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

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 ε-amino groups of lysine residues. For this purpose, we generated antibodies that recognized the 3α-hydroxy-5β-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-α-(t-butoxycarbonyl)-L-lysineε-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. Supplementary key words: Lithocholic acid; antibody, rat liver, cytosol fractions, immunoprecipitation, 2-DE, MALDI-TOFMS, Rab proteins. 2 by gest, on S etem er 1, 2017 w w w .j.org D ow nladed fom INTRODUCTORY STATEMENT During the past decades, considerable attention has been focused on the possible role of covalent coupling of bile acids to proteins in the pathogenesis of disorders such as cholestasis and colon cancer (1-7). The bile acid that has attracted most interest is lithocholic acid (LCA), a secondary bile acid formed by bacterial 7α-dehydroxylation of chenodeoxycholic acid. LCA, a major fecal bile acid in man, is a hydrophobic bile acid that is highly toxic in some experimental animals (8). A protein bound-form of LCA was found in human livers (1, 2), and its concentration was reported to be elevated in the livers of rat treated with a carcinogen, methylazoxymethanol (3). Furthermore, LCA was detected in normal and neoplastic human mammary tissues and neoplasms of the uterus, kidney, lung and the colon (3). It has also been proposed that LCA acts as a promoter of colon cancer (9-17). One mechanism that has been suggested to explain the carcinogenic properties of LCA is that cellular proteins are covalently modified by the chemically reactive species that are formed during metabolism of LCA (18). LCA metabolism by the hepatocyte is complex. On entering the hepatocyte, LCA is converted to its Coenzyme A thioester, which is then transferred to the amino group of glycine or taurine. In addition to this conjugation (or N-acyl amidation) process, LCA also may undergo hydroxylation at C-6, C-7, or C-15 (19). In man and mouse, LCA undergoes sulfation at C-3 in addition to N-acyl amidation. When incubated with microsomes, LCA may undergo acyl glucuronidation (20-23). Finally adenylation with AMP has also been observed under in vitro conditions (24). In principle, the Coenzyme A thioester, the acyl glucuronide, or the adenylate should be capable of binding irreversibly to proteins (18, 25, 26). However, no detailed study on the structure 3 by gest, on S etem er 1, 2017 w w w .j.org D ow nladed fom analysis of protein bound-LCA formed in the liver has been reported. Therefore, identification of the cellular proteins chemically modified with LCA is essential as a step in elucidation of the mechanism of LCA-induced cytotoxicity. Proteomics is the identification of the total proteins from a particular organelle, cell line, tissue, or organism. The most commonly used experimental techniques in proteomics are two-dimensional electrophoresis (2-DE) for separating proteins and mass spectrometry (MS) for the identification of separated proteins. Due to the complexity of the proteome of many biological samples, it is desirable to concentrate the desired analytes from biological samples prior to the MS analysis. Immunoaffinity capture is a powerful protein separation method. This method is based on the specific interaction between an antibody and the target proteins to be captured. The aim of this work was to show the potential of the immunoaffinity capture of the protein bound-LCA in the liver of bile duct-ligated rat using a specific antibody with high affinity for the 3α-hydroxy-5β-steroid moiety of LCA. The proteins isolated by this technique could be subjected to structural analysis by the basic proteomic technique of 2-DE, combined with the use of matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOFMS) and computer assisted programs, in order to identify which proteins had formed LCA adducts. 4 by gest, on S etem er 1, 2017 w w w .j.org D ow nladed fom