Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins

Cystic fibrosis: An inherited disease affecting mucin-producing organs

Our current understanding of cystic fibrosis (CF) has revealed that the biophysical properties of mucus play a considerable role in the pathogenesis of the disease in view of the fact that most mucus-producing organs are affected in CF patients. In this review, we discuss the potential causal relationship between altered cystic fibrosis transmembrane conductance regulator (CFTR) function and the production of mucus with abnormal biophysical properties in the intestine and lungs, highlighting what has been learned from cell cultures and animal models that mimic CF pathogenesis. A similar cascade of events, including mucus obstruction, infection and inflammation, is common to all epithelia affected by impaired surface hydration. Hence, the main structural components of mucus, namely the polymeric, gel-forming mucins, are critical to the onset of the disease. Defective CFTR leads to epithelial surface dehydration, altered pH/electrolyte composition and mucin concentration. Further, it can influence mucin transition from the intracellular to extracellular environment, potentially resulting in aberrant mucus gel formation. While defective HCO3− production has long been identified as a feature of CF, it has only recently been considered as a key player in the transition phase of mucins. We conclude by examining the influence of mucins on the biophysical properties of CF sputum and discuss existing and novel therapies aimed at removing mucus from the lungs

Munc13-2 −/− baseline secretion defect reveals source of oligomeric mucins in mouse airways: Muc5b secretion defect in Munc13-2−/−mouse airways

Since the airways of control mouse lungs contain few alcian blue/periodic acid–Schiff's (AB/PAS)+ staining ‘goblet’ cells in the absence of an inflammatory stimulus such as allergen sensitization, it was surprising to find that the lungs of mice deficient for the exocytic priming protein Munc13-2 stain prominently with AB/PAS under control conditions. Purinergic agonists (ATP/UTP) stimulated release of accumulated mucins in the Munc13-2-deficient airways, suggesting that the other airway isoform, Munc13-4, supports agonist-regulated secretion. Notably, however, not all of the mucins in Munc13-2-deficient airways were secreted, suggesting a strict Munc13-2 priming requirement for a population of secretory granules. AB/PAS+ staining of Munc13-2-deficient airways was not caused by an inflammatory, metaplastic-like response: bronchial–alveolar lavage leucocyte numbers, Muc5ac and Muc5b mRNA levels, and Clara cell ultrastructure (except for increased secretory granule numbers) were all normal. A Muc5b-specific antibody indicated the presence of this mucin in Clara cells of wildtype (WT) control mice, and increased amounts in Munc13-2-deficient mice. Munc13-2 therefore appears to prime a regulated, baseline secretory pathway, such that Clara cell Muc5b, normally secreted soon after synthesis, accumulates in the gene-deficient animals, making them stain AB/PAS+. The defective priming phenotype is widespread, as goblet cells of several mucosal tissues appear engorged and Clara cells accumulated Clara cell secretory protein (CCSP) in Munc13-2-deficient mice. Additionally, because in the human airways, MUC5AC localizes to the surface epithelium and MUC5B to submucosal glands, the finding that Muc5b is secreted by Clara cells under control conditions may indicate that it is also secreted tonically from human bronchiolar Clara cells

Chronic Exposure to Beta-Blockers Attenuates Inflammation and Mucin Content in a Murine Asthma Model

Single-dose administration of beta-adrenoceptor agonists produces bronchodilation and inhibits airway hyperresponsiveness (AHR), and is the standard treatment for the acute relief of asthma. However, chronic repetitive administration of beta-adrenoceptor agonists may increase AHR, airway inflammation, and risk of death. Based upon the paradigm shift that occurred with the use of beta-blockers in congestive heart failure, we previously determined that chronic administration of beta-blockers decreased AHR in a murine model of asthma. To elucidate the mechanisms for the beneficial effects of beta-blockers, we examined the effects of chronic administration of several beta-adrenoceptor ligands in a murine model of allergic asthma. Administration of beta-blockers resulted in a reduction in total cell counts, eosinophils, and the cytokines IL-13, IL-10, IL-5, and TGF-β1 in bronchoalveolar lavage, and attenuated epithelial mucin content and morphologic changes. The differences in mucin content also occurred if the beta-blockers were administered only during the ovalbumin challenge phase, but administration of beta-blockers for 7 days was not as effective as administration for 28 days. These results indicate that in a murine model of asthma, chronic administration of beta-blockers reduces inflammation and mucous metaplasia, cardinal features of asthma that may contribute to airflow obstruction and AHR. Similar to heart failure, our results provide a second disease model in which beta-blockers producing an acutely detrimental effect may provide a therapeutically beneficial effect with chronic administration

