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Niklaus H. Mueller, Spencer LaFasto, Meredith Kolloen, David Ammar, Mark Petrash; Strategies For Transduction Of α-crystallin Into Mammalian Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):1618.
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© ARVO (1962-2015); The Authors (2016-present)
Cataract is a protein aggregation disease of the lens. Loss of native α-crystallin, a major lens protein that has been shown to prevent protein aggregation in vitro, is associated with increased risk of cataract development. We hypothesize that introduction of additional α-crystallin protein into cells, facilitated by a protein transduction domain, will lead to suppressed protein aggregation. This study was carried out to identify the optimal strategy to modify α-crystallin to maximize its delivery into mammalian cells while at the same time minimizing its loss of chaperone-like activity (CLA).
Recombinant human αA- and αB-crystallins were modified by addition of putative transduction sequences at their N- or C-termini. These proteins were expressed in E. coli and isolated to apparent homogeneity. Purified proteins were analyzed by size exclusion chromatography to determine size heterogeneity. CLA was assessed by measuring the ability of candidate proteins to suppress chemically-induced protein aggregation. Selected crystallins were fluorescently-labeled using Alexa-based dyes and were incubated with human lens epithelial cells (HLE B3) and human embryonic kidney cells (HEK293). Time-dependent uptake was measured by fluorescent microscopy.
We examined the effects of different transduction sequences, including an Arginine-rich nonapeptide (ARN) as well as a peptide predicted from the HSV-1 gC gene (gC). We found that α-crystallin subunits expressed in E. coli were insoluble if they were fused to either transduction peptide at the C-terminus. Attempts to refold these proteins by dialysis following denaturant treatment were unsuccessful. In addition, when ARN was fused to the amino terminus of α-crystallin, it was recovered in low levels from E. coli. In contrast, α-crystallin subunits modified by addition of the gC peptide at the N-terminus were soluble. These modified α-crystallin subunits formed large molecule weight complexes of ~650 kDa, similar to wild-type α-crystallins, based on size exclusion chromatography. These proteins also displayed CLA, as evidenced by suppressing the chemically-induced aggregation of substrate proteins. When incubated with cultured cells, wild-type α-crystallin proteins resulted in limited protein uptake. In contrast, uptake was markedly improved by addition of the gC peptide sequence.
N-terminal gC tagged α-crystallin proteins are soluble, form oligomer complexes that display chaperone like activity, and have increased cellular uptake compared to the wild-type form of the protein.
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