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Y.V. Sergeev, P.S. Backlund, L.V. Soustov, E.V. Chelnokov, M.A. Ostrovsky, J.F. Hejtmancik; Increased UV–Light Sensitivity of Amino–Arm Truncated ßA3–Crystallin: Specific Structural Modifications . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3878.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose. Exposure to UV–B light is a risk factor for age–related cataracts. As the lens ages, ß–crystallins tend to undergo proteolytic cleavage of their terminal extensions. In order to delineate the effects of loss of terminal arms on ß–crystallin function, the sensitivity of recombinant wild type (rßA3) to UV–irradiation induced aggregation was compared with that of ßA3–crystallin missing the N–terminal arm (rßA3tr). Methods: Proteins were expressed, purified by chromatography and their solutions irradiated using a 308 nm excimer laser. UV–treated and control samples of ßA3tr were digested with trypsin o/n at 37 oC. The reduced and alkylated peptides of irradiated crystallins were analyzed by MALDI and LC–MS/MS. Results: UV–Irradiation of both rßA3 and rßA3tr resulted in major loss of soluble protein with concomitant formation of insoluble aggregates producing light scattering. As compared to wild type rßA3, rßA3tr shows a significant tendency to begin scattering light at lower UV–dose. The MALDI spectra suggest that certain regions of rßA3tr are particularly sensitive to fragmentation by UV irradiation. Extensive fragmentation of rßA3tr at the peptide bonds at positions 193–210 has been observed. The Met–oxidation in tryptic peptides containing M45 and M110 increased significantly with UV exposure. In contrast, at low UV doses little increase in Trp–oxidation was observed, but at higher doses (>150 J/cm2) Trp–oxidation in peptides 95–109 and 193–210 also increased dramatically. Conclusions: Although the specific mechanism for peptide fragmentation by UV treatment is not clear from these experiments, regions containing tryptophans appear to be particularly prone to fragmentation. Observed accessibility to photolysis of residues in positions from 200 to 204 is consistent with the prediction that this loop is exposed at the protein surface which might make it more susceptible to the UV treatment. An increase in Met–oxidation under UV–treatment suggests that in certain conditions oxidized methionines may promote the formation of higher molecular weight aggregates. Loss of ß–crystallin terminal arms appears to increase their tendency to aggregate in response to UV irradiation, suggesting that this loss in the maturing lens might increase susceptibility to age–related cataract.
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