Covalent Modification of Lysine Residues by Allyl Isothiocyanate in

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Covalent Modification of Lysine Residues by Allyl Isothiocyanate in Physiological Conditions: Plausible Transformation of Isothiocyanate from Thiol to Amine Toshiyuki Nakamura,† Yoshichika Kawai,‡ Noritoshi Kitamoto,† Toshihiko Osawa,§ and Yoji Kato*,† Graduate School of Human Science and EnVironment, UniVersity of Hyogo, Himeji 670-0092, Japan, Department of Food Science, Graduate School of Nutrition and Biosciences, The UniVersity of Tokushima, Tokushima 770-8503, Japan, and Nagoya UniVersity Graduate School of Bioagricultural Sciences, Nagoya 464-8601, Japan ReceiVed October 21, 2008

We investigated the reactivity of allyl isothiocyanate (AITC) with amino groups under physiological conditions. First, the chemical reaction of AITC with bovine serum albumin (BSA) was investigated. When BSA was incubated with AITC in a phosphate buffer (pH 7.4), the loss of Lys residues was observed. Second, the Lys residue NR-benzoyl-glycyl-L-lysine (BGK) was reacted with AITC in the buffer, and a novel peak was detected using high performance liquid chromatography (HPLC). The peak was purified and identified as AITC-modified BGK with a Nε-thiocarbamoyl linkage. However, a thiol residue is known to be a predominant target of an isothiocyanate (ITC). Although AITC may react with a thiol moiety in vivo, a thiocarbamoyl linkage between ITC and thiol is unstable, and an AITC molecule may be regenerated. To prove the plausible transformation of ITC from thiol to amine, synthetic AITCconjugated NR-acetyl-L-cysteine (NAC) was incubated with BGK at 37 °C in physiological buffer, and the generation of AITC-Lys was analyzed. The loss of the AITC-NAC adduct corresponded to the formation of the AITC-BGK adduct. Furthermore, using a novel monoclonal antibody (A4C7mAb) specific for AITC-Lys, we found that the AITC-Lys residue was generated from the reaction between AITCNAC and BSA. Although AITC preferentially reacts with thiol rather than with Lys, AITC can be liberated from thiols and can then react with amino groups. The ITC-Lys adduct may be a useful marker for ITC target molecules. Introduction Isothiocyanates (ITCs1) are derived from cruciferous plants such as Wasabia japonica (wasabi), mustard, cabbage, and broccoli. Of these ITC-containing foods, wasabi is a very popular spice in Japanese traditional meals such as Sushi. Wasabi rhizomes contain 0.247% allyl isothiocyanate (AITC, Figure 1), 0.038% 6-methylsulfinylhexyl isothiocyanate (6MSITC), and 0.003% 6-methylthiohexyl isothiocyanate (6MTITC) (1). The major component in wasabi, AITC, has bactericidal activities (2, 3) and can induce glutathione Stransferase (GST) (4-6). 6-MSITC acts as a potential inhibitor of human platelet aggregation (7) and has inhibitory effects on cancer cell growth (8, 9). Apart from wasabi, sulforaphane (4* Corresponding author. Fax: +81 (79) 293 5710. E-mail: yojikato@ shse.u-hyogo.ac.jp. † University of Hyogo. ‡ The University of Tokushima. § Nagoya University Graduate School of Bioagricultural Sciences. 1 Abbreviations: ITCs, isothiocyanates; BITC, benzyl isothiocyanate; PEITC, phenethyl isothiocyanate; AITC, allyl isothiocyanate; 6-MSITC, 6-methylsulfinylhexyl isothiocyanate; 6-MTITC, 6-methylthiohexyl isothiocyanate; GST, glutathione S-transferase; SFN, sulforaphane; BSA, bovine serum albumin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; KLH, keyhole limpet hemocyanin; BGK, NR-benzoyl-glycyl-L-lysine; NAC, NRacetyl-L-cysteine; DMSO, dimethyl sulfoxide; BCA, bicinchoninic acid assay; TNBS, trinitrobenzenesulfonic acid; HFBA, heptafluorobutyric acid; SIM, selected ion monitoring; ELISA, enzyme-linked immunosorbent assay; MS/MS, tandem mass spectrometry; MRM, multiple reaction monitoring; Keap1, kelch-like erythroid cell-derived protein with cap’n’collar homologyassociated protein 1; Nrf2, nuclear factor-E2-related factor 2; Boc-Lys, NRtert-butoxycarbonyl-L-lysine.

