Mucins MUC5B Research Articles

Requirement for Fucosyltransferase 2 in Allergic Airway Hyperreactivity and Mucus Obstruction.

Mucus hypersecretion is an important pathological problem in respiratory diseases. Mucus accumulates in the airways of people with asthma, and it contributes to airflow limitation by forming plugs that occlude airways. Current treatments have minimal effects on mucus or its chief components, the polymeric mucin glycoproteins MUC5AC and MUC5B. This treatment gap reflects a poor molecular understanding of mucins that could be used to determine how they contribute to airway obstruction. Due to the prominence of glycosylation as a defining characteristic of mucins, we investigated characteristics of mucin glycans in asthma and in a mouse model of allergic asthma. Mucin fucosylation was observed in asthma, and in healthy mice it was induced as part of a mucous metaplastic response to allergic inflammation. In allergically inflamed mouse airways, mucin fucosylation was dependent on the enzyme fucosyltransferase 2 (Fut2). Fut2 gene deficient mice were protected from asthma-like airway hyperreactivity and mucus plugging. These findings provide mechanistic and translational links between observations in human asthma and a mouse model that may help improve therapeutic targeting of airway mucus.

Spatiotemporal characteristics of eustachian tube development in C57BL/6 mice: Correlation between morphological and functional maturation.

The eustachian tube (ET), a critical conduit connecting the middle ear and nasopharynx, is essential for normal middle ear function. However, it remains one of the least understood anatomical structures due to its complexity and the challenges of in vitro manipulation. Historically, these challenges have hindered research into the morphology and function development of the ET. This study elucidates the spatiotemporal relationship of ET morpho-functional maturation in mice, identifying key periods and factors that lay the theoretical foundation for exploring the molecular mechanisms of ET-related diseases. We comprehensively characterized the ET development in C57BL/6 mice from embryonic day (E) 12.5 to postnatal day (P) 30, focusing on the development of cilia, secretory cells, surrounding glands, and macrophages. Immunostaining identified the localization and secretion patterns of the mucins Muc5b and Muc5ac within the ET. Additionally, using improved ET function assessment tools, we evaluated the developmental features of ET mucociliary clearance and ventilation functions. In C57BL/6 mice, E16.5 marks a critical period for middle ear cavity and ET formation. Muc5b plays a foundational role during early stages, while Muc5ac enhances function in later stages. During P7-11, despite morphological maturity, ET function remains underdeveloped but continues to improve with growth.

Structural and genetic diversity in the secreted mucins MUC5AC and MUC5B

The secreted mucins MUC5AC and MUC5B are large glycoproteins that play critical defensive roles in pathogen entrapment and mucociliary clearance. Their respective genes contain polymorphic and degenerate protein-coding variable number tandem repeats (VNTRs) that make the loci difficult to investigate with short reads. We characterize the structural diversity of MUC5AC and MUC5B by long-read sequencing and assembly of 206 human and 20 nonhuman primate (NHP) haplotypes. We find that human MUC5B is largely invariant (5,761-5,762 amino acids [aa]); however, seven haplotypes have expanded VNTRs (6,291-7,019 aa). In contrast, 30 allelic variants of MUC5AC encode 16 distinct proteins (5,249-6,325 aa) with cysteine-rich domain and VNTR copy-number variation. We group MUC5AC alleles into three phylogenetic clades: H1 (46%, ∼5,654 aa), H2 (33%, ∼5,742 aa), and H3 (7%, ∼6,325 aa). The two most common human MUC5AC variants are smaller than NHP gene models, suggesting a reduction in protein length during recent human evolution. Linkage disequilibrium and Tajima's D analyses reveal that East Asians carry exceptionally large blocks with an excess of rare variation (p<0.05) at MUC5AC. To validate this result, we use Locityper for genotyping MUC5AC haplogroups in 2,600 unrelated samples from the 1000 Genomes Project. We observe a signature of positive selection in H1 among East Asians and a depletion of the likely ancestral haplogroup (H3). In Europeans, H3 alleles show an excess of common variation and deviate from Hardy-Weinberg equilibrium (p<0.05), consistent with heterozygote advantage and balancing selection. This study provides a generalizable strategy to characterize complex protein-coding VNTRs for improved disease associations.

