title: Impact of Subunit Composition on the Uptake of α-Crystallin by Lens and Retina.
abstract: Misfolded protein aggregation, including cataract, cause a significant amount of blindness worldwide. α-Crystallin is reported to bind misfolded proteins and prevent their aggregation. We hypothesize that supplementing retina and lens with α-crystallin may help to delay disease onset. The purpose of this study was to determine if αB-crystallin subunits containing a cell penetration peptide (gC-tagged αB-crystallin) facilitate the uptake of wild type αA-crystallin (WT-αA) in 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). Recombinant gC-αB and wild-type αA-crystallin (WT-αA) were purified from E. coli over-expression cultures. After Alexa-labeling of WT-αA, these proteins were mixed at ratios of 1:2, 1:5 and 1:10, respectively, and incubated at 37°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 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). Chaperone-like activity assays demonstrated that hetero-oligomeric complex of gC-αB to WT-αA (in 1:5 ratio) retained protein aggregation protection. We observed a significant increase in protein uptake when optimized (gC-αB to WT-αA (1:5 ratio)) hetero-oligomers were used in mouse lens and retinal organ cultures. Increased levels of α-crystallin were found in lens and retina following intravitreal injection of homo- and hetero-oligomers in rats.
authors.affiliation: Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Niklaus H
authors.initials: NH
authors.lastname: Mueller
authors.affiliation: Department of Pharmaceutical Sciences, Skaggs School of Pharmacy, University of Colorado, Aurora, Colorado, United States of America.
authors.forename: Uma
authors.initials: U
authors.lastname: Fogueri
authors.affiliation: Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Michelle G
authors.initials: MG
authors.lastname: Pedler
authors.affiliation: Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: Kameron
authors.initials: K
authors.lastname: Montana
authors.affiliation: Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado, United States of America; Department of Pharmaceutical Sciences, Skaggs School of Pharmacy, University of Colorado, Aurora, Colorado, United States of America.
authors.forename: J Mark
authors.initials: JM
authors.lastname: Petrash
authors.affiliation: Department of Ophthalmology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
authors.forename: David A
authors.initials: DA
authors.lastname: Ammar
mesh_terms: D000818:Animals; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism*; D051379:Mice; D055503:Protein Multimerization; D051381:Rats; D012160:Retina / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D038202:alpha-Crystallin A Chain / Q000737:chemistry / Q000378:metabolism; D038203:alpha-Crystallin B Chain / Q000737:chemistry / Q000378:metabolism; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D038202:alpha-Crystallin A Chain; D038203:alpha-Crystallin B Chain; D038201:alpha-Crystallins