Mucin Glycosylation Research Articles

Expression of core 3 synthase in human pancreatic cancer cells suppresses tumor growth and metastasis

Core 3-derived glycans, a major type of O-glycan expressed by normal epithelial cells of the gastrointestinal tract, are downregulated during malignancy because of loss of expression of functional β3-N-acetylglucosaminyltransferase-6 (core 3 synthase). We investigated the expression of core 3 synthase in normal pancreas and pancreatic cancer and evaluated the biological effects of re-expressing core 3 synthase in pancreatic cancer cells that had lost expression. We determined that pancreatic tumors and tumor cell lines have lost expression of core 3 synthase. Therefore, we re-expressed core 3 synthase in human pancreatic cancer cells (Capan-2 and FG) to investigate the contribution of core 3 glycans to malignant progression. Pancreatic cancer cells expressing core 3 synthase showed reduced in vitro cell proliferation, migration and invasion compared to vector control cells. Expression of core 3 O-glycans induced altered expression of β1 integrin, decreased activation of focal adhesion kinase, led to the downregulation of expression of several genes including REG1α and FGFR3 and altered lamellipodia formation. The addition of a GlcNAc residue by core 3 synthase leads to the extension of the tumor-associated Tn structure on MUC1. Orthotopic injection of FG cells expressing core 3 synthase into the pancreas of nude mice produced significantly smaller tumors and decreased metastasis to the surrounding tissues compared to vector control FG cells. These findings indicate that expression of core 3-derived O-glycans in pancreatic cancer cells suppresses tumor growth and metastasis through modulation of glycosylation of mucins and other cell surface and extracellular matrix proteins.

Virulent Shigella flexneri Affects Secretion, Expression, and Glycosylation of Gel-Forming Mucins in Mucus-Producing Cells

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.

The mucin-type glycosylating enzyme polypeptide N-acetylgalactosaminyltransferase 14 promotes the migration of ovarian cancer by modifying mucin 13

A high expression of O-glycosylated proteins is one of the prominent characteristics of ovarian carcinoma cells associated with cell migration, which would be attributed to the upregulated expression of glycosyltransferases. Therefore, elucidating glycosyltransferases and their substrates may improve our understanding of their roles in tumor metastasis. In the present study, we reported that knockdown of polypeptide N-acetylgalactosaminyltransferase 14 (GALNT14) by small interfering RNA significantly suppressed the cell migration and altered cellular morphology. Immunoprecipitation and western blot analyses indicated that GALNT14 contributed to the glycosylation of transmembrane mucin 13 (MUC13), which was significantly higher in ovarian cancer cells compared with the normal/benign ovary tissues. Furthermore, interleukin-8 (IL-8), which could regulate the migration ability of epithelial ovarian cancer (EOC) cells, had no remarkable effect on the expression of GALNT14 and the tumor-associated carbohydrate epitope Tn antigen. In addition, extracellular signal-regulated kinase 1/2 (ERK1/2) inhibitor modulated the expression levels of GALNT14. Our findings provide evidence that GALNT14 may contribute to ovarian carcinogenesis through aberrant glycosylation of MUC13, but not through the IL-8 pathway. These data provide novel insights into understanding the function of MUC13 on neoplasm metastasis and may aid in the development of new anticancer drugs for EOC.

Bacteroides thetaiotaomicron and Faecalibacterium prausnitziiinfluence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent

BackgroundThe intestinal mucus layer plays a key role in the maintenance of host-microbiota homeostasis. To document the crosstalk between the host and microbiota, we used gnotobiotic models to study the influence of two major commensal bacteria, Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii, on this intestinal mucus layer. B. thetaiotaomicron is known to use polysaccharides from mucus, but its effect on goblet cells has not been addressed so far. F. prausnitzii is of particular physiological importance because it can be considered as a sensor and a marker of human health. We determined whether B. thetaiotaomicron affected goblet cell differentiation, mucin synthesis and glycosylation in the colonic epithelium. We then investigated how F. prausnitzii influenced the colonic epithelial responses to B. thetaiotaomicron.ResultsB. thetaiotaomicron, an acetate producer, increased goblet cell differentiation, expression of mucus-related genes and the ratio of sialylated to sulfated mucins in mono-associated rats. B. thetaiotaomicron, therefore, stimulates the secretory lineage, favoring mucus production. When B. thetaiotaomicron was associated with F. prausnitzii, an acetate consumer and a butyrate producer, the effects on goblet cells and mucin glycosylation were diminished. F. prausnitzii, by attenuating the effects of B. thetaiotaomicron on mucus, may help the epithelium to maintain appropriate proportions of different cell types of the secretory lineage. Using a mucus-producing cell line, we showed that acetate up-regulated KLF4, a transcription factor involved in goblet cell differentiation.ConclusionsB. thetaiotaomicron and F. prausnitzii, which are metabolically complementary, modulate, in vivo, the intestinal mucus barrier by modifying goblet cells and mucin glycosylation. Our study reveals the importance of the balance between two main commensal bacteria in maintaining colonic epithelial homeostasis via their respective effects on mucus.

264 Bacteroides Thetaiotaomicron and Faecalibacterium prausnitzii Shape the Mucus Production and Mucin O-Glycosylation in Colon Epithelium

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.

265 An International Patient Registry and Database for Microvillus Inclusion Disease and Associated MYO5B Mutations

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.

Aberrant Expression of Mucin Core Proteins and O-Linked Glycans Associated with Progression of Pancreatic Cancer

Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed in pancreatic adenocarcinoma-sialyl Tn (STn), Tn, T antigen, sialyl Lewis A (CA19-9), sialyl Lewis C (SLeC), Lewis X (LeX), and sialyl LeX (SLeX)-during the progression of pancreatic cancer from early stages to metastatic disease. Immunohistochemical analyses of mucin and associated glycan expression on primary tumor and liver metastatic tumor samples were conducted with matched sets of tissues from 40 autopsy patients diagnosed with pancreatic adenocarcinoma, 14 surgically resected tissue samples, and 8 normal pancreata. There were significant changes in mucin expression patterns throughout disease progression. MUC1 and MUC4 were differentially glycosylated as the disease progressed from early pancreatic intraepithelial neoplasias to metastatic disease. De novo expression of several mucins correlated with increased metastasis indicating a potentially more invasive phenotype, and we show the expression of MUC6 in acinar cells undergoing acinar to ductal metaplasia. A "cancer field-effect" that included changes in mucin protein expression and glycosylation in the adjacent normal pancreas was also seen. There are significant alterations in mucin expression and posttranslational processing during progression of pancreatic cancer from early lesions to metastasis. The results are presented in the context of how mucins influence the biology of tumor cells and their microenvironment during progression of pancreatic cancer.

Abstract 4511: An evaluation of the role of Mucin in nano drug delivery.

Abstract Introduction: Solid tumors comprise more than 85% of cancers that affect humans. The presence of the extracellular matrix, namely mucin, on the tumor cell is considered one of the barriers against effective treatment of solid tumors. In tumor cells, mucin is aberrantly overexpressed producing a mucin mesh which may impede the rate of entry of chemotherapy medications. The type O-glycosylation inhibitor, benzyl-α-GalNAc, significantly reduces mucin type O-glycosylation in mucin-producing tumor cells resulting in enhanced cytotoxic drug action. We next seek to determine the role of mucin in the delivery of nano-sized drug delivery systems. Methods: The cell lines used in our studies were derived from tumor or normal tissues representing multiple different tissue environments. A fluorescence microplate reader was used to evaluate the MUC1 expression in the cell lines by using CD227 (anti-MUC1 antibody). We investigated the cellular uptake of the fluorescently labeled polystyrene microsphere beads in Capan-1 (a human pancreatic cancer cell line). The size of these beads was determined by particle size analysis. FACS analysis was used to determine cellular uptake following inhibition of mucin type O-glycosylation and compared to control. Finally, fluorescence microscopy was used to show the effect of inhibitor on the uptake of the beads into ZR-75-1 (a human breast cancer cell line) and Capan-1 cells. Results: Different levels of mucin expression were observed among the various cell lines. By investigating the antibody association with the MUC1, Capan-1and ZR-75-1have shown the greatest cellular uptake following inhibition of glycosylation. The microspheres used in this study were 920 ±70 nm, 100 ±36 nm, and 52 ±8 nm in size. Cellular uptake studies of the microsphere beads into Capan-1 showed a significant increase in the uptake of the 920 nm beads in cells when compared to control cells. However, in case of the smaller beads, we did not observe a significant increase of the cellular uptake following inhibition of the mucin glycosylation. FACS analysis of the beads with Capan-1 cell line showed a red shift in the fluorescence peak of the 920 nm beads in cells treated with inhibitor compared to control. The smaller beads (52 nm and 100 nm), however, did not show any shift indicating that the uptake of the beads was hindered by the mucin mesh. Conclusion: Our results suggest that inhibiting the mucin type O-glycosylation alters the uptake of relatively large microsphere beads. This might be helpful in developing a better therapeutic approach to treat cancer. Citation Format: Musaed Allkholief, Robert Campbell. An evaluation of the role of Mucin in nano drug delivery. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4511. doi:10.1158/1538-7445.AM2013-4511

