Normal murine respiratory tract has its mucus concentrated in clouds based on the Muc5b mucin.
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ABSTRACT:
SUBMITTER: Fakih D
PROVIDER: S-EPMC7347266 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.
| S-EPMC3094342 | biostudies-literature
Project description:Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites on each MUC5B monomer. Self-diffusion studies with intact MUC5B showed that calcium binding at the protein site catalyzed reversible cross-links between MUC5B chains to form networks. The site of cross-linking was identified in the MUC5B D3-domain as it was specifically blocked by D3 peptide antibodies. Biophysical analysis and single particle EM of recombinant MUC5B N terminus (D1D2D'D3; NT5B) and subdomains (D1, D1-D2, D2-D'-D3, and D3) generated structural models of monomers and disulfide-linked dimers and suggested that MUC5B multimerizes by disulfide linkage between D3-domains to form linear polymer chains. Moreover, these analyses revealed reversible homotypic interactions of NT5B at low pH and in high calcium, between disulfide-linked NT5B dimers, but not monomers. These results enable a model of MUC5B to be derived, which predicts mechanisms of mucin intracellular assembly and storage, which may be common to the other major gel-forming polymeric mucins.
| S-EPMC4047408 | biostudies-literature
Project description:To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles.
| S-EPMC5596833 | biostudies-literature
Project description:We have developed a new approach to study the molecular organization of salivary mucus and salivary mucins using confocal fluorescence recovery after photobleaching (confocal-FRAP). MUC5B mucin, its reduced subunit and T-domains were prepared from saliva and fluorescently labelled. The translational self-diffusion coefficients were determined up to 3.6 mg/ml by confocal-FRAP. The results suggest that, in solutions of purified MUC5B mucin, at concentrations at which the hydrodynamic domains overlap, the intermolecular interactions are predominantly due to dynamic entanglements, and there was no evidence of specific self-association of MUC5B mucin, or of its subunits, or T-domains. The analysis of the salivary mucus gel also showed no specific interactions with the purified MUC5B components, but it was much less permeable than expected from its MUC5B content. The saliva was completely permeable to microspheres of 207 nm diameter, but showed size-dependent effects on the diffusion of larger microspheres (499 nm and 711 nm diameter). From these analyses the salivary mucus was shown to be both permeable and dynamic, and with the characteristics of a semi-dilute transient network at physiological concentration. Comparison of the results from saliva and purified MUC5B mucin solutions showed that the network properties of saliva were equivalent to a solution of purified MUC5B mucin of 10-20 times higher concentration. This showed that saliva has additional structure and organization not present in the purified MUC5B mucin and suggests there are other interactions and/or components within saliva that combine with MUC5B to produce its complete properties.
| S-EPMC1222388 | biostudies-other
Project description: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.
| S-EPMC6851316 | biostudies-literature
Project description:Mucins from human whole saliva, as well as from respiratory- and cervical-tract secretions, were subjected to density-gradient centrifugation in CsCl/0.5 M guanidinium chloride. A polydisperse population of MUC5B mucins was demonstrated in all samples using anti-peptide antisera (LUM5B-2, LUM5B-3 and LUM5B-4) raised against sequences within the MUC5B mucin. The sequences recognized by the LUM5B-2 and LUM5B-3 antisera are located within the domains flanking the highly glycosylated regions of MUC5B, and reduction increased the reactivity with these antibodies, suggesting that the epitopes are partially shielded and that these regions are folded and stabilized by disulphide bonds. Rate-zonal centrifugation before and after reduction showed MUC5B to be a large oligomeric mucin composed of disulphide-linked subunits. In saliva and respiratory-tract secretions, populations of MUC5B mucins with different charge densities were identified by ion-exchange HPLC, suggesting the presence of MUC5B 'glycoforms'. In trachea, the F2 monoclonal antibody against the sulpho-Lewis C structure reacted preferentially with the later-to-be-eluted populations. An antibody (LUM5B-4) recognizing a sequence in the C-terminal domain of MUC5B identified, after reduction, the mucin subunits as well as smaller fragments, suggesting that some of the MUC5B mucins are cleaved within the C-terminal domain. Immunohistochemistry revealed that MUC5B is produced by cells dispersed throughout the human submandibular and sublingual glands, in the airway submucosal glands as well as the goblet cells, and in the epithelium and glands of the endocervix. The F2 antibody stained a subpopulation of the MUC5B-producing cells in the airway submucosal glands, suggesting that different cells may produce different glycoforms of MUC5B in this tissue.
| S-EPMC1219739 | biostudies-other
Project description: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.
