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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 Ikegawa^2,*, Tetsushi Yamamoto^1,*, Hiromi Ito^*, Shunji Ishiwata^*, Toshihiro Sakai^*, Kuniko Mitamura^* and Masako Maeda

^* Faculty of Pharmaceutical Sciences, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan
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

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
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 -amino group of lysine residues. Antibodies recognizing the 3-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--(t-butoxycarbonyl)-L-lysine--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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Over 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 7-dehydroxylation of chenodeoxycholic acid (CDCA). 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 rats 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 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 CoA 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 CoA thioester, the acyl glucuronide, or the adenylate should be capable of binding irreversibly to proteins (18, 25, 26). However, no detailed study on the structural 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 the 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. Because of 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 is to show the potential of the immunoaffinity capture of the protein-bound LCA in the liver of the bile duct-ligated rat using a specific antibody with high affinity for the 3-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-TOF-MS) and computer-assisted programs, in order to identify which proteins had formed LCA adducts.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
Materials
Cholic acid, CDCA, deoxycholic acid, and LCA were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), and ursodeoxycholic acid was kindly donated by Mitsubishi Tanabe Pharma Co. (Osaka, Japan). Glycine-conjugated LCA and taurine-conjugated LCA were prepared by the carbodiimide method (27). N--(t-butoxycarbonyl)-L-lysine--LCA (LCA-N-BOC-lysine) was synthesized by the activated ester method (28). Lysozyme from chicken egg white (EC 3.2.1.17), BSA, ovalbumin, 3,5-dimethoxy-4-hydroxycinnamic acid [sinapinic acid (SA)], and -cyano-4- hydroxycinnamic acid (-CHCA) were purchased from Sigma Chemical Co. (St. Louis, MO). The BCA protein assay kit was from Pierce (Rockford, IL). Immobiline DryStrip gel and imobilized pH gradient (IPG) buffer were obtained from GE Healthcare UK Ltd. Endoproteinase Lys-C (lysylendopeptidase) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Alkaline phosphatase (ALP) and Zip-Tip C₁₈ were purchased from Toyobo (Tokyo, Japan) and Millipore (Billerica, MA), respectively. Freund's complete adjuvant was purchased from Rockland (Gillbertsville, PA). HRP-conjugated anti-rabbit IgG antibody (Fc-specific) and goat anti-rabbit IgG antibody (Fc-specific) were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). HRP-conjugated protein G was purchased from Zymet (East Hanover, NJ). Immobilized protein G beads and normal rabbit IgG protein G beads were obtained from Pierce. Ninety-six-well EIA/RIA plates (No. 3590) were purchased from Corning, Inc. (Corning, MA). EIA/RIA plates coated with goat anti-rabbit IgG antibody were kindly donated by Eiken Chemical Co. Ltd. (Tokyo, Japan). Nitrocellulose membrane (0.45 µm) was obtained from Toyo Roshi Kaisha Ltd. (Tokyo, Japan). All other reagents were of analytical reagent grade and all solvents were purified by distillation prior to use. Water from a Millipore Ultra Water Purification System (Milli-Q Synthesis A10) was used for the preparation of the mobile phase and the aqueous solutions described below.

Buffers
The buffers used for this work were as follows: buffer A, 50 mM NaH₂PO₄-Na₂HPO₄ (pH 7.3); buffer B, buffer A containing gelatin (0.1%, w/v) and NaCl (0.9%, w/v); buffer C, buffer A containing NaCl (0.9%, w/v); buffer D, buffer C containing Tween 20 (0.05%, v/v); buffer E, 1.5 mM KH₂PO₄-8.1 mM Na₂HPO₄ (pH 7.4) containing NaCl (0.8%, w/v) and KCl (0.02%, w/v).

