Weinreb O, van Rijk A F, Dovrat A, Bloemendal H
Department of Biochemistry, University of Nijmegen, The Netherlands.
Invest Ophthalmol Vis Sci. 2000 Nov;41(12):3893-7.
To determine whether alpha-crystallin is capable of forming filament-like structures with other members of the crystallin family.
Water-soluble crystallins were isolated from calf lenses and fractionated into alpha-, betaH-, betaL-, and gamma-crystallins according to standard procedures. Chaperone-like activity of alpha-crystallin was determined in control and UV-A-irradiated lenses by the heat-induced aggregation assay of betaL-crystallin. Protein samples from this assay were analyzed by electron microscopy. In vitro filament formation was examined by transmission immunoelectron microscopy using specific antibodies directed against the crystallins. Involvement of intermediate filament constituents was excluded by the results of Western blot analysis, which were all negative. Moreover, the in vitro amyloid fibril interaction test using thioflavin T (ThT) was also performed.
At the supramolecular level heating at 60 degrees C has no effect on the morphologic appearance of alpha-crystallin as observed by transmission electron microscopy. Moreover alpha-crystallin obtained from UV-A-irradiated lenses shows a virtually identical shape. However, heating in the presence of betaL-crystallin results in the formation of filament-like alphabeta-hybrids as demonstrated by immunoelectron microscopy using specific antibodies directed either against alpha- or betaL-crystallin. Parallel experiments with alpha-crystallin derived from UV-A-irradiated lenses showed even more pronounced filamentous structures, compared with the controls. Nonetheless, we were able to show that the UV-light treatment affected the chaperone-like capacity of alpha-crystallin, as revealed by a diminished ability to inhibit in vitro denaturation of betaL-crystallin. To exclude the presence of cytoskeletal contamination in the crystallin preparations, vimentin antibodies were also tested. These latter experiments were negative. The filamentous nature of the hybrids was further confirmed by the results obtained with the ThT assay earlier applied for the detection of amyloid fibrils.
Crystallin hybrids have previously been detected in the water-soluble lens crystallin fraction. Our findings indicate that such endogenous hybrids, formerly called "rods," may result from stress-induced interaction between alpha-crystallin and other lens constituents such as betaL-crystallin. Because the hybrid formation is enhanced when alpha-crystallin from UV-A-irradiated lenses is used as one of the two components of the hybrid, one can only speculate that this formation may be one of the factors leading to UV-A cataract.
确定α-晶状体蛋白是否能够与晶状体蛋白家族的其他成员形成丝状结构。
从小牛晶状体中分离出水溶性晶状体蛋白，并按照标准程序将其分为α-、βH-、βL-和γ-晶状体蛋白。通过βL-晶状体蛋白的热诱导聚集试验，在对照和紫外线A照射的晶状体中测定α-晶状体蛋白的伴侣样活性。该试验的蛋白质样品通过电子显微镜进行分析。使用针对晶状体蛋白的特异性抗体，通过透射免疫电子显微镜检查体外丝状结构的形成。蛋白质印迹分析结果均为阴性，排除了中间丝成分的参与。此外，还进行了使用硫黄素T（ThT）的体外淀粉样原纤维相互作用试验。
在超分子水平上，通过透射电子显微镜观察，60℃加热对α-晶状体蛋白的形态外观没有影响。此外，从紫外线A照射的晶状体中获得的α-晶状体蛋白显示出几乎相同的形状。然而，在βL-晶状体蛋白存在下加热会导致形成丝状的αβ-杂化物，这通过使用针对α-或βL-晶状体蛋白的特异性抗体的免疫电子显微镜得到证实。与对照相比，用来自紫外线A照射晶状体的α-晶状体蛋白进行的平行实验显示出更明显的丝状结构。尽管如此，我们能够证明紫外线处理影响了α-晶状体蛋白的伴侣样能力，这通过抑制βL-晶状体蛋白体外变性的能力降低得以揭示。为了排除晶状体蛋白制剂中细胞骨架污染的存在，还测试了波形蛋白抗体。后一项实验结果为阴性。ThT试验早期用于检测淀粉样原纤维，其结果进一步证实了杂化物的丝状性质。
此前在水溶性晶状体晶状体蛋白组分中检测到晶状体蛋白杂化物。我们的研究结果表明，这种以前称为“杆状体”的内源性杂化物可能是由α-晶状体蛋白与其他晶状体成分（如βL-晶状体蛋白）之间的应激诱导相互作用导致的。由于当将来自紫外线A照射晶状体的α-晶状体蛋白用作杂化物的两个组分之一时，杂化物的形成会增强，因此只能推测这种形成可能是导致紫外线A性白内障的因素之一。