Mining the “glycocode”—exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy

Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.—Gunning, A. P., Kirby, A. R., Fuell, C., Pin, C., Tailford L. E., Juge, N. Mining the “glycocode”—exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy.

Protective immunity against Echinostoma caproni in rats is induced by Syphacia muris infection

Syphacia muris (Nematoda: Oxyuridae) is a ubiquitous nematode that commonly infects rats in the laboratory and can interfere with the development of unrelated biological assays. In this context, we analysed the effect of a patent S. muris infection in Wistar rats on a superimposed infection with the intestinal trematode, Echinostoma caproni (Trematoda: Echinostomatidae). The results indicate that in the rats, infection with S. muris induces an immunity against a subsequent infection with E. caproni, although each parasite occupies different niches in the host. Echinostoma caproni worm recovery was significantly decreased in the rats primarily infected with S. muris and, at 3 and 4weeks post-primary infection, the rats primarily infected with S. muris were refractory to the challenge infection with E. caproni. We observed that the main alterations induced by S. muris in the niche of E. caproni (ileum) that may be the cause of the resistance are: (i) a local antibody response with elevated levels of mucosal IgA, IgE, IgG, IgG1 and IgG2a that cross-react with E. caproni antigens; (ii) development of a biased Th17/Th2 phenotype; and (iii) changes in the glycosylation of ileal mucins. This indicates that S. muris induces distant alterations to the ileum of rats affecting the development of other helminth species. Apart from the interest of these results in the study of the interactions between helminths in a single host, it has been demonstrated that pinworm infections may interfere in non-related experiments.

Cancer Associated Aberrant Protein O-Glycosylation Can Modify Antigen Processing and Immune Response

Aberrant glycosylation of mucins and other extracellular proteins is an important event in carcinogenesis and the resulting cancer associated glycans have been suggested as targets in cancer immunotherapy. We assessed the role of O-linked GalNAc glycosylation on antigen uptake, processing, and presentation on MHC class I and II molecules. The effect of GalNAc O-glycosylation was monitored with a model system based on ovalbumin (OVA)-MUC1 fusion peptides (+/− glycosylation) loaded onto dendritic cells co-cultured with IL-2 secreting OVA peptide-specific T cell hybridomas. To evaluate the in vivo response to a cancer related tumor antigen, Balb/c or B6.Cg(CB)-Tg(HLA-A/H2-D)2Enge/J (HLA-A2 transgenic) mice were immunized with a non-glycosylated or GalNAc-glycosylated MUC1 derived peptide followed by comparison of T cell proliferation, IFN-γ release, and antibody induction. GalNAc-glycosylation promoted presentation of OVA-MUC1 fusion peptides by MHC class II molecules and the MUC1 antigen elicited specific Ab production and T cell proliferation in both Balb/c and HLA-A2 transgenic mice. In contrast, GalNAc-glycosylation inhibited the presentation of OVA-MUC1 fusion peptides by MHC class I and abolished MUC1 specific CD8+ T cell responses in HLA-A2 transgenic mice. GalNAc glycosylation of MUC1 antigen therefore facilitates uptake, MHC class II presentation, and antibody response but might block the antigen presentation to CD8+ T cells.