Figure 1. Chemical structure of AITC.

methylsulfinylbutyl ITC, SFN), which is one of the bioactive compounds in broccoli sprouts, can induce the activity of detoxification enzymes (10). Benzyl isothiocyanate (BITC) has been shown to induce cell cycle arrest and apoptosis (11), and phenethyl isothiocyanate (PEITC) has also been shown to trigger apoptosis (12). The biological activities of ITCs seem to be derived from the reaction of ITC with thiols to form an unstable thiocarbamoyl adduct (13, 14). It has been reported that 6-MSITC and SFN can induce phase II detoxification enzymes through the reaction of ITCs with protein thiols (15, 16). Furthermore, it has been shown that BITC inhibited cell growth and reacted with the Cys residue at 347 in R-tubulin (17). However, the thiol conjugate is known to be unstable at physiological conditions, especially in the presence of other free thiols (14). However, ITCs also react with amino moieties under alkaline pH forming a stable ITC-amine conjugate. This reaction has been utilized for protein sequencing (Edman degradation) and protein labeling with a fluorescent dye. However, the reaction of ITCs with amino moieties under physiological conditions has not been adequately explored. Therefore, in this study, we investigated the reactivity of AITC with Lys in a protein under neutral conditions. During this study, we prepared a monoclonal antibody against AITC-Lys to clarify the formation of the ITC-

10.1021/tx8003906 CCC: $40.75  2009 American Chemical Society Published on Web 02/13/2009

Reaction of Allyl Isothiocyanate with Lysine

amine adduct in proteins. We found that ITC-thiol conjugates generate ITC-amine conjugates under physiological conditions.

Materials and Methods Materials. AITC was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan). Bovine serum albumin (BSA, A7511, g97%), glyceraldehyde-3-phosphate dehydrogenase (GAPDH from rabbit muscle), peptidase (from porcine intestinal mucosa) and protease (from Streptomyces griseus) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Keyhole limpet hemocyanin (KLH) and standard amino acid (20088, Amino Acid Standard H) were purchased from Pierce (Rockford, IL). 13C- and 15N-labeled amino acid mixtures (13C g98% and 15N g98% of 20 amino acids) were purchased from Spectra Gases Inc. The composition of the amino acid mixture was as follows: alanine, 6.9%; arginine, 5.1%; asparagine, 3.6%; aspartic acid, 8.7%; cysteine, 3.6%; glutamine, 3.6%; glutamic acid, 9.2%; glycine, 5.8%; histidine, 2.1%; isoleucine, 2.8%; leucine, 7.6%; lysine, 10.9%; methionine, 1.4%; phenylalanine, 7.5%; proline, 5.1%; serine, 4.0%; threonine, 4.3%; tryptophan, 0.2%; tyrosine, 3.4%; valine, 4.1%. NR-Benzoyl-glycyl-L-lysine (BGK) was purchased from Peptide Institute, Inc. (Osaka, Japan). NR-Acetyl-L-cysteine (NAC) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). NR-tert-butoxycarbonyl-Llysine (Boc-Lys) was purchased from Novabiochem. All other chemicals were purchased from Wako Chemical Co. Ltd. (Osaka, Japan). Modification of Proteins by AITC or AITC-Modified NAC. Prior to the reaction, AITC was dissolved in dimethyl sulfoxide (DMSO) to a concentration of 1 M and then diluted in 0.1 M phosphate buffer (pH 7.4) to achieve concentrations of AITC ranging from 2.5-20 mM. BSA (5 mg/mL) was dissolved in 0.1 M phosphate buffer (pH 7.4) and exposed to various concentrations of AITC for 24 h at 37 °C. The modified protein was separated from unreacted AITC by a spin column (Bio-Rad, Micro Bio-Spin 6). The protein concentration was determined using a bicinchoninic acid assay (BCA protein assay; Pierce, Rockford, IL) and then adjusted by the addition of 0.1 M phosphate buffer (pH 7.4) to achieve a concentration of 3.5 mg/mL. The protein was stored at -20 °C until analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 0.7 mg/mL) was exposed to various concentrations AITC (0.78-12.5 µΜ) in 0.1 M phosphate buffer (pH 7.4) for 24 h at 37 °C. The modified protein was separated from unreacted AITC using a spin column, and the protein concentrations were determined using the BCA protein assay. The protein concentration was adjusted to 0.4 mg/mL by the addition of 0.1 M phosphate buffer (pH 7.4), and stored at -20 °C until analysis. Modification of the Lys residue by the AITC-thiol conjugate was examined as follows. AITC-NAC adduct (30-1000 µM), which had been synthesized as described below, was incubated with BSA (1 mg/mL) in 0.1 M phosphate buffer (pH 7.4) for 24 h at 37 °C. The modified protein was separated from the low molecular weight compound using a spin column, and the protein concentrations were determined using the BCA protein assay. The protein concentration was adjusted to 0.8 mg/mL by the addition of 0.1 M phosphate buffer (pH 7.4). Determination of Free Amino Groups in Protein. The modification of Lys was confirmed by quantitation of amino residues using the trinitrobenzenesulfonic acid (TNBS) method (18) with some modifications. The protein samples (150 µL) were incubated with 1.2 mL of 4% NaHCO3 and 60 µL of 0.1% TNBS for 1 h at 37 °C. This mixture was treated with 120 µL of 1 N HCl and 10% sodium dodecyl sulfate (SDS), and the

