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.

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.

Systemic AA-amyloidosis is a protein-misfolding disease that is characterized by fibril deposition of serum amyloid-A protein (SAA) in several organs in humans and many animal species. Fibril deposits originate from abnormally high serum levels of SAA during chronic inflammation. In domestic short-hair cats, AA-amyloidosis has only been anecdotally reported and is considered a rare disease. Here we report that an astonishing 57-73% of early deceased short-hair cats kept in three independent shelters suffer from amyloid deposition in the liver, spleen, or kidney. Histopathology and mass spectrometry of post-mortem extracted deposits identified SAA as the major protein source. The duration of stay in the shelters was positively associated with a histological score of AA-amyloidosis (B=0.026, CI95%=0.007-0.046; p=0.010). Presence of SAA fragments in bile secretions raises the possibility of fecal-oral transmission of the disease.

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B cell/plasma cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood.Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis.Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the Nglycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs.Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies.