| 1. |
- Arike, Liisa, et al.
(författare)
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Identifying transglutaminase reaction products via mass spectrometry as exemplified by the MUC2 mucin - Pitfalls and traps
- 2020
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 0003-2697. ; 597
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Tidskriftsartikel (refereegranskat)abstract
- In order to demonstrate transglutaminase activity in biological samples immunological as well as glutamine- and amine-donor based assays are commonly used. However, the identification of the transglutaminase reaction product, i. e. the isopeptide cross-linked peptides/proteins or the deamidated protein/peptide are often neglected. This article describes a workflow for the detection of the products of transglutaminase-catalyzed reactions. In particular, possible pitfalls and traps that can arise during the mass spectrometry-based identification of isopeptide cross-links are addressed and characterised on actual samples.
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| 2. |
- Arike, Liisa, et al.
(författare)
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Intestinal Muc2 mucin O-glycosylation is affected by microbiota and regulated by differential expression of glycosyltranferases
- 2017
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 27:4, s. 318-328
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal cells are covered by mucus. In the small intestine, a single unattached mucus is present whereas the colon has both an inner attached mucus layer and an outer loose mucus. The attached mucus of the colon is impenetrable to bacteria while the loose mucus acts as a habitat for commensal bacteria. In germ-free (GF) mice, small intestinal mucus is attached to the epithelium and the inner colon mucus is penetrable. O-glycosylation plays an important role in the host-microbiota interactions as the commensal bacteria use glycans as nutrient sources and attachment sites. While mucus protein composition is relatively homogenous along the intestine, its main component the Muc2 mucin shows regiospecific O-glycan patterns. We have now analyzed the glycosyltransferase relative concentrations in the epithelial cells along the intestine in GF and conventionally raised mice and compared this with the O-glycans formed. As Muc2 is the main O-glycosylated product in mucus, we made the simplified assumption that most of the glycosyltransferases found in the epithelial cells are involved in Muc2 O-glycan biosynthesis. The O-glycosyltransferase abundances along the intestine correlated well with the Muc2 O-glycan patterns. Some of the glycosyltransferases involved in the O-glycan elongation were decreased in GF mice, something that is in concordance with the observed shorter Muc2 O-glycans.
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| 3. |
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| 4. |
- Arike, Liisa, et al.
(författare)
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Protein Turnover in Epithelial Cells and Mucus along the Gastrointestinal Tract Is Coordinated by the Spatial Location and Microbiota
- 2020
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 30:4, s. 1077-1087
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 5. |
- Arike, Liisa, et al.
(författare)
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The Densely O-Glycosylated MUC2 Mucin Protects the Intestine and Provides Food for the Commensal Bacteria.
- 2016
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Ingår i: Journal of molecular biology. - : Elsevier BV. - 1089-8638 .- 0022-2836. ; 428:16, s. 3221-3229
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Forskningsöversikt (refereegranskat)abstract
- All mucins are highly O-glycosylated by variable glycans depending on species, histoblood group and organ. This makes the intestinal main mucin MUC2 non-degradable by the host digestive system but well by both commensal and pathogenic bacteria. The MUC2 glycans are important for selection of the commensal bacteria and act as a nutritional source for the bacteria; this also helps the host to recover some of the energy spent on constantly renewing the protective mucus layer. Glycosylation is the most diverse and common posttranslational modification of cell surfaces and secreted proteins. N-Glycosylation is most well studied and predictable, whereas O-glycosylation is more diverse and less well understood. O-Glycosylation is also often called mucin-type glycosylation as it is typical for mucins that often have more than 80% of the mass as O-glycans. This review will discuss the mucin-type O-glycosylation and especially the O-glycosylation of human and mice intestinal mucin MUC2 in relation to bacteria and disease.
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| 6. |
- Birchenough, George M. H., et al.
