AA-amyloidosis. Tissue component-specific association of various protein AA subspecies and evidence of a fourth SAA gene product.

Protein AA, the major repetitive protein subunit present in fibrils deposited in AA-amyloidosis, is an N-terminal cleavage product of a 104-amino acid precursor, serum amyloid A (SAA). Protein AA subspecies varying between 45 and 94 amino acids in length have been described. In this study it is shown that the different protein AA subspecies are not evenly distributed in amyloid deposits and that in single patients, certain subspecies of protein AA are deposited in specific tissue component sites. Thus larger protein AA subspecies occur in lower concentration in amyloid in the glomeruli compared to other sites and are especially found in amyloid in vessel walls. Three different SAA forms have been predicted from genomic and complementary DNA studies. The existence of a fourth type has been suspected from amino acid sequence studies of purified SAA. Protein AA derived from this fourth type of SAA is now shown to be present in amyloid fibrils in one of the patients studied in this paper.     

Is the serum amyloid A protein in acute phase plasma high density lipoprotein the precursor of AA amyloid fibrils?

Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is generally considered to be the precursor of AA protein, which forms the fibrils in reactive systemic amyloidosis in man and animals. This view is based on amino acid sequence identity between AA and the amino-terminal portion of SAA. However, in extensive and well-controlled studies of experimentally induced murine AA amyloidosis, we were unable to demonstrate a direct precursor-product relationship between SAA, in SAA-rich HDL preparations from acute phase or amyloidotic mouse or human serum, and AA protein in the amyloid deposits. This raises the possibility that SAA in its usual form, as an apolipoprotein of HDL synthesized during the acute phase response, may not be the major precursor of AA fibrils. The amyloidogenic forms of circulating SAA molecules may not be isolated during the preparation of HDL. Alternatively, particularly in the light of recent evidence that SAA mRNA is expressed in many different tissues throughout the body of appropriately stimulated animals, amyloidogenic SAA may be derived from sources other than the liver cells in which SAA-rich HDL is synthesized.     

The pathogenesis and biochemistry of amyloidosis

The transformation of serum proteins into Congo red-sensitive fibrillar material is requisite for the onset and progression of amyloid disease. All the mechanisms which lead to the disease itself have not been elucidated, but our knowledge has increased significantly. It is apparent that in all types of amyloid fibrils, three common features are displayed by the major protein constituents. These are that the fibril protein has a serum precursor, a high degree of anti-parallel beta-sheet conformation and a distinctive ultrastructure on electron microscopy. In the AL and AA forms of amyloidosis, the putative precursors appear to undergo limited degradation to form the protein component of amyloid fibrils. It has been suggested that there may be certain primary structural characteristics inherent in precursor molecules which make them amyloidogenic, thus predisposing them to amyloid fibril formation. This would include certain subtypes of immunoglobulin light chains, possibly kappa I and lambda VI, in the AL type of amyloidosis and one of the polymorphic SAA species, SAA2, which has been identified as the predominating isotype found in AA amyloid fibrils. In AH amyloidosis, the mechanism of amyloid fibril formation appears to be simply a concentration phenomenon where elevated concentrations of B2-M are not handled normally and amyloid deposition is the result. Amyloidogenesis in the hereditary form of systemic amyloidosis may involve other factors in addition to the presence of a variant precursor prealbumin as indicated by the delayed onset of the disease. It is evident that the elucidation of the mechanism(s) which governs the onset and progression of the amyloidoses will allow future regulation and treatment of these all too often complex disorders.     

A serum AA-like protein as a common constituent of secondary amyloid fibrils

Amyloid fibrils, purified from the spleen of four patients with amyloidosis associated with rheumatoid arthritis, had protein AA as a major protein. Besides this protein, all four amyloid fibril preparations contained a protein which in size, amino acid composition and N-terminal amino acid sequence was the same as the postulated serum precursor of protein AA, serum AA (SAA). The SAA-like amyloid fibril protein had a tendency to aggregate in neutral conditions, a phenomenon which is also seen in SAA but not in protein AA.     

