glycyllysine and 3-hydroxykynurenine

3-Hydroxykynurenine (3OHKyn) is present in the mammalian lens as a UV filter and is formed from kynurenine in the tryptophan metabolic pathway. 3OHKyn is a readily autoxidized o-aminophenol which binds to proteins in vitro. The lens, particularly its central region, the nucleus, becomes increasingly oxidized with age. Under such conditions, the oxidation products of 3OHKyn may bind to lens proteins and contribute to nuclear cataract formation. The purpose of this study was to determine the structures of in vitro reaction products of 3OHKyn with model peptides as a general model for 3OHKyn modification of proteins. 3OHKyn was incubated with the dipeptide glycylglycine (GG) and the tetrapeptide tuftsin (sequence TKPR) under oxidizing conditions, and the reaction products were characterized by a variety of spectroscopic techniques. The major 3OHKyn-GG reaction product involves formation of a benzimidazole moiety between the GG N-terminus and the oxidized amino and/or phenol groups of 3OHKyn. In contrast, tuftsin, which has an N-terminal threonine, forms predominantly a cross-linked dimer with oxidized 3OHKyn. This product is analogous in structure to the dimeric reaction product, quinilinobenzoxamine, formed between oxidized 3OHKyn and glycyllysine [Aquilina, J. A., et al. (1999) Biochemistry 38, 11455-11464], which contains a benzoxazole moiety. The identification of a tuftsin dimer suggests that 3OHKyn can react with any peptide having a free alpha-amino group, via a general side chain elimination mechanism. The identification of both benzimidazole and benzoxazole adducts in peptides with a free N-terminus suggests that peptide amino groups can react initially at either the aromatic amino or hydroxyl group of oxidized 3OHKyn. The proportion of each adduct may change, however, depending on the amino acid sequence at the N-terminus.

Topics: Chromatography, High Pressure Liquid; Cross-Linking Reagents; Dipeptides; Glycine; Humans; Kynurenine; Mass Spectrometry; Nuclear Magnetic Resonance, Biomolecular; Oxidation-Reduction; Peptides; Spectrometry, Fluorescence; Tuftsin

3-Hydroxykynurenine, a metabolite of tryptophan, is a powerful antioxidant and neurotoxin. The neurotoxicity results from the oxidation of 3-hydroxykynurenine, and hydroxyl radicals, formed via H(2)O(2), may also be implicated [Okuda, S., Nishiyama, N., Saito, H. , and Katsuki, H. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12553-12558]. Oxidation of o-aminophenols, such as 3-hydroxykynurenine, also results in the formation of highly reactive quinonimines. Thus, one possible consequence of 3-hydroxykynurenine oxidation may be covalent modification of cellular macromolecules. Such a process could contribute to the neurotoxicity and may potentially be important in other tissues, such as the human lens, where 3-hydroxykynurenine functions as a UV filter. In this work, we demonstrate that 3-hydroxykynurenine can bind to protein amino groups and, further, that under oxidative conditions, 3-hydroxykynurenine can function to cross-link polypeptide chains. The structure of the cross-linked moiety, using the peptide glycyllysine, has been elucidated. The cross-link, which is both colored and fluorescent, involves the peptide alpha-amino groups. Proteins modified by 3-hydroxykynurenine become colored and fluorescent as well as cross-linked. LC-MS studies indicate that the cross-link is also present in gamma-crystallin, following incubation of this lens protein in the presence of 3-hydroxykynurenine. Similar posttranslational modifications of lens proteins accompany cataract formation, and knowledge of the precise mode of reaction of 3-hydroxykynurenine with proteins will assist in determining if 3-hydroxykynurenine is involved in degenerative conditions in which oxidation of such aminophenols is implicated.

Topics: Animals; Cattle; Cross-Linking Reagents; Crystallins; Dipeptides; Electrophoresis, Polyacrylamide Gel; Humans; Kynurenine; Mass Spectrometry; Nuclear Magnetic Resonance, Biomolecular; Peptides; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet