Mapping the protein domain structures of the respiratory mucins: a mucin proteome coverage study.

Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides that greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion, and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel-forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from 10 different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or "spontaneous cleavages". Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20, which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.

Mucociliary interactions and mucus dynamics in ciliated human bronchial epithelial cell cultures.

The airway epithelial surface liquid is generally considered to be composed of two layers, a periciliary layer and a continuous thick mucus layer moving in bulk. This view may not be appropriate for all areas of the lung. Our hypothesis, that mucus may form a discontinuous layer with dynamic attachments to the surface, is investigated using a culture system. We used live-cell confocal microscopy to investigate thin mucus layers and fluorescent beads and exogenous MUC5B to visualize mucus dynamics on ciliated human bronchial cultures. A continuous mucus layer was not observed. In sparsely ciliated cultures, mucus attached to ciliated cells; however, in highly ciliated cultures, mucus formed strands several hundred micrometers long. As with increases in ciliation, increases in bead concentration caused the appearance of mucus strands. We confirmed the involvement of mucins in the binding of mucus to cilia by adding labeled purified MUC5B to the cultures. These data suggest that mucins may have an intrinsic ability to form attachments to cilia. The significance of these findings is that aberrant modulation of such an intrinsic property may explain the initiation of highly adherent mucus in cystic fibrosis lung disease.

Airway and lung pathology due to mucosal surface dehydration in {beta}-epithelial Na+ channel-overexpressing mice: role of TNF-{alpha} and IL-4R{alpha} signaling, influence of neonatal development, and limited efficacy of glucocorticoid treatment.

Overexpression of the epithelial Na(+) channel beta subunit (Scnn1b gene, betaENaC protein) in transgenic (Tg) mouse airways dehydrates mucosal surfaces, producing mucus obstruction, inflammation, and neonatal mortality. Airway inflammation includes macrophage activation, neutrophil and eosinophil recruitment, and elevated KC, TNF-alpha, and chitinase levels. These changes recapitulate aspects of complex human obstructive airway diseases, but their molecular mechanisms are poorly understood. We used genetic and pharmacologic approaches to identify pathways relevant to the development of Scnn1b-Tg mouse lung pathology. Genetic deletion of TNF-alpha or its receptor, TNFR1, had no measurable effect on the phenotype. Deletion of IL-4Ralpha abolished transient mucous secretory cell (MuSC) abundance and eosinophilia normally observed in neonatal wild-type mice. Similarly, IL-4Ralpha deficiency decreased MuSC and eosinophils in neonatal Scnn1b-Tg mice, which correlated with improved neonatal survival. However, chronic lung pathology in adult Scnn1b-Tg mice was not affected by IL-4Ralpha status. Prednisolone treatment ablated eosinophilia and MuSC in adult Scnn1b-Tg mice, but did not decrease mucus plugging or neutrophilia. These studies demonstrate that: 1) normal neonatal mouse airway development entails an IL-4Ralpha-dependent, transient abundance of MuSC and eosinophils; 2) absence of IL-4Ralpha improved neonatal survival of Scnn1b-Tg mice, likely reflecting decreased formation of asphyxiating mucus plugs; and 3) in Scnn1b-Tg mice, neutrophilia, mucus obstruction, and airspace enlargement are IL-4Ralpha- and TNF-alpha-independent, and only MuSC and eosinophilia are sensitive to glucocorticoids. Thus, manipulation of multiple pathways will likely be required to treat the complex pathogenesis caused by airway surface dehydration.

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.

Characterization of exosome-like vesicles released from human tracheobronchial ciliated epithelium: a possible role in innate defense.

