Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

Stahl, Mark; Uemura, Kazuhide; Ge, Changrong; Shi, Shaolin; Tashima, Yohtalou; Stanley, Pamela

doi:10.1074/jbc.m802027200

Cited by 158 publications

(187 citation statements)

References 69 publications

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“…LADII patient and control human fibroblasts were kindly provided by Robert Haltiwanger (SUNY Stonybrook) (29), rat intestinal epithelial cells (IEC) cells were kindly provided by Len Augenlicht (Albert Einstein College of Medicine), and Slc35c1 Ϫ/Ϫ and Slc35c1 ϩ/ϩ mouse embryonic fibroblasts (MEF) prepared from mouse embryos (27) were cultured at 37°C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% FBS (Gemini), penicillin, streptomycin, and glutamine (Millipore, Billerica, MA). L cells expressing Delta1 or Jagged1 were sorted for high ligand expression as described previously (14). Plasmids-pCMV6-Kan/Neo-mSlc35c1 and pCMV6-Kan/ Neo-mSlc35c2 were purchased from OriGene Technologies, Inc. (Rockville, MD).…”

Section: Cells and Cell Culture-parental Cho Cells Promentioning

confidence: 99%

“…Ligand-dependent Notch Signaling Assays-Notch signaling co-culture assays were performed as previously described (13,14,40). Briefly, duplicate cell cultures were plated at 2 ϫ 10 5 cells per well of a six-well dish and, after ϳ24 h, were cotransfected using FuGENE 6 (Roche Applied Science) with 0.2 g of the Notch reporter plasmid TP-1 that carries eight copies of a RBP-J DNA binding sequence driving a firefly luciferase reporter gene (41), 0.05 g of pRL-TK, or 0.01 g of pRL-CMV Renilla luciferase (Promega, Madison, WI), and 1.5 g of pMIRB empty vector.…”

Section: Cells and Cell Culture-parental Cho Cells Promentioning

confidence: 99%

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Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Hou

Shi

et al. 2010

Journal of Biological Chemistry

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Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1-and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.

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Section: Cells and Cell Culture-parental Cho Cells Promentioning

confidence: 99%

Section: Cells and Cell Culture-parental Cho Cells Promentioning

confidence: 99%

Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Hou

Shi

et al. 2010

Journal of Biological Chemistry

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“…The molecular mechanism for the effects of these sugars on Notch activity is not yet clear. The presence of O-fucose can enhance ligand binding by mammalian Notch receptors (22,23), whereas O-glucose does not appear to affect ligand binding but a subsequent step before receptor cleavage (20,21).…”

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confidence: 99%

“…The simplest case appears in flies, where Fringe modification has a positive effect on Delta-mediated signaling and a negative effect on Serrate-mediated signaling. Prior studies have reported variable, and sometimes contradictory, effects of mammalian Fringes on Notch signaling (22,(26)(27)(28)(29)(30)) (SI Appendix, Table S1). This variability may be the result of the different cell systems used in these studies, which could cause cell-based variations in the extent of Notch glycosylation.…”

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confidence: 99%

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

Taylor

Takeuchi

Sheppard

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

123

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The Notch signaling pathway is essential for many aspects of development, cell fate determination, and tissue homeostasis. Notch signaling can be modulated by posttranslational modifications to the Notch receptor, which are known to alter both ligand binding and receptor activation. We have modified the ligand-binding region (EGF domains 11-13) of human Notch1 (hN1) with O-fucose and O-glucose glycans and shown by flow cytometry and surface plasmon resonance that the Fringe-catalyzed addition of GlcNAc to the O-fucose at T466 in EGF12 substantially increases binding to Jagged1 and Delta-like 1 (DLL1) ligands. We have subsequently determined the crystal structures of EGF domains 11-13 of hN1 modified with either the O-fucose monosaccharide or the GlcNAc-fucose disaccharide at T466 of EGF12 and observed no change in backbone structure for each variant. Collectively, these data demonstrate a role for GlcNAc in modulating the ligand-binding site in hN1 EGF12, resulting in an increased affinity of this region for ligands Jagged1 and DLL1. We propose that this finding explains the Fringe-catalyzed enhancement of Notch-Delta signaling observed in flies and humans, but suggest that the inhibitory effect of Fringe on Jagged/Serrate mediated signaling involves other regions of Notch.glycosylation | mass spectrometry | X-ray

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“…Although the O-fucosylation of N is required for its further GlcNAc modification by Fng, conflicting results have been reported for the specific contribution of O-fucose monosaccharide to N signaling in Drosophila (5,(17)(18)(19).…”

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confidence: 96%

Rescue of Notch signaling in cells incapable of GDP- l -fucose synthesis by gap junction transfer of GDP- l -fucose in Drosophila

Ayukawa¹,

Matsumoto

Ishikawa

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Notch (N) is a transmembrane receptor that mediates cell-cell interactions to determine many cell-fate decisions. N contains EGF-like repeats, many of which have an O-fucose glycan modification that regulates N-ligand binding. This modification requires GDP-L-fucose as a donor of fucose. The GDP-L-fucose biosynthetic pathways are well understood, including the de novo pathway, which depends on GDP-mannose 4,6 dehydratase (Gmd) and GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase/4-reductase (Gmer). However, the potential for intercellularly supplied GDP-L-fucose and the molecular basis of such transportation have not been explored in depth. To address these points, we studied the genetic effects of mutating Gmd and Gmer on fucose modifications in Drosophila. We found that these mutants functioned cell-nonautonomously, and that GDP-L-fucose was supplied intercellularly through gap junctions composed of Innexin-2. GDP-L-fucose was not supplied through body fluids from different isolated organs, indicating that the intercellular distribution of GDP-L-fucose is restricted within a given organ. Moreover, the gap junction-mediated supply of GDP-L-fucose was sufficient to support the fucosylation of N-glycans and the O-fucosylation of the N EGF-like repeats. Our results indicate that intercellular delivery is a metabolic pathway for nucleotide sugars in live animals under certain circumstances.sugar metabolism | intercellular transport

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Roles of Pofut1 and O-Fucose in Mammalian Notch Signaling

Cited by 158 publications

References 69 publications

Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Fringe-mediated extension of O -linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands

Rescue of Notch signaling in cells incapable of GDP- l -fucose synthesis by gap junction transfer of GDP- l -fucose in Drosophila

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