Effect of Ca++ on the structure and rheology of canine tracheal mucin

Mucin glycoproteins are known to be the principal determinants of epithelial mucus rheology and hence of mucociliary transport rates. We are studying the structure of such glycoproteins using a model mucin purified from canine tracheal pouch secretions. Of particular interest is the effect on mucin structure of increased Ca++ such as occurs in certain disease states. Quasielastic laser light scattering was used to study the effect of Ca++ on the hydrodynamic radius of the mucin molecules. Scattering data from 0.3mg/ml mucin solutions in physiological phosphate buffer containing 0, 5 X 10(-5)M, and 5 X 10(-4)M Ca++ were analyzed to obtain an average translational diffusion coefficient and the distribution of molecular radii for the dispersion. The effect of Ca++ was to decrease the average Stokes radius. The light scattering results are supported by rheologic measures of mucin gel viscoelasticity.     

Hydrophobic interaction of fluorescent probes with fetuin, ovine submaxillary mucin, and canine tracheal mucins.

The presence of hydrophobic sites in fetuin, ovine submaxillary mucin and two homogeneous canine tracheal mucins was established by fluorescence probe techniques. The interaction between the above-mentioned glycoproteins and two hydrophobic fluorescent compounds, sodium mansate and mansylphenylalanine, was accompanied by an enhancement in fluorescence and a shift of the fluorescence maxima to shorter wavelengths. The introduction of a phenylalanine residue to the mansyl group enhanced the binding affinity of the probe for the hydrophobic sites of these glycoproteins as evidenced by lower values for the dissociation constants. The high molecular weight (581 600) tracheal mucin, which had the highest carbohydrate content (80%) of all the glycoproteins investigated, exhibited the highest fluorescence enhancement and the largest number of binding sites for these fluorescent probes.     

Deglycosylation studies on tracheal mucin glycoproteins

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.     

Colorimetric determination of N-acetylhexosamine-terminating O-glycosidically linked saccharides in mucins and glycoproteins

A sensitive colorimetric assay for detecting mucins and glycoproteins rich in O-glycosidically linked saccharides is reported. The method combines the susceptibility of N-acetylgalactosamine terminating O-glycosidically linked saccharides to beta-elimination with the Morgan-Elson reaction for N-acetylhexosamines with free reducing ends. All mucin and mucin-type glycoproteins but none of the serum-type glycoproteins tested resulted in characteristic color production. All mucins tested gave linear responses in the range 5 to 200 micrograms and the assay was also adapted to the microscale involving the use of 96-well microtiter plates. The microassay in which the volumes of samples and reagents are scaled down 2.5-fold was particularly useful in monitoring of mucins, in the presence of other glycoconjugates, in large numbers of samples obtained during fractionation procedures. Cesium chloride, cesium bromide, potassium thiocyanate, and various detergents do not interfere with the colorimetric determination. Guanidine hydrochloride, cesium trifluoroacetate, and beta-mercaptoethanol decreased color by 30 to 45%; however, the interference was not serious to prevent the use of the method for detection of mucins in their presence. The use of the method for the specific detection of mucin during fractionation by gel filtration and density gradient centrifugation of cystic fibrosis sputum samples is demonstrated.     

Membrane differentiation markers of airway epithelial secretory cells

We describe here a system for culturing epithelial cells isolated from hamster trachea, which results in a highly enriched population of mucus-secreting cells. The culture system has enabled us to study the process of secretory cell differentiation in vitro. We found that epithelial secretory cells, in vivo and after 5 days in vitro, selectively bind the lectin Helix pomatia agglutinin (HPA) to apical and, to a lesser extent, basolateral surfaces as well as to mucin granules and intracellular secretory organelles. SDS-PAGE gels of detergent extracts of secretory cells cultured for 5 days reveal three HPA-binding glycoproteins with MW of 120 KD, 220 KD, and greater than 400 KD. The high-MW glycoprotein appears identical to mucin, since it is found in secretions from intact trachea and in spent media from 5-day cultures. It does not appear in spent media from 3-day cultures when cells contain few mucous granules and secrete little mucin. The 220 KD HPA-binding glycoprotein is also present in 5-day but not in 3-day cultures. In contrast, the 120 KD glycoprotein is present at both times. HPA-gp120 is a hydrophobic integral membrane protein, whereas HPA-gp220 and mucin are hydrophilic and are membrane associated. These studies define three membrane glycoproteins, one of which is specific for the tracheal epithelial secretory cell regardless of its mucous content, whereas the other two glycoproteins correlate with mucin secretion. They also demonstrate that, in the fully differentiated state, mucin is bound in a non-covalent fashion to the apical plasma membrane of the tracheal epithelial secretory cell.     

