AFM TO MONITOR FIBRIL FORMATION FROM AMYLOID IG LIGHT CHAINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 淀粉样纤维是由异常折叠的蛋白质组成的。原子力显微镜已经证明了它能够解释体外纤维的形成过程。淀粉样蛋白如淀粉样蛋白和重组免疫球蛋白轻链的纤维形成已有报道。Ionescu-Zanetti等人提出的模型表明，淀粉样蛋白形成片状结构，形成直径约2.4 nm的单丝。然后，两根或两根以上的细丝可以缠绕在一起，直接形成更大尺寸的原丝或原纤维。 该AFM项目与我们的其他淀粉样质谱项目相关，重点是从患者器官提纯并在溶液中悬浮的免疫球蛋白轻链，以及直接从人体器官提取的纤维。纯化的尿免疫球蛋白轻链蛋白(01-029)在37℃，0.03 mM的缓冲液和100 mM的氯化钠溶液中，适度搅拌。标准缓冲液包括20 mM盐酸、20 mM Tris和50 mM醋酸盐。在攻丝模式下，于不同时间取材进行AFM分析。我们的初步数据表明，纤维形成的速度非常依赖于培养条件，如pH，搅拌。在pH=2时可观察到纤维，而在pH=5.5和7.5时未观察到纤维。还测量了从患者器官中提纯的淀粉样纤维的构象，并测定了每种淀粉样纤维及其蛋白水解物的质谱学特征。不同的实验条件将测试抗体轻链原纤维的形成。目前正在研究包括肝素、硫酸肝素在内的糖胺多聚糖在纤维形成过程中的作用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Amyloid fibrils are composed of abnormally refolded proteins. AFM has demonstrated its capability to elucidate the in vitro fibril formation process. The fibril formation of amyloid protein such as Amyloid-¿ and recombinant IgG lightchain has been reported. The model proposed by Ionescu-Zanetti et al shows that the amyloid proteins form ¿-sheet structures to become a single filament in the diameter around 2.4 nm. Then two or more filaments can intertwine to form larger size protofilbrils or fibrils directly. This AFM project is related to our other amyloid mass spectrometry projects, and focuses on IgG light-chains purified from patient organs and resuspended in solution, as well as the fibrils taken directly from human organs. Purified urinary IgG light chain protein (01-029) was incubated at 0.03 mM in the presence of buffer and 100 mM NaCl solution at 37 deg C with moderate stirring. Standard buffers included 20 mM HCl, 20 mM TRIS, and 50 mM acetate. Aliquots were taken at different times for AFM analysis under tapping mode. Our preliminary data showed that the rate of fibril formation is very dependent on the incubation conditions, such as pH, stirring. Fibrils were observed at pH 2 with stirring, but not at pH 5.5 and 7.5. The conformation of the amyloid fibrils purified from patient organs were also measured, and the mass spectral characteristics of each of these and their proteolytic digests were also determined. Different experimental conditions will be tested for IgG light-chain fibril formation. The effects of glycosaminoglycan including heparin, heparin sulfate during the fibril formation are currently being explored.