Airway mucus forms the structural basis of the local innate immune defense mechanism. It is an integrated, active, viscoelastic gel matrix evolved to protect the exposed lung from physical, chemical, and pathological erosion. Exosomes are biologically active vesicles secreted by different cell types including epithelial, hematopoietic, and some tumor cells. They are also present in some biological fluids such as serum, urine, breast milk, and bronchoalveolar lavage fluid. In this study, we demonstrate for the first time that exosome-like vesicles with antiviral properties are present in human tracheobronchial epithelial (HTBE) cell culture secretions. These vesicles have been isolated by differential centrifugation and are characterized further by mass spectrometry, flow cytometry, immunoblotting, electron microscopy, and light-scattering methods. HTBE vesicles exhibited characteristic exosomal size (30-100 nm) and morphology (cup-shaped) with a buoyant density in sucrose of 1.12-1.18 g/ml. Biochemical characterization further revealed typical surface, cytoskeletal, and cytoplasmic proteins characteristic of exosomes, including the multivesicular and late endosomal membrane markers Tsg101 and CD63. The presence of RNA was also observed. The epithelial mucins MUC1, MUC4, and MUC16 also contributed to the vesicles' structure. Notably, alpha-2,6-linked sialic acid was associated with these mucin molecules and subsequent functional analysis showed that these vesicles have a neutralizing effect on human influenza virus, which is known to bind sialic acid. Taken together, these findings suggest that airway epithelial cells release exosome-like vesicles and that these structures may be involved in diverse physiological processes in airway biology, including innate mucosal defense.

Identification of salivary mucin MUC7 binding proteins from Streptococcus gordonii.

BACKGROUND: The salivary mucin MUC7 (previously known as MG2) can adhere to various strains of streptococci that are primary colonizers and predominant microorganisms of the oral cavity. Although there is a growing interest in interaction between oral pathogens and salivary mucins, studies reporting the specific binding sites on the bacteria are rather limited. Identification and characterization of the specific interacting proteins on the bacterial cell surface, termed adhesins, are crucial to further understand host-pathogen interactions. RESULTS: We demonstrate here, using purified MUC7 to overlay blots of SDS-extracts of Streptococcus gordonii cell surface proteins, 4 MUC7-binding bands, with apparent molecular masses of 62, 78, 84 and 133 kDa from the Streptococcus gordonii strain, PK488. Putative adhesins were identified by in-gel digestion and subsequent nanoLC-tandem mass spectrometry analysis of resultant peptides. The 62 kDa and 84 kDa bands were identified as elongation factor (EF) Tu and EF-G respectively. The 78 kDa band was a hppA gene product; the 74 kDa oligopeptide-binding lipoprotein. The 133 kDa band contained two proteins; alpha enolase and DNA-directed RNA polymerase, beta' subunit. Some of these proteins, for example alpha enolase are expected to be intracellular, however, flow cytometric analysis confirmed its location on the bacterial surface. CONCLUSION: Our data demonstrated that S. gordonii expressed a number of putative MUC7 recognizing proteins and these contribute to MUC7 mucin binding of this streptococcal strain.

Munc13-2-/- baseline secretion defect reveals source of oligomeric mucins in 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.

Analyzing the functions of large glycoconjugates through the dissipative properties of their absorbed layers using the gel-forming mucin MUC5B as an example.

Glyconjugates such as mucins, proteoglycans, and polysaccharides form the structural basis of protective cell-surface layers. In particular gel-forming mucins define a zone between the epithelial cell layer and the environment. Such molecules are of extreme molecular weight 5-100 x 10(6) and size (Rg 20-300 nm). On this account their biochemistry is inseparable from their physical biochemistry. Combining laser light scattering and quartz crystal mass balance with dissipation methods (QCM-D) we have investigated the properties of the MUC5B mucin and its cognate fragments when bound to a hydrophobic surface. MUC5B forms the basis of gels responsible for the protection of the oral cavity, lung, and cervical canal surfaces. Here we show, by analyzing dissipative interactions of hydrophobic, gold, and polystyrene surfaces, with the intact MUC5B molecule, its reduced subunits, and glycosylated tryptic fragments (obtained after reduction), the formation of 40- to 100-nm-thick highly structured, hydrated interfaces. These interfaces are dominated in their geometry and dissipative properties by the negatively charged carbohydrate-rich domains of the molecule, the naked protein domains being responsible for attachment. These carbohydrate-rich surfaces have well-defined absorptive properties and permit the entry and entrapment of albumin-coated micro-beads into the absorbed layer at and below a size of 60 nm. However beads larger than 100 nm are completely excluded from the surfaces. These absorptive phenomena correlate with large changes in film dissipation and thus may not only be important in biological functions, e.g. binding viruses, but could also be informative to the surfaces (often ciliated) onto which such mucus films are attached.

