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Kyle A. Floyd, David R. Stella, Landon S. Wilson, Matthew B. Renfrow, Om P. Srivastava, Stephen Barnes; Analysis Of Uva-induced Post-translational Modifications Of The αB-crystallin With Antioxidant Supplementation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4739.
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UVA radiation passes through the lens and can lead to protein post-translational modification (PTM). While the lens contains antioxidants to prevent this, some are capable of forming advanced glycation endproducts (AGE) PTMs. This study aimed to determine the effects of UVA light exposure on the αB-crystallin when supplemented with antioxidants.
Recombinant human αB-crystallin was exposed to UVA light and supplemented with ascorbate (AA), dehydroascorbate (DHA), glutathione (GSH), separately and in combinations. After exposure, the samples were analyzed by SDS-PAGE to determine the effects of UVA light. Samples were also digested with trypsin and analyzed using high-resolution mass spectrometry (MS) to identify PTMs. Separately, a triple-quadropole instrument was used to quantify specific AGEs.
SDS-PAGE analysis of UVA exposed and dark control samples showed that UVA exposure induces multimerization of αB-crystallin. Treatment with AA or GSH did not alter multimerization, while supplementation with DHA increased it. PTM analysis revealed three AGE modifications; carboxymethyl-lysine, carboxyethyl-lysine, and malondialdehyde adducts (MDA) on His residues. High-resolution MS showed these AGEs were found in samples treated with AA, DHA, or AA/DHA, but were absent from GSH-treated samples. Quantitation of the MDA modification indicated that it was present with or without UVA exposure, and that supplementation with GSH decreased MDA modification on His83 and prevented it on His101.
UVA light induces multimerization of the αB-crystallin that was not reduced with the presence of tested antioxidants. Supplementation with AA, DHA, or AA/DHA leads to AGE modifications on lysine and histidine residues, regardless of UVA exposure, while GSH decreased or prevented MDA adducts on analyzed histidine residues.
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