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.     

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.     

Effect of pH on the association behavior in aqueous solutions of pig gastric mucin

In this study, dynamic light scattering (DLS), turbidity, and rheo-small angle light scattering (rheo-SALS) methods have been utilized to examine the impact of pH (1 < or = pH < or = 7) on aqueous solutions of noncommercial purified pig gastric mucin. The asymmetric flow field-flow fractionation (AFFFF) measurements established that the mucin sample has a high molecular weight and is polydisperse. DLS measurements on dilute solutions of mucin disclosed large interchain aggregates at pH 2, where the polymer has a low charge density or is uncharged. At lower or higher values of pH, mucin is charged and the tendency of forming interpolymer complexes is affected. In the semidilute concentration regime, pronounced junction zones ('lumps' of polymer) are evolved and a heterogeneous connected network is formed at pH 2, whereas the association structures are disintegrated (smaller 'lumps') at lower or higher pH values due to electrostatic repulsive interactions, and a more homogeneous network is evolved. The DLS and viscosity results at pH 1 indicate the development of a fragmented network, composed of contracted chains that are decorated by some positive charges. The effect of shear flow on the structure of semidilute solutions of mucin was investigated with the aid of rheo-SALS methods. The scattered intensity revealed a strong upturn at low values of the wave vector (q) for mucin solutions at pH 2 and pH 4, which suggests the evolution of large association domains. At these pH values, a flow-induced anisotropy in the 2D SALS patterns in the form of elliptical shapes was observed at high shear rates.     

Molecular structure and rheological properties of short-side-chain heavily glycosylated porcine stomach mucin

The current accepted model for high-molecular-weight gastric mucins of the MUC family is that they adopt a polydisperse coil conformation in bulk solutions. We develop this model using well-characterized highly purified porcine gastric mucin Orthana that is genetically close to the human MUC6 type. It has short side chains and low levels of sialic acid residues and includes minute amounts of cysteine residues that, if abundant, can be responsible for the self-polymerization of mucin. We have established that the mucin structure in bulk solutions corresponds to a daisy-chain random coil. Dynamic light scattering experiments probe the internal dynamics of globular subunits (individual daisies) at the approximately 9 nm length scale, whereas viscosity and light scattering measurements indicate that the size of the whole mucin chains is much larger, approximately 50 nm. The bulk viscosity (eta) scales with mucin concentration (c) in a manner similar to that found for short-side-chain synthetic comb polyelectrolytes and is characterized by a transition between semidilute (eta approximately c1/2) and entangled (eta approximately c3/2) regimes.     

Particle tracking microrheology of purified gastrointestinal mucins

The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins (MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol, DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal mucins formed self‐assembled semi‐dilute networks above a certain critical mucin concentration (c*) with the viscosity (η) scaling as for MUC5AC and for MUC2, where c is the mucin concentration. Above an even higher mucin concentration threshold (c_e, the entanglement concentration) reptation occurs and there is a dramatic increase in the viscosity scaling, for MUC5AC and for MUC2. The dynamics of the self‐assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred‐fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi‐dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol–gel transition in semi‐dilute mucin solutions at neutral pH. © 2013 The Authors. Biopolymers published by Wiley Periodicals, Inc. Biopolymers 101: 366–377, 2014.     

The Mucolytic Activity of Amides: A New Approach to Mucus Dispersion

A method which will reduce significantly the viscosity of epithelial mucus is essential to the physiological mechanisms involved in the mobilization and removal of such secretions. The life expectancy of patients with chronic pulmonary conditions and cystic fibrosis has been considerably increased and consequently the problem of liquefying mucin acquires new importance. In view of these considerations, as well as to facilitate research into the structural relationship of the glycoprotein macromolecule, a systematic investigation of mucolysis was undertaken using gastric mucin. Three amides, carbamide, acetamide and formamide, were found to dissolve gastric gel mucin with minimal degradation, and rapidly disperse the viscous secretions produced in pathological conditions of the tracheobronchial tree. Their effect on secretions from patients with cystic fibrosis and bronchiectasis is dramatic, and within five minutes of adding the reagent the flow time was reduced by at least 95%. Clinical studies were carried out with carbamide (urea in anhydrous, lyophilized, sterile powder form) in 32 patients with a variety of bronchial conditions, including chronic bronchitis, cystic fibrosis, asthma, bronchiectasis and emphysema. With the concentrations used, no irritant, bronchospastic or other reactions were observed.It is concluded that amides of this type have at least two actions on the epithelial mucous secretion: (1) breakage of the three-dimensional gel structure and (2) a slower reduction in viscosity followed by solution of the solid material.     