Pulmonary Fibrosis Ferret Model Demonstrates Sustained Fibrosis, Restrictive Physiology, and Aberrant Repair.

The role of MUC5B mucin expression in IPF pathogenesis is unknown. Bleomycin-exposed rodent models do not exhibit sustained fibrosis or airway remodeling. Unlike mice, ferrets have human-like distribution of MUC5B expressing cell types and natively express the risk-conferring variant that induces high MUC5B expression in humans. We hypothesized that ferrets would consequently exhibit aberrant repair to propagate fibrosis similar to human IPF. Bleomycin (5U/kg) or saline-control was micro-sprayed intratracheally then wild-type ferrets were evaluated through 22 wks. Clinical phenotype was assessed with lung function. Fibrosis was assessed with µCT imaging and comparative histology with Ashcroft scoring. Airway remodeling was assessed with histology and quantitative immunofluorescence. Bleomycin ferrets exhibited sustained restrictive physiology including decreased inspiratory capacity, decreased compliance, and shifted Pressure-Volume loops through 22 wks. Volumetric µCT analysis revealed increased opacification of the lung bleomycin-ferrets. Histology showed extensive fibrotic injury that matured over time and MUC5B-positive cystic structures in the distal lung suggestive of honeycombing. Bleomycin ferrets had increased proportion of small airways that were double-positive for CCSP and alpha-tubulin compared to controls, indicating an aberrant 'proximalization' repair phenotype. Notably, this aberrant repair was associated with extent of fibrotic injury at the airway level. Bleomycin-exposed ferrets exhibit sustained fibrosis through 22 wks and have pathologic features of IPF not found in rodents. Ferrets exhibited proximalization of the distal airways and other pathologic features characteristic of human IPF. MUC5B expression through native cell types may play a key role in promoting airway remodeling and lung injury in IPF.

Investigation of Hydrocolloid Plant Polysaccharides as Potential Candidates to Mimic the Functions of MUC5B in Saliva

The successful substitution of complex physiological fluids, such as human saliva, remains a major challenge in drug development. Although there are a large number of saliva substitutes on the market, their efficacy is often inadequate due to short residence time in the mouth, unpleasant mouthfeel, or insufficient protection of the teeth. Therefore, systems need to be identified that mimic the functions of saliva, in particular the salivary mucin MUC5B and the unique physiological properties of saliva. To this end, plant extracts known to contain hydrocolloid polysaccharides and to have mucus-forming properties were studied to evaluate their suitability as saliva substitutes. The aqueous plant extracts of Calendula officinalis, Fucus sp. thalli, and lichenan from Lichen islandicus were examined for composition using a range of techniques, including GC-MS, NMR, SEC, assessment of pH, osmolality, buffering capacity, viscoelasticity, viscoelastic interactions with human saliva, hydrocolloid network formation, and in vitro cell adhesion. For this purpose, a physiologically adapted adhesive test was developed using human buccal epithelial cells. The results show that lichenan is the most promising candidate to mimic the properties of MUC5B. By adjusting the pH, osmolality, and buffering capacity with K2HPO4, it was shown that lichenan exhibited high cell adhesion, with a maximum detachment force that was comparable to that of unstimulated whole mouth saliva.