(författare)
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Muc2-dependent microbial colonization of the jejunal mucus layer is diet sensitive and confers local resistance to enteric pathogen infection
- 2023
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Ingår i: Cell Reports. - Cambridge : Elsevier BV. - 2211-1247. ; 42:2
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Tidskriftsartikel (refereegranskat)abstract
- Intestinal mucus barriers normally prevent microbial infections but are sensitive to diet-dependent changes in the luminal environment. Here we demonstrate that mice fed a Western-style diet (WSD) suffer regiospe-cific failure of the mucus barrier in the small intestinal jejunum caused by diet-induced mucus aggregation. Mucus barrier disruption due to either WSD exposure or chromosomal Muc2 deletion results in collapse of the commensal jejunal microbiota, which in turn sensitizes mice to atypical jejunal colonization by the enteric pathogen Citrobacter rodentium. We illustrate the jejunal mucus layer as a microbial habitat, and link the re-giospecific mucus dependency of the microbiota to distinctive properties of the jejunal niche. Together, our data demonstrate a symbiotic mucus-microbiota relationship that normally prevents jejunal pathogen colo-nization, but is highly sensitive to disruption by exposure to a WSD.
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| 7. |
- Blacher, E., et al.
(författare)
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Potential roles of gut microbiome and metabolites in modulating ALS in mice
- 2019
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Ingår i: Nature. - : Springer Science and Business Media LLC. - 0028-0836 .- 1476-4687. ; 572:7770
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Tidskriftsartikel (refereegranskat)abstract
- Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder, in which the clinical manifestations may be influenced by genetic and unknown environmental factors. Here we show that ALS-prone Sod1 transgenic (Sod1-Tg) mice have a pre-symptomatic, vivarium-dependent dysbiosis and altered metabolite configuration, coupled with an exacerbated disease under germ-free conditions or after treatment with broad-spectrum antibiotics. We correlate eleven distinct commensal bacteria at our vivarium with the severity of ALS in mice, and by their individual supplementation into antibiotic-treated Sod1-Tg mice we demonstrate that Akkermansia muciniphila (AM) ameliorates whereas Ruminococcus torques and Parabacteroides distasonis exacerbate the symptoms of ALS. Furthermore, Sod1-Tg mice that are administered AM are found to accumulate AM-associated nicotinamide in the central nervous system, and systemic supplementation of nicotinamide improves motor symptoms and gene expression patterns in the spinal cord of Sod1-Tg mice. In humans, we identify distinct microbiome and metabolite configurations-including reduced levels of nicotinamide systemically and in the cerebrospinal fluid-in a small preliminary study that compares patients with ALS with household controls. We suggest that environmentally driven microbiome-brain interactions may modulate ALS in mice, and we call for similar investigations in the human form of the disease.
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| 8. |
- Erickson, N. A., et al.
(författare)
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The Goblet Cell Protein Clca1 (Alias mClca3 or Gob-5) Is Not Required for Intestinal Mucus Synthesis, Structure and Barrier Function in Naive or DSS-Challenged Mice
- 2015
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Ingår i: Plos One. - : Public Library of Science (PLoS). - 1932-6203. ; 10:7
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Tidskriftsartikel (refereegranskat)abstract
- The secreted, goblet cell-derived protein Clca1 (chloride channel regulator, calcium-activated-1) has been linked to diseases with mucus overproduction, including asthma and cystic fibrosis. In the intestine Clca1 is found in the mucus with an abundance and expression pattern similar to Muc2, the major structural mucus component. We hypothesized that Clca1 is required for the synthesis, structure or barrier function of intestinal mucus and therefore compared wild type and Clca1-deficient mice under naive and at various time points of DSS (dextran sodium sulfate)-challenged conditions. The mucus phenotype in Clca1-deficient compared to wild type mice was systematically characterized by assessment of the mucus protein composition using proteomics, immunofluorescence and expression analysis of selected mucin genes on mRNA level. Mucus barrier integrity was assessed in-vivo by analysis of bacterial penetration into the mucus and translocation into sentinel organs combined analysis of the fecal microbiota and ex-vivo by assessment of mucus penetrability using beads. All of these assays revealed no relevant differences between wild type and Clca1-deficient mice under steady state or DSS-challenged conditions in mouse colon. Clca1 is not required for mucus synthesis, structure and barrier function in the murine colon.
