Mucins: structure, function, and associations with malignancy.

Mucins are a family of high molecular weight, highly glycosylated glycoproteins found in the apical cell membrane of human epithelial cells from the mammary gland, salivary gland, digestive tract, respiratory tract, kidney, bladder, prostate, uterus and rete testis. Increased synthesis of the core protein and alterations in the carbohydrates attached to these glycoproteins are believed to play important roles in the function and proliferation of tumour cells. Aberrant glycosylation leads not only to the production of novel carbohydrate structures, but also to the exposure of the core peptide. These novel epitopes may be candidates for diagnosis or therapy, by using either synthetic mucin fragments as vaccines, or monoclonal antibody-based reagents which detect these structures.     

Physiological changes in the gastrointestinal tract and host protective immunity: learning from the mouse-Trichinella spiralis model

Infection and inflammation in the gastrointestinal (GI) tract induces a number of changes in the GI physiology of the host. Experimental infections with parasites represent valuable models to study the structural and physiological changes in the GI tract. This review addresses research on the interface between the immune system and GI physiology, dealing specifically with 2 major components of intestinal physiology, namely mucin production and muscle function in relation to host defence, primarily based on studies using the mouse-Trichinella spiralis system. These studies demonstrate that the infection-induced T helper 2 type immune response is critical in generating the alterations of infection-induced mucin production and muscle function, and that this immune-mediated alteration in gut physiology is associated with host defence mechanisms. In addition, by manipulating the host immune response, it is possible to modulate the accompanying physiological changes, which may have clinical relevance. In addition to enhancing our understanding of immunological control of GI physiological changes in the context of host defence against enteric infections, the data acquired using the mouse-T. spiralis model provide a basis for understanding the pathophysiology of a wide range of GI disorders associated with altered gut physiology.     

Purification and characterization of a human pancreatic adenocarcinoma mucin.

Pancreatic mucins consist of core proteins that are decorated with carbohydrate structures. Previous studies have identified at least two physically distinct populations of mucins produced by a pancreatic adenocarcinoma cell line (HPAF); one is the MUC1 core protein, which includes an oligosaccharide structure identified by a monoclonal antibody (MAb) recognizing the DU-PAN-2 epitope. In this study, we purified and characterized a second mucin fraction, which also shows reactivity with the DU-PAN-2 antibody, but which has an amino acid composition that is not consistent with the MUC1 core protein. This new mucin was purified by ammonium sulfate precipitation, molecular sieve chromatography, and density gradient centrifugation. It eluted in the void volume of a Sepharose 4B column together with an associated low molecular weight protein, which could be further resolved. The mucin is highly polyanionic due to numerous sulfated and sialylated saccharide chains. Carbohydrate analyses of the purified mucin showed the presence of galactose, glucosamine, galactosamine, and sialic acid, but no mannose, glucose, or uronic acid. The purified and deglycosylated mucin shows no reactivity with anti-MUC1 apomucin antibody, but reacts with antiserum against deglycosylated tracheal mucins and antiserum against the MUC4 tandem repeat peptide. Analysis of mucin expression in HPAF cells revealed high levels of MUC1 and MUC4 mRNA, and moderate levels of MUC5AC and MUC5B mRNA. The amino acid composition of the purified mucin shows a high degree of similarity to the MUC4 core protein.     

