Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death

AbstractIn addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically

Impact of Subunit Composition on the Uptake of α-Crystallin by Lens and Retina

<div><p>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.</p></div

Analysis of mixed oligomers chaperone-like activity on thermally (A, B and C) and chemically (D and E) induced aggregating client proteins.

<p>In A, B and C, 2.5 μM recombinant human aldose reductase (HAR) was incubated with 2.5, 1.25, or 0.625 μM α-crystallin, respectively. The α-crystallin proteins used in A, B, or C were WT-αA, WT-αB, or 5:1 mixed oligomers of WT-αA with, WT-αB or gC-αB. In D and E, 10 μM lysozyme was incubated with equimolar WT-αA, WT-αB, or 5:1 mixed oligomers of WT-αA with, WT-αB or gC-αB. Increase in absorbance at 360 nm is proportional to the level of protein aggregation. (A) Client protein, HAR, with 1 mM DTT was incubated at 52°C for 30 minutes at 1:1 with α-crystallin. (B) Client protein, HAR, with 1 mM DTT was incubated at 52°C for 30 minutes at 1:0.5 with α-crystallin and percent protection determined. (C) Client protein, HAR, with 1 mM DTT was incubated at 52°C for 30 minutes at 1:0.25 with α-crystallin and percent protection determined. (D and E)Client protein, lysozyme, along with 2 mM DTT were incubated at 37°C for 1 hr and percent protection determined.</p

Uptake and quantification of Alexa-647 labeled αA-crystallin by mouse retina organ culture.

<p>5:1 hetero-oligomers of Alexa-647 labeled αA-crystallin to unlabeled WT-αA, WT-αB, or gC-αB were incubated with extracted C57 mouse retinas for 1 hr at 37°C. Retinas were analyzed for protein uptake by confocal microscopy (A-D), and quantitated by flow cytometry (E). Retinas harvested for confocal microscopy, were imaged for uptake of Alexa-647 labeled αA-crystallin (red) and nuclei stained with Hoechst (blue). In (A) retina were cultured with no protein (PBS). In (B-D) lenses were cultured with Alexa-647 labeled αA-crystallin plus (B) unlabeled WT-αA (C) unlabeled WT-αB (D) unlabeled gC-αB. Flow cytometry of retina in (E) show the number of cells that internalized various hetero-oligomers of exogenous αA-crystallin quantitated by selecting cells positive for both Hochest and for Alexa-647 label αA-crystallin. In each experimental replicate, αA-only oligomers were set to 1. Samples having more Alexa-647 labeled αA-crystallin were greater than αA-only oligomers, while those with less were smaller than it. Experiments were repeated in triplicate and the normalized mean ±S.E. determined. As αA-only oligomers were all set to 1, no error bars are noted. Results were statistically compared by ANOVA on repeated measure with Tukey’s multiple comparison, where *** = P<0.001, ** = P<0.01. Scale bar = 50 μm. PR = photoreceptor layer, ONL = outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer.</p

In vivo uptake of Alexa-647 labeled αA-crystallin in the rat eye.

<p>5:1 hetero-oligomers of Alexa-647 labeled αA-crystallin to unlabeled WT-αA, WT-αB, or gC-αB were injected intravitreally into adult Sprague-Dawley rats. After 2 hours, lenses (Panel A) and retina (Panel B) were digested to produce cells for with flow cytometry analysis. The number of cells that internalized various hetero-oligomers of exogenous crystallin were quantitated by selecting cells positive for both Hochest and for Alexa-647 label αA-crystallin. In each experimental replicate, αA-only oligomers were set to 1. Samples having more Alexa-647 labeled αA-crystallin were greater than αA-only oligomers, while those with less were smaller than it. Experiments were repeated in triplicate and the normalized mean ±S.E. determined. As αA-only oligomers were all set to 1, no error bars are noted. Results were statistically compared by ANOVA on repeated measure with Tukey’s multiple comparison.</p

Uptake and quantification of Alexa-647 labeled αA-crystallin by mouse lens organ culture.

<p>5:1 hetero-oligomers of Alexa-647 labeled αA-crystallin to unlabeled WT-αA, WT-αB, or gC-αB were incubated with lenses extracted from C57 mice for 1 hr at 37°C. Lens were analyzed for protein uptake by confocal microscopy (A-D), and quantitated by flow cytometry (E). Lenses harvested for confocal microscopy, were imaged for uptake of Alexa-647 labeled αA-crystallin (red) and nuclei stained with Hoechst (blue). For illustration purposes 647, the red Alexa-647 αA-crystallin fluorescence (A’,B’,C’,D’) and corresponding blue Hoechst staining (A”,B”,C”,D”) are shown as separate images. In (A) lenses were cultured with no protein (PBS). In (B-D) lenses were cultured with Alexa-647 labeled αA-crystallin plus (B) unlabeled WT-αA (C) unlabeled WT-αB (D) unlabeled gC-αB. Flow cytometry of lenses in (E) show the number of cells that internalized various hetero-oligomers of exogenous crystallin was quantitated by selecting Hochest positive (nucleated) cells that were also positive for Alexa-647 label αA-crystallin. In each experimental replicate, αA-only oligomers were set to 1. Samples having more Alexa-647 labeled αA-crystallin were greater than αA-only oligomers, while those with less were smaller than it. Experiments were repeated in triplicate and the normalized mean ±S.E. determined. As αA-only oligomers were all set to 1, no error bars are noted. Results were compared statistically by ANOVA on repeated measure with Tukey’s multiple comparison, where *** = P<0.001, ** = P<0.01. Scale bar = 50 μm.</p