Innate immunity and mucus structure and function.

Many of the proteins associated with innate immunity in the upper respiratory tract are to be found localized into mucus gels and the mucin-rich surface layers of the epithelium and the cilia. Mucus is a relatively dilute suspension of such macromolecules being around 2-4% solids in normal induced sputum. These proteins scavenge, immobilise and/or kill pathogens and at the same time immobilize them into the mucus. Mucus is moved from the lung by the mucociliary clearance mechanisms or by cough. Some 190 proteins are readily detectable in sputum by proteomics methods and about 100 in bronchial air-liquid interface culture secretions. This cell culture system mimics the surface ciliated phenotype of the large airways very well and about 85 secreted proteins are common to both culture and sputum secretions. The major single protein by weight in cell culture secretions is MUC5B and in sputum a mixture of MUC5B and MUC5AC. The three epithelial mucins MUC1, 4 and 16 are also detectable in both secretions. In this paper the roles that these molecules play in protecting and stabilising the ciliated surface and building the gel will be discussed. The role of water and ion homeostasis is particularly crucial in mucus gel formation and evidence is gathering that it is perturbation of hydration mechanisms that may play into defective mucus leading subsequently to stasis and mechanical problems.

Interaction of mucus with freshly neutralised aluminium in freshwater.

This study examined the interaction of mollusc trail mucus, and its biochemical constituents, with environmentally relevant concentrations of freshly neutralised aluminium (Al) in freshwater. Upon neutralisation Al starts to polymerise. In the presence of mucus the metal is rapidly localised into the hydrated mucus gel resulting in a likely reduction of its overall degree of polymerisation. A simple Al binding assay identified large-M(r) glycoconjugates as major Al-complexing molecules in mucus. Subsequent isolation and purification of these mucus glycoconjugates showed the metal readily bound to the carbohydrate portion and, in particular, to acidic components such as those containing carboxyl functionality. It is suggested gel-forming extracellular glycoconjugates play a crucial role in preventing the diffusion of Al into biological systems and thus serve to maintain metabolic homeostasis.

Partial characterisation of high-molecular weight glycoconjugates in the trail mucus of the freshwater pond snail Lymnaea stagnalis.

We have studied the glycoconjugates in trail mucus of the pond snail Lymnaea stagnalis. The mucus was dissolved with 6 M guanidinium hydrochloride (GuHCl) and the major component was comprised of very high-M(r) glycoconjugates that were eluted in the void volume of a Sepharose CL-4B gel-filtration column. This high-M(r) material was pooled and thereafter subjected to density gradient centrifugation first in 4 M GuHCl/CsCl and subsequently 0.2 M GuHCl/CsCl to further remove non-glycosylated proteins and DNA. The harvested glycoconjugate pool chromatographed in the void volume of Sepharose CL-2B. However, reduction of disulfide bonds lowered the molecular size of approximately 80% of the void material yielding a major fragment and some minor smaller fragments in gel chromatography. The reduced glycoconjugates were digested with papain and yielded high molecular weight, proteinase-resistant glycopeptides. This fragmentation pattern is similar to that found for oligomeric gel-forming mucins in mammals and the amino acid composition (60% Ser/Thr) and sugar analysis of the glycopeptides is consistent with mucin-like molecules, there being no significant amounts of xylose or uronic acids. The residual 20% of the preparation, which apparently resisted reduction and protease digestion, had a similar amino acid composition to the bulk, but was somewhat different in sugar composition, containing some xylose and a significant amount of glucuronic acid. The two groups of molecules had very different morphologies in the electron microscope. Taken together, these data suggest that trail mucus is a complex mixture of at least two families of protein-glycoconjugate molecules based upon the gel-forming mucin and proteoglycan families, though we cannot rule out that polysaccharides may also be present.

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.

Mass spectrometric analysis of mucin core proteins.

