Wang Steven S-S, Wen Wen-Sing
Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan.
Mol Vis. 2010 Dec 16;16:2777-90.
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).
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.
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.
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.
人γD晶状体蛋白是人类眼球晶状体的主要蛋白质成分，与青少年和成年期白内障的发生有关。紫外线（UV）照射被认为会扰乱蛋白质结构并最终导致聚集。本研究旨在探讨UV-C照射对重组人γD晶状体蛋白（HGDC）的影响。
重组HGDC蛋白在携带质粒pEHisHGDC的大肠杆菌BL21(DE3)菌株中表达，并采用色谱方法进行纯化。然后将蛋白质暴露于强度为420、800或1850 μW/cm²的UV-C光（λ(max)=254 nm，15 W）下。通过SDS-PAGE、浊度测量、圆二色光谱（CD）、色氨酸荧光光谱、丙烯酰胺荧光猝灭分析和巯基测量等方法对未暴露于UV-C的样品、暴露于UV-C后的上清液部分以及重新溶解的UV-C暴露沉淀物部分进行表征。
发现HGDC样品溶液的浊度与HGDC浓度、UV-C照射强度和UV-C照射持续时间呈正相关。暴露于UV-C时，HGDC样品溶液明显变浑浊，长时间照射后可观察到肉眼可见的大量较大蛋白质颗粒。发现照射后的HGDC样品沉淀物部分可被盐酸胍重新溶解。电泳、丙烯酰胺荧光猝灭和光谱分析揭示了未照射的HGDC、照射后的HGDC上清液部分以及照射后的HGDC重新溶解沉淀物部分之间结构上的差异。通过使用L-半胱氨酸、巯基含量测量以及还原和非还原SDS-PAGE，我们的数据进一步表明二硫键的形成和/或断裂可能在UV-C照射引起的HGDC聚集和/或沉淀中起重要作用。
我们的研究结果突出了二硫键断裂和/或形成、分子间相互作用以及UV-C照射诱导的HGDC聚集体形成之间的密切联系。本研究结果不仅有助于理解导致蛋白质聚集的环境因素，还对阐明白内障发生的分子机制具有重要意义。
