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Increased Sensitivity of Amino-Arm Truncated ßA3-Crystallin to UV-Light–Induced Photoaggregation

¹From the National Eye Institute, the ³National Institute of Child Health and Human Development, and the ⁴National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland; the ²Institute of Applied Physics, Nizhnii Novgorod, Russia; and the ⁵Institute of Biochemical Physics, Moscow, Russia.

PURPOSE. Exposure to UV-B light (wavelength, 290–320 nm) is a well-documented risk factor for age-related cataracts. As the lens ages, ß-crystallins tend to undergo proteolytic cleavage of their terminal extensions. To delineate the effects of loss of terminal arms on ß-crystallin function, the sensitivity of purified recombinant wild-type (rßA3) to UV-irradiation induced aggregation was compared with that of ßA3-crystallin missing the N-terminal extension (rßA3tr).

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 rßA3 and rßA3tr 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 rßA3, rßA3tr 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 ß-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.