Apparatus
Melting points were measured on an electric microhot stage and are uncorrected. Proton ¹H-NMR analysis was performed using a JNM-GS 270 (JEOL, Tokyo) at 270.05 MHz. Chemical shifts are given as the value with tetramethylsilane as an internal standard (s, singlet; d, doublet; t, triplet; m, multiplet). The absorbance for ELISA was measured using an MPR A4i microplate reader (TOSOH, Tokyo, Japan). SDS-PAGE and immunoblotting were performed with NA-1013 mini gel slab electrophoresis equipment and an NA-1510B double cassette mini-transfer instrument (Nihon Eido, Tokyo, Japan), respectively. Centrifugal filtration was performed with a 1.5 ml ULTRACENT-10 microconcentrator (TOSOH). The filtrate has a nominal molecular weight cutoff of 10,000, and was used without preconditioning. The chromatograph used in this study was an LC-10 Advp (Shimadzu, Kyoto, Japan) equipped with an ultraviolet (UV) spectrophotometer (215 nm). A CAPCELL PAK C₁₈ UG 120 column [5 µm, 150 x 1 mm inner diameter (ID)] (Shiseido Co., Tokyo, Japan) was used at ambient temperature.

MALDI-TOF-MS analysis
Analysis by MALDI-TOF-MS in the positive-ion detection mode was carried out with a Voyager RP (PerSeptive Biosystems, Framingham, MA) equipped with a pulsed nitrogen laser (337 nm). The 5 µl aliquot of the samples in acetonitrile and water (1:1) containing 0.1% (v/v) trifluoroacetic acid was mixed with 5 µl of SA (10 mg/ml) for the protein and/or -CHCA (10 mg/ml) for the peptide in the same solvent, and 1 µl of the mixture was applied to the stainless steel sample plate and allowed to dry at room temperature and then subjected to MALDI-TOF-MS analysis. The measured m/z values were the average mass for the fragment ions. Peptides were initially analyzed in the linear mode with external calibration using the protonated ion [M+H]⁺ of angiotensin I (1,296.7), ACTH (clip 18–39) (2,465.2), ACTH (clip 7–38) (3,657.9), and insulin (5,733.6). The data were obtained from 256 laser shots using the following parameters: 20.0 kV accelerating voltage, 19.0 kV grid voltage, and 1 kV guide wire voltage.

Synthesis of N-(3-hydroxy-5β-cholan-24-oyl)-6-aminohexanoic acid
To a stirred solution of LCA (1 g) in dioxane (15 ml) were added p-nitrophenol (0.74 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbonyldiimide hydrochloride (1.1 g) at room temperature, and the mixture was stirred for 24 h at room temperature. The resulting mixture was diluted with ethyl acetate, washed with H₂O, and dried over anhydrous Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by column chromatography on a silica gel using toluene-acetone (10:1; v/v) as an eluent, to give LCA p-nitrophenyl ester (1.2 g). To the ester (300 mg) dissolved in pyridine (6 ml) was added a solution of 6-aminohexanoic acid (158 mg) in 1% NaOH (1 ml). The mixture, after having been kept stirred overnight at room temperature, was acidified to pH 2.0 with 5% HCl. The resulting precipitates were collected by filtration and then purified by column chromatography on a silica gel using chloroform-methanol (10:1; v/v) as an eluent. Recrystallization of the eluate from methylene chloride-methanol gave the 6-aminohexanoic acid derivative (261 mg) as colorless crystalline product. Melting point 86–92°C. Anal. Calcd for C₃₀H₅₁O₄N·1/4H₂O: C, 72.90; H, 10.50; N, 2.83. Found: C, 72.53; H, 10.25; N, 2.79. ¹H-NMR (CDCl₃) :0.65 (3H, s, 18-H), 0.92 (3H, s, 19-H), 0.92 (3H, d, J = 6.1 Hz, 21-H), 2.37 (2H, t, J = 8.1 Hz, -NHCH₂CH₂CH₂CH₂CH₂CO-), 3.25 (2H, m, -NHCH₂CH₂-), 3.63 (1H, m, 3β-H), 5.67 (1H, t, J = 7.6 Hz, -CONHCH₂-).