Effects of calcium and bound sialic acid on the viscosity of mucin

A viscosity drop was observed when pig gastric mucin was titrated with HCl below pH 3.0. When EDTA was present, addition of HCl resulted in an increase in viscosity between pH 3.0 and pH 1.0. Previous treatment of the mucin solution with neuraminidase abolished the pH-dependent viscosity rise in the presence of EDTA.     

Effects of rabbit gastrointestinal mucins and dextran on hydrochloride diffusion in vitro

We compared a viscous fingering formation of hydrochloric acid (HCl) in rabbit corpus, antral and duodenal mucins and with dextran under neutral and acidic conditions with respect to relative viscosity, molecular mass, and carbohydrate composition. The effect of desialyzation of duodenal mucin on the viscous fingering formation of HCl was also examined. HCl (0.1 N) was injected into 1% solutions of mucins and dextran and a subsequent viscous fingering formation was assessed based on an influx volume rate of HCl. A low influx volume rate indicates a high ability of the solutions to produce viscous fingers. The influx volume rate of HCl was lowest in duodenal mucin followed bl corpus mucin, antral mucin, and dextran at pH 7. The influx volume rate of HCl was inversely correlated with the relative viscosity of the solution. Maximum molecular masses were large in the order of corpus, antral, and duodenal mucins, and they were larger than dextran T2000. Rabbit gastrointestinal mucins were very polydisperse system. Duodenal mucin contains more sialic acid than gastric mucins; the influx volume rate of HCl increased in desialylated duodenal mucin. It is suggested that the higher ability of gastric mucins to prevent HCl diffusion than dextran were due to the differences in the molecular mass. The ability of duodenal mucin to prevent HCl diffusion was probably attributed to its high sialic acid content, which may reflect a physiological role of duodenal mucin in the duodenum that has to deal with HCl influx from the stomach.     

Flexibility and length of human bronchial mucin studied using low-shear viscometry, birefringence relaxation analysis, and electron microscopy

The glycoprotein mucin was isolated from the sputum of patients with chronic obstructive bronchitis. The fractionation procedure included treatment with 6M urea at pH 12.5 followed by gel filtration in 6M urea at neutral pH. (1) Using a low-shear Cartesian diver viscometer, we found that the mucin intrinsic viscosity equals (0.32 ± 0.03) L/g in 1000 mM NaCl solution increasing to (12 ± 3) L/g in 0.1 mM NaCl (pH 7 and 20°C). (2) The relaxation of electrically induced birefringence in mucin solutions was measured and the relaxation spectrum calculated using a Fourier-transform deconvoltion method. We found that the dominant relaxation time increased from 1 to 150 μs when the exitation pulse duration used was increased from 2 to 300 μs. (3) Mucin was vacuum-dried from glycerol-containing solutions followed by low-angle rotary shadowing and electron microscopy. Mucin was found to be unbranched, with contour lengths ranging from 300 to 2500 nm and with an average of 900 nm. Our result indicate that mucin is an extended and flexible molecule with Kuhn length 0.3–0.5% of the contour length.     

