Examining the influence of ultraviolet C irradiation on recombinant human γD-crystallin

PURPOSE Human γD crystallin is a principal protein component of the human eye lens and associated with the development of juvenile and mature-onset cataracts. Exposure to ultraviolet (UV) light is thought to perturb protein structure and eventually lead to aggregation. This work is aimed at exploring the effects of UV-C irradiation on recombinant human γD-crystallin (HGDC). METHODS Recombinant HGDC proteins were expressed in E. coli strain BL21(DE3) harboring plasmid pEHisHGDC and purified using chromatographic methods. The proteins were then exposed to UV-C light (λ(max)=254 nm, 15 W) at the intensity of 420, 800, or 1850 μW/cm(2). The UV-C-unexposed, supernatant fraction of UV-C-exposed, and re-dissolved precipitated fraction of UV-C exposed preparations were characterized by SDS-PAGE, turbidity measurement, CD spectroscopy, tryptophan fluorescence spectroscopy, acrylamide fluorescence quenching analysis, and sulfhydryl group measurements. RESULTS The turbidity of the HGDC sample solution was found to be positively correlated with HGDC concentration, UV-C irradiation intensity, and UV-C irradiation duration. When exposed to UV-C, HGDC sample solutions became visibly turbid and a noticeable amount of larger protein particle, perceptible to the naked eye, was observed upon prolonged irradiation. The precipitated fraction of irradiated HGDC sample was found to be re-dissolved by guanidine hydrochloride. Electrophoresis, acrylamide fluorescence quenching, and spectroscopic analyses revealed differences in structures among the non-irradiated HGDC, the supernatant fraction of irradiated HGDC, and the re-dissolved precipitated fraction of irradiated HGDC. Through the use of L-cysteine, the measurements of sulfhydryl contents, and the reducing as well as non-reducing SDS-PAGE, our data further suggested that disulfide bond formation and/or cleavage probably play an important role in aggregation and/or precipitation of HGDC elicited by UV-C irradiation. CONCLUSIONS Our findings highlight the close connections among disulfide bond cleavage and/or formation, intermolecular interactions, and the resultant formation of aggregates of HGDC induced by UV-C irradiation. The results from this research may not only contribute to the understanding of the environmental factors causing protein aggregation but also have implications for deciphering the molecular mechanism of cataractogenesis.