Title: Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling
Abstract: Mammalian Notch receptors contain 29–36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1–/– ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1–/– cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose. Mammalian Notch receptors contain 29–36 epidermal growth factor (EGF)-like repeats that may be modified by protein O-fucosyltransferase 1 (Pofut1), an essential component of the canonical Notch signaling pathway. The Drosophila orthologue Ofut1 is proposed to function as both a chaperone required for stable cell surface expression of Notch and a protein O-fucosyltransferase. Here we investigate these dual roles of Pofut1 in relation to endogenous Notch receptors of Chinese hamster ovary and murine embryonic stem (ES) cells. We show that fucosylation-deficient Lec13 Chinese hamster ovary cells have wild type levels of Pofut1 and cell surface Notch receptors. Nevertheless, they have reduced binding of Notch ligands and low levels of Delta1- and Jagged1-induced Notch signaling. Exogenous fucose but not galactose rescues both ligand binding and Notch signaling. Murine ES cells lacking Pofut1 also have wild type levels of cell surface Notch receptors. However, Pofut1–/– ES cells do not bind Notch ligands or exhibit Notch signaling. Although overexpression of fucosyltransferase-defective Pofut1 R245A in Pofut1–/– cells partially rescues ligand binding and Notch signaling, this effect is not specific. The same rescue is achieved by an unrelated, inactive, endoplasmic reticulum glucosidase. Therefore, mammalian Notch receptors require Pofut1 for the generation of optimally functional Notch receptors, but, in contrast to Drosophila, Pofut1 is not required for stable cell surface expression of Notch. Importantly, we also show that, under certain circumstances, mammalian Notch receptors are capable of signaling in the absence of Pofut1 and O-fucose. Notch signaling controls growth and determines cell fate in the metazoa through direct cell-cell contact (1Bray S.J. Nat. Rev. Mol. Cell Biol. 2006; 7: 678-689Crossref PubMed Scopus (1898) Google Scholar, 2Stanley P. Curr. Opin. Struct. Biol. 2007; 17: 530-535Crossref PubMed Scopus (121) Google Scholar). The four mammalian Notch receptors are single pass transmembrane glycoproteins, whose extracellular domains (NECDs) 8The abbreviations used are: NECD, Notch extracellular domain; PE, phycoerythrin; EGF, epidermal growth factor; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; CHO, Chinese hamster ovary; ES, embryonic stem; MFI, mean fluorescence intensity; siRNA, small interfering RNA; MES, 4-morpholineethanesulfonic acid; RNAi, RNA interference; Ab, antibody. 8The abbreviations used are: NECD, Notch extracellular domain; PE, phycoerythrin; EGF, epidermal growth factor; BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid; DAPT, N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester; CHO, Chinese hamster ovary; ES, embryonic stem; MFI, mean fluorescence intensity; siRNA, small interfering RNA; MES, 4-morpholineethanesulfonic acid; RNAi, RNA interference; Ab, antibody. contain 29–36 tandemly organized N-terminal epidermal growth factor (EGF)-like repeats. Interaction of Notch receptors with canonical Delta or Serrate/Jagged Notch ligands expressed on neighboring cells triggers regulated intramembrane proteolytic processing that releases Notch intracellular domain (3Mumm J.S. Kopan R. Dev. Biol. 2000; 228: 151-165Crossref PubMed Scopus (831) Google Scholar). Upon translocation to the nucleus, Notch intracellular domain binds to CSL (CBF1/Su(H)/Lag-1), a transcriptional repressor that recruits the co-activator Mastermind, and the complex activates the