BIOCHEM AND MOLECULAR BIOL STUDIES OF AIRWAY MUCINS

气管支气管粘液分泌物起着重要的保护作用

Tracheobronchial mucus secretions play an important protective role in the normal functioning of the airways. Mucus glycoproteins present in the mucus secretions are responsible, to a large extent, for the physical properties of the secretions. In the disease states, the mucins may have altered chemical and physical properties and this in turn may influence the clearance of the secretions by ciliated epithelium. Although, there is a considerable information on the oligosaccharide side chains of airway mucins, the structure, including amino acid sequence of the protein backbone is lacking. The objectives of this research are to investigate the molecular size, amino acid sequence, and physical properties of the protein core of the airway mucins. The specific aims of this proposal are to a) further purify and biochemically characterize the native (non-reduced) and deglycosylated mucins from canine tracheal mucus secretions, b) physically characterize the native and deglycosylated mucins to determine molecular size, radius of gyration, aggregation behavior and presence of hydrophobic domains, c) prepare immunological probes against native and deglycosylated mucins in order to detect specific apomucin(s) generated during in vitro cell free translation of mRNA from canine tracheal epithelial (CTE) cells and to screen the production of apomucin(s) in the cDNA library constructed in the lambda gt11 vector and d) isolate and characterize cDNA clones of apomucin(s) from canine tracheal epithelial cells in order to determine the primary amino acid sequence and amino acid sequences which signal transport and processing. The mucins will be purified using protocols established in this laboratory. Biochemical characterization will include: determination of carbohydrate, sulfate, amino acid composition, thiol and disulfide content of the mucin molecules. The molecular size, radius of gyration and agregation behavior will be determined using light scattering and sedimentation equilibrium methods. Poly- and monoclonal antibodies directed against the mucins will be prepared using standard established protocols. Isolation, characterization, detection of mucin cDNA clones and nucleotide sequencing will be carried out using well established molecular biological techniques. The knowledge gained from the proposed studies should eventually lead to more effective treatment strategies for obstructive lung disease.