John K. Sheehan

A Periciliary Brush Promotes the Lung Health by Separating the Mucus Layer from Airway Epithelia

Sticky Mucus? Mucus—experienced, for example, in the form of a runny nose or productive cough—is one of the tools the body uses to expel or prevent the uptake of foreign matter. In a number of diseases, a failure of the normal mucus-control system leads to obstructions of the airways and respiratory problems. Button et al. (p. 937; see the Perspective by Dickey) examine the existing gel-on-liquid model, where the mucus is thought to sit on a watery periciliary layer around the beating lung cilia that has been used to explain the flow of mucus. A gel-on-brush model is proposed where the mucus sits on a brushlike periciliary layer. The key elements of this layer are membrane-tethered macromolecules that cause normal flow and clearance of mucus. When dehydrated, the interface is disrupted, preventing normal mucus motion. The lung is protected by a brushlike biopolymer that contributes to mucus flow and can trigger muco-obstructive diseases. Mucus clearance is the primary defense mechanism that protects airways from inhaled infectious and toxic agents. In the current gel-on-liquid mucus clearance model, a mucus gel is propelled on top of a “watery” periciliary layer surrounding the cilia. However, this model fails to explain the formation of a distinct mucus layer in health or why mucus clearance fails in disease. We propose a gel-on-brush model in which the periciliary layer is occupied by membrane-spanning mucins and mucopolysaccharides densely tethered to the airway surface. This brush prevents mucus penetration into the periciliary space and causes mucus to form a distinct layer. The relative osmotic moduli of the mucus and periciliary brush layers explain both the stability of mucus clearance in health and its failure in airway disease.

Heterogeneity of airways mucus: variations in the amounts and glycoforms of the major oligomeric mucins MUC5AC and MUC5B.

Respiratory mucus contains a mixture of gel-forming mucins but the functional significance of these different mucin species is unknown. To help gain a better understanding of mucus in airways we therefore need to ascertain the concentration of each of the gel-forming mucins within respiratory secretions. Thus the aim of this study was to determine the amounts of specific gel-forming mucins directly from solubilized secretions of the airways and purified mucin preparations. We investigated the feasibility of using direct-binding ELISA employing mucin-specific antisera but were unable to obtain reliable data owing to interference with the immobilization of the mucins on the assay surface by 6 M urea and high levels of non-mucin proteins. We therefore developed an alternative approach based on quantitative Western blotting after agarose-gel electrophoresis, which was not subject to these problems. Here we demonstrate that this procedure provides reliable and reproducible data and have employed it to determine the amounts of the MUC2, MUC5AC and MUC5B mucins in saline-induced sputa from healthy airways and spontaneous sputa from asthmatic airways. Additionally we have used this procedure to analyse these glycoproteins in mucin preparations purified from cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) mucus. Our findings indicate that MUC5AC and MUC5B are the major oligomeric mucins and that airways mucus contains variable amounts of these glycoproteins. By contrast, the MUC2 mucin comprised, at most, only 2.5% of the weight of the gel-forming mucins, indicating that MUC2 is a minor component in sputum. Finally, we show that the amounts and glycosylated variants of the MUC5AC and MUC5B mucins can be altered significantly in diseased airways with, for instance, an increase in the low-charge form of the MUC5B mucin in CF and COPD mucus.

Hyaluronan: polysaccharide chaos to protein organisation

Molecular dynamics simulations of hyaluronan have revealed the inherent flexibility of this glycosaminoglycan in solution. Crystal structures of hyaluronan-digesting enzymes have provided the first direct insights into the molecular basis of hyaluronan-protein interactions. Various studies on hyaluronan-binding proteins suggest there is considerable diversity in their mode of interaction with hyaluronan, which might result in many different bound conformations of the polysaccharide.

Reduced Three-Dimensional Motility in Dehydrated Airway Mucus Prevents Neutrophil Capture and Killing Bacteria on Airway Epithelial Surfaces

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus (“normal” model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated (“thick”) mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.

Tracheobronchial air-liquid interface cell culture: A model for innate mucosal defense of the upper airways?

Human tracheobronchial epithelial cells grown in air-liquid interface culture have emerged as a powerful tool for the study of airway biology. In this study, we have investigated whether this culture system produces “mucus” with a protein composition similar to that of in vivo, induced airway secretions. Previous compositional studies of mucous secretions have greatly underrepresented the contribution of mucins, which are major structural components of normal mucus. To overcome this limitation, we have used a mass spectrometry-based approach centered on prior separation of the mucins from the majority of the other proteins. Using this approach, we have compared the protein composition of apical secretions (AS) from well-differentiated primary human tracheobronchial cells grown at air-liquid interface and human tracheobronchial normal induced sputum (IS). A total of 186 proteins were identified, 134 from AS and 136 from IS; 84 proteins were common to both secretions, with host defense proteins being predominant. The epithelial mucins MUC1, MUC4, and MUC16 and the gel-forming mucins MUC5B and MUC5AC were identified in both secretions. Refractometry showed that the gel-forming mucins were the major contributors by mass to both secretions. When the composition of the IS was corrected for proteins that were most likely derived from saliva, serum, and migratory cells, there was considerable similarity between the two secretions, in particular, in the category of host defense proteins, which includes the mucins. This shows that the primary cell culture system is an important model for study of aspects of innate defense of the upper airways related specifically to mucus consisting solely of airway cell products.

