Characterization of covalent multimers of crystallins in aging human lenses.
The purpose of this study was to characterize covalent multimers with molecular mass of >90 kDa in the water-insoluble (WI) proteins of aging human lenses. The experimental approach was to first separate the multimers (molecular mass >90 kDa) as individual spots by two-dimensional gel electrophoresis and next analyze compositions of each multimers by matrix-assisted laser desorption ionization-time of flight and electrospray ionization-tandem mass spectrometric (ES-MS/MS) methods. The WI proteins from lenses of 25- and 41-year-old subjects showed distinct 5- and 16-multimer spots on two-dimensional gels, respectively, but the spots from 52- and 72-year-old lenses were non-descript and diffused. ES-MS/MS analyses showed two types of covalent multimers in 25- and 41-year-old lenses, i.e. the first type composed of fragments of eight different crystallins (i.e. alphaA, alphaB, betaA3, betaA4, betaB1, betaB2, gammaS, and gammaD), and the second type of alpha-, beta-, and gamma-crystallins (possibly fragments) and two beaded filament proteins (phakinin and filensin). The most commonly identified species in the complexes of 41-year-old lenses were: alphaA-fragment (C-terminally truncated, residues 1-157), alphaB-fragment (residues 83-90), betaB1-crystallin (residues 60-71), betaA3 (residues 33-44), betaA4 (residues 106-117), filensin (residues 78-90), and phakinin (residues 77-89). Three post-translational modifications (i.e. oxidation of Met and Trp, conversion of Ser to dehydroalanine, and formylation of His) were observed in alphaA-crystallin fragment, and the first two modifications could cross-link proteins. Together, the results suggested that covalent multimers appeared early in life (i.e. 25 years of age) and increased in number with aging, and the two beaded filament proteins form covalent complexes with crystallin fragments in vivo.
Srivastava
Om P
OP
Department of Physiological Optics,University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. Srivasta@uab.edu
Kirk
Marion C
MC
Srivastava
Kiran
K
D000595:Amino Acid Sequence; D003459:Crystallins / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007908:Lens, Crystalline / Q000737:chemistry*; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D003459:Crystallins