Synthesis of N-(3-hydroxy-5β-cholan-24-oyl)-6-aminohexanoic acid succinimidyl ester
To a stirred solution of the 6-aminohexanoic acid derivative (261 mg) in 95% dioxane (13 ml) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (204 mg) and N- hydroxysuccinimide (123 mg). The mixture, after having been kept stirred overnight at room temperature, was diluted with ethyl acetate, washed with H₂O, and dried over anhydrous Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by column chromatography on a silica gel using chloroform-methanol (10:1; v/v) as an eluent to give the succinimidyl ester (95 mg) as colorless crystalline product. mp 83.5–88°C. Anal. Calcd for C₃₄H₅₄O₅N₂·H₂O: C, 69.35; H, 9.59; N, 4.91. Found: C, 68.69; H, 9.14; N, 4.88. ¹H-NMR (CDCl₃) :0.64 (3H, s, 18-H), 0.92 (3H, s, 19-H), 0.92 (3H, d, J = 6.0 Hz, 21-H), 2.63 (2H, t, J = 7.0 Hz, -NHCH₂CH₂CH₂CH₂CH₂CO-), 3.26 (2H, m, -NHCH₂CH₂-), 3.61(1H, m, 3β-H), 5.66 (1H, t, J = 5.5 Hz, -CONHCH₂-).

Synthesis of 3-formyloxy-24-hydroxy-5β-cholane
To a stirred solution of LCA (2.0 g) in formic acid (20 ml) was added dropwise perchloric acid (60%) (0.4 ml) at 0°C, whereupon it was allowed to stir at the same temperature for 20 min. After addition of acetic anhydride (2 ml) under ice cooling, the resulting mixture was poured into ice water. The resulting precipitates were collected by filtration and dried in vacuo under P₂O₅. To the crude product (1.99 g) dissolved in anhydrous tetrahydrofurane (10 ml) were added dropwise triethylamine (1.36 ml) and ethyl chlorocarbonate (0.94 ml) under ice cooling, whereupon it was allowed to stir at the same temperature for 30 min, after which NaBH₄ (0.6 g) in H₂O (0.2 ml) was added dropwise to it. Stirring was continued for 1 h, after which acetic acid was added dropwise to decompose the excess reagent and extracted with ethyl acetate. The extract was washed with H₂O, 5% NaHCO₃, and saturated brine, and then dried over anhydrous Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by column chromatography on a silica gel using toluene-acetone (20:1; v/v) as an eluent to give the 3-formyloxy-24-alcohol (1.09 g). ¹H-NMR (CDCl₃) : 0.65 (3H, s, 18-H), 0.91 (3H, s, 19-H), 0.92 (3H, d, J = 6.0 Hz, 21-H), 3.61 (2H, t, J = 7.0 Hz, 24-H), 4.85 (1H, m, 3β-H), 8.00 (1H, s, -OCOH).

Synthesis of 3-formyloxy-24-hemisuccinoyloxy-5β-cholane
A mixed solution of 3-formyloxy-24-alcohol (765 mg) and succinic anhydride (392 mg) in pyridine (4 ml) was heated at 90°C for 9 h, after which the reaction mixture was diluted with ethyl acetate, washed with H₂O, and dried over anhydrous Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by column chromatography on a silica gel using n-hexane-ethyl acetate (7:1; v/v) as an eluent. Recrystallization of the eluate from acetone-hexane gave the 3-formyloxy-24-hemisuccinate (1.04 g) as colorless plate. mp 111–114°C. ¹H-NMR (CDCl₃) : 0.65 (3H, s, 18-H), 0.95 (3H, s, 19-H), 0.93 (3H, d, J = 6.0 Hz, 21-H), 2.62 (4H, m, -CO(CH₂)₂COOH), 4.06 (2H, m, 24-H), 4.85 (1H, m, 3β-H), 8.05 (1H, s, -OCOH).

Synthesis of 3-hydroxy-24-hemisuccinoyloxy-5β-cholane
To a solution of 3-formyloxy-24-hemisuccinate (1.04 g) in methanol (20 ml) was added 5% NaHCO₃ (5 ml), after which the mixture was stirred at 60°C for 2 h. After evaporation of the solvent in vacuo, the residue was diluted with ethyl acetate, washed with H₂O, and dried over anhydrous Na₂SO₄. The solvent was evaporated to dryness in vacuo to give the 3-hydroxy-24-hemisuccinate (157 mg) as colorless crystalline. mp 111–114°C. Anal. Calcd for C₂₈H₄₆O₅: C, 72.69; H, 10.02. Found: C, 72.44; H, 10.06. ¹H-NMR (CDCl₃) : 0.64 (3H, s, 18-H), 0.92 (3H, s, 19-H), 0.91 (3H, d, J = 6.0 Hz, 21-H), 2.65 (4H, m, -CO(CH₂)₂COOH), 3.64 (1H, m, 3β-H), 4.06 (2H, m, 24-H).

