Creation of amyloid fibrils from mutant Asn187 gelsolin peptides.

The amyloid protein in familial amyloidosis, Finnish type, is a 71 amino acid long fragment of the inner region of mutant Asp187----Asn gelsolin. The mechanism of gelsolin amyloid formation was tested with synthetic 11 and 30 residue peptides corresponding to the normal and mutant sequence of gelsolin. Fibrils meeting the morphologic criteria of amyloid were formed from the mutant Asn187 peptides. Substitution of the normal Asp187 residue with the mutant Asn residue resulted in a 9-fold increase in fibrillogenicity as determined by quantitative fluorometry. The present study demonstrates the first successful in vitro creation of amyloid-like fibrils from Asn187 gelsolin peptides and provides evidence that amyloid formation in Finnish amyloidosis is a direct consequence of the Asp187----Asn substitution in gelsolin.     

Gelsolin variant (Asn-187) in familial amyloidosis, Finnish type.

Familial amyloidosis, Finnish type (FAF), is an inherited form of systemic amyloidosis clinically characterized by cranial neuropathy and lattice corneal dystrophy. We have demonstrated that the protein subunit isolated from amyloid fibrils shows considerable sequence identity with gelsolin, an actin-binding protein. We have purified the amyloid subunit from a second case and further analysed different fractions from the previous one. Sequence analysis shows that, in both cases, the amyloid subunit starts at position 173 of the mature molecule; it has a heterogeneous N-terminus and contains one amino acid substitution, namely asparagine for aspartic acid, at position 15 (gelsolin residue 187), that is due to a guanine-to-adenine transversion corresponding to nucleotide-654 of human plasma gelsolin cDNA. The substitution maps in a fragment with actin-binding activity and is located in a repetitive motif highly conserved among species. Thus FAF is the first human disease known to be caused by an internal abnormal degradation of a gelsolin variant. We designate this variant of gelsolin-associated amyloidosis 'Agel Asn-187'.     

Amyloid protein in familial amyloidosis (Finnish type) is homologous to gelsolin, an actin-binding protein

Familial amyloidosis, Finnish type, is clinically characterized by cranial neuropathy and lattice corneal dystrophy. It is an autosomal dominant form of systemic amyloidosis with small deposits of congophilic material occurring in most tissues, particularly in association with blood vessel walls and basement membranes. Amyloid fibrils were extracted from the kidney of patient VUO, and rabbit antiserum raised against the 12 kDa purified amyloid subunit displayed strong immunohistochemical reactivity with the amyloid deposits. The amino terminal sequence of this 12 kDa amyloid protein (ATEVPVSWESFNNGD) showed homology with gelsolin (or actin depolymerizing factor), a 93 kDa plasma protein. The amyloid peptide is a degradation product, starting at position 173, of the gelsolin molecule.     

Variant apolipoprotein AI as a major constituent of a human hereditary amyloid

Amyloid fibrils were isolated from spleen and liver of a patient who died with Familial Amyloidotic Polyneuropathy Type III (Iowa). The major protein constituent of the fibrils was found to be the amino terminal portion (residues 1-83) of apolipoprotein AI with an arginine for glycine substitution at position 26. This is the first report of an apolipoprotein as a major amyloid constituent in a form of autosomal dominant hereditary amyloidosis in humans.     

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.     

Effect of amino acid variations in the central region of human serum amyloid A on the amyloidogenic properties

Human serum amyloid A (SAA) is a precursor protein of the amyloid fibrils that are responsible for AA amyloidosis. Of the four human SAA genotypes, SAA1 is most commonly associated with AA amyloidosis. Furthermore, SAA1 has three major isoforms (SAA1.1, 1.3, and 1.5) that differ by single amino acid variations at two sites in their 104-amino acid sequences. In the present study, we examined the effect of amino acid variations in human SAA1 isoforms on the amyloidogenic properties. All SAA1 isoforms adopted α-helix structures at 4 °C, but were unstructured at 37 °C. Heparin-induced amyloid fibril formation of SAA1 was observed at 37 °C, as evidenced by the increased thioflavin T (ThT) fluorescence and β-sheet structure formation. Despite a comparable increase in ThT fluorescence, SAA1 molecules retained their α-helix structures at 4 °C. At both temperatures, no essential differences in ThT fluorescence and secondary structures were observed among the SAA1 isoforms. However, the fibril morphologies appeared to differ; SAA1.1 formed long and curly fibrils, whereas SAA1.3 formed thin and straight fibrils. The peptides corresponding to the central regions of the SAA1 isoforms containing amino acid variations showed distinct amyloidogenicities, reflecting their direct effects on amyloid fibril formation. These findings may provide novel insights into the influence of amino acid variations in human SAA on the pathogenesis of AA amyloidosis.     