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absorbance at 340 nm was measured with a spectrophotometer (Shimadzu, UV mini 1240). A standard curve was prepared using glycine at concentrations ranging from 0.125-2.5 mM. Amino Acid Analysis of AITC-Modified BSA. AITCmodified BSA was hydrolyzed by 6 N HCl for 24 h at 105 °C. An internal standard comprising a stable amino acid mixture (13C, 15N) was added to the hydrolysates as described below and then measured by liquid chromatography-mass spectrometry (LC-MS) using a quadrupole tandem mass spectrometer (API-3000; Applied Biosystems Co.) connected to an Agilent 1100 high performance liquid chromatography (HPLC) system. The final concentration of 13C- and 15N-amino acid mixtures of the internal standard was determined on the basis of 25 µM stable methionine, which was the lowest concentration in the 13 C- and 15N-amino acid mixtures. This means that the final concentration of all internal standards was greater than 25 µM. In this condition, the Lys internal standard was approximately 200 µM. The separation was performed by gradient systems using solvent A (5 mM heptafluorobutyric acid (HFBA) in water) and solvent B (5 mM HFBA in CH3CN/water ) 9/1) with a Develosil RPAQUEOUS-AR-5 (2.0 × 150 mm) column at a flow rate of 0.2 mL/min. The gradient program was as follows: 0 min (A 100%), 20 min (A 80%), 30 min (A 80%), 31 min (A 100%), and 40 min (A 100%). The positive mode was used for the electrospray ionization. The amount of amino acids was estimated by selected ion monitoring (SIM) and compared with the known concentration of the amino acid standards. Analysis and Identification of AITC-Modified Lys. AITC was dissolved in DMSO at a concentration of 100 mM. BGK (1 mM) was incubated with 10 mM AITC for 0-24 h at 37 °C in 90 mM phosphate buffer (pH 7.4). The reaction was analyzed by reversed-phase HPLC with a photodiode array (PDA) detector using a Develosil ODS-HG-5 (4.6 × 150 mm) column. The HPLC was done by a gradient system using solvent A (0.1% acetic acid) and solvent B (CH3CN) at a flow rate of 0.8 mL/ min. The gradient program was as follows: 0 min (A 100%), 5 min (A 100%), 20 min (A 50%), 25 min (A 50%), 26 min (A 100%), and 40 min (A 100%). To identify the adduct, an isocratic separation of AITC-modified BGK was done by a Develosil Combi-RP (20 × 100 mm) column using 0.1% acetic acid/CH3CN (7/3) as the eluent at a flow rate of 5.0 mL/min. The major peak was collected, concentrated, and further purified by HPLC. The purified AITC-BGK adduct was identified by LC-MS and 1H nuclear magnetic resonance (1H NMR) analysis using the JNM-AL series AL300 (JEOL, Tokyo, Japan) in CD3OD. The spectral data were as follows: 1H NMR (ppm) 1.43 (m, 2H), 1.59 (m, 2H), 1.75 (m, 1H), 1.91 (m, 1H), 3.45 (m, 2H), 4.06 (m, 2H), 4.09 (m, 2H), 4.44 (m, 1H), 5.13 (m, 2H), 5.85 (m, 1H), 7.46 (t, J ) 7.7, 2H), 7.54 (t, J ) 7.3, 1H), 7.87 (d, J ) 6.9, 2H); LC-MS (ESI+) m/z 407.0 [M + H]+. Similarly, AITC-BGK, Nε-AITC-modified NR-Boc-Lys was also prepared and purified by HPLC. To remove the NR-Boc moiety, trifluoroacetic acid (2 mL) was added and then incubated for 30 min at room temperature. After drying the solution, the free AITC-Lys was identified by LC-MS (ESI+) m/z 246.0 [M + H]+, as described below. Estimation of AITC-Modified Lys in AITC-BSA. AITCmodified BSA (0.5 mg/mL) was enzymatically hydrolyzed by peptidase (0.01 mg/mL) in 0.1 M phosphate buffer (pH 7.4) containing 0.025% NaN3 at 37 °C overnight (19). Furthermore, additional peptidase (0.01 mg/mL) was used and reacted for 6 h at 37 °C. Protease (0.01 mg/mL) was then added, and the mixture was incubated at 37 °C overnight. The sample was

