Monoclonal antibody against protein-bound glutathione: use of glutathione conjugate of acrolein-modified proteins as an immunogen.
Acrolein shows a facile reactivity with the ε-amino group of lysine to form N(ε)-(3-formyl-3,4-dehydropiperidino)lysine (FDP-lysine) as the major product. In addition, FDP-lysine generated in the acrolein-modified protein could function as an electrophile, reacting with thiol compounds, to form an irreversible thioether adduct. In the present study, to establish the utility of this irreversible conjugate, we attempted to use it as an immunogen to raise a monoclonal antibody (mAb), which specifically recognized protein-bound thiol compounds. Using the glutathione (GSH) conjugate of the acrolein-modified protein as an immunogen, we raised the mAb 2C4, which cross-reacted with the GSH conjugate of acrolein-modified proteins. Specificity studies revealed that mAb 2C4 recognized both the GSH conjugate of an acrolein-lysine adduct, FDP-lysine, and oxidized GSH (GSSG). In addition, mAb 2C4 cross-reacted not only with the GSH conjugates of the acrolein-modified protein but also with the GSH-treated, oxidized protein (S-glutathiolated protein), suggesting that the antibody significantly recognized the protein-bound GSH as the epitope. An immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2C4 were indeed present in the macrophage-derived foam cells and migrating smooth muscles. In addition, using mAb 2C4, we analyzed the GSH-treated, oxidized low-density lipoproteins by agarose gel electrophoresis under reducing or nonreducing conditions followed by immunoblot analysis and found that the majority of the GSH was irreversibly incorporated into the proteins. The results of this study not only showed the utility of the antibody raised against the GSH conjugate of the acrolein-modified proteins but also suggested that the irreversible binding of GSH and other redox molecules to the oxidized LDL might represent the process common to the modification of LDL during atherogenesis.
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Atsunori
A
Furuhata
Kazuya
K
Honda
Takahiro
T
Shibata
Miho
M
Chikazawa
Yoshichika
Y
Kawai
Noriyuki
N
Shibata
Koji
K
Uchida
D000171:Acrolein / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D050197:Atherosclerosis / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008077:Lipoproteins, LDL / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives / Q000737:chemistry; D010084:Oxidation-Reduction; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
C481039:2C4 antibody; D000911:Antibodies, Monoclonal; D008077:Lipoproteins, LDL; C113380:N(epsilon)-(3-formyl-3,4-dihydropiperidino)lysine; D011506:Proteins; C099252:oxidized low density lipoprotein; D000171:Acrolein; D005978:Glutathione; D008239:Lysine