Composition of human airway mucins and effects after inhalation of acid aerosol.

Characterization of normal airway mucus is required to elucidate mechanisms protecting the airways and to understand changes associated with disease and environmental insult. Toward this goal, we collected bronchial washes (10 ml saline) from healthy human subjects to 1) evaluate the yield of high-density material (delta > or = 1.35 g/ml), and 2) characterize glycoconjugates associated with collected secretions. Samples were lipid extracted followed by CsCl density gradient centrifugation. The yield of high-density material from individual subjects was variable but sufficient to demonstrate that mucin glycoproteins are a major constituent of mucus from healthy airways and that proteoglycans are absent. Next, we investigated whether inhalation of H2SO4 aerosol (1,000 microgram/m3), an environmental insult associated with alterations in mucociliary clearance, changes the composition of high-density glycoproteins in airway secretions. In a paired, double-blinded study, high-density fractions of bronchial secretions from 12 subjects were collected 18 h after exposures of 2 h to aerosolized NaCl and H2SO4. In all cases the high-density material displayed characteristics of mucin glycoproteins. In addition, a unique 150-kDa glycoprotein was detected in most but not all samples and may represent a small mucin glycoprotein differentially expressed in humans. No differences were noted between the two exposure conditions in the profiles of the glycoproteins or proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Statistically, large changes with acid exposure in the composition of carbohydrates and amino acids were absent. Thus no substantial systematic changes in airway mucin glycoproteins or closely associated proteins and glycoproteins were correlated with H2SO4 exposure. Alternatively, statistical analysis of the differences between exposures in glycoprotein constituents among subjects denoted greater variability in carbohydrates compared with amino acids with repeated sampling, suggesting normal daily variations in the mucin composition of individual airway mucus.

Silver staining of extensively glycosylated proteins on sodium dodecyl sulfate-polyacrylamide gels: enhancement by carbohydrate-binding dyes.

Two methods are described for detecting less than 1 microgram of highly glycosylated proteins, such as mucins, on sodium dodecyl sulfate-polyacrylamide gels. They combine commonly employed periodic acid-Schiff (PAS) and Alcian blue dyes with silver stain. Carbohydrate prestaining renders mucins more cationic and favors greater complexation with ionic silver. Comparisons of different mucin samples stained either with PAS-silver or alcian blue-silver indicate differential staining between the two techniques. Such differences may, in part, be due to an affinity of Alcian blue for sulfated glycoproteins. These two staining protocols when used in conjunction with silver staining alone are particularly valuable for assessing sample purity and for detecting contaminating proteins during mucin purification protocols.

Characterization of link protein(s) from human intervertebral-disc tissues.

Proteoglycan aggregates (A1) were prepared from the anulus fibrosus, nucleus pulposus and cartilage-endplate tissues of postnatal (0-6-month-old)-and young-adult (20-30-year-old)-human intervertebral discs. The A1 fractions from young-adult disc contained a greater proportion of non-aggregating proteoglycans than did postnatal tissues. After dissociative CsCl-density-gradient fractionation of the A1, more than 90% of the uronic acid was found in the postnatal A1D1, whereas only 60-80% of the hexuronate was present in the A1D1 isolated from young-adult disc tissues. These results indicated that more lower-buoyant-density proteoglycans occur in the young-adult disc. Link-protein-rich fractions (A1D3) were subjected to SDS/polyacrylamide-gel electrophoresis and immunolocation analyses using monoclonal antibodies specific for epitopes on link protein or proteoglycan. Under non-reducing conditions, the major link protein present in postnatal disc tissues was link protein 1. By contrast, all three link proteins (1, 2 and 3) were detected in young-adult tissues, with the smaller link protein 3 predominating. Analyses of the A1D3 fractions under reducing conditions also indicated the presence of link-protein-degradation peptides (Mr approx. 26,000) from young-adult disc tissues, but not from postnatal tissues. Sequential Sepharose CL-6B and Sephacryl S-300 chromatography in 4 M-guanidinium chloride was employed to separate the link proteins of the A1D3 fraction from protein-rich proteoglycan. Immunolocation analyses indicated that postnatal samples contained no detectable contaminating proteoglycan fragments. However, young-adult link-protein preparations could not be separated from hyaluronic acid-binding region and other proteoglycan fragments by means of these chromatographic procedures. The studies indicate that, compared with hyaline articular cartilage, degraded link protein and proteoglycan accumulate at an early age in young-adult disc tissues. These partially degraded proteoglycan aggregate components may significantly alter the biomechanical properties of disc tissues.

Proteoglycans of the human intervertebral disc. Electrophoretic heterogeneity of the aggregating proteoglycans of the nucleus pulposus.