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| 9. |
- Fakih, Dalia, et al.
(författare)
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Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs
- 2018
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Ingår i: Jci Insight. - : American Society for Clinical Investigation. - 2379-3708. ; 3:17
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 10. |
- Jabbar, Karolina S., et al.
(författare)
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Highly Accurate Identification of Cystic Precursor Lesions of Pancreatic Cancer Through Targeted Mass Spectrometry: A Phase IIc Diagnostic Study
- 2018
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Ingår i: Clinical Oncology. - : American Society of Clinical Oncology (ASCO). - 0936-6555 .- 1433-2981. ; 36:4, s. 367-375
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Tidskriftsartikel (refereegranskat)abstract
- Purpose Pancreatic cystic lesions are common incidental findings on imaging, but up to half may be forerunners of pancreatic cancer. Therefore, accurate differential diagnosis is crucial for correct patient management. Unfortunately, currently available diagnostic methods cannot robustly identify premalignant and malignant pancreatic cystic lesions. Cyst fluid samples obtained by routine endoscopic ultrasound-guided aspiration were used for the analyses. In a cohort of 24 patients, eight biomarker candidates for malignant potential and high-grade dysplasia/cancer were identified by an explorative proteomic approach. Subsequently, a quantitative analysis, using 30 heavy-labeled peptides from the biomarkers and parallel reaction monitoring mass spectrometry, was devised, tested in a training cohort of 80, and prospectively evaluated in a validation cohort of 68 patients. End points were surgical pathology diagnosis/clinical follow-up. Diagnostic assessments were blinded to mass spectrometry results. The optimal set of markers for detecting malignant potential was a panel of peptides from mucin-5AC and mucin-2, which could discriminate premalignant/malignant lesions from benign with an accuracy of 97% (95% CI, 89% to 99%) in the validation cohort. This result compared favorably with the accuracy of standard analyses: cyst fluid carcinoembryonic antigen (61%; 95% CI, 46% to 74%; P <.001) and cytology (84%; 95% CI, 71% to 92%; P =.02). A combination of proteins mucin-5AC and prostate stem-cell antigen could identify high-grade dysplasia/cancer with an accuracy of 96% (95% CI, 90% to 99%), and detected 95% of malignant/severely dysplastic lesions, compared with 35% and 50% for carcinoembryonic antigen and cytology (P <.001 and P =.003, respectively). Targeted mass spectrometry analysis of just three cyst fluid biomarkers provides highly accurate identification and assessment of cystic precursors to pancreatic adenocarcinoma. Additional studies should determine whether the method can facilitate timely cancer diagnosis, successful intervention, and prevention.
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| 11. |
- Johansson, Malin E V, 1971, et al.
(författare)
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Normalization of Host Intestinal Mucus Layers Requires Long-Term Microbial Colonization
- 2015
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Ingår i: Cell Host & Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 18:5, s. 582-592
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 12. |
- Layunta, Elena, et al.
(författare)
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IL-22 promotes the formation of a MUC17 glycocalyx barrier in the postnatal small intestine during weaning
- 2021
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Ingår i: Cell Reports. - : Elsevier BV. - 2211-1247. ; 34:7
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Tidskriftsartikel (refereegranskat)abstract
- The intestine is under constant exposure to chemicals, antigens, and microorganisms from the external environment. Apical aspects of transporting epithelial cells (enterocytes) form a brush-border membrane (BBM), shaped by packed microvilli coated with a dense glycocalyx. We present evidence showing that the glycocalyx forms an epithelial barrier that prevents exogenous molecules and live bacteria from gaining access to BBM. We use a multi-omics approach to investigate the function and regulation of membrane mucins exposed on the BBM during postnatal development of the mouse small intestine. Muc17 is identified as a major membrane mucin in the glycocalyx that is specifically upregulated by IL-22 as part of an epithelial defense repertoire during weaning. High levels of IL-22 at time of weaning reprogram neonatal postmitotic progenitor enterocytes to differentiate into Muc17-expressing enterocytes, as found in the adult intestine during homeostasis. Our findings propose a role for Muc17 in epithelial barrier function in the small intestine.
