The "edge strand" hypothesis: Prediction and test of a mutational "hot-spot" on the transthyretin molecule associated with FAP amyloidogenesis

The amyloid fibrils of familial amyloidotic polyneuropathy (FAP) are composed of genetic variants of transthyretin (TTR). Over 40 amino-acid variants have been identified associated with amyloidosis. We have performed an analysis of the effect of the different substitutions, such as location and changes in side-chain volume, hydrophilicity and charge on the TTR molecule. We have also analyzed the distribution of variants along the peptide chain. While we could demonstrate no pattern of change associated with the bulk properties of the amyloidogenic variant residues, the distribution analysis suggested a significant correlation with the position of variant residues along the polypeptide chain. We identified a significant peak in the distribution of variants which, in the folded protein chain, was associated with the edge strands (residues 45-58) of the two ß-sheets that form the structural framework of the TTR molecule. In contrast, the regions of the molecule associated with the subunit interactions are almost devoid of amyloidogenic variants. To test this finding, we constructed two variant TTRs using recombinant techniques, one with a triple substitution and one with a triple deletion in the edge strand region. On expression, the variants spontaneously formed material that produced a characteristic redshift upon Congo red staining, was composed of fibrils 80-110Å in diameter and gave ß-structure X-ray patterns suggesting the formation of amyloid fibrils. We conclude that the effect of amyloidogenic variants may be to alter the structure or stability of the edge strands of the ß-sheets of TTR, thus promoting the growth of inter-molecular ß-sheet structures characteristic of amyloid.