Abstract
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-N(epsilon)-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.
Highlights
Saturated fatty acids has been implicated in the pathogenesis of many types of liver injuries and especially in the hepatic damage induced by several toxic substances
Monoclonal Antibody against Protein-bound 2-Nonenal—To assess the reactivity of 2-nonenal to proteins, human serum albumin (HSA) (1 mg/ml) was exposed to 2-nonenal (0 –10 mM) under physiological conditions in vitro, and changes in the amino acid composition of the protein were examined by an amino acid analysis
During the preparation of the monoclonal antibody (mAb), hybridomas were selected by comparing the reactivities of the culture supernatant to the 2-nonenal-modified bovine serum albumin (BSA)
Summary
Fe3ϩ-NTA, ferric nitrilotriacetate; AcLDL, acetylated LDL; BSA, bovine serum albumin; DiD, 1,1Ј-dioctadecyl-3, 3,3Ј,3Ј-tetramethylindodicarbocyanine perchlorate; ELISA, enzyme-linked immunosorbent assay; HHP-lysine, N⑀-3-[(hept-1-enyl)-4-hexylpyridinium]lysine; HMBC, 1H-detected multiple-bond connectivity; KLH, keyhole limpet hemocyanin; LDL, low density lipoprotein; LOX-1, lectin-like oxidized LDL receptor; HSA, human serum albumin; LC/ESI/MS/MS, high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry; mAb, monoclonal antibody; MRM, multiple reaction monitoring; PBS, phosphate-buffered saline; HPLC, high pressure liquid chromatography; CFP, cyan fluorescent protein; CHO, Chinese hamster ovary; Fmoc, N-(9-fluorenyl)methoxycarbonyl; HSA, human serum albumin. Lipid Peroxidation Generates Protein-bound 2-Nonenal recently, Haze et al [6] 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 suggested that cis-2-nonenal and trans-2-nonenal are formed from the oxidative degradation of polyunsaturated fatty acids, such as palmitoleic acid. 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
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