Evidence of hydrophobic domains in human respiratory mucins. Effect of sodium chloride on hydrophobic binding properties

Abstract

Hydrophobic binding properties of purified human respiratory mucins were studied by the fluorescence probe technique using mansylphenylalanine (Mns-Phe) as the fluorescent probe. Mucins were purified from tracheobronchial secretions of cystic fibrosis (CF) and asthmatic patients, as well as from individuals with normal lungs, according to a protocol earlier established in our laboratory. Purified mucins were subjected to reduction-alkylation and Pronase digestion to study the effects of these treatments on the hydrophobic properties of the mucins. In addition, the effects of increased NaCl concentration on the hydrophobic properties of native and reduced-alkylated mucins were also investigated. Native mucins showed evidence of a large number of low-affinity (KD approximately 10(-5) M) binding sites for the hydrophobic ligand Mns-Phe and had between 40 and 50 binding sites/mg of mucin. Reduction of mucin using dithiothreitol in the presence of 6 M guanidine hydrochloride and subsequent alkylation with iodoacetamide apparently caused marked conformational changes in the mucin molecules as revealed by the presence of both high-affinity (KD approximately 10(-6) M) and low-affinity (KD approximately 10(-5) M) binding sites for the probe and an increase in the number of probe binding sites. Pronase digestion of the native and reduced-alkylated mucins almost completely eliminated binding of the fluorescent probe to the mucins, showing that the binding sites are on the nonglycosylated, Pronase-sensitive portion of the mucin molecules. Increasing NaCl concentrations (0.03-1.0 M) did not appreciably alter the native mucin-induced Mns-Phe fluorescence, while that of the reduced-alkylated mucin-induced Mns-Phe fluorescence was progressively increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Respiratory Tract Mucin Genes and Mucin Glycoproteins in Health and Disease

“…The large, cysteine-rich, gel-forming mucins MUC5AC (116,194,271), MUC5B (273), and MUC2 (116) mucins have been identified immunochemically in induced mucus or bronchial washings from normal airways, while MUC5AC (179,194,223,271), MUC5B (57, 273, 300), MUC8 (241,242), and MUC2 (57) mucins have been identified in sputum samples or bronchial washings from patients with chronic airway diseases.…”

Biophysical Approaches to Salivary Mucin Structure, Conformation and Dynamics

“…Short-lived entanglements of the longer carbohydrate side chains typical of gel-forming mucins have been proposed to provide the necessary gel-forming cross-links (Sellers etal., 1988;Soby et al, 1990). Hydrophobic interactions involving nonglycosylated portions of the peptide core, also common to the gel-forming mucins, may be another mechanism for cross-link formation (Shankar et al, 1990; see Sheehan et al, 1991). The actual basis of the differing gel-forming properties needs to be further elucidated.…”

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).…”