Transgenic mouse model of AA amyloidosis

AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.     

Morphologic and chemical variation of the kidney lesions in amyloidosis secondary to rheumatoid arthritis

The kidneys of 20 patients who died of secondary systemic amyloidosis due to rheumatoid arthritis were studied histologically, and four of these were shown to have an uncommon pattern of deposition with almost no glomerular involvement but heavy deposits in the outer zone of the medulla. In three of the four patients frozen tissue was available. Immunochemical characterization of amyloid fibrils from these three cases showed that the major subunit amyloid fibril protein was protein AA, typical of secondary amyloidosis. Gel chromatography of fibrils revealed an uncommon elution pattern with two retarded major protein peaks. Both these proteins showed immunologic identity with protein AA and had N-terminal amino acid sequences identical with that protein but differed in size obviously due to a shortening of the C-terminal in one of the proteins. The reason for the correlation between the pattern of deposition of amyloid and alterations in protein AA is unclear but might be due to variations in enzymes responsible for the cleavage of the amyloid fibril subunit precursor protein SAA.     

Induction of murine AA amyloidosis by various homogeneous amyloid fibrils and amyloid-like synthetic peptides

We investigated amyloid-enhancing factor (AEF) activity of amyloid fibrils extracted from amyloid-laden livers of mice, cow, cheetah, cat and swan. All amyloid fibrils were confirmed to be amyloid protein A (AA) by an immunohistochemical analysis. We found that these fibrils accelerated the deposition of amyloid in an experimental mouse model of AA amyloidosis. Furthermore, the degree of deposition was dependent on the concentration of fibrils. When we compared the minimal concentration of amyloid fibrils needed to induce deposition, we found that these fibrils showed different efficiencies. Murine amyloid fibril induced amyloid deposition more efficiently than cow, cat, cheetah or swan amyloid fibrils. These data suggest that amyloid deposition is preferentially induced by amyloid fibrils with the same primary sequence as the endogenous amyloid protein. We then analysed the AEF activity of synthetic peptides, synthesized corresponding to amino acids 1-15 of mouse SAA (mSAA), 2-15 of cow SAA (bSAA), 1-15 of cat SAA (cSAA), which was the same as cheetah, and the common amino acids 33-45 of these four SAA (aSAA). We found that mSAA, bSAA and cSAA formed amyloid-like fibrils in morphology and showed similar AEF properties to those of native amyloid fibrils. Although aSAA also formed highly ordered amyloid-like fibrils, it showed weaker AEF activity than the other synthetic fibrils. Our results indicate that amyloidosis is transmissible between species under certain conditions; however, the efficiency of amyloid deposition is species-specific and appears to be related to the primary amino acid sequence, especially the N-terminal segment of the amyloid protein.     

Secondary (AA) amyloidosis in cystic fibrosis. A report of three cases

The authors report the pathologic features of three cases of amyloidosis associated with cystic fibrosis. Renal biopsy led to the diagnosis (case 1) or suspicion (case 2) of amyloidosis in patients who were 23 and 21 years old, respectively. The third patient died at age 22 years, and amyloidosis was not discovered until autopsy. Immunohistochemical staining and potassium-permanganate pretreatment of histologic sections in all three cases provided evidence that the amyloid seen in these patients is of the secondary (AA) type. Congo red staining in each case and electron microscopy in case 1 confirmed the initial diagnosis of amyloidosis. A markedly elevated serum amyloid A protein (160 micrograms/mL; normal less than 1 microgram/mL) in case 1 indicated the presence of large quantities of the precursor protein from which the AA fibrils of secondary amyloid are derived. The kidneys, spleen, and liver contained amyloid deposits in autopsy material from all three cases. Involvement of other organs by amyloid was variable. Review of autopsy material in Boston from 23 additional cystic fibrosis patients with long-term survival did not reveal any evidence of amyloidosis. It appears that secondary amyloidosis is emerging as a significant, although rare, complication of cystic fibrosis as greater numbers of these patients survive into adulthood.     

An Experimental Model in Mink for Studying the Relation Between Amyloid Fibril Protein AA and the Related Serum Protein, SAA

Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amyloids. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.     

An Experimental Model in Mink for Studying the Relation Between Amyloid Fibril Protein AA and the Related Serum Protein, SAA

Experimental amyloidosis was induced in mink by repeated injections with endotoxin. Amyloid fibrils extracted from liver and spleen were fractionated by gel filtration after treatment with guanidine-hydrochloride and a reducing agent, dithiothreitol. An elution profile very similar to that of human amyloid fibrils, having protein AA as a major component, was obtained. The mink amyloid protein eluted at a position similar to that of human protein AA was by amino acid composition and partial sequence studies shown to be very similar to the latter protein and was called mink protein AA. In addition, a protein AA-related component (protein SAA) was found in increased amounts in serum of amyloidotic mink, providing further evidence of the homology with human amy-loidosis. Experimental amyloidosis in mink represents a suitable model for studying amyloid proteins and related serum components.     

A novel localized amyloidosis associated with lactoferrin in the cornea

We report a novel localized amyloidosis associated with lactoferrin. To elucidate the precursor protein of corneal amyloidosis associated with trichiasis, we analyzed amyloid deposits from three patients by histopathology and biochemistry. Amyloid deposits showed immunoreactivity, confirmed by electron microscopy, for only anti-human lactoferrin antibody. Electrophoresis of amyloid fibrils revealed lactoferrin with and without sugar chains; N-terminal sequence analysis revealed full-length lactoferrin and a truncated tripeptide of N-terminal amino acids, Gly-Arg-Arg. Carboxymethylated wild-type lactoferrin formed amyloid fibrils in vitro. Lactoferrin gene analysis in the three patients revealed a Glu561Asp mutation in all of the patients and a compound heterozygote of Ala11Thr and Glu561Asp mutations in one patient. A heterozygotic Glu561Asp mutation appeared in 44.8% of healthy Japanese volunteers, suggesting that the mutation may not be an essential mutation for amyloid formation (p = 0.104). Results thus suggest that lactoferrin is this precursor protein.     

Amyloid and nonfibrillar deposits in mice transgenic for wild-type human transthyretin: a possible model for senile systemic amyloidosis

The human serum protein transthyretin (TTR) is highly fibrillogenic in vitro and is the fibril precursor in both autosomal dominant (familial amyloidotic polyneuropathy [FAP] and familial amyloidotic cardiomyopathy [FAC]) and sporadic (senile systemic amyloidosis [SSA]) forms of human cardiac amyloidosis. We have produced mouse strains transgenic for either wild-type or mutant (TTRLeu55Pro) human TTR genes. Eighty-four percent of C57BI/6xDBA/2 mice older than 18 months, transgenic for the wild-type human TTR gene, develop TTR deposits that occur primarily in heart and kidney. In most of the animals, the deposits are nonfibrillar and non-Congophilic, but 20% of animals older than 18 months that bear the transgene have human TTR cardiac amyloid deposits identical to the lesions seen in SSA. Amino terminal amino acid sequence analysis and mass spectrometry of the major component extracted from amyloid and nonamyloid deposits revealed that both were intact human TTR monomers with no evidence of proteolysis or codeposition of murine TTR. This is the first instance in which the proteins from amyloid and nonfibrillar deposits in the same or syngeneic animals have been shown to be identical by sequence analysis. It is also the first time in any form of amyloidosis that nonfibrillar deposits have been shown to systematically occur temporally before the appearance of fibrils derived from the same precursor in the same tissues. These findings suggest, but do not prove, that the nonamyloid deposits represent a precursor of the fibril. The differences in the ultrastructure and binding properties of the deposits, despite the identical sizes and amino terminal amino acid sequences of the TTR and the dissociation of deposition and fibril formation, provide evidence that in vivo factors, perhaps associated with aging, impact on both systemic precursor deposition and amyloid fibril formation.     

