Structural Basis of Protein-bound Endogenous Aldehydes

Hashimoto, Mika; T, Sibata; Wasada, Hiroaki; Toyokuni, Shinya; Uchida, Koji

doi:10.1074/jbc.m210129200

Cited by 79 publications

(44 citation statements)

References 31 publications

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“…In contrast to the nuclear staining with mAb21, the acrolein-lysine adduct immunoreactive with mAb5F6 was mainly detected in the cytoplasm. This pattern of distribution in the rat kidney was consistent with that of the distribution of other lipid peroxidation products and their conjugates with cytosolic proteins (15,23,24). In addition, these and the observation that the acrolein-derived adducts co-localized with 8-oxo-2Ј-deoxyguanosine, a marker of oxidative stress, strongly suggest that acrolein was generated under oxidative stress.…”

Section: Formation Of Acrolein-2ј-deoxyadenosine Adduct In the Renal supporting

confidence: 61%

Formation of Acrolein-derived 2′-Deoxyadenosine Adduct in an Iron-induced Carcinogenesis Model

Kawai

Furuhata

Toyokuni

et al. 2003

Journal of Biological Chemistry

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Acrolein is a representative carcinogenic aldehyde found ubiquitously in the environment and formed endogenously through oxidation reactions, such as lipid peroxidation and myeloperoxidase-catalyzed amino acid oxidation. It shows facile reactivity toward DNA to form an exocyclic DNA adduct. To verify the formation of acrolein-derived DNA adduct under oxidative stress in vivo, we raised a novel monoclonal antibody (mAb21) against the acrolein-modified DNA and found that the antibody most significantly recognized an acroleinmodified 2 -deoxyadenosine. On the basis of chemical and spectroscopic evidence, the major antigenic product of mAb21 was the 1,N 6 -propano-2 -deoxyadenosine adduct. The exposure of rat liver epithelial RL34 cells to acrolein resulted in a significant accumulation of the acrolein-2 -deoxyadenosine adduct in the nuclei. Formation of this adduct under oxidative stress in vivo was immunohistochemically examined in rats exposed to ferric nitrilotriacetate, a carcinogenic iron chelate that specifically induces oxidative stress in the kidneys of rodents. It was observed that the acrolein-2 -deoxyadenosine adduct was formed in the nuclei of the proximal tubular cells, the target cells of this carcinogenesis model. The same cells were stained with a monoclonal antibody 5F6 that recognizes an acrolein-lysine adduct, by which cytosolic accumulation of acrolein-modified proteins appeared. Similar results were also obtained from myeloperoxidase knockout mice exposed to the iron complex, suggesting that the myeloperoxidase-catalyzed oxidation system might not be essential for the generation of acrolein in this experimental animal carcinogenesis model. The data obtained in this study suggest that the formation of a carcinogenic aldehyde through lipid peroxidation may be causally involved in the pathophysiological effects associated with oxidative stress.Lipid peroxidation leads to the formation of a broad array of different products with diverse and powerful biological activities. Among them are a variety of different aldehydes (1). The primary products of lipid peroxidation, lipid hydroperoxides (2), can undergo carbon-carbon bond cleavage via alkoxyl radicals in the presence of transition metals, giving rise to the formation of short chain, unesterified aldehydes (2, 3) or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including DNA, leading to disruption of important cellular functions and mutations. The important agents that give rise to the modification of DNA may be represented by ␣,␤-unsaturated aldehydic intermediates, such as 2-alkenals and 4-hydroxy-2-alkenals (4, 5). 2-Alkenals represent a group of highly reactive aldehydes containing two electrophilic reaction centers. A partially positive carbon 1 or 3 in such molecules can attack nucleophiles, such as protein and DNA, which leads to the formation of cyclic adducts or cross-links. Among all of the ␣,␤-unsaturated aldehydes, acro...

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Section: Formation Of Acrolein-2ј-deoxyadenosine Adduct In the Renal supporting

confidence: 61%

Formation of Acrolein-derived 2′-Deoxyadenosine Adduct in an Iron-induced Carcinogenesis Model

Kawai

Furuhata

Toyokuni

et al. 2003

Journal of Biological Chemistry

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“…We have previously described the preparation and characterization of the monoclonal antibody mAbR310 (Hashimoto et al, 2003) and its Fab fragment (Akagawa et al, 2006). The antibody was cleaved into its Fab and Fc fragments using an ImmunoPure Fab Preparation kit (Pierce).…”

Section: Crystallization and Data Collection Of Mabr310 Fab Fragmentmentioning

confidence: 99%

“…The novel monoclonal antibody mAbR310 against (R)-HNEmodified keyhole limpet haemocyanin showed the highest affinity for the (R)-HNE-treated protein and had a higher affinity for the mixture of 2R,4S,5R and 2S,4S,5R HNE-histidine adduct isomers than for HNE-lysine and HNE-cysteine adducts (Hashimoto et al, 2003). An immunohistochemical study using mAbR310 and anti-(S)-HNEmodified protein monoclonal antibody mAbS412 revealed that the (R)-and (S)-HNE epitopes are differentially distributed in the kidney of rats exposed to Fe 3+ -NTA (Hashimoto et al, 2003;Toyokuni et al, 1994). Very recently, the bispecific character of mAbR310 against modified protein and DNA and the structure of mAbR310 complexed with antigen were reported (Akagawa et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

“…These antibodies have been used for assessing oxidative stress in vitro and in vivo (Hashimoto et al, 2003;Kondo et al, 2001;Okada et al, 1999;Toyokuni et al, 2000). Because HNE generated in lipid peroxidation is a racemic mixture of 4R and 4S enantiomers, the HNE-histidine Michael adduct possesses three chiral centres at C-2, C-4 and C-5 in the tetrahydrofuran moiety (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein

et al. 2006

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4-Hydroxy-2-nonenal (HNE), a major racemic product of lipid peroxidation, reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centres in the cyclic hemiacetal structure. Monoclonal antibodies against HNE-modified protein have been widely used for assessing oxidative stress in vitro and in vivo. Here, the purification, crystallization and preliminary crystallographic analysis of a Fab fragment of novel monoclonal antibody R310 (mAbR310), which recognizes (R)-HNEmodified protein, are reported. The Fab fragment of mAbR310 was obtained by digestion with papain, purified and crystallized. Using hanging-drop vapourdiffusion crystallization techniques, crystals of mAbR310 Fab were obtained. The crystal belongs to the monoclinic space group C2 (unit-cell parameters a = 127.04, b = 65.31, c = 64.29 Å , = 118.88 ) and diffracted X-rays to a resolution of 1.84 Å . The asymmetric unit contains one molecule of mAbR310, with a corresponding crystal volume per protein weight of 2.51 Å 3 Da À1 and a solvent content of 51.0%.

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“…Upon reaction with protein, HNE specifically reacts with nucleophilic amino acids, such as cysteine, histidine, and lysine, to form stable Michael addition adducts possessing three chiral centers at C-2, C-4, and C-5 in the cyclic hemiacetal structure (Fig. 1A) (5). Because of this chirality, the HNE adduct is composed of eight configurational isomers.…”

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confidence: 99%

Bispecific Abs against modified protein and DNA with oxidized lipids

Akagawa

Ito

Toyoda

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R-and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress. HNE reacts with histidine to form a stable HNE-histidine Michael addition-type adduct possessing three chiral centers in the cyclic hemiacetal structure. We have previously raised the mAbs, anti-R mAb 310 and anti-S mAb S412, that enantioselectively recognized the R-HNE-histidine and R-HNEhistidine adducts, respectively, and demonstrated the presence of both epitopes in vivo. In the present study, to further investigate the anti-HNE immune response, we analyzed the variable genes and primary structure of these Abs and found that the sequence of R310 was highly homologous to anti-DNA autoantibodies, the hallmark of systemic lupus erythematosus. An x-ray crystallographic analysis of the R310 Fab fragment showed that the R-HNEhistidine adduct binds to a hydrophobic pocket in the antigenbinding site. Despite the structural identity to the anti-DNA autoantibodies, however, R310 showed only a slight crossreactivity with the native double-stranded DNA, whereas the Ab immunoreactivity was dramatically enhanced by the treatment of the DNA with 4-oxo-2-nonenal (ONE), an analog of HNE. Moreover, the 7-(2-oxo-heptyl)-substituted 1,N 2 -etheno-type ONE-2 -deoxynucleoside adducts were identified as alternative epitopes of R310. Molecular mimicry between the R-HNE-histidine configurational isomers and the ONE-DNA base adducts is proposed for the dual crossreactivity.4-hydroxy-2-nonenal ͉ anti-DNA autoantibody ͉ lipid peroxidation ͉ oxidative stress ͉ systemic lupus erythematosus I t is estimated that most of the proteins in the human body are posttranslationally modified. Such modifications include phosphorylation, methylation, and glucosylation. They are enzymemediated and homeostatically important, either to carry out a particular structural or functional role or to allow the efficient recycling of the amino acid constituents. However, several lines of evidence indicate that the nonenzymatic oxidative modification of proteins and the subsequent accumulation of the modified proteins have been found in cells during aging and oxidative stress and in various pathological states, including premature diseases, muscular dystrophy, rheumatoid arthritis, and atherosclerosis (1). The important agents that give rise to the modification of proteins are represented by oxidized lipid metabolites, such as 2-alkenals, 4-hydroxy-2-alkenals, and ketoaldehydes (2, 3). These metabolites are considered important mediators of cell damage because of their ability to covalently modify biomolecules, which can disrupt important cellular functions and can cause mutations (2).4-Hydroxy-2-nonenal (HNE), a racemic mixture of 4R-and 4S-enantiomers, is a major product of lipid peroxidation and is believed to be largely responsible for the cytopathological effects observed during oxidative stress (2). HNE exerts these effects because of its...

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Structural Basis of Protein-bound Endogenous Aldehydes

Cited by 79 publications

References 31 publications

Formation of Acrolein-derived 2′-Deoxyadenosine Adduct in an Iron-induced Carcinogenesis Model

Formation of Acrolein-derived 2′-Deoxyadenosine Adduct in an Iron-induced Carcinogenesis Model

Crystallization and molecular-replacement studies of the monoclonal antibody mAbR310 specific for the (R)-HNE-modified protein

Bispecific Abs against modified protein and DNA with oxidized lipids

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