Increased sensitivity of amino-arm truncated betaA3-crystallin to UV-light-induced photoaggregation.
| Autor: | Sergeev YV; National Eye Institute, National Institutes of Health, Building 10, Room 10B10, 10 Center Drive, Bethesda, MD 20892, USA. sergeev@helix.nih.gov, Soustov LV, Chelnokov EV, Bityurin NM, Backlund PS Jr, Wingfield PT, Ostrovsky MA, Hejtmancik JF |
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| Jazyk: | angličtina |
| Zdroj: | Investigative ophthalmology & visual science [Invest Ophthalmol Vis Sci] 2005 Sep; Vol. 46 (9), pp. 3263-73. |
| DOI: | 10.1167/iovs.05-0112 |
| Abstrakt: | Purpose: Exposure to UV-B light (wavelength, 290-320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, beta-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on beta-crystallin function, the sensitivity of purified recombinant wild-type (rbetaA3) to UV-irradiation induced aggregation was compared with that of betaA3-crystallin missing the N-terminal extension (rbetaA3tr). 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 rbetaA3 and rbetaA3tr 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 rbetaA3, rbetaA3tr 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 beta-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. |
| Databáze: | MEDLINE |
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