The inner of the two Muc2 mucin-dependent mucus layers in colon is devoid of bacteria Malin E. V. Johansson*, Mia Phillipson†, Joel Petersson†, Anna Velcich§, Lena Holm†, and Gunnar C. Hansson*‡ *Department of Medical Biochemistry, University of Gothenburg, 405 30 Gothenburg, Sweden; †Department of Medical Cell Biology, Uppsala University, 751 23 Uppsala, Sweden; and §Department of Oncology, Montefiore/Albert Einstein Cancer Center, Bronx, NY 10467 Edited by E. Peter Greenberg, University of Washington School of Medicine, Seattle, WA, and approved August 12, 2008 (received for review March 30, 2008) We normally live in symbiosis with ϳ1013 bacteria present in the colon in vivo in C57BL/6 mice by a micropipette that can colon. Among the several mechanisms maintaining the bacteria/host penetrate the mucus layer down to the epithelial cells (5). In the balance, there is limited understanding of the structure, function, and mouse colon, the mucus extended Ϸ150 ␮m above the epithelial properties of intestinal mucus. We now demonstrate that the mouse cells and was composed of two layers with distinct physical colonic mucus consists of two layers extending 150 ␮m above the properties (Fig. 1B,FϩL). Most of the mucus could be aspirated epithelial cells. Proteomics revealed that both of these layers have off and represented the loose layer (Ϸ100 ␮m thick); the similar protein composition, with the large gel-forming mucin Muc2 as remaining fraction was adhering firmly to the epithelia and was the major structural component. The inner layer is densely packed, firmly identified as firm (Ϸ50 ␮m thick) (Fig. 1B,F0min).This firmly attached to the epithelium, and devoid of bacteria. In contrast, the outer adherent layer could, however, be removed by gentle scraping. layer is movable, has an expanded volume due to proteolytic cleavages Similar measurements in the rat colon have shown firm and loose of the Muc2 mucin, and is colonized by bacteria. Muc2؊/؊ mice have mucus layers of Ϸ100- and 700-␮m thickness, respectively (5). After bacteria in direct contact with the epithelial cells and far down in the aspiration, the loose layer was slowly replenished, as shown by the crypts, explaining the inflammation and cancer development observed increasing mucus thickness when measurements were taken 15 and in these animals. These findings show that the Muc2 mucin can build a 30 min after the initial aspiration (Fig. 1B,Fϩ 15 and F ϩ 30 min). mucus barrier that separates bacteria from the colon epithelia and To prove that the newly formed layer was indeed loosely adherent suggest that defects in this mucus can cause colon inflammation. mucus, we performed a second aspiration that decreased the thickness to the firm level (Fig. 1B,F30min). commensal bacteria ͉ proteomics ͉ ulcerative colitis ͉ large intestine ͉ colon cancer Composition of the Two Mucus Layers. We next investigated the components of the two colonic mucus layers by separating he digestive tract is home to 10 times as many bacteria as reduced and alkylated samples of mucus by composite agarose- Thuman cells in an ecosystem that has evolved to ensure PAGE (AgPAGE). Upon gel staining with Alcian blue to homeostasis. How we can manage this enormous bacterial load visualize mucin bands, we detected two major bands for Muc2, without overt immune responses from the adaptive and innate corresponding to Muc2 monomer (the smallest detectable com- systems is not well understood. When the equilibrium is altered, ponent of Muc2, marked M) and a likely dimer (3) that were as in the disease ulcerative colitis, inflammatory responses are similarly represented in the loose- and firm-mucus layer prep- initiated against the commensal bacteria. An important compo- arations (Fig. 1C). Mass spectrometry of the tryptic peptides nent, often neglected due to lack of understanding, is the mucus showed Muc2 as the only gel-forming mucin detected in these layer that covers the epithelial cells. The major components of bands. The identified peptides encompassed 30% of the protein mucus, the gel-forming mucins, are part of the innate immunity (excluding the PTS domains) and included most of the peptides expected not to be glycosylated [Fig. 1A and supporting infor- and are well preserved in evolution (1). The secreted gel-forming mation (SI) Table S1]. Only minor peptide differences, localized mucin, MUC2, is a major mucin of the colon mucus (2). This is to the N-terminal part, were observed between Muc2 from the a large glycoprotein characterized by abundant and variable firm and loose mucus. The intensity of the bands and peptide O-linked glycans attached to hydroxy amino acids clustered in representation suggest that the Muc2 mucin is a major constit- PTS or