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.