Mucus hypersecretion is a hallmark of obstructive lung diseases, including chronic bronchitis, asthma, and cystic fibrosis. This condition is the result of hypertrophy and hyperplasia of mucus cells. Secreted from goblet cells on the surface epithelium and mucus cells in the submucosal glands, mucins not only are the major determinant of the viscoelastic properties of mucus secretion but also can serve as the receptors for pathogens. The functions of mucins reside primarily in the carbohydrates, which constitute 70-90% of airway mucins by weight. In addition, mucin carbohydrates are very heterogeneous, which allow them to trap many different inhaled pathogens and facilitate their removal from the airways. Mucin carbohydrate structures and their functional potential can be expanded by core 2, core 4, and blood group I branch structures. All three structures can be formed by mucus tissue-specific core 2 N-acetylglucosaminyltransferase-M (C2GnT-M). Modulation of C2GnT-M gene expression can greatly affect the physicochemical properties of airway mucins and functions of airway mucus. Expression of C2GnT-M gene can be inhibited by epidermal growth factor but enhanced by retinoic acid and Th2 cytokines. C2GnT-M activity also can be regulated at the substrate level. Loss of C2GnT-M has been reported in colorectal cancer and its reexpression can inhibit tumorigenicity of colonic cancer cells. Thus, alteration of C2GnT-M can have a significant impact on health as well as diseases. The objective of this application is to characterize the modulation of C2GnT-M at the levels of enzyme activity and gene expression. We propose to: 1. Determine the active site of C2GnT-M by X-ray crystallography and then confirm the amino acids involved in catalysis by site-directed mutagenesis followed by measurement of enzyme activities using core 1, core 3, and blood group i disaccharide acceptors and their homologues. 2. Characterize C2GnT-M gene regulation by mapping cis-regulatory elements and identifying the cognate transcription factors under basal conditions. These transcription factors will be identified by transfection with cDNAs of known transcription factors and pull-down with biotinylated promoter followed by assay with transcription factor protein array. They will be characterized by electrophoresis mobility shift assay and chromatin immunoprecipitation assay. Current studies could facilitate the development of therapy for mucus hypersecretory diseases through identification of small carbohydrate inhibitors and mucus cell-specific promoter.
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