Mucin glycoproteins in neoplasia

Mucins are high molecular weight glycoproteins that are heavily glycosylated with many oligosaccharide side chains linked O-glycosidically to the protein backbone. With the recent application of molecular biological methods, the structures of apomucins and regulation of mucin genes are beginning to be understood. At least nine human mucin genes have been identified to date. Although a complete protein sequence is known for only three human mucins (MUC1, MUC2, and MUC7), common motifs have been identified in many mucins. The pattern of tissue and cell-specific expression of these mucin genes are emerging, suggesting a distinct role for each member of this diverse mucin gene family. In epithelial cancers, many of the phenotypic markers for pre-malignant and malignant cells have been found on the carbohydrate and peptide moieties of mucin glycoproteins. The expression of carbohydrate antigens appears to be due to modification of peripheral carbohydrate structures and the exposure of inner core region carbohydrates. The expression of some of the sialylated carbohydrate antigens appears to correlate with poor prognosis and increased metastatic potential in some cancers. The exposure of peptide backbone structures of mucin glycoproteins in malignancies appears to be due to abnormal glycosylation during biosynthesis. Dysregulation of tissue and cell-specific expression of mucin genes also occurs in epithelial cancers. At present, the role of mucin glycoproteins in various stages of epithelial cell carcinogenesis (including the preneoplastic state and metastasis), in cancer diagnosis and immunotherapy is under investigation.     

Understanding the mechanisms of chilling injury in bell pepper fruits using the proteomic approach

In order to advance in the understanding of CI in pepper fruits, the cell ultrastructure alterations induced by CI and the physiological and metabolic changes have been studied along with the proteomic study. When stored at low temperatures bell pepper (Capsicum annuum) fruits exhibited visual CI symptoms and important alterations within the cell ultrastructure, since peroxisomes and starch grains were not detected and the structure of the chloroplast was seriously damaged in chilled tissues. Physiological and metabolic disorders were also observed in chilled fruits, such as higher ethylene production, increased MDA content, changes in sugar and organic acids and enzymatic activities. The comparative proteomic analysis between control and chilled fruits reveals that the main alterations induced by CI in bell pepper fruits are linked to redox homeostasis and carbohydrate metabolism. Thus, protein abundance in the ascorbate-glutathione cycle is altered and catalase is down-regulated. Key proteins from glycolysis, Calvin cycle and Krebs cycle are also inhibited in chilled fruits. Enolase and GAPDH are revealed as proteins that may play a key role in the development of chilling injury. This study also provides the first evidence at the protein level that cytosolic MDH is involved in abiotic stress.     

Different Roles for Lactococcal Aggregation Factor and Mucin Binding Protein in Adhesion to Gastrointestinal Mucosa

Adhesion of bacteria to mucosal surfaces and epithelial cells is one of the key features for the selection of probiotics. In this study, we assessed the adhesion property of Lactococcus lactis subsp. lactis BGKP1 based on its strong autoaggregation phenotype and the presence of the mucin binding protein (MbpL). Genes involved in aggregation (aggL) and possible interaction with mucin (mbpL), present on the same plasmid pKP1, were previously separately cloned in the plasmid pAZIL. In vivo and in vitro experiments revealed potentially different physiological roles of these two proteins in the process of adherence to the intestine during the passage of the strain through the gastrointestinal tract. We correlated the in vitro and in vivo aggregation of the BGKP1-20 carrying plasmid with aggL to binding to the colonic mucus through nonspecific hydrophobic interactions. The expression of AggL on the bacterial cell surface significantly increased the hydrophobicity of the strain. On the other hand, the presence of AggL in the strain reduced its ability to adhere to the ileum. Moreover, MbpL protein showed an affinity to bind gastric type mucin proteins such as MUC5AC. This protein did not contribute to the binding of the strain to the ileal or colonic part of the intestine. Different potential functions of lactococcal AggL and MbpL proteins in the process of adhesion to the gastrointestinal tract are proposed.     

Expression of pro-apoptotic Bfk isoforms reduces during malignant transformation in the human gastrointestinal tract

Reduced expression of pro-apoptotic Bcl-2 family proteins has been described in many gastrointestinal cancers, and may play a role in tumourigenesis. The human homologue of the pro-apoptotic Bcl-2 protein, Bfk, is predominantly expressed in tissues of the gastrointestinal tract. In colon, four alternatively spliced isoforms were identified; of which two are pro-apoptotic when overexpressed. In the transition from normal tissue to tumour, pro-apoptotic Bfk isoform expression is substantially reduced in up to 80% of tumours isolated from the human gastrointestinal tract (8/10 colonic tumours and 26/37 of all gastrointestinal tumours) compared to 3/117 tumours from outside the gastrointestinal tract. These data suggest that pro-apoptotic isoforms of Bfk may help to protect against the development of human gastrointestinal malignancy.     

