A specific test for transthyretin 122 (Val----Ile), based on PCR-primer-introduced restriction analysis (PCR-PIRA): confirmation of the gene frequency in blacks.

The variant transthyretin (TTR) allele, TTR (122 Val----Ile), associated with cardiac amyloidosis in blacks, is caused by a G----A transition which destroys a MaeIII site. This variant has previously been detected by PCR around codon 122, followed by MaeIII digestion, but this test is not specific: any of 12 mutations in the MaeIII recognition site, each of which yields a different amino acid change, would also destroy this site. A modification of PCR, termed "PCR-primer-introduced restriction analysis," was used to introduce a new FokI site into the PCR products derived from the variant (122 Ile) but not wild-type (122 Val) allele. This test demonstrated that each of six previously identified MaeIII(-) alleles had lost its MaeIII site because of a G----A transition encoding TTR (122 Val----Ile), confirming that the same TTR variant was present both in 4/177 healthy black individuals and as a homozygous variant in an individual with cardiac amyloidosis.     

Frequency and genetic background of the position 122 (Val----Ile) variant transthyretin gene in the black population.

Transthyretin (TTR) (122 Val----Ile), caused by a point mutation which destroys a MaeIII restriction site, is associated with cardiac amyloidosis in black individuals. To estimate the frequency of the MaeIII(-) gene in the black population without overt cardiac disease, DNA from 177 black individuals without amyloidosis was amplified by the PCR around TTR codon 122 and was digested with MaeIII. The MaeIII(-) gene frequency was 4/354 (1.1%; 95% confidence interval 0.32%2.7%), suggesting that the variant is relatively common in blacks. HLA genotype testing did not suggest that the TTR (122 Val----Ile) heterozygotes were of a closely related genetic background.     

Restriction fragment analysis confirms the position 33 mutation in transthyretin from an Israeli patient (SKO) with familial amyloidotic polyneuropathy

Transthyretin isolated from amyloid fibrils from an Israeli patient with Familial Amyloidotic Polyneuropathy was sequenced by two research groups. One laboratory reported a position 49 Thr----Gly substitution, while the other noted a Phe for Ile interchange at amino acid 33. We used a transthyretin cDNA probe to study DNA from this patient by Southern blotting. The DNA displayed the unique Bcl I restriction site predicted by the mutation in codon 33. Because of the close size of the normal (6.40 kb), and variant (6.27 kb) fragments, the variant was more easily demonstrated after digestion with both Bcl I and Sph I, which generated two easily resolvable fragments of 2.39 and 2.27 kb.     

The modulation of transthyretin tetramer stability by cysteine 10 adducts and the drug diflunisal. Direct analysis by fluorescence-detected analytical ultracentrifugation

Transthyretin (TTR) is normally a stable plasma protein. However, in cases of familial TTR-related amyloidosis and senile systemic amyloidosis (SSA), TTR is deposited as amyloid fibrils, leading to organ dysfunction and possibly death. The mechanism by which TTR undergoes the transition from stable, soluble precursor to insoluble amyloid fibril and the factors that promote this process are largely undetermined. Most models involve the dissociation of the native TTR tetramer as the initial step. It is largely accepted that the TTR gene mutations associated with TTR-related amyloidosis lead to the expression of variant proteins that are intrinsically unstable and prone to aggregation. It has been suggested that amyloidogenicity may be conferred to wild-type TTR (the form deposited in SSA) by chemical modification of the lone cysteine residue (Cys(10)) through mixed disulfide bonds. S-Sulfonation and S-cysteinylation are prevalent TTR modifications physiologically, and studies have suggested their ability to modulate the structure of TTR under denaturing conditions. In the present study, we have used fluorescence-detected sedimentation velocity to determine the effect of S-sulfonate and S-cysteine on the quaternary structural stability of fluorophore-conjugated recombinant TTR under nondenaturing conditions. We determined that S-sulfonation stabilized TTR tetramer stability by a factor of 7, whereas S-cysteinylation enhanced dissociation by 2-fold with respect to the unmodified form. In addition, we report the direct observation of tetramer stabilization by the potential therapeutic compound diflunisal. Finally, as proof of concept, we report the sedimentation of TTR in serum and the qualitative assessment of the resulting data.     

Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis

Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to improve our understanding of the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria: dissociation of tetramers into folded monomers and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by globally fitting them to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild-type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to those of WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR, it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stabilities. The observed differences in stability among the disease-associated TTR variants highlight the complexity and heterogeneity of TTR amyloid disease, an observation that has important implications for the treatment of these maladies.     

