Covalent cross-linking of glutathione and carnosine to proteins by 4-oxo-2-nonenal.
The lipid oxidation product 4-oxo-2-nonenal (ONE) derived from peroxidation of polyunsaturated fatty acids is a highly reactive protein cross-linking reagent. The major family of cross-links reflects conjugate addition of side chain nucleophiles such as sulfhydryl or imidazole groups to the C triple bond C of ONE to give either a 2- or 3-substituted 4-ketoaldehyde, which then undergoes Paal-Knorr condensation with the primary amine of protein lysine side chains. If ONE is intercepted in biological fluids by antielectrophiles such as glutathione (GSH) or beta-alanylhistidine (carnosine), this would lead to circulating 4-ketoaldehydes that could then bind covalently to the protein Lys residues. This phenomenon was investigated by SDS-PAGE and mass spectrometry (matrix-assisted laser desorption/ionization time-of-flight and LC-ESI-MS/MS with both tryptic and chymotryptic digestion). Under the reaction conditions of 0.25-2 mM ONE, 1 mM GSH or carnosine, 0.25 mM bovine beta-lactoglobulin (beta-LG), and 100 mM phosphate buffer (pH 7.4, 10% ethanol) for 24 h at 37 degrees C, virtually every Lys of beta-LG was found to be fractionally cross-linked to GSH. Cross-linking of Lys to carnosine was less efficient. Using cytochrome c and RNase A, we showed that ONE becomes more protein-reactive in the presence of GSH, whereas protein modification by 4-hydroxy-2-nonenal is inhibited by GSH. Stable antielectrophile-ONE-protein cross-links may serve as biomarkers of oxidative stress and may represent a novel mechanism of irreversible protein glutathionylation.
Zhu
Xiaochun
X
Departments of Chemistry, Pharmacology, and Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Gallogly
Molly M
MM
Mieyal
John J
JJ
Anderson
Vernon E
VE
Sayre
Lawrence M
LM
D000447:Aldehydes / Q000737:chemistry* / Q000633:toxicity; D000595:Amino Acid Sequence; D000818:Animals; D002336:Carnosine / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000633:toxicity; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000737:chemistry* / Q000378:metabolism; D007782:Lactoglobulins / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
C403894:4-oxo-2-nonenal; D000447:Aldehydes; D003432:Cross-Linking Reagents; D007782:Lactoglobulins; D010446:Peptide Fragments; D002336:Carnosine; D005978:Glutathione