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure

The pathogenesis of mucoinfective lung disease in cystic fibrosis (CF) patients likely involves poor mucus clearance. A recent model of mucus clearance predicts that mucus flow depends on the relative mucin concentration of the mucus layer compared with that of the periciliary layer; however, mucin concentrations have been difficult to measure in CF secretions. Here, we have shown that the concentration of mucin in CF sputum is low when measured by immunologically based techniques, and mass spectrometric analyses of CF mucins revealed mucin cleavage at antibody recognition sites. Using physical size exclusion chromatography/differential refractometry (SEC/dRI) techniques, we determined that mucin concentrations in CF secretions were higher than those in normal secretions. Measurements of partial osmotic pressures revealed that the partial osmotic pressure of CF sputum and the retained mucus in excised CF lungs were substantially greater than the partial osmotic pressure of normal secretions. Our data reveal that mucin concentration cannot be accurately measured immunologically in proteolytically active CF secretions; mucins are hyperconcentrated in CF secretions; and CF secretion osmotic pressures predict mucus layer–dependent osmotic compression of the periciliary liquid layer in CF lungs. Consequently, mucin hypersecretion likely produces mucus stasis, which contributes to key infectious and inflammatory components of CF lung disease

SARS-CoV-2 infection of airway cells causes intense viral and cell shedding, two spreading mechanisms affected by IL-13

Muco-obstructive lung diseases are typically associated with high risks of COVID-19 severity; however, allergic asthma showed reduced susceptibility. To investigate viral spread, primary human airway epithelial (HAE) cell cultures were infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and host–virus interactions were examined via electron microscopy, immunohistochemistry, RNA in situ hybridization, and gene expression analyses. In HAE cell cultures, angiotensin-converting enzyme 2 (ACE2) expression governed cell tropism and viral load and was up-regulated by infection. Electron microscopy identified intense viral egress from infected ciliated cells and severe cytopathogenesis, culminating in the shedding of ciliated cells packed with virions, providing a large viral reservoir for spread and transmission. Intracellular stores of MUC5AC, a major airway mucin involved in asthma, were rapidly depleted, likely to trap viruses. To mimic asthmatic airways, HAE cells were treated with interleukin-13 (IL-13), which reduced viral titers, viral messenger RNA, and cell shedding, and significantly diminished the number of infected cells. Although mucus hyperproduction played a shielding role, IL-13–treated cells maintained a degree of protection despite the removal of mucus. Using Gene Expression Omnibus databases, bulk RNA-sequencing analyses revealed that IL-13 up-regulated genes controlling glycoprotein synthesis, ion transport, and antiviral processes (albeit not the typical interferon-induced genes) and down-regulated genes involved in cilial function and ribosomal processing. More precisely, we showed that IL-13 reduced ACE2 expression, intracellular viral load, and cell-to-cell transmission while increasing the cilial keratan sulfate coating. In conclusion, intense viral and cell shedding caused by SARS-CoV-2 infection was attenuated by IL-13, which affected viral entry, replication, and spread

Genomewide CRISPR knockout screen identified PLAC8 as an essential factor for SADS-CoVs infection

Zoonotic transmission of coronaviruses poses an ongoing threat to human populations. Endemic outbreaks of swine acute diarrhea syndrome coronavirus (SADS-CoV) have caused severe economic losses in the pig industry and have the potential to cause human outbreaks. Currently, there are no vaccines or specific antivirals against SADS-CoV, and our limited understanding of SADS-CoV host entry factors could hinder prompt responses to a potential human outbreak. Using a genomewide CRISPR knockout screen, we identified placenta-associated 8 protein (PLAC8) as an essential host factor for SADS-CoV infection. Knockout of PLAC8 abolished SADS-CoV infection, which was restored by complementing PLAC8 from multiple species, including human, rhesus macaques, mouse, pig, pangolin, and bat, suggesting a conserved infection pathway and susceptibility of SADS-CoV among mammals. Mechanistically, PLAC8 knockout does not affect viral entry; rather, knockout cells displayed a delay and reduction in viral subgenomic RNA expression. In a swine primary intestinal epithelial culture (IEC) infection model, differentiated cultures have high levels of PLAC8 expression and support SADS-CoV replication. In contrast, expanding IECs have low levels of PLAC8 expression and are resistant to SADS-CoV infection. PLAC8 expression patterns translate in vivo; the immunohistochemistry of swine ileal tissue revealed high levels of PLAC8 protein in neonatal compared to adult tissue, mirroring the known SADS-CoV pathogenesis in neonatal piglets. Overall, PLAC8 is an essential factor for SADS-CoV infection and may serve as a promising target for antiviral development for potential pandemic SADS-CoV

