Detection of kynurenine modifications in proteins using a monoclonal antibody.
N-formylkynurenine and kynurenine are oxidation products of tryptophan formed from the reaction catalyzed by indoleamine 2,3-dioxygenase. These kynurenines react with proteins to produce chemical modifications in the lens. We developed a novel monoclonal antibody that detects a kynurenine modification in proteins. The antibody recognized proteins (human lens proteins, RNase A and BSA) that were modified by either kynurenine or N-formylkynurenine. The antibody also reacted strongly with N-formylkynurenine-modified N(alpha)-acetyl histidine and weakly with N-formylkynurenine-modified N(alpha)-acetyl lysine, N(alpha)-acetyl cysteine and N(alpha)-acetyl arginine. The antibody recognized kynurenine and N-formylkynurenine but not other tryptophan oxidation products. We isolated and purified a major antigen from the reaction mixture of N(alpha)-acetyl histidine and N-formylkynurenine and identified the product as N-acetyl-1-[3-(2-aminophenyl)-1-carboxy-3-oxopropyl]-histidine. We then used our purified antibody to detect kynurenine modifications in kynurenine-treated human lens epithelial cells and human lens. We found epithelial immunoreactivity in a lens from an aged donor but not in one from a very young donor. This would suggest that the antibody detects age-related changes in lens proteins altered by kynurenines. We believe that our antibody could be used to establish the importance of kynurenine modifications in diseases where tryptophan oxidation is enhanced.
Staniszewska
Magdalena
M
Department of Ophthalmology, Cleveland, OH 44106, USA.
Nagaraj
Ram H
RH
D000368:Aged; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002675:Child, Preschool; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000276:immunology* / Q000378:metabolism*; D006801:Humans; D007737:Kynurenine / Q000031:analogs & derivatives / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology; D007908:Lens, Crystalline / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008807:Mice, Inbred BALB C
D000911:Antibodies, Monoclonal; D005136:Eye Proteins; C007772:N'-formylkynurenine; D007737:Kynurenine