title: Identification of Kynoxazine, a Novel Fluorescent Product of the Reaction between 3-Hydroxykynurenine and Erythrulose in the Human Lens, and Its Role in Protein Modification.
abstract: Kynurenine pathway metabolites and ascorbate degradation products are present in human lenses. In this study, we showed that erythrulose, a major ascorbate degradation product, reacts spontaneously with 3-hydroxykynurenine to form a fluorescent product. Structural characterization of the product revealed it to be 2-amino-4-(2-hydroxy-3-(2-hydroxyethyl)-2H-benzo[b][1,4]oxazin-5-yl)-4-oxobutanoic acid, which we named kynoxazine. Unlike 3-hydroxykynurenine, 3-hydroxykynurenine glucoside and kynurenine were unable to form a kynoxazine-like compound, which suggested that the aminophenol moiety in 3-hydroxykynurenine is essential for the formation of kynoxazine. This reasoning was confirmed using a model compound, 1-(2-amino-3-hydroxyphenyl)ethan-1-one, which is an aminophenol lacking the amino acid moiety of 3-hydroxykynurenine. Ultra-performance liquid chromatography-tandem mass spectrometry analyses showed that kynoxazine is present in the human lens at levels ranging from 0 to 64 pmol/mg lens. Kynoxazine as well as erythrulose degraded under physiological conditions to generate 3-deoxythreosone, which modified and cross-linked proteins through the formation of an arginine adduct, 3-deoxythreosone-derived hydroimidazolone, and a lysine-arginine cross-linking adduct, 3-deoxythreosone-derived hydroimidazolimine cross-link. Ultra-performance liquid chromatography-tandem mass spectrometry quantification showed that 32-169 pmol/mg protein of 3-deoxythreosone-derived hydroimidazolone and 1.1-11.2 pmol/mg protein of 3-deoxythreosone-derived hydroimidazolimine cross-link occurred in aging lenses. Taken together, these results demonstrate a novel biochemical mechanism by which ascorbate oxidation and the kynurenine pathway intertwine, which could promote protein modification and cross-linking in aging human lenses.
authors.affiliation: From the Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado 80045.
authors.forename: Stefan
authors.initials: S
authors.lastname: Rakete
authors.affiliation: From the Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado 80045 ram.nagaraj@ucdenver.edu.
authors.forename: Ram H
authors.initials: RH
authors.lastname: Nagaraj
mesh_terms: D000375:Aging / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism*; D006801:Humans; D007737:Kynurenine / Q000031:analogs & derivatives* / Q000378:metabolism; D007908:Lens, Crystalline / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013780:Tetroses / Q000378:metabolism*
chemical_list: D005136:Eye Proteins; D013780:Tetroses; C005045:3-hydroxykynurenine; D007737:Kynurenine; C100073:erythrulose
keywords: ascorbic acid; cataract; crystallin; glycation; oxidation-reduction (redox); protein aggregation; protein chemical modification