Sex-Dependent Regulation of Mucin Gene Transcription and Goblet Cell Secretion Machinery Following Intra-Airway IL-13 in Mice with Conditional Loss of Club Cell Creb1

IL-13 is an important effector molecule in allergic asthma, converting secretoglobin-positive club cells into mucin-secreting goblet cells and promoting mucin hypersecretion. IL-13-mediated conversion of club cells to goblet cells requires decreased gene expression of forkhead box A2 ( FOXA2) and increased gene expression of SAM pointed domain containing ETS transcription factor ( SPDEF). In addition, IL-13-mediated mucin hypersecretion may include modulation of purinergic and muscarinic receptors that control basal and stimulated mucus secretion, respectively. We recently found that the cAMP response element-binding protein (CREB), a ubiquitous transcription factor, directly binds to FOXA2 and modulates mucus secretion mechanisms in mice. Here we tested the hypothesis that conditional loss of club cell Creb1 in murine airways modulates the pro-mucin effects of IL-13. Loss of club cell Creb1 augmented IL-13-mediated increases in gene expression of the major gel-forming secreted mucins Muc5ac and Muc5b in male murine airways, with no effect on IL-13-mediated increases in Muc5ac and Muc5b in female murine airways. Examination of mucin secretion mechanisms revealed that IL-13 decreased muscarinic 3 receptor ( M3R) mRNA expression in male murine airways, whereas loss of club cell Creb1 prevented this, resulting in a significant treatment by genotype interaction. Interestingly, in female airways, a main effect of genotype was observed, with loss of club cell Creb1 reducing M3R mRNA expression in the airway. Examination of the purinergic receptor P2Y ( P2ry2) mRNA revealed a main treatment effect for IL-13 to increase P2ry2 expression in male murine airways with no impact of loss of club cell Creb1. In the females, IL-13 increased P2ry2 mRNA in both genotypes, with loss of club cell Creb1 significantly blunting the effect of IL-13 on P2ry2 mRNA. Lastly, we examined goblet cell density and mucin secretion mechanisms using Alcian Blue-PAS staining post in vivo stimulation with a muscarinic agent. We found no major effect of treatment or genotype in the density of goblet cells containing acidic mucins in either male or female airways. In contrast, both males and females showed IL-13-mediated increases in the density of goblet cells containing neutral mucins that was not impacted by genotype. Our preliminary findings suggest that loss of club cell Creb1 decreases molecular mucin secretion mechanisms in male and female airways, and increases Muc5ac and Muc5b mRNA in male airways, though a corresponding genotype-dependent increase in goblet cell density post cholinergic stimulation was not observed. Thus, we conclude that loss of club cell Creb1 causes changes to molecules important for IL-13-mediated mucin hypersecretion, without apparent functional benefit or detriment to the murine airway. This work was supported by the National Institutes of Health (OD026582, HL152101), the Cystic Fibrosis Foundation (REZNIKO20I0, REZNIKO19I0), the Katie Rose Foundation (AWD08203), and the University of Florida McKnight Career Accelerator Award. Angelina Bonilla was funded by the National institutes of Health (R25GM115298). Pedro Trevizan Bau receives the University of Florida McKnight Brain Institute Gator NeuroScholar Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-dependent regulation of mucin gene transcription and airway secretion and mechanics following intra-airway IL-13 in mice with conditional loss of club cell Creb1.

