J. Lipid Res. Neurobiology of Lipids (ISSN1683-5506)
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Originally published In Press as doi:10.1194/jlr.M800350-JLR200 on July 19, 2008

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Journal of Lipid Research, Vol. 49, 2463-2473, November 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats

Shigeo Ikegawa2,*, Tetsushi Yamamoto1,*, Hiromi Ito*, Shunji Ishiwata*, Toshihiro Sakai*, Kuniko Mitamura* and Masako Maeda{dagger}

* Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
{dagger} School of Pharmaceutical Sciences of Showa University, 1-5-8, Hatanodai, Shinagawa, Tokyo 142-8555, Japan
1 Present address of T. Yamamoto: Department of Pathology, Integrative Oncological Pathology, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-Ku, Tokyo 113-8602, Japan.

Published, JLR Papers in Press, July 19, 2008.

This work was supported in part by a SUNBOR grant from the Suntory Institute for Bioorganic Research (2007), a Grant-in-Aid for Scientific Research (C) (Grant 19590165) from the Japan Society for the Promotion of Science for 2007–2008, and High-Tech Research Center Project for Private Universities: matching fund subsidy from Ministry of Education, Culture, Sports, Science and Technology. (2007–2009).

2 To whom correspondence should be addressed: e-mail: ikegawa{at}phar.kindai.ac.jp

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the {varepsilon}-amino group of lysine residues. Antibodies recognizing the 3{alpha}-hydroxy-5β-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-{alpha}-(t-butoxycarbonyl)-L-lysine-{varepsilon}-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were 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 influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.

Supplementary key words antibody • rat liver • cytosol fractions • two-dimensional electrophoresis • matrix-assisted laser desorption ionization time-of-flight mass spectrometry • Rab proteins


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Copyright © 2008 by the American Society for Biochemistry and Molecular Biology.