Interaction of Eimeria tenella with intestinal mucin in vitro

The mucus gel layer overlying the gastrointestinal epithelium plays an important role in host-pathogen interactions. The initial interaction between the coccidian parasite Eimeria tenella and host cells of the intestinal epithelium must occur across this mucus interface. In this study, we examined the relationship between E. tenella and avian mucin, in particular the effect of purified intestinal regional mucin on parasite adherence and invasion in vitro. Secreted mucin from the chicken duodenum and cecum was purified by density gradient centrifugation and gel chromatography. Parasite invasion studies were performed in the Madin-Darby bovine kidney cell model. Eimeria tenella adherence to chicken duodenal mucin was detected, whereas adherence to cecal or bovine mucin was not shown. Parasite invasion into epithelial cells was not influenced by bovine mucin, whereas chicken mucin purified from the duodenum and cecum significantly inhibited invasion. Inhibition of E. tenella invasion into cells by mucin from the duodenum was marginally greater than that of the cecum, but this was not significant. This study demonstrated E. tenella interaction with native chicken intestinal mucin, which in turn inhibited parasite invasion into epithelial cells in vitro.     

The rheology of pig small intestinal and colonic mucus: weakening of gel structure by non-mucin components.

Mechanical spectroscopy has been used to study the structure and properties of pig small intestinal and colonic adherent mucus gel. Both mucus secretions had properties of viscoelastic gels, but that from the small intestine was substantially weaker in quality. Small intestinal mucus gel was disrupted by acid (pH 1), detergents (bile) and protein denaturants while that from the colon remained stable following these treatments. Concentration of purified colonic mucin produced a gel with the same rheological properties as the native secretion. Purified small intestinal mucin when concentrated produced a stronger gel than the native secretion and, in contrast to the latter, one which was not disrupted by acid or denaturants. The instability of native small intestinal mucus was shown not to be a function of the mucin components (which alone could account for the gel-forming properties), but to arise from the presence of insoluble material largely from sloughed mucosal cells. These studies show (1) that mucus gels from the colon and small intestine have similar mechanical behaviour and properties to those from the stomach and duodenum, and (2) emphasise the caution that should be exercised when interpreting the rheological properties of mucus preparations, particularly with respect to their content of mucosal cellular material.     

Effect of pepsin on partially purified pig gastric mucus and purified mucin

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid-Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (less than 10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.     

Protein structure and hydration probed by SANS and osmotic stress

Interactions governing protein folding, stability, recognition, and activity are mediated by hydration. Here, we use small-angle neutron scattering coupled with osmotic stress to investigate the hydration of two proteins, lysozyme and guanylate kinase (GK), in the presence of solutes. By taking advantage of the neutron contrast variation that occurs upon addition of these solutes, the number of protein-associated (solute-excluded) water molecules can be estimated from changes in both the zero-angle scattering intensity and the radius of gyration. Poly(ethylene glycol) exclusion varies with molecular weight. This sensitivity can be exploited to probe structural features such as the large internal GK cavity. For GK, small-angle neutron scattering is complemented by isothermal titration calorimetry with osmotic stress to also measure hydration changes accompanying ligand binding. These results provide a framework for studying other biomolecular systems and assemblies using neutron scattering together with osmotic stress.     

Accounting for thermodynamic non-ideality in the Guinier region of small-angle scattering data of proteins

Hydrodynamic studies of the solution properties of proteins and other biological macromolecules are often hard to interpret when the sample is present at a reasonably concentrated solution. The reason for this is that solutions exhibit deviations from ideal behaviour which is manifested as thermodynamic non-ideality. The range of concentrations at which this behaviour typically is exhibited is as low as 1–2 mg/ml, well within the range of concentrations used for their analysis by techniques such as small-angle scattering. Here we discuss thermodynamic non-ideality used previously used in the context of light scattering and sedimentation equilibrium analytical ultracentrifugation and apply it to the Guinier region of small-angle scattering data. The results show that there is a complementarity between the radially averaged structure factor derived from small-angle X-ray scattering/small-angle neutron scattering studies and the second virial coefficient derived from sedimentation equilibrium analytical ultracentrifugation experiments.     