Expression of a normal and variant Alzheimer's beta-protein gene in amyloid of hereditary cerebral hemorrhage, Dutch type: DNA and protein diagnostic assays

Amyloid fibrils deposited in cerebral vessel walls in Dutch patients with hereditary cerebral hemorrhage with amyloidosis (HCHWA-D) are formed by polymerization of a 39-residue peptide similar to the beta-protein of Alzheimer's disease, Down syndrome, sporadic cerebral amyloid angiopathy and normal aging. Sequence analysis of genomic DNA in HCHWA-D patients demonstrated a point mutation, cytosine for guanine at position 1852 of the precursor beta-protein gene, which causes a single amino acid substitution (glutamine for glutamic acid) corresponding to position 22 of the amyloid protein. The normal allele was also present in these patients. To examine the expression of normal and variant beta-protein alleles in HCHWA-D we analyzed all the tryptic peptides obtained from several amyloid fractions from leptomeningeal vascular walls. Amino acid sequence of two peptides (T3a and T3b) with identical amino acid composition revealed that T3a had glutamine and T3b had glutamic acid at position 22. Thus both the normal and variant Alzheimer's beta-protein alleles are expressed in vascular amyloid in HCHWA-D and may be detected by tryptic peptide mapping. Moreover, we have developed a diagnostic assay for high risk populations and prenatal evaluation that is based on the existence of the mutation.     

Structural diversity of ex vivo amyloid fibrils studied by cryo-electron microscopy

Cryo-electron microscopy studies are presented on amyloid fibrils isolated from amyloidotic organs of two patients with different forms of hereditary non-neuropathic systemic amyloidosis, caused, respectively, by Leu60Arg apolipoprotein AI and Asp67His lysozyme. Although ex vivo amyloid fibrils were thought to be more uniform in structure than those assembled in vitro, our findings show that these fibrils are also quite variable in structure. Structural disorder and variability of the fibrils have precluded three-dimensional reconstruction, but averaged cryo-electron microscopy images suggest models for protofilament packing in the lysozyme fibrils. We conclude that ex vivo amyloid fibrils, although variable, assemble as characteristic structures according to the identity of the precursor protein.     

Systemic AA amyloidosis in the red fox (Vulpes vulpes)

Amyloid A (AA) amyloidosis occurs spontaneously in many mammals and birds, but the prevalence varies considerably among different species, and even among subgroups of the same species. The Blue fox and the Gray fox seem to be resistant to the development of AA amyloidosis, while Island foxes have a high prevalence of the disease. Herein, we report on the identification of AA amyloidosis in the Red fox (Vulpes vulpes). Edman degradation and tandem MS analysis of proteolyzed amyloid protein revealed that the amyloid partly was composed of full‐length SAA. Its amino acid sequence was determined and found to consist of 111 amino acid residues. Based on inter‐species sequence comparisons we found four residue exchanges (Ser31, Lys63, Leu71, Lys72) between the Red and Blue fox SAAs. Lys63 seems unique to the Red fox SAA. We found no obvious explanation to how these exchanges might correlate with the reported differences in SAA amyloidogenicity. Furthermore, in contrast to fibrils from many other mammalian species, the isolated amyloid fibrils from Red fox did not seed AA amyloidosis in a mouse model.     

Structural stability of amyloid fibrils of beta(2)-microglobulin in comparison with its native fold

Among various amyloidogenic proteins, beta(2)-microglobulin (beta2-m) responsible for dialysis-related amyloidosis is a target of extensive study because of its clinical importance and suitable size for examining the formation of amyloid fibrils in comparison with protein folding to the native state. The structure and stability of amyloid fibrils have been studied with various physicochemical methods, including H/D exchange of amyloid fibrils combined with dissolution of fibrils by dimethylsulfoxide and NMR analysis, thermodynamic analysis of amyloid fibril formation by isothermal calorimetry, and analysis of the effects of pressure on the structure of amyloid fibrils. The results are consistent with the view that amyloid fibrils are a main-chain-dominated structure with larger numbers of hydrogen bonds and pressure-accessible cavities in the interior, in contrast to the side-chain-dominated native structure with the optimal packing of amino acid residues. We consider that a main-chain dominated structure provides the structural basis for various conformational states even with one protein. When this feature is combined with another unique feature, template-dependent growth, propagation and maturation of the amyloid conformation, which cannot be predicted with Anfinsen's dogma, take place.     

Proteomic Analysis of Highly Prevalent Amyloid A Amyloidosis Endemic to Endangered Island Foxes

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.     