Increased colonic expression of ACE2 associates with poor prognosis in Crohn’s disease

The host receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2), is highly expressed in small intestine. Our aim was to study colonic ACE2 expression in Crohn's disease (CD) and non-inflammatory bowel disease (non-IBD) controls. We hypothesized that the colonic expression levels of ACE2 impacts CD course. We examined the expression of colonic ACE2 in 67 adult CD and 14 NIBD control patients using RNA-seq and quantitative (q) RT-PCR. We validated ACE2 protein expression and localization in formalin-fixed, paraffin-embedded matched colon and ileal tissues using immunohistochemistry. The impact of increased ACE2 expression in CD for the risk of surgery was evaluated by a multivariate regression analysis and a Kaplan–Meier estimator. To provide critical support for the generality of our findings, we analyzed previously published RNA-seq data from two large independent cohorts of CD patients. Colonic ACE2 expression was significantly higher in a subset of adult CD patients which was defined as the ACE2-high CD subset. IHC in a sampling of ACE2-high CD patients confirmed high ACE2 protein expression in the colon and ileum compared to ACE2-low CD and NIBD patients. Notably, we found that ACE2-high CD patients are significantly more likely to undergo surgery within 5 years of CD diagnosis, and a Cox regression analysis found that high ACE2 levels is an independent risk factor for surgery (OR 2.17; 95% CI, 1.10–4.26; p = 0.025). Increased intestinal expression of ACE2 is associated with deteriorated clinical outcomes in CD patients. These data point to the need for molecular stratification that can impact CD disease-related outcomes

SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo

The spike D614G substitution is prevalent in global SARS-CoV-2 strains, but its effects on viral pathogenesis and transmissibility remain unclear. We engineered a SARS-CoV-2 variant containing this substitution. The variant exhibits more efficient infection, replication, and competitive fitness in primary human airway epithelial cells, but maintains similar morphology and in vitro neutralization properties, compared with the ancestral wild-type virus. Infection of human angiotensin-converting enzyme 2 (ACE2) transgenic mice and Syrian hamsters with both viruses resulted in similar viral titers in respiratory tissues and pulmonary disease. However, the D614G variant transmits significantly faster and displayed increased competitive fitness than the wild-type virus in hamsters. These data show that the D614G substitution enhances SARS-CoV-2 infectivity, competitive fitness, and transmission in primary human cells and animal models

Prevalence and Mechanisms of Mucus Accumulation in COVID-19 Lung Disease

Rationale: The incidence and sites of mucus accumulation, and molecular regulation of mucin gene expression, in COVID-19 lung disease have not been reported. Objectives: Characterize incidence of mucus accumulation and the mechanisms mediating mucin hypersecretion in COVID-19 lung disease. Methods: Airway mucus and mucins were evaluated in COVID-19 autopsy lungs by AB-PAS and immunohistochemical staining, RNA in situ hybridization, and spatial transcriptional profiling. SARS-CoV-2-infected human bronchial epithelial (HBE) cultures were utilized to investigate mechanisms of SARS-CoV-2-induced mucin expression and synthesis and test candidate countermeasures. Measurements and Main Results: MUC5B and variably MUC5AC RNA levels were increased throughout all airway regions of COVID-19 autopsy lungs, notably in the sub-acute/chronic disease phase following SARS-CoV-2 clearance. In the distal lung, MUC5B-dominated mucus plugging was observed in 90% of COVID-19 subjects in both morphologically identified bronchioles and microcysts, and MUC5B accumulated in damaged alveolar spaces. SARS-CoV-2-infected HBE cultures exhibited peak titers 3 days post inoculation, whereas induction of MUC5B/MUC5AC peaked 7-14 days post inoculation. SARS-CoV-2 infection of HBE cultures induced expression of EGFR ligands and inflammatory cytokines (e.g., IL-1α/β) associated with mucin gene regulation. Inhibiting EGFR/IL-1R pathways, or dexamethasone administration, reduced SARS-CoV-2-induced mucin expression. Conclusions: SARS-CoV-2 infection is associated with a high prevalence of distal airspace mucus accumulation and increased MUC5B expression in COVID-19 autopsy lungs. HBE culture studies identified roles for EGFR and IL-1R signaling in mucin gene regulation post SARS-CoV-2 infection. These data suggest that time-sensitive mucolytic agents, specific pathway inhibitors, or corticosteroid administration may be therapeutic for COVID-19 lung disease. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org.libproxy.lib.unc.edu/licenses/by-nc-nd/4.0/)