BIOSYNTHESIS OF TRACHEAL MUCOUS GLYCOPROTEINS

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Chronic obstructive pulmonary diseases, such as cystic fibrosis, chronic bronchitis and asthma, are manifested by hypersecretion and accumulation of tenacious mucus in the airways. Mucins, a major determinant of the rheological properties of mucus, are secreted by secretory cells in surface epithelium and submucosal glands. Mucin carbohydreate, which constitutes 80-90% of the molecule, is heterogeneous and its structure may be regulated by relative activities of glycosyltransferases. Mucin core 2 beta6 G1cNAc transferase (TF) and alpha2,6 (or 60) NeuAc TF are known to compete for the first branch point of mucin oligosaccharides, resulting in oligosaccharides with different structures. We hypothesize that relative activities of these two enzymes in the secretory cells regulate mucin acidic properties as reflected in differential histochemical staining of these cells. It has been noted that same glycosyl TF from different animal species is highly homologous in amino acid and nucleotide sequences. We propose to clone bovine tracheal beta6 G1cNAc TF, which can form core 2, core 4 and I beta6 G1cNAc structures, and 60 NeuAc TF by cross-species hybridization using recently cloned cDNAs of these two enzymes in other animal species. We will verify the cloned cDNAs by characterizing acceptor specificities of these two recombinant blycosyl TF's. Monoclonal antibodies (MABs) against these two recombinant glycosyl TFs will be generated and used with {S- 35}riboprobes for each enzyme to probe bovine tracheal epithelial secretory cells by immunocytochemistry and in situ hybridization, respectively. These results will be correlated with histochemical straining properties of these cells. We will ascertain whether these two TFs differentially express in different secretory cell populations, and whether predominance of beta6G1cNAc TF is associated with elaboration of more acidic mucins containing longer, sulfated olgosaccharides and 60 NeuAc TF with elaboration of less acidic mucins containing shorter, sialylated oligosaccharides. Same studies will also be performed in primary cell and organ cultures of bovine tracheal epithelium by modulating relative levels of these two enzymes by antisense oligonucleotide approach and by gene transfer mediated by an adenoviruspolylysine conjugate using a vector containing pCMV promoter. These studies should provide a molecular basis at the glycosyl TF level for understanding the regulation of the biosynthesis of mucin-type carbohydrates and the heterogeneity of secretory cells in healthy and diseased airway epithelium.