A recombinant bovine gallbladder mucin polypeptide binds biliary lipids and accelerates cholesterol crystal appearance time.
OBJECTIVE
Mucin has a central role in the pathogenesis of cholesterol gallstones, in part because of its ability to bind biliary lipids and accelerate cholesterol crystal appearance time. Previous studies have localized 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 polypeptide containing these repeats and investigate its lipid-binding and pronucleating properties.


METHODS
A recombinant mucin polypeptide was expressed as a glutathione S-transferase fusion protein in Escherichia coli, purified by affinity chromatography, and compared with native bovine gallbladder mucin in lipid-binding and cholesterol crystal appearance time assays.


RESULTS
The recombinant mucin polypeptide bound a hydrophobic fluorescent probe and cholesterol in a concentration-dependent manner. It accelerated the appearance of cholesterol crystals from lithogenic model bile, an effect that was both time and concentration dependent.


CONCLUSIONS
The cysteine-rich repeats in the recombinant mucin polypeptide correspond to the protease-sensitive 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 growth.
Nunes
D P
DP
Section of Gastroenterology, Department of Medicine, Boston University School of Medicine and Boston Medical Center, Boston, Massachusetts 02118, USA.
Afdhal
N H
NH
Offner
G D
GD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001646:Bile / Q000378:metabolism*; D002417:Cattle; D002769:Cholelithiasis / Q000209:etiology; D002784:Cholesterol / Q000737:chemistry*; D003460:Crystallization; D005704:Gallbladder / Q000737:chemistry*; D050356:Lipid Metabolism; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D009077:Mucins; D010455:Peptides; D011994:Recombinant Proteins; D002784:Cholesterol