167 results on '"Gunnar C. Hansson"'
Search Results
2. Transglutaminase 3 crosslinks the secreted gel-forming mucus component Mucin-2 and stabilizes the colonic mucus layer
- Author
-
Jack D. A. Sharpen, Brendan Dolan, Elisabeth E. L. Nyström, George M. H. Birchenough, Liisa Arike, Beatriz Martinez-Abad, Malin E. V. Johansson, Gunnar C. Hansson, and Christian V. Recktenwald
- Subjects
Mucin-2 ,Transglutaminases ,Multidisciplinary ,Colon ,Science ,Glycobiology ,General Physics and Astronomy ,General Chemistry ,Colitis ,Article ,General Biochemistry, Genetics and Molecular Biology ,digestive system diseases ,Isoenzymes ,Mice ,Mucus ,Transferases ,Animals ,Intestinal diseases - 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 factors contributing 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., The colonic mucus layer is an organized system providing a physical barrier against pathogens and simultaneously harbouring the commensal flora. Here the authors report that transglutaminase 3 activity contributes to homeostasis of the colonic mucus layer and the lack of this enzymatic activity leads to increased susceptibility against DSS-induced colitis in mice.
- Published
- 2022
3. Mucus threads from surface goblet cells clear particles from the airways
- Author
-
Gunnar C. Hansson, Andrea Bähr, Lauren N. Meiss, Lars Ewaldsson, Nikolai Klymiuk, Brendan Dolan, Anna Ermund, and Florian Jaudas
- Subjects
Mucociliary clearance ,Swine ,Respiratory tract ,Video microscopy ,Bronchi ,Mucus bundle ,Diseases of the respiratory system ,fluids and secretions ,Bronchoscopy ,medicine ,Animals ,Goblet cells ,Submucosal glands ,Lung ,Microscopy, Video ,biology ,RC705-779 ,Chemistry ,Research ,Mucin ,Mucins ,respiratory system ,Mucus ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Trachea ,medicine.anatomical_structure ,Mucociliary Clearance ,Models, Animal ,biology.protein - Abstract
Background The mucociliary clearance system driven by beating cilia protects the airways from inhaled microbes and particles. Large particles are cleared by mucus bundles made in submucosal glands by parallel linear polymers of the MUC5B mucins. However, the structural organization and function of the mucus generated in surface goblet cells are poorly understood. Methods The origin and characteristics of different mucus structures were studied on live tissue explants from newborn wild-type (WT), cystic fibrosis transmembrane conductance regulator (CFTR) deficient (CF) piglets and weaned pig airways using video microscopy, Airyscan imaging and electron microscopy. Bronchoscopy was performed in juvenile pigs in vivo. Results We have identified a distinct mucus formation secreted from the surface goblet cells with a diameter less than two micrometer. This type of mucus was named mucus threads. With time mucus threads gathered into larger mucus assemblies, efficiently collecting particles. The previously observed Alcian blue stained mucus bundles were around 10 times thicker than the threads. Together the mucus bundles, mucus assemblies and mucus threads cleared the pig trachea from particles. Conclusions These results demonstrate that normal airway mucus is more complex and has a more variable structural organization and function than was previously understood. These observations emphasize the importance of studying young objects to understand the function of a non-compromised lung.
- Published
- 2021
4. Calcium-activated chloride channel regulator 1 (CLCA1) forms non-covalent oligomers in colonic mucus and has mucin 2–processing properties
- Author
-
Liisa Arike, Gunnar C. Hansson, Erik Ehrencrona, Malin E. V. Johansson, and Elisabeth E. L. Nyström
- Subjects
0301 basic medicine ,Proteases ,metalloprotease ,Colon ,colitis ,medicine.medical_treatment ,Mucin 2 ,mucin-2 (MUC2) ,Biochemistry ,law.invention ,Mice ,03 medical and health sciences ,Protein Domains ,mucin ,Gob-5 ,Chloride Channels ,law ,Disintegrin ,medicine ,Animals ,Humans ,Amino Acid Sequence ,Intestinal Mucosa ,Protein Structure, Quaternary ,intestine ,Molecular Biology ,Mucin-2 ,Metalloproteinase ,Protease ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Mucin ,mouse Clca3 ,von Willebrand type D domain (VWD) ,protease ,Cell Biology ,Mucus ,030104 developmental biology ,Proteolysis ,Recombinant DNA ,biology.protein ,gastrointestinal tract ,Protein Multimerization - 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.
- Published
- 2019
5. A single sulfatase is required to access colonic mucin by a gut bacterium
- Author
-
Robert W. P. Glowacki, Dominic P. Byrne, Mark Reihill, Edwin A. Yates, James A. London, Arnaud Baslé, Nicholas A. Pudlo, Eric C. Martens, Mirjam Czjzek, Ana S. Luis, Stefan Oscarson, Sadie R. Gugel, Patrick A. Eyers, Alan Cartmell, Tristan Barbeyron, Gunnar C. Hansson, Chunsheng Jin, Gabriel V. Pereira, Niclas G. Karlsson, Gurvan Michel, Shaleni Singh, Department of Microbiology and Immunology, University of Michigan, Department of Medical Biochemistry, Institute for Biomedicine, Sahlgrenska Academy, University of Gothenburg, Department of Microbiology and Immunology, University of Michigan Medical School, University of Michigan [Ann Arbor], University of Michigan System-University of Michigan System, Department of Biochemistry and Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Institute for Cell and Molecular Biosciences, Newcastle University, Centre for Synthesis and Chemical Biology, University College Dublin, Laboratoire de Biologie Intégrative des Modèles Marins (LBI2M), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Station biologique de Roscoff (SBR), and Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Male ,Models, Molecular ,Glycan ,Acetylgalactosamine ,Colon ,[SDV]Life Sciences [q-bio] ,Crystallography, X-Ray ,Article ,Substrate Specificity ,Mice ,03 medical and health sciences ,Animals ,Bacteroides ,Humans ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Multidisciplinary ,biology ,Glycobiology ,Sulfatase ,030302 biochemistry & molecular biology ,Mucin ,Mucins ,Galactose ,biology.organism_classification ,Mucus ,Gastrointestinal Microbiome ,Biochemistry ,chemistry ,biology.protein ,Female ,Sulfatases ,Glycoprotein ,Bacteroides thetaiotaomicron ,Bacteria - Abstract
International audience; Humans have co-evolved with a dense community of microbial symbionts that 34 inhabit the lower intestine. In the colon, secreted mucus creates a physical barrier that 35 separates these microbes from the intestinal epithelium. Some gut bacteria are able to 36 utilize mucin glycoproteins, the main mucus component, as a nutrient source. However, 37 it remains unclear which enzymes initiate the degradation of the highly complex O-38 glycans found in mucins. In the colon, these glycans are heavily sulfated, but sulfatases active on colonic mucins have not been identified. Here, we show that sulfatases are essential to the utilization of colonic mucin O-linked glycans by the human gut symbiont Bacteroides thetaiotaomicron. We characterized the activity of 12 different sulfatases encoded by this species, showing that these enzymes collectively are active on all of the known sulfate linkages in colonic O-glycans and even possess the ability to cleave additional linkages not yet known to occur in host glycans. Crystal structures of 3 enzymes provide mechanistic insight into the molecular basis of substrate-specificity. Unexpectedly, we found that a single sulfatase is essential for utilization of sulfated Oglycans in vitro and also plays a major role in vivo. Our results provide insight into the mechanisms of mucin degradation by gut bacteria, an important process for both normal microbial gut colonization and diseases like inflammatory bowel disease (IBD). Sulfatase activity is likely to be a keystone step in bacterial mucin degradation and inhibition of these enzymes may therefore represent a viable therapeutic path for treatment of IBD and other diseases.
- Published
- 2021
6. The IgGFc-binding protein FCGBP is secreted with all GDPH sequences cleaved but maintained by interfragment disulfide bonds
- Author
-
Erik Ehrencrona, Christian V. Recktenwald, Sergio Trillo-Muyo, Malin Bäckström, Pablo Gallego, Malin Johansson, Maria-Jose Garcia-Bonete, Ana M. Rodríguez-Piñeiro, Sjoerd van der Post, and Gunnar C. Hansson
- Subjects
0301 basic medicine ,MUC5AC, mucin-5AC ,MUC2, mucin-2 (Muc2, mouse) ,vWF, von Willebrand factor ,Biochemistry ,von Willebrand domain ,chemistry.chemical_compound ,PVDF, polyvinylidene difluoride ,Mice ,Cricetinae ,Disulfides ,Intestinal Mucosa ,Peptide sequence ,EndoH, endoglycosidase H ,biology ,Chemistry ,respiratory system ,GDPH, Gly-Asp-Pro-His ,Chaotropic agent ,WB, Western blot ,Iodoacetamide ,GuHCl, guanidinium chloride ,Research Article ,IgG, immunoglobulin G ,vWD, von Willebrand D domain ,CHO Cells ,CHO, Chinese hamster ovary ,03 medical and health sciences ,Endoglycosidase H ,Cricetulus ,Protein Domains ,mucus ,von Willebrand Factor ,Animals ,Humans ,intestinal epithelium ,Molecular Biology ,intestine ,FCGBP, IgGFc-binding protein (Fcgbp, mouse) ,GAPH, Gly-Ala-Pro-His ,Mucin-2 ,030102 biochemistry & molecular biology ,colon ,Binding protein ,Endoplasmic reticulum ,Mucin ,ITH3, inter-alpha-trypsin inhibitor heavy chain 3 ,Cell Biology ,Mucus ,Mice, Inbred C57BL ,030104 developmental biology ,MUC2 ,Proteolysis ,biology.protein ,Immunoglobulin G (IgG) ,IAA, iodoacetamide ,Cell Adhesion Molecules ,disulfide - Abstract
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.
- Published
- 2021
7. Obesity-associated microbiota contributes to mucus layer defects in genetically obese mice
- Author
-
Bjoern O, Schroeder, George M H, Birchenough, Meenakshi, Pradhan, Elisabeth E L, Nyström, Marcus, Henricsson, Gunnar C, Hansson, and Fredrik, Bäckhed
- Subjects
Blood Glucose ,obesity ,gut microbiota ,Colon ,Mice, Obese ,microbiome ,metabolic disease ,Microbiology ,Gastrointestinal Microbiome ,Mice, Inbred C57BL ,Mice ,fluids and secretions ,Glucose ,Phenotype ,host defense ,Mice, Inbred NOD ,mucus ,mucosal immunology ,Animals ,Female ,intestinal epithelium ,Intestinal Mucosa ,barrier dysfunction ,metabolism - Abstract
The intestinal mucus layer is a physical barrier separating the tremendous number of gut bacteria from the host epithelium. Defects in the mucus layer have been linked to metabolic diseases, but previous studies predominantly investigated mucus function during high-caloric/low-fiber dietary interventions, thus making it difficult to separate effects mediated directly through diet quality from potential obesity-dependent effects. As such, we decided to examine mucus function in mouse models with metabolic disease to distinguish these factors. Here we show that, in contrast to their lean littermates, genetically obese (ob/ob) mice have a defective inner colonic mucus layer that is characterized by increased penetrability and a reduced mucus growth rate. Exploiting the coprophagic behavior of mice, we next co-housed ob/ob and lean mice to investigate if the gut microbiota contributed to these phenotypes. Co-housing rescued the defect of the mucus growth rate, whereas mucus penetrability displayed an intermediate phenotype in both mouse groups. Of note, non-obese diabetic mice with high blood glucose levels displayed a healthy colonic mucus barrier, indicating that the mucus defect is obesity- rather than glucose-mediated. Thus, our data suggest that the gut microbiota community of obesity-prone mice may regulate obesity-associated defects in the colonic mucosal barrier, even in the presence of dietary fiber.
