In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

alpha A-crystallin and alpha B-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in alpha A-R49C and alpha B-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old alpha A-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of alpha A-crystallin; increased association between alpha A-crystallin and filensin, actin, or creatine kinase B; increased acidification of beta B1-crystallin; increased levels of grifin; and an association between beta A3/A1-crystallin and alpha A-crystallin. Homozygous alpha A-R49C mutant lenses exhibited increased associations between alpha A-crystallin and beta B3-, beta A4-, beta A2-crystallins, and grifin, whereas levels of beta B1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, alpha-enolase, and cytochrome c increased more than 50-fold in homozygous alpha A-R49C mutant lenses. In alpha B-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several beta- and gamma-crystallins, and degradation of alpha A- and alpha B-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal alpha-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of alpha A- and alpha B-crystallin.