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 LC 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 LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.
Highlights
Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins
We have characterized, using MS coupled with derivatization of free N-terminal amino groups and free C-terminal carboxyl groups, the termini of the immunoglobulin light chain (LC) proteoforms deposited as amyloid fibrils in the hearts of two patients affected by AL l amyloidosis, containing deposits of LCs named AL-H7 and AL
With the extensive use of protease inhibitors and in the absence of enzymatic treatments, heart-derived amyloid fibrils are tightly entangled to collagen and the yield of waterextracted material is low, because most fibrils remain in the insoluble pellet
Summary
Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. The presence of the entire variable domain (VL) in the most abundant fragments [7] and the increased amyloidogenic potential of the isolated VL compared with the full-length LCs lead to postulate that VL may have a crucial importance in amyloid fibril formation [14,15,16] This hypothesis has been recently supported by two fibril structures [17, 18], determined by cryo-EM, in which VL was shown to constitute the rigid core of the fibril, which is largely resistant to limited proteolysis. Light chains’ cleavage sites in AL amyloid fibrils hypothesis that proteolysis may largely occur after fibril deposition
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