Detailed O-glycomics of the Muc2 mucin from colon of wild-type, core 1- and core 3-transferase-deficient mice highlights differences compared with human MUC2

Abstract

The heavily O-glycosylated mucin MUC2 constitutes the major protein in the mucosal layer that acts as a physical barrier protecting the epithelial layer in the colon. In this study, Muc2 was purified from mucosal scrapings from the colon of wild-type (WT) mice, core 3 transferase knockout (C3Gnt(-/-)) mice and intestinal epithelial cell-specific core 1 knockout (IEC C1Galt1(-/-)) mice. The Muc2 O-glycans were released by reductive β-elimination and analyzed with liquid chromatography-mass spectrometry in the negative-ion mode. Muc2 from the distal colon of WT and C3Gnt(-/-) knockout mice carried a mixture of core 1- or core 2-type glycans, whereas Muc2 from IEC C1Galt1(-/-) mice carried highly sialylated core 3- and core 4-type glycans. A large portion of NeuAc in all mouse models was positioned on disialylated N-acetyllactosamine units, an epitope not reported on human colonic MUC2. Mass spectra and proton NMR spectroscopy revealed an abundant NeuAc linked to internally positioned N-acetylglucosamine on colonic murine Muc2, which also differs markedly from human MUC2. Our results highlight that murine colonic Muc2 O-glycosylation is substantially different from human MUC2, which could be one explanation for the different commensal microbiota of these two species.

Fig. 1.

Core structures commonly found on mucins (A). Work flow for methodology applied for structural O-glycan analysis from murine Muc2 from the mucosal layer of colon (B).

Fig. 2.

Negative-ion mode capillary-LC/MS base peak chromatograms of O-glycan alditols from colonic Muc2 mucin from WT mouse (A) and from the core 1 knockout (IEC C1Galt^−/−) mouse (B). Major components are annotated with m/z ([M–H⁺]⁻) and proposed sequences interpreted from MS² spectra. The structural data are compiled in Table 1, with more details in Supplementary data, Table S1.

Fig. 3.

Negative-ion mode capillary-LC/MS base peak chromatograms of mucin O-glycan alditols from colonic Muc2 from WT mouse (A) and from the core 1 knockout (IEC C1Galt1^−/−) mouse (B) after neuraminidase treatment. Major components are annotated with m/z ([M–H⁺]) and proposed sequences interpreted from MS² data.

Fig. 4.

MS² spectra from negative-ion mode capillary LC/MS of colonic Muc2 glycan alditols labeled 878a with the sequence Gal-(NeuAc-)GlcNAc-GalNAcol from the core 1 knockout (IEC C1Galt1^−/−) mouse (A) and 1040a with the sequence Gal-(Gal-(NeuAc-)GlcNAc-)GalNAcol from the WT mouse (B). Corresponding mass chromatograms are inserted.

Fig. 5.

Chemical shift values for NeuAc H3eq and H3ax signals for reduced sialylated standards analyzed with one-dimensional proton NMR spectroscopy at 25°C (A). Selected region of the two-dimensional proton NMR spectrum encompassing NeuAc H3eq/H3ax cross-peaks from the analysis of colonic Muc2 O-glycan alditols from WT mouse (B). The areas where cross-peaks from (α2-3)-linked NeuAc, (α2-6)-linked NeuAc and in Sd^a epitopes were expected according to our reference samples A–D and also published by others (Capon et al. 2001; Robbe et al. 2003) are highlighted by ellipses.

Fig. 6.

Negative-ion mode capillary-LC/MS base peak chromatograms of Muc2 O-glycan alditols purified from a mucosal scraping of the distal colon from a WT mouse (A), from human biopsies from sigmoideum (B) and from colon of the core 1 (IEC C1Galt1^−/−) knockout mouse. The sequences of major components are annotated. A general structure of the oligosaccharides is depicted adjacent to each chromatogram.