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Modulation of the NOTCH1 pathway by LUNATIC FRINGE is dominant over that of MANIC or RADICAL FRINGE

ABSTRACT: Fringes are glycosyltransferases that transfer a GlcNAc to O-fucose residues on Epidermal Growth Factor-like (EGF) repeats. Three Fringes exist in mammals: LUNATIC FRINGE (LFNG), MANIC FRINGE (MFNG) and RADICAL FRINGE (RFNG). Fringe modification of O-fucose on EGF repeats in the NOTCH1 (N1) extracellular domain modulates the activation of N1 signaling. Not all O-fucose residues of N1 are modified by all Fringes; some are modified by one or two Fringes and others not modified at all. The distinct effects on N1 activity depend on which Fringe is expressed in a cell. However, little data is available on the effect that more than one Fringe has on the modification of O-fucose residues and the resulting downstream consequence on Notch activation. Using mass spectral glycoproteomic site mapping and cell-based N1 signaling assays, we compared the effect of co-expression of N1 with one or more Fringes on modification of O-fucose and activation of N1 in three cell lines. Individual expression of each Fringe with N1 in the three cell lines revealed differences in modulation of the Notch pathway dependent on the presence of endogenous Fringes. Despite these cell-based differences, co-expression of several Fringes with N1 demonstrated a dominant effect of LFNG over MFNG or RFNG. MFNG and RFNG appeared to be co-dominant but strongly dependent on the ligands used to activate N1 and on the endogenous expression of Fringes. These results show a hierarchy of Fringe activity and indicate that the effect of MFNG and/or RFNG could be small in the presence of LFNG.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Cell Culture

SUBMITTER: FLORIAN PENNARUBIA

LAB HEAD: Robert S Haltiwanger

PROVIDER: PXD027619 | Pride | 2021-09-28

REPOSITORIES: Pride

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O-Fucose glycans on mouse NOTCH1 from activated T cells of Fringe mutant mice

Project description:NOTCH1 (N1) is a transmembrane receptor that initiates a cell-cell signaling pathway controlling various cell fate specifications in metazoans. The addition of O-fucose by Protein O-fucosyltransferase 1 (POFUT1) to Epidermal Growth Factor-like (EGF) repeats in the N1 extracellular domain is essential for N1 function, and modification of O-fucose with GlcNAc by the Fringe family of glycosyltransferases modulates Notch activity. Prior cell-based studies showed that POFUT1 modifies EGF repeats containing the appropriate consensus sequence at high stoichiometry, while Fringe GlcNAc-transferases (LFNG, MFNG and RFNG) modify O-fucose on only a subset of NOTCH1 EGF repeats. Previous in vivo studies showed that each FNG affects naïve T cell development. To examine Fringe modifications of N1 expressed at a physiological level, we used mass spectral glycoproteomic methods to analyze O-fucose glycans of endogenous N1 from activated T cells obtained from mice lacking all Fringe enzymes, or expressing only a single FNG. While most O-fucose sites were modified at high stoichiometry, only EGF6, EGF16, EGF26, and EGF27 were extended in control T cells. Cell-based assays of N1 lacking fucose at each of those O-fucose sites revealed minor functional correlations in the EGF16 and EGF27 mutant with Notch ligand binding. In activated T cells expressing only LFNG, MFNG or RFNG alone, the extension of O-fucose with GlcNAc in the same EGF repeats was diminished, consistent with cooperative interactions when all three Fringes were present. The combined data open the door for the analysis of O-glycans on endogenous N1 derived from different cell types.

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