Synthesis of mucin glycoproteins by epithelial cells isolated from swine trachea by specific proteolysis

Mucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease from Bacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation , and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [35S]O4 and [I-14C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium. The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration of Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by beta-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 columns. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions.     

Quantitation of mucus glycoproteins blotted onto nitrocellulose membranes

A sensitive assay for mucus glycoproteins (mucins) and fragments thereof is presented. The macromolecules are blotted onto nitrocellulose membranes and visualized using a periodate-Schiff (PAS) reaction and the color yield quantitated with an image analysis system used as a reflectance densitometer. At least 50 ng of the macromolecules was detected. "Whole" mucins and subunits were assayed on 0.2-micron pore size nitrocellulose membranes whereas immobilization of the high-molecular-weight mucin glycopeptides (Mr 300-500,000) required pretreatment of membranes with poly-L-lysine. Binding of the glycopeptides to the polylysine-treated membranes was found to decrease with increasing salt concentration suggesting an electrostatic interaction. The data obtained with this method and a solution PAS assay are in good agreement but the former is more sensitive and can be performed on samples dissolved in chaotropic solvents.     

Isolation and characterization of human and canine gastric mucosal glycoproteins and their degradation by proteases and acid hydrolases

High molecular weight glycoproteins were isolated and purified from canine antral and fundic mucosal tissue by means of non-degrading techniques. The results disclosed the advantage of urea extraction technique over the culture method in isolating the native glycoproteins. The glycoproteins were susceptible to degradation by protease, thus yielding low molecular weight glycopeptides. Chemical analysis of these glycopeptides and their parent macromolecules revealed that the oligosaccharide residues are attached to threonine, serine and proline residues of the protein chains. Similarly, high molecular weight glycoproteins isolated from human gastric gel mucin showed the same characteristics of canine gastric glycoproteins. Canine fundic glycoprotein or glycopeptide released their prosthetic carbohydrate groups under the lytic effect of fundic acid hydrolases.     

Inhibition and promotion of cholesterol crystallization by protein fractions from normal human gallbladder bile

Pooled, normal human gallbladder biles were initially separated on a molecular sieving chromatography column to remove soluble mucin glycoproteins as well as high molecular weight proteins (greater than 200,000). The remaining lower molecular weight proteins and other bile components were then examined by lectin affinity chromatography with four different types of lectin. The separated bound fractions were compared for inhibiting and promoting activities with a newly devised sensitive cholesterol crystal growth assay and for differences in electrophoretic patterns on SDS-gels. Protein factors (presumably glycoproteins) were found to have both inhibiting and promoting activities, even in the absence of cholesterol gallstone disease. The promoting effect was indicated by shortened crystal detection times and increases in crystal growth rate; whereas the inhibiting effect was indicated by decreases in crystal growth rate and reductions in the final crystal concentration as determined by the growth assay. Affinity chromatography mitigated the major problems of removing both lipids and pigment from the glycoproteins. In addition, partial purification of bound fractions with potent cholesterol crystal nucleation-altering activity can be obtained by this technique.     

Synthesis of mucin glycoproteins by epithelial cells isolated from swine trachea by specific proteolysis

Summary Mucus-producing cells were isolated from swine trachea mucosa by a method that included enzymatic digestion of the epithelial surface with Dispase, a neutral protease fromBacillus polymyxa, and differential attachment of the washed cells to culture flasks coated with collagen. Epithelial cells were the major cell type isolated by these procedures. Ciliated cells that did not attach to the flasks were removed by decantation, and fibroblasts were destroyed by the bacterial protease. The isolated cells synthesized respiratory mucins and the rate of secretion was increased about threefold when tracheas were exposed to sulfur dioxide. The cultured cells incorporated both [³⁵S]O₄ and [I-¹⁴C]N-acetylglucosamine into secreted mucin glycoproteins. The secretion of glycoprotein increased for about 3 d until the cells became confluent, and then a constant rate was observed for a period of at least 7 d. This increase in the output of mucin glycoprotein during the initial 3 d of culture was accompanied by a corresponding increase in the number of mucus-producing cells in the flasks. The results obtained in these and subsequent studies suggest that the rate of formation of mucus-producing cells may be a rate limiting step in the regulation of mucin glycoprotein synthesis in tracheal epithelium. The chemical, physical, and immunological properties of the glycoprotein secreted by isolated tracheal epithelial cells were very similar to the mucin glycoprotein purified from washes of swine trachea epithelium. The purified mucin glycoproteins showed complete cross-reaction with antibodies to trachea mucin glycoprotein. They were eluted near the void volume during gel filtration on Sepharose CL-6B columns. The glycoprotein isolated from culture media under the standard assay conditions had nearly the same carbohydrate composition as samples purified from washes of trachea epithelium. Reduced oligosaccharides released by β-elimination with dilute alkaline borohydride showed similar elution profiles during chromatography on Bio Gel P-6 colums. Taken collectively, these results suggest that the isolated epithelial cells secreted mucin glycoproteins that were very similar to those synthesized by the intact trachea epithelium under standard incubation conditions. This investigation was supported by United States Public Health Service Grant HL 20868 from the National Heart, Lung and Blood Institute and AM 28187 from the National Institute of Arthritis, Diabetes and Digestive and Kidney Diseases.     

