Abstract
Purpose: :
Previous reports have linked cataractogenesis to oxidative protein modifications. These modifications of lens proteins are believed to result from UV-induced singlet oxygen generation. Our current study aims to correlate the effect of an oxidative environment with the structural and functional properties of the lens protein, αB crystallin. We propose that oxidative modifications can be tolerated by these proteins while maintaining their ability to function properly.
Methods: :
Purified recombinant αB crystallin was exposed to 50 J/cm2 in a near-UV calibrated crosslinking instrument in the presence or absence of the antioxidant ascorbate. Treated αB crystalline samples were analyzed using SDS-PAGE and mass spectrometry to determine the UV-induced modifications of αB crystallin at the amino acid level. These studies were followed by a quantitative mass spectrometric assay to determine the extent of oxidative modifications of αB crystallins.
Results: :
After exposure of αB crystallins to a dose of near-UV light, a dimer and higher oligomer bands were observed by SDS-PAGE. Tandem mass spectrometry experiments of these samples demonstrated that the αB crystallin undergoes oxidation events (+16 Da mass shift) on methionines and tryptophans in two regions consisting of tryptic peptides 1-11 and 57-69. Upon further analysis of peptide 57-69, our assay revealed +32 Da additions present in three isobaric forms: single oxidation upon both residues or double oxidation on a single residue. As expected, the addition of ascorbate reduced both the levels of oligomerization and oxidatively modified amino acids.
Conclusions: :
Our data suggests that an oxidative environment is generated during near-UV light irradiation. This environment affects the αB crystallin through oxidative modifications that alter the amino acid residues and oligomerization states. It is known that the alpha crystallins function by first forming a dimeric subunit. From our data, a stable dimer is formed after near-UV irradiation. Previous reports have confirmed chaperone activity after irradiation and our data suggest that this may be the cause. Current studies are focused on the functional effects of these oxidatively modified αB crystallins in relation to these characterized structural modifications.
Keywords: cataract • oxidation/oxidative or free radical damage • chaperones