CHARACTERIZATION OF 2 HIGHLY AMYLOIDOGENIC MUTANTS OF TRANSTHYRETIN

The plasma protein transthyretin (TTR) has the potential to form amylo id under certain conditions, More than 50 different point mutations ha ve been associated with amyloid formation that occurs only in adults. It is not known what structural changes are introduced into the struct ure of this otherwise stable molecule that results in its aggregation into insoluble amyloid fibrils. On the basis of calculations of the fr equency of known mutations over the polypeptide, we have constructed t wo mutants in the D-strand of the polypeptide. These molecules, contai ning either a deletion or a substitution at amino acid positions 53-55 , were unstable and spontaneously formed aggregates upon storage in TB S (pH 7.6). The precipitates were shown to be amyloid by staining with thioflavin T and Congo Red. Their ultrastructure was very similar to that of amyloid fibrils deposited in the vitreous body of patients wit h familial amyloidotic polyneuropathy type 1 with an amino acid replac ement in position 30 (TTRmet30), Like amyloid isolated from the vitreo us body of the eye, the amyloid precipitates generated from the TTR mu tants exposed a trypsin cleavage site between amino acid residues 48 a nd 49, while plasma TTRmet30 isolated from amyloidosis patients as wel l as wild-type TTR only showed minor trypsin sensitivity. Our data ind icate that the mutants we have constructed are similar to amyloid prec ursors or may share structural properties with intermediates on a path way leading to amyloid deposits of plasma TTR.