Introduction: Interleukin 13 (IL-13) is an important effector molecule in allergic asthma. IL-13-mediated mucin hypersecretion requires conversion of secretoglobin-positive club cells into goblet cells through suppression of forkhead box A2 (FOXA2) and induction of SAM pointed domain containing ETS transcription factor (SPDEF). IL-13-mediated mucin hypersecretion may also include modulation of purinergic and muscarinic receptors that control basal and stimulated mucin secretion. We recently found that the transcription factor cAMP response element-binding protein (Creb1) inhibits FOXA2 and modulates mucus secretion in mice. Methods: We tested the hypothesis that loss of club cell Creb1 mitigates the pro-mucin effects of IL-13. We challenged male and female mice with conditional loss of club cell Creb1 and wild type littermates with intra-airway IL-13 or vehicle. We also studied human "club cell-like" NCI-H322 cells. Results: Loss of club cell Creb1 augmented IL-13-mediated increases in mRNA for the gel-forming mucins Muc5ac and Muc5b and prevented IL-13-mediated decreases in muscarinic 3 receptor (M3R) mRNA in male airways. In female airways, loss of club cell Creb1 reduced M3R mRNA and significantly blunted IL-13-mediated increases in purinergic receptor P2Y2 (P2ry2) mRNA but did not impact Muc5ac and Muc5b mRNA. Despite changes in mucins and secretion machinery, goblet cell density following cholinergic stimulation was not impacted by loss of club cell Creb1 in either sex. IL-13 treatment decreased basal airway resistance across sexes in mice with loss of club cell Creb1, whereas loss of club cell Creb1 augmented IL-13-mediated increases in airway elastance in response to methacholine. NCI-H322 cells displayed IL-13 signaling components, including IL-13Rα1 and IL-4Rα. Pharmacologic inhibition of CREB reduced IL-13Rα1 mRNA, whereas recombinant CREB decreased IL-4Rα mRNA. Application of IL-13 to NCI-H322 cells increased concentrations of cAMP in a delayed manner, thus linking IL-13 signaling to CREB signaling. Conclusion: These data highlight sex-specific regulation of club cell Creb1 on IL-13-mediated mucin hypersecretion and airway mechanics.

The combination of propylene glycol and vegetable glycerin e-cigarette aerosols induces airway inflammation and mucus hyperconcentration

Despite concerns over their safety, e-cigarettes (e-cigs) remain a popular tobacco product. Although nicotine and flavors found in e-cig liquids (e-liquids) can cause harm in the airways, whether the delivery vehicles propylene glycol (PG) and vegetable glycerin (VG) are innocuous when inhaled remains unclear. Here, we investigated the effects of e-cig aerosols generated from e-liquid containing only PG/VG on airway inflammation and mucociliary function in primary human bronchial epithelial cells (HBEC) and sheep. Primary HBEC were cultured at the air–liquid interface (ALI) and exposed to e-cig aerosols of 50%/50% v/v PG/VG. Ion channel conductance, ciliary beat frequency, and the expression of inflammatory markers, cell type-specific markers, and the major mucins MUC5AC and MUC5B were evaluated after seven days of exposure. Sheep were exposed to e-cig aerosols of PG/VG for five days and mucus concentration and matrix metalloproteinase-9 (MMP-9) activity were measured from airway secretions. Seven-day exposure of HBEC to e-cig aerosols of PG/VG caused a significant reduction in the activities of apical ion channels important for mucus hydration, including the cystic fibrosis transmembrane conductance regulator (CFTR) and large conductance, Ca2+-activated, and voltage-dependent K+ (BK) channels. PG/VG aerosols significantly increased the mRNA expression of the inflammatory markers interleukin-6 (IL6), IL8, and MMP9, as well as MUC5AC. The increase in MUC5AC mRNA expression correlated with increased immunostaining of MUC5AC protein in PG/VG-exposed HBEC. On the other hand, PG/VG aerosols reduced MUC5B expression leading overall to higher MUC5AC/MUC5B ratios in exposed HBEC. Other cell type-specific markers, including forkhead box protein J1 (FOXJ1), keratin 5 (KRT5), and secretoglobin family 1A member 1 (SCGB1A1) mRNAs, as well as overall ciliation, were significantly reduced by PG/VG exposure. Finally, PG/VG aerosols increased MMP-9 activity and caused mucus hyperconcentration in sheep in vivo. E-cig aerosols of PG/VG induce airway inflammation, increase MUC5AC expression, and cause dysfunction of ion channels important for mucus hydration in HBEC in vitro. Furthermore, PG/VG aerosols increase MMP-9 activity and mucus concentration in sheep in vivo. Collectively, these data show that e-cig aerosols containing PG/VG are likely to be harmful in the airways.

