A variant prealbumin-related low molecular weight amyloid fibril protein in familial amyloid polyneuropathy of Japanese origin

Amyloid fibril protein with a molecular weight of 8K daltons, in addition to one of 16K daltons, has been isolated and characterized from an autopsy sample of a patient with familial amyloid polyneuropathy in a Japanese family from Ogawa Village, Nagano Prefecture. The component was shown to react with an antiserum to normal plasma prealbumin, as did the other. Following the purification by reverse phase liquid chromatography, it was digested with trypsin and the peptides, after the purification by HPLC were sequenced. The data showed that the component was a distinct fragment whose sequence was identical with that of the residues from Gly-6 to Tyr-78 of the prealbumin, except that it had a methionine for a valine at position 25. This corresponded with the position 30 where a valine residues has been reported for the sequence of the normal plasma prealbumin.     

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.     

Isolation and characterization of cardiac amyloid in familial amyloid polyneuropathy type IV (Finnish): relation of the amyloid protein to variant gelsolin

Amyloid subunit protein was isolated from familial amyloid polyneuropathy type IV (Finnish type) cardiac tissue and purified to homogeneity. N-terminal amino acid sequence analysis shows that the amyloid protein is a fragment of the inner region of human gelsolin. When compared with the predicted sequence of human plasma gelsolin, the amyloid protein contains an asparagine-for-aspartic acid substitution at position 15 corresponding to residue 187 of the secreted protein. Antibodies raised against the amyloidogenic region of gelsolin specifically stained the amyloid deposited in tissues in familial amyloidosis type IV. The results show that the subunit amyloid protein in familial amyloid polyneuropathy type IV represents a unique type of amyloid derived from a variant (Asn-187) gelsolin molecule by limited proteolysis.     

Identification of a prealbumin variant in the serum of a Japanese patient with familial amyloidotic polyneuropathy

Serum prealbumin isolated from a Japanese patient with familial amyloidotic polyneuropathy (FAP) has been found to consist of a mixture of normal prealbumin and a prealbumin variant which contains a methionine for valine substitution at position 30. The prealbumin variant in the serum is identical to the prealbumin variant derived from amyloid fibrils of a Japanese FAP patient. FAP likely results from the deposition of abnormal serum prealbumin in various organs as amyloid fibrils.     

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.     

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.     

Capture of a dimeric intermediate during transthyretin amyloid formation

Point mutations in the human plasma protein transthyretin are associated with the neurological disorder familial amyloidosis with polyneuropathy type 1. The disease is characterized by amyloid fibril deposits causing damage at the site of deposition. Substitution of two amino acids in the hydrophobic core of transthyretin lead to a mutant that was very prone to form amyloid. In addition, this mutant has also been shown to induce a toxic response on a neuroblastoma cell line. Renaturation of the transthyretin mutant at low temperature facilitated the isolation of an amyloid-forming intermediate state having the apparent size of a dimer. Increasing the temperature effectively enhanced the rate of interconversion from a partly denatured protein to mature amyloid. Using circular dichroism the beta-sheet content of the formed mature fibrils was significantly lower than that of the native fold of transthyretin. Morphology studies using electron microscopy also indicated a temperature-dependent transformation from amorphous aggregates toward mature amyloid fibrils. In addition, 1-anilino-8-naphtalenesulfonate fluorescence studies suggested the loss of the thyroxin-binding channel within both the isolated intermediate and the mature fibrils.     

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.     

Inhibition of transthyretin amyloid fibril formation by 2,4-dinitrophenol through tetramer stabilization

Transthyretin (TTR), a homotetrameric thyroxine transport protein found in the plasma and cerebrospinal fluid, circulates normally as a innocuous soluble protein. In some individuals, TTR polymerizes to form insoluble amyloid fibrils. TTR amyloid fibril formation and deposition have been associated with several diseases like familial amyloid polyneuropathy and senile systemic amyloidosis. Inhibition of the fibril formation is considered a potential strategy for the therapeutic intervention. The effect of small water-soluble, hydrophobic ligand 2,4-dinitrophenol (2,4-DNP) on TTR amyloid formation has been tested. 2,4-DNP binds to TTR both at acidic and physiological pH, as shown by the quenching of TTR intrinsic fluorescence. Interestingly, 2,4-DNP not only binds to TTR at acidic pH but also inhibits amyloid fibril formation as shown by the light scattering and Congo red-binding assay. Inhibition of fibril formation by 2,4-DNP appears to be through the stabilization of TTR tetramer upon binding to the protein, which includes active site. These findings may have implications for the development of mechanism based small molecular weight compounds as therapeutic agents for the prevention/inhibition of the amyloid 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.     