Effects of bovine alpha-lactalbumin on gastric defense mechanisms in naive rats

We recently investigated the effects of the major proteins in cow's milk on gastric mucosal injuries in rat ulcer models. We found that alpha-lactalbumin (alpha-LA) has marked preventive effects against gastric mucosal injuries and that prostaglandin (PG) synthesis may contribute to these effects [Matsumoto et al., Biosci. Biotechnol. Biochem., 65, 1104-1111, 2001]. In this study, we investigated the effects of alpha-LA on several defense mechanisms of gastric mucosa by evaluating gastric PGE2 content, gastric mucin content, gastric luminal pH, gastric fluid volume, and gastric emptying in naive rats. Oral administration of alpha-LA (200, 500, and 1000 mg/kg) elevated endogenous PGE2 levels in gastric tissue and increased the gastric mucin contents of both the gastric fluid and the adherent mucus gel layer. In addition to these PG-related responses, alpha-LA also caused PG-independent responses such as elevation of gastric luminal pH, increase in gastric fluid volume, and delay in gastric emptying. These responses were observed to be dose-dependent (200-1000 mg/kg of alpha-LA). Thus, we demonstrated that alpha-LA enhances both PG-dependent and PG-independent gastric defense mechanisms in naive rats. Both of these mechanisms are probably involved in its gastroprotective action.     

Rheology of gastric mucin exhibits a pH-dependent sol-gel transition

Gastric mucin, a high molecular weight glycoprotein, is responsible for providing the gel-forming properties and protective function of the gastric mucus layer. Bulk rheology measurements in the linear viscoelastic regime show that gastric mucin undergoes a pH-dependent sol-gel transition from a viscoelastic solution at neutral pH to a soft viscoelastic gel in acidic conditions, with the transition occurring near pH 4. In addition to pH-dependent gelation behavior in this system, further rheological studies under nonlinear deformations reveal shear thinning and an apparent yield stress in this material which are also highly influenced by pH.     

Why is chitosan mucoadhesive?

Chitosan is a biocompatible and biodegradable amino polysaccharide, which is soluble in aqueous solutions at pH < 6.5. It has been widely used for developing drug delivery systems because of its excellent mucoadhesive properties. Although many studies report on chitosan being mucoadhesive, the nature of interactions between chitosan and mucin remains poorly defined. Here, we have examined the role of primary amino groups and the role of electrostatic attraction, hydrogen bonding, and hydrophobic effects on aggregation of gastric mucin in the presence of chitosan. Reducing the number of amino groups through their half acetylation results in expansion of chitosan's pH-solubility window up to pH 7.4 but also reduces its capacity to aggregate mucin. We demonstrated that electrostatic attraction forces between chitosan and gastric mucin can be suppressed in the presence of 0.2 mol/L sodium chloride; however, this does not prevent the aggregation of mucin particles in the presence of this biopolymer. The presence of 8 mol/L urea or 10% v/v ethanol in solutions also affects mucin aggregation in the presence of chitosan, demonstrating the role of hydrogen bonding and hydrophobic effects, respectively, in mucoadhesion.     

Aggregation of mucin by chromium(III) complexes as revealed by electrokinetic and rheological studies: influence on the tryptic and O-glycanase digestion of mucin

In the present study, the impact of chromium(III) complexes ([Cr(salen)(H2O)2](+) (1), [Cr(en)3]3+ (2) and [Cr(EDTA)(H2O)]- (3)) on the biophysical properties of mucin like specific viscosity, zeta potential and particle size has been investigated. It is evident from the present investigation that the nature of the coordinated ligand has a major role to play in bringing about the changes in the physical characteristics of the glycoprotein. It was observed that (1) and (3) because of their coordinate mode of binding lead to decrease in the specific viscosity of mucin, whereas (2) on the other hand was found to bring about drastic increase in the mucin viscosity due to sol-gel transition in the mucin conformation. Complex (2) was found to gradually lower the zeta potential value of mucin (particle size=51.5 nm) from -24.8 +/- 1.31 mV to -0.58 +/- 0.30 mV, which reveals aggregation (particle size=216 nm) and subsequent sedimentation of mucin with an increase in the average diameter of mucin particles. The binding of (2) to mucin was found to impart resistance to mucin against both tryptic and O-glycanase digestion, suggesting that, the aggregation of mucin causes conformational as well as configurational changes in the glycoprotein; thus perturbing the location of carbohydrate domains.     

Enhancement of the viscosity of mucin by serum albumin.

