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R.E. Neal, E. An, K.C. Winn, M. Datiles, N. Congdon, D. Garland; Site–Specific Oxidation of Tryptophan and Methionine Residues in ßB1–Crystallin From Human Lens Nucleus: Normal, Nuclear Cataract, and Nuclear/PSC Cataract . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3889.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To compare oxidative post–translational modifications of ßB1–crystallin in human lens nuclear regions from nuclear, nuclear/PSC cataract subtypes and from normal lenses. Methods: Cataracts were removed by intracapsular (Indian) or extracapsular (US) extraction. Normal lenses were obtained from NDRI. The nuclear regions from all lenses (5/group) were dissected and the proteins were separated by 2D gel electrophoresis resulting in the clear separation of ßB1–crystallin from the other proteins. Multiple ßB1–crystallin spots from each gel were subjected to in–gel tryptic digestion. Samples were analyzed by MALDI–TOF–MS and the positions of potential oxidative modification of peptides were predicted by the FindMod search algorithm. The identification of the modified amino acid in the predicted tryptic peptides was confirmed by LC–ESI–MS/MS. Results: Similar two dimensional gel patterns demonstrating increased acidification of ßB1–crystallin were observed in all cataracts. ESI–MS/MS data documenting the conversion of tyrptophan to kynurenine was found for 3 out of 8 possible tryptophans, W101, W175, and W193, in the normal lens nucleus, the Indian nuclear and Indian nuclear/PSC cataract samples. In the US nuclear cataracts, kynurenine was primarily observed in the position of W175. The results demonstrate the oxidative modification of tryptophan in the polypeptide chains which coexist in many cases with the unmodified peptide. No evidence for the addition of kynurenine to lysine, histidine or cysteine was found by LC–ESI–MS/MS. Evidence for the oxidation of methionine to methionine sulfoxide was found at one of the five potential methionines in all ßB1–crystallin spots from all groups examined, M226. Oxidation of M113 was observed in a limited number of ßB1–crystallin spots from both the US and Indian nuclear cataracts. The unmodified peptides were also observed in many cases. Conclusions: These results are the first evidence of specific tryptophan residue conversions to the oxidation product kynurenine in ßB1–crystallin from human lens. The results also demonstrate the site–specificity of tryptophan and methionine oxidation in human lenses, both normal and cataractous. Unique locations of these modifications in nuclear cataracts relative to normal lenses were not observed. These modifications do not provide a ready explanation for cataract formation or the acidification of ßB1–crystallin.
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