Assembly and organization of the N-terminal region of mucin MUC5AC: Indications for structural and functional distinction from MUC5B.

Elevated levels of MUC5AC, one of the major gel-forming mucins in the lungs, are closely associated with chronic obstructive lung diseases such as chronic bronchitis and asthma. It is not known, however, how the structure and/or gel-making properties of MUC5AC contribute to innate lung defense in health and drive the formation of stagnant mucus in disease. To understand this, here we studied the biophysical properties and macromolecular assembly of MUC5AC compared to MUC5B. To study each native mucin, we used Calu3 monomucin cultures that produced MUC5AC or MUC5B. To understand the macromolecular assembly of MUC5AC through N-terminal oligomerization, we expressed a recombinant whole N-terminal domain (5ACNT). Scanning electron microscopy and atomic force microscopy imaging indicated that the two mucins formed distinct networks on epithelial and experimental surfaces; MUC5B formed linear, infrequently branched multimers, whereas MUC5AC formed tightly organized networks with a high degree of branching. Quartz crystal microbalance-dissipation monitoring experiments indicated that MUC5AC bound significantly more to hydrophobic surfaces and was stiffer and more viscoelastic as compared to MUC5B. Light scattering analysis determined that 5ACNT primarily forms disulfide-linked covalent dimers and higher-order oligomers (i.e., trimers and tetramers). Selective proteolytic digestion of the central glycosylated region of the full-length molecule confirmed that MUC5AC forms dimers and higher-order oligomers through its N terminus. Collectively, the distinct N-terminal organization of MUC5AC may explain the more adhesive and unique viscoelastic properties of branched, highly networked MUC5AC gels. These properties may generate insight into why/how MUC5AC forms a static, "tethered" mucus layer in chronic muco-obstructive lung diseases.

Pregnancy-associated decrease of Siaα2-3Gal-linked glycans on salivary glycoproteins affects their binding ability to avian influenza virus.

Salivary glycoproteins are known as an important barrier to inhibit influenza infection by presenting sialic acid (Sia) ligands that can bind with viral hemagglutination. Here, to further understand why pregnant women are more vulnerable to avian influenza virus (AIV), we investigated the alteration of protein sialylation in the saliva of women during pregnancy and postpartum, and its impact on the saliva binding affinity to AIV. Totally 1200 saliva samples were collected, the expression levels of terminal α2-3/6-linked Sia on salivary proteins were tested and validated, and the binding activities of salivary proteins were assessed against 3 strains of AIV and the H1N1 vaccine. Result showed that the expression of terminal α2-3-linked Sia in the saliva of women decreased dramatically during pregnancy compared to that of non-pregnancy control, especially for women in the second or third trimester (fold change = 0.53 and 0.37, p < 0.001). And their salivary protein binding ability to AIV declined accordingly. The variation of terminal α2-3-linked Sia on salivary MUC5B and IgA was consistent with the above results. This study indicates that the decrease of terminal α2-3-linked Sia on salivary glycoproteins of pregnant women affects their binding ability to AIV, which may provide new insights into AIV prevention and control.

Mucin O-glycans suppress quorum-sensing pathways and genetic transformation in Streptococcus mutans.

Mucus barriers accommodate trillions of microorganisms throughout the human body while preventing pathogenic colonization1. In the oral cavity, saliva containing the mucins MUC5B and MUC7 forms a pellicle that coats the soft tissue and teeth to prevent infection by oral pathogens, such as Streptococcus mutans2. Salivary mucin can interact directly with microorganisms through selective agglutinin activity and bacterial binding2-4, but the extent and basis of the protective functions of saliva are not well understood. Here, using an ex vivo saliva model, we identify that MUC5B is an inhibitor of microbial virulence. Specifically, we find that natively purified MUC5B downregulates the expression of quorum-sensing pathways activated by the competence stimulating peptide and the sigX-inducing peptide5. Furthermore, MUC5B prevents the acquisition of antimicrobial resistance through natural genetic transformation, a process that is activated through quorum sensing. Our data reveal that the effect of MUC5B is mediated by its associated O-linked glycans, which are potent suppressors of quorum sensing and genetic transformation, even when removed from the mucin backbone. Together, these results present mucin O-glycans as a host strategy for domesticating potentially pathogenic microorganisms without killing them.

The comparative inhibitory potency of salivary mucins against human immunodeficiency virus type 1.