- Published
- 2020
8. Mucins and the Microbiome
- Author
-
Gunnar C. Hansson
- Subjects
Glycosylation ,Gene Expression ,Glycocalyx ,Biochemistry ,Article ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,Carbohydrate Conformation ,Animals ,Humans ,Outer mucus layer ,Symbiosis ,030304 developmental biology ,Inner mucus layer ,chemistry.chemical_classification ,0303 health sciences ,Chemistry ,Mucin ,Mucins ,Glycosyltransferases ,Golgi apparatus ,Mucus ,Transmembrane protein ,Cell biology ,Gastrointestinal Microbiome ,Carbohydrate Sequence ,030220 oncology & carcinogenesis ,symbols ,Goblet Cells ,Glycoprotein - Abstract
Generating the barriers that protect our inner surfaces from bacteria and other challenges requires large glycoproteins called mucins. These come in two types, gel-forming and transmembrane, all characterized by large, highly O-glycosylated mucin domains that are diversely decorated by Golgi glycosyltransferases to become extended rodlike structures. The general functions of mucins on internal epithelial surfaces are to wash away microorganisms and, even more importantly, to build protective barriers. The latter function is most evident in the large intestine, where the inner mucus layer separates the numerous commensal bacteria from the epithelial cells. The host's conversion of MUC2 to the outer mucus layer allows bacteria to degrade the mucin glycans and recover the energy content that is then shared with the host. The molecular nature of the mucins is complex, and how they construct the extracellular complex glycocalyx and mucus is poorly understood and a future biochemical challenge.
- Published
- 2020
9. Membrane mucins of the intestine at a glance
- Author
-
Gunnar C. Hansson and Thaher Pelaseyed
- Subjects
Population ,Antimicrobial peptides ,Biology ,Glycocalyx ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Cell Science at A Glance ,Animals ,Humans ,Intestinal Mucosa ,education ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,education.field_of_study ,Host Microbial Interactions ,Mucin ,Mucins ,Epithelial Cells ,Cell Biology ,Mucus ,Epithelium ,Small intestine ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Enterocytes ,chemistry ,030220 oncology & carcinogenesis ,Glycoprotein - Abstract
Membrane mucins cover most mucosal surfaces throughout the human body. The intestine harbors complex population of microorganisms (the microbiota) and numerous exogenous molecules that can harm the epithelium. In the colon, where the microbial burden is high, a mucus barrier forms the first line of defense by keeping bacteria away from the epithelial cells. In the small intestine where the mucus layer is less organized, microbes are kept at bay by peristalsis and antimicrobial peptides. Additionally, a dense glycocalyx consisting of extended and heavily glycosylated membrane mucins covers the surface of enterocytes. Whereas many aspects of mucosal barriers are being discovered, the function of membrane mucins remains a largely overlooked topic, mainly because we lack the necessary reagents and experimental animal models to investigate these large glycoproteins. In this Cell Science at a Glance article and accompanying poster, we highlight central concepts of membrane mucin biology and the role of membrane mucins as integral components of intestinal mucosal barriers. We also present the current consensus concerning the role of membrane mucins in host–microbe interactions. Moreover, we discuss how regulatory circuits that govern membrane mucins in the healthy gut display strong overlap with pathways that are perturbed during chronic inflammation. Finally, we review how dysregulation of intestinal membrane mucins may contribute to human diseases, such as inflammation and cancer.
- Published
- 2020
10. Normal murine respiratory tract has its mucus concentrated in clouds based on the Muc5b mucin
- Author
-
Ana M. Rodríguez-Piñeiro, Christopher M. Evans, Dalia Fakih, Anna Ermund, Sergio Trillo-Muyo, and Gunnar C. Hansson
- Subjects
0301 basic medicine ,Pulmonary and Respiratory Medicine ,Larynx ,Pathology ,medicine.medical_specialty ,Physiology ,Mucociliary clearance ,Respiratory System ,Mucin 5AC ,Fluorescence ,03 medical and health sciences ,0302 clinical medicine ,fluids and secretions ,Physiology (medical) ,medicine ,Animals ,Submucosal glands ,Mucous Membrane ,Chemistry ,Mucin ,Biological Transport ,Cell Biology ,respiratory system ,Mucus ,Mucin-5B ,Mice, Inbred C57BL ,Trachea ,030104 developmental biology ,medicine.anatomical_structure ,030228 respiratory system ,sense organs ,Goblet Cells ,Airway ,Ex vivo ,Respiratory tract ,Research Article - Abstract
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.
- Published
- 2020
- Full Text
- View/download PDF
11. New generation ENaC inhibitors detach cystic fibrosis airway mucus bundles via sodium/hydrogen exchanger inhibition
- Author
-
Robert Tarran, Anna Ermund, Melania Giorgetti, Gunnar C. Hansson, Annika Åstrand, Nikolai Klymiuk, Martin Hemmerling, Lisa Jinton, Anna Malmgren, and Andrea Bähr
- Subjects
Male ,0301 basic medicine ,Epithelial sodium channel ,Sodium-Hydrogen Exchangers ,Cystic Fibrosis ,Swine ,Sodium ,Cystic Fibrosis Transmembrane Conductance Regulator ,chemistry.chemical_element ,Inflammation ,Ileum ,Cystic fibrosis ,Article ,Mice ,03 medical and health sciences ,fluids and secretions ,0302 clinical medicine ,Epithelial Sodium Channel Blockers ,medicine ,Animals ,Epithelial Sodium Channels ,Lung ,Pharmacology ,biology ,Chemistry ,Hydrogen-Ion Concentration ,respiratory system ,medicine.disease ,Mucus ,Epithelial sodium channel blocker ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Bicarbonates ,Sodium–hydrogen antiporter ,030104 developmental biology ,medicine.anatomical_structure ,Animals, Newborn ,biology.protein ,Female ,medicine.symptom ,030217 neurology & neurosurgery - Abstract
Background and Purpose: Cystic fibrosis (CF) is a recessive inherited disease caused by mutations affecting anion transport by the epithelial ion channel cystic fibrosis transmembrane conductance regulator (CFTR). The disease is characterized by mucus accumulation in the airways and intestine, but the major cause of mortality in CF is airway mucus accumulation, leading to bacterial colonization, inflammation and respiratory failure. One of the drug targets under evaluation to alleviate airway mucus obstruction in CF is the epithelial sodium channel, ENaC. Experimental Approach: To explore effects of ENaC inhibitors on mucus properties, we used two model systems to investigate mucus characteristics, mucus attachment in mouse ileum and mucus bundle transport in piglet airways. We quantified mucus attachment in explants from CFTR null (CF) mice and tracheobronchial explants from newborn CFTR null (CF) piglets to evaluate effects of ENaC or sodium/hydrogen exchange (NHE) inhibitors on mucus attachment. Key Results: ENaC inhibitors detached mucus in the CF mouse ileum, although the ileum lacks ENaC expression. This effect was mimicked by two sodium/proton exchange (NHE) inhibitors. Airway mucus bundles were immobile in untreated newborn CFTR null (CF) piglets but were detached by the therapeutic drug candidate AZD5634. Conclusion and Implications: These results suggest that the ENaC inhibitor AZD5634 causes detachment of CF mucus in the ileum and airway via NHE inhibition and that drug design should focus on NHE instead of ENaC inhibition.
- Published
- 2021
12. Postnatal development of the small intestinal mucosa drives age-dependent, regio-selective susceptibility to Escherichia coli K1 infection
- Author
-
Peter W. Taylor, Luci A. Witcomb, Gunnar C. Hansson, George M. H. Birchenough, Alex J. McCarthy, Fatma Dalgakiran, and Malin E. V. Johansson
- Subjects
0301 basic medicine ,Paneth Cells ,Enterocyte ,Science ,030106 microbiology ,Antimicrobial peptides ,Biology ,Article ,Microbiology ,03 medical and health sciences ,Submucosa ,Intestine, Small ,Escherichia coli ,medicine ,Animals ,Intestinal Mucosa ,Cells, Cultured ,Escherichia coli Infections ,Multidisciplinary ,biology.organism_classification ,Mucus ,Epithelium ,Small intestine ,Rats ,Disease Models, Animal ,Blood ,medicine.anatomical_structure ,Animals, Newborn ,Organ Specificity ,Medicine ,Intracellular ,Bacteria - Abstract
The strong age dependency of neonatal systemic infection with Escherichia coli K1 can be replicated in the neonatal rat. Gastrointestinal (GI) colonization of two-day-old (P2) rats leads to invasion of the blood within 48 h of initiation of colonization; pups become progressively less susceptible to infection over the P2-P9 period. We show that, in animals colonized at P2 but not at P9, E. coli K1 bacteria gain access to the enterocyte surface in the mid-region of the small intestine and translocate through the epithelial cell monolayer by an intracellular pathway to the submucosa. In this region of the GI tract, the protective mucus barrier is poorly developed but matures to full thickness over P2-P9, coincident with the development of resistance to invasion. At P9, E. coli K1 bacteria are physically separated from villi by the mucus layer and their numbers controlled by mucus-embedded antimicrobial peptides, preventing invasion of host tissues.
- Published
- 2017
13. Gram-positive bacteria are held at a distance in the colon mucus by the lectin-like protein ZG16
- Author
-
Bjoern O. Schroeder, Joakim H. Bergström, Gunnar C. Hansson, Anna Ermund, George M. H. Birchenough, André Schütte, Gergely Katona, and Malin E. V. Johansson
- Subjects
Proteomics ,0301 basic medicine ,Glycosylation ,Colon ,Gram-positive bacteria ,Motility ,Biology ,Gram-Positive Bacteria ,Bacterial cell structure ,Microbiology ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Lectins ,medicine ,Animals ,Symbiosis ,Inner mucus layer ,Mice, Knockout ,Multidisciplinary ,Membrane Proteins ,Epithelial Cells ,Biological Sciences ,biology.organism_classification ,Mucus ,Epithelium ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,030220 oncology & carcinogenesis ,Host-Pathogen Interactions ,Peptidoglycan ,Digestive System ,Bacteria - Abstract
The distal colon functions as a bioreactor and harbors an enormous amount of bacteria in a mutualistic relationship with the host. The microbiota have to be kept at a safe distance to prevent inflammation, something that is achieved by a dense inner mucus layer that lines the epithelial cells. The large polymeric nets made up by the heavily O-glycosylated MUC2 mucin forms this physical barrier. Proteomic analyses of mucus have identified the lectin-like protein ZG16 (zymogen granulae protein 16) as an abundant mucus component. To elucidate the function of ZG16, we generated recombinant ZG16 and studied Zg16−/− mice. ZG16 bound to and aggregated Gram-positive bacteria via binding to the bacterial cell wall peptidoglycan. Zg16−/− mice have a distal colon mucus layer with normal thickness, but with bacteria closer to the epithelium. Using distal colon explants mounted in a horizontal perfusion chamber we demonstrated that treatment of bacteria with recombinant ZG16 hindered bacterial penetration into the mucus. The inner colon mucus of Zg16−/− animals had a higher load of Gram-positive bacteria and showed bacteria with higher motility in the mucus close to the host epithelium compared with cohoused littermate Zg16+/+. The more penetrable Zg16−/− mucus allowed Gram-positive bacteria to translocate to systemic tissues. Viable bacteria were found in spleen and were associated with increased abdominal fat pad mass in Zg16−/− animals. The function of ZG16 reveals a mechanism for keeping bacteria further away from the host colon epithelium.
