Cataract-specific posttranslational modifications and changes in the composition of urea-soluble protein fraction from the rat lens

Purpose To determine age-related changes in the composition of the urea-soluble (US) protein fraction from lenses of senescence-accelerated OXYS (cataract model) and Wistar (control) rats and to establish posttranslational modifications (PTMs) occurring under enhanced oxidative stress in OXYS lenses. Methods The identity and the relative abundance of crystallins in the US fractions were determined using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization–time of flight mass spectrometry (MS). The identities and the positions of PTMs were established using MS/MS measurements. Results Two-dimensional gel electrophoresis maps of US protein fractions were obtained for lenses of 3-, 12-, and 62-week-old Wistar and OXYS rats, and the relative abundance of different isoforms of α-, β-, and γ-crystallins was determined. β-Crystallins were the major contributor of the US fraction in 3-week-old lenses (above 50%), γ-crystallins in 12-week-old lenses (50–60%), and in 62-week-old lenses, the contributions from all three crystallin families leveled out. The major interstrain difference was the elevated level of α-crystallins in the US fraction from 12-week-old OXYS lenses. Spots with increased relative abundance in OXYS maps were attributed to the cataract-specific spots of interest. The crystallins from these spots were subjected to MS/MS analysis, and the positions of acetylation, oxidation, deamidation, and phosphorylation were established. Conclusions The increased relative abundance of α-crystallins in the US fraction from 12-week-old OXYS lenses points to the fast insolubilization of α-crystallins under oxidative stress. Most of the PTMs attributed to the cataract-specific modifications also correspond to α-crystallins. These PTMs include oxidation of methionine residues, deamidation of asparagine and glutamine residues, and phosphorylation of serine and threonine residues.