Synthesis of 3-hydroxy-24-hemisuccinyloxy-5β-cholane succinimidyl ester
To a solution of 3-hydroxy-24-hemisuccinate (300 mg) in dioxane (5 ml) were added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (250 mg), N-hydroxysuccinimide (150 mg) and H₂O (0.5 ml). The mixture, after having been kept stirred overnight at room temperature, was diluted with ethyl acetate, washed with H₂O, and dried over anhydrous Na₂SO₄. After evaporation of the solvent in vacuo, the crude product was purified by column chromatography on a silica gel using toluene-acetone (3:1; v/v) as an eluent to give the 24-succinimidyl ester (150 mg) as colorless crystalline product. mp 145–154°C. Anal. Calcd for C₃₂H₅₀NO₇·3/2H₂O: C, 65.39; H, 9.08; N, 2.38. Found: C, 65.02; H, 8.96; N, 2.37. ¹H-NMR (CDCl₃) : 0.64 (3H, s, 18-H), 0.91 (3H, d, J = 6.0 Hz, 21-H), 0.92 (3H, s, 19-H), 2.74 (2H, t, J = 7.0 Hz, -COCH₂CH₂COOH), 2.96 (2H, t, J = 7.0 Hz, -COCH₂CH₂COOH), 3.61 (1H, m, 3β-H), 4.09 (2H, m, 24-H).

Synthesis of LCA acyl-adenylate
LCA acyl-adenylate (LCA-AMP) was chemically synthesized by the method previously reported (26). The synthetic products were purified by column chromatography on Sephadex LH-20 (40 x 1.5 cm ID) using 40% methanol as the eluent at a flow rate of 18 ml/h. Fractions (each 0.3 ml) having a spot with a rate of flow value of 0.55 for LCA-AMP on TLC were collected, and the solvent was removed in vacuo, to give the desired compound (4% yield) as colorless amorphous substances that were kept at –80°C. The structures of the purified LCA-AMP were confirmed by ¹H-NMR (CD₃OD) : 0.50 (3H, s, 18-H), 0.87 (3H, s, 19-H), 0.95 (3H, d, J = 5.4 Hz, 21-H) 3.65 (3H, m, 3β-H), 4.16 and 4.30 (each 1H, m, 4'- and 5'-H), 4.44 and 4.63 (each 1H, t, J = 5.4 Hz, 2'- and 3'-H), 6.11 (1H, d, J = 5.4 Hz, 1'-H), 8.28 and 8.59 (each 1H, s, 2''- and 8''-H), and the negative-ion ESI mass spectrum: [M-H]^– at m/z 704.

Preparation of immunogen and enzyme-labeled antigen
Two kinds of hapten-carrier conjugates (10 and 11) were prepared by the reaction of the succinimidyl esters (4 and 8) with BSA (60 mol equiv. to BSA). The hapten/BSA molar ratio was determined to be 19 for the 10 and 28 for the 11 by titration of the residual free amino groups on BSA using trinitrobenzensulfonic acid. To obtain enzyme-labeled antigen, 4 was reacted with 0.2 mg of ALP in 0.1 M carbonate buffer (pH 9.0)-dioxane (1:1; v/v, 400 µl) at 4°C for 4 h. The molar ratio of the activated ester to enzyme in this reaction was adjusted to 40. Removal of unreacted steroid by dialysis afforded the desirable enzyme-labeled LCA, which was stored at 4°C until use.

Immunization of rabbit for production of anti-LCA antiserum
The hapten-BSA conjugate (1.5 mg) was dissolved in sterile saline (0.5 ml) and emulsified with Freund's complete adjuvant (0.5 ml). The emulsion was injected into domestic female albino rabbits (1.5–2 kg body weight) subcutaneously at multiple sites over the back. This procedure was repeated at 1 week intervals during the first month and then once every month. The antisera prepared from blood by centrifugation at 3,000 rpm for 10 min was stored at 4°C with NaN₃ (0.1%, w/v).