MUC5B and MUC7 salivary mucins are reported to inhibit HIV-1 entry into target cells in vitro; however, their relative inhibitory potencies have not been quantitively compared. There is also conflicting evidence regarding whether HIV-1 infection diminishes mucins' inhibitory efficacy. We explored the effect of donor HIV-1 status upon the anti-HIV-1 potency of purified MUC5B and MUC7 while comparing their relative inhibitory potential using a pseudovirus-based neutralization assay. HIV status of sample donors had no detectable effect on HIV-1 inhibition by salivary mucins. MUC5B (median IC50 50 µg/ml, IQR 10-116 µg/ml) exhibited significantly more potent HIV-1 inhibition than MUC7 (median IC50 458 µg/ml, IQR 192->2000 µg/ml; Mann-Whitney U p < 0.0001). We suggest that larger size, gel-forming properties and extensive glycosylation of MUC5B allow more effective binding and aggregation of viral particles. MUC5B is also more abundant in the saliva and is therefore likely to make a substantially greater contribution to it's anti-HIV-1 properties.

The MUC5B mucin polymer is dominated by repeating structural motifs and its topology is regulated by calcium and pH.

The polymeric mucin MUC5B provides the structural and functional framework of respiratory mucus, conferring both viscoelastic and antimicrobial properties onto this vital protective barrier. Whilst it is established that MUC5B forms disulfide-linked linear polymers, how this relates to their packaging in secretory granules, and their molecular form in mucus remain to be fully elucidated. Moreover, the role of the central heavily O-glycosylated mucin domains in MUC5B conformation is incompletely described. Here we have completed a detailed structural analysis on native MUC5B polymers purified from saliva and subsequently investigated how MUC5B conformation is affected by changes in calcium concentration and pH, factors important for mucin intragranular packaging and post-secretory expansion. The results identify that MUC5B has a beaded structure repeating along the polymer axis and suggest that these repeating motifs arise from distinct glycosylation patterns. Moreover, we demonstrate that the conformation of these highly entangled linear polymers is sensitive to calcium concentration and changes in pH. In the presence of calcium (Ca2+, 10 mM) at pH 5.0, MUC5B adopted a compact conformation which was lost either upon removal of calcium with EGTA, or by increasing the pH to 7.4. These results suggest a pathway of mucin collapse to enable intracellular packaging and mechanisms driving mucin expansion following secretion. They also point to the importance of the tight control of calcium and pH during different stages of mucin biosynthesis and secretion, and in the generation of correct mucus barrier properties.

The C-terminal dimerization domain of the respiratory mucin MUC5B functions in mucin stability and intracellular packaging before secretion.

Mucin 5B (MUC5B) has an essential role in mucociliary clearance that protects the pulmonary airways. Accordingly, knowledge of MUC5B structure and its interactions with itself and other proteins is critical to better understand airway mucus biology and improve the management of lung diseases such as asthma, cystic fibrosis, and chronic obstructive pulmonary disease (COPD). The role of an N-terminal multimerization domain in the supramolecular organization of MUC5B has been previously described, but less is known about its C-terminal dimerization domain. Here, using cryogenic electron microscopy (cryo-EM) and small-angle X-ray scattering (SAXS) analyses of recombinant disulfide-linked dimeric MUC5B dimerization domain we identified an asymmetric, elongated twisted structure, with a double globular base. We found that the dimerization domain is more resistant to disruption than the multimerization domain suggesting the twisted structure of the dimerization domain confers additional stability to MUC5B polymers. Size-exclusion chromatography-multiangle light scattering (SEC-MALS), SPR-based biophysical analyses and microscale thermophoresis of the dimerization domain disclosed no further assembly, but did reveal reversible, calcium-dependent interactions between the dimerization and multimerization domains that were most active at acidic pH, suggesting that these domains have a role in MUC5B intragranular organization. In summary, our results suggest a role for the C-terminal dimerization domain of MUC5B in compaction of mucin chains during granular packaging via interactions with the N-terminal multimerization domain. Our findings further suggest that the less stable multimerization domain provides a potential target for mucin depolymerization to remove mucus plugs in COPD and other lung pathologies.

A glycopolymer improves vascoelasticity and mucociliary transport of abnormal cystic fibrosis mucus.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca2+ upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca2+-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

Higher Energy Collisional Dissociation Mass Spectrometry of Sulfated O-Linked Oligosaccharides.

