Degradation of native and oxidized beta-and gamma-crystallin using bovine lens epithelial cell and rabbit reticulocyte extracts

In many types of cells, modified proteins are selectively and rapidly removed by various proteolytic systems. In eye lens, as in most cells, there appears to be a multiplicity of proteolytic pathways, including ubiquitin-dependent, ATP-dependent and ATP-independent pathways. Each of these appears to be involved in the degradation of α-crystallins. The objective of this study was to determine if oxidized β-and γ-crystallins would be selectively degraded and which proteolytic systems might be involved, β-and γ-crystallins were oxidized by exposure to 137Cs radiation under N2O. This system generates •OH primarily. Oxidation of β-and γ-crystallins was indicated by decreased protein sulfhydryl content and tryptophan fluorescence, as well as by increased levels of carbonyl and high molecular weight aggregates with increasing radiation dose, γ-crystallin was more susceptible than β-crystallin to oxidation based on loss of native crystallin, increase in aggregates and fragmentation products, and loss of tryptophan. Low molecular mass polymers (dimers) appear to be the precursors of high mass aggregates induced upon oxidation. At a specific level of oxidative insult interchain covalent bonds in addition to disulfides were more extensive in the polymers of γ-crystallin as compared to β-crystallin. Except for β-crystallin irradiated with 1 krad, the degradation rate of crystallins using both reticulocyte and bovine lens epithelial cells (BLEC) proteolytic systems increased in proportion to the extent of oxidation. Proteolysis of oxidized γ-crystallins increased 1775% and 900%, respectively, using reticulocytes and BLEC supernatants as the source of proteases. Using the same proteolytic systems, oxidation of β-crystallins resulted in only a 160% and 130% increase in proteolysis, respectively. However, the absolute rate of proteolysis was higher with β-crystall ins. Mest of these oxidized crystallins were degraded by an ATP-independent pathway, but ATP-dependent degradation was also observed. These results indicate that BLEC, as well as the reticulocytes, contain proteases which selectively degrade oxidized crystallins. Unlike proteolysis of specifically oxidized α-crystallins, the degradation of oxidized β-and γ-crystallins did not appear to involve ubiquitination.