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Niklaus Mueller, Karen Christopher, David Ammar, Jonathan Petrash; Cell transduction of αA-crystallin is negatively impacted by phosphorylation mimics. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5751.
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The chaperone-like activity and solubility of α-crystallins is thought to be affected by post-translational modifications such as phosphorylation, deamination, and truncation. The current studies were carried out to investigate the functional effect of phosphorylation of αA-crystallin at each of three serine residues: S45, S59 and S122, and to determine if human αA- and αB-crystallins differ in the functional consequences of phosphorylation at similar residues.
Recombinant αA-crystallin was mutated by serine to aspartate substitution to generate αA-S45D, αA-S59D, αA-S122D, and a triple D3 mutant. Additionally, the D3-αA mutant was modified by the addition of a cell penetration peptide (CPP) we showed previously to enhance uptake of α-crystallins into eukaryotic cells. Mutant proteins were over-expressed in E. coli and purified by column chromatography. Crystallins were analyzed by size exclusion chromatography (SEC) to characterize oligomeric complexes (OC). Chaperone-like activity (CLA) was assessed by measuring the ability of αA-crystallin mutants to suppress heat-induced and chemically-induced protein aggregation. Alexa488-labeled αA-crystallins were used to evaluate protein uptake by HLE-B3 cells using fluorescence microscopy.
As evidenced by SEC elution profiles, αA-crystallin mutant proteins tested in this study, as with the wild type protein, form OC ranging in apparent molecular weight from 0.6-1.4 MDa. All αA-crystallins displayed CLA by protecting client proteins from heat and chemically induced protein aggregation in vitro. Analysis of gC-tagged αA-TM indicated that addition of aspartate residues resulted in decreased uptake into HLE B3 cells.
Substitution of serines 45, 59, 122 with aspartate in αA-crystallin is not detrimental to OC formation or CLA in vitro. However, the addition of acidic residues to αA-crystallin inhibits uptake by HLE-B3 cells. These results suggest that phosphorylation of serine residues would negatively regulate the uptake of crystallins into target cells.
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