Identification of S-sulfonation and S-thiolation of a novel transthyretin Phe33Cys variant from a patient diagnosed with familial transthyretin amyloidosis

Familial transthyretin amyloidosis (ATTR) is an autosomal dominant disorder associated with a variant form of the plasma carrier protein transthyretin (TTR). Amyloid fibrils consisting of variant TTR, wild-type TTR, and TTR fragments deposit in tissues and organs. The diagnosis of ATTR relies on the identification of pathologic TTR variants in plasma of symptomatic individuals who have biopsy proven amyloid disease. Previously, we have developed a mass spectrometry-based approach, in combination with direct DNA sequence analysis, to fully identify TTR variants. Our methodology uses immunoprecipitation to isolate TTR from serum, and electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry (MS) peptide mapping to identify TTR variants and posttranslational modifications. Unambiguous identification of the amino acid substitution is performed using tandem MS (MS/MS) analysis and confirmed by direct DNA sequence analysis. The MS and MS/MS analyses also yield information about posttranslational modifications. Using this approach, we have recently identified a novel pathologic TTR variant. This variant has an amino acid substitution (Phe --> Cys) at position 33. In addition, like the Cys10 present in the wild type and in this variant, the Cys33 residue was both S-sulfonated and S-thiolated (conjugated to cysteine, cysteinylglycine, and glutathione). These adducts may play a role in the TTR fibrillogenesis.     

Transthyretin fibrillogenesis entails the assembly of monomers: a molecular model for in vitro assembled transthyretin amyloid-like fibrils

Extracellular accumulation of transthyretin (TTR) variants in the form of fibrillar amyloid deposits is the pathological hallmark of familial amyloidotic polyneuropathy (FAP). The TTR Leu55Pro variant occurs in the most aggressive forms of this disease. Inhibition of TTR wild-type (WT) and particularly TTR Leu55Pro fibril formation is of interest as a potential therapeutic strategy and requires a thorough understanding of the fibril assembly mechanism. To this end, we report on the in vitro assembly properties as observed by transmission electron microscopy (TEM), atomic force microscopy (AFM) and quantitative scanning transmission electron microscopy (STEM) for both TTR WT fibrils produced by acidification, and TTR Leu55Pro fibrils assembled at physiological pH. The morphological features and dimensions of TTR WT and TTR Leu55Pro fibrils were similar, with up to 300 nm long, 8 nm wide fibrils being the most prominent species in both cases. Other species were evident; 4-5 nm wide fibrils, 9-10 nm wide fibrils and oligomers of various sizes. STEM mass-per-length (MPL) measurements revealed discrete fibril types with masses of 9.5 and 14.0(+/-1.4) KDa/nm for TTR WT fibrils and 13.7, 18.5 and 23.2(+/-1.5) kDa/nm for TTR Leu55Pro fibrils. These MPL values are consistent with a model in which fibrillar TTR structures are composed of two, three, four or five elementary protofilaments, with each protofilament being a vertical stack of structurally modified TTR monomers assembled with the 2.9 nm axial monomer-monomer spacing indicated by X-ray fibre diffraction data. Ex vivo TTR amyloid fibrils were examined. From their morphological appearance compared to these, the in vitro assembled TTR WT and Leu55Pro fibrils examined may represent immature fibrillar species. The in vitro system operating at physiological pH for TTR Leu55Pro and the model presented for the molecular arrangement of TTR monomers within fibrils may, therefore, describe early fibril assembly events in vivo.     

Production of recombinant human transthyretin with biological activities toward the understanding of the molecular basis of familial amyloidotic polyneuropathy (FAP).

Transthyretin (TTR) is a plasma protein interacting with thyroxine T4 and retinol binding protein (RBP). Several variants of TTR with single amino acid substitutions have been identified as the major components of the amyloid fibrils of familial amyloidotic polyneuropathy (FAP), a fetal, autosomal dominant genetic disease. The elucidation of the molecular nature of the variants distinct from that of the wild-type TTR is crucial for understanding the amyloidogenesis in FAP, but our understanding is very poor mainly because of the unavailability of pure variant TTRs. In the present study, we used an Escherichia coli OmpA secretion vector (Ghrayeb et al., 1984) and achieved an effective production of the variant TTRs related to FAP including Met-30, Ile-33, Ala-60, Tyr-77, Met-111, and Ile-122 types. The variant TTRs produced in this system were efficiently secreted to the culture media. The chemical analysis showed that the secreted TTR (Met-30 type) has the same N-terminus as the native one. IEF analyses also indicated that the secreted product is properly processed as assessed by its pI. Furthermore, the secreted TTR was shown to have biological activities, namely, the thyroxin binding activity and the ability to associate with retinol binding protein, indicating that the secreted TTR polypeptide is properly folded. The present work also demonstrated that the processing/secretion of the recombinant TTR molecules in E. coli was strongly affected by single amino acid substitutions.     

