The overproduction of mucus is common in many pulmonary diseases, including asthma, cystic fibrosis and chronic bronchitis. Excess mucus clearly contributes to the symptoms of and may play a role in producing the airway obstruction of these disorders. Even though mucus expectoration is a cardinal feature of these diseases very little is known about the macromolecules which constitute this secretion; without this essential knowledge it is unlikely that our appreciation of the pathobiology of these and related disorders will increase. A major factor which has prevented further understanding of the role of mucus in lung biology is that the major protein component of mucus, mucus glycoprotein (MGP) is not well characterized. In this proposal we outline studies designed to provide novel structural definition of mucus glycoprotein, and to study the regulation of its synthesis and secretion. In order to achieve these aims we plan to extend protein chemical studies in which peptide segments useful for the production of synthetic oligonucleotides have been identified; these oligonucleotide probes will be used to screen human lung cDNA libraries for clones containing MGP cDNA. Antisera directed against deglycosylated human MGP will also be used as a second degree screening method to identify and/or confirm MGP cDNA clones. The cDNA sequence for MGP will be determined. Further, the topology of human respiratory mucus glycoproteins will be studied to determine if the MGP(s) are products of single or multiple genes or undergo post-translational proteolytic processing. Antibodies produced against native or deglycosylated MGP, as well as against synthetic peptides derived from the MGP structure, will be used to purify mucus glycoprotein and to identify the presence or absence of subunits or """"""""link"""""""" proteins within the MGP molecule. Finally the primary structural information determined above will be used to assess the regulation of glycoprotein synthesis and secretion at the cellular level. We will determine the effects of known secretory agonists, including adrenergic and cholinergic stimuli as well as inflammatory mediators, on the expression of mucus glycoprotein mRNA in explant cultures.
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