Mucins are difficult to handle for their identification and characterization via proteomic applications due to their heavily glycosylated nature (up to 90%), high molecular weight (200 kDa-200 MDa), and size (Rg 10-300 nm). Their core proteins are extremely large and highly substituted with oligosaccharides, which only allow access to a highly restricted portion of their protein. For this reason, conventional 1D or 2D polyacrylamide gel-based proteomic approaches are not effective for identification and characterization of mucin molecules. In this chapter, we present our current protocol employing a modified shotgun proteomic approach to identify these complex glycoproteins.

Studying mucin secretion from human bronchial epithelial cell primary cultures.

Mucin secretion is regulated by extracellular signaling molecules emanating from local, neuronal, or endocrine sources. Quantifying the rate of this secretion is important to understanding how the exocytic process is regulated, and also how goblet/mucous cells synthesize and release mucins under control and pathological conditions. Consequently, measuring mucins in a quantitatively accurate manner is the key to many experiments addressing these issues. This paper describes procedures used to determine agonist-induced mucin secretion from goblet cells in human bronchial epithelial (HBE) cell cultures. It begins with primary epithelial cell culture, offers methods for purifying MUC5AC and MUC5B mucins for standards, and describes five different microtiter plate binding assays which use various probes for mucins. A polymeric mucin-specific antibody is used in standard and sandwich ELISA formats for two assays while the others target the extensive glycosylated domains of mucins with lectin, periodate oxidation, and antibody-based probes. Comparing the data derived from the different assays applied to the same set of samples of HBE cell cultures indicates a qualitative agreement between baseline and agonist stimulated mucin release; however, the polymeric mucin-specific assays yield substantially lower values than the assays using non-specific molecular reporters. These results indicate that the more nonspecific assays are suitable to assess overall secretory responses by goblet cells, but are likely unsuited for specific measurements of polymeric mucins, per se.

The NAD(+)-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status.

SIRT1, a NAD(+)-dependent protein deacetylase, is an important regulator in cellular stress response and energy metabolism. While the list of SIRT1 substrates is growing, how the activity of SIRT1 is regulated remains unclear. We have previously reported that SIRT1 is activated by phosphorylation at a conserved Thr522 residue in response to environmental stress. Here we demonstrate that phosphorylation of Thr522 activates SIRT1 through modulation of its oligomeric status. We provide evidence that nonphosphorylated SIRT1 protein is aggregation-prone in vitro and in cultured cells. Conversely, phosphorylated SIRT1 protein is largely in the monomeric state and more active. Our findings reveal a novel mechanism for environmental regulation of SIRT1 activity, which may have important implications in understanding the molecular mechanism of stress response, cell survival, and aging.

A periciliary brush promotes the lung health by separating the mucus layer from airway epithelia.

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.

Investigating the molecular basis for the virulence of Escherichia coli K5 by nuclear magnetic resonance analysis of the capsule polysaccharide.

The capsular polysaccharide of Escherichia coli K5 has been hypothesised to promote virulence through its molecular mimicry of host heparan sulphate. To test this hypothesis, we have produced pure oligosaccharides from K5 capsular polysaccharide and investigated their conformational properties with ultra-high-field nuclear magnetic resonance (NMR) (900 MHz). Ultra-high-field affords a significant resolution enhancement over previous studies and allowed a full-atomic assignment of the K5 hexasaccharide for the first time. All carbohydrate rings adopt a (4)C(1) conformation, the amide sidechains have a trans orientation and the hydroxymethyl group is freely exposed to bulk solvent. Initial models of the glycosidic linkage conformation based upon simple interpretation of NOE cross-peaks suggests that the beta1-->4 linkage adopts a 3D geometry of phi approximately 60 degrees , psi approximately 0 degrees and the alpha1-->4 linkage prefers phi approximately -30 degrees , psi approximately -30 degrees (phi and psi being defined by dihedral angles involving linkage protons). In this conformation the overall molecular geometries of K5 polysaccharide, heparan sulphate and even fully-sulphated heparin are remarkably similar. These results substantiate the hypothesis that the K5 capsular polysaccharide confers virulence to E. coli K5 by being a 3D molecular mimetic of host heparan sulphate, helping it to evade detection by the mammalian immune system.

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.

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-angstroms 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 T(m) of 45-46 degrees C but refolds completely upon cooling to 25 degrees 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 beta sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.

The cystic fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.