Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis

Abstract

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N- and C-termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N- and C-terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LCs’ variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling, by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LCs processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, they show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.

Universal amyloidogenicity of patient-derived immunoglobulin light chains

“…In our fibrils formed in vitro we only detected a fragment of almost the entire variable domain in P006 (pH 3), in addition to shorter fragments of the same region. We could find many similar C-terminal fragments, which is in accordance with the heterogeneous fragmentation positions reported recently 71 . The fragments we have detected are mostly rather short with an average molecular weight of 3.8 kDa.…”

“…Previous studied already demonstrated that short peptides of 12 residues of the variable and constant domain of an amyloidogenic λ LC can form fibrillar structures in vitro 72 . It has been proposed that at least some proteolytic cleavage events happen after the assembly into amyloid fibrils in vivo 71,73 . This is likely also to be the case for our amyloid fibrils assembled in vitro.…”

Dissecting the Molecular Features of Systemic Light Chain (AL) Amyloidosis: Contributions from Proteomics

“…In particular, the N-and C-terminal residues of fragments were chemically derivatized, and the labeled residues were identified by LC-MS/MS and bioinformatics [87]. The data showed that fragmentations mainly occurs in the C L ; the cleavage sites that generate LC fragments are mostly located in poorly structured regions of the fibrillar structure, suggesting that the ensemble of proteolytic events observed in mature ex vivo fibrils largely reflects extensive proteolytic remodeling of the preformed aggregates [87]. Amyloidogenic LCs, however, are not the only molecular species present in the deposits.…”

“…The pathogenetic role of proteolysis in AL (i.e., whether it precedes or follows LC deposition) is still debated, and the discussion of this topic is beyond the scope of this review. Indeed, new findings on the features of amyloid LC fragments come from a recent proteomic study performed by our group, dedicated to the investigation of the termini of these proteoforms [ 87 ] in ex vivo cardiac deposits. In particular, the N- and C-terminal residues of fragments were chemically derivatized, and the labeled residues were identified by LC-MS/MS and bioinformatics [ 87 ].…”

Barriers to Small Molecule Drug Discovery for Systemic Amyloidosis

“…Full-length LCs are more stable in vitro and less prone to aggregation under native-like conditions than their component VL-domains [9,11]. AL fibrils appear to be primarily composed of fragments of LCs, although the nature of this cleavage is unknown and may happen after deposition as amyloid [10,[12][13][14][15][16]. The nascent amyloid fibrils must be stable enough to recruit new protein molecules and grow into mature fibrils, rather than dissociate.…”