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centrifuged at 8000 rpm for 10 min at 4 °C, and the supernatant was filtered with a centrifugal filter device (Millipore, YM-10). The AITC-Lys residues were detected by LC-MS/MS with MRM of 246.0/84.0 [M + H]+. HPLC was done with a gradient system using solvent A (0.1% acetic acid) and solvent B (CH3CN) using a Develosil ODS-HG-3 (2.0 × 50 mm) column at a flow rate of 0.2 mL/min. The gradient program was as follows: 0 min (A 100%), 2 min (A 100%), 7 min (A 50%), 7.1 min (A 100%), 15 min (A 100%). Preparation of AITC-Modified NAC. The AITC-NAC adduct, N-acetyl-S-(N-allylthiocarbamoyl)-L-cysteine, was prepared as described previously with some modifications (14). Briefly, AITC (6.6 mg/mL) was dissolved in ethanol, and NAC (10.9 mg/mL) was dissolved in 50% aqueous ethanol. The NAC solution was adjusted to pH 7.8 using 1 N NaOH, and 0.6 mL of AITC solution was added to 1.2 mL of NAC solution. The mixture was stirred under N2 for 3 h at room temperature. The sample was dried by evaporation and purified by HPLC. The major peak was collected using the Develosil CombiRP (20 × 100 mm) column with 0.1% acetic acid/CH3CN (3/ 1) at a flow rate of 5.0 mL/min. The purified AITC-NAC adduct was identified by 1H NMR and LC-MS. The spectral data were as follows: 1H NMR (ppm) 1.84 (s, 3H), 3.40 (d, J ) 14.1, 1H), 3.86 (d, J ) 14.1, 1H), 4.18 (m, 2H), 4.55 (d, J ) 8.5, 1H), 5.05 (m, 2H), 5.78 (m, 1H); LC-MS (ESI+) m/z 263.0 [M + H]+. Coincubation of AITC-NAC with the Lys Residue. AITCNAC (1 mM) was incubated with 1 mM BGK in 0.1 M phosphate buffer (pH 7.4) for 24 h at 37 °C. The formation of AITC-BGK in the reaction mixture was analyzed by HPLC using the gradient system described in the section Analysis and Identification of AITC-Modified Lys. The AITC-BGK was detected by LC-MS/MS with MRM of 407.0/308.0 [M + H]+. The HPLC was done with a gradient system using solvent A (0.1% acetic acid) and solvent B (CH3CN) using the Develosil ODS-HG-3 (2.0 × 50 mm) column at a flow rate of 0.2 mL/ min. The gradient program was as follows: 0 min (A 100%), 7 min (A 30%), 7.5 min (A 30%), 7.6 min (A 100%), and 15 min (A 100%). AITC-NAC was quantified by LC-MS with a SIM of 263.0 [M + H]+ using isocratic separation by 0.1% acetic acid/CH3CN (8/2) with the ODS-HG-3 column. Preparation of the AITC-Lys Monoclonal Antibody. The KLH (5 mg/mL) was reacted with 10 mM AITC in DMSO in 65 mM borate buffer (pH 9.0) for 24 h at 37 °C. The AITCmodified KLH was dialyzed against phosphate-buffered saline (PBS) for 3 days at 4 °C with several exchanges of PBS. Similarly, the AITC-modified BSA was prepared to check the antibody titer. The obtained AITC-modified KLH (0.3 mg/mL in PBS) was emulsified with an equal volume of Freund’s complete adjuvant (20), and 250 µL of the emulsion was injected intraperitoneally into BALB/c mice. The mice were repeatedly boosted with the immunogens (0.3 mg/mL) emulsified with an equal volume of Freund’s incomplete adjuvant every 2 weeks. For the final boost, 100 µL of the immunogens (0.3 mg/mL) without adjuvant was injected intravenously. Three days after the final booster dose, the mice were sacrificed and spleens removed. Spleen cells were fused with myeloma cells in polyethylene glycol #6000 (Kanto Chem. Co., Tokyo, Japan) and cultured in hypoxanthine/aminopterin/thymidine medium for the selection of the hybridomas. After repeated screening and cloning, the immunoglobulin type of the obtained monoclonal antibody (A4C7mAb) was determined as IgG1 using an Isotyping Kit (Sigma-Aldrich Co.).

