Inherited defect in ST6GalNAc1 reveals roles of sialylation in intestinal homeostasis
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE183821 | GEO | 2022/03/16
REPOSITORIES: GEO
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Project description:Chronic inflammatory bowel disease (IBD) causes disability, suffering and risk of colon cancer in over 30 million people globally. Here, we investigate 3 kindreds of IBD with mutations in the N-acetylgalactosamide alpha-2,6-sialyltransferase 1/ST6GALNAC1 (ST6) glycosyltransferase gene that is uniquely expressed in the goblet cells (GCs). These mutations cause defective sialic acid (SA) conjugation, elimination of the onco-antigen S-Tn (Sialyl-Tn), and altered glycosylation of the MUC2 protein, a key component of intestinal mucus. Decreased MUC2 sialylation increases susceptibility to bacterial proteolytic degradation and compromises the gastrointestinal (GI) mucus barrier. Mice harboring the patient ST6 mutations recapitulate human colitis and reveal that defective sialylation causes dysbiosis, butyrate overproduction, and impaired GI stem cell proliferation during injury. Thus, this new genetic disease illustrates how sialylation controls host-microbe homeostasis and reveals several new potential approaches to IBD treatment.
2022-03-17 | PXD028239 | Pride
Project description:Background; MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. MUC2 homo-oligomerizes intracellularly into large secreted polymers which give mucus its viscous properties. The inflammatory bowel disease (IBD) ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, whereas goblet cells and the mucus layer are increased in the other major inflammatory bowel disease, Crohnâs disease. Methods and Findings; By murine N-ethyl-N-nitrosourea-mutagenesis we identified two distinct non-complementing missense mutations in Muc2 exons encoding N- and C-terminal homo-oligomerization domains causing an ulcerative colitis-like phenotype. Both strains developed mild spontaneous distal intestinal inflammation, chronic diarrhea, rectal bleeding and prolapse, increased susceptibility to acute and chronic colitis induced by a luminal toxin, aberrant Muc2 biosynthesis, smaller goblet cell thecae (less stored mucin) and a diminished mucus barrier. Enhanced local production of IL-1beta, TNF-alpha and IFN-gamma was seen in the distal colon. The number of leukocytes within mesenteric lymph nodes was increased five-fold and leukocytes cultured in vitro produced both Th1 and Th2 cytokines (IFN-gamma, TNF-alpha and IL-13). Intestinal permeability was increased and the luminal bacterial flora were more heavily coated with immunoglobulin as occurs in IBD. This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis and wound repair. Expression of mutated Muc2 oligomerization domains in vitro demonstrated that aberrant Muc2 oligomerization underlies the ER stress. These models show that mutations in Muc2 oligomerization domains can lead to aberrant assembly of the Muc2 complex leading to ER stress, a depleted mucus barrier and intestinal inflammation. In ulcerative colitis we demonstrate similar accumulation of non-glycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in non-inflamed intestinal tissue. Conclusions; The observations that mucin misfolding and ER stress lead directly to intestinal inflammation and that ER stress and goblet cell pathology occur in ulcerative colitis suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of colitis. Experiment Overall Design: 3 individual mice from the Eeyore, Winnie or Wild-type strains were compared as groups. An Affymetrix ID was compared between groups if the ID was Present within two of the three mice within each grouping. IDs were compared by calculating the log2 of Group One average signal divided by Group 2 average signal.
2008-04-06 | E-GEOD-9913 | biostudies-arrayexpress
Project description:Background MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. MUC2 homo-oligomerizes intracellularly into large secreted polymers which give mucus its viscous properties. The inflammatory bowel disease (IBD) ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, whereas goblet cells and the mucus layer are increased in the other major inflammatory bowel disease, Crohn’s disease. Methods and Findings By murine N-ethyl-N-nitrosourea-mutagenesis we identified two distinct non-complementing missense mutations in Muc2 exons encoding N- and C-terminal homo-oligomerization domains causing an ulcerative colitis-like phenotype. Both strains developed mild spontaneous distal intestinal inflammation, chronic diarrhea, rectal bleeding and prolapse, increased susceptibility to acute and chronic colitis induced by a luminal toxin, aberrant Muc2 biosynthesis, smaller goblet cell thecae (less stored mucin) and a diminished mucus barrier. Enhanced local production of IL-1beta, TNF-alpha and IFN-gamma was seen in the distal colon. The number of leukocytes within mesenteric lymph nodes was increased five-fold and leukocytes cultured in vitro produced both Th1 and Th2 cytokines (IFN-gamma, TNF-alpha and IL-13). Intestinal permeability was increased and the luminal bacterial flora were more heavily coated with immunoglobulin as occurs in IBD. This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis and wound repair. Expression of mutated Muc2 oligomerization domains in vitro demonstrated that aberrant Muc2 oligomerization underlies the ER stress. These models show that mutations in Muc2 oligomerization domains can lead to aberrant assembly of the Muc2 complex leading to ER stress, a depleted mucus barrier and intestinal inflammation. In ulcerative colitis we demonstrate similar accumulation of non-glycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in non-inflamed intestinal tissue. Conclusions The observations that mucin misfolding and ER stress lead directly to intestinal inflammation and that ER stress and goblet cell pathology occur in ulcerative colitis suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of colitis. Keywords: Single gene, multiple mutant comparison
2008-03-01 | GSE9913 | GEO
Project description:Mucus/MUC2 from TGM3-deficient mice is less cross-linked with isopeptide-bond cross-links compared to Mucus/MUC2 from WT mice. This results in protease-labile MUC2-molecules that become degraded in the colonic lumen
2022-02-09 | PXD029071 | Pride
Project description:Gastrointestinal (GI) mucus is continuously secreted and lines the entire length of the GI tract. Essential for health, it keeps the noxious luminal content away from the epithelium and propels forward the digesta. The aim of our study was to characterize the composition and structures of mucus throughout the various GI segments in dog. Mucus from the stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, proximal and distal colon) was collected from 5 dogs. pH and water content of GI mucus and digesta were analyzed. Composition of all GI-tract segments from a domestic and a laboratory dog was determined by label-free global proteomics. A colonic-focussed composition analysis with TMT-labelled proteomics was used on jenunal and proximal and distal colonic mucus samples from 3 laboratory and 1 domestic dog. Finally, the composition of jejunal and colonic mucus samples of 3 laboratory and 1 domestic dog was evaluated with lipidomics and metabolomics. Structural properties were investigated using cryoSEM and rheology. The proteome was similar across the different GI segments. The highest abundant secreted gel-forming mucin in the gastric mucus was mucin 5AC, whether mucin 2 had highest abundance in the intestinal mucus. Lipid and metabolite abundance was generally higher in the jejunal mucus than the colonic mucus. In conclusion, the mucus is a highly viscous and hydrated material. The proteins, lipids and metabolites were similar throughout the GI tract, although abundances depended on location. These data provide an important baseline for future studies on human and canine intestinal diseases and the dog model in drug absorption.