Codeposition of apolipoprotein A-IV and transthyretin in senile systemic (ATTR) amyloidosis

Protein material was extracted from amyloid-rich sections of formalin-fixed and paraffin-embedded heart tissue from an individual with senile systemic amyloidosis, known to contain wild-type transthyretin as major amyloid fibril protein. Amino acid sequence analysis of tryptic peptides of this material revealed in addition to transthyretin sequences, also amino acid sequence corresponding to an N-terminal fragment of apolipoprotein A-IV. In immunohistochemistry, an antiserum to a synthetic apolipoprotein A-IV peptide labeled amyloid specifically. This peptide formed spontaneously amyloid-like fibrils in vitro and enhanced fibril formation from wild-type transthyretin. We conclude that several apolipoproteins, including apolipoprotein A-IV, may be important minor amyloid constituents, promoting fibril formation.     

The heterogeneity of protein AA in secondary (reactive)systemic amyloidosis

In secondary systemtic amyloidosis, amyloid fibrils have protein AA as a main subunit protein. As judged from gel chromatography and electrophoresis, this protein is rather homogeneous. In the present paper it is shown, however, that protein AA is very heterogeneous and composed of many peptides with different isoelectric points. However, their antigenic properties and amino acid compositions vary only little. It is concluded that protein AA is as heterogeneous as its postulated precursor, the acute phase reactant serum AA and that a theory that only one or a few serum protein AA's can give rise to amyloid fibrils, might be wrong.     

Structural stability of amyloid fibrils of β2-microglobulin in comparison with its native fold

Among various amyloidogenic proteins, β₂-microglobulin (β2-m) responsible for dialysis-related amyloidosis is a target of extensive study because of its clinical importance and suitable size for examining the formation of amyloid fibrils in comparison with protein folding to the native state. The structure and stability of amyloid fibrils have been studied with various physicochemical methods, including H/D exchange of amyloid fibrils combined with dissolution of fibrils by dimethylsulfoxide and NMR analysis, thermodynamic analysis of amyloid fibril formation by isothermal calorimetry, and analysis of the effects of pressure on the structure of amyloid fibrils. The results are consistent with the view that amyloid fibrils are a main-chain-dominated structure with larger numbers of hydrogen bonds and pressure-accessible cavities in the interior, in contrast to the side-chain-dominated native structure with the optimal packing of amino acid residues. We consider that a main-chain dominated structure provides the structural basis for various conformational states even with one protein. When this feature is combined with another unique feature, template-dependent growth, propagation and maturation of the amyloid conformation, which cannot be predicted with Anfinsen's dogma, take place.     

Finnish type of familial amyloidosis: cosegregation of Asp187----Asn mutation of gelsolin with the disease in three large families.

Familial amyloidosis of Finnish type (FAF) is one of the familial amyloidotic polyneuropathy (FAP) syndromes, a group of inherited disorders characterized by extracellular accumulation of amyloid and by clinical symptoms and signs of polyneuropathy. FAF, an autosomal dominant trait, belongs to those rare monogenic disorders which occur with increased frequency in the Finnish population: only single FAF cases have been reported from other populations. In most types of FAP syndromes the accumulating protein is a transthyretin variant. However, recent evidence has suggested that the amyloid peptides in FAF are related to gelsolin, an actin modulating protein. The gelsolin fragments isolated from at least one patient with amyloidosis have been reported to have an amino acid substitution, with asparagine replacing aspartic acid at position 187 of the plasma gelsolin. In this study allele-specific oligonucleotides were used to analyze three large FAF families with multiple affected individuals as well as healthy family members. We found the corresponding G-A mutation in nucleotide 654 of the plasma gelsolin gene to cosegregate with the disease. The result was confirmed by sequencing and strongly suggests that the mutation has caused all the FAF cases of these families. Since the disease is clustered in restricted areas on the southern coast of Finland, this mutation most probably causes the majority, if not all, of FAF cases in Finland.     

The protofilament substructure of amyloid fibrils

Tissue deposition of normally soluble proteins, or their fragments, as insoluble amyloid fibrils causes the usually fatal, acquired and hereditary systemic amyloidoses and is associated with the pathology of Alzheimer's disease, type 2 diabetes and the transmissible spongiform encephalopathies. Although each type of amyloidosis is characterised by a specific amyloid fibril protein, the deposits share pathognomonic histochemical properties and the structural morphology of all amyloid fibrils is very similar. We have previously demonstrated that transthyretin amyloid fibrils contain four constituent protofilaments packed in a square array. Here, we have used cross-correlation techniques to average electron microscopy images of multiple cross-sections in order to reconstruct the sub-structure of ex vivo amyloid fibrils composed of amyloid A protein, monoclonal immunoglobulin lambda light chain, Leu60Arg variant apolipoprotein AI, and Asp67His variant lysozyme, as well as synthetic fibrils derived from a ten-residue peptide corresponding to the A-strand of transthyretin. All the fibrils had an electron-lucent core but the packing arrangement comprised five or six protofilaments rather than four. The structural similarity that defines amyloid fibres thus exists principally at the level of beta-sheet folding of the polypeptides within the protofilament, while the different types vary in the supramolecular assembly of their protofilaments.     

