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Identification of polyphenol-specific innate epitopes originated from a resveratrol analogue Mai Furuhashi, Yukinori Hatasa, Sae Kawamura, Takahiro Shibata, Mitsugu Akagawa, and Koji Uchida Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00409 • Publication Date (Web): 10 Aug 2017 Downloaded from http://pubs.acs.org on August 14, 2017

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Biochemistry

Identification of polyphenol-specific innate epitopes originated from a resveratrol analogue Mai Furuhashi†, Yukinori Hatasa†, Sae Kawamura†, Takahiro Shibata†, §, Mitsugu Akagawa‡, and Koji Uchida†, ¶, * †

Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan

§

PRESTO, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

‡

Department of Biological Chemistry, Division of Applied Life Science, Graduate School of

Life and Environmental Sciences, Osaka Prefecture University, Sakai, Japan ¶

Laboratory of Food Chemistry, Graduate School of Agricultural and Life Sciences, The

University of Tokyo, Tokyo 113-8657, Japan *

To whom correspondence should be addressed. Koji Uchida, Ph.D., Laboratory of Food

Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. Tel: 81-3-5841-5127, Fax: 81-3-5841-8026, E-mail: [email protected]

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ACS Paragon Plus Environment

Biochemistry

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ABSTRACT

Polyphenols have received significant attention in disease prevention due to their unique chemical and biological properties. However, the underlying molecular mechanism for their beneficial effects remains unclear. We have now identified a polyphenol as a source of innate epitopes detected in natural IgM and established a unique gain-of-function mechanism in the formation of innate epitopes by polyphenol via the polymerization of proteins. Upon incubation with bovine serum albumin (BSA) under physiological conditions, several polyphenols converted the protein into the innate epitopes recognized by the IgM Abs. Of interest, piceatannol, a naturally-occurring hydroxylated analogue of a red wine polyphenol, resveratrol, mediated the modification of BSA, whose polymerized form was specifically recognized by the IgMs. The piceatannol-mediated polymerization of the protein was associated with the formation of a lysine-derived cross-link, dehydrolysinonorleucine. In addition, an oxidatively deaminated product, α-aminoadipic semialdehyde, was detected as a potential precursor for the cross-link in the piceatannol-treated BSA, suggesting that the polymerization of the protein might be mediated by the oxidation of a lysine residue by piceatannol followed by a Schiff-base reaction with the ε-amino group of an unoxidized lysine residue. The results of this study established a novel mechanism for the formation of innate epitopes by small dietary molecules and support the notion that many of the beneficial effects of polyphenols could be attributed, at least in part, to their lysyl oxidase-like activity. They also suggest that resveratrol may show its beneficial effects on human health by conversion to piceatannol.

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Biochemistry

INTRODUCTION

Phytochemicals, non-nutritive plant chemicals in fruits and vegetables, are believed to play a role in their beneficial effects on human health. Polyphenols are the most diverse group of phytochemicals and have attracted significant attention as potential chemopreventive agents against chronic diseases, such as heart disease, cancer, diabetes, stroke, and arthritis. They are considered as antioxidants in general; however, their roles in health and in disease still remain unclear. Dietary small molecules, including polyphenols, are absorbed across the intestinal epithelium into the bloodstream and are widely distributed in mammalian tissues. 1-4 The most abundant carrier proteins for these small molecules in the circulatory system are serum albumins. They can bind a wide variety of small molecules and even undergo covalent modification by electrophilic small molecules and gain new function in contributing to biological processes associated with the immune and inflammatory responses.5, 6 Natural antibodies (NAbs), essentially antibodies (Abs) of the IgM isotype present in the circulation of mammalian species, are mainly produced by B-1 lymphocytes independently of external antigenic stimulation. NAbs provide immediate protection against pathogens and play an important role in the host defense mechanism against various stresses. Most of NAbs are poly-reactive (multi-specific or cross-reactive) in nature and recognize specific molecular patterns with no apparent structural similarity.7-9 It has been demonstrated that the modified self-proteins, such as oxidized low-density lipoproteins, are important targets of NAbs.10, 11 In addition, based on the findings that these modified proteins have several properties different from native proteins, including an elevated electronegative potential due to modification of the positively charged amino acid residues, we have proposed that the electronegative potential of antigens might be involved, at least in part, in the recognition by NAbs.6 Lysyl oxidase, a copper-dependent amine oxidase, plays an important role in the biogenesis of connective tissue matrices by cross-linking the extracellular matrix proteins, such as collagen and elastin. The enzyme catalyzes the oxidation of lysine residues to α-aminoadipic semialdehyde (AAS), which form covalent cross-links in collagens and elastin. It is known that some polyphenols, catechol-type polyphenols in particular, gain the lysyl oxidase-like activity via oxidation to the corresponding o-quinone derivatives.12, 13 (-)-Epigallocatechin-3-O-gallate (EGCG), among them, is the most studied polyphenol component with the lysyl oxidase-like activity, mediating the oxidative deamination of lysine residues in proteins. Strikingly, our recent study provided multiple lines of evidence 3


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suggesting that these polyphenols with the lysyl oxidase-like activity could be an endogenous source of innate epitopes recognized by NAbs (Fig. 1A).14 In addition, we suggested a mechanism, in which the electronegative potential of the polyphenol-treated proteins might be involved, at least in part, in the recognition by NAbs. However, the underlying molecular mechanism for the formation of the innate epitopes by polyphenols remains unclear. Here we further extended our study on the formation of polyphenol-derived innate epitopes and discover a unique gain-of-function mechanism via the polymerization of proteins. Moreover, we provided the first mechanistic details of the formation of a structural element in the polymerized proteins essential for the recognition by NAbs. These findings support the notion that many of the beneficial effects of polyphenols could be attributed, at least in part, to their lysyl oxidase-like activity.

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Biochemistry

MATERIALS AND METHODS

Materials Piceatannol was obtained from Tokyo Chemical Industry Co., Ltd. BSA was obtained from Wako Pure Chemicals (Japan). The IgM mAbs, DDL17 and ADL19, were established from the MRL-lpr mice.6 The anti-citrulline rabbit polyclonal antibody (ab100932) was obtained from Abcam. All of the other reagents used in the study were of analytical grade and obtained from commercial sources.

Animals Female BALB/c, female MRL-MpJ, and female MRL-lpr mice, which carry a defective Fas gene and develop a spontaneous systemic lupus erythematosus-like disease as they age, were purchased from Japan SLC (Hamamatsu, Japan). For the ELISA analysis, the mice were used as sera donors at 10-12 weeks old. Blood was collected from tail vein and allowed to stand for 2 h at room temperature, after which the sera were collected by centrifugation at 3,500 rpm for 10 min and stored at -20 °C until use. All procedures were approved by the Animal Experiment Committee in the Graduate School of Bioagricultural Sciences, Nagoya University (Permit Number: 2015030216 and 2015051301).

Preparation of modified proteins in vitro Polyphenols were dissolved in dimethyl sulfoxide (DMSO) to the concentration of 10 mM. BSA (1.0 mg/ml) was incubated with 1.0 mM polyphenols in PBS (pH 7.4) at 37 °C under atmospheric oxygen. After 24 h, aliquots were collected and dialyzed against PBS. The glutaraldehyde-modified BSA was prepared upon incubation of BSA (1.0 mg/ml) with 19 mM glutaraldehyde for 24 h in PBS (pH 7.4) at 4 °C.

