Aggregation of Mouse Serum Amyloid A Protein Was Promoted by Amyloid-Enhancing Factors with the More Genetically Homologous Serum Amyloid A.

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-ß structure accumulate and are deposited extracellularly in organs, resulting in chronic inflammatory diseases and infections. The incidence of AA amyloidosis is high in humans and several animal species. Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis. Amyloid enhancing factor (AEF) serves as a seed for fibril formation and shortens the onset of AA amyloidosis sharply. In this study, we examined whether AEFs extracted and purified from five animal species (camel, cat, cattle, goat, and mouse) could promote mouse SAA (mSAA) protein aggregation in vitro using quantum-dot (QD) nanoprobes to visualize the aggregation. The results showed that AEFs shortened and promoted mSAA aggregation. In addition, mouse and cat AEFs showed higher mSAA aggregation-promoting activity than the camel, cattle, and goat AEFs. Interestingly, homology analysis of SAA in these five animal species revealed a more similar amino acid sequence homology between mouse and cat than between other animal species. Furthermore, a detailed comparison of amino acid sequences suggested that it was important to mSAA aggregation-promoting activity that the 48th amino acid was a basic residue (Lys) and the 125th amino acid was an acidic residue (Asp or Glu). These data imply that AA amyloidosis exhibits higher transmission activity among animals carrying genetically homologous SAA gene, and may provide a new understanding of the pathogenesis of amyloidosis.

Increasing fatal AA amyloidosis in hunting falcons and how to identify the risk: a report from the United Arab Emirates.

In hunting falcons, a fatal syndrome of wasting, weight loss, green mutes and, finally, sudden death of emaciated birds has been observed in the United Arab Emirates (UAE). Histological examination using Congo red has revealed amyloid in most organs, in particular in the liver, spleen, kidney, and adrenal glands. Moreover, a retrospective study revealed amyloidosis in 100 cases among a total of 623 necropsied falcons between August 1995 and March 2004 in Dubai/UAE (16%; varying from 8 to 30% in different raptor bird species). The amyloid was immunohistochemically classified as amyloid A (AA), which was confirmed by Western blot analysis and N-terminal amino acid sequence analysis, suggesting it to be secondary to a chronic inflammatory process. Retrospective analysis has indicated a significantly increased prevalence of bumble foot and visceral gout among falcons with amyloidosis. In addition, a significant increase of amyloidosis from 5.6% of necropsied falcons with amyloidosis in 1995 to 40.0% in 2004 has been noticed. Finally, a semi-quantitative serum test for falcon serum amyloid A (f-SAA) has been developed. Among 38 falcons with fatal AA amyloidosis, f-SAA was increased pathologically in 36, whereas f-SAA was elevated in only one of 15 apparently disease-free falcons (p < 0.001). This significant result indicates that a normal f-SAA will indicate a minimal or even absent risk of succumbing to AA amyloidosis.

Novel truncated isoforms of constitutive serum amyloid A detected by MALDI mass spectrometry.

The main focus of the serum amyloid A (SAA) family has been on the acute phase isoforms. However, the constitutive isoform (SAA4) may have a strong effect on the metabolism of human serum lipoproteins. In this study, the SAA4 protein was examined in the high-density lipoprotein fraction of both healthy and diseased individuals. Novel isoforms of SAA4 were detected using ultracentrifugation combined with solid-phase extraction and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three truncated isoforms were identified as well as two glycosylated isoforms. Patterns of isoform distribution may be significant for assessment of cardiovascular risk as well as direction of patient treatment.

Both acute phase and constitutive serum amyloid A are present in atherosclerotic lesions.

The polymorphic protein, serum amyloid A (SAA), consists of acute phase isotypes and a constitutive isotype. Both are associated mostly with high density lipoproteins (HDL) in the circulation. In the present study, both SAA isotypes were detected by immunohistochemistry and immunoblotting using monoclonal antibodies in atherosclerotic lesions. As the distribution of SAA was identical with that of apolipoprotein B and SAA is known to be associated also with low density lipoproteins (LDL), SAA may also be delivered to the artery wall by LDL.

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.

Serum amyloid A protein (SAA) subtypes in acute and chronic inflammatory conditions.

Serum amyloid A (SAA), a polymorphic high density lipoprotein associated plasma protein, is the putative circulating precursor of tissue AA protein fibrils in reactive (secondary) amyloidosis. In the present study we examined the SAA subtype pattern in various acute and chronic inflammatory states in order to find out whether disease-specific SAA isoform profiles exist. The method used to study the subtype pattern is based on electrofocusing of serum followed by immunoblotting. Our results show that the SAA subtype pattern is similar in patients with rheumatoid arthritis with or without amyloid. In addition, in amyloidotic subjects the SAA subtype response to acute tissue injury (arthroplasty) did not differ from that in patients without amyloidosis. Analysis of patients with acute and chronic infectious diseases and non-rheumatic inflammatory conditions showed similar SAA patterns in all subjects. These results suggest that the SAA subtype response to tissue injury and inflammation is similar irrespective of the initiating stimulus.

Immunohistochemical typing of amyloid on hydroxyethyl-methacrylate-embedded renal biopsies.

Hydroxyethyl-methacrylate (GMA)-embedded renal biopsies containing amyloid were tested by the indirect immunoperoxidase method with antibodies against the following purified amyloid fibril proteins: AA, three different A lambda preparations, Ak and AF. The anti-AA reagent was monoclonal (mc13), all the others were polyclonal. In four biopsies from cases with generalized amyloidosis, two were found to be of the AA and two of the A lambda type. Two control cases of membranous glomerulonephritis showed only a marginal reaction with anti-A lambda. These results demonstrate that GMA-embedded tissue sections are suitable for immunohistochemical classification of amyloid diseases, because proteinaceous antigenic determinants of amyloid fibril are preserved.

Further structural and antigenic studies of light-chain amyloid proteins.

The major subunit protein of amyloid fibrils (758) isolated from a patient with systemic amyloidosis and studied by N-terminal amino acid sequence analysis was found to be almost identical to the sequence of a V lambda IV Bence-Jones protein and a previously described A lambda IV amyloid protein. The two A lambda IV amyloid proteins showed strong antigenic cross-reaction, appearing as antigenic identity in double immunodiffusion tests using anti-A lambda IV antiserum raised against one or the other of the two proteins. In addition, another new A lambda V amyloid fibril protein (R.S.) showed strong amino acid sequence homology and antigenic identity in double immunodiffusions with the prototype of the A lambda V subgroup (the AR protein). Finally, 20 primary or myeloma-associated amyloid proteins were characterized using antisera against the AA and several Ig light-chain-derived amyloid proteins.

Structure and antigenic behaviour of kappa I-immunoglobulin light-chain amyloid proteins.

N-terminal amino acid sequence analysis of three amyloid fibril subunit proteins from three different patients revealed a primary structure homologous to KI immunoglobulin light chains. In double immunodiffusion an antiserum against one of these amyloid proteins reacted with all three amyloids showing one line of identity, while an antiserum against one of the other amyloid proteins reacted with only one of the nonhomologous amyloids, appearing as partial identity, These two antisera did not react with any amyloid of lambda I, lambda IV or lambda VI type. The results confirm previous experiments, in which it has been shown that some antisera against amyloid proteins of immunoglobulin origin give a reaction of identity with other amyloids in the same immunoglobulin subgroup, probably owing to subgroup specific antigenic determinants in the variable segments. It was found that the distribution of amyloid varied widely among the three patients. There does not seem to be any correlation between the type of manifestation of amyloidosis and the type of immunoglobulin light chain forming the amyloid fibril.