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.

UVA Light‐mediated Ascorbate Oxidation in Human Lenses

“…This could be due to the short half‐life of DHA under physiological conditions of about 30 min and the irreversibility of 2,3‐DKG to DHA or ASC . Although 3‐DT can also be derived from glucose, we recently showed that it is mainly derived from 2,3‐DKG in the human lens . An increase in the 3‐DT levels also coincided with an increase in ASC oxidation, although this increase was modest (Fig.…”

“…α ‐Dicarbonyls are major precursors of AGEs , they primarily react with lysine and arginine residues to generate AGEs and can also form cross‐linking adducts between arginine and lysine residues in proteins . Thus, our results suggest that UVA‐mediated ASC oxidation in the human lens could lead to lens protein modification by AGEs . However, we were not able to demonstrate AGE formation from ASC oxidation in the lens because frozen lenses are not suitable for longer incubation after irradiation, which is necessary for AGE formation.…”

“…Acetonitrile, formic acid, heptafluorobutyric acid, N ‐ethylmaleimide, 2,2’‐bipyridyl, ferric chloride, trichloroacetic acid sodium phosphate, sodium biphosphate and PBS were obtained from Fisher Scientific (Waltham, MA). The quinoxalines of DHA, 2,3‐DKG and 3‐DT were prepared as previously described .…”

“…UPLC-MS 2 analysis for a-dicarbonyls in lens homogenates. a-Dicarbonyls were analyzed as previously described (11).…”

“…Upon oxidation, ASC is converted to dehydroascorbic acid (DHA), which can be directly reduced by glutathione (GSH) . However, the ASC levels in aged and cataractous lenses are either very low or nonexistent, and low ASC levels are associated with higher levels of DHA . The loss of GSH along with the loss in the activity of enzymes needed for GSH synthesis and the conversion of GSSG to GSH could favor ASC oxidation in aged and cataractous lenses .…”