TNF regulates sialyl-Lewisx and 6-sulfo-sialyl-Lewisx expression in human lung through up-regulation of ST3GAL4 transcript isoform BX

Bronchial mucins from severely infected patients suffering from lung diseases such as chronic bronchitis or cystic fibrosis exhibit increased amounts of sialyl-Lewis(x) (NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAc-R, sLe(x)) glycan structures. In cystic fibrosis, sLe(x) and its sulfated form 6-sulfo-sialyl-Lewis(x) (NeuAcα2-3Galβ1-4[Fucα1-3](HO(3)S-6)GlcNAc-R, 6-sulfo-sLe(x)) serve as receptors for Pseudomonas aeruginosa and are involved in the chronicity of airway infection. However, little is known about the molecular mechanisms regulating the changes in glycosylation and sulfation of mucins in airways. Herein, we show that the pro-inflammatory cytokine TNF increases the expression of α2,3-sialyltransferase gene ST3GAL4, both in human bronchial mucosa and in A549 lung carcinoma cells. The role of sialyltransferase ST3Gal IV in sLe(x) biosynthesis was confirmed by siRNA silencing of ST3GAL4 gene. BX is the major transcript isoform expressed in healthy bronchial mucosa and in A549 cells, and is up-regulated by TNF in both models. Bioinformatics analysis and luciferase assays have confirmed that the 2kb genomic sequence surrounding BX exon contains a promoter region regulated by TNF-related transcription factors. These results support further work aiming at the development of anti-inflammatory strategy to reduce chronic airway infection in diseases such as cystic fibrosis.

Human Gastric Mucins Differently Regulate Helicobacter pylori Proliferation, Gene Expression and Interactions with Host Cells

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

Almost all human gastric mucin O-glycans harbor blood group A, B or H antigens and are potential binding sites for Helicobacter pylori

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.

Construction of a Natural Mucin Microarray and Interrogation for Biologically Relevant Glyco-Epitopes

Mucins are the principal components of mucus, and mucin glycosylation has important roles in defense, microbial adhesion, immunomodulation, inflammation, and cancer. Mucin expression and glycosylation are dynamic, responding to changes in local environment and disease. Potentially hundreds of heterogeneous glycans can substitute one mucin molecule, and it is difficult to identify biologically accessible glyco-epitopes. Thirty-seven mucins, from the reproductive and gastrointestinal (GI) tracts of six species (bovine, ovine, equine, porcine, chicken, and deer) and from two human-derived cell lines, were purified. Following optimization of mucin printing and construction of a novel mucin microarray, the glycoprofiles of the whole mucins on the microarray were compared using a panel of lectins and one antibody. Accessible glyco-motifs of GI mucins varied according to species and localization of mucin origin, with terminal fucose, the sialyl T-antigen, and N-linked oligosaccharides identified as potentially important. The occurrence of T- and sialyl T-antigen varied in bovine and ovine reproductive tract mucins, and terminal N-acetylgalactosamine (GalNAc) and sulfated carbohydrates were detected. This study introduces natural mucin microarrays as an effective tool for profiling mucin glyco-epitopes and highlights their potential for discovery of biologically important motifs in bacterial-host interactions and fertility.

The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer

There has been considerable interest in understanding the epitopes that bind the lectin Helix pomatia agglutinin (HPA) in breast cancer as the lectin has been shown to identify glycosylation changes associated with the development of metastatic disease. HPA has previously been shown to recognize aberrant O-linked α-N-acetylgalactosamine (GalNAcα)/mucin glycosylation in cancer, including exposed Tn epitopes. However, recent glycan-array analysis reported that diverse epitopes are also recognized by the lectin, e.g. consortium for functional glycomics (CFG) data: GalNAcα1,3Gal; β-GalNAc; GlcNAcβ1,4Gal. The intriguing observations from the CFG array led to this study, in which HPA-binding epitopes were localized and characterized in an in vitro model of breast cancer metastasis. HMT3522 (benign disease), BT474 (primary cancer) and T47D/MCF7 (metastatic cancer) cells were assessed in confocal microscopy-based co-localization studies and a glycoproteomic analysis based on 2-dimensional electrophoresis (2DE), western blotting and mass spectrometry was adopted. HPA binding correlated with levels of integrin α6, transcription factors heterogeneous nuclear ribonuclear protein (HnRNP) H1, HnRNP D-like, HnRNP A2/B1 as well as heat shock protein 27 (Hsp27), glial fibrillary acidic protein and enolase 1 (ENO1). These glycoproteins were non-detectable in the non-metastatic breast cancer cell lines. The recognition of HnRNPs, Hsp27 and ENO1 by HPA correlated with O-GlcNAcylation of these proteins. Integrin α6 was the most abundant HPA glycoprotein in the breast cancer cells with a metastatic phenotype; this concurred with previous findings in colorectal cancer. This is the first report in which HPA has been shown to bind O-GlcNAcylated transcription factors. This class of proteins represents a new means by which HPA differentiates cancer cells with an aggressive metastatic phenotype.

