Review. Reflections on amyloidosis in Papua New Guinea

The amyloidoses comprise a heterogeneous group of diseases in which 1 out of more than 25 human proteins aggregates into characteristic beta-sheet fibrils with some unique properties. Aggregation is nucleation dependent. Among the known amyloid-forming constituents is the prion protein, well known for its ability to transmit misfolding and disease from one individual to another. There is increasing evidence that other amyloid forms also may be transmissible but only if certain prerequisites are fulfilled. One of these forms is systemic AA-amyloidosis in which an acute-phase reactant, serum AA, is over-expressed and, possibly after cleavage, aggregates into amyloid fibrils, causing disease. In a mouse model, this disorder can easily be transmitted from one animal to another both by intravenous and oral routes. Also, synthetic amyloid-like fibrils made from defined small peptides have this property, indicating a prion-like transmission mechanism. Even some fibrils occurring in the environment can transmit AA-amyloidosis in the murine model. AA-amyloidosis is particularly common in certain areas of Papua New Guinea, probably due to the endemicity of malaria and perhaps genetic predisposition. Now, when kuru is disappearing, more interest should be focused on the potentially lethal systemic AA-amyloidosis.     

Immunoelectron microscopic classification of amyloid in renal biopsies

Recent classification of amyloidosis is based on the chemical type of amyloid protein involved. In this study, routinely embedded kidney biopsies from nine patients with generalized amyloidosis and renal involvement were tested by immunoelectron microscopy, using the protein A-gold technique, with a panel of antibodies against the following amyloid proteins: AA, A lambda, A kappa and AF. Among the antibodies, the anti-AA was monoclonal (mc1) and the others polyclonal. In all nine cases, only one type of antibody reacted with each amyloid type. Six cases were classified as AA and three cases as A lambda type. These classifications were in agreement with the clinical data and the results of serum and urine immunoelectrophoresis. The gold particles were always associated with amyloid fibrils. No reaction was evident when an amyloid type was stained by a non-corresponding antibody, or in the four control cases without amyloid. The results show that antigenic classification of amyloid is feasible on routinely processed ultra-thin epoxy sections by immunoelectron microscopy, and thus affords the possibility of retrospective studies.     

Diffusible amyloid oligomers trigger systemic amyloidosis in mice

AA (amyloid protein A) amyloidosis in mice is markedly accelerated when the animals are given, in addition to an inflammatory stimulus, an intravenous injection of protein extracted from AA-laden mouse tissue. Previous findings affirm that AA fibrils can enhance the in vivo amyloidogenic process by a nucleation seeding mechanism. Accumulating evidence suggests that globular aggregates rather than fibrils are the toxic entities responsible for cell death. In the present study we report on structural and morphological features of AEF (amyloid-enhancing factor), a compound extracted and partially purified from amyloid-laden spleen. Surprisingly, the chief amyloidogenic material identified in the active AEF was diffusible globular oligomers. This partially purified active extract triggered amyloid deposition in vital organs when injected intravenously into mice. This implies that such a phenomenon could have been inflicted through the nucleation seeding potential of toxic oligomers in association with altered cytokine induction. In the present study we report an apparent relationship between altered cytokine expression and AA accumulation in systemically inflamed tissues. The prevalence of serum AA monomers and proteolytic oligomers in spleen AEF is consistent to suggest that extrahepatic serum AA processing might lead to local accumulation of amyloidogenic proteins at the serum AA production site.     

Serum amyloid A and protein AA: Molecular mechanisms of a transmissible amyloidosis

Systemic AA-amyloidosis is a complication of chronic inflammatory diseases and the fibril protein AA derives from the acute phase reactant serum AA. AA-amyloidosis can be induced in mice by an inflammatory challenge. The lag phase before amyloid develops can be dramatically shortened by administration of a small amount of amyloid fibrils. Systemic AA-amyloidosis is transmissible in mice and may be so in humans. Since transmission can cross species barriers it is possible that AA-amyloidosis can be induced by amyloid in food, e.g. foie gras. In mice, development of AA-amyloidosis can also be accelerated by other components with amyloid-like properties. A new possible risk factor may appear with synthetically made fibrils from short peptides, constructed for tissue repair.     

