Proteome of airway surface liquid and mucus in newborn wildtype and cystic fibrosis piglets.

BACKGROUND: The respiratory tract is protected from inhaled particles and microbes by mucociliary clearance, mediated by the mucus and the cilia creating a flow to move the mucus cephalad. Submucosal glands secrete linear MUC5B mucin polymers and because they pass through the gland duct before reaching the airway surface, bundled strands of 1000-5000 parallel molecules exit the glands. In contrast, the surface goblet cells secrete both MUC5AC and MUC5B. METHODS: We used mass-spectrometry based proteomic analysis of unstimulated and carbachol stimulated newborn wild-type (WT) and cystic fibrosis transmembrane conductance regulator (CFTR) null (CF) piglet airways to study proteins in the airway surface liquid and mucus, to investigate if levels of MUC5AC and MUC5B were affected by carbachol stimulation and whether the proteins clustered according to function. RESULTS: Proteins in the first four extracted fractions clustered together and the fifth fraction contained the mucus cluster, mucins and other proteins known to associate with mucins, whereas the traditional airway surface liquid proteins clustered to fraction 1-4 and were absent from the mucus fraction. Carbachol stimulation resulted in increased MUC5AC and MUC5B. CONCLUSIONS: These results indicate a distinct separation between proteins in the washable surface liquid and the mucus fraction. In fractions 1-4 from newborn CF piglets an additional cluster containing acute phase proteins was observed, suggesting an early inflammatory response in CF piglets. Alternatively, increased levels of these proteins could indicate altered lung development in the CF piglets. This observation suggests that CF airway disease is present at birth and thus, treatment should commence directly after diagnosis.

The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

Normal murine respiratory tract has its mucus concentrated in clouds based on the Muc5b mucin.

The organization of the normal airway mucus system differs in small experimental animals from that in humans and large mammals. To address normal murine airway mucociliary clearance, Alcian blue-stained mucus transport was measured ex vivo on tracheal tissues of naïve C57BL/6, Muc5b-/-, Muc5ac-/-, and EGFP-tagged Muc5b reporter mice. Close to the larynx with a few submucosal glands, the mucus appeared as thick bundles. More distally in the trachea and in large bronchi, Alcian blue-stained mucus was organized in cloud-like formations based on the Muc5b mucin. On tilted tissue, the mucus clouds moved upward toward the larynx with an average velocity of 12 µm/s compared with 20 µm/s for beads not associated with clouds. In Muc5ac-/- mice, Muc5b formed mucus strands attached to the tissue surface, while in Muc5b-/- mice, Muc5ac had a more variable appearance. The normal mouse lung mucus thus appears as discontinuous clouds, clearly different from the stagnant mucus layer in diseased lungs.

Protein Turnover in Epithelial Cells and Mucus along the Gastrointestinal Tract Is Coordinated by the Spatial Location and Microbiota.

The gastrointestinal tract is covered by a single layer of epithelial cells that, together with the mucus layers, protect the underlying tissue from microbial invasion. The epithelium has one of the highest turnover rates in the body. Using stable isotope labeling, high-resolution mass spectrometry, and computational analysis, we report a comprehensive dataset of the turnover of more than 3,000 and the expression of more than 5,000 intestinal epithelial cell proteins, analyzed under conventional and germ-free conditions across five different segments in mouse intestine. The median protein half-life is shorter in the small intestine than in the colon. Differences in protein turnover rates along the intestinal tract can be explained by distinct physiological and immune-related functions between the small and large intestine. An absence of microbiota results in an approximately 1 day longer protein half-life in germ-free animals.

Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs.

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.

The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin.

To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.

Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization.

The intestinal mucus layer provides a barrier limiting bacterial contact with the underlying epithelium. Mucus structure is shaped by intestinal location and the microbiota. To understand how commensals modulate gut mucus, we examined mucus properties under germ-free (GF) conditions and during microbial colonization. Although the colon mucus organization of GF mice was similar to that of conventionally raised (Convr) mice, the GF inner mucus layer was penetrable to bacteria-sized beads. During colonization, in which GF mice were gavaged with Convr microbiota, the small intestine mucus required 5 weeks to be normally detached and colonic inner mucus 6 weeks to become impenetrable. The composition of the small intestinal microbiota during colonization was similar to Convr donors until 3 weeks, when Bacteroides increased, Firmicutes decreased, and segmented filamentous bacteria became undetectable. These findings highlight the dynamics of mucus layer development and indicate that studies of mature microbe-mucus interactions should be conducted weeks after colonization.

