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 αA- and αB-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 αA- and αB-crystallin increased when the lens was viewed under 1 µM and 10 µM Dex, which was identical with the results of western-blot, immuno histochemistry or fluorescence; βB2- and βA3-crystallin increased when lens was viewed under 1 µM Dex and 100 µM Dex. βA1-, βA4-, and βB1-crystallins decreased under 0.1–100 µM Dex. Electronic microscope figures showed the condition of the lens center gradually worsened and cracked between fiber cells that became larger under 1–100 µM Dex. Moreover, αA-crystallins were associated with increased phosphorylation (PI decreased).The newly protein spots: βA2-, βA3-, βB1-, and γs-crystallin appeared under 0.1–100 µM Dex. Native-page showed α-crystallin increased when the lens was exposed to 1 µM Dex; however, β-crystallin did not decrease under 0.1–100 µM Dex. The percentage of α-crystallin gradually decreased, however β-crystallin gradually increased, perhaps because the emergence of newly appeared β-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.