- Published
- 2016
14. Potential roles of gut microbiome and metabolites in modulating ALS in mice
- Author
-
Yotam Cohen, Michal Zabari, Gunnar C. Hansson, Noam Bar, Adrian Israelson, Hagit Shapiro, Izhak Levi, Stavros Bashiardes, Alon Harmelin, Alexander Brandis, Niv Zmora, Yael Kuperman, Rotem Ben-Zeev Brik, Daphna Rothschild, Mally Dori-Bachash, Marc Gotkine, Nira Amar, Michael Tsoory, Yotam Harnik, Eran Blacher, Claudia Moresi, Michal Schwartz, Eran Elinav, Uria Mor, Denise Kviatcovsky, Tevie Mehlman, Malin E. V. Johansson, Inbal E. Biton, Liisa Arike, Eran Segal, Leenor Alfahel, Christian Kleimeyer, and Maya Zur
- Subjects
0301 basic medicine ,Male ,Niacinamide ,Transgene ,SOD1 ,Longevity ,Mice, Transgenic ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,0302 clinical medicine ,Superoxide Dismutase-1 ,Verrucomicrobia ,medicine ,Animals ,Germ-Free Life ,Humans ,Microbiome ,Amyotrophic lateral sclerosis ,Symbiosis ,Multidisciplinary ,biology ,Nicotinamide ,Amyotrophic Lateral Sclerosis ,Akkermansia ,biology.organism_classification ,medicine.disease ,3. Good health ,Anti-Bacterial Agents ,Gastrointestinal Microbiome ,Survival Rate ,Disease Models, Animal ,030104 developmental biology ,chemistry ,Immunology ,Dysbiosis ,Female ,030217 neurology & neurosurgery ,Akkermansia muciniphila - 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.
- Published
- 2019
15. The Nlrp6 inflammasome is not required for baseline colonic inner mucus layer formation or function
- Author
-
Joana K, Volk, Elisabeth E L, Nyström, Sjoerd, van der Post, Beatriz M, Abad, Bjoern O, Schroeder, Åsa, Johansson, Frida, Svensson, Sofia, Jäverfelt, Malin E V, Johansson, Gunnar C, Hansson, and George M H, Birchenough
- Subjects
Mice, Knockout ,Mucin-2 ,Colon ,Inflammasomes ,Interleukin-18 ,Mice, Transgenic ,Receptors, Cell Surface ,Inflammatory Bowel Diseases ,digestive system ,Article ,Gastrointestinal Microbiome ,Mice, Inbred C57BL ,stomatognathic diseases ,Mucus ,fluids and secretions ,Animals ,Goblet Cells ,Intestinal Mucosa ,Research Articles ,Signal Transduction - Abstract
It is thought that the Nlrp6 inflammasome regulates formation of the inner mucus layer (IML) barrier that prohibits contact between the microbiota and colonic epithelial cells. Using microbiota-controlled mice and combined ex vivo/in vivo IML analytical approaches, Volk et al. delineate the relative roles of the inflammasome and microbiota in shaping IML formation and function., The inner mucus layer (IML) is a critical barrier that protects the colonic epithelium from luminal threats and inflammatory bowel disease. Innate immune signaling is thought to regulate IML formation via goblet cell Nlrp6 inflammasome activity that controls secretion of the mucus structural component Muc2. We report that isolated colonic goblet cells express components of several inflammasomes; however, analysis of IML properties in multiple inflammasome-deficient mice, including littermate-controlled Nlrp6−/−, detect a functional IML barrier in all strains. Analysis of mice lacking inflammasome substrate cytokines identifies a defective IML in Il18−/− mice, but this phenotype is ultimately traced to a microbiota-driven, Il18-independent effect. Analysis of phenotypic transfer between IML-deficient and IML-intact mice finds that the Bacteroidales family S24-7 (Muribaculaceae) and genus Adlercrutzia consistently positively covary with IML barrier function. Together, our results demonstrate that baseline IML formation and function is independent of inflammasome activity and highlights the role of the microbiota in determining IML barrier function.
- Published
- 2019
16. Assembly, Release, and Transport of Airway Mucins in Pigs and Humans
- Author
-
Sergio Trillo-Muyo, Gunnar C. Hansson, and Anna Ermund
- Subjects
0301 basic medicine ,Pulmonary and Respiratory Medicine ,Lung Diseases ,Mucociliary clearance ,Swine ,Respiratory Mucosa ,Cystic fibrosis ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Humans ,Secretion ,Respiratory system ,Submucosal glands ,AnnalsATS Supplements: Thirty-Third Transatlantic Airway Conference. Secreted Mucins in Lung Diseases ,business.industry ,Cilium ,Secretory Vesicles ,Mucin ,Mucins ,respiratory system ,medicine.disease ,Mucus ,Cell biology ,Disease Models, Animal ,030104 developmental biology ,Mucociliary Clearance ,business ,030217 neurology & neurosurgery - Abstract
The respiratory system is protected from inhaled particles and microbes by the mucociliary system. This system differs between animal species, where pigs and humans have numerous submucosal glands. The polymer-forming mucin, MUC5B, is packed in a highly organized way in granules of the mucus-secreting cells in the glands. Upon secretion, the packed MUC5B is flushed out by a chloride- and bicarbonate-rich fluid from the cystic fibrosis transmembrane conductance regulator-expressing serosal cells located at the most distal part of the gland. The bicarbonate raises the pH and removes calcium from the N terminus of MUC5B, allowing the mucin to be pulled out into a linear polymer. Thousands of such polymers gather in bundles in the submucosal gland duct, and these bundles appear at the opening of the glands. They are moved by the beating cilia, and sweep over the airway surface and are patchily coated with the MUC5AC mucin from the surface goblet cells. The movement of these bundles is controlled by the MUC5AC mucin attachment/detachment to the goblet cells. Thus, higher animals with submucosal glands and large diameters of the proximal airways are efficiently cleaned by the thick mucus bundles sweeping the airway surface and moving particles and bacteria toward the larynx.
- Published
- 2018
17. Immunological aspects of intestinal mucus and mucins
- Author
-
Gunnar C. Hansson and Malin E. V. Johansson
- Subjects
0301 basic medicine ,History ,digestive system ,Article ,Education ,03 medical and health sciences ,0302 clinical medicine ,Immune system ,Antigen ,medicine ,Animals ,Humans ,Intestinal Mucosa ,Inflammation ,Lamina propria ,Goblet cell ,Chemistry ,Mucin ,Mucins ,respiratory system ,Mucus ,Small intestine ,Gastrointestinal Microbiome ,Computer Science Applications ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Mucosal immunology ,030220 oncology & carcinogenesis ,Immunology ,Goblet Cells - Abstract
A number of mechanisms ensure that the intestine is protected from pathogens and also against our own intestinal microbiota. The outermost of these is the secreted mucus, which entraps bacteria and prevents their translocation into the tissue. Mucus contains many immunomodulatory molecules and is largely produced by the goblet cells. These cells are highly responsive to the signals they receive from the immune system and are also able to deliver antigens from the lumen to dendritic cells in the lamina propria. In this Review, we will give a basic overview of mucus, mucins and goblet cells, and explain how each of these contributes to immune regulation in the intestine.
- Published
- 2016
18. The Reduction-insensitive Bonds of the MUC2 Mucin Are Isopeptide Bonds
- Author
-
Gunnar C. Hansson and Christian V. Recktenwald
- Subjects
0301 basic medicine ,Colon ,Tissue transglutaminase ,Lysine ,Glycobiology and Extracellular Matrices ,CHO Cells ,Mucin 2 ,Biology ,digestive system ,Biochemistry ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,Cricetulus ,Protein Domains ,Biosynthesis ,Intestinal mucosa ,Cricetinae ,Animals ,Humans ,Intestinal Mucosa ,Molecular Biology ,Mucin-2 ,Mucin ,Epithelial Cells ,Cell Biology ,respiratory system ,Mucus ,digestive system diseases ,Isopeptidase activity ,030104 developmental biology ,chemistry ,biology.protein ,Oxidation-Reduction ,Protein Processing, Post-Translational - Abstract
The main structural component of the mucus in the gastrointestinal tract is the MUC2 mucin. It forms large networks that in colon build the loose outer mucous layer that provides the habitat for the commensal flora and the inner mucous layer that protects the epithelial cells by being impenetrable to bacteria. The epithelial cells in mice lacking MUC2 are not adequately protected from bacteria, resulting in inflammation and the development of colon cancer as found in human ulcerative colitis. Correct processing of the MUC2 mucin is the basis for the building of these protective networks. During the biosynthesis of the MUC2 mucin, post-translational modifications are formed resulting in reduction-insensitive bonds between MUC2 monomers. By the use of γ-glutamyltranspeptidase and isopeptidase activity in leech saliva, we could show that the molecular nature of these reduction-insensitive bonds is isopeptide bonds formed between side chains of lysine and glutamine. Transglutaminase 2 has an affinity to the MUC2 CysD2 domain in the nanomolar range and can catalyze its cross-linking. By using mass spectrometry, we identified MUC2 residues involved in this cross-linking. This shows for the first time that transamidation is not only stabilizing the skin and the fibrin clot, but is also important for the correct intracellular processing of MUC2 to generate protective mucus.
- Published
- 2016
19. Mucus Architecture and Near-Surface Swimming Affect Distinct Salmonella Typhimurium Infection Patterns along the Murine Intestinal Tract
- Author
-
Markus, Furter, Mikael E, Sellin, Gunnar C, Hansson, and Wolf-Dietrich, Hardt
- Subjects
Mice, Knockout ,Salmonella typhimurium ,Mucin-2 ,Colon ,Ubiquitin-Protein Ligases ,live imaging ,respiratory system ,baceterial infections ,digestive system ,Epithelium ,Article ,mucus layer ,DNA-Binding Proteins ,Intestines ,Mice, Inbred C57BL ,intestinal infection ,Mice ,Mucus ,fluids and secretions ,bacteria tracking ,intestinal mucus ,Salmonella Infections ,Animals ,near-surface swimming ,Cecum - Abstract
Summary Mucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis., Graphical Abstract, Highlights • Live imaging of Salmonella near-surface swimming on mouse colon inner mucus layer • Colon inner mucus layer traversal requires mucus breaches and flagellar propulsion • The mouse cecum lacks a continuous mucus layer, leaving epithelium tips uncovered • Exposed cecum epithelium tips are a hotspot for Salmonella infection, Using live microscopy, Furter et al. describe how the enteropathogen Salmonella Typhimurium crosses the protective intestinal mucus layer of its murine host. Flagella-driven motility, the mucus architecture, and its distribution determine where the pathogen preferentially infects the gut epithelium.
