[14C]GlcNAc. The addition of ∼4.5 mol of GlcNAc/mol of N-EGF:FLAG was catalyzed by FNG in this reaction. Although there are 23 potential sites of O-fucosylation on Drosophila Notch, it is not known whether all of them can be modified by FNG. In addition, because FNG requires a properly folded EGF domain (29Shao L. Moloney D.J. Haltiwanger R. J. Biol. Chem. 2003; 278: 7775-7782Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), if any Notch on the beads was misfolded or aggregated, the calculated value of ∼5 sites/Notch could be an underestimate. Regardless, these results suggest that purified Notch can be s" @default.
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• W1986322003 date "2007-11-01" @default.
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• W1986322003 title "In Vitro Reconstitution of the Modulation of Drosophila Notch-Ligand Binding by Fringe" @default.
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