Mucin Monomers Research Articles

Water Sorption and Structural Properties of Human Airway Mucus in Health and Muco-Obstructive Diseases.

Muco-obstructive diseases change airway mucus properties, impairing mucociliary transport and increasing the likelihood of infections. To investigate the sorption properties and nanostructures of mucus in health and disease, we investigated mucus samples from patients and cell cultures (cc) from healthy, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) airways. Atomic force microscopy (AFM) revealed mucin monomers with typical barbell structures, where the globule to spacer volume ratio was the highest for CF mucin. Accordingly, synchrotron small-angle X-ray scattering (SAXS) revealed more pronounced scattering from CF mucin globules and suggested shorter carbohydrate side chains in CF mucin and longer side chains in COPD mucin. Quartz crystal microbalance with dissipation (QCM-D) analysis presented water sorption isotherms of the three types of human airway mucus, where, at high relative humidity, COPD mucus had the highest water content compared to cc-CF and healthy airway mucus (HAM). The higher hydration of the COPD mucus is consistent with the observation of longer side chains of the COPD mucins. At low humidity, no dehydration-induced glass transition was observed in healthy and diseased mucus, suggesting mucus remained in a rubbery state. However, in dialyzed cc-HAM, a sorption-desorption hysteresis (typically observed in the glassy state) appeared, suggesting that small molecules present in mucus suppress the glass transition.

Mucus, mucins, and cystic fibrosis.

Cystic fibrosis (CF) is both the most common and most lethal genetic disease in the Caucasian population. CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene and is characterized by the accumulation of thick, adherent mucus plaques in multiple organs, of which the lungs, gastrointestinal tract and pancreatic ducts are the most commonly affected. A similar pathogenesis cascade is observed in all of these organs: loss of CFTR function leads to altered ion transport, consisting of decreased chloride and bicarbonate secretion via the CFTR channel and increased sodium absorption via epithelial sodium channel upregulation. Mucosa exposed to changes in ionic concentrations sustain severe pathophysiological consequences. Altered mucus biophysical properties and weakened innate defense mechanisms ensue, furthering the progression of the disease. Mucins, the high-molecular-weight glycoproteins responsible for the viscoelastic properties of the mucus, play a key role in the disease but the actual mechanism of mucus accumulation is still undetermined. Multiple hypotheses regarding the impact of CFTR malfunction on mucus have been proposed and are reviewed here. (a) Dehydration increases mucin monomer entanglement, (b) defective Ca2+ chelation compromises mucin expansion, (c) ionic changes alter mucin interactions, and (d) reactive oxygen species increase mucin crosslinking. Although one biochemical change may dominate, it is likely that all of these mechanisms play some role in the progression of CF disease. This article discusses recent findings on the initial cause(s) of aberrant mucus properties in CF and examines therapeutic approaches aimed at correcting mucus properties.

Site‐specific N‐linked glycosylation analysis on the human salivary mucin MUC5B using Precursor Ion Discovery on the CAPLC Q‐TOF system