O-linked glycosylation of mucins

O-linked glycosylation of mucins

O-linked glycosylation of mucins

O-linked glycosylation of mucins

Analysis of mucosal mucins separated by SDS‐urea agarose polyacrylamide composite gel electrophoresis

Efficient separation of mucins (200 kDa-2 MDa) was demonstrated using gradient SDS agarose/polyacrylamide composite gel electrophoresis (SDS-AgPAGE). Inclusion of urea (SDS-UAgPAGE) in the gels casting were shown to have no effect on the migration of mucins in the gel and allowed casting of gel at room temperature. This simplified the procedure for multiple casting of agarose polyacrylamide gradients and increased reproducibility of these gels. Hence, the implementation of urea makes the technique applicable for high throughput isolation and screening of mucin oligosaccharides by LC-MS after releasing the oligosaccharides from isolated, blotted mucin subpopulations. It was also shown that the urea addition had no effect on other supporting applications such as western and lectin blotting. In addition, identification of the mucin protein after tryptic digestion and LC-MS was possible and no protein carbamylation due to the presence of urea in the gel was detected. LC-MS software developed for metabolomic analysis was used for O-linked oligosaccharide detection and differential display of various mucin samples. Using this method, heterogeneous glycosylation of mucins and mucin-type molecules isolated by SDS-AgPAGE and SDS-UAgPAGE was shown to consist of more than 80 different components in a single band, and in the extreme cases, up to 300-500 components (MUC5B/AC from saliva and sputum and). Metabolomic software was also used to show that the migration of mucin isoforms within the gel is due to heterogeneous size distribution of the oligosaccharides, with the slower migrating bands enriched in high-molecular-weight oligosaccharides.

Decreased salivary sulphotransferase activity correlated with inflammation and autoimmunity parameters in Sjogren's syndrome patients

To determine the expression and enzymatic activities of sulphotransferases involved in mucin hyposulphation in labial salivary glands (LSGs) from SS patients and to correlate sulphotransferase activity with clinical parameters such as secretion, inflammation and serology. LSG from 31 SS patients and 31 control subjects were studied. Relative mRNA and protein levels of Gal3-O-sulphotransferases (Gal3STs) and β1,3-galactosyltransferase-5 (β3GalT5) were determined by quantitative RT-PCR and western blotting, respectively. Enzymatic activities were quantified using radioactively labelled donor substrates and specific acceptor substrates. Products were purified by chromatography. Spearman's correlation analysis was used to compare data. The levels of Gal3ST activity were significantly decreased in SS patients, without changes in mRNA and protein levels, while the enzymatic activities of glycosyltransferases involved in mucin glycosylation were similar in both groups. An inverse correlation was observed between Gal3ST activity and glandular function measured by scintigraphy, but not with unstimulated salivary flow. Gal3ST activity was inversely correlated with focus score, TNF-α levels and presence of the autoantibodies Ro/SS-A and La/SS-B. The decrease in sulphotransferase activity provides an explanation for mucin hyposulphation observed in the LSGs from SS patients. The decrease in Gal3STs activity was not a consequence of reduced gene expression, but probably due to alterations in the enzyme activity regulation. Interestingly, the levels of sulphotransferase activity detected correlated well with secretory function, inflammation and serology. Finally, we postulate that pro-inflammatory cytokines induced by autoantibodies, such as Ro/SS-A and La/SS-B in SS patients, may modulate Gal3ST activity, thereby altering mucin quality and leading to mouth dryness.