AFM TO MONITOR FIBRIL FORMATION FROM AMYLOID IG LIGHT CHAINS

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。该项目的目标是对尿免疫球蛋白轻链进行成像，并确定是否可以检测到来自轻链的原纤维形成，并与从淀粉样蛋白患者组织中分离的原纤维进行比较。我们假设，了解淀粉样蛋白的结构和随时间的变化将增强我们在体外和体内淀粉样蛋白形成的知识。为了评价水提取的原纤维的特性，将它们在水中培养，蛋白酶消化并在云母上干燥或在云母上活体成像。计算来自许多组织的原纤维的大小，并与硫磺素T相关。此外，使用DMMB测定总GAG的百分比，并在特异性多糖酶消化后评价类型。 纯化所有轻链，用质谱法推导每条轻链的序列。轻链的一般质量为23 kDa。进行大部分分析的轻链是后修饰的。在AFM之前，将轻链超声处理并置于云母上并干燥。最初的一组实验涉及在室温下在pH 4、6和8下在一个月的时间内评价原纤维的结构而不旋转。我们证明了在第一周内存在一些环状环和形成原纤维，但是随着时间的推移，没有检测到原纤维形成。在若干放大倍数下进行扫描，得到1和2微米以及500 nm的512 × 512图像。 还进行了实验，其中轻链孵育超过1个月，并向轻链中添加糖胺聚糖（硫酸乙酰肝素、硫酸软骨素和肝素）并进行评价。添加糖胺聚糖可提高原纤维的形成速率，并使原纤维的宽度和高度增加，形成箭头状形态。同时，进行硫磺素T检测原纤维，在所有时间点均为阴性。 然后通过将GAG和轻链添加到云母上并监测随时间的变化来进行实时成像。同样，添加GAG增强了原纤维的有序结构。未检测到原纤维形成。

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. The goal of the project is to image urinary immunoglobulin light chains and to determine if fibril formation from the light chains can be detected and compared to fibrils isolated from tissues of patients with Amyloid. We hypothesized that understanding the structure and change over time would enhance our knowledge in amyloid formation in vitro and in vivo. To evaluate the characteristics of the water extracted fibrils, they were brought up in water, proteinase digested and dried on or imaged live on mica. The size of the fibrils from a number of tissues was calculated and correlated with thioflavin T. Furthermore, the percentage of total GAG was determined using DMMB and the type was also evaluated following specific polysaccaridase digestion. All light chains were purified and the sequence of each light chain was deduced with Mass Spectrometry. Light chains have a general mass of 23 kDa. The light chains that the majority of the analysis was conducted were post-translationally modified. Prior to AFM, the light chains were sonicated and placed on mica and dried. The initial set of experiments involved evaluating the structure of the fibrils over a period of one month at pH 4, 6 and 8 at room temperature without rotation. We demonstrated the presence of some annular rings and the formation of protofibrils within the first week however over time, no fibril formation was detected. Scans were taken at several magnifications yielding 512x512 images of 1 and 2 microns and 500 nm. Experiments were also performed where light chains were incubated over 1 month and glycosaminoglycans were added (heparan sulfate, chondrotin sulfate and heparin) to the light chains and evaluated. The addition of GAGs enhanced the rate of protofibril formation and the width and height of them yielding arrowhead morphology. Concurrently, thioflavin T was performed to detect fibrils and was negative at all time points. Live imaging was then performed with the addition of GAGs and light chains onto the mica and monitoring changes over time. Again, the addition of GAGs enhanced the ordered structure of the protofibrils. Fibril formation was not detected.