Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Abstract

Edited by Paul E. FraserIntervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo. We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ϳ50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-␤ and ␣-synuclein. Systemic light chain amyloidosis (AL)2 is the most common form of systemic amyloidosis (1), but it is still a rare disease with an incidence of 6 -7 in 1,000,000 people (2). Underlying AL pathology is a clonal plasma cell disorder, which produces an immunoglobulin light chain in the bone marrow with a unique sequence. These light chains are released into the blood (monoclonal gammopathy). When elevated LC levels in serum overwhelm renal absorption, LC is also found in the urine. Fulllength LC can be isolated from the urine of these patients (3-5).Clonal plasma cell disorders can present different types of pathologies; light chains form amyloid deposits in AL patients. Here, LC fibrils adopt a conformation characterized by highly stable, intramolecular cross-␤-sheets that stain with Congo red (6) and thioflavin T (ThT) (7-9).In contrast, amyloid deposits are not observed in patients suffering from multiple myeloma (MM), a malignant disease characterized by bone marrow failure and bone destruction. The two contrasting pathologies of LC gammopathies raise the question of which factors determine amyloid formation. Amyloid formation could be initiated (a) by the sequences of the individual light chains, (b) by high levels of light chain expression, (c) by p...

Kinetic stability and sequence/structure studies of urine-derived Bence-Jones proteins from multiple myeloma and light chain amyloidosis patients

“…1C & D), without significant differences between BJP from MM or AL patients belonging to the same isotype. We confirmed that the 7 BJPs studied did not refold reversibly, in agreement with previous reports from our laboratory and others [7–11]. …”

“…Bence Jones proteins from patients with AL amyloidosis or MM have been compared for their thermodynamic stability. Consistent results from these comparative studies have indicated that the former are less thermodynamically stable than MM BJPs from the same or similar germline sequence with a high level of sequence identity [7, 9, 11]. One important characteristic of the thermodynamic properties of BJPs is that these proteins do not refold reversibly, in contrast to the reversible behavior observed for immunoglobulin light chain variable domains (V L ) from AL amyloidosis proteins (for a review, please see [2]).…”

“…Recent experimental evidence [8, 10, 11] indicate that the folding of free light chains (and BJPs) is kinetically controlled, facilitating sampling/populating amyloidogenic conformations. To determine whether the stability of the BJPs is governed by kinetic or thermodynamic stability, we measured scan rate dependency of the unfolding reactions.…”

“…Bence Jones proteins are monoclonal and readily isolated from patients’ urine, and they are available in sufficient quantities for detailed biophysical analysis. Because of this, these proteins have been an invaluable research material for biochemical and biophysical investigation of immunoglobulin molecules [6–11]. Early studies assessing BJP stability were conducted studying protease resistance of these proteins [12].…”

“…It is interesting to note that in most of the studies, the thermal unfolding of Bence-Jones Proteins, free MM LCs, AL LC, and recombinant LCs (encompassing the V L and the C L domain, also called FL) has been described as an apparent 2-state process, showing a single irreversible sigmoidal transition, often followed by protein aggregation [7–11, 14–16]. There is only one report of multiple unfolding transitions for the LC thermal unfolding performed at pH 3.23 [17].…”