Experimentally derived structural constraints for amyloid fibrils of wild-type transthyretin

Transthyretin (TTR) is a largely β-sheet serum protein responsible for transporting thyroxine and vitamin A. TTR is found in amyloid deposits of patients with senile systemic amyloidosis. TTR mutants lead to familial amyloidotic polyneuropathy and familial amyloid cardiomyopathy, with an earlier age of onset. Studies of amyloid fibrils of familial amyloidotic polyneuropathy mutant TTR suggest a structure similar to the native state with only a simple opening of a β-strand-loop-strand region exposing the two main β-sheets of the protein for fibril elongation. However, we find that the wild-type TTR sequence forms amyloid fibrils that are considerably different from the previously suggested amyloid structure. Using protease digestion with mass spectrometry, we observe the amyloid core to be primarily composed of the C-terminal region, starting around residue 50. Solid-state NMR measurements prove that TTR differs from other pathological amyloids in not having an in-register parallel β-sheet architecture. We also find that the TTR amyloid is incapable of binding thyroxine as monitored by either isothermal calorimetry or 1,8-anilinonaphthalene sulfonate competition. Taken together, our experiments are consistent with a significantly different configuration of the β-sheets compared to the previously suggested structure.     

Organ-specific (localized) synthesis of Ig light chain amyloid

Ig amyloidosis is usually a systemic disease with multisystem involvement. However, in a significant number of cases amyloid deposition is limited to one specific organ. It has not been determined if the Ig light chain (LC) amyloid precursor protein in localized amyloidosis is synthesized by circulating plasma cells with targeting of the amyloid fibril-forming process to one specific organ, or whether the synthesis of Ig LC and fibril formation occurs entirely as a localized process. In the present study local synthesis of an amyloid fibril precursor LC was investigated. Amyloid fibrils were isolated from a ureter that was obstructed by extensive infiltration of the wall with amyloid. Amino acid sequence analysis of the isolated fibril subunit protein proved it to be derived from a lambdaII Ig LC. Plasma cells within the lesion stained positively with labeled anti-lambda Ab and by in situ hybridization using an oligonucleotide probe specific for lambda-LC mRNA. RT-PCR of mRNA extracted from the tumor and direct DNA sequencing gave the nucleotide sequence coding specifically for the lambdaII amyloid subunit protein, thus confirming local synthesis of the LC protein.     

Molecular genetics of familial amyloidotic polyneuropathy

We established a diagnosis of familial amyloidotic polyneuropathy (FAP) based on DNA and demonstrated a direct link between prealbumin gene mutation and FAP. The individuals with FAP, so far examined, were heterozygous for the prealbumin gene, carrying one normal and one mutant gene. To investigate the molecular pathogenesis of FAP, we isolated a normal prealbumin gene (7 kb in length) and also a mutant prealbumin gene associated with FAP. We compared their nucleotide sequences and found that they matched except for a single-base substitution present in the second exon. In an effort to construct mouse model systems for the FAP, we developed strains of transgenic mice carrying human mutant prealbumin genes.     

The association of amyloid P-component (AP) with the amyloid fibril: an updated method for amyloid fibril protein isolation

An amyloid fibril isolation procedure is proposed which uses citrate as well as saline washes to dissociate the calcium dependent linkage of amyloid P-component (AP) from the amyloid fibril. In two amyloid rich tissues, the amount of AP was quantitated in each saline and citrate wash and totalled 13.8% and 20.8% of the amyloid fibrils isolated. The amount of AP removed from these and 22 additional amyloid rich tissues was greater than had previously been recognized. AP protein was present in tissue only when amyloid fibrils were present. It could not be found in normal non-amyloidotic tissue, nor could it be found in tissue sediment after the fibrils were removed.     

Effect of nitric oxide in amyloid fibril formation on transthyretin-related amyloidosis

Although oxidative stress is said to play an important role in the amyloid formation mechanism in several types of amyloidosis, few details about this role have been described. Amyloid is commonly deposited around the vessels that are the primary site of action of nitric oxide generated from endothelial cells and smooth muscle cells, so nitric oxide may be also implicated in amyloid formation. For this study, we examined the in vitro effect of S-nitrosylation on amyloid formation induced by wild-type transthyretin, a precursor protein of senile systemic amyloidosis, and amyloidogenic transthyretin V30M, a precursor protein of amyloid deposition in familial amyloidotic polyneuropathy. S-Nitrosylation of amyloidogenic transthyretin V30M via the cysteine at position 10 was 2 times more extensive than that of wild-type transthyretin in a nitric oxide-generating solution. Both wild-type transthyretin and amyloidogenic transthyretin V30M formed amyloid fibrils under acidic conditions, and S-nitrosylated transthyretins exhibited higher amyloidogenicity than did unmodified transthyretins. Moreover, S-nitrosylated amyloidogenic transthyretin V30M formed more fibrils than did S-nitrosylated wild-type transthyretin. Structural studies revealed that S-nitrosylation of amyloidogenic transthyretin V30M induced a change in its conformation, as well as instability of the tetramer conformation. These results suggest that the nitric oxide-mediated modification of transthyretin, especially variant transthyretin, may play an important role in amyloid formation in senile systemic amyloidosis and familial amyloidotic polyneuropathy.     

