The long term goals of the studies outlined in this proposal are to understand the mechanisms involved in the regulation of mucin biosynthesis and secretion and relationship of these processes to the pathology of hypersecretion associated with chronic obstructive pulmonary diseases, such as, cystic fibrosis (CF). Excess mucus secretions with altered viscoelastic properties clearly contributes to the clinical symptoms. To date, little is known about the mucin macromolecules that constitute the secretion; especially knowledge regarding the primary structures of their core protein(s) is lacking. The objectives of this proposal are to gain insights into organization of the macrostructure of human tracheobronchial mucins (HTMs) and to investigate regulation of their gene expression.
The specific aims are to, a) purify and compare physicochemical properties of mucins HTM-1 and HTM-2 isolated from CF and normal lung secretions, b) prepare and characterize poly- and monoclonal antibodies against deglycosylated mucins, c) prepare synthetic oligonucleotide probes based on select peptide sequences of the mucins, d) construct cDNA library in lambda gt22A from poly(A) +RNA isolated from human tracheal epithelial (HTE) cells, e) screen the library using immunological and synthetic oligonucleotide probes, f) isolate, characterize and sequence cDNA clones encoding for protein cores of the mucins, g) using cDNAs, thus obtained, isolate genomic clones which will also sequenced and g) to investigate the mechanism(s) and effects of select secretagogue(s) on regulation of transcription of the HTM apomucin gene. The mucins will be purified according to protocols established in our laboratory and their physicochemical properties will be determined using established techniques. mRNA will be isolated from fresh HTE cells and cultured mucus secreting HTE cells. These preparations will be used to generate cDNA libraries which will be screened using immunological and synthetic oligonucleotide probes. The cDNA inserts will be sequenced. Clone identity will be confirmed by amino acid sequence analysis of selected peptide fragments. The cDNAs will be utilized to isolate genomic clones which will also be sequenced. Effects of secretagogue(s) on mucin related mRNA synthesis will be detected by Northern blot analysis using mucin cDNA probe(s). This knowledge will be essential in order to develop a rational approach to the treatment of chronic obstructive lung diseases including CF which will ultimately require an ability to control the viscosity and rate of secretion.
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