Proteomics analysis of water insoluble-urea soluble crystallins from normal and dexamethasone exposed lens.
OBJECTIVE
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.
Eye Hospital, The First Affiliated Hospital, Harbin Medical University, Harbin, China.
Lin
L
Wang
DongRui
D
Liu
Ping
P
Liu
YongBin
Y
Yu
D000595:Amino Acid Sequence; D000818:Animals; D002386:Cataract / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D003459:Crystallins / Q000032:analysis* / Q000145:classification; D003907:Dexamethasone / Q000009:adverse effects*; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D005938:Glucocorticoids / Q000009:adverse effects; D007908:Lens, Crystalline / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012995:Solubility; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea; D014867:Water
D003459:Crystallins; D005938:Glucocorticoids; D014867:Water; D003907:Dexamethasone; D014508:Urea