Abstract
Intervention 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, ∼50% 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-β and α-synuclein.
Highlights
Inhibition of amyloid deposition is emerging as a potential secondary treatment option in AL amyloidosis
A number of studies have scrutinized the factors governing LC amyloid formation, most of which have been performed on recombinant variable domains from five different LC proteins: Wil and Jto [18, 19], AL-09 and AL-103 (20 –24), and SMA [25, 26]
When systematically reviewing EGCG mechanisms in various proteins, we found that EGCG binding to aggregation intermediates may inhibit amyloid formation very effectively in cases where these species are rate-limiting to amyloid formation [57]
Summary
The significance of full-length LC for amyloid formation is not yet fully understood [27,28,29,30]. For the first time, quantifies stabilities and amyloid formation propensities of nine authentic, full-length LC proteins that we isolated from the urine of patients (4 with AL, 5 with MM). Our study tested whether AL and MM light chains had inherently different stabilities or amyloid formation propensities. We found that both AL and MM full-length light chains formed amyloid aggregates in vitro, but only under conditions that cleaved intermolecular disulfide bonds. A reduction in cardiac pathology was observed in some but not all patients treated with EGCG [35]. Our study tested the effect of EGCG on amyloid formation kinetics of our nine patient-derived LC proteins. We found that EGCG inhibited the conversion of oligomeric LC into ThT-positive fibrils and induced the formation of SDSstable aggregates in all LC proteins
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