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* This work was supported by a grant-in-aid for scientific research on innovative areas (research in a proposed research area), from the Ministry of Education, Culture, Sports, Science and Technology, Japan (to K. U.). The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S22.
trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-Nϵ-3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.
Lipid peroxidation in tissue and in tissue fractions represents a degradative process, which is the consequence of the production and propagation of free radical reactions primarily involving membrane polyunsaturated fatty acids, and has been implicated in the pathogenesis of numerous diseases, including atherosclerosis, diabetes, cancer, and rheumatoid arthritis, as well as in drug-associated toxicity, postischemic reoxygenation injury, and aging (
). The peroxidative breakdown of polyunsaturated fatty acids has also been implicated in the pathogenesis of many types of liver injuries and especially in the hepatic damage induced by several toxic substances. The 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 (
). The primary products of lipid peroxidation, lipid hydroperoxides, 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 or a second class of aldehydes still esterified to the parent lipid. These reactive aldehydic intermediates readily form covalent adducts with cellular macromolecules, including protein, leading to the disruption of important cellular functions. The important agents that give rise to the modification of protein may be represented by α,β-unsaturated aldehydic intermediates, such as 2-alkenals, 4-hydroxy-2- alkenals, and 4-oxo-2-alkenals (
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. It has been suggested that these aldehydes primarily react with the sulfhydryl group of cysteine, the ϵ-amino group of lysine, and the imidazole group of histidine in the proteins (
). Among the 2-alkenals, 2-nonenal (Fig. 1A) has a characteristically unpleasant greasy and grassy odor. It is also a major contributor to the unpleasant cardboard flavor in aged beer. It was previously shown that 2-nonenal could be formed through lipid peroxidation as a minor product in peroxide- mediated oxidation of high concentrations of linoleic acid hydroperoxide or from liver microsomes treated with ADP/iron in vitro (
) analyzed the body odor components that adhered to the shirts of the subjects by gas chromatography/mass spectrometry and demonstrated that 2-nonenal is present in increasing amounts in the body odors of persons 40 years or older. They have also suggested that cis-2-nonenal and trans-2-nonenal are formed from the oxidative degradation of polyunsaturated fatty acids, such as palmitoleic acid. However, it is still not clear how 2-nonenal could be formed in vivo. Because of its insolubility in water, 2-nonenal is less reactive with proteins than other 2-alkenals, such as acrolein and crotonaldehyde, and therefore has received relatively little attention as a causative agent for modification of proteins. Only inhibition of enzymes, such as platelet membrane-bound phosphotyrosine phosphatase (
A structurally diverse protein supergroup called scavenger receptors mediates the cellular uptake of modified lipoproteins. Scavenger receptors are expressed by endothelial cells, macrophages, and smooth muscle cells and mediate recognition, internalization, and physiological responses to a wide range of ligands, including phospholipids, lipoprotein particles, apoptotic cells, and pathogens. Several oxidized low density lipoprotein (LDL) receptors have been identified so far, including SR-A I/II, CD36, SR-BI, FcγRII, lectin-like oxidized LDL receptor (LOX-1), macrosialin, SR expressed by endothelial cells, etc. Among them, LOX-1 is characterized as the major receptor for oxidized LDL in the endothelial cells of large arteries. Its inducible expression (
) might be involved in the oxidized LDL-mediated endothelial dysfunction. LOX-1 exhibits a binding activity for multiple ligands. LOX-1 is capable of interacting with a variety of structurally and functionally distinct ligands, including oxidized LDL, platelets, aged red blood cells, apoptotic cells, advanced glycation end products, heat shock protein 70, bacteria, and phosphatidylserine (
). LOX-1 binds and internalizes a diverse array of macromolecules, although their structures are not always related to each other.
In this study, to understand the mechanism underlying the formation of a covalently modified protein with 2-nonenal in vivo, we raised a monoclonal antibody (mAb) against protein-bound 2-nonenal and identified a novel 2-nonenal-lysine adduct as the epitope. Our immunohistochemical studies demonstrated the localization of the immunoreactive materials in the kidney of rats exposed to Fe3+-NTA, an iron chelate that induces acute renal proximal tubular necrosis, a consequence of free radical-mediated oxidative tissue damage, eventually leading to a high incidence of renal adenocarcinoma in rodents. Importantly, using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS), we also showed evidence that the 2-nonenal-lysine adduct is indeed accumulated during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, to evaluate the biological implication of the 2-nonenal modification of protein, we examined the involvement of LOX-1, a member of the scavenger receptor family, in the recognition of the 2-nonenal-lysine adducts.
