The long term objectives of this project are to investigate the structure and function of epithelial mucins and to investigate the contributions of these glycoproteins to disease states, particularly gallstones and peptic ulcer.
The specific aims of this application are focussed on the peptide core (apomucin) of gastric and gallbladder mucin in humans and experimental animals. We propose to: 1. Purify apomucin, obtain partial amino acid sequences and prepare apomucin-specific antibodies, 2. Use gallbladder and gastric RNA from several species to direct synthesis of apomucin in cell- free translating systems 3. Study lipid binding to apomucin and whole mucin, and determine the effects of bound lipid viscosity, barrier function, and cholesterol monohydrate nucleation. Our experiments will allow us to test the hypothesis that hydrophobic areas of apomucin are important for lipid binding which in turn influences functional parameter. Highly purified mucin from human stomachs and gallbladders, pig stomach and prairie dog gallbladder will be deglycosylated with trimethanesulfonic acid and endoglycosidases and purified by HPLC. Tryptic peptides of apomucin will be sequenced and antibodies to apomucin prepared in rabbits. Apomucin specific antibody will be used to identify translated apomucin in cell-free systems primed with gastric gallbladder and RNA. Purified apomucins from pig and human stomach will be evaluated for ability to bind lipids of various classes. These studies will provide for the first time the molecular weight, partial amino acid sequence and amino acid composition of intestinan and biliary apomucins and will allow comparison of apomucins from stomach and gallbladder in several species. Studies of lipid binding to apomucin will allow us to determine the site of binding, and the effects of bound lipid on functional parameters such as viscosity, ion diffusion and cholesterol monohydrate nucleation in model and native biles. Because mucin is thought to be involved in gallstone nucleation in man and experimental animals, these results should provide new insights into gallstone pathophysiology. These experiments should also provide a better understanding of how the mucus barrier protects the stomach from auto digestion.
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