Book of Abstracts: Albany 2005
Systematic Investigation of the Kinetics of Amyloid Formation for Immunoglobulin Light Chains
Monoclonal immunoglobulin light chains are responsible for the characteristic pathological features found in patients with light chain-related diseases that include Multiple Myeloma (MM) and Light Chain amyloidosis (AL). AL is characterized by the extracellular deposition of monoclonal immunoglobulin light chains as amyloid fibrils in vital organs leading to organ failure and death. The organ most frequently affected is the kidney followed by the heart and liver. MM is characterized by bone lesions, hypercalcemia, renal failure, and anemia; however, no amyloid deposits are found in MM patients. Proteins from both MM and AL patients have accumulated somatic mutations with respect to their germline genes. The goal of this study is to understand the effect that the mutations have on protein stability, structure and kinetics of aggregate/amyloid formation for AL, using MM proteins as non-amyloidogenic light chain controls. We have mapped the mutations from the germline sequence of 8 proteins and found 4 mutational regions: mutations in the top and bottom of the immunoglobulin beta-barrel; mutations in the N and C terminus beta strands; mutations in the beta-hairpin between strands D and E, and mutations in the region that binds to the immunoglobulin heavy chain. MM-01 protein has mutations in the N and C termini strands of the domain (Group 2). AL-09 presents most of the mutations in the interface that interacts with the heavy chain to form an immunoglobulin molecule (group 4). Proteins from the same mutational region have similar structure and thermodynamic stability as followed by Circular Dichroism Spectroscopy. We followed the kinetics of aggregate/amyloid formation for AL-09, a cardiac AL protein, under a number of experimental conditions. AL-09 follows a fast rate of amyloid formation when incubated at the melting temperature (49°C) in the presence of 500 mM sodium sulfate, conditions in which a beta-sheet rich intermediate seems to be stabilized. Congo red acts as an inhibitor of amyloid formation even in the presence of 500 mM sodium sulfate. AL-09 follows a slow rate of amyloid formation under physiological conditions. Seeded reactions accelerate amyloid formation. Most glycosaminoglycans, present in the extracellular matrix, accelerate the seeded reactions. Surprisingly, Chondroitin sulfate A, a glycosaminoglycan that is very abundant in heart tissues inhibits amyloid formation for AL-09 and stabilizes a spherical intermediate. MM-01 does not form amyloid fibrils when incubated at its melting temperature, instead it forms spherical intermediates. These structures are not inhibited or enhanced in the presence of Chondroitin sulfate A. We are interested in understanding the kinetics of amyloid formation with both AL and MM proteins to gain new insight into these fibril/aggregate pathways.
Department of Biochemistry and Molecular Biology