Sulfation is the final decoration of mucin-type O-linked oligosaccharides before mucins are released into the lumen of the gastrointestinal, respiratory, and genital tracts. Because only a fraction of oligosaccharides undergo this type of modifications in the Golgi apparatus, sometimes also only by dedicated cells, the glycobiology of these low abundant sulfated oligosaccharides is often overlooked. At the same time, the technology to consistently identify and characterize them has been lagging. We adopted higher energy collisional dissociation to characterize sulfated oligosaccharides from porcine gastric and human salivary MUC5B mucins. With this approach we could generate conclusive spectra up to nonasaccharides. Both singly and doubly sulfated oligosaccharides were characterized. By comparing the fragmentation of low-mass fragments of m/ z 100-320 with standards for six-linked and three-linked sulfate, it could be shown that characteristic fragmentation exists, verifying that porcine gastric mucin contains mostly six-linked sulfate to GlcNAc, whereas human MUC5B contains mostly three-linked Gal. When performing ion-trap MS2 fragmentation, these low-molecular-mass fragments are usually not detected. Hence it can be concluded that to be able to address biological questions of sulfation low-mass fragments are important for the assignment of sulfate position.

Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers.

Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

Secondary Structure and Glycosylation of Mucus Glycoproteins by Raman Spectroscopies.

The major structural components of protective mucus hydrogels on mucosal surfaces are the secreted polymeric gel-forming mucins. The very high molecular weight and extensive O-glycosylation of gel-forming mucins, which are key to their viscoelastic properties, create problems when studying mucins using conventional biochemical/structural techniques. Thus, key structural information, such as the secondary structure of the various mucin subdomains, and glycosylation patterns along individual molecules, remains to be elucidated. Here, we utilized Raman spectroscopy, Raman optical activity (ROA), circular dichroism (CD), and tip-enhanced Raman spectroscopy (TERS) to study the structure of the secreted polymeric gel-forming mucin MUC5B. ROA indicated that the protein backbone of MUC5B is dominated by unordered conformation, which was found to originate from the heavily glycosylated central mucin domain by isolation of MUC5B O-glycan-rich regions. In sharp contrast, recombinant proteins of the N-terminal region of MUC5B (D1-D2-D'-D3 domains, NT5B), C-terminal region of MUC5B (D4-B-C-CK domains, CT5B) and the Cys-domain (within the central mucin domain of MUC5B) were found to be dominated by the ß-sheet. Using these findings, we employed TERS, which combines the chemical specificity of Raman spectroscopy with the spatial resolution of atomic force microscopy to study the secondary structure along 90 nm of an individual MUC5B molecule. Interestingly, the molecule was found to contain a large amount of α-helix/unordered structures and many signatures of glycosylation, pointing to a highly O-glycosylated region on the mucin.

Biosynthesis of the polymeric gel-forming mucin MUC5B.

MUC5B is a major polymeric mucin in the airway mucus gel and is an essential component of innate defense of the respiratory epithelium. Knowledge of the synthesis and intracellular processing of MUC5B is incomplete. We investigated the molecular details of MUC5B assembly in primary human bronchial epithelial cells (HBECs) grown at an air-liquid interface (ALI). Electrophoretic and centrifugal separations of intracellular forms of MUC5B probed with antibodies specific for non-O-glycosylated and O-glycosylated forms of the mucin identified three major intracellular populations of MUC5B (non-O-glycosylated monomer and dimer, and O-glycosylated polymers). Biophysical analysis of recombinant MUC5B COOH-terminus (CT5B; D4-B-C-CK) expressed in 293-EBNA cells showed that MUC5B dimerizes by disulfide linkage. Pulse-chase studies in the HBEC ALI cultures showed that non-O-glycosylated MUC5B was synthesized within 20 min of metabolic labeling and O-glycosylated, polymeric mucin within 2 h. Radiolabeled O-glycosylated mucin polymers were secreted within 2 h and the majority were released by 48 h. These data indicate that MUC5B follows a similar assembly to the related glycoprotein, von Willebrand factor (vWF); however, unlike vWF the MUC5B polypeptide shows no evidence of major proteolytic processing of D-domains during the production of the mature secreted polymeric mucin in normal and cystic fibrosis (CF) primary bronchial epithelial cells. In contrast, MUC5B D-domains were modified by neutrophil elastase, a protease commonly found in CF sputum, demonstrating that proteolytic degradation of MUC5B is an extracellular event in CF sputum. These results define the pathway for synthesis of MUC5B in primary human goblet cells.

Reassessment of the importance of mucins in determining sputum properties in cystic fibrosis.

