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Approximately 1 billion people worldwide suffer from zinc deficiency, with severe consequences for their well-being, such as critically impaired intestinal health. In addition to an extreme degeneration of the intestinal epithelium, the intestinal mucus is seriously disturbed in zinc-deficient (ZD) animals. The underlying cellular processes as well as the relevance of zinc for the mucin-producing goblet cells, however, remain unknown. To this end, this study examines the impact of zinc deficiency on the synthesis, production, and secretion of intestinal mucins as well as on the zinc homeostasis of goblet cells using the in vitro goblet cell model HT-29-MTX. Zinc deprivation reduced their cellular zinc content, changed expression of the intestinal zinc transporters ZIP-4, ZIP-5, and ZnT1 and increased their zinc absorption ability, outlining the regulatory mechanisms of zinc homeostasis in goblet cells. Synthesis and secretion of mucins were severely disturbed during zinc deficiency, affecting both MUC2 and MUC5AC mRNA expression with ongoing cell differentiation. A lack of zinc perturbed mucin synthesis predominantly on the post-translational level, as ZD cells produced shorter O-glycans and the main O-glycan pattern was shifted in favor of core-3-based mucins. The expression of glycosyltransferases that determine the formation of core 1-4 O-glycans was altered in zinc deficiency. In particular, B3GNT6 mRNA catalyzing core 3 formation was elevated and C2GNT1 and C2GNT3 elongating core 1 were downregulated in ZD cells. These novel insights into the molecular mechanisms impairing intestinal mucus stability during zinc deficiency demonstrate the essentiality of zinc for the formation and maintenance of this physical barrier.

Mucus is the first biological barrier encountered by particles and pathogenic bacteria at the surface of secretory epithelia. The viscoelasticity of mucus is governed in part by low energy interactions that are difficult to assess. The CYS domain is a good candidate to support low energy interactions between GFMs and/or mucus constituents. Our aim was to stiffen the mucus from HT29-MTX cell cocultures and the colon of mice through the delivery of a recombinant protein made of hydrophobic CYS domains and found in multiple copies in polymeric mucins. The ability of the delivery of a poly-CYS molecule to stiffen mucus gels was assessed by probing cellular motility and particle diffusion. We demonstrated that poly-CYS enrichment decreases mucus permeability and hinders displacement of pathogenic flagellated bacteria and spermatozoa. Particle tracking microrheology showed a decrease of mucus diffusivity. The empirical obstruction scaling model evidenced a decrease of mesh size for mouse mucus enriched with poly-CYS molecules. Our data bring evidence that enrichment with a protein made of CYS domains stiffens the mucin network to provide a more impermeable and protective mucus barrier than mucus without such enrichment.

Mucus is a major component of the intestinal barrier involved both in the protection of the host and the fitness of commensals of the gut. Streptococcus thermophilus is consumed world-wide in fermented dairy products and is also recognized as a probiotic, as its consumption is associated with improved lactose digestion. We determined the overall effect of S. thermophilus on the mucus by evaluating its ability to adhere, degrade, modify, or induce the production of mucus and/or mucins. Adhesion was analyzed in vitro using two types of mucins (from pig or human biopsies) and mucus-producing intestinal HT29-MTX cells. The induction of mucus was characterized in two different rodent models, in which S. thermophilus is the unique bacterial species in the digestive tract or transited as a sub-dominant bacterium through a complex microbiota. S. thermophilus LMD-9 and LMG18311 strains did not grow in sugars used to form mucins as the sole carbon source and displayed weak binding to mucus/mucins relative to the highly adhesive TIL448 Lactococcus lactis. The presence of S. thermophilus as the unique bacteria in the digestive tract of gnotobiotic rats led to accumulation of lactate and increased the number of Alcian-Blue positive goblet cells and the amount of the mucus-inducer KLF4 transcription factor. Lactate significantly increased KLF4 protein levels in HT29-MTX cells. Introduction of S. thermophilusvia transit as a sub-dominant bacterium (103 CFU/g feces) in a complex endogenous microbiota resulted in a slight increase in lactate levels in the digestive tract, no induction of overall mucus production, and moderate induction of sulfated mucin production. We thus show that although S. thermophilus is a poor mucus-adhesive bacterium, it can promote mucus pathway at least in part by producing lactate in the digestive tract. 9

