Radioimmunoassay for detecting abnormal prealbumin in the serum for diagnosis of familial amyloidotic polyneuropathy (Japanese type)

In the serum of a Japanese patient with familial amyloidotic polyneuropathy (FAP), we demonstrated the presence of a prealbumin variant having a single amino acid substitution of a methionine residue for a valine at position 30. We have developed a highly sensitive and specific method for quantitative analysis of the prealbumin variant in the sera of FAP patients by using radioimmunoassay for a nonapeptide corresponding to subsequence [22-30] of the prealbumin variant. This peptide is produced from the prealbumin variant by cyanogen bromide cleavage followed by tryptic digestion. The serum concentration of the prealbumin variant in five Japanese FAP patients ranges from 4.0 mg/dl to 7.8 mg/dl, which is 100 times or even higher than normal controls. This method should be helpful for an early diagnosis of this hereditary disease.     

Identification of carriers of mutant prealbumin gene associated with familial amyloidotic polyneuropathy type I by Southern blot procedures: study of six pedigrees in the Arao district of Japan

Summary Fifty-six Japanese individuals from six pedigrees with familial amyloidotic polyneuropathy (FAP), together with 2 individuals with symptomatic FAP from an unknown pedigree were analyzed, using the Southern blot procedures for the prealbumin gene structure. A human prealbumin cDNA was used as the probe. Altogether, these individuals included 20 with symptomatic FAP, 30 who were asymptomatic, and 8 disease-free spouses. Twenty individuals with symptomatic FAP were all heterozygous for the prealbumin genes, carrying one normal and one mutant gene. We confirmed the direct linkage between the mutant prealbumin gene and the Japanese type of FAP. Moreover, 10 of the 30 asymptomatic individuals from pedigrees with FAP were also heterozygous for the prealbumin gene. The number of asymptomatic individuals with the mutant prealbumin gene showed age-related decreases, and none was over 40 years. A linkage between the mutant prealbumin gene and serum levels of the prealbumin variant was also evident.     

Role of variant prealbumin in the pathogenesis of familial amyloidotic polyneuropathy: fate of normal and variant prealbumin in the circulation

According to recent studies on protein chemistry and genetic engineering, replacement of the Val30 residue of prealbumin by methionine is believed to play a critical role in the formation of amyloid deposit and the pathogenesis of familial amyloidotic polyneuropathy (FAP). However, only limited information is available concerning the behavior of prealbumin in the circulation. To obtain the molecular insight into the mechanism of amyloid deposition, it is indispensable to know the fates of normal and variant prealbumin in vivo. Thus, the fates of prealbumin samples from normal and FAP patients were studied in normal rats as well as in animals that were challenged with acute inflammation induced by turpentine. The effect of in vitro photooxidation of prealbumin samples on their behavior was also examined in vivo. Kinetic analysis revealed no appreciable difference between prealbumin samples from normal and FAP patients. These results suggest that factors other than the rate of transfer of the variant form prealbumin from plasma to an extravascular compartment may play a critical role in the pathogenesis of amyloid deposition in FAP patients.     

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.     

Identification of amyloid prealbumin variant in familial amyloidotic polyneuropathy (Japanese type)

Structural studies on an amyloid fibril protein of 14 K daltons (AFj(INO] isolated from a Japanese patient who suffered from familial amyloidotic polyneuropathy were carried out to unambiguously identify its difference from normal human serum prealbumin. Sequence analyses performed by comparing peptide maps prepared from cyanogen bromide fragments and tryptic peptides of purified RCM-amyloid protein with those from RCM-prealbumin indicate that only a valine residue at position 30 in prealbumin is replaced by a methionine residue. Furthermore, it was also proved that AFj(INO) consists of four components; the prealbumin variant and its three related proteins, which are derived by successively accumulated deletion of the N-terminal three amino acid residues (Gly1, Pro2 and Thr3) from the prealbumin variant.     

Genetic basis for familial amyloidotic polyneuropathy

Familial amyloidotic polyneuropathy (FAP) is an inherited systemic amyloidosis, characterized by the extracellular deposition of fibrillar amyloid protein, i.e. a variant type of prealbumin, and by prominent peripheral nerve involvement. We recently established the basis of FAP, using a cloned human prealbumin cDNA, restriction endonuclease(s) and Southern blot procedures. This approach clearly revealed a direct link between mutation in the prealbumin gene and FAP; individuals with FAP are heterozygous for the prealbumin gene, carrying one normal and one mutant gene. Molecular analysis of the prealbumin gene yielded pertinent data on the genetic basis for FAP.     

Haplotype analysis of familial amyloidotic polyneuropathy

Summary Familial amyloidotic polyneuropathy (FAP) is an autosomal dominant genetic disease characterized by systemic accumulation of amyloid fibrils. A major component of FAP anyloid has been identified as variant transthyretin (TTR, also called prealbumin). In particular, a variant with the substitution ³⁰ValMet has been commonly found in FAP of various ethnic groups. To understand the origin and spread of the ValMet mutation, we analyzed DNA polymorphisms associated with the TTR gene in six Japanese FAP families and several Portuguese FAP patients. Three distinct haplotypes associated with the ValMet mutation were identified in Japanese FAP families, one of which was also found in Portuguese patients. On the other hand, it was found that the ValMet mutation can be explained by a C-T transition at the C_pG dinucleotide sequence of a mutation hot spot. Thus, our findings indicate that the ValMet mutation has probably recurred in the human population, to generate FAP families of independent origin.     

