Oxidative Deamination Activity of EGCG

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG in vivo. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies

Identification of Polyphenol-Specific Innate Epitopes That Originated from a Resveratrol Analogue

Polyphenols have received a significant amount of attention in disease prevention because of 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. Interestingly, 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 have beneficial effects on human health because of its conversion to piceatannol

Identification of C1q as a Binding Protein for Advanced Glycation End Products

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway

Oxidative Deamination of Serum Albumins by (-)-Epigallocatechin-3-<i>O</i>-Gallate: A Potential Mechanism for the Formation of Innate Antigens by Antioxidants

<div><p>(-)-Epigallocatechin-3-<i>O</i>-gallate (EGCG), the most abundant polyphenol in green tea, mediates the oxidative modification of proteins, generating protein carbonyls. However, the underlying molecular mechanism remains unclear. Here we analyzed the EGCG-derived intermediates generated upon incubation with the human serum albumin (HSA) and established that EGCG selectively oxidized the lysine residues via its oxidative deamination activity. In addition, we characterized the EGCG-oxidized proteins and discovered that the EGCG could be an endogenous source of the electrically-transformed proteins that could be recognized by the natural antibodies. When HSA was incubated with EGCG in the phosphate-buffered saline (pH 7.4) at 37°C, the protein carbonylation was associated with the formation of EGCG-derived products, such as the protein-bound EGCG, oxidized EGCG, and aminated EGCG. The aminated EGCG was also detected in the sera from the mice treated with EGCG <i>in vivo</i>. EGCG selectively oxidized lysine residues at the EGCG-binding domains in HSA to generate an oxidatively deaminated product, aminoadipic semialdehyde. In addition, EGCG treatment results in the increased negative charge of the protein due to the oxidative deamination of the lysine residues. More strikingly, the formation of protein carbonyls by EGCG markedly increased its cross-reactivity with the natural IgM antibodies. These findings suggest that many of the beneficial effects of EGCG may be partly attributed to its oxidative deamination activity, generating the oxidized proteins as a target of natural antibodies.</p></div

LC-ESI-MS/MS analysis of oxidized amino acids in the EGCG-treated HSA.

<p>(<b>A</b>) Chemical structures of aminoadipic semialdehyde (AAS) and glutamic semialdehyde (GGS). (<b>B</b>) Collision-induced dissociation of the [M+H]⁺ of ABA-AAS at <i>m/z</i> 267 at a collision energy of 25 V and the proposed structures of individual ions. (<b>C</b>) Collision-induced dissociation of the [M+H]⁺ of ABA-GGS at <i>m/z</i> 253 at a collision energy of 25 V and the proposed structures of individual ions. (<b>D</b>) The ion current tracings of ABA-AAS (<i>left three tracings</i>) and ABA-GGS (<i>right three tracings</i>) using LC-ESI-MS/MS with SRM. (<b>E</b>) Determination of AAS in the EGCG-treated HSA. HSA (1 mg/ml) was incubated with EGCG (1 mM) in 0.1 ml of PBS (pH 7.4) for 24 h at 37°C. The yield of AAS was semi-quantitatively determined based on a calibration curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153002#pone.0153002.s003" target="_blank">S3 Fig</a>). (<b>F</b>) Schematic illustration of the transformation of a lysine residue to AAS by EGCG.</p

LC-ESI-MS/MS analysis of EGCG and NH₂-EGCG in the sera of mice treated with EGCG.

<p>(<b>A</b>) The ion current tracings of EGCG (<i>left five tracings</i>) and NH₂-EGCG (<i>right five tracings</i>). BALB/c mice were intraperitoneally injected with 0.1 ml of EGCG (10 mM) or PBS. After injection for 10 or 30 min, the sera were collected. After removing proteins by precipitation with cold acetone, the samples were analyzed by LC-ESI-MS/MS with SRM mode. (<b>B</b>) Quantitative analysis of EGCG (<i>upper panel</i>) and NH₂-EGCG (<i>lower panel</i>) in the sera of the mice treated with EGCG.</p

Docking simulations of EGCG to HSA.

<p>(<b>A</b>) Surface representation of HSA with the lysine residues sensitive to oxidation highlighted in yellow. (<b>B</b>) <i>Left</i>, schematic representation of the EGCG binding site in subdomains IIA and IIIA. Ribbon model is colored in orange. <i>Right</i>, close up view of the EGCG-binding pocket from its entrance located in subdomains IIA and IIIA. The electrostatic potential is represented on a color scale from blue for a positive potential, white for neutral, to red for a negative potential. (<b>C</b>) <i>Left</i>, schematic representation of the predicted EGCG binding site in subdomain IIIB. Ribbon model is colored in orange. <i>Right</i>, close up view of the predicted EGCG-binding pocket in subdomain IIIB. In panels <b>B</b> and <b>C</b>, selected residues shown in stick and color-coded by atom type: carbon in <i>dark green</i>; oxygen in <i>red</i>; and nitrogen in <i>blue</i>. The electrostatic potential is represented on a color scale from blue for a positive potential, white for neutral, to red for a negative potential. The EGCG molecule is shown in stick and color-coded by atom type: carbon in <i>right green</i>; oxygen in <i>red</i>.</p

Formation of electrically-charged proteins by EGCG.

<p>(<b>A</b>) Changes in the zeta potential of HSA treated with the catechins. HSA (1 mg/ml) was incubated with 1 mM catechins in 0.1 ml of PBS (pH 7.4) for 24 h at 37°C. (<b>B</b>) Changes in the zeta potential of BSA treated with the metal-catalyzed oxidation reactions. BSA (1 mg/ml) was incubated with 200 μM PQQ or 1 mM H₂O₂ in the presence and absence of 100 μM Cu²⁺ in 0.1 ml of PBS (pH 7.4) for 24 h at 37°C. (<b>C</b>) Schematic illustration of the EGCG-mediated transformation of HSA into electronegative molecules via oxidative deamination.</p