Lipid peroxidation proceeds by a free radical chain reaction mechanism and yields lipid hydroperoxides as major initial reaction products. Subsequently, the decomposition of the lipid hydroperoxides generates a number of breakdown products that display a wide variety of damaging actions. A number of reactive aldehydes derived from lipid peroxidation have been implicated as causative agents in cytotoxic processes initiated by the exposure of biological systems to oxidizing agents (
). On the basis of a large number of reports concerning the detection of lipid peroxidation-specific adducts as biomarkers in human diseases, there is no doubt that the steady-state levels of lipid peroxidation products increase in pathophysiological states associated with oxidative stress. Considerable progress has also recently been made toward understanding the mechanisms of action of lipid peroxidation products. The quantitative and analytical importance of lipid peroxidation-specific adducts has prompted the development of methods to specifically analyze these adducts to understand their chemical nature, formation pathway, and distribution level in vivo.
The results presented herein show that 2-nonenal, a body odorant originating from lipid peroxidation, covalently modifies proteins. A spontaneous chemical reaction was observed in vitro. In addition, we obtained a murine monoclonal antibody, mAb 27Q4, that clearly distinguished the 2-nonenal-modified protein from the native protein. This antibody appeared to be highly specific for the protein-bound 2-alkenals, including 2-heptenal, 2-octenal, and 2-nonenal. Using the antibody, the formation of the 2-nonenal-modified proteins in vivo was tested in the kidney of rats exposed to Fe3+-NTA. The iron chelate was originally used for an experimental model of iron overload (
). Repeated intraperitoneal injections of Fe3+-NTA were reported to induce acute and subacute renal proximal tubular necrosis and a subsequent high incidence (60–92%) of renal adenocarcinoma in male rats and mice (
). A single injection of Fe3+-NTA causes a number of time-dependent morphological alterations in the structure and the function of the renal proximal tubular cells and their mitochondria. During the early stage of injury, typical cellular changes are the loss of the brush border, cytoplasmic vesicles, mitochondrial disorganization, and dense cytoplasmic deposits in the proximal tubular cells. Most of the damaged epithelia show the typical appearance of necrotic cells, and more than half of the proximal tubular cells are gone. It has been suggested that oxidative stress is one of the basic mechanisms of Fe3+-NTA-induced acute renal injury and is closely associated with renal carcinogenesis (
). The present in vivo study has shown that lipid peroxidation generates 2-nonenal covalently bound to proteins in the renal proximal tubules of rats treated with Fe3+-NTA (Fig. 3). To the best of our knowledge, this is the first report of the in vivo formation of protein-bound 2-nonenal/2-alkenals in the target organ of the carcinogenic protocol. It was also striking that the immunoreactivities with mAb 27Q4 were detected even 48 h after the administration of Fe3+-NTA (Fig. 3E). Long retention of this aldehyde may play a role in the Fe3+-NTA-induced renal carcinogenesis.
Upon investigation of an antigenic adduct recognized by the antibody (mAb 27Q4), we unexpectedly identified novel lysine-pyridinium adducts, cis- and trans-HHP-lysines. Of interest, the monoclonal antibodies raised against protein-bound 2- alkenals, such as acrolein and crotonaldehyde, recognize pyridinium-containing adducts as the major epitopes (
). It is likely that, due to the placement of a fixed, positive charge on the ϵ-amino group, the pyridinium-containing adducts could be an important immunological epitope generated in 2-alkenal-modified proteins. The formation of the 3,4-substituted pyridinium adducts has also been reported to be a dominant pathway for modification of the primary amine with 2-alkenals, such as 2-hexenal and 2-octenal (
). Based on these studies, HHP-lysine is likely to be formed through the formation of the 2-nonenal-lysine Schiff base (Fig. 7). After the formation of the Schiff base adduct, the C3 position of the initial Schiff base may be attacked by the C2 of the enolate anion from the second aldehyde, followed by dehydration and cyclization to form the isomeric pyridinium adduct. On the other hand, trans-cis isomerization of α,β-unsaturated compounds is known to be catalyzed by amines (addition-elimination). Thus, the isomerization required for the formation of cis-HHP-lysine may occur at the stage of the free 2-nonenal or following Schiff base formation.