Evidence that the amyloid fibril protein in senile systemic amyloidosis is derived from normal prealbumin

Familial amyloidosis in different kindreds is associated with a variety of point mutations in the prealbumin gene, resulting in prealbumin variants which are believed to be amyloidogenic, i.e. prone to form amyloid fibrils. In the most common amyloid-associated variant, there is a methionine for valine substitution in position 30. We have studied the prealbumin-derived amyloid protein ASc1 in the common age-related senile systemic amyloidosis. Evidence is presented that there is no abnormality in the primary structure of prealbumin in this disease and that, in addition to complete prealbumin, fibrils contain prealbumin fragments lacking a significant part of the N-terminus.     

The prealbumin nature of the amyloid protein in familial amyloid polyneuropathy (FAP)-Swedish variety

Familial amyloid polyneuropathy (FAP) is a dominant hereditary type of amyloidosis affecting kinships originating in many countries. We have isolated a 15,000 dalton protein from the amyloid laden tissue of a patient of Swedish origin with familial amyloid polyneuropathy. By N-terminal sequence analysis it is homologous to the normal plasma protein, prealbumin. An antiserum prepared to the isolated protein confirms this by reacting identically with the amyloid protein and prealbumin. The normal plasma protein, prealbumin, is linked to a disease syndrome for the first time.     

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.     

Enhancement of AA-amyloid formation in mice by transthyretin amyloid fragments and polyethylene glycol

The mechanism behind amyloid formation is unknown in all types of amyloidosis. Several substances can enhance amyloid formation in animal experiments. To induce secondary systemic amyloid (AA-type amyloid) formation, we injected silver nitrate into mice together with either amyloid fibrils obtained from patients with familial polyneuropathy (FAP) type I or polyethylene glycol (PEG). Mice injected with silver nitrate only served as controls. Amyloid deposits were detectable at day 3 in animals injected with amyloid fibrils and in those injected with PEG, whereas in control mice, deposits were not noted before day 12. Our results indicate that amyloid fibrils from FAP patients and even a non-sulfate containing polysaccharide (PEG) have the potential to act as amyloid-enhancing factors.     

Transthyretin amyloidosis: a tale of weak interactions.

Over 70 transthyretin (TTR) mutations have been associated with hereditary amyloidoses, which are all autosomal dominant disorders with adult age of onset. TTR is the main constituent of amyloid that deposits preferentially in peripheral nerve giving rise to familial amyloid polyneuropathy (FAP), or in the heart leading to familial amyloid cardiomyopathy. Since the beginning of this decade the central question of these types of amyloidoses has been why TTR is an amyloidogenic protein with clinically heterogeneous pathogenic consequences. As a result of amino acid substitutions, conformational changes occur in the molecule, leading to weaker subunit interactions of the tetrameric structure as revealed by X-ray studies of some amyloidogenic mutants. Modified soluble tetramers exposing cryptic epitopes seem to circulate in FAP patients as evidenced by antibody probes recognizing specifically TTR amyloid fibrils, but what triggers dissociation into monomeric and oligomeric intermediates of amyloid fibrils is largely unknown. Avoiding tetramer dissociation and disrupting amyloid fibrils are possible avenues of therapeutic intervention based on current molecular knowledge of TTR amyloidogenesis and fibril structure.     

Systemic amyloidosis in transgenic mice carrying the human mutant transthyretin (Met 30) gene

To analyze the pathologic processes of amyloid deposition in type I familial amyloidotic polyneuropathy (FAP), mice were made transgenic by introducing the human mutant transthyretin (TTR) gene(MT-hMet 30). An inbred strain of mouse, C57 BL/6, was chosen. Transgenic mice were killed using ether anesthesia at 3-mo intervals up to 24 mo after birth. In these transgenic mice, amyloid deposition started in the gastrointestinal tract, cardiovascular system, and kidneys and extended to various other organs and tissues with advancing age. The pattern of amyloid deposition was similar to that observed in human autopsy cases of FAP, except for its absence in the choroid plexus and in the peripheral and autonomic nervous systems. We extracted the amyloid fibrils from kidneys of these mice with a human mutant TTR gene and analyzed them immunochemically and electronmicroscopically. Deposited amyloid was shown to be composed of human mutant TTR and mouse serum amyloid P component. Amyloid fibril from transgenic mice was morphologically and immunohistochemically similar to that of human FAP. The most striking pathologic feature of the transgenic mice was the absence of amyloid deposition in the peripheral and autonomic nervous tissues. Thus, other intrinsic factors may be involved in amyloid deposition in the nervous tissues of human FAP.     

The tetrameric protein transthyretin dissociates to a non-native monomer in solution. A novel model for amyloidogenesis.

