AFM TO MONITOR FIBRIL FORMATION FROM AMYLOID IG LIGHT CHAINS

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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.