Med-X Research Institute, Shanghai Jiao Tong University, Shanghai, China.
Invest Ophthalmol Vis Sci. 2010 Feb;51(2):672-8. doi: 10.1167/iovs.09-3987. Epub 2009 Aug 13.
Mature-onset cataract results from the formation of light-scattering aggregates of lens crystallins. Although oxidative or mutational damage may be a prerequisite, little is known of the initiation or nucleation of these aggregated states. In mice carrying mutations in gamma-crystallin genes, a truncated form of gamma-crystallin formed intranuclear filamentous inclusions within lens fiber cells. Previous studies have shown that bovine crystallins and human gammaD-crystallin form amyloid fibrils under denaturing conditions in vitro. The amyloid fibril formation of human gammaC-crystallin (HgammaC-Crys) induced by low pH, together with characterization of a partially unfolded intermediate in the process were investigated.
HgammaC-Crys was expressed and purified from Escherichia coli. Partially unfolded intermediates were detected by tryptophan fluorescence spectroscopy and UV resonance Raman spectroscopy. The aggregation into amyloid fibrils was monitored by solution turbidity and fluorescence assay. The morphology of aggregates was characterized using transmission electron microscopy (TEM). Secondary structure of the peptides in their fibrillar state was characterized using Fourier transform infrared spectroscopy (FTIR).
The structure of HgammaC-Crys was perturbed at low pH. Partially unfolded intermediates were detected when solution pH was lowered to pH 3. At pH 3, HgammaC-Crys aggregated into amyloid fibrils. The kinetics and extent of the reaction was dependent on protein concentration, pH, and temperature. TEM images of aggregates revealed aggregation stages from short to long fibrils and from long fibrils to light-scattering fibril networks. FTIR spectroscopy confirmed the cross-beta character of the secondary structure of these fibrils.
HgammaC-Crys formed amyloid fibrils on incubation at low pH via a partially unfolded intermediate. This process could contribute to the early stages of the formation of light-scattering species in the eye lens.
老年性白内障是由于晶状体晶体蛋白形成光散射聚集体而导致的。尽管氧化或突变损伤可能是前提条件，但对于这些聚集状态的起始或成核知之甚少。在携带 γ-晶体蛋白基因突变的小鼠中，γ-晶体蛋白的截断形式在晶状体纤维细胞内形成核内丝状包涵体。先前的研究表明，牛晶体蛋白和人γD-晶体蛋白在体外变性条件下形成淀粉样纤维。研究了低 pH 诱导的人γC-晶体蛋白（HgammaC-Crys）的淀粉样纤维形成，以及该过程中部分展开中间体的特征。
从大肠杆菌中表达和纯化 HgammaC-Crys。通过色氨酸荧光光谱法和紫外共振拉曼光谱法检测部分展开的中间体。通过溶液浊度和荧光测定监测聚集形成淀粉样纤维。使用透射电子显微镜（TEM）对聚集体的形态进行了表征。使用傅立叶变换红外光谱（FTIR）对肽在其纤维状态下的二级结构进行了表征。
在低 pH 下，HgammaC-Crys 的结构受到干扰。当溶液 pH 降低至 pH 3 时，检测到部分展开的中间体。在 pH 3 时，HgammaC-Crys 聚集形成淀粉样纤维。该反应的动力学和程度取决于蛋白质浓度、pH 和温度。聚集体的 TEM 图像显示了从短到长纤维以及从长纤维到光散射纤维网络的聚集阶段。FTIR 光谱证实了这些纤维的二级结构具有交叉-β特征。
HgammaC-Crys 在低 pH 孵育时通过部分展开的中间体形成淀粉样纤维。该过程可能有助于眼晶状体中光散射物质形成的早期阶段。
