Proteomic analysis of water insoluble proteins from normal andcataractous human lenses

[摘要] Purpose: The purpose of the study was to compare and analyze thecomposition of crystallin species that exist in the water insoluble-ureasoluble (WI-US) and water insoluble-urea insoluble (WI-UI) proteinfractions of a human cataractous lens and an age-matched normal lens.Methods: The water soluble (WS) and water insoluble (WI) proteinfractions from a 68-year-old normal lens and a 61-year-old cataractouslens were isolated, and the WI proteins were further solubilized in ureato separate WI-US and WI-UI protein fractions. The WI-US and WI-UIprotein fractions from normal and cataractous lenses were individuallyanalyzed by two-dimensional (2D) gel electrophoresis. The protein spotswere excised from 2D gels, digested with trypsin, and analyzed by thematrix-assisted laser desorption ionization-time of flight (MALDI-TOF)method. The tryptic peptides from individual spots were further analyzedby the electrospray tandem mass spectrometry (ES-MS/MS) method todetermine their amino acid sequences.Results: The comparative 2D gel electrophoretic analyses of WI-USproteins of normal and cataractous lenses showed that the majority ofspecies in a normal lens (68 years old) and a cataractous lens (61 yearsold) had Mr between 20 to 30 kDa. The ES-MS/MS analyses showedthat the individual WI-US protein spots from normal and cataractouslenses contained mostly either αA- or αB-crystallin withβ-crystallins, or α- and β-crystallins with filensin aswell as vimentin. Similar sequence analyses of tryptic fragments of 2Dgel spots of WI-UI proteins revealed that the normal lens showed eitherindividual αA- and αB-crystallins, a mixture of βA3/A1-, βB1-, and βB2-crystallins and filensin, βA4-,βB1-, βB2-, βS-crystallins and filensin, or αA-,αB1-, filensin, and vimentin or αB-, βA3-, βA4-,βB1-, βB2-, and βS-crystallins. In contrast, the WI-UIproteins from a cataractous lens showed three intact crystallins (αB-, γS-, and βB2-crystallins), and three spots containing amixture of β-crystallins (the first containing βB1- and βB2-crystallins, the second γS-, βB1-, and βB2-crystallins, and the third βA3-, βA4-, and βB1-crystallins).Conclusions: The compositions of WI-US and WI-UI proteins, isolatedfrom one normal and one cataractous lens, were different. The absence ofαA- but not of αB-crystallin and preferentialinsolubilization mostly of β-crystallins in the WI-US proteinfraction from the cataractous lens but not in the normal lens wasobserved. Similarly, in contrast to the normal lens, the WI-UI proteinsof the cataractous lens contained αB-crystallin while αA-crystallin was absent, which suggested a major role of αB-crystallin in the insolubilization process of crystallins.

[学科分类] 生物化学/生物物理