The colonic mucus protection depends on the microbiota.

The intestinal mucus is a pivotal part of our intestinal protection. It provides slow diffusion of protective molecules, trapping of luminal material as bacteria and smooth transport in the small intestine. In colon it restricts bacterial access to the epithelium limiting the responses to the enormous bacterial load present at this location. The development of these systems depends on the microbiota composition as seen in our recent study comparing the mucus phenotype in 2 colonies kept in different husbandries within the same SPF animal facility. One colony had impenetrable colonic mucus while the other colony had more penetrable mucus. The mucus phenotypes were transmitted via the microbiota and clear differences in its composition could be detected. Candidates associated with the different colonies were identified but the observed mucus difference could not be assigned to a specific bacterium.

The composition of the gut microbiota shapes the colon mucus barrier.

Two C57BL/6 mice colonies maintained in two rooms of the same specific pathogen-free (SPF) facility were found to have different gut microbiota and a mucus phenotype that was specific for each colony. The thickness and growth of the colon mucus were similar in the two colonies. However, one colony had mucus that was impenetrable to bacteria or beads the size of bacteria-which is comparable to what we observed in free-living wild mice-whereas the other colony had an inner mucus layer penetrable to bacteria and beads. The different properties of the mucus depended on the microbiota, as they were transmissible by transfer of caecal microbiota to germ-free mice. Mice with an impenetrable mucus layer had increased amounts of Erysipelotrichi, whereas mice with a penetrable mucus layer had higher levels of Proteobacteria and TM7 bacteria in the distal colon mucus. Thus, our study shows that bacteria and their community structure affect mucus barrier properties in ways that can have implications for health and disease. It also highlights that genetically identical animals housed in the same facility can have rather distinct microbiotas and barrier structures.

Postoperative serum levels of sCD26 for surveillance in colorectal cancer patients.

One of the main aims of the follow-up after curative resection of colorectal cancer is the early detection and treatment of tumor recurrence. We previously demonstrated decreased preoperative soluble CD26 (sCD26) levels in serum from colorectal cancer patients. We extended now the study to investigate if sCD26 levels in postoperative serum serve as marker of recurrence of the disease during surveillance. Soluble sCD26 was measured in pre- and postoperative serum samples of 43 patients with primary colorectal cancer. Carcinoembryonic antigen, carbohydrate antigen 19.9 and 72.4 levels were also measured during surveillance. The average follow-up period was 41.8 ± 20.8 months. sCD26 levels during follow-up showed well-defined patterns in patients without disease (n = 28), and in patients with tumor persistence (n = 2), local recurrence (n = 3) or distant metastasis (n = 10). Disease-free patients showed stable levels between 460-850 ng/mL during follow-up, while high (over 850 ng/mL) and unstable sCD26 levels were found before recurrence was diagnosed. The mean maximum/minimum sCD26 ratios during surveillance were 1.52, 2.12 and 2.63 for patients with no recurrence, local recurrence and metastasis, respectively (p = 0.005). From the cut-off obtained from a receiver operator characteristics (ROC) curve built with the maximum/minimum sCD26 ratios and the upper and lower cut-offs of sCD26, we were able to discriminate patients with and without recurrent disease. We propose that the measurement of serum sCD26 during the follow-up of patients diagnosed of colorectal cancer could be valuable for the early detection of local and distant recurrence. A large, randomized, prospective trial should be performed to confirm our findings.

Microbial-induced meprin ß cleavage in MUC2 mucin and a functional CFTR channel are required to release anchored small intestinal mucus.

The mucus that covers and protects the epithelium of the intestine is built around its major structural component, the gel-forming MUC2 mucin. The gel-forming mucins have traditionally been assumed to be secreted as nonattached. The colon has a two-layered mucus system where the inner mucus is attached to the epithelium, whereas the small intestine normally has a nonattached mucus. However, the mucus of the small intestine of meprin ß-deficient mice was now found to be attached. Meprin ß is an endogenous zinc-dependent metalloprotease now shown to cleave the N-terminal region of the MUC2 mucin at two specific sites. When recombinant meprin ß was added to the attached mucus of meprin ß-deficient mice, the mucus was detached from the epithelium. Similar to meprin ß-deficient mice, germ-free mice have attached mucus as they did not shed the membrane-anchored meprin ß into the luminal mucus. The ileal mucus of cystic fibrosis (CF) mice with a nonfunctional cystic fibrosis transmembrane conductance regulator (CFTR) channel was recently shown to be attached to the epithelium. Addition of recombinant meprin ß to CF mucus did not release the mucus, but further addition of bicarbonate rendered the CF mucus normal, suggesting that MUC2 unfolding exposed the meprin ß cleavage sites. Mucus is thus secreted attached to the goblet cells and requires an enzyme, meprin ß in the small intestine, to be detached and released into the intestinal lumen. This process regulates mucus properties, can be triggered by bacterial contact, and is nonfunctional in CF due to poor mucin unfolding.

