Detection of Kynurenine-Modified Proteins in the Human Lens Using a Monoclonal Antibody

Abstract

Purpose:: N-formylkynurenine (NFK) and kynurenine (KYN) are oxidation products of tryptophan formed from the reaction catalyzed by indoleamine 2,3-dioxygenase. These kynurenines react with proteins and produce chemical modifications. Such modifications have been detected in the human lens. A monoclonal antibody was developed for detection and quantification of such modifications in human lenses.

Methods:: A monoclonal antibody was generated by immunizing mice with NFK-modified keyhole limpet hemocyanin (KLH). Human lens proteins (HLP), BSA and RNAse A were incubated with tryptophan and tryptophan oxidation products at pH 7.4 and 37 ⁰C to determine the reactivity with the antibody. NFK was incubated with N^α-acetyl amino acids in order to determine reaction with specific amino acid modification. A major antigen was isolated from the incubation of N^α-acetyl histidine and NFK and was characterized by spectrometry. HLE-B3 cells were treated with KYN to determine if exogenous KYN modified cellular proteins. The presence of NFK/KYN-modification in the human lens was determined by immunohistochemistry.

Results:: KYN- or NFK-modified HLP, RNase A and BSA, but not the proteins incubated with other tryptophan oxidation products, showed strong reactivity against the antibody. The antibody strongly reacted with NFK-modified N^α-acetyl histidine and weakly with NFK-modified N^α-acetyl lysine, N^α-acetyl cysteine and N^α-acetyl arginine. The antibody was also found to react with protein-free KYN and NFK. The major antigen purified from the reaction mixture of N^α-acetyl histidine and NFK was identified to be N-acetyl-1-[3-(2-aminophenyl)-1-carboxy-3-oxopropyl]-histidine. KYN-derived modifications were observed in the cytoplasmic and nuclear proteins of cells incubated with KYN. KYN-modifications were detected mostly in the epithelium of an aged (77 Y) lens but not in a young lens (3 Y).

Conclusions:: We have generated a novel monoclonal antibody, which detects KYN-modifications in NFK and KYN modified proteins. The presence of KYN-modifications in HLE-B3 cells coupled with high levels of KYN-modifications in the aged lens suggests that NFK and KYN might damage the lens epithelium during lens aging and senile cataractogenesis.

Keywords: protein modifications-post translational • aging • cataract