- Published
- 2018
20. Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs
- Author
-
Sonya Jackson, Christopher M. Evans, Joan Antoni Fernández-Blanco, Dalia Fakih, Dave Singh, Annika Åstrand, Gunnar C. Hansson, Liisa Arike, Christopher McCrae, Anna Ermund, Ana M. Rodríguez-Piñeiro, Elin Skansebo, Beatriz Martínez-Abad, and James Root
- Subjects
0301 basic medicine ,Cystic Fibrosis ,Respiratory Mucosa ,Cystic fibrosis ,Microbiology ,03 medical and health sciences ,Mice ,Pulmonary Disease, Chronic Obstructive ,0302 clinical medicine ,fluids and secretions ,medicine ,Animals ,Humans ,Lung ,Mice, Knockout ,COPD ,biology ,Bacteria ,Pancreatic Elastase ,Chemistry ,Elastase ,Epithelial Cells ,General Medicine ,respiratory system ,medicine.disease ,biology.organism_classification ,Mucus ,Mucin-5B ,Hypertonic saline ,respiratory tract diseases ,Mice, Inbred C57BL ,Disease Models, Animal ,030104 developmental biology ,medicine.anatomical_structure ,030228 respiratory system ,Pseudomonas aeruginosa ,Respiratory epithelium ,Female ,Bronchoalveolar Lavage Fluid ,Respiratory tract ,Research Article - 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.
- Published
- 2018
21. Calcium-activated Chloride Channel Regulator 1 (CLCA1) Controls Mucus Expansion in Colon by Proteolytic Activity
- Author
-
Elisabeth E L, Nyström, George M H, Birchenough, Sjoerd, van der Post, Liisa, Arike, Achim D, Gruber, Gunnar C, Hansson, and Malin E V, Johansson
- Subjects
Mice, Knockout ,Proteome ,Colon ,respiratory system ,Asthma ,Mice ,Mucus ,fluids and secretions ,mClca3 ,Gob-5 ,Chloride Channels ,Proteolysis ,MUC2 ,Metalloproteases ,Animals ,Homeostasis ,Humans ,COPD ,Research Paper - 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., Graphical Abstract Unlabelled Image, Highlights • Endogenous metalloprotease activity is important for intestinal mucus dynamics. • CLCA1 acts as a metalloprotease in intestinal mucus and this function is independent of ion and mucus secretion. • CLCA1 is involved in the transition from the inner to outer mucus layer in colon. In this article we provide evidence that endogenous enzyme activity is important for normal processing of the intestinal mucus layer, which creates a protective barrier against the vast number of bacteria in the large intestine. CLCA1, a highly abundant intestinal mucus protein, seems to be a key contributor to mucus processing. This role for CLCA1 is different from what was previously described. As mucus clearance is of importance for several diseases, better understanding of mucus processing could be of great importance to develop new therapies.
- Published
- 2018
22. Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers
- Author
-
Sergio, Trillo-Muyo, Harriet E, Nilsson, Christian V, Recktenwald, Anna, Ermund, Caroline, Ridley, Lauren N, Meiss, Andrea, Bähr, Nikolai, Klymiuk, Jeffrey J, Wine, Philip J B, Koeck, David J, Thornton, Hans, Hebert, and Gunnar C, Hansson
- Subjects
Swine ,regulated secretory pathway ,Glycobiology and Extracellular Matrices ,Hydrogen-Ion Concentration ,Mucin-5B ,Recombinant Proteins ,lung ,secretion ,mucin bundle ,Protein Domains ,mucin ,mucus ,EM ,Animals ,Humans ,Calcium ,Protein Multimerization ,Protein Structure, Quaternary ,submucosal gland - Abstract
Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1–D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.
- Published
- 2017
23. Quantitative Imaging of Gut Microbiota Spatial Organization
- Author
-
Gabriel Billings, Michael Sigal, Joshua E. Elias, Gunnar C. Hansson, Manuel R. Amieva, Kristen A. Earle, Justin L. Sonnenburg, Kerwyn Casey Huang, and Joshua S. Lichtman
- Subjects
Cancer Research ,Gut flora ,Immunofluorescence ,Microbiology ,Mice ,Immunology and Microbiology(all) ,Virology ,medicine ,Animals ,Progenitor cell ,Molecular Biology ,Spatial organization ,Bacteriological Techniques ,Helicobacter pylori ,biology ,medicine.diagnostic_test ,Optical Imaging ,Gastrointestinal Microbiome ,biology.organism_classification ,Mucus ,Epithelium ,Diet ,Cell biology ,Gastrointestinal Tract ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Host-Pathogen Interactions ,Carbohydrate Metabolism ,Parasitology - Abstract
SummaryGenomic technologies have significantly advanced our understanding of the composition and diversity of host-associated microbial populations. However, their spatial organization and functional interactions relative to the host have been more challenging to study. Here we present a pipeline for the assessment of intestinal microbiota localization within immunofluorescence images of fixed gut cross-sections that includes a flexible software package, BacSpace, for high-throughput quantification of microbial organization. Applying this pipeline to gnotobiotic and human microbiota-colonized mice, we demonstrate that elimination of microbiota-accessible carbohydrates (MACs) from the diet results in thinner mucus in the distal colon, increased proximity of microbes to the epithelium, and heightened expression of the inflammatory marker REG3β. Measurements of microbe-microbe proximity reveal that a MAC-deficient diet alters monophyletic spatial clustering. Furthermore, we quantify the invasion of Helicobacter pylori into the glands of the mouse stomach relative to host mitotic progenitor cells, illustrating the generalizability of this approach.
- Published
- 2015
24. Colitogenic Bacteroides thetaiotaomicron Antigens Access Host Immune Cells in a Sulfatase-Dependent Manner via Outer Membrane Vesicles
- Author
-
Nicole P. Malvin, Gerard E. Kaiko, Elizabeth A. Cameron, Haerin Jung, Christina A. Hickey, David L. Donermeyer, Marta Wegorzewska, Eric C. Martens, Kristine A. Kuhn, Chunsheng Jin, Thaddeus S. Stappenbeck, Nathan T. Porter, Gunnar C. Hansson, Robert W. P. Glowacki, and Paul M. Allen
- Subjects
Cancer Research ,Inflammation ,Biology ,Microbiology ,Mice ,Immune system ,Antigen ,Virology ,Immunology and Microbiology(all) ,medicine ,Animals ,Bacteroides ,Colitis ,Molecular Biology ,chemistry.chemical_classification ,Antigens, Bacterial ,Secretory Vesicles ,Sulfatase ,medicine.disease ,3. Good health ,Disease Models, Animal ,Enzyme ,chemistry ,Genes, Bacterial ,Host-Pathogen Interactions ,Parasitology ,Sulfatases ,medicine.symptom ,Bacterial outer membrane ,Bacteroides thetaiotaomicron ,Gene Deletion - Abstract
Summary Microbes interact with the host immune system via several potential mechanisms. One essential step for each mechanism is the method by which intestinal microbes or their antigens access specific host immune cells. Using genetically susceptible mice ( dnKO ) that develop spontaneous, fulminant colitis, triggered by Bacteroides thetaiotaomicron ( B. theta ), we investigated the mechanism of intestinal microbial access under conditions that stimulate colonic inflammation. B. theta antigens localized to host immune cells through outer membrane vesicles (OMVs) that harbor bacterial sulfatase activity. We deleted the anaerobic sulfatase maturating enzyme (anSME) from B. theta , which is required for post-translational activation of all B. theta sulfatase enzymes. This bacterial mutant strain did not stimulate colitis in dnKO mice. Lastly, access of B. theta OMVs to host immune cells was sulfatase dependent. These data demonstrate that bacterial OMVs and associated enzymes promote inflammatory immune stimulation in genetically susceptible hosts.
- Published
- 2015
- Full Text
- View/download PDF
25. Hypertonic saline releases the attached small intestinal cystic fibrosis mucus
- Author
-
Lauren N. Meiss, Gunnar C. Hansson, Anna Ermund, Bob J. Scholte, and Cell biology
- Subjects
Male ,Pathology ,medicine.medical_specialty ,Cystic Fibrosis ,Physiology ,Ileum ,Mice, Transgenic ,Biology ,Cystic fibrosis ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Organ Culture Techniques ,fluids and secretions ,mucin ,Physiology (medical) ,Intestine, Small ,medicine ,Animals ,hypertonic saline solution ,ΔF508 ,030304 developmental biology ,Pharmacology ,Saline Solution, Hypertonic ,0303 health sciences ,Mucin ,Original Articles ,respiratory system ,medicine.disease ,Mucus ,Cystic fibrosis transmembrane conductance regulator ,Epithelium ,Hypertonic saline ,Mice, Inbred C57BL ,medicine.anatomical_structure ,biology.protein ,Female ,ileum ,030217 neurology & neurosurgery - Abstract
Hypertonic saline inhalation has become a cornerstone in the treatment of cystic fibrosis (CF), but its effect on CF mucus is still not understood. In CF, mucus stagnates in the airways, causing mucus plugging, and forming a substrate for bacterial invasion. Using horizontal Ussing-type chambers to allow easy access to the tissue, we have recently shown that the small intestinal mucus of CF mice is attached to the epithelium and not freely movable as opposed to normal mucus, thus pointing to a similarity between the CF mucus in the ileum and airways. In the same type of system, we investigated how hypertonic saline affects mucus thickness, attachment and penetrability to fluorescent beads the size of bacteria in ileal explants from the cystic fibrosis transmembrane conductance regulator mutant (ΔF508) mouse, in order to characterize how this common therapy affects mucus properties. Hypertonic saline (1.75–5%) detached the mucus from the epithelium, but the mucus remained impenetrable to beads the size of bacteria. This approach might be used to test other mucolytic interventions in CF.
- Published
- 2015
26. The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin
- Author
-
Anna, Ermund, Lauren N, Meiss, Ana M, Rodriguez-Pineiro, Andrea, Bähr, Harriet E, Nilsson, Sergio, Trillo-Muyo, Caroline, Ridley, David J, Thornton, Jeffrey J, Wine, Hans, Hebert, Nikolai, Klymiuk, and Gunnar C, Hansson
- Subjects
Swine ,Respiratory tract ,Respiratory Mucosa ,respiratory system ,Mucin 5AC ,MUC5AC ,Mucin-5B ,Article ,Trachea ,Mucus ,fluids and secretions ,Exocrine Glands ,Mucociliary Clearance ,Animals ,Airway surface liquid ,Lung - Abstract
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., Highlights • Submucosal glands in the piglet trachea form bundles of MUC5B mucin. • The mucus bundles are coated with MUC5AC mucin produced by surface goblet cells. • The mucus bundles are transported 10-times slower than the airway surface liquid. • The surface goblet cells are suggested to control the mucus bundle movement.
- Published
- 2017
27. Bacteria Tell Us How to Protect Our Intestine
- Author
-
Gunnar C. Hansson and George M. H. Birchenough
- Subjects
0301 basic medicine ,Colon ,Metabolite ,Cell ,Microbial metabolism ,Mucin 2 ,Microbiology ,Epithelium ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Intestinal mucosa ,Virology ,medicine ,Animals ,Humans ,Intestinal Mucosa ,Mucin-2 ,biology ,Bacteria ,Host (biology) ,biology.organism_classification ,Intestines ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Cytokines ,Parasitology - Abstract
The inner colon mucus layer capacity to separate bacteria from the epithelium is dependent on bacterial colonizers signaling to the host epithelium. In this issue of Cell Host & Microbe, Wlodarska et al. (2017) demonstrate that the mucin-utilizing Peptostreptococcus russellii protects the host from inflammatory disease via metabolite signals.
