A recombinant bovine gallbladder mucin polypeptide binds biliary lipids and accelerates cholesterol crystal appearance time

Background & Aims: Mucin has a central role in the pathogenesis of choleste rol gallstones, in part because of its ability to bind biliary lipids and a ccelerate cholesterol crystal appearance time. Previous studies have locali zed these properties to nonglycosylated mucin domains, and we have recently shown that these domains contain a series of 127-amino acid, cysteine-rich repeats. The aim of this study was to express a recombinant mucin polypept ide containing these repeats and investigate its lipid-binding and pronucle ating properties. Methods: A recombinant mucin polypeptide was expressed as a glutathione S-transferase fusion protein in Escherichia coli, purified b y affinity chromatography, and compared with native bovine gallbladder muci n in lipid-binding and cholesterol crystal appearance time assays, Results: The recombinant mucin polypeptide bound a hydrophobic fluorescent probe an d cholesterol in a concentration-dependent manner, It accelerated the appea rance of cholesterol crystals from lithogenic model bile, an effect that wa s both time and concentration dependent. Conclusions: The cysteine-rich rep eats in the recombinant mucin polypeptide correspond to the protease-sensit ive hydrophobic domains identified in earlier biochemical studies. further delineation of the lipid-binding site(s) in these repeats will provide new insights into the mechanism of cholesterol crystal nucleation and stone gro wth.