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

Purpose. To determine the formation of imidazolysine, a Maillard reaction d erived protein crosslink in the human lens in relation to aging and catarac t by immunochemical methods. Methods. Antibodies against RNase-imidazolysine were raised in rabbits. The antibodies were tested for their specificity for imidazolysine by using va rious imidazolysine-like compounds and imidazoles. A competitive ELISA test ed human lens water-soluble proteins and enzyme-digested water-insoluble pr oteins for immunoreactivity against the antibodies. Results. The antibodies strongly reacted with structurally related imidazol ysine and GOLD (glyoxal-lysine dimer) and thus precluded us from distinguis hing imidazolysine from GOLD in the human lens. We assumed that the detecte d immunoreactivity is due to a combination of GOLD and imidazolysine. The a ntibodies did not react with histidine. The immunoreactivity in lens protei ns was expressed as units of imidazolium crosslinks per unit of protein (1 unit = 1% inhibition of antibody binding to microplate well, 1 unit of prot ein = approximately 0.3 mg protein). The levels in the water-insoluble prot eins were 8.4 +/- 4.5 units (mean +/- SD) and 40.4 +/- 8.5 units per unit o f 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 c ataractous 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 com pared to the water-insoluble proteins from the same lenses. Western blot an alysis of lens proteins showed that the antigens are primarily present in t he high molecular weight protein aggregates. Conclusions. This study provides additional evidence for alpha-dicarbonyl-m ediated protein crosslinking in the human lens and suggests that such react ions could play a role in lens aging and cataractogenesis.