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.

Characterization of the Transthyretin Acid Denaturation Pathways by Analytical Ultracentrifugation: Implications for Wild-Type, V30M, and L55P Amyloid Fibril Formation

“…The faster the rate of tetramer dissociation is relative to the rates governing the assembly of the monomeric amyloidogenic intermediate, the easier intermediate detection is. The importance of the formation of the monomeric amyloidogenic intermediate is also consistent with earlier data from our laboratory showing that several of the single site amyloidogenic variants that cause FAP behave similarly, in that they destabilize tetrameric TTR in favor of the monomeric amyloidogenic intermediate (23,24). Ultracentrifuge data described within also imply that the rate of formation of the monomeric amyloidogenic intermediate for L55P is fast relative to the assembly rate under acidic conditions; hence, a ladder of quaternary structural intermediates are prevalent and easily observed for L55P by sedimentation velocity studies, Figure 6A,B.…”

“…The activation barriers for the formation of the monomeric amyloidogenic intermediate appear to increase in the order of L55P < V30M < wild-type TTR, employing either acid or thermal denaturation on the basis of time-dependent analysis (23,24,29). These results are strictly consistent with recent kinetic GdnHCl-mediated denaturation studies of wild-type, V30M, and L55P TTR, demonstrating that V30M and L55P denature more quickly than wild-type TTR (30,31).…”

“…The L55P and V30M FAP-associated TTR variants, like wild-type TTR, exhibit similar amyloid fibril formation at pH 4.4, as discerned by light scattering and congo red binding (23,24). Sedimentation velocity analysis of L55P TTR samples (0.2 mg/mL incubated at 25°C or 37°C for 2 days at pH 4.4) exhibit extensive fibril formation at both temperatures as discerned by rapid sedimentation of the amyloid component of the sample (80-90% conversion of soluble L55P into amyloid at 37°C).…”

“…Normally folded wild-type, V30M, and L55P TTR can be converted into amyloid fibrils either by partial acid denaturation or during pH-mediated reconstitution (refolding), implying that an intermediate on the denaturation/ reconstitution pathway is the amyloidogenic intermediate that self-assembles into amyloid fibrils (23,24,26,29). Earlier data from our laboratory show that the V30M and L55P variants can form amyloid fibrils under mildly acidic conditions (e.g.…”

Lipid Binding to Amyloid β‐Peptide Aggregates: Preferential Binding of Cholesterol as Compared with Phosphatidylcholine and Fatty Acids

“…However, it has not been established as to how Aß produces its toxicity. Aß belongs to a group of proteins that can aggregate and make fibrils (McCutchen et al, 1995;Kelly, 1996). One of those proteins, serum amyloid A, has been shown to bind cholesterol (Banka et al, 1995;Liang and Sipe, 1995).…”