Immunolocalization of lipid peroxidation/advanced glycation end products in amyloid A amyloidosis

Chronic inflammation, superimposed by amyloid fibril deposition, is believed to trigger the cascade of oxidative stress response in the affected organs and tissues. We examined immunohistochemically the distribution of 4-hydroxy-2-nonenal (HNE) and N(epsilon)-(carboxymethyl)lysine (CML), markers of lipid peroxidation and advance glycation end products (AGE), respectively, in spleen sections and peritoneal macrophages (MPhi) from mice before and during AA amyloidosis. With time, both HNE and CML immunoreactivities increased significantly in MPhi and splenic reticuloendothelial cells, known to be associated with the clearance of serum amyloid A, the precursor of AA fibrils. HNE and CML were localized to the plasma membrane and the cytoplasmic compartment of MPhi and HNE only at the nuclear membrane. These markers were also colocalized bound to AA fibrils infiltrating the splenic sinus walls. Our results reinforce the notion that oxidative stress is an integral component of amyloidotic tissues. Both lipid peroxidation and AGE have been implicated in protein modification and amyloid fibril formation. The significance of HNE and CML associated with the monocytoid cells and implicated in SAA clearance and AA fibril formation, is discussed with the pathogenesis of AA fibrils.     

Extrahepatic production of acute phase serum amyloid A

Amyloidosis is a group of diseases characterized by the extracellular deposition of protein that contains non-branching, straight fibrils on electron microscopy (amyloid fibrils) that have a high content of beta-pleated sheet conformation. Various biochemically distinct proteins can undergo transformation into amyloid fibrils. The precursor protein of amyloid protein A (AA) is the acute phase protein serum amyloid A (SAA). The concentration of SAA in plasma increases up to 1000-fold within 24 to 48 h after trauma, inflammation or infection. Individuals with chronically increased SAA levels may develop AA amyloidosis. SAA has been divided into two groups according to the encoding genes and the source of protein production. These two groups are acute phase SAA (A-SAA) and constitutive SAA (C-SAA). Although the liver is the primary site of the synthesis of A-SAA and C-SAA, extrahepatic production of both SAAs has been observed in animal models and cell culture experiments of several mammalian species and chicken. The functions of A-SAA are thought to involve lipid metabolism, lipid transport, chemotaxis and regulation of the inflammatory process. There is growing evidence that extrahepatic A-SAA formation may play a crucial role in amyloidogenesis and enhances amyloid formation at the site of SAA production.     

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2–76 (or 75) of SAA2α and the other corresponded to positions 2–76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1α has valine and alanine and SAA1β has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA1^52,57Ala.     

Identification of two novel amyloid A protein subsets coexisting in an individual patient of AA-amyloidosis.

Amyloid A protein (AA), the major fibril protein in AA-amyloidosis, is an N-terminal cleavage product of the precursor protein, serum amyloid A (SAA). Using mass spectrometry and amino-acid sequencing, we identified and characterized two novel AA protein subsets co-deposited as amyloid fibrils in an patient having AA-amyloidosis associated with rheumatoid arthritis. One of the AA proteins corresponded to positions 2-76 (or 75) of SAA2 alpha and the other corresponded to positions 2-76 (or 75) of known SAA1 subsets, except for position 52 or 57, where SAA1 alpha has valine and alanine and SAA1 beta has alanine and valine in position 52 and 57, respectively, whereas the AA protein had alanine at the both positions. Our findings (1), demonstrate that not only one but two SAA subsets could be deposited together as an AA-amyloid in a single individual and (2), support the existence of a novel SAA1 allotype, i.e., SAA152,57Ala.     

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.     

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.     

Inflammation protein SAA2.2 spontaneously forms marginally stable amyloid fibrils at physiological temperature

For nearly four decades, the formation of amyloid fibrils by the inflammation-related protein serum amyloid A (SAA) has been pathologically linked to the disease amyloid A (AA) amyloidosis. However, here we show that the nonpathogenic murine SAA2.2 spontaneously forms marginally stable amyloid fibrils at 37 °C that exhibit cross-beta structure, binding to thioflavin T, and fibrillation by a nucleation-dependent seeding mechanism. In contrast to the high stability of most known amyloid fibrils to thermal and chemical denaturation, experiments monitored by glutaraldehyde cross-linking/SDS-PAGE, thioflavin T fluorescence, and light scattering (OD(600)) showed that the mature amyloid fibrils of SAA2.2 dissociate upon incubation in >1.0 M urea or >45 °C. When considering the nonpathogenic nature of SAA2.2 and its ~1000-fold increased concentration in plasma during an inflammatory response, its extreme in vitro amyloidogenicity under physiological-like conditions suggest that SAA amyloid might play a functional role during inflammation. Of general significance, the combination of methods used here is convenient for exploring the stability of amyloid fibrils that are sensitive to urea and temperature. Furthermore, our studies imply that analogous to globular proteins, which can possess structures ranging from intrinsically disordered to extremely stable, amyloid fibrils formed in vivo might have a broader range of stabilities than previously appreciated with profound functional and pathological implications.     