- Published
- 2017
28. Functional mucous layer and healing of proximal colonic anastomoses in an experimental model
- Author
-
Marion J.J. Gijbels, Audrey C. H. M. Jongen, Elisabeth E. L. Nyström, Joanna W. A. M. Bosmans, Joep P. M. Derikx, George M. H. Birchenough, Nicole D. Bouvy, Gunnar C. Hansson, ACS - Atherosclerosis & ischemic syndromes, Medical Biochemistry, Paediatric Surgery, AGEM - Amsterdam Gastroenterology Endocrinology Metabolism, RS: NUTRIM - R1 - Metabolic Syndrome, Promovendi NTM, Surgery, RS: NUTRIM - R2 - Liver and digestive health, Pathologie, Moleculaire Genetica, RS: CARIM - R3.06 - The vulnerable plaque: makers and markers, MUMC+: MA Heelkunde (9), and Pediatrics
- Subjects
Pathology ,medicine.medical_specialty ,Genotype ,Colon ,SURGERY ,Anastomotic Leak ,Enzyme-Linked Immunosorbent Assay ,Inflammation ,INTESTINAL DAMAGE ,MUCUS LAYERS ,Anastomosis ,GOBLET CELL ,Fatty Acid-Binding Proteins ,Real-Time Polymerase Chain Reaction ,Dinoprostone ,COLORECTAL-CANCER ,Mice ,03 medical and health sciences ,0302 clinical medicine ,LEAKAGE ,Intestinal mucosa ,Colon surgery ,ANTERIOR RESECTION ,medicine ,Animals ,Mesenteric lymph nodes ,Intestinal Mucosa ,Mucin-2 ,Wound Healing ,Gastrointestinal tract ,Goblet cell ,business.industry ,Anastomosis, Surgical ,Models, Theoretical ,respiratory system ,Mucus ,digestive system diseases ,medicine.anatomical_structure ,Bacterial Translocation ,030220 oncology & carcinogenesis ,RISK-FACTORS ,GASTROINTESTINAL-TRACT ,030211 gastroenterology & hepatology ,medicine.symptom ,business ,RAT COLON ,Colorectal Surgery - Abstract
Background: Anastomotic leakage (AL) is the most dreaded complication after colorectal surgery, causing high morbidity and mortality. Mucus is a first line of defence against external factors in the gastrointestinal tract. In this study, the structural mucus protein Muc2 was depleted in genetically engineered mice and the effect on healing of colonic anastomoses studied in an experimental model. Methods: Mice of different Muc2 genotypes were used in a proximal colonic AL model. Tissues were scored histologically for inflammation, bacterial translocation was determined by quantitative PCR of bacterial 16S ribosomal DNA, and epithelial cell damage was determined by assessing serum levels of intestinal fatty acid-binding protein. Results: Of 22 Muc2-deficient (Muc2−/−) mice, 20 developed AL, compared with seven of 22 control animals (P < 0·001). Control mice showed normal healing, whereas Muc2−/− mice had more inflammation with less collagen deposition and neoangiogenesis. A tendency towards higher bacterial translocation was seen in mesenteric lymph nodes and spleen in Muc2−/− mice. Intestinal fatty acid-binding protein levels were significantly higher in Muc2−/− mice compared with controls (P = 0·011). Conclusion: A functional mucous layer facilitates the healing of colonic anastomoses. Clinical relevance Colorectal anastomotic leakage remains the most dreaded complication after colorectal surgery. It is known that the aetiology of anastomotic leakage is multifactorial, and a role is suggested for the interaction between intraluminal content and mucosa. In this murine model of proximal colonic anastomotic leakage, the authors investigated the mucous layer at the intestinal mucosa, as the first line of defence, and found that a normal, functioning mucous layer is essential in the healing process of colonic anastomoses. Further research on anastomotic healing should focus on positively influencing the mucous layer to promote better postoperative recovery.
- Published
- 2017
29. Intestinal Muc2 mucin O-glycosylation is affected by microbiota and regulated by differential expression of glycosyltranferases
- Author
-
Jessica Holmén-Larsson, Gunnar C. Hansson, and Liisa Arike
- Subjects
0301 basic medicine ,Glycan ,Glycosylation ,Colon ,030106 microbiology ,Biochemistry ,Mice ,03 medical and health sciences ,chemistry.chemical_compound ,fluids and secretions ,Biosynthesis ,Polysaccharides ,Intestine, Small ,Glycosyltransferase ,medicine ,Animals ,Intestinal Mucosa ,Mucin-2 ,biology ,Chemistry ,Glycosyltransferases ,respiratory system ,Original articles ,biology.organism_classification ,Mucus ,digestive system diseases ,Epithelium ,Small intestine ,Gastrointestinal Microbiome ,Cell biology ,Intestines ,carbohydrates (lipids) ,030104 developmental biology ,medicine.anatomical_structure ,biology.protein ,Bacteria - 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.
- Published
- 2017
30. '''Mucus Detachment by Host Metalloprotease Meprin β Requires Shedding of Its Inactive Pro-form, which Is Abrogated by the Pathogenic Protease RgpB'''
- Author
-
Jan Potempa, Frederike Wilkens, Christoph Becker-Pauly, Jörg W. Bartsch, Philipp Arnold, Miroslaw Ksiazek, Susanna Nikolaus, Stefan Rose-John, Barbara Potempa, Marit Stirnberg, Anna Ermund, Peter J. Dempsey, Rielana Wichert, Matthias Schweinlin, Marco Metzger, Philip Rosenstiel, Björn Rabe, Gunnar C. Hansson, Ralph Lucius, Maren Falk-Paulsen, Katharina Knittler, Stefanie Schmidt, and Publica
- Subjects
0301 basic medicine ,Male ,metalloprotease ,medicine.medical_treatment ,Cell ,Mice, Transgenic ,Mucin 2 ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,03 medical and health sciences ,mucus ,Organoid ,medicine ,intestinal mucus barrier ,Animals ,Humans ,Amino Acid Sequence ,Adhesins, Bacterial ,lcsh:QH301-705.5 ,Metalloproteinase ,Mucin-2 ,host-microbiome interaction ,Protease ,030102 biochemistry & molecular biology ,Protein ,Cell Membrane ,Metalloendopeptidases ,Epithelial Cells ,Mucus ,Cysteine protease ,Cell biology ,Cysteine Endopeptidases ,030104 developmental biology ,medicine.anatomical_structure ,HEK293 Cells ,lcsh:Biology (General) ,Cell culture ,Gingipain Cysteine Endopeptidases ,Metalloproteases ,Female ,ectodomain shedding ,Schleim - Abstract
Summary The host metalloprotease meprin β is required for mucin 2 (MUC2) cleavage, which drives intestinal mucus detachment and prevents bacterial overgrowth. To gain access to the cleavage site in MUC2, meprin β must be proteolytically shed from epithelial cells. Hence, regulation of meprin β shedding and activation is important for physiological and pathophysiological conditions. Here, we demonstrate that meprin β activation and shedding are mutually exclusive events. Employing ex vivo small intestinal organoid and cell culture experiments, we found that ADAM-mediated shedding is restricted to the inactive pro-form of meprin β and is completely inhibited upon its conversion to the active form at the cell surface. This strict regulation of meprin β activity can be overridden by pathogens, as demonstrated for the bacterial protease Arg-gingipain (RgpB). This secreted cysteine protease potently converts membrane-bound meprin β into its active form, impairing meprin β shedding and its function as a mucus-detaching protease.
- Published
- 2017
- Full Text
- View/download PDF
31. Carbachol-induced colonic mucus formation requires transport via NKCC1, K+ channels and CFTR
- Author
-
Gunnar C. Hansson, Bob J. Scholte, Jenny K. Gustafsson, Henrik Sjövall, Sara K. Lindén, Ala H. Alwan, and Cell biology
- Subjects
medicine.medical_specialty ,Potassium Channels ,Carbachol ,Colon ,Physiology ,Clinical Biochemistry ,Cystic Fibrosis Transmembrane Conductance Regulator ,Muscarinic Agonists ,Biology ,Article ,Exocytosis ,Mice ,Chlorides ,Intestinal mucosa ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Solute Carrier Family 12, Member 2 ,Secretion ,Intestinal Mucosa ,Ion transporter ,Ion Transport ,Mucin ,Mucins ,respiratory system ,Mucus ,Cystic fibrosis transmembrane conductance regulator ,Cell biology ,Mice, Inbred C57BL ,Endocrinology ,Potassium ,biology.protein ,medicine.drug - Abstract
The colonic mucosa protects itself from the luminal content by secreting mucus that keeps the bacteria at a distance from the epithelium. For this barrier to be effective, the mucus has to be constantly replenished which involves exocytosis and expansion of the secreted mucins. Mechanisms involved in regulation of mucus exocytosis and expansion are poorly understood, and the aim of this study was to investigate whether epithelial anion secretion regulates mucus formation in the colon. The muscarinic agonist carbachol was used to induce parallel secretion of anions and mucus, and by using established inhibitors of ion transport, we studied how inhibition of epithelial transport affected mucus formation in mouse colon. Anion secretion and mucin exocytosis were measured by changes in membrane current and epithelial capacitance, respectively. Mucus thickness measurements were used to determine the carbachol effect on mucus growth. The results showed that the carbachol-induced increase in membrane current was dependent on NKCC1 co-transport, basolateral K(+) channels and Cftr activity. In contrast, the carbachol-induced increase in capacitance was partially dependent on NKCC1 and K(+) channel activity, but did not require Cftr activity. Carbachol also induced an increase in mucus thickness that was inhibited by the NKCC1 blocker bumetanide. However, mice that lacked a functional Cftr channel did not respond to carbachol with an increase in mucus thickness, suggesting that carbachol-induced mucin expansion requires Cftr channel activity. In conclusion, these findings suggest that colonic epithelial transport regulates mucus formation by affecting both exocytosis and expansion of the mucin molecules.
- Published
- 2014
32. Intestinal MUC2 Mucin Supramolecular Topology by Packing and Release Resting on D3 Domain Assembly
- Author
-
Philip J.B. Koeck, Elisabeth Thomsson, Malin Bäckström, Hans Hebert, Harriet Nilsson, Daniel Ambort, and Gunnar C. Hansson
- Subjects
Colon ,Protein Conformation ,Size-exclusion chromatography ,Green Fluorescent Proteins ,Supramolecular chemistry ,Mucin 2 ,CHO Cells ,Biology ,digestive system ,Article ,Green fluorescent protein ,law.invention ,Protein structure ,Cricetulus ,Imaging, Three-Dimensional ,Structural Biology ,law ,Animals ,Humans ,Intestinal Mucosa ,Molecular Biology ,Mucin-2 ,Mucin ,respiratory system ,digestive system diseases ,Recombinant Proteins ,Protein Structure, Tertiary ,Crystallography ,Microscopy, Electron ,Electron microscope ,Myc-tag - Abstract
MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D′D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D′D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexamers upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180° against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.
- Published
- 2014
- Full Text
- View/download PDF
33. 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
- Author
-
Malin E. V. Johansson, André Schütte, Sjoerd van der Post, Ana M. Rodríguez-Piñeiro, Catharina Wising, Joakim H. Bergström, Jenny K. Gustafsson, Anna Ermund, Thaher Pelaseyed, Frida Svensson, Gunnar C. Hansson, George M. H. Birchenough, and Elisabeth E. L. Nyström
- Subjects
Enterocyte ,Immunology ,Biology ,digestive system ,Article ,Peyer's Patches ,fluids and secretions ,Immune system ,medicine ,Animals ,Humans ,Immunology and Allergy ,Inner mucus layer ,Mucous Membrane ,Mucin ,Mucins ,Mucous membrane ,Intracellular vesicle ,respiratory system ,Mucus ,Small intestine ,Cell biology ,Gastrointestinal Tract ,Enterocytes ,medicine.anatomical_structure ,Immune System ,Goblet Cells - Abstract
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.