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Enzyme-Linked Immunosorbent Assay (ELISA). To screen the hybridomas, 50 µL of AITC-BSA (0.01 mg/mL in PBS) was dispensed in wells and incubated overnight at 4 °C. After washing three times with 200 µL of PBS containing 0.05% Tween-20 (TPBS), the wells were blocked with 200 µL of 1% Block Ace aqueous solution (Dainihon Sumitomo Seiyaku, Osaka, Japan) for 1 h at 37 °C. After washing, 50 µL of the conditioned medium of hybridomas was added to the wells and incubated for 2 h at 37 °C. After washing, 100 µL of peroxidaselabeled affinity purified antibody to mouse IgA+IgG+IgM (H+L) with a 1:5000 dilution in TPBS was added and reacted for 1 h at 37 °C. After washing, color development was done by adding an o-phenylenediamine and hydrogen peroxide mixture (100 µL). The reaction was terminated by adding 50 µL of 2 N H2SO4, and the plate was measured at 490 nm. For competitive ELISA, the wells were coated with AITCmodified BSA (0.01 mg/mL). At the same time, the antibody (0.25 µg/mL) and competitor (0-100 µM) in TPBS were reacted in a tube overnight at 4 °C. After washing and blocking of the plate, the reaction mixture was added and incubated for 2 h at 37 °C. After washing, 100 µL of peroxidase-labeled antimouse IgG with a 1:5000 dilution in TPBS was added and reacted for 1 h at 37 °C. After washing, color development was performed adding 100 µL of TMB substrate solution (KPL, Gaithersburg MD, USA). The reaction was terminated by adding 100 µL of 1 M phosphoric acid, and the plate was measured at 450 nm. The cross-reactivity of the antibody to the competitors was expressed as B/B0 in which B is the amount of the antibody bound to the coating antigen in the presence of the competitor, and B0 is the amount in the absence of the competitor. Analysis of AITC-Modified Proteins by SDS-PAGE and Western Blotting. AITC-modified GAPDH and AITCNAC-modified BSA were mixed with SDS-sample buffer without 2-mercaptoethanol. The proteins were applied to two gels for SDS-polyacrylamide gel electrophoresis (SDS-PAGE). In one gel, the protein was stained with SYPRO Orange (Invitrogen, Eugene, Oregon, USA) according to the manufacturer’s recommendations. With the other gel, the protein was electro-transferred onto an Immobilon-P transfer membrane (Millipore, Bedford, USA). After washing the membrane three times, each for 10 min, with TTBS (Tris-buffered saline containing 0.05% Tween-20), the membrane was blocked with 4% Block Ace aqueous solution for 1 h at room temperature. After washing, the membrane was incubated with A4C7mAb (0.5 µg/mL dilution in TTBS) for 2 h at room temperature. After washing again, the membrane was incubated with horseradish peroxidase-conjugated antimouse IgG at a 1:20000 dilution in TTBS for 1 h at room temperature. After washing, the membrane was visualized using an ECL-Plus detection reagent (GE Healthcare, UK). The images were captured by Lumino Image Analyzer LAS-1000plus (Fujifilm) and analyzed with Image Gauge software (ver. 3.4). Measurement of Free Thiol Concentration in GAPDH. The amount of free thiol in commercial GAPDH was measured using 5,5′-dithibis-(2-nitrobenzoic acid) (DTNB). GAPDH (10 mg/mL) and DTNB (10 mM) were dissolved in 50 mM phosphate buffer (pH 7.4). Twenty microliters of the DTNB solution was mixed with 200 µL of GAPDH solution to 780 µL of the 50 mM phosphate buffer (pH 7.4). After incubation for 15 min at room temperature in the dark, the absorbance was measured at 412 nm. The free thiol GAPDH concentration was calculated from the molar extinction coefficient (14100 M-1 L-1).


