BIOCHEMICAL CHARACTERIZATION OF LIGHT CHAIN AMYLOIDOSIS PROTEINS

Clonal proliferation of monoclonal plasma cells can result in the deposition of immunoglobulin light chains and/or the excretion of Bence Jones proteins (BJPs) in the urine. Multiple Myeloma (MM), Light Chain Deposition Disease (LCDD) and Light Chain Amyloidosis (AL) are three light chain related diseases that fall into this category.

The characteristics of each light chain disease vary: MM shows bone lesions, hypercalcemia, renal failure, and anemia; LCDD localizes in the basement membrane of the kidney and it forms granular, amorphous aggregates; while AL has deposition of immunoglobulin light chains as amyloid fibrils in vital organs leading to organ failure and death. However, all of them involve clonal proliferation of light chains. Kappa light chains are more prevalent in LCDD and MM while lambda light chains are more prevalent in AL.

BENCE JONES PROTEINS AND LIGHT CHAIN DISEASES

BJPs from patients with AL, LCDD and MM have been purified in the lab from their urine and studies were performed to determine their secondary structure, thermodynamic stability and aggregate formation kinetics. Our results have shown that LCDD and MM proteins are the most stable with the lowest free energy of folding while all proteins show similar melting temperatures. Incubation of the BJPs at their melting temperature produced morphologically different aggregates: amyloid fibrils from the AL proteins, amorphous aggregates from the LCDD proteins and large spherical species from the MM proteins. This demonstrates differences in aggregation pathways between these proteins.

AL PROTEINS AND FIBRIL FORMATION STUDIES

We perform biochemical and biophysical studies on AL proteins predominantly from recombinant sources, (E.coli and Pichia pastoris), as well as urine and cell-line derived proteins. Initial characterization of protein identity, purity and oligomerization state is done using mass spectrometry, amino acid analysis, size exclusion chromatography and in some cases, analytical ultracentrifugation We study protein thermodynamic stability by thermal and chemical denaturation using Circular Dichroism and Fluorescence Spectroscopy. We study amyloid formation in vitro Thioflavine T fluorescence as well as Electron Microscopy.

We are interested in the role of salts and glycosaminoglycans (GAGs) conditions in protein structure, stability, and amyloid formation kinetics in vivo and in vitro. Our results have shown that the β-sheet structure of AL proteins is unaffected by various external factors. Salts increase the stability of AL proteins by following the rankings of the Hofmeister series and the GAGs tested so far stabilize AL proteins in different ways. Amyloid formation is promoted in the presence of salts by significantly shortening the lag time involved in the nucleation kinetics of amyloid fibril formation.

We are currently studying the possible toxic effect of AL protein oligomeric species and amyloid fibrils formed in the presence of different external factors in cardiomyocytes. We are also studying light chain internalization into cardiomyocytes by fluorescently tagging our AL proteins and following their uptake rates and intracellular localization via confocal microscopy.