mucin domains, Fig. 1A (1). The assembly process of uent of both the firm and loose mucus layers. Upon analysis of MUC2 is well documented (2–4): MUC2 dimerizes in the the small-sized protein components of the loose and firm mucus endoplasmic reticulum via its C terminus, becomes heavily by PAGE and Coomassie blue staining, identical patterns for the O-glycosylated in the Golgi and polymerizes via the N terminus two mucus layers were observed (Fig. S1). A detailed compar- by disulfide-bonded trimerization (4). The glycosylated primary ison identified proteins that were intracellular components, translation product of MUC2 has a mass Ϸ2.5 MDa, and the serum proteins, and likely mucus constituents. Out of these, the secreted polymer has a huge, net-like structure. secreted proteins and proteins with large extracellular domains Previous studies in rat colon have shown that there are two mucus as well as their association to the loose and/or firm layers is layers. An inner adherent mucus layer that is possible to remove presented in Table 1 and Tables S2 and S3, revealing that the only by gentle scraping and an outer loosely adherent mucus layer proteins were present in both the firm and loose mucus layers that is easy to remove by gentle suctioning (5). To understand how these mucus layers are formed and their function, we have analyzed the composition and properties of these two mucus layers. The Author contributions: M.E.V.J., L.H., and G.C.H. designed research; M.E.V.J., M.P., and J.P. results show that both these layers are formed largely by the Muc2 performed research; A.V. contributed new reagents/analytic tools; M.E.V.J., M.P., J.P., and mucin, that the two layers have different properties, and that the G.C.H. analyzed data; and M.E.V.J., A.V., L.H., and G.C.H. wrote the paper. inner layer excludes the bacteria. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Results ‡To whom correspondence should be addressed. E-mail: [email protected]. The organization of the large-intestinal mucus, where most of This article contains supporting information online at www.pnas.org/cgi/content/full/ the intestinal bacteria are localized, is not well understood. To 0803124105/DCSupplemental. address this, we first measured the thickness of the mucus in © 2008 by The National Academy of Sciences of the USA 15064–15069 ͉ PNAS ͉ September 30, 2008 ͉ vol. 105 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0803124105 Downloaded by guest on September 26, 2021 Table 1. The proteins of the loose (L) and firm (F) mucus were separated by PAGE (Fig. S1) and the proteins identified as tryptic peptides by LC-MS/MS Protein name Gene Loc L* F* Anterior gradient 2 Agr2 sec x x Ca-activated Cl channel 3 Clca3 sec x x Ca-activated Cl channel 6 Clca6 sec x x IgG Fc-binding protein Fcgbp sec x x ␥-Glutamyl hydrolase Ggh sec x x Immunoglobulin Ig sec x x Lumican Lum sec x x Mucin 2 Muc2 sec x x Major urinary proteins 11 and 8 Mup sec x x Oncoprotein-induced transcript 1 Oit1 sec x x Mucosal pentraxin Ptx sec x x Arginyl aminopeptidase Rnpep sec x x Seminal vesicle secretory protein IV Svs4 sec x x Zymogen granule membrane protein 16 Zg16 sec x x Mucin 3(17), orthologue hMUC17 Muc3(17) sec-tm1 x (x) Vomeroglandin Dmbt1 sec-tm1 x (x) Polymeric immunoglobulin receptor Pigr sec-tm1 x (x) Quiescinsulfhydryl oxidase 1 Qsox1 sec-tm1 x (x) Mucin13 Muc13 tm1 x x Basigin Bsg tm1 x x Fig. 1. The loose and the firm mucus layers of the colon consist mainly of the Muc2 mucin. (A) Schematic view of the domain organization of the MUC2 Uvomorulin Cdh1 tm1 x x mucin and indication of where the sites for polymerization are localized. The Cadherin 17 Cdh17 tm1 x x human apoprotein is 5,174 aa long (2); vW identifies von Willebrand C (dark CEACAM 1 Ceacam1 tm1 x x blue) or D (red) domain; PTS, the mucin domain (green); CysD (yellow), and CK, Hephaestin Heph tm1 x x the cysteine knot domain (light blue) (1). The bands in C were digested with Mucin-like protocadherin Mupcdh tm1 x x trypsin, and the peptides identified with LC-MS/MS and searched against the Nicastrin Ncstn tm1 x x mucin database. Identified Muc2 peptides are schematically marked with one Protocadherin LKC Pcdh tm1 x x line per peptide under the Muc2 protein sequence. Peptides identified in both Tumor-associated Ca signal transducer 1 Tacstd1 tm1 x x fractions are marked blue, those found only in the firm fraction are marked Maltase-glucoamylase Mgam tm2 x (x) green, and those only found in the loose fraction are marked red. A full peptide list is presented in Table S1.(B) In vivo measurements of the mucus Only secreted proteins or transmembrane proteins with a large extracel- thickness in the distal colon reveal the presence of a firm and a loose layer.