Galactose differentially modulates lunatic and manic fringe effects on Delta1-induced NOTCH signaling

Abstract

NOTCH signaling induced by Delta1 (DLL1) and Jagged1 (JAG1) NOTCH ligands is modulated by the β3N-acetylglucosaminyl transferase Fringe. LFNG (Lunatic Fringe) and MFNG (Manic Fringe) transfer N-acetylglucosamine (GlcNAc) to O-fucose attached to EGF-like repeats of NOTCH receptors. In co-culture NOTCH signaling assays, LFNG generally enhances DLL1-induced, but inhibits JAG1-induced, NOTCH signaling. In mutant Chinese hamster ovary (CHO) cells that do not add galactose (Gal) to the GlcNAc transferred by Fringe, JAG1-induced NOTCH signaling is not inhibited by LFNG or MFNG. In mouse embryos lacking B4galt1, NOTCH signaling is subtly reduced during somitogenesis. Here we show that DLL1-induced NOTCH signaling in CHO cells was enhanced by LFNG, but this did not occur in either Lec8 or Lec20 CHO mutants lacking Gal on O-fucose glycans. Lec20 mutants corrected with a B4galt1 cDNA became responsive to LFNG. By contrast, MFNG promoted DLL1-induced NOTCH signaling better in the absence of Gal than in its presence. This effect was reversed in Lec8 cells corrected by expression of a UDP-Gal transporter cDNA. The MFNG effect was abolished by a DDD to DDA mutation that inactivates MFNG GlcNAc transferase activity. The binding of soluble NOTCH ligands and NOTCH1/EGF1-36 generally reflected changes in NOTCH signaling caused by LFNG and MFNG. Therefore, the presence of Gal on O-fucose glycans differentially affects DLL1-induced NOTCH signaling modulated by LFNG versus MFNG. Gal enhances the effect of LFNG but inhibits the effect of MFNG on DLL1-induced NOTCH signaling, with functional consequences for regulating the strength of NOTCH signaling.

FIGURE 1.

DLL1-induced NOTCH signaling is enhanced by LFNG but not MFNG in CHO and Lec1 cells. A, the histogram shows the fold-activation (top) and relative NOTCH signaling of normalized luciferase units (bottom) in CHO and Lec1 cells expressing vector (Control), LFNG or MFNG as indicated, and stimulated by L cells versus JAG1/L cells. B, the same as A except that signaling reflects stimulation by DLL1/L cells versus L cells. Error bars represent S.D.; n ≥ 4. **, p < 0.01; *, p < 0.05.

FIGURE 2.

Galactose is required for LFNG enhancement of DLL1-induced NOTCH signaling. A, fold-activation (top) and relative NOTCH signaling (bottom) in Lec1, Lec2, or Lec8 cells stably expressing vector (Control) or LFNG stimulated by DLL1/L versus L cells. B, fold-activation (top) or relative NOTCH signaling (bottom) in vector control or LFNG expressing Lec2, Lec20, or Lec20 cells corrected with a B4galt1 cDNA stimulated by DLL1/L versus L cells. Error bars represent S.D.; n ≥ 4 experiments with 2–3 replicates per experiment. **, p < 0.01; *, p < 0.05. Symbols represent O-fucose glycans on NOTCH: triangle, fucose; black square, GlcNAc; gray circle, Gal; diamond, sialic acid.

FIGURE 3.

Galactose is required for MFNG to inhibit JAG1-induced signaling, but prevents MFNG enhancement of DLL1-induced NOTCH signaling. A, relative NOTCH signaling in Lec2, Lec8, or Lec20 cells expressing vector (Control) or Mfng stimulated by JAG1/L versus L cells. B, the effects of DLL1/L cells on the same cell lines. Error bars represent S.D.; n ≥ 4 experiments with 3 replicates per experiment. **, p < 0.01. Lec20/Mfng2.6 was 2 experiments with 3 replicates each. C, Western blot analysis of activated, cleaved NOTCH1 (Val¹⁷⁴⁴), full-length NOTCH1 (9E10), and β-actin from Lec2, Lec8, or Lec20 cells expressing vector or Mfng stimulated by DLL1/L, JAG1/L, or L cells, respectively, in the presence or absence of γ-secretase inhibitor (GSI). The gels are representative of replicates from two independent experiments. Symbols are as described in the legend to Fig. 2 and represent O-fucose glycans on NOTCH/EGF repeats.

FIGURE 4.

Rescue of MFNG effect in Lec8 by SLC35A2. A, flow cytometry profiles of L-PHA binding to Lec8 cells with or without transient transfection of Slc35a2. B, relative NOTCH signaling in Lec8 cells expressing vector (Control), MFNG, or MFNG + SLC35A2 stimulated by JAG1/L versus L cells. C, the same experiment as B in transfectants stimulated by DLL1/L versus L cells. Error bars represent S.D.; n ≥ 4 experiments with 2–3 replicates each. **, p < 0.01; *, p < 0.05. Symbols are as described in the legend to Fig. 2 and represent O-fucose glycans on NOTCH/EGF repeats.

FIGURE 5.

Fringe glycosyltransferase activity is required for the MFNG effect on NOTCH signaling. A, the histogram shows relative NOTCH signaling based on normalized luciferase ratios in CHO cells expressing vector (Control), wild-type MFNG (MFNG.DDD), or mutant MFNG (MFNG.DDA) stimulated by JAG1/L versus L cells. B, the same experiment in Lec8 cells expressing vector (Control), MFNG wild-type, or mutant MFNG.DDA with stimulation by DLL1/L versus L cells. Error bars represent S.D.; n ≥ 4 experiments with 2–3 replicates each. **, p < 0.01. Symbols are as described in the legend to Fig. 2 and represent O-fucose glycans on NOTCH/EGF repeats.

FIGURE 6.

NOTCH receptor expression and NOTCH ligand binding to cells expressing Mfng or Lfng. A, the surface expression of NOTCH1 (8G10) and NOTCH3 (AF1308) on Lec1 or Lec8 cells expressing vector (thin line), Lec1 or Lec8 cells expressing Lfng or Mfng as indicated (bold lines). B, the binding of soluble JAG1-Fc or DLL1-Fc to the surface of the cells shown in A. Thin lines indicate cells expressing vector. Bold lines indicate cells expressing Lfng or Mfng, respectively. Representative results of ≥2 experiments are shown.

FIGURE 7.

Binding of N1/EGF1–36-MycHis₆ carrying different O-fucose glycans to DLL1/L cells. A, structure of predominant O-fucose glycans and N-glycans on NOTCH1 ECD produced in Lec1 and Lec8 CHO cells. Symbols are as described in the legend to Fig. 2. B, Western blots of mouse N1/EGF1–36-MycHis₆ (N1/EGF1–36) prepared from Lec1 or Lec8 cells expressing vector, Lfng, or Mfng. N1/EGF1–36 was detected by anti-Myc antibody 9E10 or lectin (GSL-II and LCA detected terminal GlcNAc on N1/EGF1–36 from Lec8; ConA detected high mannose N-glycans on N1/EGF1–36 from Lec1. C, surface expression of DLL1 on L cells and DLL1/L cells. D, the binding of N1/EGF1–36 prepared from Lec1 expressing vector (thin line), Lfng (dotted line), or Mfng (bold line) to L or DLL1/L cells in the presence or absence of 5 mm EDTA. E, binding of N1/EGF1–36 prepared from Lec1 or Lec8 expressing vector (thin lines), Lfng (dotted lines), or Mfng (bold lines) to L or DLL1/L cells. Binding was detected with anti-His antibody. Profiles are representative of ≥2 experiments.