Quantitation of MUC5AC and MUC5B by Stable Isotope Labeling Mass Spectrometry.

Mucins MUC5AC and MUC5B are large glycoproteins that play an essential role in the innate defense of epithelial surfaces and their quantitation in biological samples would be informative about the health status of the tissue/samples they are derived from. However, they are difficult to study and quantify with traditional methods such as ELISA and western blot, due to their size, heterogeneity, and high degree of glycosylation. We successfully implemented a stable isotope labeling mass spectrometry approach for absolute quantification of mucin macromolecules. Here, in detail, we describe this accurate and sensitive liquid chromatography and mass spectrometry (LC-MS) method applied for both MUC5AC and MUC5B quantification in diverse and complex biological samples.

Thiolated polyglycerol sulfate as potential mucolytic for muco-obstructive lung diseases.

Increased disulfide crosslinking of secreted mucins causes elevated viscoelasticity of mucus and is a key determinant of mucus dysfunction in patients with cystic fibrosis (CF) and other muco-obstructive lung diseases. In this study, we describe the synthesis of a novel thiol-containing, sulfated dendritic polyglycerol (dPGS-SH), designed to chemically reduce these abnormal crosslinks, which we demonstrate with mucolytic activity assays in sputum from patients with CF. This mucolytic polymer, which is based on a reportedly anti-inflammatory polysulfate scaffold, additionally carries multiple thiol groups for mucolytic activity and can be produced on a gram-scale. After a physicochemical compound characterization, we compare the mucolytic activity of dPGS-SH to the clinically approved N-acetylcysteine (NAC) using western blot studies and investigate the effect of dPGS-SH on the viscoelastic properties of sputum samples from CF patients by oscillatory rheology. We show that dPGS-SH is more effective than NAC in reducing multimer intensity of the secreted mucins MUC5B and MUC5AC and demonstrate significant mucolytic activity by rheology. In addition, we provide data for dPGS-SH demonstrating a high compound stability, low cytotoxicity, and superior reaction kinetics over NAC at different pH levels. Our data support further development of the novel reducing polymer system dPGS-SH as a potential mucolytic to improve mucus function and clearance in patients with CF as well as other muco-obstructive lung diseases.

Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1β contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1β-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1β-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Goblet cell interactions reorient bundled mucus strands for efficient airway clearance.

The respiratory tract of larger animals is cleared by sweeping bundled strands along the airway surface. These bundled strands can be millimetric in length and consist of MUC5B mucin. They are produced by submucosal glands, and upon emerging from these glands, the long axis of the bundled strands is oriented along the cilia-mediated flow toward the oral cavity. However, after release, the bundled strands are found to have turned orthogonal to the flow, which maximizes their clearance potential. How this unexpected reorientation is accomplished is presently not well understood. Recent experiments suggest that the reorientation process involves bundled strands sticking to MUC5AC mucus threads, which are tethered to the goblet cells. Such goblet cells are present in small numbers throughout the airway epithelium. Here, we develop a minimal model for reorientation of bundled mucus strands through adhesive interactions with surface goblet cells. Our simulations reveal that goblet cell interactions can reorient the bundled strands within 10 mm of release-making reorientation on the length scale of the tracheal tube feasible-and can stabilize the orthogonal orientation. Our model also reproduces other experimental observations such as strong velocity fluctuations and significant slow-down of the bundled strand with respect to the cilia-mediated flow. We further provide insight into the strand turning mechanism by examining the effect of strand shape on the impulse exerted by a single goblet cell. We conclude that goblet cell-mediated reorientation is a viable route for bundled strand reorientation, which should be further validated in future experiment.

Identification of a DNA-cytosine methyltransferase that impacts global transcription to promote group B streptococcal vaginal colonization.

