Purpose: The purpose of the study was to compare and analyze the composition of crystallin species that exist in the water insoluble-urea soluble (WI-US) and water insoluble-urea insoluble (WI-UI) protein fractions of a human cataractous lens and an age-matched normal lens.
Methods: The water soluble (WS) and water insoluble (WI) protein fractions from a 68-year-old normal lens and a 61-year-old cataractous lens were isolated, and the WI proteins were further solubilized in urea to separate WI-US and WI-UI protein fractions. The WI-US and WI-UI protein fractions from normal and cataractous lenses were individually analyzed by two-dimensional (2D) gel electrophoresis. The protein spots were excised from 2D gels, digested with trypsin, and analyzed by the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) method. The tryptic peptides from individual spots were further analyzed by the electrospray tandem mass spectrometry (ES-MS/MS) method to determine their amino acid sequences.
Results: The comparative 2D gel electrophoretic analyses of WI-US proteins of normal and cataractous lenses showed that the majority of species in a normal lens (68 years old) and a cataractous lens (61 years old) had M(r) between 20 to 30 kDa. The ES-MS/MS analyses showed that the individual WI-US protein spots from normal and cataractous lenses contained mostly either alphaA- or alphaB-crystallin with beta-crystallins, or alpha- and beta-crystallins with filensin as well as vimentin. Similar sequence analyses of tryptic fragments of 2D gel spots of WI-UI proteins revealed that the normal lens showed either individual alphaA- and alphaB-crystallins, a mixture of betaA3/A1-, betaB1-, and betaB2-crystallins and filensin, betaA4-, betaB1-, betaB2-, betaS-crystallins and filensin, or alphaA-, alphaB1-, filensin, and vimentin or alphaB-, betaA3-, betaA4-, betaB1-, betaB2-, and betaS-crystallins. In contrast, the WI-UI proteins from a cataractous lens showed three intact crystallins (alphaB-, gammaS-, and betaB2-crystallins), and three spots containing a mixture of beta-crystallins (the first containing betaB1- and betaB2-crystallins, the second gammaS-, betaB1-, and betaB2-crystallins, and the third betaA3-, betaA4-, and betaB1-crystallins).
Conclusions: The compositions of WI-US and WI-UI proteins, isolated from one normal and one cataractous lens, were different. The absence of alphaA- but not of alphaB-crystallin and preferential insolubilization mostly of beta-crystallins in the WI-US protein fraction from the cataractous lens but not in the normal lens was observed. Similarly, in contrast to the normal lens, the WI-UI proteins of the cataractous lens contained alphaB-crystallin while alphaA-crystallin was absent, which suggested a major role of alphaB-crystallin in the insolubilization process of crystallins.