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Niklaus H. Mueller, David A. Ammar, J. Mark Petrash; Cell Penetration Peptides for Enhanced Entry of αB-Crystallin into Lens Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(1):2-8. doi: https://doi.org/10.1167/iovs.12-10947.
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The prevalence of cataract increases with age. Conversely, the abundance of native α-crystallin diminishes with age and cataract development. We hypothesize replenishing lens α-crystallin may delay or prevent cataract. Herein we investigated the ability of cell penetration peptides (CPP) to enhance entry of α-crystallins into lens-derived cells.
Recombinant αB-crystallins were modified by the addition of CPPs. Candidate CPP were designed with reference to the HSV-1 glycoprotein C gene (gC) or the HIV-1 TAT peptide. αB-crystallins produced by fusing gC or TAT were over-expressed in E. coli . Purified proteins were subjected to size exclusion chromatography (SEC) to characterize oligomeric complexes (OC). Chaperone-like activity (CLA) was evaluated by measuring the ability of α-crystallins to suppress chemically-induced protein aggregation. To evaluate protein uptake, labeled α-crystallins were incubated with HLE B3 cells and monitored by fluorescence microscopy for 48 hours.
We examined the effects of the addition of CPP on the structure, CLA, and cell transduction properties of αB-crystallins. C-terminal CPP fused crystallins had poor solubility. In contrast, N-terminal tagged αB-crystallins were soluble. These modified αB-crystallins formed OC that were larger than wild-type based on SEC. Wild-type and gC tagged αB-crystallin displayed robust CLA. Subunit exchange was observed when gC-fused αB-crystallin was mixed with αA. In contrast to wild-type, modified α-crystallins accumulated in HLE B3 cells.
Addition of CPP improves the uptake of αB-crystallins into HLE B3 cells. No undesirable changes to the chaperone-like abilities of α-crystallins were observed in αB-crystallin modified by the addition of the gC-derived CPP.
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