The goal of this study is to study the function and functional relationship of two important gallbladder mucosal proteins, mucin and trefoil proteins in cholesterol nucleation and gallstone pathogenesis. Gallstone disease is a major source of morbidity, health care and social cost in the United States. Strategies to prevent and modify gallstone disease have largely been ineffective. Manipulation of important mucosal proteins such as mucin is an exciting strategy for the prevention or modification of gallstone disease. Mucin plays a centra role in gallstone disease: mucin hypersecretion occurs prior to gallstone formation, mucin accelerates cholesterol nucleation and crystal growth and forms the scaffold on which mature gallstones are built. It has been shown that it is the non- glycosylated domains of both human and bovine mucin that are important in conferring these properties on mucin. Trefoil proteins are a newly recognized class of mucosal protein that are co-secreted with mucin and may have important effects on the physico-chemical properties of mucin . They are cysteine rich molecules which results in a characteristic trefoil or clover leaved shape. Recent studies have identified a cysteine rich region in bovine mucin that binds biliary lipids and accelerates cholesterol nucleation. Thus we suggest that trefoil proteins may also be potentially important pronucleating agents. The long-term objectives of this proposal are to better define the relationship between gallbladder mucin and trefoil proteins in gall stone pathogenesis.
The specific aims of this proposal are to:-l.) Clone and characterize the non-glycosylated domains of human gallbladder mucin and search for important pronucleating domains. This will be achieved by immunoscreening a cDNA library with antibodies to human deglycosylated mucin and by expressing regions of interest as recombinant proteins to study their effects on lipid binding and cholesterol nucleation. 2) To express and purify human gallbladder trefoil proteins in a yeast vector, establish an immunoassay to measure biliary trefoil proteins and to look for pronucleating activity of trefoil proteins. 3.) To examine the effects of purified trefoil proteins on the cholesterol nucleating and physico- chemical properties of gallbladder mucin in vitro. These studies will help to define the important structural domains of human gallbladder mucin and the important structural and pronucleating properties of these two important mucosal proteins.
| Nunes, D P; Afdhal, N H; Offner, G D (1999) A recombinant bovine gallbladder mucin polypeptide binds biliary lipids and accelerates cholesterol crystal appearance time. Gastroenterology 116:936-42 |
| Offner, G D; Nunes, D P; Keates, A C et al. (1998) The amino-terminal sequence of MUC5B contains conserved multifunctional D domains: implications for tissue-specific mucin functions. Biochem Biophys Res Commun 251:350-5 |
| Troxler, R F; Iontcheva, I; Oppenheim, F G et al. (1997) Molecular characterization of a major high molecular weight mucin from human sublingual gland. Glycobiology 7:965-73 |