BACKGROUND: There is conflicting evidence about the importance of airway mucins (MUC5AC and MUC5B) in determining physical properties of sputum in cystic fibrosis (CF). We studied the effects of endogenous degradation of mucins on CF sputum elasticity and apparent mucin concentrations. METHODS: Elastic shear moduli (G') and mucin concentrations in sputum of 12 CF patients were measured before and after incubation at 37°C for 60min. RESULTS: G' fell from a median of 5.98 to 4.70Pa (p=0.01). There were significant falls in MUC5AC (8.2 to 5.2µg/ml, p=0.02) and MUC5B (17.3 to 12.5µg/ml, p=0.02) over the same period, and associated decrease in molecular weight and size. CONCLUSIONS: Sputum is not inert and degradation reduces apparent mucin concentrations and sputum elasticity. Even if care is taken to process samples rapidly, sputum may therefore differ from secretions retained in airways. Previous studies may have underestimated the role of mucins in CF sputum.

Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts.

BACKGROUND: We have previously shown that MUC5B and MUC7 mucins from saliva of HIV negative individuals inhibit HIV-1 activity by 100% in an in vitro assay. The purpose of this subsequent study was to investigate whether MUC5B and MUC7 from saliva of HIV patients or with full blown AIDS had a similar inhibitory activity against the virus. METHODS: Salivary MUC5B and MUC7 from HIV patients with different CD4 counts (< 200, 200-400 and > 400) were incubated with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells). Cells were then cultured and viral replication was measured by a qualitative p24 antigen assay. The size, charge and immunoreactivity of mucins from HIV negative and positive individuals was also analysed by SDS-PAGE, Western blot and ELISA respectively. RESULTS: It was shown that irrespective of their CD4 counts both MUC5B and MUC7 from HIV patients, unlike the MUC5B and MUC7 from HIV negative individuals, did not inhibit HIV-1 activity. Size, charge and immunoreactivity differences between the mucins from HIV negative and positive individuals and among the mucins from HIV patients of different CD4 count was observed by SDS-PAGE, Western blot and ELISA. CONCLUSIONS: Purified salivary mucins from HIV positive patients do not inhibit the AIDS virus in an in vitro assay. Although the reason for the inability of mucins from infected individuals to inhibit the virus is not known, it is likely that there is an alteration of the glycosylation pattern, and therefore of charge of mucin, in HIV positive patients. The ability to inhibit the virus by aggregation by sugar chains is thus diminished.

Identification of salivary proteins at oil-water interfaces stabilized by lysozyme and beta-lactoglobulin.

In this research, we investigated the interaction occurring between oil-in-water emulsion droplets, stabilized by different emulsifiers, i.e. lysozyme and beta-lactoglobulin (beta-lg), and salivary proteins (SPs) with a molecular mass (M(r)) above about 10kDa. Different techniques, i.e. infrared spectroscopy, Western blotting, PAS staining and SDS-PAGE coupled to MS, were employed for this purpose. This study demonstrated the interaction between several salivary proteins and the emulsifiers at the oil-water interfaces. In particular, results show that the high M(r) mucin MUC5B was strongly bound to lysozyme stabilized emulsions, whereas beta-lg stabilized emulsions associated with MUC7 and, moderately, with MUC5B. Furthermore, we observed that salivary proteins in the range M(r) 10-100kDa associated differently with emulsion droplets. A large majority of SPs was found to interact with lysozyme stabilized emulsion droplets whilst in case of beta-lg stabilized emulsions, the SPs distribute more evenly between the fraction associated and non-associated with the droplets. A clear example is alpha-amylase (M(r) approximately 55kDa) which predominantly associates with lysozyme stabilized emulsion droplets, but not with beta-lg emulsion droplets. To conclude, our findings indicate that adsorption/association of salivary protein components onto the emulsion droplets is related to the type of emulsifying proteins at the oil-water interfaces and it is probably driven by the overall net charge at the droplet's oil-water interfaces, i.e. positive for lysozyme stabilized emulsions and negative for beta-lactoglobulin stabilized emulsion at neutral pH.

Unpacking a gel-forming mucin: a view of MUC5B organization after granular release.