Molecular cloning of the carboxy terminus of a canine tracheobronchial mucin.

A cDNA library constructed from canine tracheal mRNA was screened with polyclonal antiserum specific to canine tracheal apomucin (CTM-A). Eight antibody reactive clones were isolated and purified to clonality. One of the clones, designated pCTM-A, had a 1.7 kb insert and included a single open reading frame with a poly (A)+ tail. The amino acid composition of the encoded protein was consistent with that expected for CTM-A. The fusion protein produced by cloning the 1.7 kb insert in the pMALc expression vector reacted with the purified anti-apomucin CTM-A antibody. Also, polyclonal antibodies raised to the purified protein product encoded by pCTM-A reacted with deglycosylated CTM-A confirming that this clone does indeed code for apomucin CTM-A. This is the first report of a cDNA encoding the C-terminus of a canine tracheal mucin.     

Biochemical characterization of the component parts of intestinal mucin from patients with cystic fibrosis.

Previous studies have shown that human small-intestinal mucin consists of high-Mr glycoproteins and a smaller S-S-bonded protein of 118 kDa. The major antigenic determinants of the mucin were associated with the large glycoproteins, but depended for stability on intact disulphide bonds, and were destroyed by digestion with Pronase. In the present study we isolated and analysed the component parts of mucin from patients with cystic fibrosis with special attention being paid to the peptide constituents. After reduction with 0.2 M-beta-mercaptoethanol [5 min, 100 degrees C in 1% SDS (sodium dodecyl sulphate)], the large glycoproteins and smaller peptide with an apparent molecular size of 118 kDa were separated by equilibrium density-gradient centrifugation in CsCl, Sepharose 4B chromatography or preparative SDS/polyacrylamide-gel electrophoresis. The large glycoproteins contained about 70% of the protein of the native mucin. Digestion with Pronase resulted in a further loss of 'naked' protein (10% of the native mucin protein) from the C-terminal end of the glycoprotein peptide core, and left behind highly glycosylated proteins comprised mainly (70 mol%) of threonine, serine and proline. The 118 kDa component, which contained about 30% of the native mucin protein, consisted mainly of aspartic acid, serine, glutamic acid and glycine (40 mol%), plus threonine, proline, alanine, valine and leucine (35 mol%). Together with the 'naked' protein segment, the 118 kDa component contained most of the cysteine residues of the native mucin. Surprisingly, the peptide also contained carbohydrate (less than or equal to 5% of the native mucin carbohydrate but 50% by weight of the 118 kDa component), which included 9 mol% mannose, suggesting the presence of N-linked oligosaccharides. The peptide exhibited strong non-covalent interactions with the high-Mr glycoproteins and a tendency to self-aggregate in the absence of dissociating agents. Our findings therefore suggest that native mucin consists of large glycoproteins capable of forming disulphide bridges from their C-terminal 'naked' (antigenic) regions to a smaller glycopeptide having an Mr of 118 000.     

A simple enzymatic method for the analysis of macromolecular fucose in biological materials

A sensitive enzymatic method employing l-fucose dehydrogenase has been used for the measurement of the amounts of fucose in 100–500 μg of plasma membrane protein and 10–100 μg of porcine submaxillary mucin. The assay showed linearity between 0 and 20 nmol of α-l-fucose when measuring NADH fluorescence. The fucose values obtained for the plasma membrane and submaxillary mucin correspond well with those obtained by gas-liquid chromatography.     

IL-17A promotes the growth of airway epithelial cells through ERK-dependent signaling pathway

The effects of IL-17A on mucin production and growth of airway epithelial cells were examined. Histological and immunohistochemical analyses revealed that IL-17A increased the mucin production and number of tracheal epithelial cells in air-liquid interface cultures. The biological property of IL-17A to stimulate the mucin production by tracheal epithelial cells was determined using an ELISA. The mitogenic effect of IL-17A on tracheal epithelial cells was confirmed with Calcein-AM assay. The growth-stimulatory effect of IL-17A was dose-dependent and mediated via the ERK MAP kinase pathway. Inhibitors of MEK abrogated the mitogenic effect of IL-17A, whereas an inhibitor of p38 or JNK displayed no significant inhibitory effect. Moreover, relatively lower doses of IL-13 also significantly increased the growth of tracheal epithelial cells through a distinct signaling pathway from that of IL-17A. These findings provide the first evidence that IL-17A stimulates the growth of airway epithelial cells through the ERK MAP kinase pathway.     