A combined small‐angle X‐ray and neutron scattering study of the structure of purified soluble gastrointestinal mucins

The structures of purified soluble porcine gastric (Muc5ac) and duodenal (Muc2) mucin solutions at neutral and acidic pH were examined using small‐angle X‐ray scattering and small‐angle neutron scattering experiments. We provide evidence for the morphology of the network above the semidilute overlap concentration and above the entanglement concentration. Furthermore, we investigated the gelation of both types of mucin solutions in response to a reduction in pH, where we observed the formation of large‐scale heterogeneities within the polymer solutions, typical of microphase‐separated gels. The concentration dependence of the inhomogeneity length scale (Ξ) and the amplitude of the excess scattering intensity [I_ex(0)] are consistent with previously studied gelled synthetic polymeric systems. The persistence lengths of the chains were found to be similar for both Muc5ac and Muc2 from Kratky plots of the neutron data (8 ± 2 nm). © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1154–1164, 2014.     

Effect of Ca++ on the structure and rheology of canine tracheal mucin

Mucin glycoproteins are known to be the principal determinants of epithelial mucus rheology and hence of mucociliary transport rates. We are studying the structure of such glycoproteins using a model mucin purified from canine tracheal pouch secretions. Of particular interest is the effect on mucin structure of increased Ca++ such as occurs in certain disease states. Quasielastic laser light scattering was used to study the effect of Ca++ on the hydrodynamic radius of the mucin molecules. Scattering data from 0.3mg/ml mucin solutions in physiological phosphate buffer containing 0, 5 X 10(-5)M, and 5 X 10(-4)M Ca++ were analyzed to obtain an average translational diffusion coefficient and the distribution of molecular radii for the dispersion. The effect of Ca++ was to decrease the average Stokes radius. The light scattering results are supported by rheologic measures of mucin gel viscoelasticity.     

Isolation and structural analysis of rat gastric mucus glycoprotein suggests a homogeneous protein backbone.

We isolated monomeric gastric mucus glycoprotein from the rat stomach by applying three successive CsCl-density-gradient steps in the continuous presence of guanidinium chloride. The rat gastric mucin was pure as compared with mucin isolated without the chaotropic reagent. In addition, the presence of guanidinium chloride resulted in a better preservation of the protein moiety. The purified mucin was fractionated according to buoyant density and chemically radiolabelled on tyrosine or cysteine residues and digested with specific proteinases. Analysis of mucin fractions of various densities gave identical peptide patterns, suggesting that the fractions contain a common protein backbone. Electron-microscopic images of the individual mucin molecules were recorded using rotary shadowing. They showed large filamentous molecules with a mean length of 208 nm that, after proteolytic digestion, yielded glycopeptides with a mean length of 149 nm. Heterogeneity in buoyant density and electrophoretic mobility is located in this large glycopeptide which remains after proteolytic digestion. Metabolic labelling of the mucin with [35 S]sulphate and [3H]galactose, followed by purification and proteolytic digestion, revealed that this glycopeptide accounts for most of the mass and contains relatively little protein, but probably all the oligosaccharides and sulphate. As this protein part is masked by the oligosaccharides, detailed study by the methods described was not possible. The results indicate that rat gastric mucin is homogeneous in a major part of the protein backbone and that the heterogeneity of the molecule originates most likely from differences in sulphate and/or sugar composition.     

Solution structure of a short DNA fragment studied by neutron scattering

The solution structure of a DNA fragment of 130 base pairs and known sequence has been investigated by neutron small-angle scattering. In 0.1 M NaCl, the overall structure of the DNA fragment which contains the strong promoter A1 of the Escherichia coli phage T7 agrees with that expected for B-DNA. The neutron scattering curve is well fitted by that of a rigid rod with a length of 44 nm and a diameter of 2 nm. The results were confirmed by quasi-elastic light scattering and analytical centrifugation. The neutron measurements in H2O and D2O buffer reveal a cross-sectional inhomogeneity not detected by X-ray small-angle scattering. This inhomogeneity is caused by the hydration layer around the DNA core and not by the helical structure. The primary solvent shell has a density increased by at least 4-9% compared to bulk water.     

Adsorption of divalent cations on DNA

The distribution of divalent ions in semidilute solutions of high-molecular-mass DNA containing both sodium chloride and strontium chloride in near-physiological conditions is studied by small-angle x-ray scattering and by small-angle neutron scattering. Both small-angle neutron scattering and small-angle x-ray scattering reveal a continuous increase in the scattering intensity at low q with increasing divalent ion concentration, while at high q the scattering curves converge. The best fit to the data is found for a configuration in which DNA strands of cross-sectional radius 10 angstroms are surrounded by a counterion sheath of outer radius approximately 13.8 angstroms, independent of the strontium chloride concentration. When the strontium chloride is replaced by calcium chloride, similar results are obtained, but the thickness of the sheath increases when the divalent salt concentration decreases. These results correspond in both cases to partial localization of the counterions within a layer that is thinner than the effective Debye screening length.     

