Proteomics analysis of water insoluble-urea soluble crystallins from normal and dexamethasone exposed lens

Purpose: The aim of this study was to identify glucocorticoid induced cataracts (GIC)-specific modified water insoluble-urea soluble (WI-US) crystallins and related changes after rat lens were exposed to dexamethasone (Dex). Methods: We separated WI-US lens proteins by two-dimensional electrophoresis (2-DE). The crystallins were then analyzed with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Protein levels and morphological changes of alpha A- and alpha B-crystallins were also determined. Electronic microscope of lens and native-page analysis of crystallins were further determined. Results: Measured masses, isoelectric points (pIs), and amino acid sequences of all detected crystallins matched previously-reported data. Analysis by 2-DE indicated that alpha A- and alpha B-crystallin increased when the lens was viewed under 1 mu M and 10 mu M Dex, which was identical with the results of western-blot, immuno histochemistry or fluorescence; beta B2- and beta A3-crystallin increased when lens was viewed under 1 mu M Dex and 100 mu M Dex. beta A1-, beta A4-, and beta B1-crystallins decreased under 0.1-100 mu M Dex. Electronic microscope figures showed the condition of the lens center gradually worsened and cracked between fiber cells that became larger under 1-100 mu M Dex. Moreover, alpha A-crystallins were associated with increased phosphorylation (PI decreased). The newly protein spots: beta A2-, beta A3-, beta B1-, and gamma s-crystallin appeared under 0.1-100 mu M Dex. Native-page showed alpha-crystallin increased when the lens was exposed to 1 mu M Dex; however, beta-crystallin did not decrease under 0.1-100 mu M Dex. The percentage of alpha-crystallin gradually decreased, however beta-crystallin gradually increased, perhaps because the emergence of newly appeared beta-crystallin under Dex. Conclusions: Our results showed multiple WI-US crystallins may be more vulnerable to glucocorticoid stress because of diminished important roles, which will in turn provide a mechanism for GIC from a proteomics perspective.