Characterization of antibodies
A solution of antiserum suitably diluted with buffer B (100 µl) was distributed in each well of a goat anti-rabbit IgG antibody-coated microplate. After incubation at 37°C for 1 h, the solutions were aspirated off and the wells were washed three times with buffer D. The ALP-labeled LCA (200 ng) in buffer B (100 µl) and standard bile acid solutions (50 µl) diluted with 50% methanol were then added, mixed, and incubated at 37°C for 2 h. After washing in the same manner, bound enzyme activity on the plate was colorimetrically measured using a substrate solution (100 µl) containing 1 mM p-nitrophenyl phosphate disodium salt and MgCl₂ (0.01%, w/v) in 50 mM carbonate buffer (pH 10). After incubation at 37°C for 60 min, the enzymatic reaction was terminated by the addition of 0.1 M NaOH (100 µl). The absorbance at 415 nm was measured using an MPR A4i microplate reader.

Immunological detection of LCA ovalbumin adducts
A solution of ovalbumin (2 mg) diluted with buffer A (100 µl) was distributed in each well of the 96-well EIA/RIA plates, which were left overnight at room temperature. After washing three times with buffer C, LCA-AMP (0, 1, and 10 µg) in 50% methanol (100 µl) was added to the wells. After incubation at 37°C for 24 h, the solutions were aspirated off and the wells were washed three times with buffer C. The wells were blocked with 5% skim milk solution in buffer C (300 µl) at 37°C for 2 h, and washed three times with buffer C. A 1:10,000 (v/v)-diluted antiserum (100 µl) diluted with buffer B was added to the wells, which were incubated at 37°C for 1 h. After washing three times with buffer B, a solution (100 µl) of 1:20,000-diluted HRP-labeled anti-rabbit IgG antibody diluted with buffer B was added and incubated at 37°C for 1 h. After washing as already described, a citrate-phosphate buffer (pH 5.0) containing 0.04% o-phenylenediamine·2HCl and 0.018% H₂O₂ was distributed to the wells, and the plates were incubated at room temperature for 15 min. The enzymatic reaction was terminated by the addition of 1 M H₂SO₄ (100 µl), and the absorbance at 492 nm was measured using an MPR A4i microplate reader.

Preparation of lysozyme-LCA adduct and structural elucidation
LCA-AMP (500 µg) was incubated with lysozyme (1 mg) in buffer C (1 ml) at 37°C for 96 h, and the reaction was stopped by addition of acetic acid (50 µl). The mixture was subjected to centrifugal filtration to remove any low-molecular-weight substances, followed by centrifugation in buffer A. A portion of the mixture was then subjected to reductive S-alkylation and proteolytic digestion with endoproteinase Lys-C, as follows. The mixture (418 µg) was reduced with DTT (8.64 µg) in 310 µl of 0.1 M Tris-HCl buffer (pH 8.1) containing 8 M urea and 2 mM EDTA for 15 min at 50°C, and carboxymethylated with sodium iodoacetate (11.6 µg) at room temperature in the dark for 1 h under N₂ atmosphere. A solution of endoproteinase Lys-C (0.4 µg) in H₂O (10 µl) was added to the reaction mixture, and the resulting mixture was incubated at 37°C for 4 h. The reaction was terminated by addition of acetic acid (100 µl). After desalting the mixture with Zip-Tip C₁₈, 1 µl of the mixture was subjected to MALDI-TOF-MS analysis.

Immunoblotting analysis of lysozyme-bound LCA
For SDS-PAGE, 10 µg protein was diluted to 1:1 with 125 mM Tris-HCl buffer (pH 6.8) containing 4% SDS, 2% 2-mercaptoethanol, 20% glycerol, and 0.02% pyronin Y, heated at 100°C for 5 min, and resolved on 15% acrylamide gels. Proteins resolved by polyacrylamide gels were transblotted to 0.45 µm nitrocellulose membrane at 55 V for 1 h in 25 mM Tris-192 mM glycine buffer (pH 8.3). After transferring, the blots were blocked by shaking overnight at 4°C in buffer C containing 5% skim milk. The blotted membranes were incubated with 1:1,000-diluted anti-LCA antiserum (Ab₂) for 3 h at room temperature with shaking. The blots were rinsed for three 5 min washes in buffer D and two 5 min washes in buffer C. The immunoblots were incubated for 1 h in 1:1,000-diluted goat anti-rabbit IgG diluted with buffer C. After washing as above, the immunoblots were incubated for 1 h with 1:1,000-diluted HRP-labeled protein G. The nitrocellulose membranes were again washed extensively as above, and the immunoreactive proteins were visualized with 0.05% (w/v) 3,3'-diaminobenzidine containing 0.03% H₂O₂ in buffer C for 5 min.