The gastrointestinal mucosal surface is the primary interface between internal host tissues and the vast microbiota. Mucins, key components of mucus, are high-molecular-weight glycoproteins characterized by the presence of many O-linked oligosaccharides to the core polypeptide. They play many biological functions, helping to maintain cellular homeostasis and to establish symbiotic relationships with complex microbiota. Mucin O-glycans exhibit a huge variety of peripheral sequences implicated in the binding of bacteria to the mucosal tissues, thereby playing a key role in the selection of specific species and in the tissue tropism displayed by commensal and pathogenic bacteria. Bacteria have evolved numerous strategies to colonize host mucosae, and among these are modulation of expression of cell surface adhesins which allow bacteria to bind to mucins. However, despite well structurally characterized adhesins and lectins, information on the nature and structure of oligosaccharides recognized by bacteria is still disparate. This review summarizes the current knowledge on the structure of epithelial mucin O-glycans and the interaction between host and commensal or pathogenic bacteria mediated by mucins.

Helicobacter pylori infects more than half of the world's population. Although most patients are asymptomatic, persistent infection may cause chronic gastritis and gastric cancer. Adhesion of the bacteria to the gastric mucosa is a necessary prerequisite for the pathogenesis of H. pylori-related diseases and is mediated by mucin O-glycans. In order to define which glycans may be implicated in the binding of the bacteria to the gastric mucosa in humans, we have characterized the exact pattern of glycosylation of gastric mucins. We have identified that the major component was always a core 2-based glycan carrying two blood group H antigens, whatever was the blood group of individuals. We have also demonstrated that around 80% of O-glycans carried blood group A, B or H antigens, suggesting that the variation of gastric mucin glycosylation between individuals is partly due to the blood group status. This study will help better understanding the role of O-glycans in the physiology and homeostasis of gastric mucosa. Overall, the results reported here give us the necessary background information to begin studies to determine whether individuals who express certain carbohydrate epitopes on specific mucins are predisposed to certain gastric diseases.

Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed on a microarray platform to investigate whether TFF1 may play a role in locating H. pylori in gastric mucus. TFF1 bound almost exclusively to human gastric mucins and did not interact with human colonic mucins. There was a strong correlation between binding of TFF1 and H. pylori to human gastric mucins, and between binding of both TFF1 and H. pylori to gastric mucins with that of Griffonia simplicifolia lectin-II, which is specific for terminal non-reducing α- or β-linked N-acetyl-d-glucosamine. These results suggest that TFF1 may help to locate H. pylori in a discrete layer of gastric mucus and hence restrain their interactions with epithelial cells.

Cancer-associated alterations in cell surface and secreted glycoproteins have been catalogued for many years but many of the studies of alterations in mucin carbohydrate have relied on histochemical or immunohistochemical methods, with little direct chemical analysis. In this study, we analyzed the O-glycosylation pattern of MUC2 glycoprotein isolated from colorectal carcinomas, transitional mucosa and resection margins from three patients with blood group A, B and O, respectively. After alkaline borohydride treatment, the released oligosaccharides were structurally characterized by nanoESI Q-TOF tandem mass spectrometry without prior fractionation or derivatization. As expected, we found an increased expression of sialyl-Tn antigen in the colonic cancer mucins. A more interesting feature was the increased expression of a core 3 sialyl-Le(x) hexasaccharide, NeuAcalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3(NeuAcalpha2-6)GalNAc in tumor, which appeared to compete with its sulfo-Le(x) counterpart in normal tissue, SO3-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3(NeuAcalpha2-6)GalNAc. This antigen, whose structure was confirmed by NMR experiments, is based on a core 3 glycan and may be a potential marker for the malignant transformation of colonic cells. Unexpectedly, most of the glycans recovered in normal and carcinomas extracts were based on a sialylated core 3, GlcNAcbeta1-3(NeuAcalpha2-6)GalNAcol. Moreover, the pattern of glycosylation was very similar between mucins isolated from each sample, the main differences related to the level of expression of the major oligosaccharides. The data obtained in this investigation may have value for future screening studies on colorectal cancer.

