Crystallins in water soluble-high molecular weight proteinfractions and water insoluble protein fractions in aging and cataractoushuman lenses

[摘要] Purpose: The aim of the study was to comparatively analyzecrystallin 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 theidentity of crystallin species that show cataract specific changes suchas truncation and post-translational modifications. Because thesechanges were cataract specific and not aging specific, the results wereexpected to provide information regarding potential mechanisms of agerelated cataract development.Methods: The WS-α-crystallin, WS-HMW protein, and WI proteinfractions were isolated from normal lenses of different ages and fromcataractous lenses. The three fractions were subjected to twodimensional (2D) gel electrophoresis (IEF in the first dimension andSDS-PAGE in the second dimension). Individual spots from 2D gels weretrypsin digested and the tryptic fragments were analyzed bymatrix-assisted laser desorption ionization-time of flight (MALDI-TOF)mass spectrometry.Results: The 2D protein profiles of WS-α-crystallin fractionsof normal human lenses showed an age related increase in the number ofcrystallin fragments. In young normal lenses, the WS-α-crystallinfragments were mostly C-terminally truncated, but in older lenses thesewere both N- and C-terminally truncated. The WS-HMW protein fractionfrom normal lenses contained mainly fragments of αA- and αB-crystallin, whereas additional fragments of βB1- and βA3-crystallin were present in this fraction from cataractous lenses.Similarly, the WI proteins in normal lenses contained fragments ofαA- and αB-crystallin, but cataractous lenses containedadditional fragments of βA3- and βB1-crystallin. Themodifications identified in the WS-HMW and WI crystallin species ofcataractous lenses were truncation, oxidation of Trp residues, anddeamidation of Asn to Asp residues.Conclusions: The results show that the components of WS-HMW and WIprotein fractions of cataractous lenses differed from normal lenses.Selective insolubilization of fragments of βA3/A1- and βB1-crystallin occurred during cataract development compared to normallenses. Further, the crystallin species of cataractous lenses showedincreased truncation, deamidation of Asn to Asp residues, and oxidationof Trp residue.

[学科分类] 生物化学/生物物理