The interaction of serum albumin with a model epithelial mucin from pig stomach was explored by rotary viscometry. During 30 min of incubation of human serum albumin(20mg/ml) and pig gastric mucin (8mg/ml) in iso-osmotic buffers at 37 degrees C, the solution became markedly viscous. Viscosity enhancement was proportional to albumin concentration (2-40mg/ml), was most pronounced under conditions of low shear rate (less than 45S-1), and was considerably greater than the additive or multiplicative viscosity values calculated from albumin or mucin solutions measured separately. The viscous mucin-albumin complex was destroyed by high shear rates (greater than 90S-1), but slowly re-formed under zero shear conditions. Elevation of pH (7 to 9), ionic strength (0.1 to 1.0), and addition of disodium EDTA (5mM) did not cause marked or specific alterations in the viscosity of the mixture, suggesting that electrostatic interactions probably do not stabilize mucin-albumin complexes. Urea (7M) and heating (35 to 55 degrees C) caused a major increase in the viscosity of mucin and mucin-albumin mixtures, suggesting that rupture of hydrogen bonds, unfolding and partial denaturation of mucin promotes greater intertangling (possibly hydrophobic interactions) between mucin and albumin molecules. The implications of mucin-albumin interaction in diseases associated with mucus obstruction are briefly discussed.     

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.     

Heterogeneity in gastrointestinal mucins

Pig digestive tract mucins have often been used as model mucins for studying mucin structure, function and metabolism. In the present study pig gastric mucin and pig colonic mucin in the subunit form have been characterised and compared. Following Sepharose 4B or 2B-CL gel chromatography, the mucin eluant fractions were assayed colorimetrically by both the periodic acid-Schiff and the Alcian blue binding assays. Subunit colonic mucin eluted as a single unimodel peak that was easily detected by both assays. In contrast, subunit gastric mucin gave a peak primarily detected by periodic acid-Schiff that was overlapped by, but partially separated from, another peak primarily detected by Alcian blue. Subunit gastric mucin was separated into two periodic acid-Schiff staining spots when electrophoresed on cellulose acetate. Cetylpyridinium chloride (CPC) was able to precipitate only about half the subunit gastric mucin. The CPC-precipitable subunit gastric mucin corresponded to the faster running spot on electrophoresis, and the subunit gastric mucin in the CPC supernatant (which may have more than one subunit mucin type) to the slower spot(s). The former had a higher sulphate content and stained with Alcian blue. The latter had a lower sulphate content and showed very little Alcian blue reactivity. These results indicate that subunit pig gastric mucin is heterogeneous with respect to both size and charge. The differences between the types may be important in biological and physiochemical behaviour of gastric mucin. It seems likely that different laboratories may have worked on one or other of the pig gastric mucin types or a mixture, depending on the preparation method.     

Co-localization of TFF2 with gland mucous cell mucin in gastric mucous cells and in extracellular mucous gel adherent to normal and damaged gastric mucosa

Trefoil factor 2 (TFF2) is mucin associated peptide that has a mucosal barrier function in addition to participating in repair and healing. We examined the localization of TFF2 and gastric mucins in gastric mucous cells, the surface mucous gel layer (SMGL) adherent to normal gastric mucosa, and in the mucoid cap covering gastric erosions. Carnoy’s solution, or formalin/picric acid-fixed paraffin embedded materials from resected stomachs and formalin-fixed paraffin embedded gastric biopsy materials were used. Sections were immunostained for the TFF2 and histochemically stained for gastric mucins. In addition, thick sectioned gastric mucosa fixed in Carnoy’s solution were stained with FITC-labeled GSA-II lectin specific for gland mucous cell mucin and examined for three-dimensional images of the SMGL using a confocal laser scanning microscope. The TFF2 and gland mucous cell mucin were found intermixed together in the gastric gland mucous cells, in the SMGL in laminated layers, and in the mucoid cap. A laminated arrangement of continuous sheets of gland mucous cell mucin in the SMGL was demonstrated in the three-dimensional images. Co-localization of the TFF2 with gland mucous cell mucin suggests a physical interaction between the TFF2 and gland mucous cell mucin. The TFF2 trapped in the adherent mucins may be responsible for mucosal defense, healing, and repair.     

Fluid dynamics of the excretory flow of zymogenic and mucin contents in rat gastric gland processed by high-pressure freezing/freeze substitution.

