Purpose: The aim of the study was to comparatively analyze crystallin fragments in the water soluble high molecular weight (WS-HMW) and in the water insoluble (WI) protein fractions of human cataractous (with nuclear opacity) and age matched normal lenses to determine the identity of crystallin species that show cataract specific changes such as truncation and post-translational modifications. Because these changes were cataract specific and not aging specific, the results were expected to provide information regarding potential mechanisms of age related cataract development.
Methods: The WS-alpha-crystallin, WS-HMW protein, and WI protein fractions were isolated from normal lenses of different ages and from cataractous lenses. The three fractions were subjected to two dimensional (2D) gel electrophoresis (IEF in the first dimension and SDS-PAGE in the second dimension). Individual spots from 2D gels were trypsin digested and the tryptic fragments were analyzed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry.
Results: The 2D protein profiles of WS-alpha-crystallin fractions of normal human lenses showed an age related increase in the number of crystallin fragments. In young normal lenses, the WS-alpha-crystallin fragments were mostly C-terminally truncated, but in older lenses these were both N- and C-terminally truncated. The WS-HMW protein fraction from normal lenses contained mainly fragments of alphaA- and alphaB-crystallin, whereas additional fragments of betaB1- and betaA3-crystallin were present in this fraction from cataractous lenses. Similarly, the WI proteins in normal lenses contained fragments of alphaA- and alphaB-crystallin, but cataractous lenses contained additional fragments of betaA3- and betaB1-crystallin. The modifications identified in the WS-HMW and WI crystallin species of cataractous lenses were truncation, oxidation of Trp residues, and deamidation of Asn to Asp residues.
Conclusions: The results show that the components of WS-HMW and WI protein fractions of cataractous lenses differed from normal lenses. Selective insolubilization of fragments of betaA3/A1- and betaB1-crystallin occurred during cataract development compared to normal lenses. Further, the crystallin species of cataractous lenses showed increased truncation, deamidation of Asn to Asp residues, and oxidation of Trp residue.