Protease‐sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation

Abstract

Light-chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full-length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel-based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL k amyloidosis patient (AL-55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC's dimeric and fibrillar states. All tissues contain fragmented LCs along with the full-length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ-specific, likely due to a complex of proteases. Cleavage sites are concentrated in 'proteolysisprone' regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients. Abbreviations 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; ACN, acetonitrile; AL amyloidosis, immunoglobulin light-chain amyloidosis; CL, light chain's constant domain; Cryo-EM, cryo-electron microscopy; DTT, dithiothreitol; EA, ethanolamine; FA, formic acid; HCD, higher energy collisional dissociation; IEF, isoelectrofocusing; LCs, immunoglobulin light chains (note, LC in the standard hyphenated abbreviation LC-MS/MS indicates liquid chromatography); PTM, post-translational modifications; VL, light chain's variable domain.

Understanding AL amyloidosis with a little help from in vivo models

“…Accordingly, few biochemical studies showed that the full-length LCs could be involved in the initial nucleation thanks to its stability which serves as a platform for the subsequent elongation by unstable VLs ( 34 – 36 ). These experimental results are in line with the observations made by mass spectrometry both in diagnosis using laser microdissection/mass spectrometry (LMD/MS) ( 37 ) and in more careful analyses of deposited protein contents ( 38 ) showing that a complete or partially degraded C domain is always present in the amyloid deposits ( 39 ), and the degradation patterns argue for a post-deposition proteolysis. Whether C domain proteolysis occurs before or after aggregation and contributes or conversely protects from amyloidogenesis in vivo is still under debate ( 3 , 39 ), but using the sole VL to study amyloidogenesis of LCs in vitro is likely a reduced approach that do not mirror the in vivo process.…”

Role of Complementarity-Determining Regions 1 and 3 in Pathologic Amyloid Formation by Human Immunoglobulin κ1 Light Chains

“…8). In addition, two segments, [12][13][14][15][16][17][18][19][20][21][22] (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…The copyright holder for this preprint this version posted February 3, 2023. ; https://doi.org/10.1101/2023.02.01.526662 doi: bioRxiv preprint can follow the misfolding rather than precede it [4,18], and the exact sequence of steps leading to formation of AL deposits remains to be determined.…”

Light Chain Stabilization: A Therapeutic Approach to Ameliorate AL Amyloidosis

“…High resolution mapping of peptide termini by mass spectrometry recently revealed a more complex picture, consistent with multiple proteolytic events after amyloid fibril formation [ 73 , 74 ]. Proteolytic events after fibril formation are also suggested by the recent cryo-electron microscopy-delineated structures of TTR amyloid fibrils in the hearts of late-onset familial amyloidosis patients [ 75 ].…”