Characterization of mucin isolated from rat tracheal transplants

Subcutaneous rat tracheal grafts yield several milligrams of secretions from which a homogeneous mucin fraction was isolated and purified. Histological evidence demonstrated that a normal mucociliary epithelium and mucous secretion were maintained for the 4-6 weeks of the experiment. The collected secretions were initially characterized by column chromatography on Sepharose CL-6B which separated the excluded high molecular weight mucins (unpurified mucin fraction) from most of the serum-type glycoproteins and proteins, including albumin. A reductive alkylation treatment of the unpurified mucin fraction followed by Sepharose CL-4B chromatography removed contaminating protein and most of the mannose-containing material from the mucin fraction. The void volume material from this column produced a single high molecular weight band upon sodium dodecyl sulfate agarose/acrylamide gel electrophoresis. The purified mucin fraction contained 16.5% protein and primarily galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid. This fraction also underwent beta-elimination in the presence of alkaline borohydride, demonstrating the presence of O-glycosidic linkages.     

Heterogeneity of rat goblet-cell mucin before and after reduction.

Goblet-cell mucin of rat small intestine was purified from mucosal scrapings by using centrifugation, Sepharose 4B and Sepharose 2B chromatography. The mucin was applied in low concentrations (1 microgram/track) to slab gels containing 0.5% agarose/2% (w/v) polyacrylamide, and bands were detected after electrophoresis by silver stain or by fluorography of 3H-labelled mucin. Before reduction the mucin contained three distinct components: a polymeric species at the top of the gel and two large glycoproteins of higher mobility. After reduction, the polymer disappeared, the two glycoproteins remained unchanged, and two glycopeptide bands of higher mobility appeared. In addition, a non-glycosylated, heavily stained peptide of mol.wt. 118000 was detected. The individual mucin components were partially separated on Sepharose 2B, 0.2M-NaCl/1% sodium dodecyl sulphate being used as eluant. Individual amino acid and carbohydrate analyses suggested that the glycosylated components, despite their differences in size, had identical profiles. The 118000-mol.wt. peptide had a very different amino acid profile, with much less serine, threonine and proline. Glycine and aspartic and glutamic acids comprised 34% of the total amino acids. Thus the 'native' mucin is a heterogeneous structure containing at least two non-covalently associated glycoproteins plus polymeric material. The latter is stabilized by disulphide bonds and consists of several glycopeptides of different size as well as a 'link' peptide of mol.wt. 118000.     

No pathophysiologic relationship of soluble biliary proteins to cholesterol crystallization in human bile

This study explores the pathophysiologic effects of soluble biliary glycoproteins in comparison to mucin gel and cholesterol content on microscopic crystal and liquid crystal detection times as well as crystallization sequences in lithogenic human biles incubated at 37 degrees C. Gallbladder biles from 13 cholesterol gallstone patients were ultracentrifuged and microfiltered (samples I). Total biliary lipids were extracted from portions of samples I, and reconstituted with 0.15 m NaCl (pH 7.0) (samples II). Portions of samples II were supplemented with purified concanavalin A-binding biliary glycoproteins (final concentration = 1 mg/mL) (samples III), or mucin gel (samples IV), respectively, isolated from the same cholesterol gallstone biles. Samples V consisted of extracted biliary lipids from uncentrifuged and unfiltered bile samples reconstituted with 0.15 m NaCl (pH 7.0). Analytic lipid compositions of samples I through IV were identical for individual biles but, as anticipated, samples V displayed significantly higher cholesterol saturation indexes. Detection times of cholesterol crystals and liquid crystals were accelerated in the rank order of samples: IV > V > I = II = III, indicating that total soluble biliary glycoproteins in pathophysiologic concentration had no appreciable effect. Crystallization sequences (D. Q-H. Wang and M. C. Carey. J. Lipid Res. 1996. 37: 606-630; and 2539-2549) were similar among samples I through V. Crystal detection times and numbers of solid cholesterol crystals were accelerated in proportion to added mucin gel and the cholesterol saturation of bile only.For pathophysiologically relevant conditions, our results clarify that mucin gel and cholesterol content, but not soluble biliary glycoproteins, promote cholesterol crystallization in human gallbladder bile.