Introduction Mucins are large highly O‐glycosylated oligomeric glycoproteins that form the polymer matrix of mucus, which forms part of the innate immune system. MUC5B, the predominant gel‐forming mucin in saliva, is around 600 kDa in mass before glycosylation. O‐linked glycosylation is abundant in defined repeated domains and may account for 80% of the observed mass of the secreted. Not much is known about the N‐linked glycosylation, although there are at least 34 potential sites. Despite little being known about the structure, N‐glycosylation has been shown to be important but not essential for mucin synthesis (Perez‐Vilar & Hill 1999). Here, we aim to study the N‐linked glycosylation site specifically using mass spectrometryMaterials and methods A sample of purified human MUC5B mucin was digested with trypsin and separated by size exclusion chromatography (Thornton et al. 1999). The previously uncharacterized fractions were analysed by online LC Q‐Tof MS in order to characterize any N‐glycosylated peptides present. Glycopeptide discovery was steered by the Precursor Ion Discovery function, which detects potential glycopeptides by looking for sugar oxonium ions during intermittent high‐collision energy surveys. Candidate ions are then selected for MSMS, which reveals both the carbohydrate content and the glycosylation site location.Results Glycopeptides were located and site specifically analysed. Data suggests N‐linked occupancy of numerous sites, and microheterogeneity was observed.Discussion This is the first study to investigate the N‐glycans on oligomeric gel‐forming mucins. The N‐glycans are localized in the N‐ and C‐terminal portions of the MUC5B polypeptide in areas which have sparse O‐glycosylation. These regions of the molecule are known to be involved in the disulfide bond‐mediated oligomerization of the mucin monomers. Furthermore, they may be potential sites for interaction with other components of saliva that are important for biological function. It may be that they have a role in stabilizing these important regions of the molecule, or they may be sites of interaction with other salivary components.

N-linked oligosaccharides play a role in disulphide-dependent dimerization of intestinal mucin Muc2.

Within the C-terminal domain of many secretory mucins is a 'cystine knot' (CK), which is needed for dimer formation in the endoplasmic reticulum. Previous studies indicate that in addition to an unpaired cysteine, the three intramolecular cystine bonds of the knot are important for stability of the dimers formed by rat intestinal mucin Muc2. The present study was undertaken to determine whether the two N-glycans N9 and N10, located near the first and second cysteines of the knot, also play a role in dimer formation. The C-terminal domain of rat Muc2 (RMC), a truncated RMC mutant containing the CK, and mutants lacking N9 and N10 sites, were expressed in COS-1 cells and the products monitored by radioactive [(35)S]Met/Cys metabolic pulse-chase and immunoprecipitation. Mutation of N9, but not N10, caused increased synthesis of dimers over a 2-h chase period. The N9 mutant remained associated with calreticulin for a prolonged period. About 34-38% of the total labelled products of RMC and its mutants was secreted into the media by 2 h, but the proportion in dimer form was dramatically reduced for the N9 mutant, suggesting lower dimer stability relative to RMC or its N10 mutant. We conclude that under normal conditions the presence of the N9 glycan functions to maintain a folding rate for mucin monomers that is sufficiently slow to allow structural maturation and stability of Muc2 dimers. To our knowledge this report is the first demonstration that a specific N-glycan plays a definitive role in mucin dimer formation.

Chemistry of milk mucins and their anti-microbial action.

Mucins are high molecular weight glycoproteins. They are the major component of all mucus secreted from epithelial cells. As a visco-elastic layer they line the surfaces of various body cavities, including the respiratory, gastrointestinal and urogenital tracts. Depending on their site or origin, mucin monomers have molecular weights between 250 and 600 kDa, and are thus significantly larger than most other glycoproteins. At least 50% of the total molecular mass of the mucin molecule is made up of the carbohydrate chains (Feizi et al., 1984; Neutra and Forstner, 1987).KeywordsSialic AcidHuman MilkBuccal Epithelial CellHuman Mammary EpitheliumMucin ConcentrationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Mechanism by which bacterial flagellin stimulates host mucin production.

In their quest to colonize and infect host tissues, bacteria cause considerable damage to their hosts. Based on this, an evolutionary trend has occurred for host organisms to develop defense mechanisms against bacteria. One is mucin production. Mucin is a high molecular weight glycoprotein synthesized by non-keratinized epithelial cells at the boundary of the host with its environment. The size of a single mucin monomer can be as large as 1 million daltons, most of which is composed of carbohydrate residue. Mucin molecules polymerize by disulfide bonds and electrostatic interactions to form a viscoelastic gel that effectively augments the barrier between host and bacteria. In the respiratory tract, bacteria-laden mucus is conducted toward the throat by cilia and then either swallowed or expectorated. In the eye, bacteria-laden mucus is cleared by blinking.