Reorganisation of the Salivary Mucin Network by Dietary Components: Insights from Green Tea Polyphenols

The salivary mucins that include MUC5B (gel-forming) and MUC7 (non-gel-forming) are major contributors to the protective mucus barrier in the oral cavity, and it is possible that dietary components may influence barrier properties. We show how one dietary compound, the green tea polyphenol epigallocatechin gallate (EGCG), can substantially alter the properties of both the polymeric MUC5B network and monomeric MUC7. Using rate-zonal centrifugation, MUC5B in human whole saliva and MUC5B purified from saliva sedimented faster in the presence of EGCG. The faster sedimentation by EGCG was shown to be greater with increasing MUC5B concentration. Particle tracking microrheology was employed to determine the viscosity of purified MUC5B solutions and showed that for MUC5B solutions of 200–1600 µg/mL, EGCG caused a significant increase in mucin viscosity, which was greater at higher MUC5B concentrations. Visualisation of the changes to the MUC5B network by EGCG was performed using atomic force microscopy, which demonstrated increased aggregation of MUC5B in a heterogeneous manner by EGCG. Using trypsin-resistant, high-molecular weight oligosaccharide-rich regions of MUC5B and recombinant N-terminal and C-terminal MUC5B proteins, we showed that EGCG causes aggregation at the protein domains of MUC5B, but not at the oligosaccharide-rich regions of the mucin. We also demonstrated that EGCG caused the majority of MUC7 in human whole saliva to aggregate. Furthermore, purified MUC7 also underwent a large increase in sedimentation rate in the presence of EGCG. In contrast, the green tea polyphenol epicatechin caused no change in the sedimentation rate of either MUC5B or MUC7 in human whole saliva. These findings have demonstrated how the properties of the mucin barrier can be influenced by dietary components. In the case of EGCG, these interactions may alter the function of MUC5B as a lubricant, contributing to the astringency (dry puckering sensation) of green tea.     

Mucin-derived O-glycans supplemented to diet mitigate diverse microbiota perturbations

Microbiota-accessible carbohydrates (MACs) are powerful modulators of microbiota composition and function. These substrates are often derived from diet, such as complex polysaccharides from plants or human milk oligosaccharides (HMOs) during breastfeeding. Host-derived mucus glycans on gut-secreted mucin proteins serve as a continuous endogenous source of MACs for resident microbes; here we investigate the potential role of purified, orally administered mucus glycans in maintaining a healthy microbial community. In this study, we liberated and purified O-linked glycans from porcine gastric mucin and assessed their efficacy in shaping the recovery of a perturbed microbiota in a mouse model. We found that porcine mucin glycans (PMGs) and HMOs enrich for taxonomically similar resident microbes. We demonstrate that PMGs aid recovery of the microbiota after antibiotic treatment, suppress Clostridium difficile abundance, delay the onset of diet-induced obesity, and increase the relative abundance of resident Akkermansia muciniphila. In silico analysis revealed that genes associated with mucus utilization are abundant and diverse in prevalent gut commensals and rare in enteric pathogens, consistent with these glycan-degrading capabilities being selected for during host development and throughout the evolution of the host–microbe relationship. Importantly, we identify mucus glycans as a novel class of prebiotic compounds that can be used to mitigate perturbations to the microbiota and provide benefits to host physiology.Subject terms: Microbial ecology, Diseases     

Evaluation of MUC6 mucin tandem repeats

The MUC6 mucin was originally isolated from stomach mucus and is one of the major secreted mucins of the digestive tract. A full-length cDNA has not been isolated for this large molecule (greater than 15 kb) and it remains poorly studied. To circumvent the lack of reagents for investigating MUC6, we isolated a cDNA clone from a human fetal pancreatic duct cDNA library that encodes 282 amino acids of the MUC6 tandem repeat. A blast search with the sequence of this cDNA clone showed 90% homology with the original MUC6 (L07517) derived from a human stomach cDNA library and 95% homology both with AK096772, a MUC6-related protein isolated from a human prostate cDNA library and the human genome project clone AC083984. The MUC6 partial cDNA clone isolated from fetal pancreas was inserted into an epitope-tagged MUC1 mucin molecule in place of the native tandem repeat. This chimeric mucin was expressed in human pancreatic (Panc1) and colon (Caco2) carcinoma cell lines and purified for analysis of O-glycosylation by fast atom bombardment mass spectrometry (FAB-MS). The FAB-MS spectra showed O-glycans that had been detected previously on chimeric mucins carrying different tandem repeats, though the spectra for MUC1F/6TR mucins expressed in the Panc1 and Caco2 cells were very different. There was a paucity of O-glycosylation in Panc1 cells in comparison to Caco2 cells where many more structures were evident, and the most abundant glycans in Panc1 cells were sialylated.     

