The long term goals of the studies outlined in this proposal are to understand the mechanisms involved in the regulation of mucin glycoprotein biosynthesis and secretion, and the relationship of these processes to the pathology of hypersecretion associated with chronic obstructive lung diseases, such as cystic fibrosis. The primary biochemical defect in cystic fibrosis remains unknown, however many of the clinical abnormalities in this disease are the result of an alteration in the viscollastic properties of mucin glycoproteins present in mucus secretions. The current study involves an investigation of the mechanisms which regulate the transcription and translation of the polypeptide chain of mucin glycoproteins and GalNAc peptidyltransferase, an enzyme which initiates the addition of carbohydrate chains to this glycoprotein. The immediate aim of this project is to determine the primary sequence of the polypeptide chain of purified deglycosylated mucin glycoproteins and Ga1NAc peptidyltransferase by cloning and sequencing cDNA's and genes encoding these proteins. We are currently preparing CNBr, peptidic and tryptic peptides from these proteins for primary amino acid sequence analysis. Suitable sequences will be used to prepare synthetic oligonucleotide probes in order to screen cDNA libraries for recombinant which contain inserts coding for deglycosylated mucin glycoproteins or GalNAc peptidyltransferase. mRNA will be isolated from fresh trachea epithelium and cultured mucus secreting epithelial cells. These preparations will be used to generate cDNA libraries which will also be screened with the synthetic oligonucleotide probes. The cDNA inserts will be sequenced to verify the identity of the clones with known amino acid sequences and they will be used to develop expression vectors. These systems will be used to synthesize enough of these proteins for definitive chemical characterization and to evaluate the regulation of these genes and their mRNAs in mucus secreting epithelial cell cultures. In current studies we have prepared specific antibodies to purified deglycosylated human and swine trachea mucin glycoproteins and GalNAc peptidyltransferase. The antibodies have been used to isolate and identify the proteins synthesized in in vitro translation systems using mRNA isolated from fresh swine trachea epithelium. These preliminary studies provide considerable encouragement for success of the studies described in this application.