Figure 2. (A) Dose-dependent loss of free amino groups in BSA exposed to AITC in a phosphate buffer for 24 h at 37 °C. The residual free amines were measured by the TNBS assay. The value of native BSA was expressed as 100%. (B) Loss of Lys residues in BSA exposed to AITC. After acid hydrolysis, amino acids were analyzed by LC-MS with selected ion monitoring (SIM). The result is expressed as the number of residues per BSA on the constancy of 61 Leu residues.

Results Modification of Protein Lys by Allyl Isothiocyanate in Physiological Conditions. To examine the modification of protein by ITC, bovine serum albumin (BSA) was incubated with AITC in a phosphate buffer, and structural changes were analyzed. On the basis of the results of the gel experiment, aggregation and fragmentation of BSA by ITC were not observed (data not shown). However, a dose-dependent decrease in free amino groups of BSA was observed after exposure to AITC in a neutral buffer (Figure 2A). Approximately 70% of their number was reduced by exposure to 20 mM AITC, according to the TNBS method (15). To confirm that the loss of free amines was derived from conjugation of ITC to Lys, AITC-modified BSA was hydrolyzed by 6 N HCl, and the changes in amino acid compositions were then measured with LC-MS. As shown in Figure 2B, the number of Lys residues


was specifically decreased by AITC treatment and decreased from 50 to 35 residues. This means that approximately 30% of the loss of amino groups occurred with exposure to 20 mM AITC. Chemical Identification of AITC-Modified Lys. To confirm the chemical structure of the AITC-Lys conjugate, AITC was reacted with BGK, as a model Lys residue. As shown in Figure 3A, a novel peak was detected on the HPLC chromatogram at the retention time of 23.5 min. The compound had a molecular mass of 407.0 [M + H]+ (Figure 3B), which is the same as that of the predicted N-thiocarbamoyl adduct (Figure 3C). 1H NMR confirmed that the chemical structure of the isolated compound was that of the AITC-BGK conjugate. AITC-BGK was gradually generated at pH 7.4 in a time-dependent manner, and approximately 12% of BGK was transformed into the AITCLys conjugate during incubation for 9 h (Figure 3D). In addition, the adduct was stable for one week when incubated at 37 °C. When AITC-BSA was enzymatically hydrolyzed by peptidase and protease, free AITC-Lys was detected by LC-MS/MS using the MRM scan mode (246.0/84.0 [M + H]+). A novel peak was observed on the chromatogram of MRM. The retention time of the peak coincided with that of authentic AITC-Lys (Figure 3E). Preparation of the Monoclonal Antibody to AITC-Lys. We have shown the presence of the AITC-Lys with [M + H]+ 246.0 in the AITC-modified BSA hydrolyzed with peptidase and proteases (Figure 3E). To further prove the generation of AITC-Lys in protein molecules, we developed a novel monoclonal antibody using AITC-treated KLH as an immunogen. The obtained monoclonal antibody (A4C7mAb) was characterized by ELISA. As shown in Figure 4A, the antibody significantly recognized AITC-modified BSA but not native BSA. Moreover, A4C7mAb specifically recognized the AITC-Lys

Figure 3. (A) HPLC chart of reaction mixtures in the phosphate buffer. The inset number shows the combination of reactants: (1), BGK (2), AITC, and (3) AITC+BGK. (B) Total ion scanning for the novel peak from 23.3 to 23.7 min by LC-MS. The inset numbers indicate the type of the reaction mixture, as described in A. (C) Chemical structure of AITC-modified BGK. (D) Time-dependent generation of AITC-BGK adducts. Reaction of AITC (10 mM) with BGK (1 mM) was done in phosphate buffer (pH 7.4) for 0-24 h at 37 °C and analyzed by HPLC. (E) Detection of the AITC-Lys conjugate in the enzymatic hydrolysates of AITC-treated BSA. MRM 246.0/84.0 [M + H]+ for AITC-Lys was scanned by LC-MS/MS.