Functional domain motions in proteins on the ~1-100 ns timescale: comparison of neutron spin-echo spectroscopy of phosphoglycerate kinase with molecular-dynamics simulation

Protein function often requires large-scale domain motion. An exciting new development in the experimental characterization of domain motions in proteins is the application of neutron spin-echo spectroscopy (NSE). NSE directly probes coherent (i.e., pair correlated) scattering on the ~1-100 ns timescale. Here, we report on all-atom molecular-dynamics (MD) simulation of a protein, phosphoglycerate kinase, from which we calculate small-angle neutron scattering (SANS) and NSE scattering properties. The simulation-derived and experimental-solution SANS results are in excellent agreement. The contributions of translational and rotational whole-molecule diffusion to the simulation-derived NSE and potential problems in their estimation are examined. Principal component analysis identifies types of domain motion that dominate the internal motion's contribution to the NSE signal, with the largest being classic hinge bending. The associated free-energy profiles are quasiharmonic and the frictional properties correspond to highly overdamped motion. The amplitudes of the motions derived by MD are smaller than those derived from the experimental analysis, and possible reasons for this difference are discussed. The MD results confirm that a significant component of the NSE arises from internal dynamics. They also demonstrate that the combination of NSE with MD is potentially useful for determining the forms, potentials of mean force, and time dependence of functional domain motions in proteins.     

Atomic force microscopy reveals aggregation of gastric mucin at low pH

Mammalian gastric mucin, at high concentration, is known to form a gel at low pH, behavior essential to the protection of the stomach from auto-digestion. Atomic force microscopy (AFM) measurements of dilute solutions of porcine gastric mucin in an aqueous environment in the pH range 6-2 provide a direct visualization of extended fiberlike molecules at pH 6 that aggregate at pH 4 and below forming well-defined clusters at pH 2. The clusters consist of 10 or less molecules. AFM images of mucin at high concentration at pH 2 reveal clusters similar to those seen in the dilute solutions at low pH. We also imaged human gastric mucus revealing a network having a "pearl necklace" structure. The "pearls" are similar in size to the clusters found in the purified porcine gastric mucin gels. AFM images of deglycosylated mucin reveal that the deglycosylated portions of the molecule re-fold into compact, globular structures suggesting that the oligosaccharide chains are important in maintaining the extended conformation of mucin. However, the oligosaccharides do not appear to be directly involved in the aggregation at low pH, as clusters of similar size are observed at pH 2 in both native and deglycosylated mucin.     

Solution scattering approaches to dynamical ordering in biomolecular systems

Clarification of solution structure and its modulation in proteins and protein complexes is crucially important to understand dynamical ordering in macromolecular systems. Small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS) are among the most powerful techniques to derive structural information. Recent progress in sample preparation, instruments and software analysis is opening up a new era for small-angle scattering. In this review, recent progress and trends of SAXS and SANS are introduced from the point of view of instrumentation and analysis, touching on general features and standard methods of small-angle scattering. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” edited by Dr. Koichi Kato.     

The C0C1 fragment of human cardiac myosin binding protein C has common binding determinants for both actin and myosin

The N-terminal domains of cardiac myosin binding protein C (MyBP-C) play a regulatory role in modulating interactions between myosin and actin during heart muscle contraction. Using NMR spectroscopy and small-angle neutron scattering, we have determined specific details of the interaction between the two-module human C0C1 cMyBP-C fragment and F-actin. The small-angle neutron scattering data show that C0C1 spontaneously polymerizes monomeric actin (G-actin) to form regular assemblies composed of filamentous actin (F-actin) cores decorated by C0C1, similar to what was reported in our earlier four-module mouse cMyBP-C actin study. In addition, NMR titration analyses show large intensity changes for a subset of C0C1 peaks upon addition of G-actin, indicating that human C0C1 interacts specifically with actin and promotes its assembly into filaments. During the NMR titration, peaks corresponding to cardiac-specific C0 domain are the first to be affected, followed by those from the C1 domain. No peak intensity or position changes were detected for peaks arising from the disordered proline/alanine-rich (P/A) linker connecting C0 with C1, despite previous suggestions of its involvement in binding actin. Of considerable interest is the observation that the actin-interaction “hot-spots” within the C0 and C1 domains, revealed in our NMR study, overlap with regions previously identified as binding to the regulatory light chain of myosin and to myosin ΔS2. Our results suggest that C0 and C1 interact with myosin and actin using a common set of binding determinants and therefore support a cMyBP-C switching mechanism between myosin and actin.     