ELISA (enzyme-linked immunosorbent assay) ELISA was performed as previously reported.14

Preparation of IgM mAbs against polyphenol-specific epitopes Preparation of IgM mAbs against polyphenol-specific epitopes was previously described.14 Briefly, hybridoma were obtained from fusions between P3/U1 mouse myeloma cells and spleen cells from SPF-maintained BALB/c mice. The hybridoma were screened for the 5


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production of antibodies using an ELISA with EGCG-treated BSA as an antigen. After repeated screenings and limited dilutions, six clones showing the most distinctive recognition of the EGCG-treated BSA were obtained.

Polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the polyphenol-treated proteins was performed according to Laemmli.15 Mobility shifts of the modified proteins were also analyzed by nondenaturing polyacrylamide gel electrophoresis (native PAGE). After electrophoresis, the gel was stained with Coomassie Brilliant Blue.

MALDI-TOF MS analysis of polyphenol-treated proteins MALDI-TOF MS analysis of the native and piceatannol-treated BSA was performed using an ABI 4700 Proteomics Analyzer MALDI-TOF-TOF mass spectrometer with version 3.6 software (AB-Sciex) operated in the linear mode.

Gel filtration Gel filtration chromatography was carried out using the ÄKTA Prime supplied by GE Healthcare Life Sciences (Sweden). The native and piceatannol-treated BSAs were eluted with PBS at the flow rate of 0.2 ml/min at 4°C with monitoring of the absorbance at 280 nm. The column system was composed of Hi Prep 16/60 Sephacryl S-300.

Particle size and zeta potential The measurements of the average particle size and zeta potential of the piceatannol-treated proteins were carried out by a Malvern Zetasizer Nano-ZS dynamic light-scattering (DLS) analyzer (Malvern Instruments, Ltd., Malvern, Worcestershire, UK).

Comprehensive analysis of modified amino acids using LC-ESI-MS/MS Acid hydrolyzed proteins used for comprehensive analysis of the modified lysines were redissolved in H2O, then subjected to LC-ESI-MS/MS analysis using a TQD triple stage quadrupole mass spectrometer (Waters) equipped with an ACQUITY ultra-performance LC (UPLC) system (Waters). The sample injection volumes of 10 µl each were separated on an Intrada amino acid column (100 × 2.0 mm) (Imtakt) at the flow rate of 0.3 ml/min. A gradient was used by solvent A (acetonitrile containing 0.1% formic acid) with solvent B (100mM 6


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Biochemistry

ammonium formate) as follows: 10% B at 0 min, 10% B at 2 min, 100% B at 8 min, 100% B at 10 min. MRM was performed in the positive ion mode using nitrogen as the nebulizing gas. The experimental conditions were set as follows: ion source temperature, 120 oC; desolvation temperature, 350 oC; cone voltage, 15 eV; collison energy, 15 eV; desolvation gas flow rate, 700 l/h; cone gas flow rate, 50 l/h; collision gas, argon. The strategy was designed to detect the product ion (m/z 84.0, for lysine adducts; m/z 110.0, for histidine adducts; m/z 70, for arginine adducts) from positively ionized lysine, histidine, and arginine adducts by monitoring the samples transmitting their [M+H]+>84.0 (for lysine adducts), [M+H]+>110.0 (for His adducts), and [M+H]+>70.0 (for Arg adducts) transitions.

Synthesis of LNL LNL was prepared by reductive amination of Nα-acetyl-L-AAS using Nα-acetyl-L-lysine instead of ABA. The resulting LNL was separated by silica gel column chromatography using ethyl acetate/acetic acid/water (3:2:2, v/v/v) as elution solvent. LNL was characterized from FAB-MS, 1H-NMR, and 1H-1H COSY spectra. LNL: FAB MS 276 (M+H)+; 1H NMR (500 MHz, D2O) δ = 1.37 (m, 4H, β-CH2), 1.61 (quin, 4H, δ-CH2) 1.82 (m, 4H, γ-CH2), 2.93 (t, 4H, J=7.9 Hz, ε-CH2), 3.79 (t, 2H, J=6.2, α-CH).

LC-ESI-MS/MS analysis of LNL Acid hydrolyzed proteins used for the LC-ESI-MS/MS analysis of LNL were redissolved in ethanol, then subjected to LC-ESI-MS/MS analysis using the TQD-ACQUITY UPLC system (Waters). The sample injection volumes of 10 µl each were separated on an Intrada amino acid column (100 × 2.0 mm) (Imtakt) at the flow rate of 0.3 ml/min. A discontinuous gradient was used by solvent A (acetonitrile containing 0.1% formic acid) with solvent B (100mM ammonium formate) as follows: 10% B at 0 min, 10% B at 2 min, 100% B at 8 min, 100% B at 10 min. The mass spectrometer was operated in the MRM mode with positive electrospray ionization (ESI+) to analyze LNL by monitoring the samples transmitting m/z 276>84.0 transitions.

LC-ESI-MS/MS analysis of oxidized lysine The authentic ABA derivative of aminoadipic semialdehyde (AAS) was prepared as previously reported.16 The ABA derivatization and LC-MS-MS analysis of ABA-derivatized AAS were performed as previously described. 14 Briefly, the protein samples were reductively 7


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aminated with 25 mM ABA in 1.1 M HCl and 16 mM NaCNBH3 in H2O at 37oC in the dark. After reacting for 2 h, the protein was precipitated by trichloroacetic acid (TCA), and the resulting protein was then acid-hydrolyzed for 24 h at 110 °C. The hydrolysate was dried followed by reconstitution in H2O. Mass spectrometric analyses were performed using the TQD-ACQUITY UPLC system (Waters) with an ACQUITY UPLC BEH C18 column (150 mm × 2.1 mm, Waters) at the flow rate of 0.3 ml/min. A gradient was used by solvent A (H2O containing 0.1% formic acid) with solvent B (acetonitrile containing 0.1% formic acid) as follows: 1% B at 0 min, 1% B at 1min, 100% B at 6min, 100% B at 8 min. The monitored MRM transitions (positive ion mode, 20 eV cone potential /15 eV collision energy) were as follows: ABA-AAS, m/z 267>84; ABA-GGS, m/z 253>70.

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Biochemistry

RESULTS

Recognition of polyphenol-specific epitopes by IgM Abs As shown in Figure 1B (left panel), the protein that had been treated with polyphenols (Figure S1), such as ellagic acid, piceatannol, malvidin, and eriodictyol, showed a modest cross-reactivity with the sera from the specific pathogen-free (SPF)-maintained BALB/c mice. The data suggest that the basal IgM titers against the polyphenol-derived antigens are largely independent of noncommensal exposure to microbial pathogens. On the other hand, the production of immunoglobulins, IgM in particular, is highly upregulated in the autoimmune or autoinflammatory diseases, such as systemic lupus erythematosus and rheumatoid arthritis, by inducing innate immune activation. Indeed, the epitopes prepared upon incubation of bovine serum albumin (BSA) with the polyphenols, such as malvidin, genistein, curcumin and piceatannol, showed a significant cross-reactivity with the sera from the MRL-lpr mice, a spontaneous murine model of autoimmune disease (Figure 1B, right panel). These polyphenol-derived epitopes were also recognized by the IgM monoclonal Abs (mAbs), DDL17 and ADL19, established from the MRL-lpr mice (Figure 2A). In addition, the anti-citrullinated protein autoantibodies (autoAbs), an important serological marker in the A

B Serum (BALB/c)

Polyphenols

Albumin

IgM Albumin

Electronegative proteins

Serum (MRL-lpr)

Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannnol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Luteolin Chrysin Kaempferol Quercitrin Quercetin Control 0

0.5

1.0 0 Absorbance (490 nm)

0.5

1.0

Figure 1. Recognition of polyphenol-specific epitopes by innate IgM Abs. (A) Schematic illustration of the polyphenol-mediated transformation of serum albumins into innate epitopes. (B) Cross-reactivity of polyphenol-treated BSAs with the sera from the SPF-maintained BALB/c (left panel) and MRL-lpr (right panel) mice. “Control” represents a sample containing only protein (BSA).