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.

Molecular organization of the mucins and glycocalyx underlying mucus transport over mucosal surfaces of the airways.

Mucus, with its burden of inspired particulates and pathogens, is cleared from mucosal surfaces of the airways by cilia beating within the periciliary layer (PCL). The PCL is held to be “watery” and free of mucus by thixotropic-like forces arising from beating cilia. With radii of gyration ∼250 nm, however, polymeric mucins should reptate readily into the PCL, so we assessed the glycocalyx for barrier functions. The PCL stained negative for MUC5AC and MUC5B, but it was positive for keratan sulfate (KS), a glycosaminoglycan commonly associated with glycoconjugates. Shotgun proteomics showed KS-rich fractions from mucus containing abundant tethered mucins, MUC1, MUC4, and MUC16, but no proteoglycans. Immuno-histology by light and electron microscopy localized MUC1 to microvilli, MUC4 and MUC20 to cilia, and MUC16 to goblet cells. Electron and atomic force microscopy revealed molecular lengths of 190–1,500 nm for tethered mucins, and a finely textured glycocalyx matrix filling interciliary spaces. Adenoviral particles were excluded from glycocalyx of the microvilli, whereas the smaller adenoassociated virus penetrated, but were trapped within. Hence, tethered mucins organized as a space-filling glycocalyx function as a selective barrier for the PCL, broadening their role in innate lung defense and offering new molecular targets for conventional and gene therapies.

Identification in Vitreous and Molecular Cloning of Opticin, a Novel Member of the Family of Leucine-rich Repeat Proteins of the Extracellular Matrix

A prominent 45-kDa component was identified by protein staining following SDS-polyacrylamide gel electrophoresis of a 4 m guanidine hydrochloride extract from bovine vitreous collagen fibrils. Peptide sequences obtained from this component were used as a basis for the cloning (from human retinal cDNA) and sequencing of a novel member of the leucine-rich repeat extracellular matrix protein family that we have named opticin. Opticin mRNA was found by reverse transcription polymerase chain reaction in ligament and skin as well as in retina. An open reading frame containing 332 amino acids was identified, the first 19 amino acids representing a signal peptide. The deduced amino acid sequence of the mature protein encodes a 35-kDa protein with a calculated isoelectric point of 5.4. The central domain of this protein consists of six B-type leucine-rich repeats. This domain is flanked by cysteine clusters including a C-terminal two-cysteine cluster containing an additional leucine-rich repeat. The N-terminal region contains a cluster of potential O-glycosylation sites, and analysis of bovine vitreous opticin demonstrated the presence of sialylatedO-linked oligosaccharides substituting the core protein. Opticin shows highest protein sequence identity to epiphycan (42%) and osteoglycin (35%) and belongs to Class III of the leucine-rich repeat extracellular matrix protein family.

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.

Crystal Structure of the Biglycan Dimer and Evidence That Dimerization Is Essential for Folding and Stability of Class I Small Leucine-rich Repeat Proteoglycans

Biglycan and decorin are two closely related proteoglycans whose protein cores contain leucine-rich repeats flanked by disulfides. We have previously shown that decorin is dimeric both in solution and in crystal structures. In this study we determined whether biglycan dimerizes and investigated the role of dimerization in the folding and stability of these proteoglycans. We used light scattering to show that biglycan is dimeric in solution and solved the crystal structure of the glycoprotein core of biglycan at 3.40-Å resolution. This structure reveals that biglycan dimerizes in the same way as decorin, i.e. by apposition of the concave inner surfaces of the leucine-rich repeat domains. We demonstrate that low concentrations of guanidinium chloride denature biglycan and decorin but that the denaturation is completely reversible following removal of the guanidinium chloride, as assessed by circular dichroism spectroscopy. Furthermore, the rate of refolding is dependent on protein concentration, demonstrating that it is not a unimolecular process. Upon heating, decorin shows a single structural transition at a Tm of 45-46 °C but refolds completely upon cooling to 25 °C. This property of decorin enabled us to show both by calorimetry and light scattering that dimer to monomer transition coincided with unfolding and monomer to dimer transition coincided with refolding; thus these processes are inextricably linked. We further conclude that folded monomeric biglycan or decorin cannot exist in solution. This implies novel interrelated functions for the parallel β sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.