Modified αA Crystallin in the Retina:  Altered Expression and Truncation with Aging

Abstract

Crystallins are small heat shock proteins with chaperone function that prevent heat- and oxidative stress-induced aggregation of proteins. This is the first report describing modifications of alphaA crystallin in the sensory retina, including altered content and truncation with aging. Proteins from adult, middle age, and old Fischer 344 Brown Norway rats were compared. Western immunoblotting was used to evaluate alphaA crystallin content and identify protein spots on two-dimensional gels containing alphaA crystallin. The type and site of multiple post-translational modifications were identified by mass spectrometry. We found the content of alphaA crystallin was significantly decreased in the oldest rats. On two-dimensional gels, retinal crystallins resolved into multiple spots with altered migration, indicative of changes in intrinsic charge and/or truncation. Post-translational modifications that were identified included oxidation, phosphorylation, deamidation, acetylation, and truncation. In samples from rats of all ages, a highly modified N-terminus containing these modifications was found. We also observed an age-dependent difference in the extent of N- and C-terminal truncation. These results suggest that protection against stress-induced protein aggregation is compromised in the aged retina.

Changes in Select Redox Proteins of the Retinal Pigment Epithelium in Age-related Macular Degeneration

“…Western immunoblotting using the BSP/NBIT (5-bromo-4-chlor-3′-iodolyl phosphate p-toluidine/nitro blue tetrazolium chloride) substrate (Bio-Rad) was performed as previously described. 44 Primary antibodies and their dilutions are provided in Table 1. Densitometric analysis was performed on the immunoreaction of individual protein bands to determine the relative content of the different proteins in the RPE preparations (SigmaScan Pro, Systat Software, Inc, San Jose, California, USA).…”

Exacerbation of retinal degeneration in the absence of alpha crystallins in an in vivo model of chemically induced hypoxia

“…Among the molecular chaperones, α-crystallins are utilized during protein synthesis and to prevent aggregation of misfolded or damaged proteins during oxidative stress (Horwitz, 1992). Loss of chaperone activity occurs in ocular tissues following a significant decrease in the crystallin content (Kapphahn et al, 2003). However, their chaperone activity is enhanced by stress and they can act at key regulatory steps in apoptosis.…”

New Biomarkers in the Retina and RPE Under Oxidative Stress

“…Phosphorylated B-crystallin directly interacts with Bax and caspase-3 to suppress their pro-apoptotic action, and thus exerts a cytoprotective effect in the retina [Kim YH et al, 2007]. Light-induced up-regulation of A-crystallin and increased phosphorylation of A-crystallin in the aged retina have been reported [Kapphahn et al, 2003]. The specific function of each crystallin, particularly / -crystallin in the RPE, is still largely unknown, although levels of B-crystallin are increased after heat shock and oxidative stress, and B-crystallin immunoreactivity has been found in both rod outer segments and the RPE after light exposure [Sakaguchi et al, 2003].…”

“…A-and B-crystallin are located in the ganglion cell layer and photoreceptor layer, whereas -crystallin is detected in all nuclear layers of the retina [Xi et al, 2003]. The functional roles of crystallins as chaperones, anti-apoptotic proteins, or signal transducers in the retina have also been described [Xi et al, 2003;Sakaguchi et al, 2003;Kapphahn et al, 2003;. Phosphorylated B-crystallin directly interacts with Bax and caspase-3 to suppress their pro-apoptotic action, and thus exerts a cytoprotective effect in the retina [Kim YH et al, 2007].…”