AUTHORS: B.K. Arendt,1 G.J. Ahmann,2 R. Fonseca,2 E.M. Mulvihill,1 A. Dispenzieri,3 L.A. Sikkink,4 M. Ramirez-Alvarado,4 S.R. Zeldenrust3 D.F. Jelinek1
1Dept. of Immunology, Mayo Cinic Rochester, MN; 2Dept. of Internal Medicine, Mayo Clinic Scottsdale, AZ; 3Dept. of Internal Medicine, Mayo Clinic Rochester, MN; 4Dept. of Biochemistry and Molecular Biology, Mayo Clinic Rochester, MN, USA
Primary amyloidosis (AL) is a rare monoclonal plasma cell disorder in which insoluble immunoglobulin light chains are produced. These proteins undergo conformational changes because of protein misfolding and become deposited in vital organs throughout the body. Because of the typically small numbers and low proliferative capability of plasma cells associated with AL, advances in the field have been limited by a lack of cell line model systems. To our knowledge, there is no human cell line in existence that has been established from a patient diagnosed with AL. In this study, we have established 2 novel AL cell lines from the same patient that have been shown to secrete free lambda light chain which exhibits the ability to form amyloid fibrils. Methods. The ALMC-1 and ALMC-2 cell lines were established from a patient diagnosed with amyloidosis before and after a peripheral blood stem cell transplant and were extensively characterized for phenotypic markers, cytokine responsiveness, and genetic abnormalities. To determine whether these cell lines could be used as tools to study the amyloidogenic LC, LC was purified from cell supernatants using size exclusion chromatography. Secondary structure was measured by circular dichroism spectroscopy (CD) and thermal denaturation was calculated to determine melting temperatures. Fibril formation was verified using Thioflavin T (ThT) to measure plaques composed of beta sheets. Electron microscopy (EM) was used to visually identify the presence of amyloid fibrils. Results. CD spectroscopy determined that the LC purified from ALMC-1 and ALMC-2 cell lines contained a beta structure, expected for an immunoglobulin molecule. Fibril formation was achieved by incubating these proteins at their melting temperature and was confirmed using ThT. In addition, EM visibly proved that production of amyloid fibrils was feasible. Long, straight, unbranching fibrils consistent with the size and shape of amyloid fibrils were observed. Conclusion. The formation of amyloid fibrils from these naturally secreting human LC cell lines is unprecedented and will clearly prove to be an invaluable resource to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics and cell biology.