3–Hydroxykynurenine–Mediated Modification of Lens Proteins: Detection by a Monoclonal Antibody

Abstract

Abstract: : Purpose: Tryptophan undergoes oxidation by enzymatic and non–enzymatic mechanisms to produce kynurenines. Kynurenines are highly reactive molecules; they react with proteins induce crosslinking and generate yellow brown pigments in the lens. In this study we have investigated effects of glycation on tryptophan oxidation and the role of kynurenines in lens protein modification.Methods:A monoclonal antibody was generated against 3–hydroxykynurenine (3–OHK)–modified KLH. The antibody was characterized using 3–OHK–modified amino acids and proteins. Effect of early and advanced glycation products on kynurenine–modification was studied using glycated amino acids and glycated lysine–Sepharose. A major antigen for the antibody was purified from the reaction of 3–OHK and N^α–acetyl lysine by reversed phase HPLC. Immunohistochemistry, Western blotting and a competitive ELISA were used to detect or quantify 3–OHK–derived products in human lens proteins. 3–OHK–mediated modification was also investigated in human lens epithelial cells incubated in the presence of 3–OHK. Results: In competitive ELISAs, the monoclonal antibody reacted similarly with 3–OHK–modified N^α–acetyl lysine, N^α–acetyl histidine, N^α–acetyl arginine and N^α–acetyl cysteine. Addition of tryptophan during glycation or incubation of proteins with early glycation products did not generate kynurenine modifications on proteins. Among several tryptophan oxidation products tested, 3–OHK produced the highest antigen level when reacted with human lens proteins. A major antigen was isolated from the reaction of 3–OHK and N^α–acetyl lysine and was characterized by spectroscopy. Western blotting detected 3–OHK–modifications in enzyme digested proteins from cataractous but not non–catarctous lenses. Immunohistochemistry revealed 3–OHK modifications in proteins associated with the fiber cell plasma membrane in cataractous lenses. Low molecular products (<10,000 kDa) isolated from cataractous lenses after treatment with glucosidase and incubation with proteins generated 3–OHK–modifications. Human lens epithelial cells (HLE B–3) incubated with 3–OHK showed intense immunoreactivity. Western blotting revealed 3–OHK modifications in high molecular weight proteins of the cytosolic fraction. Conclusions: Tryptophan oxidation to kynurenines does not occur during glycation. 3–OHK–derived products crosslinked to basic amino acids and cysteine are likely to be present in cataractous lenses, which may contribute to protein aggregation and browning during cataract formation.

Keywords: cataract • protein modifications-post translational • oxidation/oxidative or free radical damage