Murine amyloid protein AA in casein-induced experimental amyloidosis.

Amyloidosis was induced in mice by 25 subcutaneous injections of casein. The splenic amyloid fibrils were identified by electron microscopy to be closely associated with reticular cells. After isolation of the fibrils by simple physical techniques, their ultrastructure revealed single filaments of 80 to 100 A width, which were rigid, nonbranching, and of indeterminate length. This is comparable to previous studies on human preparations. The amyloid fibrils were dissociated by solution in guanidine and chromatography. The resultant amyloid fibril protein was characterized as to its molecular weight, amino acid analysis, and amino-terminal sequence. It was thus definitely identified as protein AA, the major component of secondary amyloidosis. An antibody to this protein, murine AA, identified a cross-reacting mouse serum protein SAA and indicated a species specificity when tested against human preparations. A comparison is made with the AA protein in another murine model as well as AA proteins from human, guinea pig, monkey, and mink amyloidosis.     

Coexistence of Protein AA and Immunoglobulin Light-Chain Fragments in Amyloid Fibrils

Coexistence of amyloid fibril protein AA and homogeneous immunoglobulin light-chain fragments was found in the isolated amyloid fibrils of two patients with amyloidosis secondary to rheumatoid arthritis. The light-chain amyloid fibril protein showed antigenic identity with a light-chain amyloid from a patient with primary amyloidosis, which was identified as the VlambdaIV subgroup by amino acid sequence analysis. In the amyloid fibrils isolated from another patient with primary amyloidosis there was a mixture of VlambdaIV and VlambdaV homogeneous immunoglobulin light chains. Thus, a mixture of protein AA had lambda light chains or two different types of homogeneous light chains may be found in the amyloid fibrils of some patients.     

Isolation and Structural Properties of Murine SAA-The Acute Phase Serum Precursor of Amyloid AA

The murine serum protein SAA, has been found to have a structure similar to human SAA, the precursor of human secondary amyloid fibril protein AA. SAA is detected by its cross-reaction in radioimmunoassay with antibodies raised to denatured amyloid fibrils of protein AA isolated from tissues of mice with amyloidosis. Murine SAA exists in the native state as a 160,000 molecular weight species, and can be isolated as a 12,500 molecular weight moiety, SAAL, by gel filtration in 10% formic acid. The quaternary structure of SAA is such that its AA determinants are relatively inaccessible for immunoreaction. Unfolding of these determinants can occur spontaneously; however, it is promoted by dissociation of SAA to SAAL.     

Amyloid fibril protein AA. Characterization of uncommon subspecies from a patient with rheumatoid arthritis

Protein AA, the main fibril protein in secondary systemic amyloidosis, is a mixture of protein fragments (subspecies) of different length, probably arising by enzymatic cleavage of a serum precursor, SAA. We have purified amyloid fibril protein AA from a patient with rheumatoid arthritis and secondary amyloidosis with an unusual amyloid distribution in organs. This protein AA contained two major subspecies of which one consisted of 50 amino acid residues shown by complete amino acid sequence analysis. The other major AA subspecies, characterized by N- and C-terminal sequence analysis and amino acid determination of proteolytic peptides, contained 45 amino acid residues. The pI of these AA-variants differed considerably, 8.1 to 5.5, respectively. Several minor protein AA subspecies were also identified, among them one with a blocked N-terminal. The findings indicate that AA proteins of different length are connected to varying AA amyloid syndromes.     

Transformation of Amyloid Precursor SAA to Protein AA and Incorporation in Amyloid Fibrils in Vivo

Experimental amyloidosis was induced in mice by intraperitoneal injections of endotoxin (lipopolysaccharide (LPS)). In addition to LPS, a group of mice received high-density lipoprotein (HDL)-SAA complexes isolated from human acute-phase serum, whereas a group of control mice received saline in addition to LPS. Isolated amyloid fibrils from the mice given HDL-SAA contained human AA protein, as shown by immunodiffusion, immunoblot, and enzyme-linked immunosorbent assay techniques, in addition to mouse AA. In contrast, amyloid from the control mice contained exclusively AA of mouse origin. Thus, the experiments provided solid evidence that SAA is the precursor for amyloid fibril protein AA.     

