Abstract
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 epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-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-epsilon-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.
Highlights
Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCAs known carcinogenic properties and hepatotoxicity
Preparation of anti-LCA antibody To capture protein-bound LCA by immunoprecipitation, the antibody should have the ability to bind to the steroidal moiety of protein-bound LCA, where LCA is coupled via the e-amino group of lysine residue on the proteins
If the chemical group involved in the linkage between the hapten and the carrier is bound to the recognition site, it is desirable to increase the distance between the steroidal moiety and the carrier protein to increase the probability of raising antibodies with high recognition [29,30,31,32]
Summary
Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCAs known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the e-amino group of lysine residues. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats. LCA is converted to its CoA thioester, which is transferred to the amino group of glycine or taurine. In addition to this conjugation (or N-acyl amidation) process, LCA may undergo hydroxylation at C-6, C-7, or C-15 [19].
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