2022-03-07 | PXD029356 | Pride
Project description:The multifunctional intestinal mucus layer plays a crucial role in human health. Our understanding of the human colonic mucus layer in terms of structure, function and has been largely dependent on expensive and advanced ex vivo or in vitro models, which often require high expertise. The mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used in more simple in vitro models, but produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured in Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2 and had a thicker mucus layer compared to conventional culturing methods. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased mucus production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell inserts for 15 days were subject to transcriptome analysis to investigate underlying molecular pathways at gene expression level. We also further characterized the model by measuring transepithelial resistance and pH and lactate production of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms in which mucus production is enhanced under SWMS and propose potential applications of this model in future studies.
2021-12-06 | GSE173729 | GEO
Project description:Bladder carcinogenesis and tumour progression is accompanied by profound alterations in protein glycosylation. These include the presence of short chain O-glycans such as Tn and T antigens and their sialylated counterparts sialyl-Tn(STn) and sialyl-T(ST). The presence of such structures is commonly associated with poor prognosis. In search for prognosis biomarkers and novel therapeutic targets we have screened, using immunohistochemistry, a series of bladder tumours with differing clinicopathology for these short-chain O-glycans. Aiming at identifying protein targets, we have studied the O-glycoproteome of a pool of STn positive tumours from muscle invasive bladder cancer (MIBC) patients.
2017-08-17 | PXD006813 | Pride
Project description:Mucus has an important protective function and forms a barrier that minimizes the bacterial contact with the colonic epithelium. It is organized as a complex network with several specific proteins of which most are still poorly understood. Abundant proteins in intestinal mucus, in addition to the mucin MUC2, are CLCA1 and FCGBP. FCGBP is expressed in all intestinal goblet cells and secreted into the mucus. It is comprised of repeated von Willebrand Factor D (vWD) domain assemblies, of which most have a GDPH amino acid sequence. This sequence can be autocatalytically cleaved, as previously found in MUC2 and MUC5AC. We show that all vWD with a GDPH sequence are cleaved, and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments remained connected by a disulfide bond within each vWD domain. The cleavage can generate a C-terminal reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by mass spectrometry showed that, in chaotropic solutions, FCGBP was mainly soluble whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. As its name indicates, FCGBP has been suggested to bind IgG, something that was not possible to reproduce in vitro using purified proteins. The function of FCGBP is still unknown, but our results suggest that the Asp-anhydrides do not contribute to covalent crosslinking in the mucin framework nor does FCGBP incorporate IgG in mucus.
2021-06-09 | PXD025493 | Pride
Project description:Mucus has an important protective function and forms a barrier that minimizes the bacterial contact with the colonic epithelium. It is organized as a complex network with several specific proteins of which most are still poorly understood. Abundant proteins in intestinal mucus, in addition to the mucin MUC2, are CLCA1 and FCGBP. FCGBP is expressed in all intestinal goblet cells and secreted into the mucus. It is comprised of repeated von Willebrand Factor D (vWD) domain assemblies, of which most have a GDPH amino acid sequence. This sequence can be autocatalytically cleaved, as previously found in MUC2 and MUC5AC. We show that all vWD with a GDPH sequence are cleaved, and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments remained connected by a disulfide bond within each vWD domain. The cleavage can generate a C-terminal reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by mass spectrometry showed that, in chaotropic solutions, FCGBP was mainly soluble whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. As its name indicates, FCGBP has been suggested to bind IgG, something that was not possible to reproduce in vitro using purified proteins. The function of FCGBP is still unknown, but our results suggest that the Asp-anhydrides do not contribute to covalent crosslinking in the mucin framework nor does FCGBP incorporate IgG in mucus.
2021-06-09 | PXD025496 | Pride
Project description:CD11b+ cells isolated from IBD patient biopsies have distinct transcriptomic signatures based on their location along the GI tract, inflammation status, IBD type, and medication response. These cells also respond to JAK inhibitors by decreasing cytokine output.
2022-08-23 | GSE183620 | GEO