| S-EPMC4896102 | biostudies-literature
Project description:Gel-forming mucins are macromolecules produced by goblet cells and responsible for the mucus gel formation. Changes in goblet cell density and in gel-forming mucin production have emerged as sensitive indicators for mucosal diseases. A Muc5b-GFP tagged reporter mouse was used to assess Muc5b production in mouse tissues by immunofluorescence microscopy and fluorescent activity using stereromicroscopy and probe-based confocal laser endomicroscopy. Muc5b production was followed longitudinally by recording the fluorescent activity in vagina and in embryonic lung explants under stimulation by interleukin 13. We show that the GFP is easily visualized in the mouse adult ear, nose, trachea, gallbladder, and cervix. Live Muc5b is also easily monitored in the nasal cavity, trachea and vagina where its production varies during the estrus cycle with a peak at the proestrus phase and in pregnant mice. Explant culture of reporter mouse embryonic whole lung shows that interleukin 13 stimulates Muc5b production. The transgenic Muc5b-GFP mouse is unique and suitable to study the mechanisms that regulate Muc5b production/secretion and mucous cell differentiation by live imaging and can be applied to test drug efficacy in mucosal disease models.
| S-EPMC5353722 | biostudies-literature
Project description:Objective:Paraquat (PQ), a widely used toxic herbicide, induces lung inflammation through mechanisms that remain incompletely understood. In a previous study, we found that the plasma MUC5B mucin level was implicated in PQ poisoning in patients. Here, we hypothesize that MUC5B is a critical mediator in PQ-induced cell inflammation. Methods:A mouse model of PQ-induced lung injury was used to examine the MUC5B expression level. A549 cells (alveolar epithelial cells line) were exposed to PQ in dose-dependent and time-dependent manners. Cell viability was detected by CCK-8 assays. The expression levels of MUC5B were examined by dot blot enzyme-linked immunosorbent assay (ELISA) and RT-qPCR. Western blotting was used to detect the levels of proteins in the MAPK and NF-?B pathways. Inflammatory factors in the cell culture medium were measured by ELISA. NF-?B and MAPK pathway inhibitors and MUC5B siRNA (siMUC5B) were used to determine the function of MUC5B. Finally, N-acetyl-cysteine (NAC) was added and its regulatory effect on the MAPK-NF-?B-MUC5B pathway was examined in PQ-induced cell inflammation. Results:MUC5B was significantly upregulated accompanying the increases in TNF-? and IL-6 secretion following PQ treatment in mouse and also in A549 cells after treatment with 50??M PQ at 24 hours. Furthermore, MAPK and NF-?B pathway inhibitors could dramatically decrease the expression of MUC5B and the secretion of TNF-? and IL-6. Importantly, siMUC5B could significantly attenuate the secretion of TNF-? and IL-6 induced by PQ. As expected, the addition of NAC efficiently suppresses the TNF-? and IL-6 secretion stimulated from PQ and also downregulated ERK, JNK, and p65 phosphorylation (ERK/JNK MAPK and NF-?B pathways) as well as MUC5B expression. Conclusion:Our findings suggest that MUC5B participates in the process of PQ-induced cell inflammation and is downstream of the NF-?B and MAPK pathways. NAC can attenuate PQ-induced cell inflammation at least in part by suppressing the MAPK-NF-?B-MUC5B pathway. These results nominate MUC5B as a new biomarker and therapeutic target for PQ-induced lung inflammation.
| S-EPMC7381986 | biostudies-literature
Project description:Chronic otitis media (COM), e.g. "glue" ear is characterized by middle ear effusion and conductive hearing loss. Although mucous glycoproteins (mucins), which contribute to increased effusion viscosity, have been analyzed in ear tissue specimens, no studies have been reported that characterize the molecular identity of secreted mucin proteins present in actual middle ear fluid. For this study, effusions from children with COM undergoing myringotomy at Children's National Medical Center, Washington, DC were collected. These were solubilized and gel fractionated, and the protein content was identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS) proteomics approach. Western blot analyses with mucin specific antibodies and densitometry were performed to validate the mass spectrometry findings. LC-MS/MS results identified mucin MUC5B by >26 unique peptides in six of six middle ear effusion samples, whereas mucin MUC5AC was only identified in one of six middle ear effusions. These findings were validated by Western blot performed on the same six and on an additional 11 separate samples where densitometry revealed on average a 6.4-fold increased signal in MUC5B when compared with MUC5AC (p = 0.0009). In summary, although both MUC5AC and MUC5B mucins are detected in middle ear effusions, MUC5B seems to be predominant mucin present in COM secretions.
| S-EPMC3679171 | biostudies-literature