Proteomic analysis of water insoluble proteins from normal and cataractous human lenses.

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.