Abstract
Purpose:
αA-crystallin is a major lens structural protein that functions as a molecular chaperone. The Arg 49 to Cys (R49C) mutation in αA-crystallin is linked with human hereditary cataract. Knock-in mice carrying this mutation develop cataracts at an early age. To determine whether the αA-R49C mutation causes a change in other proteins, we analyzed the abundance of proteins in the postnatal lenses of αA-R49C mutant mice by proteomics, mass spectrometry, and immunohistochemical analysis.
Methods:
Lenses of two day old wild type, αAR49C heterozygous and homozygous mutant mice on a C57BL6 background were compared by two dimensional difference gel electrophoresis and mass spectrometric analysis. Protein identifications were validated by Mascot software. Immunohistochemical analysis and confocal microscopy were performed with antibodies to MIP, E-cadherin, N-cadherin and F-actin.
Results:
Proteomic analysis of two-day-old αA-R49C heterozygous lenses demonstrated increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin as compared with the age-matched wild type lens. In addition, complexes of αA-crystallin with filensin, actin and creatine kinase B increased in heterozygous lenses, and βB1-crystallin became more acidic. βA3/A1-crystallin complexed with αA-crystallin and grifin also increased in the heterozygous lenses. Homozygous mutant lenses demonstrated an increase in αA-crystallin complexes with βB3-, βA4, βA2-crystallin and grifin, whereas βB1-crystallin decreased. Increase in more acidic forms of filensin and phakinin, and in several other cytoskeletal proteins complexed with αA-crystallin, as well as degraded grifin were observed. Spectrin α, hemoglobin and histones also increased in homozygous lenses whereas gelsolin and calpain 3 decreased. Degraded glutamate dehydrogenase, α-enolase and cytochrome c increased >50-fold in homozygous lenses. Immunohistochemical analysis showed a decrease in N- and E-cadherin staining in the homozygous mutant lenses as compared with the wild type.
Conclusions:
Our analysis identified specific crystallins, enzymes and cytoskeletal proteins that change with expression of mutant αA-crystallin in the mouse lens. We identified for the first time changes in abundance of hemoglobin and histones. Our data suggest that these proteins may play an important role in the biochemical mechanims of hereditary cataracts.
Keywords: 488 crystallins •
445 cataract •
663 proteomics