Gel-forming mucins are the largest complex glycoprotein macromolecules in the body. They form the matrix of gels protecting all the surface epithelia and are secreted as disulfide-bonded polymeric structures. The mechanisms by which they are formed and organized within cells and thereafter released to form mucus gels are not understood. In particular, the initial rate of expansion of the mucins after release from their secretory granules is very rapid (seconds), but no clear mechanism for how it is achieved has emerged. Our major interest is in lung mucins, but most particularly in MUC5B, which is the major gel-forming mucin in mucus, and which provides its major protective matrix. In this study, using OptiPrep density gradient ultracentrifugation, we have isolated a small amount of a stable form of the recently secreted and expanding MUC5B mucin, which accounts for less than 2% of the total mucin present. It has an average mass of approximately 150 x 10(6) Da and size Rg of 150 nm in radius of gyration. In transmission electron microscopy, this compact mucin has maintained a circular structure that is characterized by flexible chains connected around protein-rich nodes as determined by their ability to bind colloidal gold. The appearance indicates that the assembled mucins in a single granular form are organized around a number of nodes, each attached to four to eight subunits. The organization of the mucins in this manner is consistent with efficient packing of a number of large heavily glycosylated monomers while still permitting their rapid unfolding and hydration. For the first time, this provides some insight into how the carbohydrate regions might be organized around the NH(2)- and COOH-terminal globular protein domains within the granule and also explains how the mucin can expand so rapidly upon its release.

Differential metabolic activity by dental plaque bacteria in association with two preparations of MUC5B mucins in solution and in biofilms.

Salivary mucin, MUC5B, is an oligomeric glycoprotein, heterogeneous in size and with a diverse repertoire of oligosaccharides, which differ in composition and charge. Since complex salivary glycoproteins are considered to be the major source of nutrients for the oral supragingival microbiota, the major aim of the current study was to determine whether different preparations of non-denatured MUC5B could be isolated exhibiting different biological properties in relation to the microflora associated with the surfaces of the oral cavity. Two preparations, solMUC5B and gelMUC5B, were isolated by density-gradient centrifugation and were shown to have different buoyant densities, carbohydrate content and surface-adsorbing characteristics. To ascertain differences in biological activity, the two mucin preparations, both in solution and adsorbed to a model surface, were incubated with freshly isolated dental plaque and assayed for metabolic (dehydrogenase) activity with the fluoresecent substrate CTC (5-cyano-2,3-ditolyl tetrazolium chloride). The plaque bacteria exhibited higher metabolism with the solMUC5B preparation in solution, with 79.4 % active plaque cells compared to the controls without mucin (9.6 %), while gelMUC5B showed 48.2 % active cells with the same plaque population. In contrast, the same mucins adhered to a surface elicited a significantly lower metabolic response, with surface-associated plaque cells showing only 12.1 % active cells with solMUC5B and 29.2 % with gelMUC5B. These results suggested that the metabolism by the plaque cells adsorbed to surface-associated mucins was downregulated compared to the same cells suspended in mucin solution. This was confirmed in an experiment where active dispersed plaque/solMUC5B suspensions were shown to lose significant metabolic activity (e.g. 74.9 to 19.3 %) when allowed to interact with gelMUC5B adsorbed to a surface. Clearly, the solMUC5B and gelMUC5B preparations exhibited different biological activity when assayed with freshly plaque bacteria in suspension and in a biofilm.

Potential use of mucins as biomaterial coatings. I. Fractionation, characterization, and model adsorption of bovine, porcine, and human mucins.

Previously, we presented evidence that mucins have potential as biomaterial coatings. Here, we reveal substantial batch-to-batch variations for a frequently used commercial bovine salivary mucin preparation (BSM) and stress the importance of standardizing mucins intended for comparative purposes. "Mild" fractionation strategies, aiming at preserving natural mucin functions, were used to prepare two more defined BSM fractions as well as three mucin fractions from porcine gastric (PGM) and human salivary (MG1) sources. While the BSM and PGM were highly purified and mainly adopted random coil conformations in solution, the MG1 contained mucin-bound components (1.6 wt% albumin) and appeared compact. Average molar masses and root-mean-square radii for the predominant BSM, PGM, and MG1 species spanned 0.8-4.2 MDa and 46-86 nm, respectively. An ellipsometric evaluation, using hydrophilic and hydrophobic silica, showed the mucin adsorption to be slow and related to mucin charge, size, conformation, and compositional complexity. The mass uptakes on hydrophobic silica averaged 2.6, 2.6, and 5.0 mg/m(2), for BSM, PGM, and MG1, respectively. Finally, we find that stable mucin coatings can be formed on polymers of different wettability. The reported mucin preparations serve as platforms for a series of studies on the biocompatibility of mucin coatings.