Nuclei pulposi were dissected from lumbar discs of radiologically normal human spines of cadavers aged 17, 20 and 21 years. Proteoglycans were extracted with 4 M guanidine hydrochloride (dissociative conditions) with proteinase inhibitors and isolated as A1 fractions by associative density-gradient centrifugation. Aggregating and non-aggregating proteoglycans were separated by Sepharose 2B chromatography. Both aggregating and non-aggregating proteoglycans contained a keratan sulphate-rich region as isolated by chondroitinase/trypsin/chymotrypsin digestion and Sepharose CL-6B chromatography. Agarose/acrylamide-gel electrophoresis of individual fractions of a Bio-Gel A-50m dissociative-column separation of the aggregating proteoglycans revealed two, well-separated bands: S and F, the slower and faster migrating bands respectively. The non-aggregating proteoglycan fractions were eluted under associative conditions (0.5 M-sodium acetate, pH 6.8) and migrated as a single band in the electrophoretic system. The gel-electrophoretic heterogeneity of the aggregating proteoglycans was still evident after hydroxylamine fragmentation and removal of the hyaluronate-binding portion of the molecule. Dissociative density-gradient centrifugation of the aggregating proteoglycans partially separated the Band-S proteoglycans from the Band-F population. Subsequent dissociative chromatography of the high-buoyant-density Band F proteoglycans permitted discrimination of this band into two gel-electrophoresis-distinguishable populations (Bands F-1 and F-2). Enzyme-linked immunosorbent assays with a monoclonal antibody that recognized keratan sulphate demonstrated that the D1 fraction containing the Band F-1 proteoglycans was enriched in keratan sulphate compared with the total aggregating or non-aggregating pool of proteoglycans. The proteoglycans of young adult nucleus pulposus could then be ascribed to one of four structurally and/or electrophoretically distinct populations: (1) the non-aggregating population, which comprised about 70% of the total extractable proteoglycans; (2) the aggregating pool, comprising: (a) Band F-1 proteoglycans, which had a relatively large hydrodynamic size, uronate/protein weight ratio, were enriched in keratan sulphate and had a high buoyant density; (b) Band S proteoglycans, which migrated slower in agarose/acrylamide gels, had a smaller hydrodynamic size, lower buoyant density and a lower uronate/protein ratio than the Band F-1 population; (c) Band F-2 proteoglycans, which were lower in buoyant density, smaller in hydrodynamic size and slightly faster in electrophoretic mobility than the Band F-1 proteoglycans.

Isolation and partial characterization of surface components of cell line MDA-MB-231 derived from a human metastatic breast carcinoma.

Neuraminidase (Vibrio cholerae) treatment of human metastatic mammary carcinoma MDA-MB-231 cells grown in culture released 0.60-0.63 mg of N-acetylneuraminic acid from 10(9) cells. Incubation of intact cells with a modified trypsin and fractionation by gel filtration gave mainly O-glycopeptides. The presence of O-glycosyl-linked chains having one or two carbohydrate residues was confirmed by treatment of the glycopeptide fractions with galactose oxidase, followed by reduction with alkaline sodium borotritide and fractionation. The major glycopeptide fraction, which consisted of 53% carbohydrate and 47% protein, and a minor glycopeptide fraction each inhibited hemagglutination by peanut lectin. These results suggest the presence of O-beta-D-galactopyranosyl-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 leads to 3)-L-serine (threonine) residues. The absorptive capacities for anti-HLA-A2 and anti-HLA-B8 antisera were slightly greater for intact than for lyophilized cells, which suggested that masking of these major histocompatibility antigens did not occur in intact cells.

Comparative lectin-binding and agglutination properties of the strain-specific, TA-3-St, and the non-strain-specific, TA3-Ha, murine mammary carcinoma ascites sublines. Further studies of receptors in epiglycanin.

The complex carbohydrates at the cell surfaces of two TA3, murine mammary carcinoma ascites sublines (the strain-specific, TA3-St subline and the nonstrain-specific. TA3-Ha line) were compared by binding studies with 125I-labelled concanavalin A (con A), Ricinis communis agglutinin (RCA), and eel-serum agglutinin (ESA). The TA3-Ha cell bound equal amounts of con A, 1.5-fold more RCA, and 4-fold more ESA than the TA3-St cell. Binding-inhibition studies by these lectins and two others [wheat-germ agglutinin (WGA) and potato lectin (STA)] suggest complementary binding-sites between con A and both RCA and ESA. Quantitative agglutination studies with the five lectins, and inhibition determinations by both neuraminidase-treated and untreated epiglycanin revealed that TA3-St, but not TA3-Ha, cells were agglutinated by con A, and that epiglycanin inhibited this agglutination, as well as the agglutination of rabbit erythrocytes by con A. The presence of a con A receptor on epiglycanin was also suggested by the binding of epiglycanin to con A-Sepharose, and its specific elution with methyl alpha-D-mannopyranoside. TA3-St cells were agglutinated at a 10-15-fold lower concentration of either STA or RCA than TA3-Ha cells, but both cells were agglutinated by the same concentration of WGA and ESA. Inhibition by epiglycanin of agglutination of TA3-St cells by either STA or ESA occurred at a concentration lower than that of TA3-Ha cells, but epiglycanin inhibited RCA agglutination of TA3-Hs cells at a concentration lower than that of TA3-St cells. Epiglycanin, but not asialoepiglycanin, inhibited the agglutination of TA3-Ha cells by WGA.