AGR2, an endoplasmic reticulum protein, is secreted into the gastrointestinal mucus.

The MUC2 mucin is the major constituent of the two mucus layers in colon. Mice lacking the disulfide isomerase-like protein Agr2 have been shown to be more susceptible to colon inflammation. The Agr2(-/-) mice have less filled goblet cells and were now shown to have a poorly developed inner colon mucus layer. We could not show AGR2 covalently bound to recombinant MUC2 N- and C-termini as have previously been suggested. We found relatively high concentrations of Agr2 in secreted mucus throughout the murine gastrointestinal tract, suggesting that Agr2 may play extracellular roles. In tissue culture (CHO-K1) cells, AGR2 is normally not secreted. Replacement of the single Cys in AGR2 with Ser (C81S) allowed secretion, suggesting that modification of this Cys might provide a mechanism for circumventing the KTEL endoplasmic reticulum retention signal. In conclusion, these results suggest that AGR2 has both intracellular and extracellular effects in the intestine.

The mucus and mucins of the goblet cells and enterocytes provide the first defense line of the gastrointestinal tract and interact with the immune system.

The gastrointestinal tract is covered by mucus that has different properties in the stomach, small intestine, and colon. The large highly glycosylated gel-forming mucins MUC2 and MUC5AC are the major components of the mucus in the intestine and stomach, respectively. In the small intestine, mucus limits the number of bacteria that can reach the epithelium and the Peyer's patches. In the large intestine, the inner mucus layer separates the commensal bacteria from the host epithelium. The outer colonic mucus layer is the natural habitat for the commensal bacteria. The intestinal goblet cells secrete not only the MUC2 mucin but also a number of typical mucus components: CLCA1, FCGBP, AGR2, ZG16, and TFF3. The goblet cells have recently been shown to have a novel gate-keeping role for the presentation of oral antigens to the immune system. Goblet cells deliver small intestinal luminal material to the lamina propria dendritic cells of the tolerogenic CD103(+) type. In addition to the gel-forming mucins, the transmembrane mucins MUC3, MUC12, and MUC17 form the enterocyte glycocalyx that can reach about a micrometer out from the brush border. The MUC17 mucin can shuttle from a surface to an intracellular vesicle localization, suggesting that enterocytes might control and report epithelial microbial challenge. There is communication not only from the epithelial cells to the immune system but also in the opposite direction. One example of this is IL10 that can affect and improve the properties of the inner colonic mucus layer. The mucus and epithelial cells of the gastrointestinal tract are the primary gate keepers and controllers of bacterial interactions with the host immune system, but our understanding of this relationship is still in its infancy.

Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucin O-glycan patterns reveal a regiospecific distribution.

The mouse intestinal mucus is mainly made up by the gel-forming Muc2 mucin and the stomach surface mucus Muc5ac, both extensively O-glycosylated. The oligosaccharide diversity provides a vast library of potential recognition sites for both commensal and pathogenic organisms. The mucin glycans are thus likely very important for the selection and maintenance of a stable intestinal flora. Here we have explored the O-glycan patterns of the mouse gastrointestinal tract mucins. The mucins from the mucus of the distal and proximal colon, ileum, jejunum, duodenum, and stomach of conventionally raised wild-type (C57BL/6) mice were separated by composite gel electrophoresis. The O-linked glycans were released by reductive elimination and structurally characterized by liquid chromatography-mass spectrometry. The mucins glycans were mostly core 2 type [Galß1-3(GlcNAcß1-6)GalNAcol], but also core 1 (Galß1-3GalNAcol). In the stomach about half of the Muc5ac mucin O-glycans were neutral and many monosulfated, but with a low grade of sialylation and fucosylation. Mouse ileum, jejunum, and duodenum had similar glycan patterns dominated by sialylated and sulfated core 2 glycans, but few fucosylated. Colon was on the other hand dominated by highly charged fucosylated glycans. The distal colon is different from the proximal colon because different biosynthetic pathways are utilized, although sialylated and sulfated glycans were highly abundant in both parts. The sulfation was higher in the distal colon, whereas sialic acid was more common in the proximal colon. Many fucosylated glycans were found in both the proximal and distal colon. Thus the mucin O-glycans vary along the mouse gastrointestinal tract.