Our studies provide direct evidence that O-glycosylation pathways play a role in the regulation of cell growth through apoptosis and proliferation pathways. A series of small molecular weight analogs of the GalNAc-alpha-1-O-serine/threonine structure based on 1-benzyl-2-acetamido-2-deoxy-alpha-O-d-galactopyranoside have been synthesized and tested in the human colorectal cancer cell lines PC/AA/C1/SB10C and HCA7/C29. Three inhibitors, 1-benzyl-2-acetamido-2-deoxy-alpha-O-D-galactopyranoside, and the corresponding 2-azido- and C-glycoside analogs were screened in these colorectal cancer cell lines at 0.5 mM and showed induction of apoptosis and downregulation of proliferation. Treatment of both cell lines with inhibitors led to changes in glycosylation detected with peanut lectin. The inhibition of glycosyltransferase activity in cell homogenates from human colorectal mucosal cells and cultured cell lines could be shown. The competitive action of the inhibitors resulted in the intracellular formation of 28 aryl-glycan products which were identified by MALDI and electrospray mass spectroscopy. The structures showed a differential pattern for each of the inhibitors in both cell lines. Gene array analysis of the glycogenes illustrated a pattern of glycosyltransferases that matched the glycan structures found in glycoproteins and aryl-glycans formed in the PC/AA/C1/SB10C cells; however, there was no action of the three inhibitors on glycogene transcript levels. The inhibitors act at both intermediary metabolic and genomic levels, resulting in altered protein glycosylation and aryl-glycan formation. These events may play a part in growth arrest.

The gastrointestinal tract is lined by a thick and complex layer of mucus that protects the mucosal epithelium from biochemical and mechanical aggressions. This mucus barrier confers protection against pathogens but also serves as a binding site that supports a sheltered niche of microbial adherence. The carcinogenic bacteria Helicobacter pylori colonize the stomach through binding to host glycans present in the glycocalyx of epithelial cells and extracellular mucus. The secreted MUC5AC mucin is the main component of the gastric mucus layer, and BabA-mediated binding of H. pylori to MUC5AC confers increased risk for overt disease. In this study we unraveled the O-glycosylation profile of Muc5ac from glycoengineered mice models lacking the FUT2 enzyme and therefore mimicking a non-secretor human phenotype. Our results demonstrated that the FUT2 determines the O-glycosylation pattern of Muc5ac, with Fut2 knock-out leading to a marked decrease in a1,2-fucosylated structures and increased expression of the terminal type 1 glycan structure Lewis-a. Importantly, for the first time, we structurally validated the expression of Lewis-a in murine gastric mucosa. Finally, we demonstrated that loss of mucin FUT2-mediated fucosylation impairs gastric mucosal binding of H. pylori BabA adhesin, which is a recognized feature of pathogenicity. The authors thank Nuno Mendes for excellent technical support at the animal facility of IPATIMUP, University of Porto; Dr. Jacques Bara from the U-673 INSERM, CNRS, Paris, France for providing the PM7, 45M1 and 7LE antibodies; Dr Ingemar Carlstedt, University of Lund, Sweden for providing LUM6-3 and LUM5-1 antibodies and Dr. Ola Söderberg from the Uppsala Universitet, Sweden for support with the PLA experiments. This work was financed by FEDER - Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020 - Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT - Fundação para a Ciência e a Tecnologia/Ministério da Ciência, Tecnologia e Inovação in the framework of the project “Institute for Research and Innovation in Health Sciences” (POCI-01-0145-FEDER-007274) and the projects with the references FCOMP-01-0124-FEDER 028188 and FCOMP-01-0124-FEDER041276 (EXPL/CTM-BIO/0762/2013, PTDC/BBB-EBI/0786/2012 and PTDC/BBB-EBI/0567/2014). The authors acknowledge the support by Gastric Glyco Explorer Initial Training Network (Seventh Framework Programme GastricGlycoExplorer project, grant number 316929). AM received an individual grant (SFRH/BPD/75871/2011) from FCT, POPH (Programa Operacional Potencial Humano) and FSE (Fundo Social Europeu) and acknowledges EMBO for a Short-Term Fellowship (EMBO ASTF 330-212). The resources provided by the Consortium for Functional Glycomics were funded by NIGMS-GM62116. TB is supported by grants from Vetenskapsrådet/VR, Cancerfonden, and the J.C. Kempe and Seth M. Kempe Memorial Foundation and this work was in part performed within the Umeå Centre for Microbial Research (UCMR), and the Biochemical Imaging Center Umeå (BICU).

