Proteomics

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


ABSTRACT: 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.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Spleen, T Cell

SUBMITTER: Kenjiroo Matsumoto  

LAB HEAD: Kenjiroo Matsumoto

PROVIDER: PXD031297 | Pride | 2022-08-12

REPOSITORIES: Pride

Dataset's files

Source:
Action DRS
KM020719_1.raw Raw
KM020719_3.raw Raw
KM040921-2.raw Raw
KM040921_1.raw Raw
KM040921_3.raw Raw
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Publications

Fringe GlcNAc-transferases differentially extend O-fucose on endogenous NOTCH1 in mouse activated T cells.

Matsumoto Kenjiroo K   Kumar Vivek V   Varshney Shweta S   Nairn Alison V AV   Ito Atsuko A   Pennarubia Florian F   Moremen Kelley W KW   Stanley Pamela P   Haltiwanger Robert S RS  

The Journal of biological chemistry 20220525 7


NOTCH1 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 NOTCH1 extracellular domain is essential for NOTCH1 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 c  ...[more]

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