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| 13. |
- Nyström, Elisabeth E. L., et al.
(författare)
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An intercrypt subpopulation of goblet cells is essential for colonic mucus barrier function
- 2021
-
Ingår i: Science. - : American Association for the Advancement of Science (AAAS). - 0036-8075 .- 1095-9203. ; 372:6539
-
Tidskriftsartikel (refereegranskat)abstract
- The intestinal mucus layer, an important element of epithelial protection, is produced by goblet cells. Intestinal goblet cells are assumed to be a homogeneous cell type. In this study, however, we delineated their specific gene and protein expression profiles and identified several distinct goblet cell populations that form two differentiation trajectories. One distinct subtype, the intercrypt goblet cells (icGCs), located at the colonic luminal surface, produced mucus with properties that differed from the mucus secreted by crypt-residing goblet cells. Mice with defective icGCs had increased sensitivity to chemically induced colitis and manifested spontaneous colitis with age. Furthermore, alterations in mucus and reduced numbers of icGCs were observed in patients with both active and remissive ulcerative colitis, which highlights the importance of icGCs in maintaining functional protection of the epithelium.
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| 14. |
- Nyström, Elisabeth E. L., et al.
(författare)
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Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity
- 2018
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Ingår i: Ebiomedicine. - : Elsevier BV. - 2352-3964. ; 33, s. 134-143
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Tidskriftsartikel (refereegranskat)abstract
- Many epithelial surfaces of the body are covered with protective mucus, and disrupted mucus homeostasis is coupled to diseases such as ulcerative colitis, helminth infection, cystic fibrosis, and chronic obstructive lung disease. However, little is known how a balanced mucus system is maintained. By investigating the involvement of proteases in colonic mucus dynamics we identified metalloprotease activity to be a key contributor to mucus expansion. The effect was mediated by calcium-activated chloride channel regulator 1 (CLCA1) as application of recombinant CLCA1 on intestinal mucus in freshly dissected tissue resulted in increased mucus thickness independently of ion and mucus secretion, but dependent on its metallohydrolase activity. Further, CLCA1 modulated mucus dynamics in both human and mouse, and knock-out of CLCA1 in mice was compensated for by cysteine proteases. Our results suggest that CLCA1 is involved in intestinal mucus homeostasis by facilitating processing and removal of mucus to prevent stagnation. In light of our findings, we suggest future studies to investigate if upregulation of CLCA1 in diseases associated with mucus accumulation could facilitate removal of mucus in an attempt to maintain homeostasis. (C) 2018 The Authors. Published by Elsevier B.V.
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| 15. |
- Nyström, Elisabeth E. L., et al.
(författare)
-
Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2-processing properties
- 2019
-
Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 294:45, s. 17075-17089
-
Tidskriftsartikel (refereegranskat)abstract
- Calcium-activated chloride channel regulator 1 (CLCA1) is one of the major nonmucin proteins found in intestinal mucus. It is part of a larger family of CLCA proteins that share highly conserved features and domain architectures. The CLCA domain arrangement is similar to proteins belonging to the ADAM (a disintegrin and metalloproteinase) family, known to process extracellular matrix proteins. Therefore, CLCA1 is an interesting candidate in the search for proteases that process intestinal mucus. Here, we investigated CLCA1's biochemical properties both in vitro and in mucus from mouse and human colon biopsy samples. Using immunoblotting with CLCA1-specific antibodies and recombinant proteins, we observed that the CLCA1 C-terminal self-cleavage product forms a disulfide-linked dimer that noncovalently interacts with the N-terminal part of CLCA1, which further interacts to form oligomers. We also characterized a second, more catalytically active, N-terminal product of CLCA1, encompassing the catalytic domain together with its von Willebrand domain type A (VWA). This fragment was unstable but could be identified in freshly prepared mucus. Furthermore, we found that CLCA1 can cleave the N-terminal part of the mucus structural component MUC2. We propose that CLCA1 regulates the structural arrangement of the mucus and thereby takes part in the regulation of mucus processing.