Due to the fact that the core structures of the pyridinium-containing lysine adducts are resistant to the conventional acid hydrolysis of proteins, we established a highly sensitive method for the detection of cis- and trans-HHP-lysine using LC/ESI/MS/MS. In vitro studies of the detection of HHP-lysine demonstrated that the 2-nonenal-lysine adducts were generated in the oxidized LDL (Fig. 5A). In addition, substantial amounts of the 2-nonenal-lysine adducts, mainly the trans-HHP-lysine, were detected in the metal-catalyzed peroxidation of unsaturated fatty acids in the presence of protein (Fig. 5B). These data suggest that covalent modification of proteins by 2-nonenal, generating the pyridinium-containing lysine adducts, could have diagnostic applications. Because oxidative degradation of unsaturated fatty acids, accelerated by lipid peroxides, may be involved in the formation of 2-nonenal, resulting in deterioration of the body odors for the middle-aged and the elderly (
), plasma or red blood cell levels of a long-lived 2-nonenal adduct could provide a measure of past exposure and aid in the management of one's health condition. Such a reporter function would be analogous to the use of glycated hemoglobin as a marker of past hyperglycemia and a guide to the management of diabetes.
It should also be noted that peroxidation of not only palmitoleic acid, but also ω6-polyunsaturated fatty acids, such as linoleic acid, γ-linolenic acid, and arachidonic acid, in the presence of HSA generated HHP-lysine. 2-Nonenal was previously identified as an unpleasant greasy and grassy odor component endogenously generated during the peroxidation of palmitoleic acid (
). However, until this study, bona fide unsaturated fatty acids responsible for the formation of 2-nonenal have remained unidentified. Therefore, this study has established that ω6-polyunsaturated fatty acids also represent an excellent source of 2-nonenal. Although the mechanism of the formation of 2-nonenal during lipid peroxidation has not yet been experimentally resolved, there may be no doubt that 2-nonenal could be ubiquitously generated under oxidative stress.
Scavenger receptors have been shown to bind aldehyde-modified proteins (
). These receptors are thought to provide a mechanism for the clearance of these modified proteins from the circulation through a number of cell types. Indeed, a previous study has shown that endothelial cells can bind and degrade an aldehyde-modified protein (
). In this study, to determine whether the 2-nonenal-specific epitopes enter cells though scavenger receptors, we selected LOX-1 and performed inhibition studies. We showed that BSA incubated with 2-nonenal significantly inhibited the LOX-1-mediated uptake of AcLDL (FIGURE 6, FIGURE 7). In addition, we directly assessed whether HHP-lysine might serve as a ligand and observed that both the cis- and trans-HHP-lysines competed for the uptake of AcLDL. These data suggested that LOX-1 might serve as a receptor for the 2-nonenal-lysine adducts generated on oxidized LDL molecules. Data from the mesenteric vein injection of an aldehyde-modified protein also suggest that LOX-1 may be involved to a certain degree in these processes (
). Thus, it is likely that LOX-1 is involved in the recognition of 2-nonenal-modified proteins. However, the possibility still exists for other receptors to be involved in the processing of 2-nonenal-modified proteins.
In summary, to assess the formation of 2-nonenal generation under oxidative stress in vivo, we raised a new murine monoclonal antibody, mAb 27Q4, against the 2-nonenal-modified KLH. The model system provides a detailed structural characterization of the epitopes, the cis- and trans-HHP-lysines, formed during the reaction of the lysine side-chain amino groups with 2-nonenal. We proved that the immunoreactive materials with mAb 27Q4 were indeed generated in an animal model of oxidative stress in vivo. In addition, using LC/ESI/MS/MS, the biological levels of the 2-nonenal-lysine adduct in vitro and in vivo were accurately estimated. Finally, we examined the involvement of the scavenger receptor LOX-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand. The present results not only offer structural insights into protein modification by lipid peroxidation products but also provide a platform for the chemical analysis of protein-bound aldehydes in vitro and in vivo.