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diagnosis of rheumatoid arthritis, also showed a cross-reactivity toward several polyphenol-derived epitopes (Figure 2B). Of interest, not all the polyphenols exhibited an epitope-producing activity upon incubation with BSA. The potential source of epitopes recognized by the autoAbs includes cyanidin, delphinidin, diosmetin, and piceatannol. The data suggest that these polyphenols could be an important source of innate epitopes recognized by the innate Abs.

A

mAb DDL17

B

mAb ADL19

Anti-citrulline Abs Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannnol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Diosmetin Luteolin Apigenin Chrysin Kaempferol Quercitrin Quercetin Control

Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannnol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Diosmetin Luteolin Apigenin Chrysin Kaempferol Quercitrin Quercetin Control 0

1.0

2.0

3.00

0.4

0.8

1.6

Absorbance (490 nm)

0

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1.2

Absorbance (490 nm)

Figure 2. Cross-reactivity of polyphenol-specific epitopes with autoAbs. (A) Cross-reactivity of polyphenol-treated BSAs with the sera from the MRL-lpr mice and with the IgM mAbs DDL17 and ADL19 established from MRL-lpr mice. (B) Cross-reactivity of polyphenol-treated BSAs with the anti-citrulline polyclonal Abs. In panels A and B, all the polyphenols tested were dissolved with DMSO and “control” represents a sample containing only protein (BSA).

Specificity of IgM mAbs to the polyphenol-specific epitopes We next sought to isolate the hybridoma clones, producing IgM mAbs, from the BALB/c SPF mice using the polyphenol-treated BSA as a screening antigen. Six IgM mAbs (SBM1, SBM2, SBM3, SBM4, SBM5, and SBM6), showing a recognition specificity toward the polyphenol (EGCG)-treated protein, were established from the SPF mice after screening based on the specific binding to the corresponding antigen (Figure 3A). These mAbs established from the SPF mice were found to be IgM. Of interest, the specificity study showed that the IgM mAb SBM3 cross-reacted not only with the polyphenol-treated BSA, but also with the protein that had been treated with a number of polyphenols, including piceatannol, malvidin, cyanidin, 10


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Biochemistry

and delphinidin (Figure 3B). Figure 3C shows the time-dependent formation of the IgM mAb SBM3-positive innate epitopes generated upon incubation of BSA with piceatannol, a naturally-occurring hydroxylated analogue of a red wine polyphenol, resveratrol. Piceatannol also produced innate epitopes in a dose-dependent manner (Figure S2). We also observed that the IgM titers were accelerated by the immunization of BALB/c mice with the piceatannol-modified protein (Figure 3D).

Figure 3. Specificity of the IgM mAbs to the polyphenol-specific epitopes. (A) Recognition of EGCG-treated BSA by the IgM mAbs established from the SPF-maintained BALB/c mice. (B) Recognition of polyphenol-treated BSAs by the IgM mAb SBM3. (C) Upper, chemical structure of piceatannol. Lower, transformation of BSA into innate epitopes by piceatannol. The IgM mAb SBM3 was used as the primary Ab by ELISA. (D) Age-dependent elevation of the IgM titers to the piceatannol-derived epitopes in the BALB/c mice immunized with the piceatannol-modified keyhole limpet hemocyanin. Female BALB/c mice were immunized with complete Freund adjuvant and 50 µg of the immunogen, then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection.

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Biochemistry

Polymerization of BSA by piceatannol We then sought to establish the molecular mechanism for the formation of the innate epitopes upon the reaction of proteins with polyphenols. We have previously shown that the electronegative potential of modified self-proteins is involved in the multi-specificities of the natural mAbs.14, 17 However, the native gel electrophoresis showed only a limited electrophoretic mobility shift of the protein that had been treated with piceatannol (Figure 4A, Figure S3). Instead of the increase in the negative charge, the experiments revealed time- and concentration-dependent increases in the polymerized forms of the protein upon the incubation with piceatannol. The polymerization was also confirmed by SDS-PAGE (Figure 4B), gel filtration (Figure 4C), and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Figure 4D).

A

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68771.4375(A25617,R14,S28)

60.0 mAu

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Piceatannol/ BSA

Piceatannol/ BSA

40.0 20.0 0.0 0

100 200 300 400 500 600

59928.0 63053.8 66179.6 69305.4 72431.2

Retention time (min)

Mass (m/z)

SDS-PAGE Mass (m/z) BSA

66027.0234

PiceatannolBSA

68771.4375

Figure 4. Polymerization of BSA by piceatannol. (A) Native PAGE analysis of piceatannol-treated BSA. (B) SDS-PAGE analysis of piceatannol-treated BSA. In panels A and B, BSA (1.0 mg/ml) was incubated with 1.0 mM piceatannol in PBS (pH 7.4) for 0 - 24 h at 37 °C under atmospheric oxygen. (C) Gel filtration of control and piceatannol-treated BSAs. The native and piceatannol-treated BSAs were eluted with PBS at the flow rate of 0.2 ml/min at room temperature with monitoring of the absorbance at 280 nm. The column system was composed of Hi Prep 16/60 Sephacryl S-300. (D) MALDI-TOF MS analysis of the piceatannol-treated BSA. BSA (1.0 mg/ml) was incubated with 1.0 mM piceatannol in PBS for 24 h at 37 °C under atmospheric oxygen.

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Recognition of polymerized proteins by IgM Abs To determine if a protein size is involved in the recognition by the IgM Abs, we fractionated the piceatannol-treated BSA by gel filtration (Figure 5A) and the immunoreactivity (Figure 5B) of each fraction with the IgM mAb SBM3 was characterized. The results showed that the immunoreactivity of the mAb SBM3 toward the polymerized proteins (fractions 29-34) was much greater than toward the late-eluting fractions (fractions 35-38). The particle size distribution of each fraction shows that the polymerized fractions (fractions 23-25) had a size of around 20 nm, which was twice the size of the monomer fractions (fractions 35-43) (Figure 5C). Thus, the polymerization of self-proteins is likely to be involved in the cross-reactivity of the polyphenol-derived epitopes with the Abs. This speculation was supported by the observation that the protein that had been treated with glutaraldehyde (GA), one of the most effective protein crosslinking reagents, was recognized A

Fraction numbers M.W. 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 (kDa) 250 150 100 75 50 37

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80.0 60.0

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C

40.0

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10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 Fraction numbers

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O

-GA

+GA

15 10 5

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Biochemistry

0.8

**

* 0.6

- GA + GA

0.4 0.2 0

3 8 1 4 7 0 5 -2 6-2 9-3 2-3 5-3 8-4 1-4 4 2 3 3 2 3 Fraction numbers

ADL17

DDL17

IgM mAbs

Figure 5. Recognition of polymerized proteins by innate IgM Abs. The eluates of the piceatannol-treated BSA in Fig. 3C were fractionated every 10 min and used for SDS-PAGE and ELISA analyses. (A) SDS-PAGE analysis of the fractions. (B) Cross-reactivity of the fractions with the IgM mAb SBM3. The chromatogram is the same as that in Fig. 4C (lower). (C) Particle sizes of the gel filtration fractions. (D) SDS-PAGE analysis of control and glutaraldehyde-treated BSAs. (E) Cross-reactivity of control and glutaraldehyde-treated BSAs with the IgM mAbs established from MRL-lpr mice. 13


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by the IgM mAbs (Figures 5D, E).

