Susceptibility of actin to modification by 4-hydroxy-2-nonenal.
4-Hydroxy-2-nonenal (HNE), a major lipid peroxidation product, reacts with histidine, lysine or cysteine residues of proteins to form hemiacetal Michael adducts and thus interferes with the functions of the proteins. Here we undertook to identify HNE-modified proteins in the target organ of a ferric nitrilotriacetate (Fe-NTA)-induced renal carcinogenesis model with histidine-specific HNEJ-2 antibody. Immunoaffinity column separation and sequencing identified one of the major modified proteins as actin. To further explore the characteristics of actin as an HNE acceptor, we produced four novel monoclonal antibodies against HNE-modified keyhole limpet hemocyanin. All these antibodies (HNEJ-1, 3-5) recognized histidine adducts, but were different from HNEJ-2 in recognizing lysine and cysteine adducts to some extent. Actin, albumin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), metallothionein and superoxide dismutase were treated in vitro with HNE and evaluated with these antibodies. The results revealed that actin was most sensitive to HNE modification and metallothionein most resistant. Furthermore, the residue-specificity of GAPDH was in accord with that shown by our recent mass spectrometry data. Immunohistochemistry with the antibodies revealed cytoplasmic staining with or without nuclear staining in the renal proximal tubules after Fe-NTA administration. The results suggest that actin is a major target protein for HNE modification in vivo, and that our monoclonal antibodies are useful for evaluating the HNE adducts produced.
Ozeki
Munetaka
M
Department of Pathology and Biology of Diseases, Graduate School of Medicine, Kyoto University, Japan.
Miyagawa-Hayashino
Aya
A
Akatsuka
Shinya
S
Shirase
Tomoyuki
T
Lee
Wen-Hua
WH
Uchida
Koji
K
Toyokuni
Shinya
S
D000199:Actins / Q000737:chemistry / Q000187:drug effects*; D000418:Albumins / Q000737:chemistry; D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D003545:Cysteine / Q000737:chemistry; D005290:Ferric Compounds; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D006433:Hemocyanins / Q000276:immunology; D006639:Histidine / Q000737:chemistry; D007668:Kidney / Q000187:drug effects; D007680:Kidney Neoplasms / Q000139:chemically induced / Q000503:physiopathology; D008239:Lysine / Q000737:chemistry; D008297:Male; D008668:Metallothionein / Q000737:chemistry; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives; D051381:Rats; D017208:Rats, Wistar; D013047:Specific Pathogen-Free Organisms; D013482:Superoxide Dismutase / Q000737:chemistry
D000199:Actins; D000418:Albumins; D000447:Aldehydes; D000911:Antibodies, Monoclonal; D005290:Ferric Compounds; D006639:Histidine; D006433:Hemocyanins; D008668:Metallothionein; D013482:Superoxide Dismutase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; C032808:keyhole-limpet hemocyanin; C027576:4-hydroxy-2-nonenal; D008239:Lysine; D003545:Cysteine; D009571:Nitrilotriacetic Acid; C020326:ferric nitrilotriacetate