A 25,000 molecular weight protein constituent of human amyloid fibrils related to amyloid protein AA

Amyloid fibrils from a patient with diffuse amyloid disease are dissociated in 6 m guanidine hydrochloride and fractionated by gel chromatography. Two major components are separated on Sepharose 6B. Both proteins are characterized by chromatography, immunodiffusion, discontinuous gel electrophoresis, amino acid tryptic peptide mapping and amino acid sequence analysis. The smaller of the two components is typical of the known protein AA by size (8400 daltons), amino acid composition and a 30-residue N-terminal sequence. The larger of the components (25,000 daltons) undergoes electrophoresis as a single band and appears unaffected by thiol reduction. It differs from protein AA in amino acid content and by its tryptic peptide map, although it contains an N-terminal amino acid sequence identical to protein AA when carried to 20 residues. Treatment of this larger component by mild acid hydrolysis results in the release of the 8400-dalton protein AA. Fractionation after guanidine hydrochloride treatment of this particular amyloid fibril preparation is compared to the fractionation of a typical secondary amyloid preparation that contains only protein AA as the major component. The origin and relationship of the 8,400- and 25,000-dalton protein components is discussed.     

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.     

Differential expression of the amyloid SAA 3 gene in liver and peritoneal macrophages of mice undergoing dissimilar inflammatory episodes

The three active serum amyloid A (SAA) genes of mice, SAA 1, SAA 2, and SAA 3, are coordinately expressed in liver during acute and chronic inflammatory stimulation and experimental amyloidosis. The genes, primarily SAA 3, are also expressed extrahepatically. The apoprotein SAA 2 is the precursor of the amyloid A (AA) fibril protein that is deposited as insoluble fibrils extracellularly in spleen and other organs when amyloidosis occurs secondarily to inflammation. The exact cause of AA fibril formation is unknown. Amyloid enhancing factor is a high m.w. glycoprotein extracted from amyloidotic organs. Administration of amyloid enhancing factor alters experimental inflammation to bring about accelerated deposition of amyloid A fibrils first in spleen and later in other organs. In this study, hepatic and extrahepatic expression of the SAA genes were compared during accelerated amyloidosis relative to inflammation uncomplicated by amyloidosis. Differences in kinetics and pattern of SAA gene expression by resident peritoneal macrophages and liver were detected during four dissimilar inflammatory episodes. Macrophages expressed the SAA 3 gene solely, and to a greater extent in chronic than in acute inflammation. In accelerated amyloid induction, macrophage SAA 3 expression increased as SAA 1 and SAA 2 expression in liver decreased. However, alpha-1-acid glycoprotein expression remained elevated throughout the course of amyloid induction. The greatly increased expression of the SAA 3 gene by macrophages and decreased expression of the SAA 1 and SAA 2 genes in liver during amyloidosis, suggests that altered SAA gene expression may play a pathogenetic role in experimental amyloid deposition.     

Acute-phase high-density lipoprotein in the rat does not contain serum amyloid A protein.

Serum amyloid A protein (SAA) is an acute-phase apolipoprotein of high-density lipoprotein (HDL). Its N-terminal sequence is identical with that of amyloid A protein (AA), the subunit of AA amyloid fibrils. However, rats do not develop AA amyloidosis, and we report here that neither normal nor acute-phase rat HDL contains a protein corresponding to SAA of other species. mRNA coding for a sequence homologous with the C-terminal but not with the N-terminal part of human SAA is synthesized in greatly increased amounts in acute-phase rat liver. These observations indicate that the failure of rats to develop AA amyloid results from the absence of most of the AA-like part of their SAA-like protein, and that the N-terminal portion of SAA probably contains the lipid-binding sequences.     

Mink serum amyloid A protein. Expression and primary structure based on cDNA sequences

The nucleotide sequences of two mink serum amyloid A (SAA) cDNA clones have been analyzed, one (SAA1) 776 base pairs long and the other (SAA2) 552 base pairs long. Significant differences were discovered when derived amino acid sequences were compared with data for apoSAA isolated from high density lipoprotein. Previous studies of mink protein SAA and amyloid protein A (AA) suggest that only one SAA isotype is amyloidogenic. The cDNA clone for SAA2 defines the "amyloid prone" isotype while SAA1 is found only in serum. Mink SAA1 has alanine in position 10, isoleucine in positions 24, 67, and 71, lysine in position 27, and proline in position 105. Residue 10 in mink SAA2 is valine while arginine and asparagine are at positions 24 and 27, respectively, all characteristics of protein AA isolated from mink amyloid fibrils. Mink SAA2 also has valine in position 67, phenylalanine in position 71, and amino acid 105 is serine. It remains unknown why these six amino acid substitutions render SAA2 more amyloidogenic than SAA1. Eighteen hours after lipopolysaccharide stimulation, mink SAA mRNA is abundant in liver with relatively minor accumulations in brain and lung. Genes encoding both SAA isotypes are expressed in all three organs while no SAA mRNA was detectable in amyloid prone organs, including spleen and intestine, indicating that deposition of AA from locally synthesized SAA is unlikely. A third mRNA species (2.2 kilobases) was identified and hybridizes with cDNA probes for mink SAA1 and SAA2. In addition to a major primary translation product (molecular mass 14,400 Da) an additional product with molecular mass 28,000 Da was immunoprecipitable.     