Identification of a cross-linked species The key question is the molecular mechanism by which piceatannol polymerizes the protein. The result that the incubation of BSA with piceatannol resulted in a significant loss of lysine residues (data not shown) suggested that the basic amino acid might be involved in the formation of cross-links. To gain insight into the piceatannol-mediated formation of cross-links, we comprehensively analyzed modified lysines by high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). Our previous study has shown that lysine adducts provide a common fragment ion at m/z 84, corresponding to the loss of NH3 from the lysine immonium ion.18 It was expected that the characteristic common fragment ion might allow comprehensive analysis of the amino acid adducts, including cross-linked species, using LC-ESI-MS/MS. Because the general methods for the acid hydrolysis of protein (e.g., 6N HCl at 105 oC for 24

Figure 6. Comprehensive analysis of modified lysines in the control and piceatannol-treated BSA. (A) Comprehensive analysis of modified lysines in the control (upper) and the piceatannol-treated BSA (lower). The blue bubbles indicate unknown products that are supposed to have originated from the lysine modification. The red bubble (product a) indicates a putative lysine-derived product that was detected at a high level in the piceatannol-treated BSA. (B) Chemical structures of a lysine-derived cross-link (deHLNL) and its reduced product (LNL). 14


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Biochemistry

h) were postulated to decompose the aldol and imine linkages, we sought to detect the cross-link as the NaBH3CN-reduced form. Figure 6A shows the maps of the putative lysine adducts generated upon incubation of BSA with piceatannol, in which the x-axis represents the LC retention time and the y-axis represents the mass-to-charge ratio (m/z) for the individual modified lysine detected. The relative size of a particular adduct spot across different samples reflects the relative abundance of the product in the corresponding samples. The most abundant product a, showing a molecular ion at m/z of 276 ([M+H]+), was suggested to be identical to lysinonorleucine (LNL), a reduced form of dehydrolysinonorleucine (deHLNL) (Figure 6B). A sim ilar cross-link was also detected when Nα-acetyllysine (5 mM) was incubated with an equimolar concentration of piceatannol (5 mM) in 0.1 M phosphate buffer (pH 7.4) and analyzed by LC-ESI-MS/MS in the positive ion mode using multiple reaction monitoring (MRM) (Figure 7).

Figure 7. LC-ESI-MS/MS analysis of a lysine-derived cross-link in the piceatannol-treated N -acetyllysine. (A) The control and piceatannol-treated N -acetyllysines were analyzed by LC-ESI-MS/MS in the positive ion mode using MRM following NaBH3CN reduction. N -Acetyllysine (5 mM) was incubated with an equimolar concentration of piceatannol (5 mM) in PBS for 24 h at 37 °C. Chromatograms: upper, piceatannol-treated N -acetyllysine; lower, control N -acetyllysine. (B) Collision-induced dissociation of the [M+H]+ of a cross-link generated in the reaction of N -acetyllysine with piceatannol and the proposed structures of the individual ions. α

α

α

α

α

α

15


Biochemistry

To confirm the formation of the lysine cross-link, LNL was prepared upon oxidative deamination of lysine by lysyl oxidase and analyzed by LC-ESI-MS/MS. LC-ESI-MS/MS analysis in the positive ion mode using MRM between the transition from the protonated parent ion [M+H]+ to the characteristic product ion (m/z 276→84) revealed that the cross-link generated in the piceatannol-treated BSA was indistinguishable from the authentic LNL (Figure 8A). Using the LC-ESI-MS/MS technique, we semi-quantitatively measured LNL generated in the piceatannol-modified BSA and observed that the incubation of BSA (1 mg/ml) with 1 mM piceatannol generated LNL in a time-dependent manner (Figure 8B). The yield of LNL generated after 24 h of incubation was 0.76 mol/mol protein. This high yield was in good agreement with the observation that LNL was detected as one of the most abundant modified lysines in the adductome analysis. Moreover, the observation that LNL was mainly detected in the fractions containing polymerized proteins (Figure 8C) suggested A

C

MRM mode (276.0→ →84.0)

1.13e5

9.09

5.51e6

Fr. 26-28

1.13e5

Fr. 38-40

9.04

LNL (standard)

9.07

9.03

1.08e5

9.04

Fr. 29-31

Fr. 41-43

Fr. 32-34

Fr. 44-46

Fr. 35-37

Fr. 47-49

7.00 9.00 11.00 Retention time (min)


Piceatannol/ BSA

1.08e5 BSA 9.03 5.00

9.00

7.00

11.00

13.00


B

1.0 LNL (mol/mol protein)

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0.8 9.01 0.6 0.4 0.2 0 0

4

8

12

16

20 24

Incubation time (min)

Figure 8. LC-ESI-MS/MS analysis of a lysine-derived cross-link generated in the piceatannol-treated BSA. (A) LC-ESI-MS/MS analysis of LNL generated in the piceatannol-treated BSA. The control and piceatannol-treated BSAs were analyzed by LC-ESI-MS/MS in the MRM mode (276→84) following NaBH3CN reduction and acid-hydrolysis. Chromatograms: top, standard lysinonorleucine; middle, piceatannol-treated BSA; bottom, control BSA. (B) Time-dependent formation of LNL in the piceatannol-treated BSA. (C) LC-ESI-MS/MS analysis of LNL in the gel filtration fractions. The fractions in Fig. 5B were used for the LC-ESI-MS/MS analysis of LNL.

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that the cross-link might be responsible for the formation of the polymerized proteins upon the reaction of BSA with piceatannol. Of interest, as compared to other polyphenols, piceatannol was found to be a particularly effective producer of the cross-link (Figure S4).

