title: Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease.
abstract: 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) not build up in concentration in this variant. The intermediate is undoubtedly forming in the Thr-119-Met variant, as it will form amyloid fibrils at high concentrations; however, the intermediate is only present at a low steady-state concentration which makes it difficult to detect.
authors.affiliation: Department of Chemistry, Texas A&M University, College Station 77843, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: McCutchen
authors.forename: Z
authors.initials: Z
authors.lastname: Lai
authors.forename: G J
authors.initials: GJ
authors.lastname: Miroy
authors.forename: J W
authors.initials: JW
authors.lastname: Kelly
authors.forename: W
authors.initials: W
authors.lastname: Colón
mesh_terms: D000595:Amino Acid Sequence; D000686:Amyloidosis / Q000235:genetics*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005804:Genes, Lethal; D014644:Genetic Variation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007930:Leucine; D008247:Lysosomes / Q000378:metabolism; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011228:Prealbumin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011392:Proline; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D017931:DNA Primers; D046911:Macromolecular Substances; D011228:Prealbumin; D011994:Recombinant Proteins; D011392:Proline; D007930:Leucine