Studies of mucus in mouse stomach, small intestine, and colon. II. Gastrointestinal mucus proteome reveals Muc2 and Muc5ac accompanied by a set of core proteins

Abstract

The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the gastrointestinal tract. To ensure that only mucus was analyzed, we combined collection from explant tissues mounted in perfusion chambers, liquid sample preparation, single-shot mass spectrometry, and specific bioinformatics tools, to characterize the proteome of the murine mucus from stomach to distal colon. With our approach, we identified ∼1,300 proteins in the mucus. We found no differences in the protein composition or abundance between sexes, but there were clear differences in mucus along the tract. Noticeably, mucus from duodenum showed similarities to the stomach, probably reflecting the normal distal transport. Qualitatively, there were, however, fewer differences than might had been anticipated, suggesting a relatively stable core proteome (∼80% of the total proteins identified). Quantitatively, we found significant differences (∼40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of such proteins that correlated with Muc2 (e.g., Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system.

Keywords: Muc2; Muc5ac; mass spectrometry; mucin; proteomics.

Fig. 1.

The gastrointestinal mucus proteome varies along the gastrointestinal tract (GIT). Mucus peptides obtained by tryptic digestion were analyzed by an Orbitrap mass spectrometer. A: schematic description of the preparation scheme used for mucus proteome analysis. B: scatter plots depicting correlations in observed protein relative abundances between sexes, organs, and segments. The Pearson correlation coefficient is shown for each plot. C: principal component analysis could classify the mucus samples on the basis of the total protein fingerprint. In particular, component 2 could separate samples from stomach (negative values) from large intestine (proximal and distal colon; positive values). Samples are represented as stomach (■), duodenum (+), jejunum (□), ileum (△), proximal colon (•), distal colon (▲).

Fig. 2.

Venn diagrams showing the number of shared and exclusive proteins found in the mucus of the 3 organs from the GIT (A), the small intestine (B), and the large intestine (C). The total number of proteins in these Venn diagrams is 1,471, 4 proteins less than mentioned in the text, owing to the necessary statistical filtration in the process of generation of the diagrams. The proteins removed were Niban, Hook2, Nrn1l, and Drg2.

Fig. 3.

The proteins observed by proteomic analysis of the GIT mucus were hierarchically clustered. A detail of this clustering around the node for Muc2 is displayed, with the proteins closely related to Muc2 highlighted in blue.

Fig. 4.

Boxplots of the relative levels of mucins (logarithm of the normalized intensity) along the GIT mucins and some of the proteins observed to cluster around Muc2 (Fig. 3). Circles represent outliers, i.e., values between 1.5 and 3 times the interquartile range (IQR; difference between the ends of the box). Stars represent extreme values, defined as those larger than 3 times the IQR. Sto, stomach; Duo, duodenum; Jej, jejunum; Ile, ileum; PC, proximal colon; DC, distal colon; SI, small intestine; LI, large intestine.

Fig. 5.

Localization of Muc2 and proteins that clustered together with Muc2 in the hierarchical clustering (Fig. 3). The Muc2 mucin (green; left), Clca1 (red; middle), and Zg16 (red, right) were specifically detected in tissues from the 6 locations from where mucus was sampled. Prox., proximal; Dist., distal. Bar: 100 μm.