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Niklaus Mueller, Uma Fogueri, michelle pedler, Kameron Montana, J. Petrash, David A Ammar; Uptake of α- crystallin subunits by lens and retina are affected by hetero-oligomer ratios of αA and αB. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5587.
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© ARVO (1962-2015); The Authors (2016-present)
Misfolded protein aggregation, including cataract, RP, and Stargardt’s, cause a significant amount of blindness. α-crystallin is reported to bind misfolded proteins and prevent aggregation. We hypothesize that supplementing ocular cells with α-crystallin, known to bind misfolded proteins and prevent protein aggregation, may help to delay disease. The purpose of this study was to determine if aB-crystallin having a cell penetration peptide (gC-tagged αB-crystallin) could facilitate the uptake of wild type αA-crystallin (WT-αA) complexes into lens and retina.
Recombinant human αB-crystallin was modified by the addition of a novel cell penetration peptide derived from the gC gene product of herpes simplex virus (gC-αB). Constructs encoding both gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37oC for 4 hrs for subunit exchange. Mixed oligomers were subsequently incubated with tissue culture cells or mouse organ culture. Similarly, crystallin mixtures were injected into the vitreous cavity of rats. At various times after exposure, tissues were harvested and analyzed for protein uptake by confocal microscopy or flow cytometry. Chaperone-like activity (CLA) assays were performed on α-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-αB to WT-αA was found to be optimal for uptake into retinal pigmented epithelial cells (ARPE-19). CLA assays demonstrated that hetero-oligomeric complexes composed on this 1:5 ratio of subunits were equivalent to wild type α-crystallin in preventing protein aggregation. We observed a significant increase in protein uptake when homo- or optimized hetero-oligomers were used in mouse lens and retinal organ culture experiments. A statistically significant amount of protein was taken up by cells in both tissues, with gC-αB having the greatest effect on lens cultures. Increased levels of α-crystallin were found following intravitreal injection of homo- and optimized α-crystallins hetero-oligomers in rats.
gC-αB to WT-αA ratio of 1:5 increased the cellular uptake of protein. Mouse organ culture indicates that gC-αB greatly enhances uptake of WT-αA in lens.
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