Cys10 mixed disulfides make transthyretin more amyloidogenic under mildly acidic conditions

Conservative mutation of transthyretin's surface residues can predispose an individual to familial amyloidosis by dramatically changing the energetics of misfolding. Senile systemic amyloidosis (SSA), however, cannot be explained in this fashion because wild-type (WT) transthyretin (TTR) misfolds and misassembles into amyloid. Since various modifications of the SH functionality of Cys10 have been reported in humans, we sought to understand the extent to which these modifications alter the stability and amyloidosis of WT TTR as a possible explanation for SSA. Homotetrameric Cys10 TTR variants, including TTR-Cys, TTR-GSH, TTR-CysGly, and S-sulfonated TTR, were chemically synthesized starting with WT TTR. The TTR-Cys, TTR-GSH, and TTR-CysGly isoforms are more amyloidogenic than WT at the higher end of the acidic pH range (pH 4.4-5.0), and they are similarly destabilized relative to WT TTR toward urea denaturation. They exhibit rates of urea-mediated tetramer dissociation (pH 7) and MeOH-facilitated fibril formation similar to those of WT TTR. Under mildly acidic conditions (pH 4.8), the amyloidogenesis rates of the mixed disulfide TTR variants are much faster than the WT rate. S-Sulfonated TTR is less amyloidogenic and forms fibrils more slowly than WT under acidic conditions, yet it exhibits a stability and rates of tetramer dissociation similar to those of WT TTR when subjected to urea denaturation. Conversion of the Cys10 SH group to a mixed disulfide with the amino acid Cys, the CysGly peptide, or glutathione increases amyloidogenicity and the amyloidogenesis rate above pH 4.6, conditions under which TTR probably forms fibrils in humans. Hence, these modifications may play an important role in human amyloidosis.     

Revised transthyretin Ile 122 allele frequency in African-Americans

The transthyretin (TTR) Ile 122 variant is associated with cardiac amyloidosis in individuals of African descent. To determine the prevalence of the allele encoding TTR Ile 122 in African-Americans, we have used PCR and restriction analysis to test DNA from African-Americans from various geographic areas, and found an allele frequency of 66/3376 (0.020), which is higher than the value we previously reported in a much smaller pilot study. Our data indicate that this TTR variant is present at equal frequency in African-Americans throughout the U.S., and suggest that this mutation may be a common, often unrecognized cause of cardiac disease in African-Americans. Received: 23 January 1996     

Structure and assembly-disassembly properties of wild-type transthyretin amyloid protofibrils observed with atomic force microscopy

Transthyretin (TTR) is an important human transport protein present in the serum and the cerebrospinal fluid. Aggregation of TTR in the form of amyloid fibrils is associated with neurodegeneration, but the mechanisms of cytotoxicity are likely to stem from the presence of intermediate assembly states. Characterization of these intermediate species is therefore essential to understand the etiology and pathogenesis of TTR-related amyloidoses. In the present work we used atomic force microscopy to investigate the morphological features of wild-type (WT) TTR amyloid protofibrils that appear in the early stages of aggregation. TTR protofibrils obtained by mild acidification appeared as flexible filaments with variable length and were able to bind amyloid markers (thioflavin T and Congo red). Surface topology and contour-length distribution displayed a periodic pattern of ～ 15 nm, suggesting that the protofibrils assemble via an end-binding oligomer fusion mechanism. The average height and periodic substructure found in protofibrils is compatible with the double-helical model of the TTR amyloid protofilament. Over time protofibrils aggregated into bundles and did not form mature amyloid-like fibrils. Unlike amyloid fibrils that are typically stable under physiological conditions, the bundles dissociated into component protofibrils with axially compacted and radially dilated structure when exposed to phosphate-buffered saline solution. Thus, WT TTR can form metastable filamentous aggregates that may represent an important transient state along the pathway towards the formation of cytotoxic TTR species.     

Identification of a new transthyretin variant (Ile49) in familial amyloidotic polyneuropathy using electrospray ionization mass spectrometry and nonisotopic RNase cleavage assay.