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| 16. |
- Raba, Grete, et al.
(författare)
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Metaproteomics reveals parallel utilization of colonic mucin glycans and dietary fibers by the human gut microbiota
- 2024
-
Ingår i: ISCIENCE. - 2589-0042. ; 27:6
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Tidskriftsartikel (refereegranskat)abstract
- A diet lacking dietary fibers promotes the expansion of gut microbiota members that can degrade host glycans, such as those on mucins. The microbial foraging on mucin has been associated with disruptions of the gut -protective mucus layer and colonic inflammation. Yet, it remains unclear how the co -utilization of mucin and dietary fibers affects the microbiota composition and metabolic activity. Here, we used 14 dietary fibers and porcine colonic and gastric mucins to study the dynamics of mucin and dietary fiber utilization by the human fecal microbiota in vitro . Combining metaproteome and metabolites analyses revealed the central role of the Bacteroides genus in the utilization of complex fibers together with mucin while Akkermansia muciniphila was the main utilizer of sole porcine colonic mucin but not gastric mucin. This study gives a broad overview of the colonic environment in response to dietary and host glycan availability.
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| 17. |
- Schneider, Hannah, et al.
(författare)
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The human transmembrane mucin MUC17 responds to TNF alpha by increased presentation at the plasma membrane
- 2019
-
Ingår i: Biochemical Journal. - : Portland Press Ltd.. - 0264-6021 .- 1470-8728. ; 476, s. 2281-2295
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Tidskriftsartikel (refereegranskat)abstract
- Transmembrane mucin MUC17 is an integral part of the glycocalyx as it covers the brush border membrane of small intestinal enterocytes and presents an extended O-glycosylated mucin domain to the intestinal lumen. Here, we identified two unknown phosphorylated serine residues, S4428 and S4492, in the cytoplasmic tail of human MUC17. We have previously demonstrated that MUC17 is anchored to the apical membrane domain via an interaction with the scaffolding protein PDZK1. S4492, localized in the C-terminal PDZ binding motif of MUC17, was mutated to generate phosphomimetic and phosphodeficient variants of MUC17. Using Caco-2 cells as a model system, we found that induction of an inflammatory state by long-term stimulation with the proinflammatory cytokine TNF alpha resulted in an increase of MUC17 protein levels and enhanced insertion of MUC17 and its two phospho-variants into apical membranes. Up-regulation and apical insertion of MUC17 was followed by shedding of MUC17-containing vesicles. Transmembrane mucins have previously been shown to play a role in the prevention of bacterial colonization by acting as sheddable decoys for encroaching bacteria. Overexpression and increased presentation at the plasma membrane of wild-type MUC17 and its phosphodeficient variant MUC17 S-4492A protected Caco-2 cells against adhesion of enteropathogenic Escherichia coli, indicating that C-terminal phosphorylation of MUC17 may play a functional role in epithelial cell protection. We propose a new function for MUC17 in inflammation, where MUC17 acts as a second line of defense by preventing attachment of bacteria to the epithelial cell glycocalyx in the small intestine.
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| 18. |
- Schröder, Björn O., et al.
(författare)
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Bifidobacteria or Fiber Protects against Diet-Induced Microbiota-Mediated Colonic Mucus Deterioration
- 2018
-
Ingår i: Cell Host & Microbe. - : Elsevier BV. - 1931-3128 .- 1934-6069. ; 23:1, s. 27-40
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Tidskriftsartikel (refereegranskat)abstract
- Diet strongly affects gut microbiota composition, and gut bacteria can influence the colonic mucus layer, a physical barrier that separates trillions of gut bacteria from the host. However, the interplay between a Western style diet (WSD), gut microbiota composition, and the intestinal mucus layer is less clear. Here we show that mice fed a WSD have an altered colonic microbiota composition that causes increased penetrability and a reduced growth rate of the inner mucus layer. Both barrier defects can be prevented by transplanting microbiota from chow-fed mice. In addition, we found that administration of Bifidobacterium longum was sufficient to restore mucus growth, whereas administration of the fiber inulin prevented increased mucus penetrability in WSD-fed mice. We hypothesize that the presence of distinct bacteria is crucial for proper mucus function. If confirmed in humans, these findings may help to better understand diseases with an affected mucus layer, such as ulcerative colitis.
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| 19. |
- Sharpen, Jack D. A., et al.
(författare)
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Transglutaminase 3 crosslinks the secreted gel-forming mucus component Mucin-2 and stabilizes the colonic mucus layer
- 2022
-
Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 13:1
-
Tidskriftsartikel (refereegranskat)abstract
- The colonic mucus layer is organized as a two-layered system providing a physical barrier against pathogens and simultaneously harboring the commensal flora. The factorscontributing to the organization of this gel network are not well understood. In this study, the impact of transglutaminase activity on this architecture was analyzed. Here, we show that transglutaminase TGM3 is the major transglutaminase-isoform expressed and synthesized in the colon. Furthermore, intrinsic extracellular transglutaminase activity in the secreted mucus was demonstrated in vitro and ex vivo. Absence of this acyl-transferase activity resulted in faster degradation of the major mucus component the MUC2 mucin and changed the biochemical properties of mucus. Finally, TGM3-deficient mice showed an early increased susceptibility to Dextran Sodium Sulfate-induced colitis. Here, we report that natural isopeptide cross-linking by TGM3 is important for mucus homeostasis and protection of the colon from inflammation, reducing the risk of colitis. © 2022, The Author(s).
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| 20. |
- Song, R., et al.
(författare)
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Trans-Golgi protein TVP23B regulates host-microbe interactions via Paneth cell homeostasis and Goblet cell glycosylation
- 2023
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Ingår i: Nature Communications. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- A key feature in intestinal immunity is the dynamic intestinal barrier, which separates the host from resident and pathogenic microbiota through a mucus gel impregnated with antimicrobial peptides. Using a forward genetic screen, we have found a mutation in Tvp23b, which conferred susceptibility to chemically induced and infectious colitis. Trans-Golgi apparatus membrane protein TVP23 homolog B (TVP23B) is a transmembrane protein conserved from yeast to humans. We found that TVP23B controls the homeostasis of Paneth cells and function of goblet cells, leading to a decrease in antimicrobial peptides and more penetrable mucus layer. TVP23B binds with another Golgi protein, YIPF6, which is similarly critical for intestinal homeostasis. The Golgi proteomes of YIPF6 and TVP23B-deficient colonocytes have a common deficiency of several critical glycosylation enzymes. TVP23B is necessary for the formation of the sterile mucin layer of the intestine and its absence disturbs the balance of host and microbe in vivo. Increased susceptibility to gastrointestinal infections and colitis can be driven by a failure to maintain intestinal homeostasis. Here, using a forward genetic screen, Song et al. identify and characterise the role of TVP23B in Paneth cell and goblet cell function, and its impact on colitis.
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| 21. |
- van der Post, Sjoerd, 1981, et al.
(författare)
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Metaproteomics Analysis of Host–Microbiota Interfaces
- 2021
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Ingår i: Shotgun Proteomics. - New York, NY : Springer. - 1064-3745.
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Bokkapitel (refereegranskat)abstract
- Metaproteomics of host–microbiome interfaces comprises the analysis of complex mixtures of bacteria, archaea, fungi, and viruses in combination with its host cells. Microbial niches can be found all over the host including the skin, oral cavity, and the intestine and are considered to be essential for the homeostasis. The complex interactions between the host and diverse commensal microbiota are poorly characterized while of great interest as dysbiosis is associated with the development of various inflammatory and metabolic diseases. The metaproteomics workflows to study these interfaces are currently being established, and many challenges remain. The major challenge is the large diversity in species composition that make up the microbiota, which results in complex samples that require extended mass spectrometry analysis time. In addition, current database search strategies are not developed to the size of the search space required for unbiased microbial protein identification. Here, we describe a workflow for the proteomics analysis of microbial niches with a focus on intestinal mucus layer. We will cover step-by-step the sample collection, sample preparation, liquid chromatography–mass spectrometry, and data analysis. © 2021, Springer Science+Business Media, LLC, part of Springer Nature.