Gelation and aggregation of mucin in relation to the stomach's protective barrier

AbstractDynamic light scattering was applied to study the effects of pH and concentration on pig gastric mucin. At low concentration (C = 2.0 mg/ml), the distribution of diffusion constants is independent of pH, with an average diffusion constant D = 4 × 10−8 cm2/s. At pH2, very large aggregates with 100‐time slower D (2 × 10−8cm2/s) are seen. Depolarized DLS can be observed at pH2 even at low concentration (C = 5.0 mg/ml). The rotational and translational diffusion constants were analyzed using the Kirkwood‐Riseman model for rods and show that at pH2 mucin is a rod‐like molecule of length 390 nm, diameter 55 nm, with an axial ratio (length/diameter) of 7. However, at pH7, no significant depolarized signals could be detected at any of the concentrations examined in the study. These results suggest that the mucin monomer undergoes a conformational change from a flexible to a rod‐like conformation upon lowering pH from 7 (neutral) to 2 (acidic). Such conformational change and aggregation of the gastric mucin molecule at acidic pH may provide a mechanism by which the gastric mucin layer prevents the stomach from digesting itself, since the back diffusion of acid from the lumen to the gastric epithelium is retarded by the gel layer.

Mucin: Aggregation and colloidal interactions of relevance to some biomedical problems

Mucus is the viscoelastic secretion that lines, most epithelial surfaces forming a protective, lubricating barrier. The viscoelastic properties of mucus arise from mucin, a glycoprotein of molecular weight ranging from 2–10 million. In this paper we address two problems where the aggregation and interactions of mucin with colloidal particles is of physiological relevance. The first deals with gastric mucin and the question of the mechanisms that prevent the stomach from digesting itself. Using dynamic light scattering techniques we show that solutions of gastric mucin aggregate below pH 4. Very large aggregates with 100-fold slower diffusion constants than the mucin monomer are observed at pH 2. Viscosity measurements indicate that mucin will eventually gel at low pH, thus acting as a diffusional, barrier and protecting the stomach. The second problem is concerned with the role of mucin in the nucleation of cholesterol crystals which lead to gallstone formation. Using dynamic light scattering we have shown that mucin at relatively low concentrations (4 mg/ml) promotes the fusion of phospholipid + cholesterol vesicles. The time evolution of the fusion process was measured. No changes in the aggregation state of the gallbladder mucin were observed during the fusion process, suggesting that this phenomenon is related to physico-chemical interactions between the polymer (mucin) and the colloidal particle (vesicle).

The human MUC2 intestinal mucin has cysteine-rich subdomains located both upstream and downstream of its central repetitive region.

The human MUC2 mucin is a large secretory glycoconjugate that coats the epithelia of the intestines, airways, and other mucus membrane-containing organs. Previous work has shown that this mucin contains an extended tandem repeat-containing domain rich in Thr and Pro. In the present work we describe two additional regions of this mucin located both upstream and downstream of the tandem repeat array. The carboxyl-terminal domain contains 984 residues and can be divided into mucin-like (139 residues) and cysteine-rich (845 residues) subdomains. This latter subdomain exhibits varying degrees of sequence similarity to a wide range of mucins and mucin-like proteins including those isolated from rats, pigs, cows, and frogs. We also report here the sequence of 1270 residues lying immediately upstream of the tandem repeats. This region contains a repetitive, mucin-like subdomain and a second cysteine-rich stretch of more than 700 residues. Both cysteine-rich subdomains of this mucin have sequence similarity with von Willebrand factor, a serum protein that exists as a disulfide-linked polymer. This suggests that these cysteine-rich subdomains are important in the catenation of mucin monomers into oligomers, the structures that confer viscoelasticity upon mucus.

