Immunochemical detection of dicarbonyl-derived imidazolium protein crosslinks in human lenses

Abstract

PURPOSE. To determine the formation of imidazolysine, a Maillard reaction derived protein crosslink in the human lens in relation to aging and cataract by immunochemical methods. METHODS. Antibodies against RNase-imidazolysine were raised in rabbits. The antibodies were tested for their specificity for imidazolysine by using various imidazolysine-like compounds and imidazoles. A competitive ELISA tested human lens water-soluble proteins and enzyme-digested water-insoluble proteins for immunoreactivity against the antibodies. RESULTS. The antibodies strongly reacted with structurally related imidazolysine and GOLD (glyoxal-lysine dimer) and thus precluded us from distinguishing imidazolysine from GOLD in the human lens. We assumed that the detected immunoreactivity is due to a combination of GOLD and imidazolysine. The antibodies did not react with histidine. The immunoreactivity in lens proteins was expressed as units of imidazolium crosslinks per unit of protein (1 unit = 1% inhibition of antibody binding to microplate well, 1 unit of protein = approximately 0.3 mg protein). The levels in the water-insoluble proteins were 8.4 ± 4.5 units (mean ± SD) and 40.4 ± 8.5 units per unit of protein in young and old lenses, respectively. Cataractous lenses showed significantly higher levels (58.8 ± 8.1 units, P < 0.05) when compared to age-matched normal lenses and highest levels were observed in brunescent cataractous lenses (76.6 ± 13.4 units). The levels were negligible in the water-soluble proteins of young lenses and were 5 to 14-fold lower when compared to the water-insoluble proteins from the same lenses. Western blot analysis of lens proteins showed that the antigens are primarily present in the high molecular weight protein aggregates. CONCLUSIONS. This study provides additional evidence for a-dicarbonyl-mediated protein crosslinking in the human lens and suggests that such reactions could play a role in lens aging and cataractogenesis.