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| 22. |
- van der Post, Sjoerd, 1981, et al.
(författare)
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Structural weakening of the colonic mucus barrier is an early event in ulcerative colitis pathogenesis
- 2019
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Ingår i: Gut. - : BMJ. - 0017-5749 .- 1468-3288. ; 68:12, s. 2142-2151
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Tidskriftsartikel (refereegranskat)abstract
- Objective The colonic inner mucus layer protects us from pathogens and commensal-induced inflammation, and has been shown to be defective in active UC. The aim of this study was to determine the underlying compositional alterations, their molecular background and potential contribution to UC pathogenesis. Design In this single-centre case-control study, sigmoid colon biopsies were obtained from patients with UC with ongoing inflammation (n=36) or in remission (n=28), and from 47 patients without colonic disease. Mucus samples were collected from biopsies ex vivo, and their protein composition analysed by nanoliquid chromatography-tandem mass spectrometry. Mucus penetrability and goblet cell responses to microbial stimulus were assessed in a subset of patients. Results The core mucus proteome was found to consist of a small set of 29 secreted/transmembrane proteins. In active UC, major structural mucus components including the mucin MUC2 (p<0.0001) were reduced, also in non-inflamed segments. Active UC was associated with decreased numbers of sentinel goblet cells and attenuation of the goblet cell secretory response to microbial challenge. Abnormal penetrability of the inner mucus layer was observed in a subset of patients with UC (12/40; 30%). Proteomic alterations in penetrable mucus samples included a reduction of the SLC26A3 apical membrane anion exchanger, which supplies bicarbonate required for colonic mucin barrier formation. Conclusion Core mucus structural components were reduced in active UC. These alterations were associated with attenuation of the goblet cell secretory response to microbial challenge, but occurred independent of local inflammation. Thus, mucus abnormalities are likely to contribute to UC pathogenesis.
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| 23. |
- Vega, Génesis, et al.
(författare)
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Normal calcium-activated anion secretion in a mouse selectively lacking TMEM16A in intestinal epithelium
- 2019
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Ingår i: Frontiers in Physiology. - : Frontiers Media SA. - 1664-042X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Calcium-activated anion secretion is expected to ameliorate cystic fibrosis, a genetic disease that carries an anion secretory defect in exocrine tissues. Human patients and animal models of the disease that present a mild intestinal phenotype have been postulated to bear a compensatory calcium-activated anion secretion in the intestine. TMEM16A is calcium-activated anion channel whose presence in the intestinal epithelium is contradictory. We aim to test the functional expression of TMEM16A using animal models with Cftr and/or Tmem16a intestinal silencing. Expression of TMEM16A was studied in a wild type and intestinal Tmem16a knockout mice by mRNA-seq, mass-spectrometry, q-PCR, Western blotting and immunolocalization. Calcium-activated anion secretion was recorded in the ileum and proximal colon of these animals including intestinal Cftr knockout and double mutants with dual Tmem16a and Cftr intestinal ablation. Mucus homeostasis was studied by immune-analysis of Mucin-2 (Muc2) and survival curves were recorded. Tmem16a transcript was found in intestine. Nevertheless, protein was barely detected in colon samples. Electrophysiological measurements demonstrated that the intestinal deletion of Tmem16a did not change calcium-activated anion secretion induced by carbachol or ATP in ileum and proximal colon. Muc2 architecture was not altered by Tmem16a silencing as was observed when Cftr was deleted from mouse intestine. Tmem16a silencing neither affected animal survival nor modified the lethality observed in the intestinal Cftr-null mouse. Our results demonstrate that TMEM16A function in the murine intestine is not related to electrogenic calcium-activated anion transport and does not affect mucus homeostasis and survival of animals.
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