Mucins are high molecular weight glycoproteins characterized by highly O-glycosylated tandem repeat domains. Mucin-type O-glycans exhibit a variety of terminal sequences including histo-blood group antigens that serve as counter receptors and participate in the adhesion and clearance of numerous bacteria including pathogens. In parallel, the pathological changes of mucin glycosylation modulate bacterial adhesion, often enhancing the adhesion of pathogenic bacteria. This review summarizes the current knowledge on the structure and biosynthesis of epithelial mucin O-glycans chains, the physio-pathological glycosylation repertoire of mucins and the role of mucin glycosylation in bacterial adhesion, focusing on the gastrointestinal tract and airway mucins.

International audience; Despite well-known intestinal epithelial barrier impairment and visceral hypersensitivity in irritable bowel syndrome (IBS) patients and IBS-like models, structural and physical changes in the mucus layer remain poorly understood. Using a water avoidance stress (WAS) model, we aimed at evaluating whether 1) WAS modified gut permeability, visceral sensitivity, mucin expression, biochemical structure of O-glycans, and related mucus physical properties, and 2) whether Lactobacillus farciminis treatment prevented these alterations. Wistar rats received orally L. farciminis or vehicle for 14 days; at day 10, they were submitted to either sham or 4-day WAS. Intestinal paracellular permeability and visceral sensitivity were measured in vivo. The number of goblet cells and Muc2 expression were evaluated by histology and immunohistochemistry, respectively. Mucosal adhesion of L. farciminis was determined ex situ. The mucin O-glycosylation profile was obtained by mass spectrometry. Surface imaging of intestinal mucus was performed at nanoscale by atomic force microscopy. WAS induced gut hyperpermeability and visceral hypersensitivity but did not modify either the number of intestinal goblet cells or Muc2 expression. In contrast, O-glycosylation of mucins was strongly affected, with the appearance of elongated polylactosaminic chain containing O-glycan structures, associated with flattening and loss of the mucus layer cohesive properties. L. farciminis bound to intestinal Muc2 and prevented WAS-induced functional alterations and changes in mucin O-glycosylation and mucus physical properties. WAS-induced functional changes were associated with mucus alterations resulting from a shift in O-glycosylation rather than from changes in mucin expression. L. farciminis treatment prevented these alterations, conferring epithelial and mucus barrier strengthening.

Mucin glycoproteins are secreted in large amounts by the intestinal epithelium and constitute an efficient component of innate immune defenses to promote homeostasis and protect against enteric pathogens. In this study, our objective was to investigate how the bacterial enteropathogen Shigella flexneri , which causes bacillary dysentery, copes with the mucin defense barrier. We report that upon in vitro infection of mucin-producing polarized human intestinal epithelial cells, virulent S. flexneri manipulates the secretion of gel-forming mucins. This phenomenon, which is triggered only by virulent strains, results in accumulation of mucins at the cell apical surface, leading to the appearance of a gel-like structure that favors access of bacteria to the cell surface and the subsequent invasion process. We identify MUC5AC, a gel-forming mucin, as a component of this structure. Formation of this gel does not depend on modifications of electrolyte concentrations, induction of trefoil factor expression, endoplasmic reticulum stress, or response to unfolded proteins. In addition, transcriptional and biochemical analyses of infected cells reveal modulations of mucin gene expression and modifications of mucin glycosylation patterns, both of which are induced by virulent bacteria in a type III secretion system-dependent manner. Thus, S. flexneri has developed a dedicated strategy to alter the mucus barrier by targeting key elements of the gel-forming capacity of mucins: gene transcription, protein glycosylation, and secretion.

