mucin-agarose-gel-electrophoresis-western-blotting-for-high-molecular

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 ...

rapid-viscoelastic-characterization-airway-mucus-using-benchtop

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

In muco-obstructive lung diseases (e.g., asthma, chronic obstructive pulmonary disease, cystic fibrosis) and other respiratory conditions (e.g., ...

University of North Carolina at Chapel Hill

The University of North Carolina at Chapel Hill

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. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances.

Cystic fibrosis: an inherited disease affecting mucin-producing organs.

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.

Cystic fibrosis airway secretions exhibit mucin hyperconcentration and increased osmotic pressure.

While protein therapeutics are invaluable in managing numerous diseases, many require frequent injections to maintain therapeutically effective concentrations, due to their short half-life in circulation. PolyXen™, a platform and patented technology employing biodegradable, non-immunogenic and hydrophilic Polysialic Acids (PSA) for drug delivery, is being utilized to overcome such limitations, thereby potentially enabling the clinical utility of a broad range of protein therapeutics. Here, we report the recent progress on two development candidates, polysialylated deoxyribonuclease I (PSA-DNase) and polysialylated erythropoietin (PSA-EPO).

Clinical Application of Polysialylated Deoxyribonuclease and Erythropoietin.

Cystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction ( acidic airway surface liquid (ASL) pH, low bicarbonate (HCO ) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown.

Treatment of cystic fibrosis airway cells with CFTR modulators reverses aberrant mucus properties  hydration.

The goal of this project was to validate the functional relevance and utility of mucus produced by an in vitro intestinal cell culture model. This is facilitated by the need to physiologically replicate both healthy and abnormal mucus conditions from native intestinal tissue, where mucus properties have been connected to intestinal disease models. Mucus harvested from colonic cell cultures derived from healthy donors was compared to mucus collected from surgically resected, noninflamed transverse colon tissue. The rheological and biochemical properties of these mucus samples were compared using oscillational rheometry, particle-tracking microrheology, multiangle laser light scattering, refractometry, and immunohistochemical imaging. An air-liquid interface culture of primary human colonic epithelial cells generated a continuous monolayer with an attached mucus layer that displayed increasing weight percent (wt%) of solids over 1 week (1.3 ± 0.5% at 2 days vs. 2.4 ± 0.3% at 7 days). The full range of mucus concentrations (0.9-3.3%) observed during culture was comparable to that displayed by ex vivo mucus (1.3-1.9%). Bulk rheological measurements displayed similar wt%-based complex viscosities between in vitro and ex vivo mucus, with the complex viscosity of both systems increasing with wt% of solids. Particle-tracking microrheology showed higher complex viscosities for ex vivo mucus samples than in vitro mucus which was explained by a greater fraction of water present in in vitro mucus than ex vivo, i.e., in vitro mucus is more heterogeneous than ex vivo. Refractometry, multiangle laser light scattering, and immunostaining showed increased mucus complex size in ex vivo mucus compared with in vitro mucus, which may have been due to the admixture of mucus and cellular debris during ex vivo mucus collection. The air-liquid interface culture system produced intestinal mucus with similar composition and rheology to native human gut mucus, providing a platform to analyze pathological differences in intestinal mucus.

Biochemical and rheological analysis of human colonic culture mucus reveals similarity to gut mucus.

Significance Gaining insights into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) high transmissibility and the role played by inflammatory mediators in viral proliferation are critical to investigating new therapeutic targets against COVID-19. Electron microscopy reveals important SARS-CoV-2 features, including the combination of large, rapidly released viral clusters and the massive shedding of epithelial cells packed with virions. Interleukin-13 (IL-13), a Th2 cytokine up-regulated in allergic asthma and associated with less severe COVID-19, protects against SARS-CoV-2 viral and cell shedding. Using gene expression analyses and biochemical assays, IL-13 is shown to affect viral entry, replication, and cell-to-cell transmission. Given the broad spectrum of COVID-19 clinical symptoms, it is important to elucidate intrinsic factors that modulate viral load and spreading mechanisms.

Camille Ehre

Marsico Lung Institute/CF Center,
Department of Pediatrics

University of North Carolina at Chapel Hill

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

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

Camille Ehre

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Marsico Lung Institute/CF Center,Department of Pediatrics

University of North Carolina at Chapel Hill

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

Rapid Viscoelastic Characterization of Airway Mucus Using a Benchtop Rheometer

Marsico Lung Institute/CF Center,
Department of Pediatrics