TRANSTHYRETIN MUTATION LEU-55-PRO SIGNIFICANTLY ALTERS TETRAMER STABILITY AND INCREASES AMYLOIDOGENICITY

A recently reported variant of human transthyretin (TTR), Leu-55-Pro, implicated as the causative agent in early-onset familial amyloid polyneuropathy was expressed and characterized, and its denaturation pathway and amyloidogenicity were compared to those of wild-type transthyretin. The overlap-extension polymerase chain reaction (PCR) methodology was used to introduce the Leu-55-Pro mutation into the transthyretin DNA sequence and to construct a new expression system. The Leu-55-Pro variant of transthyretin was expressed with a leader sequence to ensure secretion into the periplasmic space of Escherichia coli. Transthyretin's resistance to sodium dodecyl sulfate- (SDS-) induced denaturation was utilized to measure the quaternary stability as a function of pH employing SDS-polyacrylamide gel electrophoresis (PAGE) in the presence and absence of an amyloid fibril inhibitor, Z 3-14. These studies reveal that the Leu-55-Pro TTR tetramer is significantly less stable than wild-type TTR. This is relevant because we have previously shown that the partial denaturation of transthyretin is sufficient to effect amyloid fibril formation from a denaturation intermediate which may be a structured monomer. The ability of Leu-55-Pro TTR to denature to the amyloidogenic intermediate at pHs where the wild-type protein is stable may explain the variant's propensity to form amyloid fibrils in vitro and in vivo where the wild-type protein remains stable and nonamyloidogenic. Congo red binding, polarized light microscopy, and electron microscopy confirm the characteristic structure of amyloid fibrils produced from Leu-55-Pro TTR in vitro. The instability of the Leu-55-Pro tetramer and the capability of this protein to form amyloid fibrils at pHs close to 5.5 suggest an explanation for the extreme pathogenicity of this variant. Several lines of evidence suggest that lysosomes may be the source of amyloid fibril formation in vivo. It is interesting and potentially physiologically relevant that Leu-55-Pro TTR forms amyloid fibrils at the normal operating pH of a lysosome.