The long term objective is to elucidate the role of mucous glycoproteins (mucins) in health and in disease. Mucins are a major component of the mucosal layer lining the respiratory tract and are over-produced in chronic obstructive pulmonary diseases including cystic fibrosis (CF). Since mucins are the macromolecules responsible for the viscoelastic properties of mucus, alterations in mucin structure may effect the physiological behavior of mucus. It has become increasingly evident that the complex structure and physical properties of human mucins almost preclude obtaining definitive information on the polypeptide backbone of normal and pathological mucins by biochemical studies alone. Thus, in order to compare mucin polypeptides and determine the role of glycosylated and non-glycosylated domains in regulating the physical properties of mucins, this laboratory has begun studies designed to isolate cDNA that codes for tracheobronchial mucin (TBM).
The specific aims are (1) to prepare, purify and sequence TBM peptide fragments. The resultant primary sequences will be useful for generating polynucleotide probes to identify and confirm clones containing TBM cDNA and to confirm or correct the primary structure of human TBM deduced from the nucleotide sequence of; (2) to isolate cDNA that encodes TBM. mRNA isolated from respiratory tract tissue will be used to synthesize cDNA for the construction of recombinant libraries in the bacteriophage lambda gt11. The cDNA libraries will be screened with antibodies specific to the mucin polypeptide backbone and with polynucleotide probes generated from amino acid sequence data; (3) to sequence sufficient number of cDNA clones to deduce the primary sequence of TBM including the glycosylated and non-glycosylated segments. (4) In addition, the effect of fatty-acid acylation on respiratory tract mucin and the question of whether CF respiratory tract mucin, like gastric mucin is over-acylated will be examined. If increased covalently bound-lipid has relevance to the pathogenesis or molecular defect of CF, increased acylation should also be manifested by non-gastric mucins. Recent studies indicate that respiratory tract mucins are fatty acid-acylated. We will further examine this finding and determine whether CF mucins are over-acylated, whether lipid is attached to glycosylated or non-glycosylated mucin domains, and whether covalently found fatty acid protects respiratroy tract mucin against proteolysis.
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