Group B Streptococcus (GBS) colonizes the female reproductive tract (FRT) in one-third of women, and carriage leads to numerous adverse pregnancy outcomes including the preterm premature rupture of membranes, chorioamnionitis, and stillbirth. The presence of GBS in the FRT during pregnancy is also the largest predisposing factor for the transmission of GBS and invasive neonatal diseases, including pneumonia, sepsis, and meningitis. The factors contributing to GBS colonization are still being elucidated. Here, we show for the first time that GBS transcription is regulated by an orphan DNA cytosine methyltransferase (Dcm). Many GBS factors are regulated by Dcm, especially those involved in carbohydrate transport and metabolism. We show that GBS persistence in the FRT is dependent on the catabolism of sugars found on the vaginal mucin MUC5B. Collectively, this work highlights the regulatory importance of a DNA methyltransferase and identifies both host and bacterial factors required for GBS colonization.

Synergistic metabolism of salivary MUC5B in oral commensal bacteria during early biofilm formation.

Bacterial metabolism in oral biofilms is comprised of complex networks of nutritional chains and biochemical regulations. These processes involve both intraspecies and interspecies networks as well as interactions with components from host saliva, gingival crevicular fluid, and dietary intake. In a previous paper, a large salivary glycoprotein, mucin MUC5B, was suggested to promote a dental health-related phenotype in the oral type strain of Streptococcus gordonii DL1, by regulating bacterial adhesion and protein expression. In this study, nuclear magnetic resonance-based metabolomics was used to examine the effects on the metabolic output of monospecies compared to dual species early biofilms of two clinical strains of oral commensal bacteria, S. gordonii and Actinomyces naeslundii, in the presence of MUC5B. The presence of S. gordonii increased colonization of A. naeslundii on salivary MUC5B, and both commensals were able to utilize MUC5B as a sole nutrient source during early biofilm formation. The metabolomes suggested that the bacteria were able to release mucin carbohydrates from oligosaccharide side chains as well as amino acids from the protein core. Synergistic effects were also seen in the dual species biofilm metabolome compared to the monospecies, indicating that A. naeslundii and S. gordonii cooperated in the degradation of salivary MUC5B. A better understanding of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is meaningful for understanding oral biofilm physiology and may contribute to the development of future prevention strategies for biofilm-induced oral disease.IMPORTANCEThe study of bacterial interactions and salivary-mediated regulation of early dental biofilm activity is of interest for understanding oral microbial adaptation to environmental cues and biofilm maturation. Findings in oral commensals can prove useful from the perspectives of both oral and systemic health of the host, as well as the understanding of general microbial biofilm physiology. The knowledge may provide a basis for the development of prognostic biomarkers, or development of new treatment strategies, related to oral health and disease and possibly also to other biofilm-induced conditions. The study is also an important step toward developing the methodology for similar studies in other species and/or growth conditions.

BPIFB1 loss alters airway mucus properties and diminishes mucociliary clearance.

Airway mucociliary clearance (MCC) is required for host defense and is often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the gene Bpifb1/Lplunc1 as a regulator of airway MUC5B protein levels using genetic approaches. Here, we show that BPIFB1 is required for effective MCC in vivo using Bpifb1 knockout (KO) mice. Reduced MCC in Bpifb1 KO mice occurred in the absence of defects in epithelial ion transport or reduced ciliary beat frequency. Loss of BPIFB1 in vivo and in vitro altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and the protein mesh of secreted mucus in human airway epithelia cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.NEW & NOTEWORTHY BPIFB1, also known as LPLUNC1, was found to regulate mucociliary clearance (MCC), a key aspect of host defense in the airway. Loss of this protein was also associated with altered biophysical and biochemical properties of mucus that have been previously linked to impaired MCC.

Elucidating the molecular role of MUC5B in progressive lung adenocarcinoma: Prospects for early diagnosis.