expression of Notch target genes (4Mumm J.S. Schroeter E.H. Saxena M.T. Griesemer A. Tian X. Pan D.J. Ray W.J. Kopan R. Mol. Cell. 2000; 5: 197-206Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 5Kopan R. J. Cell Sci. 2002; 115: 1095-1097Crossref PubMed Google Scholar, 6Wu L. Griffin J.D. Semin. Cancer Biol. 2004; 14: 348-356Crossref PubMed Scopus (76) Google Scholar). Numerous modulators of the canonical Notch signaling pathway have been identified, most of which act intracellularly. The discovery that Fringe, a well established modifier of Notch signaling (7Panin V.M. Papayannopoulos V. Wilson R. Irvine K.D. Nature. 1997; 387: 908-912Crossref PubMed Scopus (505) Google Scholar), is a glycosyltransferase (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 (713) Google Scholar, 9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (588) Google Scholar) revealed that O-fucose glycans on the extracellular domain of Notch also regulate Notch signaling. EGF domains with a C2X4–5(S/T)C3 consensus are substrates for protein O-fucosyltransferase 1 (Pofut1) (10Rampal R. Luther K.B. Haltiwanger R.S. Curr. Mol. Med. 2007; 7: 427-445Crossref PubMed Scopus (76) Google Scholar). Pofut1 transfers O-fucose to Notch EGF repeats (11Wang Y. Shao L. Shi S. Harris R.J. Spellman M.W. Stanley P. Haltiwanger R.S. J. Biol. Chem. 2001; 276: 40338-40345Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar) and thereby generates the substrate of Fringe. The removal of Pofut1 leads to global Notch signaling defects during embryonic development in Drosophila (12Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 13Sasamura T. Sasaki N. Miyashita F. Nakao S. Ishikawa H.O. Ito M. Kitagawa M. Harigaya K. Spana E. Bilder D. Perrimon N. Matsuno K. Development. 2003; 130: 4785-4795Crossref PubMed Scopus (145) Google Scholar) and the mouse (14Shi S. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 5234-5239Crossref PubMed Scopus (316) Google Scholar). Reduced GDP-fucose in Lec13 Chinese hamster ovary (CHO) cells (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 (713) Google Scholar, 15Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar) or the wing disc (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 17Sasamura T. Ishikawa H.O. Sasaki N. Higashi S. Kanai M. Nakao S. Ayukawa T. Aigaki T. Noda K. Miyoshi E. Taniguchi N. Matsuno K. Development. 2007; 134: 1347-1356Crossref PubMed Scopus (68) Google Scholar, 18Okajima T. Reddy B. Matsuda T. Irvine K.D. BMC Biol. 2008; 6: 1Crossref PubMed Scopus (76) Google Scholar) also results in Notch signaling defects. However, in mice, the inability to synthesize GDP-fucose is not manifested until after birth due to maternal rescue effects and results in failure to thrive (19Smith P.L. Myers J.T. Rogers C.E. Zhou L. Petryniak B. Becker D.J. Homeister J.W. Lowe J.B. J. Cell Biol. 2002; 158: 801-815Crossref PubMed Scopus (126) Google Scholar). The absence of the Golgi GDP-fucose transporter Slc35c1 in mice results in a leukocyte adhesion deficiency (20Hellbusch C.C. Sperandio M. Frommhold D. Yakubenia S. Wild M.K. Popovici D. Vestweber D. Grone H.J. von Figura K. Lubke T. Korner C. J. Biol. Chem. 2007; 282: 10762-10772Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), and in Drosophila, mutation of the orthologous gene leads to mild, temperature-sensitive Notch signaling defects (21Ishikawa H.O. Higashi S. Ayukawa T. Sasamura T. Kitagawa M. Harigaya K. Aoki K. Ishida N. Sanai Y. Matsuno K. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 18532-18537Crossref PubMed Scopus (54) Google Scholar). There may be another GDP-fucose transporter Slc35c2 (22Ishida N. Kawakita M. Pflugers Arch. 2004; 447: 768-775Crossref PubMed Scopus (127) Google Scholar), since the complete absence of GDP-fucose transport should lead to severe Notch signaling defects and embryonic lethality. The mechanism by which O-fucose glycans modulate the level of Notch signaling appears, at least in part, to be by regulating ligand binding in mammals (23Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (333) Google Scholar, 24Shimizu 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, 25Shimizu K. Chiba S. Kumano K. Hosoya N. Takahashi T. Kanda Y. Hamada Y. Yazaki Y. Hirai H. J. Biol. Chem. 1999; 274: 32961-32969Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar, 26Wang S. Sdrulla A.D. diSibio G. Bush G. Nofziger D. Hicks C. Weinmaster G. Barres B.A. Neuron. 1998; 21: 63-75Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar) and Drosophila (9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (588) Google Scholar, 13Sasamura T. Sasaki N. Miyashita F. Nakao S. Ishikawa H.O. Ito M. Kitagawa M. Harigaya K. Spana E. Bilder D. Perrimon N. Matsuno K. Development. 2003; 130: 4785-4795Crossref PubMed Scopus (145) Google Scholar, 27Lei L. Xu A. Panin V.M. Irvine K.D. Development. 2003; 130: 6411-6421Crossref PubMed Scopus (77) Google Scholar, 28Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar, 29Xu A. Haines N. Dlugosz M. Rana N.A. Takeuchi H. Haltiwanger R.S. Irvine K.D. J. Biol. Chem. 2007; 282: 35153-35162Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Either direct recognition of different O-fucose glycans on EGF repeats by Notch ligands and/or glycan-induced changes in binding strength between ligands and NECD (30Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Mol. Biol. Cell. 2005; 16: 927-942Crossref PubMed Scopus (167) Google Scholar, 31Nichols J.T. Miyamoto A. Olsen S.L. D'Souza B. Yao C. Weinmaster G. J. Cell Biol. 2007; 176: 445-458Crossref PubMed Scopus (188) Google Scholar) may affect the initiation of downstream events leading to Notch receptor proteolysis. Although it is clear that O-fucose on Notch is an essential substrate of Fringe (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 (713) Google Scholar, 9Bruckner K. Perez L. Clausen H. Cohen S. Nature. 2000; 406: 411-415Crossref PubMed Scopus (588) Google Scholar), Fringe-independent Notch signaling defects are observed in Drosophila Ofut1 mutants (12Okajima T. Irvine K.D. Cell. 2002; 111: 893-904Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 13Sasamura T. Sasaki N. Miyashita F. Nakao S. Ishikawa H.O. Ito M. Kitagawa M. Harigaya K. Spana E. Bilder D. Perrimon N. Matsuno K. Development. 2003; 130: 4785-4795Crossref PubMed Scopus (145) Google Scholar), indicative of signaling functions that are regulated solely by O-fucose on Notch. This is consistent with the reduced Jagged1-induced Notch signaling observed in Lec13 CHO cells that have very low GDP-fucose (32Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar, 33Sullivan F.X. Kumar R. Kriz R. Stahl M. Xu G.Y. Rouse J. Chang X.J. Boodhoo A. Potvin B. Cumming D.A. J. Biol. Chem. 1998; 273: 8193-8202Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 34Ohyama C. Smith P.L. Angata K. Fukuda M.N. Lowe J.B. Fukuda M. J. Biol. Chem. 1998; 273: 14582-14587Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 35Kanda Y. Imai-Nishiya H. Kuni-Kamochi R. Mori K. Inoue M. Kitajima-Miyama K. Okazaki A. Iida S. Shitara K. Satoh M. J. Biotechnol. 