Cell Penetration Peptides for Enhanced Entry of aB-Crystallin into Lens Cells

PURPOSE. The prevalence of cataract increases with age. Conversely, the abundance of native a-crystallin diminishes with age and cataract development. We hypothesize replenishing lens a-crystallin may delay or prevent cataract. Herein we investigated the ability of cell penetration peptides (CPP) to enhance entry of a-crystallins into lens-derived cells. METHODS. Recombinant aB-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. aB-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 a-crystallins to suppress chemically-induced protein aggregation. To evaluate protein uptake, labeled a-crystallins were incubated with HLE B3 cells and monitored by fluorescence microscopy for 48 hours. RESULTS. We examined the effects of the addition of CPP on the structure, CLA, and cell transduction properties of aBcrystallins. C-terminal CPP fused crystallins had poor solubility. In contrast, N-terminal tagged aB-crystallins were soluble. These modified aB-crystallins formed OC that were larger than wild-type based on SEC. Wild-type and gC tagged aB-crystallin displayed robust CLA. Subunit exchange was observed when gC-fused aB-crystallin was mixed with aA. In contrast to wildtype, modified a-crystallins accumulated in HLE B3 cells. CONCLUSIONS. Addition of CPP improves the uptake of aBcrystallins into HLE B3 cells. No undesirable changes to the chaperone-like abilities of a-crystallins were observed in aBcrystallin modified by the addition of the gC-derived CPP. (Invest Ophthalmol Vis Sci. 2013;54:2-8