Osmoregulated periplasmic glucans (OPGs) are oligosaccharides found in the periplasm of many Gram-negative bacteria. Glucose is the sole constitutive sugar and this backbone may be substituted by various kinds of molecules depending on the species. InE. coli, OPG are substituted by phosphoglycerol and phosphoethanolamine derived from membrane phospholipids and by succinyl residues. In this study, we describe the isolation of theopgEgene encoding the phosphoethanolamine transferase by a screen previously used for the isolation of theopgBgene encoding the phosphoglycerol transferase. Both genes show structural and functional similarities without sequence similarity.

International audience; DMBT1 (deleted in malignant brain tumor 1), a human mucin-like glycoprotein, belonging to the scavenger receptor cystein-rich (SRCR) superfamily, is mainly secreted from mucosal epithelia. It has been shown previously that interaction of hensin, the rabbit ortholog of DMBT1, with galectin 3, a β-galactoside-binding lectin, induces a terminal differentiation of epithelial cells. In this paper, we have used surface plasmon resonance (SPR), to analyse the binding of galectin 3 to two purified samples of human DMBT1:recombinant DMBT1 produced in CHO cells and DMBT1 isolated from intestinal tissues. Characterization of their glycosylation profile by nano-ESI-Q-TOF tandem mass spectrometry showed significant differences in O-glycans between the two DMBT1 samples. Results obtained by SPR demonstrated that the oligosaccharide side chains of DMBT1 are recognized by the carbohydrate-recognition domain (CRD) of galectin 3 and modification in the pattern of oligosaccharides modulates the binding parameters of DMBT1 with galectin 3. Moreover, using immunohistochemistry on paraffin-embedded colonic tissue sections, we could show a co-localisation of DMBT1 and galectin 3 in human intestine, suggesting a potential physiological interaction.

International audience; Intestinal mucins are very high molecular weight glycoproteins secreted by goblet cells lining the crypt and the surface of the colonic mucosa. Profound alterations of mucin O-glycans are observed in diseases such as cancer and inflammation, modifying the function of the cell and its antigenic and adhesive properties. Based on immunohistochemical studies, certain cancer- and inflammation- associated glycans have been defined as oncofetal antigens. However, little or no chemical analysis has allowed the structural elucidation of O-glycans expressed on human fetal mucins. In this paper, mucins were isolated from different regions of the normal human intestine (ileum, right, transverse and left colon) of eight fetuses with A, B or O blood group. After alkaline borohydride treatment, the released oligosaccharides were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 117 different glycans were identified, mainly based on core 2 structures. Some core 1, 3 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly α2-6 linked to the N-acetylgalactosaminitol and sulphate residues 3-linked to galactose or 6-linked to GlcNAc. In contrast to adult human intestinal mucins, Sda/Cad determinants were not expressed on fetal mucin O-glycans and the presence of an acidic gradient along the intestinal tract was not observed. Similar patterns of glycosylation were found in each part of the intestine and the level of expression of the major oligosaccharides was in the same order of magnitude. This study could help determining new oncofetal antigens, which can be exploited for the diagnosis or the treatment of intestinal diseases.