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Figure 5. Formation of AITC-Lys in the reaction between GAPDH and AITC. GAPDH was exposed to AITC in phosphate buffer and then applied to SDS-PAGE. The gel was blotted onto a PVDF membrane. The membrane was incubated with A4C7mAb, and the antigen-antibody complex was detected by chemiluminescence using ECL-Plus. Structural changes of the protein were also measured by protein staining using SYPRO Orange according to the manufacturer’s instructions. The migrated GAPDH in the gel showed two bands, probably because an intramolecular thiol cross-linkage was generated in the GAPDH. Figure 4. Characterization of the A4C7mAb to AITC-Lys by ELISA. (A) Reactivity of A4C7mAb with immobilized native and AITC-treated BSA on a microplate. Symbols: (b) AITC-BSA and (O) native BSA. (B) Competitive ELISA using structurally related compounds of AITCBGK as competitors. Symbols: (b) AITC-BGK, (O) allylamine, (9) BGK, and (0) AITC-NAC. The cross-reactivity of the antibody to the competitors was expressed as B/B0 in which B is the amount of the antibody bound to the coating antigen in the presence of the competitor, and B0 is the amount in the absence of the competitor. (C) Chemical structure of competitors used.

between AITC and protein, BSA was exposed to AITC-NAC, and the generation of AITC-Lys in BSA was then immunochemically confirmed. As shown in Figure 6D, a positive band for BSA was increased dose dependently by AITC-NAC, suggesting that the AITC migrated between the thiol and Lys in protein.

conjugate, but not allylamine, an analogue of AITC, and BGK (Figure 4B,C). We also confirmed that the antibody did not react with BITC-Lys and 6-MSITC-Lys (unpublished data). Furthermore, the A4C7mAb did not recognize AITC-NAC. These results suggest that this monoclonal antibody was specific for AITC-Lys. Immunochemical Detection of AITC-Modified Lys in Protein. To examine the effect of free thiol residues in a protein on AITC-Lys formation, rabbit GAPDH was used as a model of thiol proteins. When GAPDH was exposed to AITC (0.78-12.5 µM) in phosphate buffer (pH 7.4), AITC-Lys was formed by AITC treatment even at micromolar concentrations (Figure 5). We have confirmed that the commercial GAPDH used has approximately 10 µM of free thiols in the reaction mixture. GAPDH was significantly stained by the antibody at the AITC concentration of 1.56 µM (6.4-fold less than the concentration of free thiols in GAPDH). Transfer Reaction of AITC from Thiol to Amine. Thiol compounds, such as glutathione, are abundant in biological tissues. ITC might preferentially and predominantly react with thiols rather than amines; however, the thiol conjugates are unstable (13, 14). To examine the possibility of the transfer of ITC from thiols to amines, a synthetic AITC-NAC adduct was incubated with BGK at physiological conditions. AITC-NAC was completely degraded within 24 h of incubating 1 mM AITC-NAC with 1 mM BGK (Figure 6A), and AITC-BGK was generated (Figure 6B). LC-MS was used to quantify the AITC adducts, and over 24 h, the formation of 150 nM AITC-BGK was estimated (Figure 6C). This suggests that the AITC moiety moved from thiol to BGK. Although the initial concentration of AITC-NAC was 1 mM, only half of the adduct could be measured. This also suggests that the thiol adduct is unstable during storage, even at 4 °C. To investigate the transformation

ITCs are a unique component in foods derived from vegetables and spices. Because ITC has a highly reactive structure, R-NdCdS, it reacts with thiols to form a thiocarbamoyl adduct (Scheme 1). This conjugation step might play a key role in their biological activities. It has been postulated that nuclear factorE2-related factor 2 (Nrf2) is dissociated from kelch-like erythroid cell-derived protein with cap‘n’collar homology-associated protein 1 (Keap1) by 6-MSITC as well as SFN through electrophilic conjugations with thiols of Keap1; Nrf2 then binds to an antioxidant response element sequence (15, 16). This leads to the induction of phase II detoxification enzymes to defend against oxidative stress. Similarly, other ITCs may activate signal transduction via reaction with thiols. It has been shown that SFN-modified Cys was generated by the reaction of SFN with Keap1 protein (13). Recently, it has been reported that Cys-347 in R-tubulin in cells was covalently modified by BITC, as determined by mass spectrometry (17). However, as shown in previous reports (13, 14), ITC-thiol conjugates have to be handled with care, because the thiolconjugated ITC molecule can easily transform into other free thiols at physiological conditions. Although AITC is known to react with Lys residues (21), this reaction has not been examined in detail at physiological conditions. We have considered that the stable adduct is produced by a reaction of ITCs with Lys in vivo. If the stable adduct is formed, it might offer a useful marker to identify ITC target molecules. In the present study, we investigated the chemical reaction of AITC with a protein and confirmed the formation of an ITC-amine adduct in physiological conditions. AITC-Lys was generated in these conditions, and the conjugate was stable. As shown in Figure 2A and B, there is a difference between the TNBS assay and amino acid analysis in terms of the decreased number of Lys residues. We found that most