pH-dependent conformational change of gastric mucin leads to sol-gel transition

We present dynamic light scattering (DLS) and hydrophobic dye-binding data in an effort to elucidate a molecular mechanism for the ability of gastric mucin to form a gel at low pH, which is crucial to the barrier function of gastric mucus. DLS measurements of dilute mucin solutions were not indicative of intermolecular association, yet there was a steady fall in the measured diffusion coefficient with decreasing pH, suggesting an apparent increase in size. Taken together with the observed rise in depolarized scattering ratio with decreasing pH, these results suggest that gastric mucin undergoes a conformational change from a random coil at pH >/= 4 to an anisotropic, extended conformation at pH < 4. The increased binding of mucin to hydrophobic fluorescent with decreasing pH indicates that the change to an extended conformation is accompanied by exposure of hydrophobic binding sites. In concentrated mucin solutions, the structure factor S(q, t) derived from DLS measurements changed from a stretched exponential decay at pH 7 to a power-law decay at pH 2, which is characteristic of a sol-gel transition. We propose that the conformational change facilitates cross-links among mucin macromolecules through hydrophobic interactions at low pH, which in turn leads to a sol-gel transition when the mucin solution is sufficiently concentrated.     

Comparison of physicochemical properties of purified mucus glycoproteins isolated from respiratory secretions of cystic fibrosis and asthmatic patients

The major nonreduced mucus glycoproteins (mucins) from sputa of cystic fibrosis (CF) and asthmatic patients have been purified to electrophoretic homogeneity and subjected to physical and chemical characterization. The sputum specimens were solubilized in buffer containing 0.22 M KSCN and fractionated on Bio-Gel A-5m, followed by digestion with DNase, rechromatography on the same column, and chromatography on hydroxylapatite. Sodium dodecyl sulfate gel electrophoresis of purified mucins gave a single band. Carbohydrate analyses of the purified mucins showed no significant differences in the sugar components from the two mucins. However, the CF mucin contained substantially higher (11%) sulfate content than that observed for the asthmatic mucin (5.9%). Amino acid analyses indicated that the CF mucin had higher levels of serine plus threonine (35%) as compared to the asthmatic mucin (29%). In contrast, CF mucin contained a lower content of aspartate, glutamate, and glycine than that observed for the asthmatic mucin. Molecular weights of 3.8 X 10(6) and 3.5 X 10(6) were obtained for CF and asthmatic mucins, respectively, from light-scattering studies of mucins in the presence of 6 M guanidine hydrochloride. Reduction of the disulfide bonds of the two mucins did not alter their molecular weights. Liquid chromatographic studies on Sepharose CL2B showed that CF mucin forms aggregates sufficiently large to be excluded from the gel. As compared to the CF mucin, the asthmatic mucin formed fewer of these large aggregates under identical experimental conditions. Reduction and alkylation of the mucins resulted in their inability to form aggregates. The higher state of aggregation of CF mucin may influence the viscoelastic properties of the CF lung's mucus secretions.     

Characterization of rat gastric mucins using a monoclonal antibody,RGM23, recognizing surface mucous cell-type mucins

A novel anti-mucin monoclonal antibody (mAb), designated RGM23, was developed against mucin purified from rat gastric mucosa. RGM23 reacted with the mucin attached to the ELISA well. The reactivity was lost by trypsin treatment, but not by periodate oxidation, indicating that RGM23 recognizes the peptide moiety of the mucin molecule. Histochemical study showed that RGM23 stained the corpus and antral surface mucosa of rat stomach, but not their glandular mucosa, nor duodenal, small intestinal or large intestinal mucosa. The area stained with RGM23 was coincident with that stained with 45M1, a mAb reacting with MUC5AC mucin. Examination of the mucin subunits extracted from rat stomach by Sepharose CL-4B and Q-Sepharose chromatography and CsTFA equilibrium centrifugation showed that RGM23 reacted with the surface mucous cell-type mucins that were stained with periodate-Schiff (PAS) and reacted with mAb RGM21. The gastric gland-type mucin, which reacted with mAb HIK1083, did not react with RGM23. On Q-Sepharose chromatography, a part of the RGM21-reactive mucins was only faintly stained with PAS and did not react with RGM23. The results together indicated that RGM23 probably reacted with the rat MUC5AC (rMuc5AC) mucin present in the surface mucosa of the stomach, and that the surface mucosal cells in rat stomach may contain mucin bearing non-rMuc5AC core protein in addition to rMuc5AC mucins.