Comparison of lethal and nonlethal transthyretin variants and their relationship to amyloid disease

Biochemistry. 1995 Oct 17;34(41):13527-36. doi: 10.1021/bi00041a032.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 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

Substances

  • DNA Primers
  • Macromolecular Substances
  • Prealbumin
  • Recombinant Proteins
  • Proline
  • Leucine