Feminizing Wolbachia: a transcriptomics approach with insights on the immune response genes in Armadillidium vulgare

Background

Clostridium thermocellum produces H₂ and ethanol, as well as CO₂, acetate, formate, and lactate, directly from cellulosic biomass. It is therefore an attractive model for biofuel production via consolidated bioprocessing. Optimization of end-product yields and titres is crucial for making biofuel production economically feasible. Relative protein expression profiles may provide targets for metabolic engineering, while understanding changes in protein expression and metabolism in response to carbon limitation, pH, and growth phase may aid in reactor optimization. We performed shotgun 2D-HPLC-MS/MS on closed-batch cellobiose-grown exponential phase C. thermocellum cell-free extracts to determine relative protein expression profiles of core metabolic proteins involved carbohydrate utilization, energy conservation, and end-product synthesis. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation was used to determine changes in core metabolic proteins in response to growth phase.

Results

Relative abundance profiles revealed differential levels of putative enzymes capable of catalyzing parallel pathways. The majority of proteins involved in pyruvate catabolism and end-product synthesis were detected with high abundance, with the exception of aldehyde dehydrogenase, ferredoxin-dependent Ech-type [NiFe]-hydrogenase, and RNF-type NADH:ferredoxin oxidoreductase. Using 4-plex 2D-HPLC-MS/MS, 24% of the 144 core metabolism proteins detected demonstrated moderate changes in expression during transition from exponential to stationary phase. Notably, proteins involved in pyruvate synthesis decreased in stationary phase, whereas proteins involved in glycogen metabolism, pyruvate catabolism, and end-product synthesis increased in stationary phase. Several proteins that may directly dictate end-product synthesis patterns, including pyruvate:ferredoxin oxidoreductases, alcohol dehydrogenases, and a putative bifurcating hydrogenase, demonstrated differential expression during transition from exponential to stationary phase.

Conclusions

Relative expression profiles demonstrate which proteins are likely utilized in carbohydrate utilization and end-product synthesis and suggest that H₂ synthesis occurs via bifurcating hydrogenases while ethanol synthesis is predominantly catalyzed by a bifunctional aldehyde/alcohol dehydrogenase. Differences in expression profiles of core metabolic proteins in response to growth phase may dictate carbon and electron flux towards energy storage compounds and end-products. Combined knowledge of relative protein expression levels and their changes in response to physiological conditions may aid in targeted metabolic engineering strategies and optimization of fermentation conditions for improvement of biofuels production.     

Ubiquitin in homeostasis,development and disease

Ubiquitin is the most phylogenetically conserved protein known. This 8,500 Da polypeptide can be covalently attached to cellular proteins as a posttranslational modification. In most cases, the addition of multiple ubiquitin adducts to a protein targets it for rapid degradation by a multisubunit protease known as the 26S proteasome. While the ubiquitin/26S proteasome pathway is responsible for the degradation of the bulk of cellular proteins during homeostasis, it may also be responsible for the rapid loss of protein during the programmed death of certain cells, such as skeletal muscle during insect metamorphosis. In addition, alterations in the expression and regulation of ubiquitin may play significant roles in pathological disorders. For example, dramatic increases in ubiquitin and ubiquitin-protein conjugates are observed in a wide variety of neurodegenerative disorders, including Alzheimer's disease. Patients suffering from the autoimmune disease systemic lupus erythematosus generate antibodies reacting with ubiquitin and ubiquitinated histones. At present, it is not known whether these changes in ubiquitin expression and regulation initiate pathological changes in these diseases or if they are altered as a consequence of these disorders.     