- Published
- 2014
34. Slc26a3 deficiency is associated with loss of colonic HCO3−secretion, absence of a firm mucus layer and barrier impairment in mice
- Author
-
Junhua Li, Qin Yu, Manoocher Soleimani, Gunnar C. Hansson, Regina Engelhardt, Malin E. V. Johansson, B Riederer, Ursula Seidler, Gang Xu, Dean Tian, Fang Xiao, Marshall H. Montrose, Weiliang Xia, and Anurag Kumar Singh
- Subjects
Male ,Sodium-Hydrogen Exchangers ,Colon ,Physiology ,Secretory Rate ,Intracellular pH ,Mucin 2 ,SLC26A3 ,Antiporters ,Mice ,In vivo ,medicine ,Animals ,Intestinal Mucosa ,Colitis ,Mice, Knockout ,Mucin-2 ,Ion Transport ,biology ,Sodium-Hydrogen Exchanger 3 ,Chemistry ,Mucin ,medicine.disease ,Molecular biology ,Mucus ,Bicarbonates ,Sulfate Transporters ,Immunology ,biology.protein ,Acidosis - Abstract
Aim Downregulated in adenoma (DRA, Slc26a3) is a member of the solute carrier family 26 (SLC26), family of anion transporters, which is mutated in familial chloride-losing diarrhoea (CLD). Besides Cl(-) -rich diarrhoea, CLD patients also have a higher-than-average incidence of intestinal inflammation. In a search for potential explanations for this clinical finding, we investigated colonic electrolyte transport, the mucus layer and susceptibility against dextran sodium sulphate (DSS)-induced colitis in Slc26a3(-/-) mice. Methods HCO3 (-) secretory (JHCO3 (-) ) and fluid absorptive rates were measured by single-pass perfusion in vivo and in isolated mid-distal colonic mucosa in Ussing chambers in vitro. Colonocyte intracellular pH (pHi ) was assessed fluorometrically, the mucus layer by immunohistochemistry and colitis susceptibility by the addition of DSS to the drinking water. Results HCO3 (-) secretory (JHCO3- ) and fluid absorptive rates were strongly reduced in Slc26a3(-/-) mice compared to wild-type (WT) littermates. Despite an increase in sodium/hydrogen exchanger 3 (NHE3) mRNA and protein expression, and intact acid-activation of NHE3, the high colonocyte pH in Slc26a3(-/-) mice prevented Na(+) /H(+) exchange-mediated fluid absorption in vivo. Mucin 2 (MUC2) immunohistochemistry revealed the absence of a firm mucus layer, implying that alkaline secretion and/or an absorptive flux may be necessary for optimal mucus gel formation. Slc26a3(-/-) mice were highly susceptible to DSS damage. Conclusions Deletion of DRA results in severely reduced colonic HCO3 (-) secretory rate, a loss of colonic fluid absorption, a lack of a firmly adherent mucus layer and a severely reduced colonic mucosal resistance to DSS damage. These data provide potential pathophysiological explanations for the increased susceptibility of CLD patients to intestinal inflammation.
- Published
- 2014
35. Studies of mucus in mouse stomach, small intestine, and colon. III. Gastrointestinal Muc5ac and Muc2 mucinO-glycan patterns reveal a regiospecific distribution
- Author
-
Hasse Karlsson, Jessica M. Holmén Larsson, Ana M. Rodríguez-Piñeiro, Gunnar C. Hansson, and Kristina A. Thomsson
- Subjects
Male ,Proteomics ,Glycan ,Glycosylation ,Colon ,Physiology ,Mucin 2 ,Mucin 5AC ,Mass Spectrometry ,Mice ,Intestinal mucosa ,Mucosal Biology ,Physiology (medical) ,Intestine, Small ,Gastric mucosa ,medicine ,Animals ,Intestinal Mucosa ,Fucose ,Mucin-2 ,Hepatology ,biology ,Sulfates ,Stomach ,Mucin ,Gastroenterology ,Mucus ,N-Acetylneuraminic Acid ,Small intestine ,Mice, Inbred C57BL ,carbohydrates (lipids) ,medicine.anatomical_structure ,Biochemistry ,Gastric Mucosa ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Female ,Chromatography, Liquid - Abstract
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 [Galβ1–3(GlcNAcβ1–6)GalNAcol], but also core 1 (Galβ1–3GalNAcol). 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.
- Published
- 2013
36. 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
- Author
-
Gunnar C. Hansson, André Schütte, Jenny K. Gustafsson, Anna Ermund, Joakim H. Bergström, Malin E. V. Johansson, and Ana M. Rodríguez-Piñeiro
- Subjects
Male ,Proteomics ,Spectrometry, Mass, Electrospray Ionization ,Colon ,Physiology ,Mucin 2 ,Mucin 5AC ,Biology ,Mice ,Intestinal mucosa ,Mucosal Biology ,Tandem Mass Spectrometry ,Physiology (medical) ,Intestine, Small ,medicine ,Animals ,Cluster Analysis ,Biotinylation ,Intestinal Mucosa ,Chromatography, High Pressure Liquid ,Chromatography, Reverse-Phase ,Mucin-2 ,Gastrointestinal tract ,Hepatology ,Mucin ,Gastroenterology ,respiratory system ,Mucus ,Small intestine ,Mice, Inbred C57BL ,medicine.anatomical_structure ,Biochemistry ,Gastric Mucosa ,Proteome ,Duodenum ,Female - 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.
- Published
- 2013
37. Mucus and the Goblet Cell
- Author
-
Malin E. V. Johansson and Gunnar C. Hansson
- Subjects
Goblet cell ,Mucin ,Mucins ,Gastroenterology ,General Medicine ,respiratory system ,Biology ,medicine.disease ,Mucus ,Ulcerative colitis ,Article ,Microbiology ,Disease Models, Animal ,fluids and secretions ,Immune system ,medicine.anatomical_structure ,medicine ,Animals ,Humans ,Colitis, Ulcerative ,Goblet Cells ,Colitis ,Outer mucus layer ,Inner mucus layer - Abstract
The discovery of an inner mucus layer normally impervious to bacteria has changed our way of understanding the interaction between commensal bacteria and the host epithelial cells. This inner colon mucus layer is rapidly renewed and converted into the outer mucus layer by host controlled endogenous proteolytic processing. The mucus characteristics esteem from the properties of the main protein component of these layers, the MUC2 mucin. This forms an enormously large net-like structure that builds the laminated inner mucus layer that largely acts as a size exclusion filter excluding bacteria. In the absence of MUC2 mucin, there is no inner mucus layer and bacteria reach the epithelial cell surface, penetrate the crypts and are also found inside epithelial cells, something that leads to severe inflammation. Other mouse models that spontaneously develop colitis due to different defects, like an absent ion channel (Nhe3) or immunological mediators (Tlr5, IL-10), all also have a defective inner colon mucus layer. Human patients with active ulcerative colitis have this layer penetrable to bacteria and beads the size of bacteria. Some of the ulcerative colitis patients in remission have a normal mucus layer whereas others have a penetrable inner mucus layer. Together, this suggests that the inner mucus layer and its integrity is important for the protection of the colon epithelium and inhibiting activation of the immune system as in ulcerative colitis.
- Published
- 2013
38. Effects of cathepsin K deficiency on intercellular junction proteins, luminal mucus layers, and extracellular matrix constituents in the mouse colon
- Author
-
Maria Arampatzidou, Paul Saftig, Gunnar C. Hansson, André Schütte, and Klaudia Brix
- Subjects
Male ,Colon ,Cathepsin L ,Cathepsin K ,Clinical Biochemistry ,Biochemistry ,Article ,Cathepsin B ,Mice ,Cathepsin O ,Cathepsin H ,Occludin ,Cathepsin L1 ,Animals ,Intestinal Mucosa ,Molecular Biology ,Cathepsin S ,Cathepsin ,biology ,Cadherins ,Extracellular Matrix ,Cell biology ,Mice, Inbred C57BL ,Intercellular Junctions ,Proteolysis ,biology.protein ,Gene Deletion - Abstract
Cathepsin K has been shown to exhibit antimicrobial and anti-inflammatory activities in the mouse colon. To further elucidate its role, we used Ctsk -/- mice and demonstrated that the absence of cathepsin K was accompanied by elevated protein levels of related cysteine cathepsins (cathepsins B, L, and X) in the colon. In principle, such changes could result in altered subcellular localization; however, the trafficking of cysteine cathepsins was not affected in the colon of Ctsk -/- mice. However, cathepsin K deficiency affected the extracellular matrix constituents, as higher amounts of collagen IV and laminin were observed. Moreover, the localization pattern of the intercellular junction proteins E-cadherin and occludin was altered in the colon of Ctsk -/- mice, suggesting potential impairment of the barrier function. Thus, we used an ex vivo method for assessing the mucus layers and showed that the absence of cathepsin K had no influence on mucus organization and growth. The data of this study support the notion that cathepsin K contributes to intestinal homeostasis and tissue architecture, but the lack of cathepsin K activity is not expected to affect the mucus-depending barrier functions of the mouse colon. These results are important with regard to oral administration of cathepsin K inhibitors that are currently under investigation in clinical trials.
- Published
- 2012
39. A sentinel goblet cell guards the colonic crypt by triggering Nlrp6-dependent Muc2 secretion
- Author
-
Elisabeth E. L. Nyström, George M. H. Birchenough, Malin E. V. Johansson, and Gunnar C. Hansson
- Subjects
0301 basic medicine ,Colon ,Inflammasomes ,Crypt ,Receptors, Cell Surface ,Mucin 2 ,Biology ,Ligands ,03 medical and health sciences ,Mice ,medicine ,Animals ,Secretion ,Intestinal Mucosa ,Inner mucus layer ,Goblet cell ,Mucin-2 ,Multidisciplinary ,Innate immune system ,Bacteria ,Mucus ,Epithelium ,Endocytosis ,Immunity, Innate ,Toll-Like Receptor 2 ,Cell biology ,Gastrointestinal Microbiome ,Mice, Inbred C57BL ,Toll-Like Receptor 4 ,Toll-Like Receptor 5 ,030104 developmental biology ,medicine.anatomical_structure ,Immunology ,Myeloid Differentiation Factor 88 ,Calcium ,Goblet Cells ,Reactive Oxygen Species ,Signal Transduction - Abstract
Mounting the intestinal barricades Gut microbiota are important for health and well-being, but they need to be kept under control and prevented from doing any harm. Birchenough et al. investigated the microbial molecules that trigger protective mucus secretion from a class of goblet cells in the colon. Once the molecules are detected, an alarm signal is transmitted from these cells via innate immune signal mediators and inflammasome components to adjacent cells, generating more mucus and repelling the invaders. Subsequently, the sentinel goblet cells are expelled from the epithelium and their remains may also add to the protective barricade. Science , this issue p. 1535
- Published
- 2016
40. The Densely O-Glycosylated MUC2 Mucin Protects the Intestine and Provides Food for the Commensal Bacteria
- Author
-
Liisa Arike and Gunnar C. Hansson
- Subjects
0301 basic medicine ,animal structures ,Glycosylation ,macromolecular substances ,Mucin 2 ,medicine.disease_cause ,Article ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Intestinal mucosa ,Structural Biology ,Polysaccharides ,medicine ,Animals ,Humans ,Intestinal Mucosa ,Molecular Biology ,Mucin-2 ,biology ,Bacteria ,Host (biology) ,Mucin ,Pathogenic bacteria ,respiratory system ,biology.organism_classification ,Mucus ,carbohydrates (lipids) ,030104 developmental biology ,chemistry ,030220 oncology & carcinogenesis ,lipids (amino acids, peptides, and proteins) - 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.