LC-ESI-MS/MS analysis of an oxidized lysine intermediate Next we sought to detect an oxidized lysine intermediate responsible for the formation of deHLNL in the piceatannol-modified BSA. The most likely candidate might be an oxidatively deaminated lysine, α-aminoadipic semialdehyde (AAS) (Figure 9A), as an intermediate for the cross-link. To this end, the piceatannol-treated and untreated BSAs were derivatized with p-aminobenzoic acid (ABA), hydrolyzed, and analyzed by LC-ESI-MS/MS in the positive ion mode using MRM. Upon incubation of BSA with 1 mM piceatannol for 24 h at 37oC, about 0.85 molecules of AAS per protein molecule was detected (Figure 9B). Thus, it appear ed that piceatannol selectively acts on the lysine residues of the protein to generate AAS. These A

m/z=84

NH 2

O

O

O

H 2N

OH

OH

ABA-AAS (267→ →84)

AAS

＋ HN O H 2N OH

OH

ABA

O

B

C 1.49e7

3.28

2.32e6

.00 2.32e6

ABA-AAS (standard)

BSA

3.22 Piceatannol/ 3.22 4.0

1.0 AAS (mol/mol protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

BSA

0.8

Piceatannol/BSA

0.6 0.4 0.2 0

BSA

0

4

8

12

16

20 24

Incubation time (h) 2.00 .00

3.00

4.00 4.0

retention time (min)

Figure 9. LC-ESI-MS/MS analysis of an oxidized lysine in the piceatannol-treated BSA. (A) Derivatization of AAS with ABA and the proposed structures of individual ions. (B) LC-ESI-MS/MS analysis of AAS generated in the piceatannol-treated BSA. BSA (1 mg/ml) was incubated with piceatannol (1 mM) in 0.1 ml of PBS for 24 h at 37 °C. Chromatograms: top, ABA derivative of AAS (standard); middle, control BSA; bottom, piceatannol-treated BSA. (C) Time-dependent formation of AAS in the piceatannol-treated BSA. BSA (1 mg/ml) was incubated with piceatannol (1 mM) in 0.1 ml of PBS for 24 h at 37 °C. In B and C, the treated and untreated BSAs were derivatized with ABA, hydrolyzed, and analyzed by LC-ESI-MS/MS in the positive ion mode using MRM (267→84). 17


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data suggest that the polymerization of the protein by piceatannol may be, at least in part, mediated by oxidation of a lysine residue, generating AAS, followed by a Schiff-base reaction with the ε-amino group of an unoxidized lysine residue (Figure 10). We also attempted to detect the piceatannol-derived intermediates, such as oxidized and aminated piceatannol, generated during the piceatannol-mediated oxidation of BSA using LC-ESI-MS. However, when monitored at m/z 243(M+H)+ and 244 (M+H)+ corresponding to the molecular mass of the oxidized and aminated products, respectively, both products were undetectable (unpublished data). This may be due to the instability of these intermediates that might be rapidly converted into other products. A

B OH

Piceatannol

HO

O2 Oxidized piceatannol

OH OH

CH2 HN

H N

H N O

Lys

O2

NH 2

O H 2N

O

NH 2

Multiple products

Albumin Lys

O

AAS - H2O IgM O N HN

Protein polymers

H N

deHLNL O

Figure 10. Lysyl oxidase-like activity of piceatannol. (A) Proposed mechanism for the formation of a lysine-derived cross-link. Oxidation of piceatannol generates O-quinone derivatives that covalently bind a lysine residue of proteins to generate an oxidatively deaminated product, AAS. The cross-link is then formed through a Schiff-base reaction of the AAS residue with the ε-amino group of an unoxidized lysine residue. (B) Schematic illustration of the piceatannol-mediated transformation of serum albumins into innate epitopes.

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Biochemistry

DISCUSSION

In the present study, to gain insight into the production of innate epitopes by small molecules, we conducted a screening of modified serum albumins treated with various dietary polyphenols and detected prominent IgM titers to the polyphenol-derived epitopes in the sera from normal (BALB/c) and MRL-lpr mice (Figure 1). Of interest, not all of the polyphenols converted serum albumins into innate antigens. The most prominent cross-reactivity with the MRL-lpr mouse sera was obtained in the proteins that had been treated with the natural phenolic antioxidants, such as anthocyanidins (cyanidin and malvidin) and piceatannol, found in numerous fruits and vegetables. In addition, the IgM mAbs established from these MRL-lpr mice showed a similar cross-reactivity to the polyphenol-treated proteins as the MRL-lpr sera. More strikingly, the patients with rheumatoid arthritis showed significant IgM titers to the polyphenol-derived epitopes (Furuhashi, M., Handa, O., Naito, Y., Shibata, T., and Uchida, K., unpublished data). These findings led us to speculate that some, but not all, dietary polyphenols might constitute a previously unrecognized, but important source of innate epitopes recognized by the NAbs. Based on the finding that the high IgM titers to the polyphenol-derived epitopes were observed in the sera from the SPF-maintained BALB/c mice, we isolated several hybridoma clones, producing the Abs specific to the antigens from the mice and characterized their specificity toward various polyphenol-treated proteins (Figure 2). Of interest, most of the IgM mAbs established from the BALB/c mice cross-reacted with a variable number of polyphenol-treated proteins. In addition, the specificity study showed that the IgM mAb cross-reacted not only with the EGCG-treated BSA, but also with the protein that had been treated with the polyphenols, including piceatannol and anthocyanidins (such as malvidin, cyanidin, and delphinidin). The IgM mAbs obtained from normal SPF-maintained BALB/c and MRL-lpr mice, therefore, showed a similar specificity toward the polyphenol-derived epitopes. Thus, the high prevalence of these epitopes as targets of the IgM mAbs is likely to reflect the ubiquitous presence of the polyphenol-modified proteins consequent to the consumption of polyphenols at dietary levels. It was also observed that the immunization of mice with the piceatannol-modified proteins in adjuvant strongly induced the IgM response (Figure 3). The data strongly suggest that the immunization of polyphenol-specific epitopes may not induce the typical B cell memory. Of interest, the epitopes generated upon incubation of serum albumins with oxidized vitamin C have been shown to induce similar innate immune 19


Biochemistry

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response, resulting in the enrichment of B-1 cells in the peritoneal cavity.6 Thus, the B-1 cells with an appropriate specificity toward oxidation-specific epitopes may not produce the high-affinity Abs that efficiently react with the self-antigens but may rather respond to these epitopes by the production of IgM and limited isotype switching. The mechanism(s) for elevation of the IgM titers associated with autoimmune diseases is presently unknown. However, the enhanced production of IgM Abs in autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis, may represent an expansion of the population of the Abs expressed ubiquitously in normal healthy individuals. Alternatively, the IgM Abs may represent the products of B lymphocyte population selected by an antigen. Indeed, the expansion of established B-1 cell clones by the exposure to modified self-antigens, leading to the elevation of IgM levels in the plasma, has been reported.19 To identify a structural element in the modified proteins responsible for the recognition by the IgMs, we characterized the molecular mechanism underlying the piceatannol-mediated formation of the innate epitopes. We have previously shown that the innate Abs could recognize the EGCG-treated proteins because the polyphenol gives rise to the increased negative charge of the protein, generating the electrically-transformed proteins.14 Therefore, we initially expected that the electronegative potential of the modified self-proteins by piceatannol might be involved in the recognition by the mAbs. However, the native gel electrophoresis showed only a limited electrophoretic mobility shift of the protein that had been treated with piceatannol (Figure 4). Instead of an increase in the negative charge, the experiments revealed time- and concentration-dependent increases in the polymerized forms of the protein upon treatment with piceatannol. The polymerization was also confirmed by MALDI-TOF MS, suggesting the involvement of covalent bonds other than the disulfide linkage. Moreover, it was revealed that, when we fractionated the piceatannol-treated BSA by gel filtration and characterized the immunoreactivity of each fraction with the IgM mAb SBM3, the polymerization of self-proteins was involved in the cross-reactivity of polyphenol-derived epitopes with the IgM mAbs. These data provided a basis for the hypothesis that the specificity of the IgM Abs toward polyphenol-derived epitopes might be ascribed, at least in part, to the polymerized proteins. To gain insight into a covalently cross-linked species generated in the piceatannol-treated BSA, we adapted a mass spectrometry-based approach. Taking advantage of the fact that the authentic lysine adducts produced specific fragment ions that were observed at m/z 84, we analyzed modified proteins with piceatannol and demonstrated that 20


