Comparison of Lethal and Nonlethal Transthyretin Variants and Their Relationship to Amyloid Disease

• Published in: Biochemistry (Easton) 1995-10, Vol.34 (41), p.13527-13536
• Main Authors: McCutchen, Sandra L., Lai, Zhihong, Miroy, Greta J., Kelly, Jeffery W., Colon, Wilfredo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amyloidosis - genetics; Base Sequence; DNA Primers; Electrophoresis, Polyacrylamide Gel; Genes, Lethal; Genetic Variation; Humans; Hydrogen-Ion Concentration; Kinetics; Leucine; Lysosomes - metabolism; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Prealbumin - chemistry; Prealbumin - genetics; Prealbumin - metabolism; Proline; Protein Denaturation; Protein Structure, Secondary; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Structure-Activity Relationship
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00041a032

The role that transthyretin (TTR) mutations play in the amyloid disease familial amyloid polyneuropathy (FAP) has been probed by comparing the biophysical properties of several TTR variants as a function of pH. We have previously demonstrated that the partial acid denaturation of TTR is sufficient to effect amyloid fibril formation by self-assembly of a denaturation intermediate which appears to be monomeric. Earlier studies on the most pathogenic FAP variant known, Leu-55-Pro, revealed that this variant is much less stable toward acid denaturation than wild-type TTR, apparently explaining why this variant can form amyloid fibrils under mildly acidic conditions where wild-type TTR remains nonamyloidogenic. The hypothesis that FAP mutations destabilize the TTR tetramer in favor of a monomeric amyloidogenic intermediate under lysosomal (acidic) conditions is further supported by the data described here. We compare the acid stability and amyloidogenicity of the most prevalent FAP variant, Val-30-Met, along with the double mutant, Val-30-Met/Thr-119-Met, which serves to model the effects of these mutations in heterozygous patients where the mutations are in different subunits. In addition, we have characterized the Thr-119-Met TTR variant, which is a common nonpathogenic variant in the Portuguese population, to further investigate the role that this mutation plays in protecting individuals who also carry the Val-30-Met mutation against the classically severe FAP pathology. This biophysical study demonstrates that Val-30-Met TTR is significantly less stable toward acid denaturation and more amyloidogenic than wild-type TTR, which in turn is less stable and more amyloidogenic than Thr-119-Met TTR. Interestingly, the double mutant Val-30-Met/Thr-119-Met is very similar to wild-type TTR in terms of its stability toward acid denaturation and its amyloidogenicity. The data suggest that the Thr-119-Met mutation confers decreased amyloidogenicity by stabilizing tetrameric TTR toward acid denaturation. In addition, fluorescence studies monitoring the acid-mediated denaturation pathways of several TTR variants reveal that the majority exhibit a plateau in the relative fluorescence intensity over the amyloid-forming pH range, i.e., ca. pH 4.3-3.3. This intensity plateau suggests that the amyloidogenic intermediate(s) is (are) being observed over this pH range. The Thr-119-Met variant does not exhibit this plateau presumably because the amyloidogenic intermediate(s) do(es