Identification of lithocholic acid-tagged proteins in the liver of bile duct-ligated rat by means of immunoaffinity capture and MALDI-TOFMS

Shigeo Ikegawa, Tetsushi Yamamoto, Hiromi Ito, Shunji Ishiwata, Toshihiro Sakai, Kuniko Mitamura, and Masako Maeda

Faculty of Pharmaceutical Sciences, Kinki University, Higashi-osaka, Osaka 577-8502

Corresponding Author: ikegawa{at}phar.kindai.ac.jp

The formation of covalently bound protein adducts with lithocholic acid (LCA) has been proposed as a possible explanation for the carcinogenicity and hepatotoxicity of LCA. However, no detailed study on the structure of the resulting protein bound-LCA formed in the liver has been reported. The aim of our study was to identify the cellular proteins in the liver of the bile duct-ligated rat that become covalently linked to LCA presumably via the $epsilon$ -amino groups of lysine residues. For this purpose, we generated antibodies that recognized the 3 $alpha$ -hydroxy-5 $beta$ -steroid moiety of LCA by immunizing rabbits with immunogens in which the side chain of LCA was coupled to bovine serum albumin via a 6-aminohexanoic acid and/or a succinic acid spacer. The resulting antibodies largely cross-reacted with the amidated and nonamidated forms of LCA as well as N- $alpha$ -(t-butoxycarbonyl)-L-lysine- $epsilon$ -LCA. The antibodies were shown to detect LCA residues bound to ovalbumin and lysozyme that were synthesized by reacting LCA acyl-adenylate, with these protein molecules. The antibodies were then used to immunoprecipitate cytosolic proteins containing bound LCA from the liver of bile duct-ligated rats. Isolated proteins were separated by two dimensional electrophoresis, and proteins containing covalently coupled LCA were analyzed using MALDI-TOFMS and in silico analysis. Proteins that were labeled with LCA consisted of Rab-3, Rab-12, Rab-16 and M-Ras. Rab proteins are Ras-like small GTP-binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may affect vesicular transport in the hepatocyte and contribute to LCA-induced liver toxicity.

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