Refolding, purification, and characterization of human and murine RegIII proteins expressed in Escherichia coli

The regenerating (Reg) family comprises an extensive, diversified group of proteins with homology to C-type lectins. Several members of this family are highly expressed in the gastrointestinal tract under normal conditions, and often show increased expression in inflammatory bowel disease. However, little is known about Reg protein function, and the carbohydrate ligands for these proteins are poorly characterized. We report here the first expression and purification of Reg proteins using a bacterial system. Mouse RegIIIgamma and its human counterpart, HIP/PAP, were expressed in Escherichia coli, resulting in the accumulation of aggregated recombinant protein. Both proteins were renatured by arginine-assisted procedures and were further purified using cation-exchange chromatography. The identities of the purified proteins were confirmed by SDS-PAGE, N-terminal sequencing, and MALDI-TOF mass spectrometry. Size exclusion chromatography revealed that both proteins exist as monomers, and circular dichroism showed that their secondary structures exhibit a predominance of beta-strands which is typical of C-type lectins. Finally, both RegIIIgamma and human HIP/PAP bind to mannan but not to monomeric mannose, giving initial insights into their carbohydrate ligands. These studies thus provide an essential foundation for further analyses of human and mouse RegIII protein function.     

Exquisite binding specificity of <Emphasis Type="Italic">Sclerotium rolfsii</Emphasis> lectin toward TF-related O-linked mucin-type glycans

Sclerotium rolfsii lectin (SRL), a secretory protein from the soil borne phytopathogenic fungus Sclerotium rolfsii, has shown in our previous studies to bind strongly to the oncofetal Thomson-Friedenreich carbohydrate (Galβ1-3GalNAc-ser/thr, T or TF) antigen. TF antigen is widely expressed in many types of human cancers and the strong binding of SRL toward such a cancer-associated carbohydrate structure led us to characterize the carbohydrate binding specificity of SRL. Glycan array analysis, which included 285 glycans, shows exclusive binding of SRL to the O-linked mucin type but not N-linked glycans and amongst the mucin type O-glycans, lectin recognizes only mucin core 1, core 2 and weakly core 8 but not to other mucin core structures. It binds with high specificity to “α-anomers” but not the “β-anomers” of the TF structure. The axial C4-OH group of GalNAc and C2-OH group of Gal is both essential for SRL interaction with TF disaccharide, and substitution on C3 of galactose by sulfate or sialic acid or N-acetylglucosamine, significantly enhances the avidity of the lectin. SRL differs in its binding to TF structures compared to other known TF-binding lectins such as the Arachis hypogea (peanut) agglutinin, Agaricus bisporus (mushroom) lectin, Jackfruit, Artocarpus integrifolia (jacalin) and Amaranthus caudatus (Amaranthin) lectin. Thus, SRL has unique carbohydrate-binding specificity toward TF-related O-linked carbohydrate structures. Such a binding specificity will make this lectin a very useful tool in future structural as well as functional analysis of the cellular glycans in cancer studies.     

Intestinal release of mucin in response to HCl and taurocholate: effect of indomethacin

1. Alkaline secretion and mucin output were analyzed along the gastrointestinal tract of a dog in response to luminal application of HCl and taurocholate with and without pretreatment with indomethacin. 2. Mucins derived from the different areas displayed similar contents of protein and carbohydrate but differed with respect to associated and covalently bound lipids. 3. Application of HCl or taurocholate in all the regions caused an increase in the output of mucins and HCO3-. However, mucins elaborated in response to HCl exhibited higher total lipid content and were richer in phospholipids. 4. Pretreatment with indomethacin prior to HCl application led to a reduction in HCO3- and caused a decrease in mucin phospholipid content, but had no effect on HCO3- secretion and the lipid content of mucins elaborated in response to taurocholate. 5. The results indicate that mucins elaborated along the gastrointestinal tract differ with respect to lipids, and that their output in response to HCl is mediated by prostaglandins.     