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Biochemistry

the data obtained from analyzing complex adduct mixtures by LC-ESI-MS/MS could be visualized as a two-dimensional plot (Figure 6). The identification of these fragment ions and the visualization of adduct patterns were crucial because they allowed the comprehensive and comparative analysis of modified lysines during the analysis of more complex samples. Based on the lysine adductome analysis of the piceatannol-modified BSA, we identified deHLNL as a cross-linked species responsible for the piceatannol-mediated formation of polymerized proteins. Strikingly, the maximum yield of LNL was about 0.8 mol/mol protein, which was one of the most abundant modified lysines detected in the piceatannol-modified protein. Thus, the lysine-derived cross-links generated in the piceatannol-treated BSA appeared to constitute the innate epitopes recognized by the IgM Abs. It is likely that the conversion of the lysine residues involved in the interaction with the resveratrol metabolite into the oxidized lysine and cross-links causes loss of the electrostatic and stereochemical interactions essential for maintaining the higher order structure of serum albumins. These global changes in the conformation of the proteins may also represent a structural element essential for the recognition by NAbs To the best of our knowledge, few if any studies have unequivocally demonstrated the lysyl oxidase-like activity of small molecules, generating the lysine-derived cross-links. It is speculated that the formation of lysine-derived cross-links by piceatannol may be mediated through the oxidative deamination of lysine residues in proteins, in which the lysine residues form Schiff-base intermediates with an oxidized piceatannol followed by their conversions to oxidatively deaminated products. The involvement of this mechanism was suggested by the detection of AAS as an oxidized lysine intermediate in the piceatannol-treated protein. The cross-link may be formed through a Schiff-base reaction of the AAS residue with the ε-amino group of an unoxidized lysine residue. These data provide the first mechanistic details of the lysyl oxidase-like activity of polyphenols, generating the lysine-derived cross-links. Our current study emphasizes the relevance of drinking wine for health. It has been suggested that a mild to moderate drinking of wine, particularly red wine, could reduce the risk of heart disease. However, the experimental basis for such health benefits is not fully understood. The cardioprotective effect of wine has been attributed, at least in part, to the wine components, i.e., polyphenols. Of all the polyphenols in wine, resveratrol is the best known for its health benefits. Piceatannol, a closely related stilbene with purported health benefits, is produced as a major metabolite of resveratrol. The results of our present study suggest that resveratrol may show its beneficial effects after conversion to its metabolites, 21


Biochemistry

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such as piceatannol, that can be oxidized much more rapidly than the original compound (resveratrol).20 Although the potential role of wine polyphenols, resveratrol and its metabolites, in health benefits needs further investigation, the interaction between wine polyphenols and proteins, such as serum albumins, may not simply be the covalent binding of exogenous small molecules to the proteins, but a possible source of innate epitopes. Thus, polyphenols and their metabolites, possessing a similar lysyl oxidase-like activity, may contribute to the protection against exogenous pathogens and damage-associated molecules via oxidative modification of proteins followed by activation of innate immune response. Lysyl oxidase and lysyl oxidase-like enzymes are known to be key players in extracellular matrix deposition and maturation.21 A number of studies have indeed shown that these enzymes are responsible for the regulation of tumor progression and metastasis. Mechanistically, they are involved in the invasive properties of cancer cells. They also play a role in the dermis during wound repair, aging, and in fibrotic disorders.22 Thus, lysyl oxidase and lysyl oxidase-like enzymes have been regarded as a potential target for preventing and treating metastases and other disease states. Our present study has established that some of the polyphenols show a lysyl oxidase-like activity, generating a lysine-derived cross-link, which is involved in the recognition by the innate IgM Abs. It may not be unlikely that these NAbs could interact with the lysyl oxidase-oxidized proteins, such as extracellular matrix, with the same antigenic cross-link, leading to the prevention of early and late stages of metastasis. General acceptance of this hypothesis will have to await future experimental confirmation. In conclusion, the results of this study raised the possibility that the polyphenol-derived epitopes might be a ubiquitous target of IgM Abs. Our most striking finding is that piceatannol generated the epitopes via a cross-linking reaction of lysine residues. The presence of the IgM Abs against the polyphenol-derived polymerized proteins in vivo suggest the ubiquitous formation of these modified proteins consequent to oxidative/antioxidative events. Moreover, because protein polymerization is a common phenomenon in fundamental biological processes, this IgM function to sense the covalently polymerized proteins may also be critical for maintaining homeostasis. Our discovery of piceatannol as a source of innate epitopes may provide a key link connecting polyphenols in foodstuffs and innate immunity and therefore lead to novel approaches exploiting mechanisms for the prevention of diseases by accelerating innate immune response by polyphenols. However, the abundance of polyphenols also suggests that additional, evolutionary conserved innate defense mechanisms

22


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Biochemistry

may exist. Future studies therefore need to evaluate the contribution of this newly discovered mechanism in the functions of polyphenols.

ASSOCIATED CONTENT Supporting Information Chemical structures of polyphenols used in this study (Figures S1), dose-dependent production of innate epitopes by piceatannol (Figures S2), dose-dependent polymerization of piceatannol-treated BSA (Figures S3), and LC-ESI-MS/MS analysis of a lysine-derived cross-link generated in the polyphenol-treated BSA (Figures S4) (PDF).

AUTHOR INFORMATION Corresponding Author *

Koji Uchida, Ph.D., Laboratory of Food Chemistry, Graduate School of Agricultural and

Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan. Tel: 81-3-5841-5127, Fax: 81-3-5841-8026, E-mail: [email protected]

Author Contributions K.U. designed the study and wrote the paper. M.F., Y.H., S.K., and T.S. performed the experiments. M.A. contributed the chemical probes. All authors analyzed the results and approved the final version of the manuscript.

Funding Sources This work was supported in part by a Grant-in-Aid for Scientific Research (A) (No. 26252018) (K.U.) and Grant-in-Aid for Scientific Research on Innovative Areas "Oxygen Biology: a new criterion for integrated understanding of life" (No. 26111011) (K.U.) of the Ministry of Education, Sciences, Sports, Technology (MEXT), Japan; a grant from the JST PRESTO program (T.S.). This work was partly supported by Nanotechnology Platform Program (Molecule and Material Synthesis) of the MEXT, Japan.

Notes The authors declare no competing financial interest. 23


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ACKNOWLEDGMENT We thank Ms Yuki Hondoh for her excellent editorial support.

ABBREVIATIONS AAS, α-aminoadipic semialdehyde; ABA, p-aminobenzoic acid; Abs, antibodies; autoAbs, autoantibodies; BSA, bovine serum albumin; deHLNL, dehydrolysinonorleucine; EGCG, (-)-Epigallocatechin-3-O-gallate; GA, glutaraldehyde; LC-ESI-MS/MS, high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry; LNL, lysinonorleucine; mAbs, monoclonal antibodies; MALDI-TOF MS, matrix-assisted laser desorption and ionization time-of-flight mass spectrometry; MRM, multiple reaction monitoring; NAbs, natural antibodies; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; SPF, specific pathogen-free.

