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

Kenjiroo Matsumoto, Vivek Kumar, Shweta Varshney, Alison V. Nairn, Atsuko Ito, Florian Pennarubia, Kelley W. Moremen, Pamela Stanley, Robert S. Haltiwanger

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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 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 NOTCH1 at a physiological level, we used mass spectral glycoproteomic methods to analyze O-fucose glycans of endogenous NOTCH1 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 WT T cells. Additionally, cell-based assays of NOTCH1 lacking fucose at each of those O-fucose sites revealed small but significant effects of LFNG on Notch-Delta binding in the EGF16 and EGF27 mutants. Finally, 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 NOTCH1 derived from different cell types.

Original languageEnglish (US)
Article number102064
JournalJournal of Biological Chemistry
Issue number7
StatePublished - Jul 2022


  • EGF repeats
  • Notch
  • O-fucose
  • T cells
  • fringe
  • glycosylation
  • mass spectrometry

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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