Physical Properties of Purified Human Respiratory Mucus Glycoproteins: Effects of Sodium Chloride Concentration on the Aggregation Properties and Shape

Abstract

The biophysical properties of purified native (nonreduced) mucus glycoproteins (mucins) isolated from lung mucus secretions of cystic fibrosis (CF) patients and subjects with normal lungs were studied using the technique of light scattering. The effects of different NaCl concentrations and 6 M guanidine hydrochloride on the molecular size of mucins, their ability to form aggregates, and their shape were investigated. Under the concentration range studied (0.05-3.5 mg/ml), in buffered 0.03 and 0.01 M NaCl, the CF mucins had higher molecular weights (12.2 x 10(6) to 17.1 x 10(6) and 9.5 x 10(6) to 10.4 x 10(6), respectively) than those observed in buffered 0.15 M NaCl (4.3 x 10(6) to 6.6 x 10(6]. These results were interpreted in terms of CF mucins self-aggregating in buffered 0.03 and 0.01 M NaCl. In contrast, in the both buffered 0.3 and 0.15 M NaCl, the normal respiratory mucins had molecular weights of 6.3 x 10(6) to 8.6 x 10(6), thus suggesting the absence of normal mucin aggregation in buffered 0.03 M NaCl. In the presence of 6 M guanidine HCl both CF and normal mucins had molecular weights of about 5 x 10(6) and showed more extended structure (i.e., larger radius of gyration) than in the presence of 0.03 or 0.15 M NaCl. Studies of the relationship of the light scattering intensity with scattering angle showed that, under the above experimental conditions studied, both CF and normal respiratory mucins were polydisperse flexible coil-shaped molecules. The increased aggregation of CF mucins observed at lower salt concentrations may alter the viscoelastic properties of CF lung mucus secretions.

Mucin Glycosylation and Sulphation in Airway Epithelial Cells Is Not Influenced by Cystic Fibrosis Transmembrane Conductance Regulator Expression

“…Others have shown abnormal rheology (7) that correlates with the degree of bacterial infection and CF disease severity (8) or that is only exhibited with increased DNA content (9) or altered salt concentration (10). Mucin abnormalities have been reported in patients with CF, and it has been suggested that these result from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein having a direct effect on mucin biosynthesis.…”

Production and Characterization of Monoclonal Antibodies to Purified Deglycosylated Cystic Fibrosis Respiratory Mucin: Evidence for the Presence of Four Immunologically Distinct Epitopes

“…The purification of mucins from human respiratory mucus secretions was carried out using a protocol established in our laboratory (3,12,13). Deglycosylation of respiratory mucins was accomplished by the method of Woodward et al (8) with slight modification.…”

“…Chemical and amino acid compositions were determined for the purified native and deglycosylated respiratory mucins as described earlier (3,12,13). In brief, sialic acid was determined by the resorcinol method using N-acetylneuraminic acid as the standard (14).…”

“…In addition, after hydrolysis (4 N HC1, 4 h at 100°C ), the amino sugars were also analyzed using an amino acid analyzer (sensitivity of detection = 0.1 fig of amino sugar). Amino acid analyses were performed essentially as described earlier (3,12,13) using a Durrum D-500 amino acid analyzer following hydrolysis of the sample with constant-boiling hydrochloric acid at 110°C for 22 h in evacuated sealed tubes. The purity of native mucins was examined by SDS composite gel electrophoresis (2% acrylamide, 0.5% agarose containing 0.1% SDS) (16).…”

“…Three CF respiratory mucins were purified usirg a protocol established in our laboratory (3,12,13). The purified mucins (50 fig each) gave a single broad band on a SDScomposite gel stained with PAS reagent (Fig.…”