Lens proteomics: the accumulation of crystallin modifications in the mouse lens with age

Abstract

Purpose: To identify modified crystallins associated with aging of lens and produce two-dimensional electrophoresis (2-DE) proteome maps of crystallins in mouse lens.

Methods: Lens proteins from mice of increasing age or different strains were separated by either chromatography or 2-DE. Masses of whole proteins or tryptic peptides were analyzed by mass spectrometry. Changes in the abundance of individual crystallins were determined by image analysis of 2-DE gels.

Results: The measured masses of all known mouse crystallins, with the exception of gammaD and gammaF, matched the masses calculated from their reported sequences. Analysis by 2-DE indicated that most posttranslational modifications took place in mice after 6 weeks of age. Partially degraded crystallins, including betaB1, betaB2, betaB3, betaA3, alphaA, and alphaB, were found in greater proportion in the insoluble fractions. gamma-Crystallins A through F also became insoluble during aging. However, insolubilization of gamma-crystallins was associated with a decrease in isoelectric point (pI). Aging was also associated with increased phosphorylation of soluble alphaA- and alphaB-crystallins, confirmed by mass measurements of these proteins eluted from 2-DE gels. Comparison of protein profiles between several strains of mice used to produce transgenic or knockout models of cataract indicated few differences, except for an additional acidic form of a gamma-crystallin, possibly due to a polymorphism.

Conclusions: These results suggest that partial degradation of alpha- and beta-crystallins and increased acidity of gamma-crystallins may cause insolubilization during aging. The 2-DE proteome maps of mouse lens proteins created in this study, using immobilized pH gradients, will be useful for comparison with maps of lens proteins of mice with cataracts so that cataract-specific modifications may be identified.