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.

Fig 1. Detection and analysis of Alexa-488 labeled αA crystallin uptake by ARPE-19 cells treated with mixed homo- and hetero- oligomers.

5 μg of total α-crystallin at ratios of Alexa-488 labeled αA-crystallin to unlabeled WT-αA, WT-αB or gC-αB at 2:1, 5:1 and 10:1, respectively, were added to ARPE-19 cells and incubated for 1 hour at 37°C. Following incubation, unbound protein was removed and fresh growth media added. 24 hours later cells were incubated with Hoechst (blue) and either imaged by confocal microscopy (A) or trypsinized and analyzed by flow cytometry (B) for uptake of Alexa-488 labeled αA (green). MIV = mean intensity value.

Fig 2. Analysis of mixed oligomers chaperone-like activity on thermally (A, B and C) and chemically (D and E) induced aggregating client proteins.

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.

Fig 3. Uptake and quantification of Alexa-647 labeled αA-crystallin by mouse lens organ culture.

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.

Fig 4. Uptake and quantification of Alexa-647 labeled αA-crystallin by mouse retina organ culture.

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.

Fig 5. In vivo uptake of Alexa-647 labeled αA-crystallin in the rat eye.

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.