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Fringe modulation of Jagged1-induced Notch signaling requires the action of beta 4galactosyltransferase-1 - PubMed

  • ️Mon Jan 01 2001

Fringe modulation of Jagged1-induced Notch signaling requires the action of beta 4galactosyltransferase-1

J Chen et al. Proc Natl Acad Sci U S A. 2001.

Abstract

Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a beta 3N-acetylglucosaminyltransferase (beta 3GlcNAcT) that transfers GlcNAc to O-fucose in epidermal growth factor-like repeats of Notch. Here we use five different Chinese hamster ovary cell glycosylation mutants to identify a key aspect of the mechanism of fringe action. Although the beta 3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAc beta 3Fuc, is not elongated. The trisaccharide, Gal beta 4GlcNAc beta 3Fuc, is the minimal O-fucose glycan to support fringe modulation of Notch signaling. Of six beta 4galactosyltransferases (beta 4GalT) in Chinese hamster ovary cells, only beta 4GalT-1 is required to add Gal to GlcNAc beta 3Fuc, identifying beta 4GalT-1 as a new modulator of Notch signaling.

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Figures

Figure 1
Figure 1

Fringe glycosyltransferase activity and fucose transfer are required for fringe modulation of Notch signaling. (A) The histogram shows the fold-activation of normalized luciferase units in control (vector), wild-type Mfng (Mfng.DDD), and mutant (Mfng.DDA) CHO and Lec1 transfectants stimulated by J1 vs. L cells (J1/L). Error bars represent SEM; n = 4. (B) The synthesis of GDP-fucose in CHO cells is blocked in Lec13 cells as a result of reduced transcription of the GDP-4,6-DH gene. (C) Immunofluorescence of FITC-pea lectin on the surface of Lec13, CHO, and Lec13 cells transiently transfected with pMirb/GDP-4,6-DH. (D) Fold-activation of normalized luciferase units in CHO, Lec13.Mfng, and Lec13.Mfng transiently transfected with pMirb/GDP-4,6-DH. Error bars represent SEM; n = 4 for each.

Figure 2
Figure 2

Galactose but not SA is required for a fringe effect. (A) Gel filtration of [3H]fucose-labeled O-glycans released by β-elimination from Notch1 EGF19–23 secreted from Lec2 or Lec8 cells expressing Lfng (filled circles) or vector (open squares). The major species (≈1,500 cpm) was O-fucose MS (fucitol). Elution positions of standard O-fucose glycans (Tetra, SAα3Galβ4GlcNAcβ3Fucitol; Tri, Galβ4GlcNAcβ3Fucitol; Di, GlcNAcβ3Fucitol; Mono, Fucitol) are indicated. (B) Fold-activation of normalized luciferase units in CHO, Lec1, Lec2, or Lec8 clones expressing vector, Mfng, or Lfng stimulated by J1 vs. L cells. Error bars represent SEM; n = 4. (C) FACS analysis with Notch1 ECD Ab. Dotted line, normal rabbit serum; dashed line, Lec2.vector or Lec8.vector; solid line, Lec2.Lfng or Lec8.Lfng.

Figure 3
Figure 3

The β4GalT-1 mutant Lec20 does not exhibit a fringe effect. (A) Fold-activation of normalized luciferase activity in Lec1 and Lec20 clones stably expressing vector, Mfng, or Lfng stimulated by J1 vs. L cells. Error bars represent SEM; n = 4. (B) Gel filtration profiles of [3H]fucose-labeled O-glycans released by β-elimination from Notch1 EGF19–23 produced by Lec20.Lfng (filled circles) or Lec20.vector (open squares). Elution positions of standard O-fucose glycans are indicated.

Figure 4
Figure 4

Rescue of a fringe effect with β4GalT-1. (A) Lectin sensitivity test. Cells surviving in E-phytohemagglutinin (PHA) were stained with methylene blue. (B) Gel filtration profiles of [3H]fucose-labeled O-glycans released by β-elimination from Notch1 EGF19–23 produced by Lec20 cells expressing Lfng and β4GalT-1. Elution positions of O-fucose glycan standards are indicated. (C) Fold-activation of normalized luciferase units in Lec2 or Lec20 clones expressing vector, Lfng, or Lfng + GT (β4GalT-1) stimulated by J1 vs. L cells. Error bars represent SEM; n = 4.

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