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Yuri V. Sergeev, Lev V. Soustov, Eugene V. Chelnokov, Nikita M. Bityurin, Peter S. Backlund, Paul T. Wingfield, Mikhail A. Ostrovsky, J. Fielding Hejtmancik; Increased Sensitivity of Amino-Arm Truncated βA3-Crystallin to UV-Light–Induced Photoaggregation. Invest. Ophthalmol. Vis. Sci. 2005;46(9):3263-3273. doi: 10.1167/iovs.05-0112.
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purpose. Exposure to UV-B light (wavelength, 290–320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, β-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on β-crystallin function, the sensitivity of purified recombinant wild-type (rβA3) to UV-irradiation induced aggregation was compared with that of βA3-crystallin missing the N-terminal extension (rβA3tr).
methods. Proteins were expressed in baculovirus-infected Sf9 cells and purified by chromatography. Purified protein solutions (pH 7.4) were reduced by using Tris (2-carboxyethyl) phosphine HCl and irradiated with a 308-nm excimer laser at physiologically relevant UV doses and wavelengths (308 nm), and light-scattering (633 nm) was measured. Irradiated crystallins were analyzed by matrix-assisted desorption ionization (MALDI) and tandem liquid chromatography/mass spectrometry (LC-MS/MS).
results. UV-irradiation of both rβA3 and rβA3tr resulted in major loss of soluble protein, as shown by absorption at 280 nm, size-exclusion chromatography (SEC) and SDS-PAGE, with concomitant formation of insoluble aggregates producing light-scattering. Compared with wild-type rβA3, rβA3tr showed a significant tendency to begin scattering light at lower UV dose and had a higher aggregation rate with increasing UV exposure. Changes in irradiated crystallins include aggregation and cross-linking, photolysis, and oxidation of methionine and tryptophan residues.
conclusions. Loss of β-crystallin terminal arms appears to increase their tendency to aggregate in response to UV irradiation, suggesting that this loss in the maturing lens may increase susceptibility to age-related cataract.
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