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.

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.

Dynamic changes in mucus thickness and ion secretion during Citrobacter rodentium infection and clearance.

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.

Changes on the Caco-2 secretome through differentiation analyzed by 2-D differential in-gel electrophoresis (DIGE).

Colorectal cancer is still a major health burden worldwide, and its diagnosis has not improved in recent years due to a lack of appropriate diagnostic serum markers. Aiming to find new diagnostic proteins, we applied the proteomic DIGE technology to analyze changes in the secretome before/after differentiation of the colon adenocarcinoma Caco-2 cell line, an accepted in vitro model to study colorectal tumorigenesis. When the secretomes from undifferentiated (tumor-like) and differentiated cells (resembling healthy enterocytes) were compared, we found 96 spots differentially expressed. After MS/MS analysis, 22 spots corresponding to 15 different proteins were identified. Principal component analysis demonstrated these 22 spots could serve as a discriminatory panel between the tumor-like and normal-like cells. Among the identified proteins, the translationally-controlled tumor protein (TCTP), the transforming growth factor-beta-induced protein ig-h3 (TGFßIp), and the Niemann-Pick disease type C2 protein (NPC2) are interesting candidates for future studies focused on their utility as serum biomarkers of colorectal cancer.

Proteomic study of the mucin granulae in an intestinal goblet cell model.

Goblet cells specialize in producing and secreting mucus with its main component, mucins. An inducible goblet-like cell line was used for the purification of the mucus vesicles stored in these cells by density gradient ultracentrifugation, and their proteome was analyzed by nanoLC-MS and MS/MS. Although the density of these vesicles coincides with others, it was possible to reveal a number of proteins that after immunolocalization on colon tissue and functional analyses were likely to be linked to the MUC2 vesicles. Most of the proteins were associated with the vesicle membrane or their outer surface. The ATP6AP2, previously suggested to be associated with vesicular proton pumps, was colocalized with MUC2 without other V-ATPase proteins and, thus, probably has roles in mucin vesicle function yet to be discovered. FAM62B, known to be a calcium-sensitive protein involved in vesicle fusion, also colocalized with the MUC2 vesicles and is probably involved in unknown ways in the later events of the MUC2 vesicles and their secretion.

Composition and functional role of the mucus layers in the intestine.

In discussions on intestinal protection, the protective capacity of mucus has not been very much considered. The progress in the last years in understanding the molecular nature of mucins, the main building blocks of mucus, has, however, changed this. The intestinal enterocytes have their apical surfaces covered by transmembrane mucins and the whole intestinal surface is further covered by mucus, built around the gel-forming mucin MUC2. The mucus of the small intestine has only one layer, whereas the large intestine has a two-layered mucus where the inner, attached layer has a protective function for the intestine, as it is impermeable to the luminal bacteria.

Selection of putative colorectal cancer markers by applying PCA on the soluble proteome of tumors: NDK A as a promising candidate.

Aiming to find new tumor markers for colorectal cancer (CRC), we applied proteomic methodologies to compare the soluble sub-proteome of healthy and tumoral colorectal mucosa. Out of 91 differentially expressed proteins, 23 were selected by principal component analysis (PCA) as the major contributors to the overall difference detected. After MS/MS analysis, 16 proteins were identified. From those, we chose 14-3-3-zeta/delta, retinoblastoma-binding protein 4 (RBBP-4), DJ-1, and nucleoside diphosphate kinase A (NDK A) for further studies, on the basis of their levels and known implication in cancer. Specific immunodetection demonstrated only the NDK A levels allowed to differentiate healthy mucosa from tumor tissue in all the patients. Hence, we used the colon cancer cell line Caco-2 to study the relationship between NDK A and colon cell tumorigenesis, finding it over-expressed in undifferentiated (tumor-like) cells regarding the differentiated ones. Noticeably, we also found increased levels of the NDK A in the secretome of tumor-like cells and, as expected, indications of higher levels of NDK A in the serum of CRC patients. In conclusion, the four validated proteins could constitute a panel of tissue markers for CRC, being the NDK A the most interesting candidate for further serum biomarker studies.