In amyloidosis, normally innocuous soluble proteins polymerize to form insoluble fibrils. Amyloid fibril formation and deposition have been associated with a wide range of diseases, including spongiform encephalopathies, Alzheimer's disease, and familial amyloid polyneuropathies (FAP). In certain forms of FAP, the amyloid fibrils are mostly constituted by variants of transthyretin (TTR), a homotetrameric plasma protein implicated in the transport of thyroxine and retinol. The most common amyloidogenic TTR variant is V30M-TTR, and L55P-TTR is the variant associated with the most aggressive form of FAP. Recently, we reported that TTR dissociates to a monomeric species at pH 7.0 and nearly physiological ionic strengths (Quintas, A., Saraiva, M. J., and Brito, R. M. (1997) FEBS Lett. 418, 297-300). Here, we show that the tetramer dissociation is apparently irreversible; and based on intrinsic tryptophan fluorescence and fluorescence quenching experiments, we show that the monomeric species formed upon tetramer dissociation is non-native. We also show, based on 1-anilino-8-naph-thalenesulfonate binding studies, that this monomeric species appears not to behave like a molten globule. These data allowed us to propose a model for TTR amyloidogenesis based on tetramer dissociation occurring naturally under commonly observed physiological solution conditions.     

The most pathogenic transthyretin variant, L55P, forms amyloid fibrils under acidic conditions and protofilaments under physiological conditions.

The L55P transthyretin (TTR) familial amyloid polyneuropathy-associated variant is distinct from the other TTR variants studied to date and the wild-type protein in that the L55P tetramer can dissociate to the monomeric amyloidogenic intermediate and form fibril precursors under physiological conditions (pH 7.0, 37 degrees C). The activation barrier associated with L55P-TTR tetramer dissociation is lower than the barrier for wild-type transthyretin dissociation, which does not form fibrils under physiological conditions. The L55P-TTR tetramer is also very sensitive to acidic conditions, readily dissociating to form the monomeric amyloidogenic intermediate between pH 5.5-5.0 where the wild-type TTR adopts a nonamyloidogenic tetrameric structure. The formation of the L55P monomeric amyloidogenic intermediate involves subtle tertiary structural changes within the beta-sheet rich subunit as discerned from Trp fluorescence, circular dichroism analysis, and ANS binding studies. The assembly of the L55P-TTR amyloidogenic intermediate at physiological pH (pH 7.5) affords protofilaments that elongate with time. TEM studies suggest that the entropic barrier associated with filament assembly (amyloid fibril formation) is high in vitro, amyloid being defined by the laterally assembled four filament structure observed by Blake upon isolation of "fibrils" from the eye of a FAP patient. The L55P-TTR protofilaments formed in vitro bind Congo red and thioflavin T (albeit more weakly than the fibrils produced at acidic pH), suggesting that the structure observed probably represents an amyloid precursor. The structural continuum from misfolded monomer through protofilaments, filaments, and ultimately fibrils must be considered as a possible source of pathology associated with these diseases.     

Mass spectrometric detection of the plasma prealbumin (transthyretin) variant associated with familial amyloidotic polyneuropathy

A plasma prealbumin variant with a methionine-for-valine substitution at position 30 is closely associated with familial amyloidotic polyneuropathy (FAP) type I. Secondary ion mass spectrometry of the tryptic digest of a carrier's prealbumin could easily detect an abnormal peptide containing the substitution besides the normal peptide. This is a sensitive and reliable method for the diagnosis of FAP.     

Chromosomal localization of the mouse prealbumin gene (Ttr) by in situ hybridization.

Prealbumin is a serum protein which plays an important role in plasma transport of retinol and thyroxine. The accumulation of a variant prealbumin is associated with a hereditary disorder, familial amyloidotic polyneuropathy (FAP). In situ hybridization with a mouse prealbumin gene cDNA probe was carried out in mouse fibroblasts. Analysis of 114 R-banded metaphases showed that 13% of the total grains were located on chromosome 4 and 46% of the grains on this chromosome were in the region C6-D1. Linkage and syntenic group analysis showed that the prealbumin gene (Ttr) is located between two syntenic groups on mouse chromosome 4, which corresponded to two syntenic groups present on human chromosomes 1 and 9.     

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.     

Study of an anti-human transthyretin immunoadsorbent: Influence of coupling chemistry on binding capacity and ligand leakage

A variant of transthyretin (TTR Val30Met) has been identified as the main protein precursor of the amyloid fibrils deposited in familial amyloidotic polyneuropathy (FAP). Specific removal of TTR in an extracorporeal immunoadsorption procedure is currently under investigation as a possible treatment of FAP. Immunoadsorbents were constructed by immobilizing murine anti-TTR monoclonal antibody 88.6.BA9 onto agarose gel supports via several different coupling chemistries. The influence of coupling conditions such as pH and antibody density, and of perfusion variables, such as antigen concentration and applied flow-rate, on the TTR capture efficiency, was determined. Cyanogen bromide-, carbonyldiimidazole- and aldehyde-activated (ALD) supports conjugated with antibody at optimal pH, provided immunoadsorbents with comparable TTR binding capacities. Regarding stability, leakage was lowest for the ALD based immunoadsorbents, particularly at high pH.     

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.