Transposition of intestinal segments is frequently used for bladder reconstruction. Following transposition, bowel segments continue to produce mucus and a correlation between excessive mucus production and complications such as urinary tract infection or catheter blockage has been observed for a long time. However, no information is currently available on the change of mucin expression and glycosylation under these abnormal conditions. In this study, the variable number tandem repeat region and the irregular repeat domain of human MUC2 were isolated as two glycopeptide populations after reduction and trypsin digestion followed by gel chromatography from urine of patients transposed with urinary bladders. After alkaline borohydride treatment, the oligosaccharides released from the whole MUC2 mucin and the two glycosylated domains were investigated by nanoESI Q-TOF MS/MS (electrospray ionization quadrupole time-of-flight tandem mass spectrometry). More than 60 different glycans were identified, mainly based on sialylated core 3 structures. Some core 1, 2 and 4 oligosaccharides were also found. Most of the structures were acidic with NeuAc residues mainly alpha2-6 linked to the N-acetylgalactosaminitol and sulphate residues exclusively 3-linked to galactose. No expression of blood group A and B or Sda/Cad determinants was observed. Similar patterns of glycosylation were found in the tandem repeat region and the irregular repeat domain and the level of expression of the major oligosaccharides were in the same order of magnitude. The most interesting feature of this study was that sialyl-Tn antigen, which is considered as a tumour antigen, was the oligosaccharide most highly expressed. This result suggests that mucins from intestinal transposed segments are abnormally glycosylated.

The intestinal mucus layer plays a key role in themaintenance of host-microbiota homeostasis. The production of goblet cells, which secrete mucus, is modulated by microbiota, but neither the species nor the mechanisms involved in this process are still unknown. We studied how two prominent commensal bacteria may influence the mucus production by goblet cells and the profile of mucin glycosylation in gnotobiotic rats. We have chosen Bacteroides thetaiotaomicron, which is characterized by its high mucus-polysaccharides degrading potential, and Faecalibacterium prausnitzii, which is a sensor of intestinal health. Germ free rats (GF) were orally inoculated with B. thetaiotaomicron either alone or with a mix of B. thetaiotaomicron and F. prausnitzii leading respectively to mono-associated (Bt-rats) and di-associated rats (Bt+Fp-rats). A panel of goblet cells markers was analyzed by histological staining, immunohistochemistry, quantitative PCR and Western blot in colon epithelium. The mucin O-glycosylation was determined by MALDI TOF mass spectrometry. In Bt-rats, the goblet cells number and the expression of mucus-related genes (muc2, muc4, klf4, c1galt1 and b4galt4 mRNAs) were increased compared to GF ones. KLF4 protein, a transcription factor involved in goblet cell terminal differentiation, was also increased in Bt-rats, whereas a decrease in Chromogranin A protein, a marker of enteroendocrine cells was observed. We propose that B. thetaiotaomicron provokes an imbalance inside the secretory lineage by favoring mucus production at the expense of enteroendocrine cells. When B. thetaiotaomicron was associated to F. prausnitzii, the effects on goblet cells were reduced/ decreased/diminished. Indeed, the number of goblet cells per crypt and the amount of KLF4 protein were lower in Bt+Fp-rats than in Bt-rats. We then analyzed the mucus quality by studying the profile of mucin O-glycosylation. In Bt-rats, a decrease in the production of sulfated (4.5% of total oligosaccharides instead of 12.9%) and neutral (40.1% instead of 52.8%) oligosaccharides was observed and was correlated to an increased proportion of sialylated O-glycans carrying NeuAc (24.2% instead of 18.9%) or NeuGc (31.2% instead of 15.4%) residues compared to GF rats. Thus, B. thetaiotaomicron impacts the composition of mucin O-glycans, with a decrease in sulfated and neutral oligosaccharides in favor of sialylated ones. Furthermore, glycosylation of mucins from Bt+Fp-rats resembled to those of GF rats. As previously observed for goblet cells, F. prausnitzii seemed to decrease the effect of B. thetaiotaomicron on mucus. Using a novel gnotobiotic model, which is the first described with F. prausnitzii, we showed how the balance between B. thetaiotaomicron and F. prausnitzii plays a key role in protecting epithelium via their respective effects on mucus.