Several mechanisms have been put forward to explain the biochemical changes that occur in cataract formation. Among them are the oxidative damage by reactive oxygen species (ROS) and non-enzymatic glycosylation (Maillard reaction) of crystallins. The Maillard reaction, the reaction between amino groups on crystallins and sugars, proceeds through a condensation product, the Amadori product, which by subsequent reactions forms structurally diverse compounds that are collectively known as advanced glycation end products (AGEs). The presence of the Amadori product as well as specific AGEs have been established in the lens. Several recent studies have shown that glycated proteins can generate ROS. It is conceivable that glycated crystallins may function as a source of ROS in the lens and induce oxidative damage. During cataract formation, the lens glutathione level decreases significantly. This may lead to a decrease in the antioxidant potential and diminished activity of glyoxalase, that converts methylglyoxal (MG) to D-lactate. As a result, MG may accumulate and induce protein damage through AGE formation. MG can also arise from autoxidation of sugars and ascorbate. Significant amounts of MG are reported to be present in the human lens. The principal investigator has recently demonstrated that MG can rapidly react with lens crystallins to form AGEs that can crosslink proteins and generate fluorescent protein adducts. The principal investigator therefore hypothesizes that formation of ROS and AGEs by the Maillard reaction may cause collateral damage to lens crystallins in cataractogenesis. In this proposal, the principal investigator plans to test this hypothesis by in vitro and in vivo experiments. The major objectives include (1) demonstration of the formation of reactive oxygen species by glycated crystallins and determination of the factors which influence their formation; (2) determination of specific modifications and changes in physical parameters in crystallins by ROS generated from the Maillard reaction products; (3) determination of the effects of Maillard reaction-induced oxidative modifications on lens opacity and cataract formation in organ cultured lens and experimental animals and (4) determination of relationship(s) between MG-mediated Maillard reactions and oxidation in cataract formation using specific probes and a novel antibody. The experiments outlined in this proposal are expected to provide a better understanding of the mechanisms of lens protein modifications in cataract formation.
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