Modifications of transthyretin in amyloid fibrils: analysis of amyloid from homozygous and heterozygous individuals with the Met30 mutation.

The finding of individuals homozygous for FAP I (familial amyloidotic polyneuropathy, transthyretin TTRMet30) with amyloid deposits in the vitreous body, gave us access to a unique material lacking wild type transthyretin and contaminating proteins. Amyloid TTR is modified in several ways. Besides the full-length protein and its dimer form, two smaller bands were identified by SDS-PAGE and protein sequencing. One corresponded to a peptide starting at amino acid Thr49, the other was a mixture of two peptides starting at positions 1 and 3 in a 3:1 ratio. Upon reduction the amount of the TTR dimer decreased, the monomer amount increased, and the resulting monomers became available for carboxymethylation. Moreover, the mobility of the small band, which includes Cys10, increased upon reduction. This cysteine seemed to be involved in an interchain disulfide bridge both between intact TTR molecules and between small fragments. The same pattern was found in heterozygous fibril material although smaller amounts of the truncated peptides were found. Fibrils were formed both from normal and mutated TTR in heterozygotes. The significance of our results for amyloid formation is discussed.     

Senile cardiac amyloid: demonstration of a unique fibril protein in tissue sections.

Antisera were raised against degrading amyloid fibrils isolated from the heart of a patient with senile cardiac amyloidosis (SCA), and from a medullary carcinoma of the thyroid (MCT). The antisera were absorbed and used in indirect immunofluorescence to identify an amyloid fibril protein (ASCA) in heart tissue from patients with senile cardiac amyloidosis and to identify the amyloid fibril protein (AMCT) found in association with medullary carcinomas of the thyroid. Absorbed anti-ASCA antiserum did not react with normal tissue such as heart, liver, spleen, and striated muscle, or with amyloid tissue known to contain amyloid fibril proteins AA, AlambdaI, AlambdaIV, AlambdaV, AMCT or with pancreatic tissue containing islet amyloid deposits. The reactions with senile amyloid he,rt tissue could be blocked completely by degraded amyloid fibrils extracted from senile amyloid heart tissue or by amyloid fibril protein ASCA isolated from such fibrils. The anti-AMCT antiserum showed a similar specific reaction restricted to amyloid associated with MCT. In addition, antisera specific for amyloid fibril proteins AA, AlambdaI, AlambdaIV, and AlambdaV failed to react with senile cardiac amyloid, pancreatic islet amyloid, or medullary thyroid amyloid.     

Direct sequencing of the gene for Maryland/German familial amyloidotic polyneuropathy type II and genotyping by allele-specific enzymatic amplification

Direct genomic DNA sequencing has been used to characterize the mutation associated with familial amyloidotic polyneuropathy in the Maryland/German kindred. A mutation of thymine to adenine in the prealbumin (transthyretin) gene at the position corresponding to the second base of codon 58 in the prealbumin mRNA gives a histidine for leucine substitution in the plasma protein. Since the mutation does not result in a change in the restriction pattern of the prealbumin gene, a new method for the direct detection of single base changes in genomic DNA was developed using the polymerase chain reaction and an allele-specific oligonucleotide primer.     

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.     

Methods for staining amyloid in tissues: a review

The traditional way of identifying amyloid in tissue sections has been staining with Congo red and demonstration of green birefringence under crossed polarizers. The original method of Congo red staining, described by Bennhold in 1922, has undergone several modifications to improve its sensitivity, specificity, and reliability. The most common modification is the alkaline Congo red method described by Puchtler and co-workers in 1962. Specificity is improved by using freshly prepared stain and a staining solution fully saturated with sodium chloride. Amyloid proteins can be further distinguished by autoclaving or by treating the tissue with potassium permanganate or alkaline guanidine. Autoclaving the tissues at 120 C for 30 min causes protein AA to lose its affinity for Congo red. Prolongation of autoclaving to 120 min abolishes the Congophilia of protein AL, but prealbumin-related amyloid shows little or no change. Treatment of the tissue with potassium permanganate causes protein AA and B2-microglobulin amyloid to lose their affinity to Congo red. Protein AA fails to stain with Congo red after treatment with alkaline guanidine for 1 min and protein AL and systemic senile amyloid protein (SSA) after 2 hr. Familial amyloid protein (FAP), prealbumin type, can stand 2 hr of alkaline guanidine treatment without losing its ability to stain with Congo red. Other methods of detection of amyloid include fluorescent stains, e.g., thioflavin T or S, and metachromatic stains such as crystal violet. Immunofluorescence and immunoperoxidase methods are used to identify and classify amyloid proteins in tissues. Antibodies against the P component, proteins AA and AL and FAP have been used with great precision. Due to cross-reactivity, these methods do not differentiate between some types of familial and senile systemic amyloidosis.