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.