Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection

Morphological and primary structural consistency of fibrils from different AA patients (common variant)

Aims: To test the hypothesis that the fibril morphology and the fibril protein primary structure are conserved across different patients suffering from the common variant of systemic Amyloid A (AA) amyloidosis. Methods: Amyloid fibrils were extracted from the renal tissue of four patients. The fibril morphology was analysed in negatively stained samples with transmission electron microscopy (TEM). The fibril protein identity and fragment length were determined by using mass spectrometry. Results: The fibrils show a consistent morphology in all four patients and exhibit an average width of similar to 9.6 nm and an average pitch of similar to 112 nm. All fibrils are composed of polypeptide chains that can be assigned to human serum amyloid A (SAA) 1.1 protein. All fragments lack the N-terminal arginine residue and are C-terminally truncated. Differences exist concerning the exact C-terminal cleavage site. The most prominent cleavage site occurs at residues 64-67. Conclusions: Our data demonstrate that AA amyloid fibrils are consistent at the level of the protein primary structure and fibril morphology in the four analysed patients