Differences Between Vascular and Plaque Core Amyloid in Alzheimer's Disease

Abstract: The predominant protein of cerebrovascular and plaque core amyloid in Alzheimer's disease, Down's syndrome, hereditary hemorrhage with amyloidosis—Dutch type, sporadic cerebral amyloid angiopathy, and age-related amyloidosis is a unique polypeptide, called β protein. The length of the plaque amyloid protein was reported to be 42–43 residues, but the complete length of the cerebral vascular amyloid is not known. To clarify this issue, amyloid fibrils from the leptomeninges of an Alzheimer's disease patient were isolated and the primary structure determined. The complete sequence of cerebrovascular β-amyloid protein, although homologous to the plaque core amyloid protein previously reported, has 39 residues instead of 42. Amino terminal heterogeneity is present but minimal, and it is three residues shorter at the carboxy terminus. These differences are similar to those found in two cases of hereditary hemorrhage with amyloidosis—Dutch type. The differences between vascular and plaque β-amyloid may reflect diverse processing of the β protein precursor in the vessel wall and brain parenchyma due to tissue-specific endopeptidases.     

A new human hereditary amyloidosis: the result of a stop-codon mutation in the apolipoprotein AII gene

Hereditary systemic amyloidosis may be caused by mutations in a number of plasma proteins including transthyretin, apolipoprotein AI, fibrinogen Aalpha-chain, lysozyme, and gelsolin. Each type of amyloidosis is inherited as an autosomal dominant disease and is associated with a structurally altered protein that aggregates to form amyloid fibrils. Here we report that the amyloid protein in a family with previously uncharacterized hereditary renal amyloidosis is apolipoprotein AII (apoAII) with a 21-residue peptide extension on the carboxyl terminus. Sequence analysis of the apoAII gene of affected individuals showed heterozygosity for a single base substitution in the apoAII stop codon. The mutation results in extension of translation to the next in-frame stop codon 60 nucleotides downstream and is predicted to give a 21-residue C-terminal extension of the apoAII protein identical to that found in the amyloid. This mutation produces a novel BstNI restriction site that can be used to identify individuals with this gene by restriction fragment length polymorphism analysis. This is the first report of apoAII amyloid in humans and the first mutation identified in apoAII protein. Amyloid fibril formation from apoAII suggests that this lipoprotein, which is predicted to have an amphipathic helical structure, must undergo a transition to a beta-pleated sheet by a mechanism shared by other lipoproteins that form amyloid.     

Analysis of Core Region from Egg White Lysozyme Forming Amyloid Fibrils

Some of the lysozyme mutants in humans cause systemic amyloidosis. Hen egg white lysozyme (HEWL) has been well studied as a model protein of amyloid fibrils formation. We previously identified an amyloid core region consisting of nine amino acids (designated as the K peptide), which is present at 54-62 in HEWL. The K peptide, with tryptophan at its C- terminus, has the ability of self-aggregation. In the present work we focused on its structural properties in relation to the formation of fibrils. The K peptide alone formed definite fibrils having β-sheet structures by incubation of 7 days under acidic conditions at 37°C. A substantial number of fibrils were generated under this pH condition and incubation period. Deletion and substitution of tryptophan in the K peptide resulted in no formation of fibrils. Tryptophan 62 in lysozyme was suggested to be especially crucial to forming amyloid fibrils. We also show that amyloid fibrils formation of the K peptide requires not only tryptophan 62 but also a certain length containing hydrophobic amino acids. A core region is involved in the significant formation of amyloid fibrils of lysozyme.     

Main-chain dominated amyloid structures demonstrated by the effect of high pressure

It has been suggested that, while the globular native forms of proteins are a side-chain-dominated compact structure evolved by pursuing a unique fold with optimal packing of amino acid residues, amyloid fibrils are a main-chain-dominated structure with an extensive hydrogen bond network. To address this issue, the effects of hydrostatic pressure on amyloid fibrils of beta2-microglobulin (beta2-m), involved in dialysis-related amyloidosis, were studied. A systematic analysis at various pressures and concentrations of guanidine hydrochloride conducted by monitoring thioflavin T fluorescence, light-scattering, and tryptophan fluorescence revealed contrasting conformational changes occurring consecutively: first, a pressure-induced reorganization of fibrils and then a pressure-induced unfolding. The changes in volume as well as the observed structural changes indicate that the beta2-m amyloid fibrils under ambient pressure are less tightly packed with a larger number of cavities, consistent with the main-chain-dominated amyloid structure. Moreover, the amyloid structure without optimal packing will enable various isoforms to form, suggesting the structural basis of multiple forms of amyloid fibrils in contrast to the unique native-fold.