Alterations to proteins in the lens of hereditary Crygs-mutated cataractous mice

Abstract

Purpose: To investigate the altered expression of proteins in the lens of mice with inherited cataracts.

Methods: Mice with inherited cataracts caused by a spontaneous mutation of the gene gamma S-crystallin (Crygs) were used as the subjects. Lens proteins were extracted and separated by two-dimensional electrophoresis (2-DE). The spots representing differential proteins were first identified by image analysis, and then further analyzed by matrix assisted laser desorption/ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS).

Results: 2-DE were conducted under high (882 microg) and low dosage (190 microg) of sample. Under each condition, the numbers of protein spots found in cataract lenses were similar to those in normal lenses (p>0.05). Seventeen proteins were identified in normal lenses, including alphaA- to alphaB-, betaA1- to betaA4-, betaB1- to betaB3-, gammaA- to gammaF-, and gammaS-crystallin, and bead-filament structure protein (BFSP/filensin). Seven differential ones were consistently identified. In the cataract lenses BFSP and gammaS-crystallin were absent; gammaF-crystallin was downregulated; and betaA1-, betaB1-, betaB2-, and alphaB-crystallin were upregulated. Those abnormally upregulated crystallins, when compared to normal ones, had smaller molecular weight, suggesting possible truncation.

Conclusions: The mutant Crygs gene can lead to changes of BFSP/filensin and other crystallins. The changes to these crystallins, together, may secondarily lead to cataract formation.

Figure 1

2-DE gels showing the proteome maps of total proteins from the lenses. 882 μg of samples were loaded. Pre-cast gels of 12 % were used for the second dimension, and were stained with colloid Coomassie brilliant blue (CBB) R-250. Low-abundance proteins were shown in the rectangle box. A: Cataract mice. BFSP/filensin spots were absent. B: Normal mice. BFSP/filensin spots were highly detectable in normal samples.

Figure 2

2-DE gel showing the differential proteins of lenses. 190 μg of samples were loaded. Pre-cast gels of 12.5 % were used for the second dimension, and were stained with colloid Coomassie brilliant blue (CBB) R-250. A: Six differential crystallins were identified from mutant mice. Red indicates absence (γS-crystallin) or reduction (γF-crystallin). Blue indicates increment (βA1-, βB1-, βB2-, and αB-crystallin). B: Sixteen crystallins were identified in normal mice, including αA~αB-, βA1~βA4-, βB1~βB3-, γA~γF-, and γS-crystallin.

Figure 3

Identification of mouse γS-crystallin by mass spectrometry. A: MALDI-MS spectrum of the γS-crystallin spot digested with trypsin. B: Tandem MS spectrum of the ion with m/z 1729.9 from a tryptic peptide.