Intraperitoneal amyloid formation by amyloid enhancing factor--rich macrophages in ascitic fluid.

Although resident peritoneal cells from amyloidotic mice (amyloidotic peritoneal cells) are capable of processing the precursor protein of secondary amyloidosis, serum amyloid A (SAA) to amyloid fibrils, the peritoneum is a rare site for amyloid deposition. This is considered to be due to a deficiency of SAA in the peritoneum. To increase the supply of SAA to the peritoneum, ascitic fluid containing about the same protein constituents as in the serum was induced in mice. Amyloidotic peritoneal cells were packed in a microchamber which was shielded with filter membranes, and cultured in ascitic fluid supplemented with additional inflammatory factors. On the 7th day, Congo red-positive structures which showed green birefringence under polarized light were found inside and occasionally outside the chamber. By anti-AA or -SAA immunostaining, amyloid deposits and the cell surfaces of macrophages were positive. Immunologic depletion of T- and B-lymphocytes from the amyloidotic peritoneal cells did not adversely effect the amyloid formation in microchambers. These results suggest that either ascitic fluid containing sufficient amounts of SAA, or peritoneal macrophages with a high amyloid enhancing factor (AEF) activity are indispensable for AA amyloid fibrillogenesis in the peritoneum.     

A temporal and ultrastructural relationship between heparan sulfate proteoglycans and AA amyloid in experimental amyloidosis

Previous histochemical studies have suggested a close temporal relationship between the deposition of highly sulfated glycosaminoglycans (GAGs) and amyloid during experimental AA amyloidosis. In the present investigation, we extended these initial observations by using specific immunocytochemical probes to analyze the temporal and ultrastructural relationship between heparan sulfate proteoglycan (HSPG) accumulation and amyloid deposition in a mouse model of AA amyloidosis. Antibodies against the basement membrane-derived HSPG (either protein core or GAG chains) demonstrated a virtually concurrent deposition of HSPGs and amyloid in specific tissue sites regardless of the organ involved (spleen or liver) or the induction protocol used (amyloid enhancing factor + silver nitrate, or daily azocasein injections). Polyclonal antibodies to AA amyloid protein and amyloid P component also demonstrated co-localization to sites of HSPG deposition in amyloid sites, whereas no positive immunostaining was observed in these locales with a polyclonal antibody to the protein core of a dermatan sulfate proteoglycan (known as "decorin"). Immunogold labeling of HSPGs (either protein core or GAG chains) in amyloidotic mouse spleen or liver revealed specific localization of HSPGs to amyloid fibrils. In the liver, heparan sulfate GAGs were also immunolocalized to the lysosomal compartment of hepatocytes and/or Kupffer cells adjacent to sites of amyloid deposition, suggesting that these cells are involved in HSPG production and/or degradation. The close temporal and ultrastructural relationship between HSPGs and AA amyloid further implies an important role for HSPGs during the initial stages of AA amyloidosis.     

Determination of amyloid core structure using chemical shifts

Amyloid fibrils are the pathological hallmark of a large variety of neurodegenerative disorders. The structural characterization of amyloid fibrils, however, is challenging due to their non‐crystalline, heterogeneous, and often dynamic nature. Thus, the structure of amyloid fibrils of many proteins is still unknown. We here show that the structure calculation program CS‐Rosetta can be used to obtain insight into the core structure of amyloid fibrils. Driven by experimental solid‐state NMR chemical shifts and taking into account the polymeric nature of fibrils CS‐Rosetta allows modeling of the core of amyloid fibrils. Application to the Y145X stop mutant of the human prion protein reveals a left‐handed β‐helix     

Amyloidosis: new strategies for treatment

Amyloidosis is a disorder of protein folding in which normally soluble proteins are deposited extracellularly as insoluble fibrils, impairing tissue structure and function. Over 20 unrelated proteins form amyloid fibrils in vivo, with fibrils sharing a lamellar cross-β sheet structure, composed of non-covalently associated protein or peptide subunits. Amyloidosis may be acquired or hereditary and local or systemic, and is defined according to the precursor protein. Of note, local amyloid deposition occurs in Alzheimer’s disease (AD) and maturity onset diabetes but their precise role in the pathogenesis of these diseases remains uncertain. Glycosaminoglycans (GAG) and the pentraxin protein, serum amyloid P (SAP) component, are universal non-fibrillar constituents of amyloid deposits that contribute to fibrillogenesis. We review potential therapies for amyloidosis, which include measures to reduce the production of amyloidogenic precursor proteins, interference with fibrillogenesis, and enhancement of amyloid clearance, either by active or passive immunisation or by destabilising deposits through removal of serum amyloid P component.