Formation of amyloid fibrils in vitro by human γD-crystallin and its isolated domains
Formation of amyloid fibrils in vitro by human γD-crystallin and its isolated domains
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发表时间：
2008-01
期刊：
影响因子：
2.2
通讯作者：
K. Papanikolopoulou;I. Mills-Henry;S. Thol;Yongting Wang;Abby A R Gross;D. Kirschner;S. Decatur;J. King
中科院分区：
文献类型：
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作者：
K. Papanikolopoulou;I. Mills-Henry;S. Thol;Yongting Wang;Abby A R Gross;D. Kirschner;S. Decatur;J. King
目的淀粉样原纤维与多种人类蛋白质错误折叠和蛋白质沉积疾病有关。先前的研究表明，牛晶状体蛋白在变性条件下形成淀粉样纤维，并且淀粉样纤维在携带晶状体蛋白基因突变的小鼠的透镜中积累。在分化的透镜纤维细胞内，晶状体蛋白可能暴露于低pH溶酶体隔室。我们研究了当暴露于低pH部分变性条件时，人γ D-晶状体蛋白是否在体外形成淀粉样纤维。方法从大肠杆菌中表达并纯化人γ D晶状体蛋白。在37 °C、pH 7下稳定且可溶，并且在这些条件下从完全变性状态再折叠回到天然状态。将纯化的人γ D-晶状体蛋白及其分离的NH 2-和COOH-末端结构域在酸性pH下孵育，随后通过透射电子显微镜、存在刚果红的吸收光谱、FTIR和低角X射线散射进行检查。在37 °C下在50 mM乙酸盐缓冲液（pH 3）中以50 mg/ml孵育完整蛋白2天，导致形成粘性凝胶样溶液。通过透射电子显微镜检查负染色的样品，发现线性的，非分支的原纤维的长度可变，宽度范围从15至35 nm。与染料刚果红孵育产生与染料结合淀粉样蛋白相关的光谱红移。样品的低角X射线散射在4.7 nm处显示出清晰的赤道反射，在10和11 nm之间的赤道上显示出更大的漫反射，这是淀粉样蛋白纤维的典型“交叉β”X射线纤维衍射图案。利用FTIR跟踪γ D-晶状体蛋白在pH 3孵育过程中二级结构随时间的演变。天然蛋白质在1640 cm-1处显示主条带，其在37 ℃孵育期间转化为1616 cm-1处的条带。在1689 cm-1处的额外带也随时间出现。在约1620 cm-1和约1680 cm-1区域中条带的存在归因于分子间β-折叠结构的形成，该结构表征纤维状淀粉样蛋白基序。分离的Hγ D-晶状体蛋白的NH 2-末端1-82和COOH-末端86-174结构域在相同条件下孵育后也形成淀粉样原纤维，但程度低于全长。结论Hγ D-晶状体蛋白及其氨基端1-82和羧基端86-174结构域在酸性pH条件下形成淀粉样纤维。需要对透镜内白内障形成的早期阶段进行研究，以评估淀粉样纤维是否在体内白内障的发生中起作用。
Purpose Amyloid fibrils are associated with a variety of human protein misfolding and protein deposition diseases. Previous studies have shown that bovine crystallins form amyloid fibers under denaturing conditions and amyloid fibers accumulate in the lens of mice carrying mutations in crystallin genes. Within differentiating lens fiber cells, crystallins may be exposed to low pH lysosome compartments. We have investigated whether human γD-crystallin forms amyloid fibrils in vitro, when exposed to low pH partially denaturing conditions. Methods Human γD-crystallin expressed and purified from E. coli, is stable and soluble at 37 °C, pH7, and refolds from the fully denatured state back to the native state under these conditions. Purified Human γD-crystallin as well as its isolated NH2- and COOH-terminal domains were incubated at acid pH and subsequently examined by transmission electron microscopy, absorption spectroscopy in the presence of Congo red, FTIR, and low-angle X-ray scattering. Results Incubation of the intact protein at 37 °C in 50 mM acetate buffer pH 3 at 50 mg/ml for 2 days, led to formation of a viscous, gel-like solution. Examination of negatively stained samples by transmission electron microscopy revealed linear, non-branching fibrils of variable lengths, with widths ranging from 15 to 35 nm. Incubation with the dye Congo red generated the spectral red shift associated with dye binding to amyloid. Low-angle X-ray scattering from samples showed clear meridional reflection at 4.7 Å and a more diffuse reflection on the equator between 10 and 11 Å which is the typical “cross-β” X-ray fiber diffraction pattern for amyloid fibers. FTIR was used to follow the evolution of the secondary structure of γD-crystallin with time during incubation of the protein at pH 3. The native protein displayed a major band at 1640 cm-1 that converted during incubation at 37 °C to a band at 1616 cm-1. An additional band at 1689 cm-1 also appeared with time. The presence of bands in the regions about 1620 cm-1 and about 1680 cm-1 has been attributed to the formation of intermolecular β-sheet structure that characterizes the fibrillar amyloid motif. The isolated NH2-terminal 1–82 and COOH-terminal 86–174 domains of HγD-crystallin also formed amyloid fibrils after incubation under the same conditions, but to a lesser extent than the full length. Conclusions HγD-crystallin, as well as its isolated NH2-terminal 1–82 and COOH-terminal 86–174 domains of HγD-crystallin formed amyloid fibrils upon incubation at acid pH. Investigations of early stages in cataract formation within the lens will be required to assess whether amyloid fibrils play a role in the initiation of cataract in vivo.