Gel-forming mucin MUC5B is significantly deregulated in lung adenocarcinoma (LUAD), however, its role in tumor progression is not yet clearly understood. Here, we used an integrated computational-pipeline-initiated with gene expression analysis followed by network, functional-enrichment, O-linked glycosylation analyses, mutational profiling, and immune cell infiltration estimation to functionally characterize MUC5B gene in LUAD. Thereafter, clinical biomarker validation was supported by the overall survival (OA) and comparative expression profiling across clinical stages using computational algorithms. The gene expression profile of LUAD identified MUC5B to be significantly up-regulated (logFC: 2.36; p-value: 0.01). Network analysis on LUAD interactome screened MUC5B-related genes, having key enrichment in immune suppression and O-linked glycosylation with serine-threonine-rich tandem repeats being highly glycosylated. Furthermore, positive correlation of mutant MUC5B with immune cells in tumor microenvironment (TME) such as cancer-associated fibroblasts and myeloid-derived suppressor cells indicates TME-mediated tumor progression. The positive correlation with immune inhibitors suggested the enhanced tumor proliferation mediated by MUC5B. Structural stability due to genetic alterations identified overall rigid N-H-backbone dynamics (S2 : 0.756), indicating an overall stable mutant protein. Moreover, the low median OA (<50 months) with a hazard ratio of 1.4 and clinical profile of MUC5B gene showed high median expression corresponding to lymph node (N2) and tumor (T3) stages. Our study concludes by highlighting the functional role of O-glycosylated and mutant MUC5B in promoting LUAD by immune suppression. Further, clinical gene expression validation of MUC5B suggests its potential role as a diagnostic biomarker for LUAD metastasis.

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface.

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry.

Immunohistochemical detection of MUC5AC and MUC5B mucins in ferrets

ObjectiveCystic fibrosis (CF) is a genetic condition that causes abnormal mucus secretions in affected organs. MUC5AC and MUC5B are gel-forming mucins and frequent targets for investigations in CF tissues. Our objective was to qualify MUC5AC and MUC5B immunohistochemical techniques to provide a useful tool to identify, localize and interpret mucin expression in ferret tissues.ResultsMUC5AC and MUC5B mucins were detected most commonly in large airways and least in small airways, consistent with reported goblet cell density in airway surface epithelia. We evaluated whether staining method affected the detection of goblet cell mucins in serial sections of bronchial surface epithelia. Significant differences between stains were not observed suggesting common co-expression MUC5AC and MUC5B proteins in goblet cells of airway surface epithelia. Gallbladder and stomach tissues are reported to have differential mucin enrichment, so we tested these tissues in wildtype ferrets. Stomach tissues were enriched in MUC5AC and gallbladder tissues enriched in MUC5B, mucin enrichment similar to human tissues. Mucin immunostaining techniques were further qualified for specificity using lung tissue from recently generated MUC5AC−/− and MUC5B−/− ferrets. Qualified techniques for MUC5AC and MUC5B immunohistochemistry will be useful tools for mucin tissue studies in CF and other ferret models.

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of β and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

The intestinal MUC2 mucin C-terminus is stabilized by an extra disulfide bond in comparison to von Willebrand factor and other gel-forming mucins

The MUC2 mucin polymer is the main building unit of the intestinal mucus layers separating intestinal microbiota from the host epithelium. The MUC2 mucin is a large glycoprotein with a C-terminal domain similar to the MUC5AC and MUC5B mucins and the von Willebrand factor (VWF). A structural model of the C-terminal part of MUC2, MUC2-C, was generated by combining Cryo-electron microscopy, AlphaFold prediction, information of its glycosylation, and small angle X-ray scattering information. The globular VWD4 assembly in the N-terminal of MUC2-C is followed by 3.5 linear VWC domains that form an extended flexible structure before the C-terminal cystine-knot. All gel-forming mucins and VWF form tail-tail disulfide-bonded dimers in their C-terminal cystine-knot domain, but interestingly the MUC2 mucin has an extra stabilizing disulfide bond on the N-terminal side of the VWD4 domain, likely essential for a stable intestinal mucus barrier.