2007; 130: 300-310Crossref PubMed Scopus (85) Google Scholar) and little Fringe activity (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 (713) Google Scholar, 15Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar) and with rescue of Notch signaling by exogenous fucose or genetic complementation of the Lec13 fucosylation defect (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 (713) Google Scholar, 15Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar). Knockdown of Ofut1 in Drosophila S2 cells causes soluble Notch extracellular domain to be secreted poorly and to have little Notch ligand binding activity (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 28Okajima T. Xu A. Irvine K.D. J. Biol. Chem. 2003; 278: 42340-42345Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). In addition, full-length Notch-expressing cells targeted for Ofut1 knockdown do not form aggregates with Delta-expressing S2 cells (36Ahimou F. Mok L.P. Bardot B. Wesley C. J. Cell Biol. 2004; 167: 1217-1229Crossref PubMed Scopus (70) Google Scholar). However, there are conflicting reports as to whether Notch is expressed at the surface of S2 cells targeted for Ofut1 (13Sasamura T. Sasaki N. Miyashita F. Nakao S. Ishikawa H.O. Ito M. Kitagawa M. Harigaya K. Spana E. Bilder D. Perrimon N. Matsuno K. Development. 2003; 130: 4785-4795Crossref PubMed Scopus (145) Google Scholar, 16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar). In vivo, Notch may be transiently observed at the surface of Drosophila epithelial cells lacking Ofut1 (37Sasaki N. Sasamura T. Ishikawa H.O. Kanai M. Ueda R. Saigo K. Matsuno K. Genes Cells. 2007; 12: 89-103Crossref PubMed Scopus (60) Google Scholar). However, there is a marked intracellular accumulation of Notch in Ofut1– mutant clones (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 17Sasamura T. Ishikawa H.O. Sasaki N. Higashi S. Kanai M. Nakao S. Ayukawa T. Aigaki T. Noda K. Miyoshi E. Taniguchi N. Matsuno K. Development. 2007; 134: 1347-1356Crossref PubMed Scopus (68) Google Scholar) that has been localized to the endoplasmic reticulum by Okajima et al. (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 18Okajima T. Reddy B. Matsuda T. Irvine K.D. BMC Biol. 2008; 6: 1Crossref PubMed Scopus (76) Google Scholar) and to novel endocytic vesicles by Sasaki et al. (37Sasaki N. Sasamura T. Ishikawa H.O. Kanai M. Ueda R. Saigo K. Matsuno K. Genes Cells. 2007; 12: 89-103Crossref PubMed Scopus (60) Google Scholar). However, there are technical concerns with the latter conclusion (38Vodovar N. Schweisguth F. J. Biol. 2008; 7: 7Crossref PubMed Scopus (20) Google Scholar). Drosophila Ofut1 binds to Notch when both are overexpressed in S2 cells (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 17Sasamura T. Ishikawa H.O. Sasaki N. Higashi S. Kanai M. Nakao S. Ayukawa T. Aigaki T. Noda K. Miyoshi E. Taniguchi N. Matsuno K. Development. 2007; 134: 1347-1356Crossref PubMed Scopus (68) Google Scholar), and Ofut1 facilitates soluble NECD secretion and ligand binding to S2 cells (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 29Xu A. Haines N. Dlugosz M. Rana N.A. Takeuchi H. Haltiwanger R.S. Irvine K.D. J. Biol. Chem. 2007; 282: 35153-35162Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The secretion and folding chaperone functions of Ofut1 are retained by a fucosyltransferase-defective mutant Pofut1/Ofut1 R245A (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar, 18Okajima T. Reddy B. Matsuda T. Irvine K.D. BMC Biol. 2008; 6: 1Crossref PubMed Scopus (76) Google Scholar). While this manuscript was in revision, Drosophila Notch synthesized in Ofut1– clones expressing Ofut1 R245A was shown to transduce a Notch signal (18Okajima T. Reddy B. Matsuda T. Irvine K.D. BMC Biol. 2008; 6: 1Crossref PubMed Scopus (76) Google Scholar). In this paper, we investigate the relative roles of Pofut1 and O-fucose in mediating the cell surface expression of mammalian Notch receptors as well as their abilities to bind Notch ligands and to transduce a Notch signal. We show that in murine embryonic stem (ES) cells or CHO