Mutation of the transthyretin (TTR) plasma protein and gene in a Japanese patient with amyloid polyneuropathy was investigated by electrospray ionization mass spectrometry (ESI-MS) and nonisotopic RNase cleavage assay (NIRCA), respectively. ESI-MS analysis showed normal TTR peaks and additionally a variant TTR with 12-dalton-higher molecular weight than normal TTR. NIRCA suggested that the mutation existed near either the 5' or 3' end of exon 3. Direct DNA sequencing revealed both a normal ACC (threonine) and a variant ATC (isoleucine) at codon 49, which was located near the 5' end of exon 3. The molecular weight shift of this mutation was 12 D, consistent with the result of ESI-MS.     

A new transthyretin mutation associated with amyloid cardiomyopathy.

In transthyretin (TTR) a new mutation (TTR-Thr45) has been identified in a patient with familial amyloidosis characterized clinically by prominent cardiomyopathy and the absence of peripheral neuropathy. Comparative peptide mapping by high-performance liquid chromatography of the patient's plasma TTR together with normal TTR showed the presence of an abnormal tryptic peptide in the patient's TTR. The sequence of this peptide (peptide 6, residues 36-48) demonstrated the presence of a threonine-for-alanine substitution at position 45. This change can be explained by a single base change of adenine for guanine in the Ala-45 codon and was demonstrated directly by DNA sequence analysis of PCR-amplified exon 2 of the TTR gene; allele-specific oligonucleotide hybridization both in the patient and in fixed heart tissue from his aunt confirmed the base change. The TTR-Thr45 mutation is a new variant TTR found associated with cardiomyopathy.     

Tetramer formation of a variant type human transthyretin (prealbumin) produced by Escherichia coli expression system

A variant of human transthyretin(TTR, prealbumin) with methionine for valine substitution at position 30 is a major component of amyloid fibrils found in patients of familial amyloidotic polyneuropathy(FAP) type I, an autosomal dominant genetic disease. But the molecular nature of the variant TTR has been obscure, because most of plasma TTR from FAP patients is a mixture of variant and wild type TTR and no pure preparation of the variant has been available. For this reason, we constructed a system in which the variant type TTR was efficiently synthesized. In this system, the recombinant variant TTR was first synthesized as a fusion protein with E. coli outer membrane protein A (ompA) signal peptide, processed to eliminate the signal peptide and finally secreted to the culture medium. The final concentration of the recombinant variant TTR in the medium was about 5 mg/l. SDS polyacrylamide gel electrophoresis and gel filtration analysis suggested that the recombinant variant TTR can form tetramer as seen for native one. Purification of the protein was accomplished by only two steps of chromatography.     

Presence of N‐glycosylated transthyretin in plasma of V30M carriers in familial amyloidotic polyneuropathy: an escape from ERAD

Familial amyloid polyneuropathy (FAP) is an autosomal dominant disease characterized by deposition of amyloid related to the presence of mutations in the transthyretin (TTR) gene. TTR is mainly synthesized in liver, choroid plexuses of brain and pancreas and secreted to plasma and cerebrospinal fluid (CSF). Although it possesses a sequon for N‐glycosylation N‐D‐S at position 98, it is not secreted as a glycoprotein. The most common FAP‐associated mutation is TTR V30M. In a screening for monoclonal antibodies developed against an amyloidogenic TTR form, we detected a distinct TTR with slower electrophoretic mobility in Western of plasma from carriers of the V30M mutation, not present in normal plasma. Mass spectrometry analyses of this slower migrating TTR (SMT) identified both wild‐type and mutant V30M; SMT was undetectable upon N‐glycosidase F treatment. Furthermore, SMT readily disappeared in the plasma of V30M ‐ FAP patients after liver transplantation and appeared in plasma of transplanted domino individuals that received a V30M liver. SMT was also detected in plasma, but not in CSF of transgenic mice for the human V30M mutation. A hepatoma cell line transduced to express human V30M did not present the SMT modification in secretion media. Glycosylated TTR was absent in fibrils extracted from human kidney V30M autopsy tissue or in TTR aggregates extracted from the intestine of human TTR transgenic mice. Studies on the metabolism of this novel, glycosylated TTR secreted from FAP liver are warranted to provide new mechanisms in protein quality control and etiopathogenesis of the disease.     

Molecular diagnosis of the transthyretin (TTR) Met111 mutation in familial amyloid cardiomyopathy of Danish origin

Summary Familial amyloid cardiomyopathy in a Danish kindred is associated with a specific mutation (Met for Leu¹¹¹) in the transthyretin (TTR) gene, causing the loss of a recognition site for the restriction enzyme DdeI in the gene. We describe a diagnostic test for the molecular detection of this mutation. A sequence of the TTR gene containing the mutation was amplified by the polymerase chain reaction from isolated genomic DNA of two affected patients and several controls. DdeI digestion of the amplified DNA from the patients revealed 3 bands by gel-electrophoresis, whereas amplified DNA of the controls showed only 2 bands, consistent with complete digestion. Thus, the assumed heterozygous TTR Met¹¹¹ mutation was confirmed in the affected patients.     