Acute phase mediator oncostatin M regulates affinity of alpha1-protease inhibitor for concanavalin A in hepatoma-derived but not lung-derived epithelial cells

Quantitative changes in plasma protein concentrations during tissue injury or inflammation (acute phase response) are often accompanied by specific alterations in the carbohydrate moieties of these proteins. The glycosylation changes comprise alterations in the type of branching of the carbohydrate structures as revealed by modulated reactivity of acute phase glycoproteins with the lectin concanavalin A. Interestingly, inflammation-induced changes in the glycosylation of acute phase proteins have been shown to affect the functional properties of these proteins. In this study we demonstrate that synthesis of acute phase protein alpha(1)-PI, the controlling inhibitor of neutrophil elastase, is significantly up-regulated in hepatic and lung-derived epithelial cells by the inflammatory mediator oncostatin M. Although oncostatin M markedly altered the concanavalin A reactivity of hepatic alpha(1)-PI, lung-derived epithelial cells did not change the pattern of alpha(1)-PI glycan branching upon stimulation with oncostatin M. These results indicate that inflammation-induced changes in glycosylation of alpha(1)-PI may have different impacts on functional properties of liver and lung-synthesized alpha(1)-PI.     

Proteomic characterization in the hippocampus of prenatally stressed rats

Rats exposed to early life stress are considered as a valuable model for the study of epigenetic programming leading to mood disorders and anxiety in the adult life. Rats submitted to prenatal restraint stress (PRS) are characterized by an anxious/depressive phenotype associated with neuroadaptive changes in the hippocampus. We used the model of PRS to identify proteins that are specifically affected by early life stress. We therefore performed a proteomic analysis in the hippocampus of adult male PRS rats. We found that PRS induced changes in the expression profile of a number of proteins, involved in the regulation of signal transduction, synaptic vesicles, protein synthesis, cytoskeleton dynamics, and energetic metabolism. Immunoblot analysis showed significant changes in the expression of proteins, such as LASP-1, fascin, and prohibitin, which may lie at the core of the developmental programming triggered by early life stress.     

Synthesis and macromolecular organization of gastrointestinal mucin.

Mucus glycoproteins (mucins), the principal determinants of mucus protective qualities and mucosal defense, are studied extensively to define pathological aberrations in the relation to gastrointestinal disease and to develop the mucous barrier strengthening agents. Recent work from our laboratory provided evidence as to the initial stages of the gastrointestinal mucin synthesis, molecular size of the apomucin, its macromolecular organization and interaction with other elements of gastrointestinal mucus. Using monoclonal antibodies against apomucin (clone 1H7), O-glycosylated with N-acetylgalactosamine apomucin (clone 2B4), and that against carboxyl terminal of the apomucin (clone 3G12), the mucin synthesizing polysomes were isolated and glycosylated peptides ranging in size from 6-60 kDa identified. The in vitro synthesis in the cell-free system also afforded 60-64 kDa products recognized by 1H7 and 3G12 antimucin MAbs. The obtained results provided evidence that the mucin core consists of 60 kDa peptide which at cotranslational stage is O-glycosylated with N-acetylgalactosamine. Studies on mucin polymer assembly revealed that mucin preparations prepared by equilibrium density gradient centrifugation and Sepharose 2B chromatography (Mantle, M., Mantle, D., and Allen, A. (1981) Biochem. J. 195, 277-285) are not completely purified and contain DNA and extraneous proteins. The evidence was obtained that so called mucin "link protein", 118 kDa glycopeptide, is a N-glycosylated fragment of fibronectin, whereas the supposedly native undegraded mucin isolated by Carlstedt et al. (Biochem. J. (1983) 211, 13-22) was found to contain mucin-fibronectin-DNA complexes. The general picture that emerged from the studies is that the pure mucin consists of 60 kDa glycosylated peptides only. The carboxyl terminal (8-12 kDa fragment) of these peptides is not glycosylated (naked) and is responsible for mucin interaction with fibronectin and other fibronectin-like extracellular matrix proteins. While the formation of the mucosal coat depends on many other factors and extracellular components, our findings on mucin structure and interaction with the extracellular matrix proteins provide explanation as to the possible mechanism of mucin adherence to the epithelial surfaces.     