cells, Pofut1 is not required for stable cell surface expression of mammalian Notch receptors. We also show that, under certain conditions, mammalian Notch receptors can bind Notch ligands and transduce a Notch signal in the absence of Pofut1 and O-fucose. However, active Pofut1 and O-fucosylation of Notch are required for optimal ligand binding and canonical Notch signaling induced by Delta1 or Jagged1. Cells and Cell Culture—CHO cells were cultured in α-modified minimal essential medium (Invitrogen) supplemented with 10% fetal bovine serum (Gemini, West Sacramento, CA) unless otherwise indicated. Pofut1+/+ or Pofut1–/– ES cell lines derived from blastocyst outgrowths obtained from mating Pofut1+/– heterozygotes as described (39Shi S. Stahl M. Lu L. Stanley P. Mol. Cell. Biol. 2005; 25: 9503-9508Crossref PubMed Scopus (50) Google Scholar) and Notch1 null ES cells (line 290-2) kindly provided by Dr. Gregory Longmore (40Hadland B.K. Huppert S.S. Kanungo J. Xue Y. Jiang R. Gridley T. Conlon R.A. Cheng A.M. Kopan R. Longmore G.D. Blood. 2004; 104: 3097-3105Crossref PubMed Scopus (183) Google Scholar) were cultured on feeder-free gelatinized plates with ES cell culture medium (α-modified minimal essential medium, 10% ES-qualified fetal bovine serum (Gemini), 1000 units/ml leukemia inhibitory factor (Chemicon, Temecula, CA), penicillin and streptomycin (Invitrogen), 50 mm β-mercaptoethanol (Sigma)). Pofut1–/– ES cells do not transfer O-fucose to EGF repeats (41Shi S. Ge C. Luo Y. Hou X. Haltiwanger R.S. Stanley P. J. Biol. Chem. 2007; 282: 20133-20141Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Pro–5Lec1.3C CHO cells (42Chen W. Stanley P. Glycobiology. 2003; 13: 43-50Crossref PubMed Scopus (92) Google Scholar) with the vector pMIRB are equivalent to parent CHO in Notch signaling activity (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 (713) Google Scholar, 15Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar) and are referred to as Lec1. Lec13.6A CHO cells were previously characterized as fucosylation-defective (32Ripka J. Adamany A. Stanley P. Arch. Biochem. Biophys. 1986; 249: 533-545Crossref PubMed Scopus (69) Google Scholar, 33Sullivan F.X. Kumar R. Kriz R. Stahl M. Xu G.Y. Rouse J. Chang X.J. Boodhoo A. Potvin B. Cumming D.A. J. Biol. Chem. 1998; 273: 8193-8202Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 34Ohyama C. Smith P.L. Angata K. Fukuda M.N. Lowe J.B. Fukuda M. J. Biol. Chem. 1998; 273: 14582-14587Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 35Kanda Y. Imai-Nishiya H. Kuni-Kamochi R. Mori K. Inoue M. Kitajima-Miyama K. Okazaki A. Iida S. Shitara K. Satoh M. J. Biotechnol. 2007; 130: 300-310Crossref PubMed Scopus (85) Google Scholar) and are referred to as Lec13. For maximum expression of the Lec13 phenotype, Lec13 cells were cultured in α-modified minimal essential medium and 10% dialyzed fetal bovine serum (Gemini). Lec1 cells expressing Lunatic fringe (Lfng) were previously described (15Chen J. Moloney D.J. Stanley P. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13716-13721Crossref PubMed Scopus (129) Google Scholar). Ligand cells for co-culture were L cells expressing rat Jagged1 (43Lindsell C.E. Shawber C.J. Boulter J. Weinmaster G. Cell. 1995; 80: 909-917Abstract Full Text PDF PubMed Scopus (538) Google Scholar) and sorted for high Jagged1 expression using goat anti-rat Jagged1 antibody AF599 (R & D Systems, Minneapolis, MN); L cells expressing rat Delta1 (23Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (333) Google Scholar) were sorted for high Delta1 expression using goat anti-human Delta1 antibody AF 1818 (R & D Systems); and control L cells were sorted for low Jagged1 expression and shown to lack Delta1 expression. Preparation