Immunoprecipitation of protein-bound LCA in the liver from bile duct-ligated rat
Male Wistar rats (230–250 g body weight) were obtained from Japan SLC. Inc. (Shizuoka, Japan), and used with approval from the Kinki University Committee for the Care and Use of Laboratory Animals, which conforms to National Institutes of Health guidelines. The rats were anesthetized with diethyl ether, and the common bile duct was ligated. Rats had free access to food and water. Animals were fasted overnight and then euthanized by decapitation under ether anesthesia. The liver (10 g wet weight) was minced and homogenized in 3 vol ice-cold 10 mM Tris-HCl buffer (pH 7.3) containing 0.25 M sucrose and 1 mM KCl by 10 rapid strokes using a motor-driven Teflon-glass homogenizer. The homogenate was centrifuged at 600 g for 10 min, followed by further centrifugation at 8,000 g for 10 min. The resulting supernatant was centrifuged at 105,000 g for 1 h. The supernatant was dialyzed against cold running water at 4°C for 48 h to remove low-molecular-weight substances, and was kept at –70°C until analysis.

One milliliter of supernatant (1 mg protein) was incubated with immobilized protein G beads (10 µl) and normal rabbit IgG-immobilized protein G beads (10 µl) at 4°C for 1 h, and then centrifuged at 15,000 rpm for 1 min. The supernatant was divided into two portions, and 1:1,000-diluted anti-LCA antiserum (Ab₂) (10 µl) and normal rabbit serum (10 µl) were added to each portion, and the resulting mixtures were incubated at 4°C for 3 h. After addition of protein G beads (10 µl), the mixture was incubated at 4°C for 1 h and then centrifuged at 2,000 rpm for 1 min. All the liquids were removed using a flat pipette tip and replaced with 400 µl of buffer C. The immunoprecipitates were washed three times with buffer C, and then subjected to 2-DE analysis.

2-DE of proteins and in-gel digestion
The immunoprecipitate (10 µl) was diluted with lysis buffer [7 M urea, 2 M thiourea, 5% CHAPS, 2% IPG buffer (pH 4–7) and 50 mM DTT] (70 µl), and allowed to stand at room temperature for 30 min. After centrifugation at 20,000 g for 5 min, the supernatant was diluted in rehydration buffer [8 M urea, 0.5% CHAPS, 0.5% IPG buffer, 20 mM DTT, and 0.04% bromophenol blue (BPB)] (70 µl), and was shaken at room temperature for 5 min. After centrifugation at 20,000 g for 5 min, the supernatant was loaded onto Immobiline DryStrip gels (pH 4–7, 7 cm) and rehydrated overnight. Isoelectric focusing was performed at 30°C using a multi-step protocol (200 V, 30 min; 400 V, 30 min; 1,000 V, 1 h; 2,000 V, 18 h). At the end of focusing, individual strips were equilibrated for 40 min with equilibration buffer [6 M urea, 30% glycerol, 1% SDS, 17.7 mM DTT, 0.04% BPB, 50 mM Tris-HCl buffer (pH 8.7)] (5 ml). For the second dimension, the proteins were separated by SDS-PAGE, which was performed at a constant voltage of 150 V using a 15% polyacrylamide separation gel for 80 min. The gel was stained with Silver Stain MS Kit (Wako Pure Chemical Industries, Japan). Protein spots were excised from the gel and placed in 1.5 ml microtubes and destained in 100 µl of destain solution containing 15 mM K₃[Fe(CN)₆] and 50 mM Na₂S₂O₃. The gel slices were washed for 30 min in 100 µl of 50% acetonitrile in 25 mM ammonium bicarbonate (pH 8.6) and then dried under a vacuum. Disulfide bonds were reduced with 10 mM DTT in the same buffer (100 µl) by incubation for 1 h at 56°C and alkylated with 55 mM iodoacetamide in the same buffer (100 µl) for 1 h in the dark at room temperature. The gel slices were washed twice. After the buffer was discarded, the gel pieces were dehydrated with 50% acetonitrile in 25 mM ammonium bicarbonate (pH 8.6) and then dried by vacuum centrifugation. The gel pieces were then reconstituted in 50 µl of digestion buffer containing 50 ng of sequencing-grade modified trypsin (Promega, Madison, WI) at 0°C for 30 min. Excess reagents were removed, and the gel slices were incubated at 37°C for 18 h. Then the gel slices were suspended in 10 µl extract buffer (3% acetonitrile, 2% trifluoroacetic acid in water) and the supernatant was desalted in Zip-Tip C₁₈ micro-columns and submitted to MALDI-TOF-MS analysis.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS AND DISCUSSION REFERENCES