A novel transthyretin mutation at position 30 (Leu for Val) associated with familial amyloidotic polyneuropathy.

A novel transthyretin (TTR) mutation associated with familial amyloidotic polyneuropathy was detected in a Japanese patient. Single-strand conformation polymorphism analysis and sequence analysis of polymerase chain reaction (PCR)-amplified exons of the patient's TTR gene revealed a point mutation resulting in a substitution of leucine for valine at position 30. As the mutation creates a Cfr13I site, it was confirmed by PCR and restriction analysis. Our finding indicates the importance of position 30 in TTR-derived amyloid fibril formation.     

Potentially amyloidogenic conformational intermediates populate the unfolding landscape of transthyretin: Insights from molecular dynamics simulations

Protein aggregation into insoluble fibrillar structures known as amyloid characterizes several neurodegenerative diseases, including Alzheimer's, Huntington's and Creutzfeldt‐Jakob. Transthyretin (TTR), a homotetrameric plasma protein, is known to be the causative agent of amyloid pathologies such as FAP (familial amyloid polyneuropathy), FAC (familial amyloid cardiomiopathy) and SSA (senile systemic amyloidosis). It is generally accepted that TTR tetramer dissociation and monomer partial unfolding precedes amyloid fibril formation. To explore the TTR unfolding landscape and to identify potential intermediate conformations with high tendency for amyloid formation, we have performed molecular dynamics unfolding simulations of WT‐TTR and L55P‐TTR, a highly amyloidogenic TTR variant. Our simulations in explicit water allow the identification of events that clearly discriminate the unfolding behavior of WT and L55P‐TTR. Analysis of the simulation trajectories show that (i) the L55P monomers unfold earlier and to a larger extent than the WT; (ii) the single α‐helix in the TTR monomer completely unfolds in most of the L55P simulations while remain folded in WT simulations; (iii) L55P forms, early in the simulations, aggregation‐prone conformations characterized by full displacement of strands C and D from the main β‐sandwich core of the monomer; (iv) L55P shows, late in the simulations, severe loss of the H‐bond network and consequent destabilization of the CBEF β‐sheet of the β‐sandwich; (v) WT forms aggregation‐compatible conformations only late in the simulations and upon extensive unfolding of the monomer. These results clearly show that, in comparison with WT, L55P‐TTR does present a much higher probability of forming transient conformations compatible with aggregation and amyloid formation.     

Search for intermediate structures in transthyretin fibrillogenesis: soluble tetrameric Tyr78Phe TTR expresses a specific epitope present only in amyloid fibrils

Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of TTR into an insoluble beta-sheet. The TTR tetramer is thought to dissociate into monomeric intermediates and subsequently polymerise into the pathogenic amyloid form. The biochemical mechanism behind this transformation is unknown. We characterised intermediate TTR structures in the in vitro amyloidogenesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the TTR tetrameric fold, based on the known crystallographic structure of a Leu55Pro transthyretin variant. We generated a soluble tetrameric form of TTR that is recognised by a monoclonal antibody, previously reported to react only with highly amyloidogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M(-1). We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway.     

Cardiac amyloid in patients with familial amyloid polyneuropathy consists of abundant wild-type transthyretin

Patients with familial amyloid polyneuropathy (FAP) are now cured by liver transplantation, but cardiac amyloidosis would further progress even after liver transplantation in some patients. To clarify the pathological mechanism of the progress of cardiac amyloidosis in FAP, we investigated cardiac tissues obtained from 6 FAP patients with 3 different types of TTR mutations. One of them had undergone liver transplantation and one year later died of cardiac amyloidosis. We determined clinical severity of cardiac involvement of those patients and characterized amyloid fibril proteins depositing in their cardiac muscles by immunohistochemistry, mass spectrometry and isoelectric focusing. All the patients had cardiac dysfunction and increased cardiac weight. Diffuse deposition of TTR-related amyloid was seen in their myocardium on microscopic examination. Amyloid fibrils of the heart were composed of wild-type TTR as well as variant TTR at a ratio of about 1:1 in 5 patients without liver transplantation. In the patient with a transplanted liver, about 80% of the cardiac amyloid consisted of wild-type TTR. Wild-type TTR contributes greatly to the development of amyloid deposition in the heart of FAP patients regardless of the types of TTR mutations.