The high-pressure freezing/freeze substitution technique followed by Lowicryl K4M embedding provided an excellent ultrastructure and retention of antigenicity of rat gastric glands as well as the intraluminal fluid contents. By taking this advantage, we histochemically investigated the excretory flow of the zymogenic and mucin contents in rat gastric glandular lumen at the ultrastructural level. The combination of KMnO(4)-UA/Pb staining for zymogenic contents and Griffonia simplicifolia agglutinin-II (GSA-II) labeling for mucous neck cell (MNC) mucin distinguished the exocytosed zymogenic contents from the MNC mucin in the glandular lumen. Interestingly, at the base and neck regions, the zymogenic contents showed a droplet-like appearance, forming a distinct interface with the MNC mucin. At the pit region, the GSA-II labeling demonstrated restricted paths, designated as MNC mucous channels, which flowed into the surface mucous gel layer. It should be noted that the interface between exocytosed zymogenic contents and MNC mucin disappeared, and that the zymogenic contents merged into the MNC mucous channels. At the top pit region, the surface mucous gel layer showed laminated arrays of three types of gastric mucins. On the basis of these ultrastructural findings, we propose a model of the excretory flow in rat gastric gland.     

Cysteine-rich regions of pig gastric mucin contain von willebrand factor and cystine knot domains at the carboxyl terminal(1).

In order to sequence the cysteine-rich regions of pig gastric mucin (PGM), we used our previously identified pig gastric mucin clone PGM-2A to screen a pig stomach cDNA library and perform rapid amplification of cDNA ends to obtain two cysteine-rich clones, PGM-2X and PGM-Z13. PGM-2X has 1071 base pairs (bp) encoding 357 amino acids containing five serine-threonine-rich 16 amino acid tandem repeats, downstream from a cysteine-rich region similar to human and mouse MUC5AC. PGM-Z13 encodes the complete 3'-terminus of PGM and is composed of 3336 bp with a 2964 bp open reading frame encoding 988 amino acids with four serine-threonine-rich tandem repeats upstream from a cysteine-rich region similar to the carboxyl terminal regions of human and rat MUC5AC and human MUC5B. This region is homologous to von Willebrand factor C and D domains involved in acid induced polymerization, and to the carboxyl terminal cystine-knot domain of various mucins, TGF-beta, vWF and norrin, which is involved in dimerization. These newly sequenced cysteine-rich regions of pig gastric mucin may be critical for its gelation and for its observed increased viscosity induced by low pH.     

Calcium binding to intestinal goblet cell mucin.

1. Binding of Ca-2+ to goblet cell mucin of rat small intestine was studied using equilibrium dialysis against 0.01 M Tris/HCl buffer (pH 7.4) and tracer amounts of 45-CaCl2. Binding was found to reach saturation at a Ca free -2+ concentration of 0.1--1.0 mM, to be independent of temperature (4-37 degrees C), and to increase with increasing pH (5.0-8.7). At low concentrations of Ca free -2+ (smaller than 0.03 mM) the binding curve was sigmoidal, suggesting positive cooperativity of binding sites and a possible change in the tertiary structure of the mucin. Binding was markedly reduced, and sigmoidicity abolished, by removal of sialic acid from the mucin, or by adding 0.14 M NaCl to the dialysis medium. This latter finding suggests that, in vivo, other cations would compete for Ca-2+ binding ligands. 2. Under conditions mimicking those used for binding studies, CaCl2 (10- minus 5 M) was found to cause a small increase (0.03 units) in the absorbance of mucin solutions, especially in the ultraviolet region, possibly indicating increased light scattering. No change in the solubility of the mucin was observed after the addition of CaCl2 (10- minus 6-10- minus 4 M). A significant decrease in viscosity of the mucin was noted, however, with the addition of CaCl2 (10- minus 6-10- minus 2 M). Together with the binding data, these findings suggested that during binding, Ca-2+ combines with negative charges on goblet cell mucin (especially those of sialic acid carboxyl groups) and induces contraction or folding of the macromolecule which promotes cooperative cation binding. No evidence was obtained to suggest that CaCl2 caused precipitation, polymerization or gelation of the mucin in 0.01 M Tris/HCl.?