The goals of this proposal are to study the polymer structure of purified gallbladder mucin, to characterize the hydrophobic binding site on gallbladder mucin and to investigate the mechanism by which gallbladder mucin enhances the nucleation of cholesterol micro-crystals from supersaturated gallbladder bile. Two sequential phenomenon are essential for cholesterol gallstone formation: 1.) the hepatic secretion of bile supersaturated with cholesterol and 2.) the nucleation of cholesterol micro-crystals from supersaturated bile in the gallbladder. The significance of this study relates to the role of gallbladder mucin as a potential nucleating agent in supersaturated gallbladder bile. Recent studies in animal models of cholesterol cholelithiasis suggests that the accumulation of a viscous mucus gel in the gallbladder lumen promotes the heterogenous nucleation of cholesterol micro-crystals from supersaturated gallbladder bile. We plan to study the relationship of hydrophobic binding and ionic interactions to the physical behavior of mucin, particularly its polymerization. Polymerization of mucin will be studied by biochemical and physical chemical methods including quasi-elastic light scattering and viscometry. The hydrophobic domain in gallbladder mucin will be structurally characterized by selective proteolysis and peptide fragment analysis, and the binding affinity of mucin for biliary lipids will be investigated by column chromatography, sucrose density ultracentrifugation and fluorescence spectroscopy. Finally, the nucleation of cholesterol micro-crystals from supersaturated model bile and from native gallbladder and hepatic bile will be studied by polarizing light microscopy and quasielastic light scattering. The relative nucleating potential of gallbladder mucin from patients with and without gallstones will be studied and the contribution of the hydrophobic domain of gallbladder mucin in nucleating will be investigated. This proposal represents a systematic biochemical and biophysical investigation of the structure-function relationship of gallbladder mucin as it relates to the pathogenesis of cholesterol cholelithiasis. Information obtained from these studies will hopefully have application in the future for clinical management of gallstones disease.