 
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 -amino group of lysine residue on the proteins. Several studies have shown that the binding of antibodies is improved when the hapten is separated spatially from the coupling site with a carrier protein such as BSA and keyhole limpet hemocyanin. 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 (29–32). Accordingly, we designed immunogens in which the terminal carbon of the side chain of LCA was coupled to BSA via a 6-aminohexanoic acid and/or a succinic acid spacer to produce antibodies specific for the steroid moiety (Fig. 1 ). Initial efforts were, therefore, directed to preparing the haptenic derivatives of LCA coupled with 6-aminohexanoic acid and succinic acid as haptenic derivatives. The former compound (LCA-6AH) was prepared by the activated ester method via LCA p-nitrophenyl ester. On the other hand, the latter compound (7) was prepared from LCA in four steps. Thus, the 3-hydroxyl group of LCA was protected as a formyl ester, and the carboxyl group was reduced to a primary alcohol by reducing the mixed anhydride with NaBH₄. The hemisuccinate (6) was then formed by esterification with succinic anhydride in pyridine. Upon treatment with NaHCO₃, the formyl group was readily removed to produce the desired hapten (LCA-HS, 7). These haptenic derivatives were then converted to their corresponding succinimidyl esters (4 and 8), which were coupled to BSA by an activated ester method to produce LCA-BSA conjugates. A satisfactory number of hapten molecules (19 for 10 and 28 for 11) were incorporated in each conjugate. These immunogens were then subcutaneously injected into individual rabbits with Fruend's complete adjuvant. The titer of the antibody was checked at each stage of the immunization regimen by a competitive ELISA system using an enzyme-labeled hapten, and ALP-labeled LCA 6-aminohexanoic acid conjugate as a probe. The antisera obtained at 3 and 6 months after the initial immunization were shown by ELISA to have optimal dilutions of 500 for Ab₁, 160,000 for Ab₂, and 90,000 for Ab_3, and feasible dose-response curves for LCA-N-BOC-lysine in the range of 20 ng–5 µg per assay. An inhibition test was further performed by the addition of bile acid derivatives to compete with an enzyme-labeled antigen for binding to the antibody, and the cross-reactivities were determined by the 50% displacement method, taking the reactivity with LCA-N-BOC-lysine as 100%. Results are shown in Table 1 . As expected, the antibody (Ab₁) obtained from LCA-6-aminohexanoic acid-derivative BSA conjugate showed high reactivity for amidated and nonamidated forms of LCA and haptenic derivatives, whereas the reactivities toward other common bile acids in human body fluids were significantly lower. Similar phenomena were observed for the antibodies (Ab₂ and Ab₃) obtained by using LCA succinic acid-derivative BSA conjugate as the immunogen. However, the magnitudes of cross-reactivities to the above-mentioned compounds was somewhat low (14–127%) except for the high reactivity (3,090% and 1,735%) with the haptenic derivatives. These results are ascribable to the site of conjugation, because LCA was coupled with BSA through the bridge at the C-24 position, remote from the steroidal moiety. Hence, in immunoblotting, the developed antisera may be useful for detection of LCA anchored on proteins.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Structures of lithocholic acid (LCA), its related compounds, and the immunogen.

View larger version (8K): [in this window] [in a new window]   Fig. 2. Immunological detection of LCA residue on ovalbumin molecule introduced by the reaction of LCA acyl-adenylate (LCA-AMP) with lysozyme. Open and closed columns represent the results of Ab2 and Ab3, respectively. Numbers at the bottom indicate the amount (µg/well) of LCA-AMP.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrum (A) and high-performance liquid chromatogram (B) of an incubation mixture of LCA-AMP with lysozyme. HPLC conditions: column, CAPCELL PAK C18 UG 120 (5 µm, 150 mm x 1.0 mm inner diameter); mobile phase, solvent A (0.1% trifluoroacetic acid in H2O) and solvent B (0.1% trifluoroacetic acid in CH3CN) from 0:100 to 70:30 over 70 min; flow rate, 50 µl/min; detection, 215 nm.

