Effect of Oxidized ß B3-crystallin Peptide (153-167) on Thermal Aggregation of Bovine -crystallins

Abstract

Abstract: : Purpose: Although the mechanism for age related cataractogenesis is not clearly understood, previous studies show that there is an association between oxidation of lens crystallins, their degradation and cataractogenesis. Recently we showed that oxidized ßB₃-crystallin peptide (153-167) can modulate the aggregation of denaturing ßL-crystallins (J. Biol. Chem. 277, 39136-39143). The purpose of this study was to investigate whether the same peptide would increase the thermal aggregation of bovine γ-crystallins. Method: Synthetic ßB₃-crystallin peptide (¹⁵³AINGTWVGYEFPGYR¹⁶⁷) was oxidized using 0.1mM CuSO₄ and 2 mM H₂O₂ for 3 hrs and dialyzed extensively. Thermal aggregation assay of γ-crystallin purified from bovine lens extract was performed at 55°C in 0.05M phosphate buffer containing 0.1M NaCl, pH 7.0 and the relative aggregation was measured as optical density at 360 nm. Assays were also performed with γ-crystallin with oxidized and unoxidized ßB₃ peptide as well as in the presence of α-crystallin. The interaction of oxidized peptides with the aggregating γ-crystallin was also investigated using Sulfo-SBED derivatized ßB3-peptide and immuno blot studies. Results: Thermal denaturation of γ-crystallins resulted in aggregation and light scattering. Oxidized ßB₃ peptide increased the aggregation of γ-crystallin compared to control with out oxidized peptide. The increase in the γ-crystallin aggregation was dependent on the concentration of the oxidized peptide. However, unoxidized ßB₃ peptide in the system did not show any increase in the aggregation of γ crystallins and self-aggregation of oxidized or unoxidized peptides per see was not observed under the experimental conditions. Increased aggregation of γ-crystallin was also observed despite the presence of α-crystallin (which has anti aggregating properties) in the assay system. The Sulfo-SBED crosslinking studies confirmed the interaction between oxidized ßB3 peptide and denaturing γ-crystallin. Conclusions: These data suggest that oxidized peptides generated from ßB₃-crystallin may interact with denaturing crystallins and facilitate their aggregation and light scattering, thus behaving like antichaperones. Further, this data suggests that the interaction of oxidized crystallin fragments with partially unfolded/modified crystallins may be contributing to the increased aggregation of proteins seen in aging lenses.

Keywords: oxidation/oxidative or free radical damage • crystallins • cataract