Ubiquitin profile in inflammatory leukocytes and binding of ubiquitin to murine AA amyloid: Immunocytochemical and immunogold electron microscopic studies

Lysosomes in activated murine monocytoid cells have been implicated in AA amyloid formation. The pathophysiology of this process is not well understood. Previous studies into the nature of the relationship between ubiquitin (UB), possessing intrinsic amyloid enhancing factor (AEF) activity; serum amyloid A (SAA), the precursor protein of AA amyloid; and activated monocytoid cells have indicated a temporal and spatial relationship between these proteins and tissue AA amyloid deposits. To extend these findings, we have examined murine peritoneal leukocytes and splenic tissues during the early amyloid deposition phase by immunocytochemical and immunogold electron microscopic methods using monospecific anti-ubiquitin and anti-mouse AA amyloid antibodies. We show here enrichment of endosome–lysosome-like (EL) vesicles in the activated monocytoid cells with UB and SAA, and the presence of UB-bound AA amyloid fibrils in the EL vesicles, perikarya, and interstitial spaces. The importance of these findings is emphasized by the fact that activated monocytoid cells, containing UB in the EL vesicles, sequester and eventually localize SAA in their EL vesicles, and that UB binds to the EL-contained AA amyloid fibrils. These findings may also have functional consequences for studies on the role of EL and UB in amyloidogenesis.     

Diagnosis and treatment in systemic amyloidosis

Systemic amyloidosis is characterized by the involvement of multiple organs and the presence of an amyloid precursor protein in serum. This disorder is classified into four major forms: immunoglobulin light chain-derived (AL), reactive AA, dialysis-related (beta2M) and hereditary transthyretin (ATTR) type. Heart, kidney, gastrointestinal tract and peripheral nerves are commonly affected by amyloid deposition in systemic amyloidosis and histopathological demonstration of amyloid deposits on any of affected organs is the first step leading to the diagnosis of this disease. Immunohistochemical analysis of amyloid protein on tissue amyloid deposits is necessary to make classification of the disease and DNA testing is also useful in a hereditary form. Amyloidosis had been considered to be an incurable disease but during the past one decade several therapeutic approaches have been employed for the amyloidosis patients with diverse pathogenetic backgrounds: intravenous large dose of melphalan accompanied by autologous peripheral blood stem cell transplantation for AL amyloidosis and liver transplantation for hereditary ATTR type amyloidosis. As a result some amyloidosis patients have been rescued and are now enjoying their own social lives. It is likely that recent advance on the research of amyloidosis has changed the concept of this disease.     

A novel tool for detecting amyloid deposits in systemic amyloidosis in vitro and in vivo

We synthesized (trans,trans)-1-bromo-2,5-bis-(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (BSB) and used this compound to detect amyloid fibrils in autopsy and biopsy samples from patients with localized amyloidosis, such as familial prion disease, and systemic amyloidosis, such as familial amyloidotic polyneuropathy, amyloid A (AA) amyloidosis, light chain (AL) amyloidosis, and dialysis-related amyloidosis. BSB showed reactions in all Congo red-positive and immunoreactive regions of the samples examined in the study, and some amyloid fibrils in the tissues could be detected more precisely with BSB than with the other methods. In the mouse model of AA amyloidosis, injected BSB reacted with amyloid in all regions in the serial sections in which Congo red staining was positive. A highly sensitive 27-MHz quartz crystal microbalance analysis revealed that BSB showed a significant affinity for amyloid fibrils purified from familial amyloidotic polyneuropathy and dialysis-related amyloidosis samples and suppressed formation of transthyretin amyloid in vitro. These results suggest that BSB may become a valuable tool for detection of amyloid deposits in amyloidosis and of the mechanism of amyloid formation.