Impact Of Subunit Composition On The Uptake Of Alpha-Crystallin By Lens And Retina

Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. alpha-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with alpha-crystallin may help to delay disease onset. The purpose of this study was to determine if alpha B-crystallin subunits containing a cell penetration peptide (gC-tagged alpha B-crystallin) facilitate the uptake of wild type alpha Acrystallin (WT-alpha A) in lens and retina. Recombinant human alpha B-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-alpha B). Recombinant gC-alpha B and wild-type alpha A-crystallin (WT-alpha A) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-alpha A, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37 degrees C for 4 hours to allow for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ cultures. Similarly, crystallin mixtures were injected into the vitreous of rat eyes. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity assays were performed on alpha-crystallins ratios showing optimal uptake using chemically-induced or heat induced substrate aggregation assays. As determined by flow cytometry, a ratio of 1:5 for gC-alpha B to WT-alpha A was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-alpha B to WT-alpha A (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-alpha B to WT-alpha A (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of alpha-crystallin were found in lens and retina following intravitreal injection of homo-and hetero-oligomers in rats.