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Anbarasu Kumarasamy, Sivakumar Jeyarajan, Victoria A. Kimler, Anthony Premceski, Jonathan Cheon, Vidhi Mishra, Frank Joseph Giblin; In vitro studies on the interaction of guinea pig αA crystallin and αA crystallin (66-80) peptide using fluorescence polarization and transmission electron microscopy. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5301.
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© ARVO (1962-2015); The Authors (2016-present)
Treatment of guinea pigs with hyperbaric oxygen produces increases in lens nuclear light scattering, crystallin aggregation and levels of truncated αA crystallin in the form of αA crystallin (66-80) peptide. This peptide is also known to be present in human nuclear cataracts. We hypothesize that binding of the peptide to αA crystallin in the oxygen-treated guinea pig lens in vivo contributes to crystallin aggregation, as has been proposed for the aging human lens by Sharma, K.K. and colleagues. Here we investigated binding properties of the peptide in vitro with recombinant guinea pig αA crystallin using fluorescence polarization (FP) and transmission electron microscopy (TEM).
Recombinant guinea pig αA crystallin was overexpressed in E. coli and purified by ion exchange and size exclusion chromatography. Synthetic human αA crystallin (66-80) peptide, fluorescently tagged with 5-FAM, was mixed with αA crystallin (0.05 mg/ml peptide and 0.20 mg/ml protein), and FP measured at 1, 2, 3, 4 and 24 h at 37°C using a BioTek Cytation 3 microplate reader. Control experiments were conducted with peptide and bovine serum albumin (BSA). TEM analysis was used to visualize proteins and peptides at 0 and 24 h following negative staining of the samples with 2% aqueous uranyl acetate.
A gradual increase in FP for peptide plus αA crystallin was observed up to 4 h; however, FP values dropped dramatically at 24 hr. In contrast, peptide plus BSA produced a continuous increase in FP up to the 24 h time period. Analysis by TEM showed that incubation of peptide with αA crystallin for 24 h produced large aggregates in the form of long spherical chains that coexisted with doughnut-like discrete structures of αA crystallin. αA crystallin plus peptide at 0 h, αA crystallin alone at 24 h, and BSA plus peptide at 24 h did not show similar aggregate formation.
αA crystallin (66-80) peptide was shown by FP and TEM analysis to bind to guinea pig αA crystallin in vitro and induce the formation of large protein aggregates. The results provide evidence that a similar mechanism may be involved in the guinea pig/hyperbaric oxygen in vivo model for human nuclear cataract.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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