Rat gastric mucin is synthesized and secreted exclusively as filamentous oligomers

Oligomeric gastric mucin was isolated from the fundic part of the rat stomach. Previously we have shown by biochemical analysis that this oligomeric mucin consists of disulphide-linked homo-oligomers, which contain no other covalently attached proteins [Dekker, Aelmans & Strous (1991) Biochem. J. 277, 423-427]. Electron-microscopic images of the oligomeric mucin revealed a heterogenous population of long filamentous molecules of 300-3000 nm length. After reduction and carboxymethylation the monomeric mucins displayed a length distribution with a single peak at about 279 nm. Length-distribution analysis of oligomeric molecules with length up to 1000 nm revealed three subpopulations with one, two or three times the length of the monomeric mucin. The oligomers displayed small globular domains of about 15 nm, which were equally spaced along the molecule's length. As the distance between these globular domains was similar to the monomer length, these domains most likely indicate attachment sites of the monomers. These results show that the mucin monomers attached end-to-end in the oligomer. Biosynthesis of the mucin oligomers was studied by labelling of stomach explants in vitro with [35S]methionine, [3H]galactose or [35S]sulphate and subsequent immunoprecipitation of the mucin with a specific antiserum. Analysis by electrophoresis and gel filtration revealed that the oligomerization takes place by formation of disulphide bonds between the 300 kDa mucin precursors. The mucin was exclusively synthesized and secreted as fully glycosylated oligomers, as neither precursor proteins nor monomeric mucin were detected in the culture medium. A model for the biosynthesis of rat gastric mucin is proposed in which the filamentous mucin monomers are linked end-to-end by disulphide bonds.

The oligomeric structure of rat and human gastric mucins

Intact oligomeric gastric mucins were isolated from the fundic part of rat and human stomach. Physicochemical properties of the oligomeric mucins from both species, such as buoyant density, molecular mass, proteinase-resistance, amino acid composition and monosaccharide composition were similar. Biochemical analysis showed that the oligomeric mucins from both species consist of disulphide-linked mucin monomers exclusively: no other covalently attached proteins were detected in purified monomeric mucin. Four major differences were found between the monomeric mucins of these species: (1) the human monomer is larger, (2) the proteolytic-digest peptides derived from proteinase-sensitive portions of the polypeptide backbone displayed no sequence similarity, (3) the human mucin was less sulphated compared with rat mucin, and (4) the proteinase-sensitive part of the human mucin was relatively larger. However, analyses of [3H]galactose-labelled mucin from both species on gel filtration revealed that both gastric mucins were exclusively synthesized as oligomers. The results indicate that the oligomeric structures of human and rat gastric mucin are similar and their biosyntheses are not affected by the differences in the subunits.

Macromolecular properties and polymeric structure of canine tracheal mucins

Two high-Mr mucus glycoproteins (mucins), CTM-A and CTM-B, were highly purified from canine tracheal pouch secretions, and their macromolecular properties as well as polymeric structure were investigated. On SDS/composite-gel electrophoresis, a diffuse band was observed for each mucin. Polyacrylamide-gel electrophoresis using 6% gels also showed the absence of low-Mr contaminants in the mucins. Comparison of chemical and amino acid compositions revealed significant differences between the two mucins. Using a static-laser-light-scattering technique, CTM-A and CTM-B were found to have weight-average Mr values of about 11.0 x 10(6) and 1.4 x 10(6) respectively. Both mucins showed concentration-dependent aggregation in buffer containing 6 M-guanidine hydrochloride. Under similar experimental conditions, reduced-alkylated CTM-A had an Mr of 5.48 x 10(6) and showed no concentration-dependent aggregation. Hydrophobic properties of the mucins, investigated by the fluorescent probe technique using mansylphenylalanine as the probe, showed the presence of a large number of low-affinity (KD approx. 10(5) M) binding sites. These sites appeared to be located on the non-glycosylated regions of the protein core, since Pronase digestion of the mucins almost completely eliminated probe binding. Reduction of disulphide bonds of CTM-A and CTM-B did not significantly alter the probe-binding properties. Also, addition of increasing NaCl concentrations (0.03-1.0 M) to the buffer caused only a small change in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable in the hydrophobic properties of native and reduced-alkylated mucins. CTM-A was deglycosylated, without notable degradation, using a combination of chemical and enzymic methods. On SDS/PAGE the protein core was estimated to have an Mr of approx. 60,000. On the basis of the protein and carbohydrate contents of the major mucin CTM-A, the mucin monomer was calculated to have an Mr of approx. 140,000. The high Mr (11 x 10(6] observed by physical methods is therefore due to self-association of the mucin monomer subunits.