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Figure Legends

Figure 1.

Recognition of polyphenol-specific epitopes by innate IgM Abs.

(A) Schematic illustration of the polyphenol-mediated transformation of serum albumins into innate epitopes. (B) Cross-reactivity of polyphenol-treated BSAs with the sera from the SPF-maintained BALB/c (left panel) and MRL-lpr (right panel) mice. “Control” represents a sample containing only protein (BSA).

Figure 2.

Cross-reactivity of polyphenol-specific epitopes with autoAbs.

(A) Cross-reactivity of polyphenol-treated BSAs with the sera from the MRL-lpr mice and with the IgM mAbs DDL17 and ADL19 established from MRL-lpr mice. (B) Cross-reactivity of polyphenol-treated BSAs with the anti-citrulline polyclonal Abs. In panels A and B, all the polyphenols tested were dissolved with DMSO and “control” represents a sample containing only protein (BSA).

Figure 3.

Specificity of the IgM mAbs to the polyphenol-specific epitopes.

(A) Recognition of EGCG-treated BSA by the IgM mAbs established from the SPF-maintained BALB/c mice. (B) Recognition of polyphenol-treated BSAs by the IgM mAb SBM3. (C) Upper, chemical structure of piceatannol. Lower, transformation of BSA into innate epitopes by piceatannol. The IgM mAb SBM3 was used as the primary Ab by ELISA. (D) Age-dependent elevation of the IgM titers to the piceatannol-derived epitopes in the BALB/c mice immunized with the piceatannol-modified keyhole limpet hemocyanin. Female BALB/c mice were immunized with complete Freund adjuvant and 50 µg of the immunogen, then boosted every 2 weeks with incomplete Freund adjuvant by emulsifying and intraperitoneal injection.

Figure 4.

Polymerization of BSA by piceatannol.

(A) Native PAGE analysis of piceatannol-treated BSA. (B) SDS-PAGE analysis of piceatannol-treated BSA. In panels A and B, BSA (1.0 mg/ml) was incubated with 1.0 mM piceatannol in PBS (pH 7.4) for 0 - 24 h at 37 °C under atmospheric oxygen. (C) Gel filtration of control and piceatannol-treated BSAs. The native and piceatannol-treated BSAs were eluted with PBS at the flow rate of 0.2 ml/min at room temperature with monitoring of the absorbance at 280 nm. The column system was composed of Hi Prep 16/60 Sephacryl 28


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biochemistry

S-300. (D) MALDI-TOF MS analysis of the piceatannol-treated BSA. BSA (1.0 mg/ml) was incubated with 1.0 mM piceatannol in PBS for 24 h at 37 °C under atmospheric oxygen.

Figure 5.

Recognition of polymerized proteins by innate IgM Abs.

The eluates of the piceatannol-treated BSA in Fig. 3C were fractionated every 10 min and used for SDS-PAGE and ELISA analyses. (A) SDS-PAGE analysis of the fractions. (B) Cross-reactivity of the fractions with the IgM mAb SBM3. The chromatogram is the same as that in Fig. 4C (lower). (C) Particle sizes of the gel filtration fractions. (D) SDS-PAGE analysis of control and glutaraldehyde-treated BSAs. (E) Cross-reactivity of control and glutaraldehyde-treated BSAs with the IgM mAbs established from MRL-lpr mice.

Figure 6.

Comprehensive analysis of modified lysines in the control and

piceatannol-treated BSA. (A) Comprehensive analysis of modified lysines in the control (upper) and the piceatannol-treated BSA (lower). The blue bubbles indicate unknown products that are supposed to have originated from the lysine modification. The red bubble (product a) indicates a putative lysine-derived product that was detected at a high level in the piceatannol-treated BSA. (B) Chemical structures of a lysine-derived cross-link (deHLNL) and its reduced product (LNL).

Figure 7.

LC-ESI-MS/MS analysis of a lysine-derived cross-link in the

piceatannol-treated Nα-acetyllysine. (A) The control and piceatannol-treated Nα-acetyllysines were analyzed by LC-ESI-MS/MS in the positive ion mode using MRM following NaBH3CN reduction. Nα-Acetyllysine (5 mM) was incubated with an equimolar concentration of piceatannol (5 mM) in PBS for 24 h at 37 °C. Chromatograms: upper, piceatannol-treated Nα-acetyllysine; lower, control Nα-acetyllysine. (B) Collision-induced dissociation of the [M+H]+ of a cross-link generated in the reaction of Nα-acetyllysine with piceatannol and the proposed structures of the individual ions.

Figure 8.

LC-ESI-MS/MS analysis of a lysine-derived cross-link generated in the

piceatannol-treated BSA.

29


Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(A) LC-ESI-MS/MS analysis of LNL generated in the piceatannol-treated BSA. The control and piceatannol-treated BSAs were analyzed by LC-ESI-MS/MS in the MRM mode (276→84) following NaBH3CN reduction and acid-hydrolysis. Chromatograms: top, standard lysinonorleucine; middle, piceatannol-treated BSA; bottom, control BSA. (B) Time-dependent formation of LNL in the piceatannol-treated BSA. (C) LC-ESI-MS/MS analysis of LNL in the gel filtration fractions. The fractions in Fig. 5B were used for the LC-ESI-MS/MS analysis of LNL.

Figure 9.

LC-ESI-MS/MS analysis of an oxidized lysine in the piceatannol-treated BSA.

(A) Derivatization of AAS with ABA and the proposed structures of individual ions. (B) LC-ESI-MS/MS analysis of AAS generated in the piceatannol-treated BSA. BSA (1 mg/ml) was incubated with piceatannol (1 mM) in 0.1 ml of PBS for 24 h at 37 °C. Chromatograms: top, ABA derivative of AAS (standard); middle, control BSA; bottom, piceatannol-treated BSA. (C) Time-dependent formation of AAS in the piceatannol-treated BSA. BSA (1 mg/ml) was incubated with piceatannol (1 mM) in 0.1 ml of PBS for 24 h at 37 °C. In B and C, the treated and untreated BSAs were derivatized with ABA, hydrolyzed, and analyzed by LC-ESI-MS/MS in the positive ion mode using MRM (267→84).

Figure 10.

Lysyl oxidase-like activity of piceatannol.

(A) Proposed mechanism for the formation of a lysine-derived cross-link. Oxidation of piceatannol generates O-quinone derivatives that covalently bind a lysine residue of proteins to generate an oxidatively deaminated product, AAS. The cross-link is then formed through a Schiff-base reaction of the AAS residue with the ε-amino group of an unoxidized lysine residue. (B) Schematic illustration of the piceatannol-mediated transformation of serum albumins into innate epitopes.