Human RegIV Protein Adopts a Typical C-Type Lectin Fold but Binds Mannan with Two Calcium-Independent Sites

Human RegIV protein, which contains a sequence motif homologous to calcium-dependent (C-type) lectin-like domain, is highly expressed in mucosa cells of the gastrointestinal tract during pathogen infection and carcinogenesis and may be applied in both diagnosis and treatment of gastric and colon cancers. Here, we provide evidence that, unlike other C-type lectins, human RegIV binds to polysaccharides, mannan, and heparin in the absence of calcium. To elucidate the structural basis for carbohydrate recognition by NMR, we generated the mutant with Pro91 replaced by Ser (hRegIV-P91S) and showed that the structural property and carbohydrate binding ability of hRegIV-P91S are almost identical with those of wild-type protein. The solution structure of hRegIV-P91S was determined, showing that it adopts a typical fold of C-type lectin. Based on the chemical shift perturbations of amide resonances, two calcium-independent mannan-binding sites were proposed. One site is similar to the calcium-independent sugar-binding site on human RegIII and Langerin. Interestingly, the other site is adjacent to the conserved calcium-dependent site at position Ca-2 of typical C-type lectins. Moreover, model-free analysis of ¹⁵N relaxation parameters and simplified Carr-Purcell-Meiboom-Gill relaxation dispersion experiments showed that a slow microsecond-to-millisecond time-scale backbone motion is involved in mannan binding by this site, suggesting a potential role for specific carbohydrate recognition. Our findings shed light on the sugar-binding mode of Reg family proteins, and we postulate that Reg family proteins evolved to bind sugar without calcium to keep the carbohydrate recognition activity under low-pH environments in the gastrointestinal tract.     

Inflammation-induced “Channelopathies” in the gastrointestinal smooth muscle

Inflammation markedly alters the motility patterns of the gastrointestinal tract, resulting mostly in decreased excitability of smooth muscle. There is emerging evidence indicating that inflammation alters ion channel expression and function of smooth muscle cells. In this review we summarize studies defining the mechanisms affecting contractile and electrical activity of gastrointestinal smooth muscle. We have focused on the evidence for decreased calcium channel conductance and alterations in the intracellular signaling mechanisms and discuss the role of muscarinic receptor activation in models of gastrointestinal inflammation. We propose that some of the clinical symptoms of altered smooth muscle contraction in pathogenesis of gut disorders such as inflammatory bowel disease may be regulated at the level of the ion channel.     

Functional modulation of enterocytes by gram-positive and gram-negative microorganisms

Clinical studies have suggested that so-called probiotic bacteria may be effective as therapy in inflammatory bowel disease. However, the molecular mechanisms of their interaction with the intestinal surface remain undefined. The influence of whole probiotic bacteria [Escherichia coli Nissle 1917 (EcN); probiotic mixture VSL#3 (PM)], bacterial cell lysates, and conditioned media on transepithelial resistance (TER), IL-8 secretion, mucin gene expression, and tight junction proteins were determined in T84 and HT-29 intestinal epithelial cells (IEC). In addition, effects on pathogen (Salmonella dublin)-induced alterations were analyzed. EcN as well as debris and cell extracts induced IL-8 secretion from IEC, whereas no such effect was observed following incubation with the PM. The PM and soluble protein(s) released from the PM increased TER, prevented pathogen-induced decrease in TER, and were shown to stabilize tight junctions. The PM induced expression of mucins in IEC, and these organisms as well as EcN diminished S. dublin-induced cell death. Inhibition of MAPKs with PD-98059 or SB-203580 significantly decreased alterations in IL-8 synthesis and mucin expression and affected the regulation of TER. Probiotics and protein(s) released by these organisms may functionally modulate the intestinal epithelium of the host by different mechanisms, including the competition of whole organisms for contact with the epithelial surface as well as stabilization of the cytoskeleton and barrier function and the induction of mucin expression. Gram-negative and gram-positive organisms differ in the mechanisms activated, and a combination of organisms might be more effective than the application of a single strain.