Fig. 2: Identification of the EatA cleavage site in the MUC2 C-terminal.

A Fragmentation spectra of the neo N-terminal after EatA cleavage followed by trypsin digestion identified the amino acid sequence SK. The reporter ion at 573.18 m/z indicates TMPP labeling, and the b-1 fragment the N-terminal serine, the peptide composition is further supported by the immonium (i) ions. B Overview of the EatA cleavage site in MUC2 and its reoccurrence in the PTS3 repeat. C Mass spectrum of the recombinant PTS3 spanning peptide after treatment with EatA. The MH⁺⁴ and MH⁺⁵ peaks represent the mass of the full-length 34-mer peptide at a mass of 3353.73 Da. D Glycopeptides identified by mass spectrometry of the recombinant GalNAc-tranferase modified PTS3-pep containing peptide, serines and threonines highlighted in red indicate residues that were found modified with GalNAc. E Mass spectrum of the GalNAc-modified recombinant TR-PTS3 peptide after treatment with EatA. The number of GalNAc residues introduced was between 7 and 13 with a majority of ten modified sites indicated by the predominant peak at 1077.91 MH⁺⁵. F Glycopeptides identified by mass spectrometry spanning the TR-PTS3 region in recombinant MUC2C, serines and threonines highlighted in red indicate residues that were found modified with GalNAc or GalNAc-Gal. G Kinetics of MUC2C digestion by EatA after sequential deglycosylation with sialidases, galactosidase and endo-N-acetylgalactosaminidase as compared to control, determined by SDS–PAGE electrophoresis and represented as percentage cleaved mean ± SEM (n = 3).