Discussion



Figure 6. Formation of AITC-Lys during the incubation of AITC-Cys with Lys. AITC-NAC was incubated with BGK or BSA in phosphate buffer. (A) SIM chart for AITC-NAC 263.0 [M + H]+. (B) MRM chart for AITC-BGK 407.0/308.0 [M + H]+. (C) Quantification of AITC-NAC and AITC-BGK. Symbols: (b) AITC-BGK and (O) AITC-NAC. (D) Generation of AITC-Lys during the incubation of AITC-NAC with BSA. The protein was stained with fluorescent dye using SYPRO Orange, and the formation of AITC-Lys was visualized by immunoblot analysis using A4C7mAb.

Scheme 1. Plausible Mechanism for the Reaction of ITC with Cys or Lys in Physiological Conditions

AITC-Lys conjugates were degraded by acid hydrolysis, and some free Lys residues were regenerated from AITC-Lys conjugates. This may explain why the content of Lys residues in the hydrolysate of AITC-treated BSA was hardly changed, as shown Figure 2B. In addition, it has been reported that only two lysines in albumin were modified by a metal-catalyzed oxidation system, while HOCl induced the modification of five lysine residues (22). TNBS might selectively react with some Lys residues in albumin. The dramatic reduction in the number of free amines by the TNBS method (compared with amino acid analysis) might indicate that the TNBS-reactive (several) amines were modified by AITC. We examined the AITC-derived modification of GAPDH, which has four thiols per molecule, using a novel monoclonal antibody to AITC-Lys. AITC-Lys was still produced in the presence of thiol residues (Figure 5), suggesting that the protein Lys residues can be a target of ITC in vivo. As described above, it has been shown that thiol could migrate between ITC-thiol and free thiol (14). Since the reaction product of ITC with thiol is unstable, ITC can be liberated from the thiols and may react with Lys. Although the transformation of AITC from Cys to Lys is a minor reaction in the peptide system (Figure 6C), the

Lys adduct should be stable enough for detection by LC-MS/ MS or by immunochemical procedures (Figure 6B and D). It has been reported that ITC released from the ITC-thiol conjugate inhibited P450 enzyme activity, but the rate of ITCthiol deconjugation was markedly reduced in the presence of physiological levels of GSH (23). In this manner, the chemopreventive activity of ITCs may occur by other mechanisms as well as the thiol-dependent reaction. Because ITC-Lys conjugation may cause structural and functional changes of the protein, the formation of ITC-Lys could, at least partly, contribute to chemoprevention by ITC in vivo. Moreover, it has been considered that ITC induced cytotoxicity and apoptosis by ITC acting directly or as liberated from cysteine or glutathione conjugates (24). The addition of the AITC-Lys conjugate to the culture medium did not induce toxicity against human leukemia HL60 cells, even at a concentration of 1 mM for 24 h (unpublished data). However, it is possible that exposure to AITC might induce cell toxicity owing to reactions with the intracellular pool of free amino acids through the conjugation between the amino moiety and the ITC. In conclusion, although some ITCs may directly react with amines, the majority of ITC preferentially reacts with thiols. However, the thiocarbamoyl linkage between thiols and ITCs gradually degrades and regenerates free ITCs. ITCs might react with Lys residues to form stable conjugates. Therefore, the ITCLys adduct may be a useful stable marker to identify ITC target molecules. Work to identify ITC-target molecules in cells using the ITC-amine adduct as a probe is now in progress. Acknowledgment. We thank Kinjirushi Wasabi, Co. Ltd. (Nagoya Japan) for kindly providing 6-MSITC, Shinsuke Hisaka for helpful technical assistance, and Dr. Yoshimasa Nakamura (Okayama University) for advice and for critically reviewing this manuscript.

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Covalent Modification of Lysine Residues by Allyl Isothiocyanate in

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