30


Page 30 of 41

Page 31 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

A

B

Polyphenols

Albumin

Albumin

Electronegative proteins

IgM

Serum (MRL-lpr)

Serum (BALB/c)

Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannnol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Luteolin Chrysin Kaempferol Quercitrin Quercetin Control 0

0.5

1.0 0

0.5

1.0

Absorbance (490 nm)

Fig. 1 ACS Paragon Plus Environment

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A

mAb DDL17

Page 32 of 41

B

mAb ADL19

Anti-citrulline Abs

Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannnol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Diosmetin Luteolin Apigenin Chrysin Kaempferol Quercitrin Quercetin Control

Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannnol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Diosmetin Luteolin Apigenin Chrysin Kaempferol Quercitrin Quercetin Control 0

1.0

2.0

0 3.0

0.4

0.8

1.6

Absorbance (490 nm)

0

0.4

0.8

1.2

Absorbance (490 nm)


Page 33 of 41

C

1.2 1.0

HO

OH

0.8

OH Piceatannol

0.6 0.4 1.6

0.2 0

B

OH

BSA EGCG/BSA

SBM1 SBM2 SBM3 SBM4 SBM5 SBM6 mAbs

Sinapic acid Coumarin o-Coumaric acid Curcumin L-DOPA Gallic acid Ellagic acid Taxiforin Caffeic acid Piceatannol Resveratrol Genistein Malvidin Cyanidin Delphinidin Eriodictyol Naringenin Hesperetin Luteolin Chrysin Kaempferol Quercitrin Quercetin Control

Absorbance at 490nm

A Absorbance (490 nm)

1.4 1.2

BSA Piceatannol/ BSA

1.0 0.8 0.6 0.4 0.2 0 0

4

8 12 16 20 24

Incubation time (h)

D Absorbance (490 nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

2.0 1.6

**

1.2 0.8 0.4 0

0

BSA Piceatannol/ BSA

0.5 1.0 1.5 2.0 2.5 Absorbance (490 nm)


5

7 9 11 Age (week)

13

Fig. 3

Biochemistry

A


1

2

4

C

8 12 24

D 70.0

mAu

60.0 50.0

BSA

BSA

40.0 30.0 20.0 10.0 0.0

Native PAGE

Final - Shots 4000 - 160706 furuhashi-2; Label H1

59930.0 63055.8 66181.6 69307.4 72433.2

100

80.0

B

73.2

90


1 2

4 8 12 24

60.0 mAu

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 34 of 41

68771.4375(A25617,R14,S28)

80

Piceatannol/ BSA

40.0

Piceatannol/ BSA

70 60 50 40

20.0

30 20

0.0 0

10

100 200 300 400 500 600

0 59928.0

63053.8

66179.6

69305.4

72431.2

75557.0

59928.0 63053.8 66179.6 69305.4 72431.2 Mass (m/z)


Mass (m/z)

SDS-PAGE Mass (m/z) BSA

66027.0234

PiceatannolBSA

68771.4375


Page 35 of 41

A M.W. (kDa)

Fraction numbers 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

250 150 100 75 50 37 0.6 0.5

80.0

0.4

60.0

0.3

40.0

0.2

mAu

Absorbance (490 nm)

B

20.0

0.1

0.0 0.0 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 Fraction numbers

D

25

E O

20

O

-GA +GA

15 10 5 0

Absorbance (490 nm)

C Particle size (nm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

0.8

**

* 0.6

- GA + GA

0.4 0.2 0 ADL17

Fraction numbers


DDL17

IgM mAbs

Fig. 5

Biochemistry

A R2

R1 N

B

O

O

OH

m/z 84

N

NH 2 H 2N

400 BSA

HO

m/z

350 300

O

Dehydrolysinonorleucine (deHLNL)

250 200

H N

150 100 0

2

4 6 8 Retention time (min)

10

12

O OH NH 2

H 2N

450 400

OH NH 2

450

Piceatannol/BSA

350 m/z

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 36 of 41

Product a (m/z=276)

HO

300

O Lysinonorleucine (LNL)

250 200 150 100 0

2

4 6 8 Retention time (min)

10

12


Page 37 of 41

A

B

m/z=130

5.06

8.13e6

O

H N Piceatannol/ N-acetyllysine

%

100

m/z=84

HN

OH O

H N O HO

0 0.00

2.00

4.00

6.00

8.00

10.00

100

O

84.23

100

N-Acetyllysine

%

8.13e6

83.92

%

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

83.74 0 0.00

2.00

4.00

6.00

8.00

10.00

0 0

50

255.23

129.98

100

50

200

250

318.37 300

350

400

m/z



Biochemistry

A

C

MRM mode (276.0→84.0)

1.13e5

9.09

5.51e6

LNL (standard)

Fr. 26-28 1.13e5

Fr. 38-40

9.04 9.07

9.03

1.08e5

9.04

Fr. 29-31

Fr. 41-43

Fr. 32-34

Fr. 44-46

Fr. 35-37

Fr. 47-49



Piceatannol/ BSA

1.08e5 BSA 9.03 5.00

9.00

7.00

11.00

13.00


B

1.0 LNL (mol/mol protein)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Page 38 of 41

0.8 9.01 0.6 0.4 0.2 0 0

4

8

12 16 20 24

Incubation time (min)


%

NH 2 O

4.00

6.00

m/z=84 O

O

H 2N

OH

OH

%

100

4.22e6

Biochemistry

0 0.00 2.00 150430 ABA-Delphinidin BSA A

4.00

HN

6.00

2

%

100

OH

ABA

B 1.49e7

8.00 10.00 4: MRM of 1 Channel ES+ TIC (ABA-AAS 84) 4.22e6

ABA-AAS (267→84)

AAS

＋ 0 O 0.00 2.00 H N 150430 ABA-Eriodictyol BSAOH

O

3.28 3.24

C ABA-AAS (standard)

1.0

8.00 10.00 4: MRM of 1 Channel ES+ TIC (ABA-AAS 84) 4.22e6

%

%

AAS (mol/mol protein)

%

150430 ABA-BSA 4: MRM of 1 Channel ES+ BSA 0.8 0 Piceatannol/BSA TIC (ABA-AAS 84) 100 0.00 2.00 4.00 8.00 10.00 0.6 6.00 4.22e6 2.32e6 BSA 150430 ABA-Piceatannnol BSA 4: MRM of 1 Channel ES+ 0.4 TIC (ABA-AAS 84) 0.2 1000 4.22e6 3.22 0.00 2.00 6.00 8.00 10.00 Piceatannol/ 3.22 4.00 2.32e6 0 BSA 0 4 8 12 4: 16 MRM 20 24 of 1 Channel ES+ 150430 ABA-L-DOPA BSA Incubation time (h) 0 TIC (ABA-AAS 84) 100 2.00 4.00 3.00 0.00 2.00 4.00 6.00 8.00 10.00 4.22e6 retention time (min) 150430 ABA-Caffeic acid BSA 4: MRM of 1 Channel ES+ TIC (ABA-AAS 84) 10000.00 2.00 4.00 6.00 8.00 10.00 4.22e6 150430 ABA-Gallic acid BSA 4: MRM of 1Fig. Channel ES+ 9 TIC (ABA-AAS 84) 0 Time 100 ACS Paragon 4.22e6 0.00 2.00 4.00Plus Environment 6.00 8.00 10.00 %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

100

%

Page 39 of 41

Biochemistry

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

A

Page 40 of 41

B Piceatannol

OH HO

O2 Oxidized piceatannol

OH OH

H 2N

O

CH2 HN

H N

H N O

Lys

O2 NH 2

O NH 2

Multiple products

Albumin Lys

O

AAS - H2O IgM O N HN

Protein polymers

H N

deHLNL O


Page 41 of 41

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Biochemistry

TOC OH HO

OH OH

Multiple products

O2

IgM

NH 2 CH2

Albumin

Protein polymers


Identification of Polyphenol-Specific Innate ... - ACS Publications

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