- Published
- 2016
41. 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
- Author
-
Jessica Holmén-Larsson, Lijun Xia, Gunnar C. Hansson, Malin E. V. Johansson, Kristina A. Thomsson, and Jonas Ångström
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Glycan ,Glycosylation ,Magnetic Resonance Spectroscopy ,Colon ,Molecular Sequence Data ,Mucin 2 ,N-Acetylglucosaminyltransferases ,digestive system ,Biochemistry ,Glycomics ,Epitopes ,Mice ,chemistry.chemical_compound ,Intestinal mucosa ,Polysaccharides ,Animals ,Humans ,Intestinal Mucosa ,Mice, Knockout ,Mucin-2 ,biology ,Mucin ,Amino Sugars ,Original Articles ,respiratory system ,Galactosyltransferases ,Molecular biology ,digestive system diseases ,Mice, Inbred C57BL ,Carbohydrate Sequence ,chemistry ,Knockout mouse ,biology.protein ,Metagenome ,C1GALT1 ,Chromatography, Liquid - 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.
- Published
- 2012
42. Role of mucus layers in gut infection and inflammation
- Author
-
Gunnar C. Hansson
- Subjects
Microbiology (medical) ,Inflammation ,Biology ,Microbiology ,Article ,Enteritis ,03 medical and health sciences ,0302 clinical medicine ,fluids and secretions ,Intestinal mucosa ,medicine ,Animals ,Humans ,Intestinal Mucosa ,030304 developmental biology ,Inner mucus layer ,0303 health sciences ,Gastrointestinal tract ,Bacteria ,Bacterial Infections ,respiratory system ,biology.organism_classification ,medicine.disease ,Mucus ,Epithelium ,3. Good health ,Gastrointestinal Tract ,Infectious Diseases ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Host-Pathogen Interactions ,medicine.symptom - Abstract
The intestinal mucus is an efficient system for protecting the epithelium from bacteria by promoting their clearance and separating them from the epithelial cells, thereby inhibiting inflammation and infection. The function of the colon inner mucus layer is especially important as this explains how we can harbor the large number of bacteria in our gut. The major component of this mucus system is the MUC2 mucin which organizes the mucus by its enormously large net-like polymers. Pathogenic microorganisms, in turn, have developed mechanisms for circumventing this well-organized mucus protective system.
- Published
- 2012
- Full Text
- View/download PDF
43. An ex vivo method for studying mucus formation, properties, and thickness in human colonic biopsies and mouse small and large intestinal explants
- Author
-
Malin E. V. Johansson, Jenny K. Gustafsson, André Schütte, Henrik Sjövall, Anna Ermund, and Gunnar C. Hansson
- Subjects
Colon ,Physiology ,Ileum ,Biology ,Mice ,03 medical and health sciences ,fluids and secretions ,0302 clinical medicine ,Intestinal mucosa ,Mucosal Biology ,Physiology (medical) ,medicine ,Animals ,Humans ,Large intestine ,Intestinal Mucosa ,030304 developmental biology ,Transepithelial potential difference ,Inner mucus layer ,0303 health sciences ,Hepatology ,Mucin ,Gastroenterology ,Epithelial Cells ,Anatomy ,respiratory system ,Mucus ,Molecular biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Ex vivo - Abstract
The colon mucus layers minimize the contact between the luminal flora and the epithelial cells, and defects in this barrier may lead to colonic inflammation. We now describe an ex vivo method for analysis of mucus properties in human colon and mouse small and large intestine. Intestinal explants were mounted in horizontal perfusion chambers. The mucus surface was visualized by adding charcoal particles on the apical side, and mucus thickness was measured using a micropipette. Mucus thickness, adhesion, and growth rate were recorded for 1 h. In mouse and human colon, the ability of the mucus to act as a barrier to beads the size of bacteria was also evaluated. Tissue viability was monitored by transepithelial potential difference. In mouse ileum, the mucus could be removed by gentle aspiration, whereas in colon ∼40 μm of the mucus remained attached to the epithelial surface. Both mouse and human colon had an inner mucus layer that was not penetrated by the fluorescent beads. Spontaneous mucus growth was observed in human (240 μm/h) and mouse (100 μm/h) colon but not in mouse ileum. In contrast, stimulation with carbachol induced a higher mucus secretion in ileum than colon (mouse ileum: Δ200 μm, mouse colon: Δ130 μm, human colon: Δ140 μm). In conclusion, while retaining key properties from the mucus system in vivo, this setup also allows for studies of the highly dynamic mucus system under well-controlled conditions.
- Published
- 2012
- Full Text
- View/download PDF
44. Loss of intestinal core 1–derived O-glycans causes spontaneous colitis in mice
- Author
-
Maomeng Tong, Malin E. V. Johansson, Thomas J. Sferra, Jerrold R. Turner, Hong Chen, Xiaowei Liu, Kristina A. Thomsson, Samuel McGee, Bo Wei, Jonathan Braun, Lijun Xia, Tao Wen, Lilah Mansour, Gunnar C. Hansson, Emily M. Bradford, Jianxin Fu, and J. Michael McDaniel
- Subjects
Mice, 129 Strain ,Myeloid ,Colon ,Mice, Transgenic ,Biology ,Mice ,Intestinal mucosa ,Polysaccharides ,medicine ,Animals ,Humans ,Antigens, Tumor-Associated, Carbohydrate ,Intestinal Mucosa ,Colitis ,DNA Primers ,Inner mucus layer ,Mice, Knockout ,Base Sequence ,Mucin ,General Medicine ,Galactosyltransferases ,medicine.disease ,Ulcerative colitis ,Molecular biology ,Gut Epithelium ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Mutation ,Immunology ,Crypt Abscess ,Research Article ,Molecular Chaperones - Abstract
Mucin-type O-linked oligosaccharides (O-glycans) are primary components of the intestinal mucins that form the mucus gel layer overlying the gut epithelium. Impaired expression of intestinal O-glycans has been observed in patients with ulcerative colitis (UC), but its role in the etiology of this disease is unknown. Here, we report that mice with intestinal epithelial cell-specific deficiency of core 1-derived O-glycans, the predominant form of O-glycans, developed spontaneous colitis that resembled human UC, including massive myeloid infiltrates and crypt abscesses. The colitis manifested in these mice was also characterized by TNF-producing myeloid infiltrates in colon mucosa in the absence of lymphocytes, supporting an essential role for myeloid cells in colitis initiation. Furthermore, induced deletion of intestinal core 1-derived O-glycans caused spontaneous colitis in adult mice. These data indicate a causal role for the loss of core 1-derived O-glycans in colitis. Finally, we detected a biosynthetic intermediate typically exposed in the absence of core 1 O-glycan, Tn antigen, in the colon epithelium of a subset of UC patients. Somatic mutations in the X-linked gene that encodes core 1 β1,3-galactosyltransferase-specific chaperone 1 (C1GALT1C1, also known as Cosmc), which is essential for core 1 O-glycosylation, were found in Tn-positive epithelia. These data suggest what we believe to be a new molecular mechanism for the pathogenesis of UC.
- Published
- 2011
45. Importance and regulation of the colonic mucus barrier in a mouse model of colitis
- Author
-
Lena Holm, Olof Schreiber, Anna Velcich, Joel Petersson, Mia Phillipson, Sandra J. Gendler, Stefan Roos, Jon O. Lundberg, and Gunnar C. Hansson
- Subjects
Lipopolysaccharides ,Male ,Lipopolysaccharide ,Colon ,Physiology ,T-Lymphocytes ,Down-Regulation ,Peptidoglycan ,Mucin 2 ,Biology ,digestive system ,Severity of Illness Index ,Inflammation/Immunity/Mediators ,Microbiology ,Mice ,chemistry.chemical_compound ,Intestinal mucosa ,Physiology (medical) ,medicine ,Animals ,Germ-Free Life ,Intestinal Mucosa ,Colitis ,Mice, Knockout ,Mucin-2 ,Hepatology ,Dextran Sulfate ,Mucin-1 ,Mucin ,Gastroenterology ,respiratory system ,medicine.disease ,Mucus ,Ulcerative colitis ,digestive system diseases ,Mice, Inbred C57BL ,chemistry ,Bacterial Translocation ,Disease Susceptibility - Abstract
The colonic mucus layer serves as an important barrier and prevents colonic bacteria from invading the mucosa and cause inflammation. The regulation of colonic mucus secretion is poorly understood. The aim of this study was to investigate the role of the mucus barrier in induction of colitis. Furthermore, regulation of mucus secretion by luminal bacterial products was studied. The colon of anesthetized Muc2−/−, Muc1−/−, wild-type (wt), and germ-free mice was exteriorized, the mucosal surface was visualized, and mucus thickness was measured with micropipettes. Colitis was induced by DSS (dextran sodium sulfate, 3%, in drinking water), and disease activity index (DAI) was assessed daily. The colonic mucosa of germ-free and conventionally housed mice was exposed to the bacterial products LPS (lipopolysaccharide) and PGN (peptidoglycan). After DSS induction of colitis, the thickness of the firmly adherent mucus layer was significantly thinner after 5 days and onward, which paralleled the increment of DAI. Muc2−/−mice, which lacked firmly adherent mucus, were predisposed to colitis, whereas Muc1−/−mice were protected with significantly lower DAI by DSS compared with wt mice. The mucus barrier increased in Muc1−/−mice in response to DSS, whereas significantly fewer T cells were recruited to the inflamed colon. Mice housed under germ-free conditions had an extremely thin adherent colonic mucus layer, but when exposed to bacterial products (PGN or LPS) the thickness of the adherent mucus layer was quickly restored to levels observed in conventionally housed mice. This study demonstrates a correlation between decreasing mucus barrier and increasing clinical symptoms during onset of colitis. Mice lacking colonic mucus (Muc2−/−) were hypersensitive to DSS-induced colitis, whereas Muc1−/−were protected, probably through the ability to increase the mucus barrier but also by decreased T cell recruitment to the afflicted site. Furthermore, the ability of bacteria to regulate the thickness of the colonic mucus was demonstrated.
