Background: We have recently shown that the actin-modulating cytoskeletal and plasma protein gelsolin is involved in the pathogenesis of familial amyloidosis of Finnish type. To define the amyloidogenic region(s) in gelsolin and clarify the mechanisms involved in amyloid formation, we tested the amyloidogenicity of synthetic gelsolin peptide analogues.
Experimental design: The in vitro amyloid fibril formation was studied using 22 synthetic peptides 7 to 30 residues long having sequence homology with wild-type or mutant gelsolins. Amyloid formation was monitored by Congo-red staining and polarization microscopy of the peptide aggregates, by negative staining electron microscopy, and by quantitative fluorometry with thioflavine T.
Results: Ultrastructurally, amyloid-like fibrils were formed from the mutant Asn-187 and Tyr-187 gelsolin peptides corresponding to the naturally occurring missense mutations found in familial gelsolin amyloidosis syndromes, as well as from a gelsolin peptide having a Val-187 substitution. The shortest peptide tested that was capable of forming amyloid-like fibrils was 9-residue mutant Asn-187 peptide. The corresponding wild-type peptide did not form amyloid. Quantitative fluorometry at the emission maximum 482 nm revealed highly accelerated amyloid fibril formation of the mutant Asn-187, Tyr-187 and Val-187 peptides as compared with the corresponding wild-type peptides.
Conclusions: We have defined the amyloidogenic region of gelsolin to a 9-residue sequence in the highly conserved repetitive motif B and showed that residue 187 represents a critical site where a substitution of an amino acid with a charged side chain (Asp) with an amino acid with an uncharged (Asn) or hydrophobic side chain (Tyr, Val) creates a conformation that is highly amyloidogenic thus providing an explanation for the amyloidogenicity of the Asn-187 and Tyr-187 gelsolin variants.