of Delta1 and Jagged1 Notch Ligands—An expression construct encoding rat Delta1-Fc kindly provided by Dr. Gerry Weinmaster (23Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (333) Google Scholar), was stably expressed in HEK293T cells, and a population producing ∼10–16 μg/ml Delta1-Fc was isolated. The expression construct for rat Jagged1-Fc also from Dr. Gerry Weinmaster (23Hicks C. Johnston S.H. diSibio G. Collazo A. Vogt T.F. Weinmaster G. Nat. Cell Biol. 2000; 2: 515-520Crossref PubMed Scopus (333) Google Scholar) was inserted into pIRES2-EGFP (Clontech). After stable expression in HEK293T cells, a population producing ∼6 μg/ml was isolated by sequential enrichment using flow cytometry sorting. For ligand preparation, HEK293T ligand-expressing cells were cultured in suspension using Pro293a serum-free medium (Cambrex Bio Science, Rockland, ME). Culture supernatants were assayed by Western blot using horseradish peroxidase-conjugated anti-human IgG (Zymed Laboratories Inc., South San Francisco, CA). Each ligand gave a single band with apparent molecular masses of ∼82 kDa (Delta1-Fc) and ∼180 kDa (Jagged1-Fc), respectively. Concentrations of Delta1-Fc and Jagged1-Fc in medium were estimated by Western blot densitometry compared with an IgG standard using NIH Image software. After sterile filtering, ligands were stable for several months at 4 °C. Notch1 ECD Fragment Preparation—Notch1 ECD fragment was produced in Lec1 cells stably transfected with mammalian expression construct pSectag mNotchEGF-(1–18)(wt)MycHis6 kindly provided by Dr. Robert Haltiwanger (44Shao L. Moloney D.J. Haltiwanger R. J. Biol. Chem. 2003; 278: 7775-7782Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar), cultured for ∼96 h in Opti-MEM I reduced serum medium (Invitrogen) supplemented with 1 mm CaCl2, penicillin, and streptomycin. The medium was clarified by centrifugation, and the supernatant was rotated with Ni2+-nitrilotriacetic acid-agarose (Qiagen, Hilden, Germany) at pH 8.0 overnight at 4 °C (750 ml of medium, 5 ml of resin). After washing, the fragment was eluted with 500 mm imidazole. Silver staining and Western blot analysis identified fractions containing the highest amount of Notch1EGF-(1–18) which was dialyzed against fragment buffer (20 mm Tris, 150 mm NaCl, 1 mm CaCl2, pH 7.4), concentrated using a Centricon YM-50 filter (50 kDa; Millipore, Billerica, MA), and stored at –80 °C. Notch Ligand and Antibody Binding Assays by Flow Cytometry—CHO cells growing in suspension were harvested by centrifugation, washed, and resuspended in ligand binding buffer (Hanks' balanced salt solution (Mediatech Inc., Herndon, VA) to which 1 mm CaCl2, 1% bovine serum albumin, and 0.05% NaN3, pH 7.4, were added). In experiments involving transient transfection prior to the binding assay, CHO cells were grown in monolayer, and ES cells were grown under feeder-free conditions on gelatinized 100-mm plates. ES cells were transfected with 3.5 μg of mouse Pofut1 cDNA (39Shi S. Stahl M. Lu L. Stanley P. Mol. Cell. Biol. 2005; 25: 9503-9508Crossref PubMed Scopus (50) Google Scholar), a mouse Pofut1 cDNA generated by site-directed mutagenesis to contain the point mutation R245A (16Okajima T. Xu A. Lei L. Irvine K.D. Science. 2005; 307: 1599-1603Crossref PubMed Scopus (201) Google Scholar), an α-glucosidase I cDNA containing the S440F point mutation (45Hong Y. Sundaram S. Shin D.J. Stanley P. J. Biol. Chem. 2004; 279: 49894-49901Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), or vector alone (pcDNA3.1; Invitrogen) using Lipofectamine 2000 (Invitrogen). Lec13 CHO cells were grown in 6-well plates and transfected using FuGENE 6 (Roche Applied Science) with 1 μgofa mouse Notch1 cDNA or empty vector (pCS2) kindly provided by Dr. Raphael Kopan. At 30–36 h post-transfection, cells were dissociated from plates using phosphate-buffered saline-based enzyme-free cell dissociation solution (Chemicon, Temecula, CA) to preserve the integrity of cell surface proteins. Cells (5 × 105) were incubated with soluble Notch ligands (Delta1-Fc or Jagged1-Fc) or anti-NECD antibodies in 0.2 ml of ligand binding buffer for 30–60 min at room temperature or at 4 °C with gentle rotation. Sodium azide blocked endocytosis that may occur at room temperature. Alternatively, cells were washed and then fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min at room temperature before adding primary antibody. After washing with binding buffer three times, cells were incubated with secondary antibody in binding buffer for 30 min at the binding temperature. Cells were washed with 1 ml of binding buffer three times, 1 μg/ml propidium iodide or 7-amino-actinomycin D (BD Pharmingen, San Diego, CA) was added, and the cells were subjected to flow cytometric analysis using a FACScan or FACSCalibur (BD Biosciences, San Jose, CA) instrument. Fluorescence-activated cell sorting data were analyzed using FlowJo software (Tree Star Inc., Ashland, OR) and are presented as profiles obtained after exclusion of dead or damaged cells that took up propidium iodide or 7-amino-actinomycin D. For Notch1 fragment inhibition studies, ligand binding was carried out as above, except that soluble Notch ligand was preincubated for 30 min at room temperature with Notch1 EGF-(1–18) fragment (100 μg) before mixing with cells. An equal amount of fragment buffer (20 mm Tris, 150 mm NaCl, 1 mm CaCl2, pH 7.4) was added to controls that did not contain the Notch1 fragment. Notch ECD antibodies and dilutions were as follows: hamster anti-Notch1 (1:10; clone 8G10; Upstate Biotechnology, Inc., Lake Placid, NY); rabbit anti-Notch2 (1:100; sc-5545 against amino acids 25–255 human NOTCH2; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); goat anti-Notch3 ECD (1:50–100; AF1308; R&D Systems, Minneapolis, MN); monoclonal antibody 5E1 against human NOTCH3 ECD (1:20 culture supernatant (46Joutel A. Andreux F. Gaulis S. Domenga V. Cecillon M. Battail N. Piga N. Chapon F. Godfrain C. Tournier-Lasserve E. J. Clin. Invest. 2000; 105: 597-605Crossref PubMed Scopus (453) Google Scholar)); goat anti-Notch4 ECD (1:100; clone L-16; sc-8645; Santa Cruz Biotechnology). Secondary antibodies were R-phycoerythrin (PE)-conjugated goat anti-human IgG F(ab′)2 fragment (Jackson ImmunoResearch, West Grove, PA), anti-hamster-Alexa488 (Molecular Probes, Inc., Carlsbad, CA), anti-rabbit IgG-PE, anti-goat IgG-PE, or anti-mouse IgG-PE (all from Jackson ImmunoResearch and used at 1:100 or a lower dilution). Fixed and Intracellular Notch Receptors—ES cells growing on gelatinized plates in ES cell medium were collected by enzyme-free cell dissociation solution (Chemicon) and washed in ligand binding buffer. To examine binding of anti-Notch3 antibodies to fixed and permeabilized ES cells, washed cells (1 × 106) were fixed with Fix & Perm Reagent A (Fixation Medium; Caltag Laboratories, Burlingame, CA) for 15 min at room temperature, centrifuged, resuspended in 1 ml of binding buffer, and divided into two aliquots. After centrifugation, half the cells were incubated in 100 μl of Reagent B (Permeabilization Medium), and the other half were incubated in 100 μl of binding buffer for 30 min at room temperature. After centrifugation, cells were resuspended in 100 μl of binding buffer or 100 μl of binding buffer containing primary anti-Notch3 antibody (1:20 clone 5E1 (46Joutel A. Andreux F. Gaulis S. Domenga V. Cecillon M. Battail N. Piga N. Chapon F. Godfrain C. Tournier-Lasserve E. J.