- Published
- 2011
46. Function of the CysD domain of the gel-forming MUC2 mucin
- Author
-
Ute Krengel, Elisabeth Thomsson, Daniel Ambort, Gunnar C. Hansson, Jenny Mackenzie, Malin E. V. Johansson, and Sjoerd van der Post
- Subjects
Glycosylation ,CID, collision-induced dissociation ,BN-PAGE, blue native PAGE ,disulfide bonds ,Mucin 2 ,Biochemistry ,chemistry.chemical_compound ,Cricetinae ,CK domain, cystine-knot domain ,MS/MS, tandem MS ,Disulfides ,Peptide sequence ,0303 health sciences ,ESI, electrospray ionization ,AGC, automatic gain control ,LC, liquid chromatography ,Chemistry ,030302 biochemistry & molecular biology ,Covalent bond ,non-covalent dimer ,Electrophoresis, Polyacrylamide Gel ,Domain of unknown function ,Dimerization ,Research Article ,Stereochemistry ,Recombinant Fusion Proteins ,Molecular Sequence Data ,Size-exclusion chromatography ,CHO Cells ,TCEP-HCl, tris(2-carboxyethyl)phosphine-HCl ,PTS domain, proline, threonine and serine domain ,03 medical and health sciences ,Cricetulus ,C-mannosylation ,FBS, fetal bovine serum ,mucus ,Animals ,Humans ,mass spectrometry (MS) ,Amino Acid Sequence ,mAb, monoclonal antibody ,Molecular Biology ,030304 developmental biology ,Mucin-2 ,EK, enterokinase ,Mucin ,IMDM, Iscove's modified Dulbecco's medium ,Cell Biology ,Fusion protein ,Protein Structure, Tertiary ,CHO, Chinese-hamster ovary - Abstract
The colonic human MUC2 mucin forms a polymeric gel by covalent disulfide bonds in its N- and C-termini. The middle part of MUC2 is largely composed of two highly O-glycosylated mucin domains that are interrupted by a CysD domain of unknown function. We studied its function as recombinant proteins fused to a removable immunoglobulin Fc domain. Analysis of affinity-purified fusion proteins by native gel electrophoresis and gel filtration showed that they formed oligomeric complexes. Analysis of the individual isolated CysD parts showed that they formed dimers both when flanked by two MUC2 tandem repeats and without these. Cleavages of the two non-reduced CysD fusion proteins and analysis by MS revealed the localization of all five CysD disulfide bonds and that the predicted C-mannosylated site was not glycosylated. All disulfide bonds were within individual peptides showing that the domain was stabilized by intramolecular disulfide bonds and that CysD dimers were of non-covalent nature. These observations suggest that CysD domains act as non-covalent cross-links in the MUC2 gel, thereby determining the pore sizes of the mucus.
- Published
- 2011
- Full Text
- View/download PDF
47. LactobacillusandBifidobacteriumspecies do not secrete protease that cleaves the MUC2 mucin which organises the colon mucus
- Author
-
Durai B. Subramani, Gunnar Dahlén, Gunnar C. Hansson, and Malin E. V. Johansson
- Subjects
Microbiology (medical) ,Colon ,digestive system ,Microbiology ,Cell Line ,Mice ,fluids and secretions ,Bacterial Proteins ,Lactobacillus ,Animals ,Humans ,Intestinal Mucosa ,Inner mucus layer ,Bifidobacterium ,Mucin-2 ,biology ,Lactobacillus brevis ,Mucin ,food and beverages ,respiratory system ,biology.organism_classification ,Mucus ,Protein Structure, Tertiary ,Biochemistry ,Extracellular Space ,Protein Processing, Post-Translational ,Bacteria ,Lactobacillus plantarum ,Peptide Hydrolases - Abstract
The colon epithelium is covered by two layers of mucus built around the MUC2 mucin. An inner dense and attached mucus layer does not allow bacteria to penetrate, thus keeping the epithelial cell surface free from bacteria. An outer loose mucus layer is the habitat for the commensal bacterial microbiota. The inner mucus layer is renewed from the epithelial side and gets converted into the outer layer due to proteolytic cleavages by host proteases. We have now analysed if potential probiotic bacteria, namely Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus bulgaricus and Bifidobacterium lactis, can secrete protease that cleaves the MUC2 mucin. We found that none of the potential probiotic bacteria Lactobacillus and Bifidobacterium could cleave the MUC2 core protein in the form of recombinant MUC2 N and C-termini although they secreted active proteases. This was in contrast to crude mixtures of oral and faecal bacteria that cleaved the MUC2 mucin. This observation further supports the view that these potential probiotic bacteria are of no harm to the host, as these bacteria cannot disrupt the mucin organised mucus as long as they are covered by glycans.
- Published
- 2010
48. O-glycosylation of MUC1 mucin in prostate cancer and the effects of its expression on tumor growth in a prostate cancer xenograft model
- Author
-
Gunnar C. Hansson, Pushpa Premaratne, Jan-Erik Damber, Malin Bäckström, and Karin Welén
- Subjects
Male ,Spectrometry, Mass, Electrospray Ionization ,medicine.medical_specialty ,Glycosylation ,Blotting, Western ,Mice, Nude ,Enzyme-Linked Immunosorbent Assay ,digestive system ,Immunoenzyme Techniques ,Mice ,Prostate cancer ,Cell Movement ,Cancer stem cell ,Internal medicine ,Pancreatic cancer ,Cell Adhesion ,Tumor Cells, Cultured ,medicine ,Animals ,Humans ,skin and connective tissue diseases ,neoplasms ,MUC1 ,Cell Proliferation ,Mice, Inbred BALB C ,biology ,Cell growth ,Mucin-1 ,Mucin ,Prostatic Neoplasms ,Cancer ,General Medicine ,Flow Cytometry ,medicine.disease ,Xenograft Model Antitumor Assays ,biological factors ,digestive system diseases ,Fibronectin ,Endocrinology ,biology.protein ,Cancer research - Abstract
MUC1 mucin is up-regulated and aberrantly glycosylated in many human epithelial carcinomas. Over-expression of MUC1 has also been implicated in prostate cancer, but neither the role of MUC1 in the cancer progression nor the mucin O-glycosylation has been fully elucidated. In this study, we characterized the O-glycans on MUC1 when over-expressed in the human prostate cancer cell line C4-2B(4). We found that the main O-glycan consisted of the neutral core 2 oligosaccharide Galβ3(Galβ3/4GlcNAcβ6)GalNAc-ol, with minor components being fucosylated and sialylated variants of the same core 2 oligosaccharide. Small amounts of the shorter core 1 O-glycans were also detected.We then used the MUC1 over-expressing cell lines to study the effects of MUC1 on prostate cancer cell behavior. The results demonstrate that over-expression of MUC1 did not affect the cells' proliferation, but led to a decreased adhesion to the extracellular matrix components fibronectin and collagen I in vitro. When inoculated in BALB/c nude mice, C4-2B(4) cells expressing MUC1 showed a tendency to form fewer tumors than wt cells and the tumors also grew more slowly, but there was a large variation between different tumors. These findings suggest that MUC1 may not have the same cancer-promoting effect in prostate cancer cells that is commonly seen in other epithelial cancers such as breast, colon, and pancreatic cancer.
- Published
- 2010
49. Proteomic Analyses of the Two Mucus Layers of the Colon Barrier Reveal That Their Main Component, the Muc2 Mucin, Is Strongly Bound to the Fcgbp Protein
- Author
-
Kristina A. Thomsson, Gunnar C. Hansson, and Malin E. V. Johansson
- Subjects
Proteomics ,Guanidinium chloride ,Colon ,Biology ,Biochemistry ,Mass Spectrometry ,Mice ,chemistry.chemical_compound ,von Willebrand Factor ,Extracellular ,medicine ,Animals ,Humans ,Large intestine ,Disulfides ,Intestine, Large ,Inner mucus layer ,Mucin-2 ,Binding protein ,Mucins ,General Chemistry ,respiratory system ,Mucus ,Mice, Inbred C57BL ,Secretory protein ,medicine.anatomical_structure ,chemistry ,Cell Adhesion Molecules ,Chromatography, Liquid - Abstract
The colon epithelium is protected from the luminal microbes as recently revealed by an inner firmly attached mucus layer impervious to bacteria and an outer loose mucus layer that is the habitat of bacteria. For an additional understanding of these layers, we analyzed the protein composition of these two mucus layers from the mouse colon. Proteomics using nano-LC-MS and MS/MS revealed more than 1000 protein entries. As the mucus layers contain detached cells, a majority of the proteins had an intracellular origin. However, at least 44 entries were described as secreted proteins and predicted to be mucus constituents together with extracellular/plasma and bacterial proteins, the latter largely in the loose mucus layer. A major protein was the Muc2 mucin that by its net-like disulfide-bonded polymer structure builds the mucus. When guanidinium chloride insoluble Muc2 units were analyzed, N-terminal parts of the Fc-gamma binding protein (Fcgbp) was found to be covalently attached in mouse and human colon, whereas its C-terminus was lost by reducing the disulfide bonds. In conclusion, the Fcgbp protein is probably cleaved at GD/PH and covalently attached to Muc2 via one or several of its von Willebrand D domains.
- Published
- 2009
50. Cervical mucins carry α(1,2)fucosylated glycans that partly protect from experimental vaginal candidiasis
- Author
-
Steven E. Domino, Kristina A. Thomsson, Jessica M. Holmén Larsson, Elisabeth Thomsson, Malin Bäckström, Gunnar C. Hansson, David Karnak, and Elizabeth A. Hurd
- Subjects
Glycan ,Fucosyltransferase ,Molecular Sequence Data ,Colony Count, Microbial ,Biology ,Hysterectomy ,Biochemistry ,Article ,Epitope ,Fucose ,Microbiology ,Epitopes ,Mice ,chemistry.chemical_compound ,Polysaccharides ,Candida albicans ,Cell Adhesion ,Animals ,Molecular Biology ,Candidiasis, Vulvovaginal ,MUC1 ,Vaginal Smears ,Mucin-1 ,Mucin ,Mucins ,Wild type ,Epithelial Cells ,Cell Biology ,Fucosyltransferases ,biology.organism_classification ,Recombinant Proteins ,Mice, Inbred C57BL ,Carbohydrate Sequence ,chemistry ,Cervix Mucus ,biology.protein ,Female ,Disease Susceptibility - Abstract
Cervical mucins are glycosylated proteins that form a protective cervical mucus. To understand the role of mucin glycans in Candida albicans infection, oligosaccharides from mouse cervical mucins were analyzed by liquid chromatography-mass spectrometry. Cervical mucins carry multiple alpha(1-2)fucosylated glycans, but alpha(1,2)fucosyltransferase Fut2-null mice are devoid of these epitopes. Epithelial cells in vaginal lavages from Fut2-null mice lacked Ulex europaeus agglutinin-1 (UEA-I) staining for alpha(1-2)fucosylated glycans. Hysterectomy to remove cervical mucus eliminated UEA-I and acid mucin staining in vaginal epithelial cells from wild type mice indicating the cervix as the source of UEA-I positive epithelial cells. To assess binding of alpha(1-2) fucosylated glycans on C. albicans infection, an in vitro adhesion assay was performed with vaginal epithelial cells from wild type and Fut2-null mice. Vaginal epithelial cells from Fut2-null mice were found to bind increased numbers of C. albicans compared to vaginal epithelial cells obtained from wild type mice. Hysterectomy lessened the difference between Fut2-null and wild type mice in binding of C. ablicans in vitro and susceptibility to experimental C. albicans vaginitis in vivo. We generated a recombinant fucosylated MUC1 glycanpolymer to test whether the relative protection of wild type mice compared to Fut2-null mice could be mimicked with exogenous mucin. While a small portion of the recombinant MUC1 epitopes displayed alpha(1-2)fucosylated glycans, the predominant epitopes were sialylated due to endogenous sialyltransferases in the cultured cells. Intravaginal instillation of recombinant MUC1 glycanpolymer partially reduced experimental yeast vaginitis suggesting that a large glycanpolymer, with different glycan epitopes, may affect fungal burden.
- Published
- 2009
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.