Effects of hydrogen peroxide, mild trypsin digestion and partial reduction on rat intestinal mucin and its disulphide-bound 118 kDa glycoprotein

The role of the disulphide-bound 118 kDa glycoprotein of rat intestinal mucin is unknown, although it has been proposed to serve as a 'link' component for the mucin monomers. The present studies investigated release or destruction of the 118 kDa glycoprotein (monitored by gel electrophoresis and Western-blot analysis) during progressive breakdown of the mucin polymer (assessed by Sepharose 2B chromatography). H2O2 gradually destroyed the 118 kDa glycoprotein and dissociated the mucin polymer into components of similar size to the monomers. After 3 h, mucin samples contained almost no 118 kDa glycoprotein or its breakdown products, but 50% of the mucin was still eluted in the void volume of a Sepharose 2B column. Although mild trypsinolysis had little effect on the Sepharose 2B elution profile of the mucin, the 118 kDa glycoprotein was completely cleaved into 54-56 kDa and 60-66 kDa fragments which remained disulphide-bound to the high-molecular-mass mucin. Increasing levels of thiol reduction resulted in progressive loss of disulphide bonds, release of the 118 kDa glycoprotein and depolymerization of the mucin. Although approx. 40% of the mucin in partially reduced samples was recovered in the Sepharose 2B void volume, this material contained no 118 kDa glycoprotein and apparently consisted of disulphide-bound mucin monomers. Thus the 118 kDa glycoprotein may be destroyed by H2O2, extensively cleaved by trypsin or released by reduction without completely dissociating the mucin into monomers. Therefore the 118 kDa glycoprotein may not function as a 'link' component for all of the mucin monomers in the native polymer.

Hydrodynamic properties of mucous glycoproteins.

AbstractIn order to understand how mucous glycoproteins, or mucins, confer such striking rheological properties on mucous secretions, it is first necessary to understand the structures of the individual glycoproteins and their components. Although significant variability of structure among mucins from different sources is evident, the “bottlebrush” structure is a common motif. For purposes of hydrodynamic calculations, we take the typical mucin monomer to have a molecular weight of 5 × 105, 20% protein and 80% carbohydrate. About 25% of the polypeptide chain is unglycosylated or bare, while the remainder is densely substituted with oligosaccharide chains, averaging about eight sugar residues long. Unless the mucins are treated with S‐S reducing agents, proteases, and denaturants, they are often found to have molecular weights around 2 × 106. Thus, we model the normally occurring form as a tetramer, crosslinked like a star in the bare region.With these structural ideas as a basis, we have confronted published hydrodynamic data with modern hydrodynamic theories to obtain a picture of mucin molecular structure. To account for the oligosaccharide side chains, we used a treatment developed for partially porous spheres. To model the composite structure of glycosylated and bare regions, we used subunit hydrodynamic theory. To consider the effects of branching, we used published theories for branched random coils and extended the broken‐rod results of Wilemski to cover branched rodlike structures.The major conclusions are (1) the bare region is probably not highly extended, (2) the glycosylated region is more like a random coil than a rigid rod, and (3) comparison of hydrodynamic properties of monomers and branched tetramers suggests that the monomer is more rigid in the tetramer. This may be due either to understimation of aggregation in the analysis of the experimental data or to a real stiffening effect imposed by polyelectrolyte and steric repulsive interactions with the other chains in the branched mucin.