DEGRADATION OF NATIVE AND OXIDIZED BETA-CRYSTALLIN 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 alpha-crystalli ns. The objective of this study was to determine if oxidized beta- and gamma-crystallins would be selectively degraded and which proteolytic systems might be involved. beta- and gamma-crystallins were oxidized by exposure to Cs-137 radiation under N2O. This system generates OH pr imarily. Oxidation of beta- and gamma-crystallins was indicated by dec reased protein sulfhydryl content and tryptophan fluorescence, as well as by increased levels of carbonyl and high molecular weight aggregat es with increasing radiation dose. gamma-crystallin was more susceptib le than beta-crystallin to oxidation based on loss of native crystalli n, increase in aggregates and fragmentation products, and loss of tryp tophan. Low molecular mass polymers (dimers) appear to be the precurso rs of high mass aggregates induced upon oxidation. At a specific level of oxidative insult interchain covalent bonds in addition to disulfid es were more extensive in the polymers of gamma-crystallin as compared to beta-crystallin. Except for beta-crystallin irradiated with 1 krad , the degradation rate of crystallins using both reticulocyte and bovi ne lens epithelial cells (BLEC) proteolytic systems increased in propo rtion to the extent of oxidation. Proteolysis of oxidized gamma-crysta llins increased 1775% and 900%, respectively, using reticulocytes and BLEC supernatants as the source of proteases. Using the same proteolyt ic systems, oxidation of beta-crystallins resulted in only a 160% and 130% increase in proteolysis, respectively. However, the absolute rate of proteolysis was higher with beta-crystallins. Most of these oxidiz ed crystallins were degraded by an ATP-independent pathway, but ATP-de pendent degradation was also observed. These results indicate that BLE C, as well as the reticulocytes, contain proteases which selectively d egrade oxidized crystallins. Unlike proteolysis of specifically oxidiz ed ci-crystallins, the degradation of oxidized beta- and gamma-crystal lins did not appear to involve ubiquitination.