View larger version (36K): [in this window] [in a new window]   Fig. 4. SDS-PAGE and immunoblot analysis of incubation mixture of LCA-AMP and lysozyme. Immunoblotting (1 and 2 µg/lane) was carried out as described in the Methods section.

Analysis of protein-bound LCA in the liver of the bile duct-ligated rat and its structural analysis by MALDI-TOF-MS
To identify proteins that had formed LCA adducts, we investigated liver tissue samples obtained from the bile duct-ligated rat as an animal model simulating cholestatic liver disease. In this study, subcellular fractions obtained by differential centrifugation, such as nuclei, microsomes, cytosol, and mitochondria, were examined by immunoprecipitation using Ab₂. The results derived from SDS-PAGE analysis indicated the presence of the target proteins in the cytosol (data not shown). We therefore carried out experiments aimed at isolating LCA-tagged proteins in the cytosol fraction by immunoprecipitation using Ab₂ antibody. The immunoprecipitation approach was a batch adsorption followed by elution, wherein cytosolic proteins from the liver of bile duct-ligated rats were captured. Proteins in the immunoprecipitates were resolved by 2-DE, and the gels were stained with silver stain to visualize protein bands (Fig. 5 ). Spots that were present in the specific immunoprecipitates captured by the anti-LCA antibody were excised, and three spots, which were not observed in the nonspecific immunoprecipitates captured by normal rabbit IgG, were destained and enzymatically digested as described in the Methods section. The resulting peptide mixtures were analyzed by MALDI-TOF-MS operating in linear mode (Fig. 6 ). The sequence of modified and unmodified peptides best matching the experimental molecule obtained with MALDI-TOF-MS was evaluated with a peptide mass database search (http://prospector.ucsf.edu/). The m/z values are the monoisotopic peaks of the protonated molecule [M+H]⁺. As depicted in Tables 3 – 5, peptide mass profiles were obtained from the database search that identified Rab-3 and M-Ras from spot 1, Rab-3 and Rab-16 from spot 2, and Rab-12, Rab-3, and Rab-16 from spot 3 with 33.5–47.1% coverage of the protein sequence. Of particular interest is the finding of Rab-3 modified with LCA from all the spots, whereby Rab-3-bound LCAs are covalently bound with different numbers and/or different binding sites. Among the ions of the identified peptides of Rab-3, ions at m/z 1,792.2 and 2,022.9 were the Lys-C peptides corresponding to residues 63–70 and 84–93, each with two molecules of LCA. In the case of M-Ras, the ions at 1,658.5, 1,741.7, 1,813.6, 2,097.9, 2,385.1, and 2,502.1 were the peptides corresponding to residues 148–158, 194–202, 128–138, 174–182, 136–147, 194–208, and 117–134, each with one or two molecules of LCA. It is also noted that several ions of the peptides linked to footnote ^a in Tables 4 and 5 were the peptides modified with one or two molecules of LCA. By using this strategy, three protein spots were successfully identified.

View larger version (54K): [in this window] [in a new window]   Fig. 5. Two-dimensional (2-DE) pattern of (left) specific immunoprecipitates and (right) nonspecific immunoprecipitates. Each 10 µg of protein of the samples was subjected to 2-DE system (first dimension, immobilized pH linear gradient, pH 3–10; second dimension, 10–20% SDS-PAGE). Proteins were visualized by silver staining. Selected proteins identified by excision and in-gel digestion followed by MS are indicated with spot numbers.

View larger version (10K): [in this window] [in a new window]   Fig. 6. MALDI-TOF mass spectra of peptides obtained from in-gel digestion of (A) protein spot 1, (B) protein spot 2, and (C) protein spot 3 excised from the gel shown in Fig. 5.

	ACKNOWLEDGMENTS

 
The authors would like to sincerely thank Professor Alan F. Hofmann for his constructive and insightful comments, which were instrumental in the preparation of this manuscript.

Submitted on July 7, 2008
Revised on July 18, 2008

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