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

Abstract

Amyloid A (AA) amyloidosis is a condition in which amyloid fibrils characterized by a linear morphology and a cross-β 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.

Keywords: amyloid A amyloidosis; amyloid enhancing factor; homology; quantum-dot; serum amyloid A.

Figure 1

Mechanism of amyloid A (AA) amyloidosis development and enhancement by amyloid enhancing factor (AEF). Serum amyloid A (SAA) protein is synthesized in the liver in normal conditions, but in AA amyloidosis, under the stimulus of AEF, SAA protein aggregates into fibrils and is deposited in the liver. Scale bar indicates 100 μm.

Figure 2

Fluorescence images of mouse serum amyloid A (mSAA) aggregation using quantum-dot (QD) nanoprobes. (A) 2D images of mSAA aggregations at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, respectively. Scale bar in 168 h fluorescent micrograph indicates 100 μm. (B) SD value of each image by time-dependent mSAA protein aggregation. mSAA protein could not spontaneously aggregate at a low concentration. Each plot represents the mean ± SEM. n = 3 separate experiments.

Figure 2

Fluorescence images of mouse serum amyloid A (mSAA) aggregation using quantum-dot (QD) nanoprobes. (A) 2D images of mSAA aggregations at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h, respectively. Scale bar in 168 h fluorescent micrograph indicates 100 μm. (B) SD value of each image by time-dependent mSAA protein aggregation. mSAA protein could not spontaneously aggregate at a low concentration. Each plot represents the mean ± SEM. n = 3 separate experiments.

Figure 3

(A) Imaging of mouse serum amyloid A (mSAA) aggregates in the presence of 20%, 40%, 60%, 80%, and 100% amyloid enhancing factor (AEFs) after 168 h of incubation. Among the AEFs from five animals, mouse and cat AEFs showed high mSAA aggregation-promoting activity. Scale bar in fluorescent micrograph indicates 100 μm. (B) The temporal increase of SD values in the presence of 100% AEFs from five animal species within 168 h. The SD values increased over time in all samples to which AEFs were added. Each plotted value represents the mean ± SEM. n = 3 separate experiments.

Figure 4

(A) 3D reconstruction images of the mouse serum amyloid A (mSAA) aggregates in the presence of camel, cattle, goat, cat, and mouse amyloid enhancing factor (AEFs) (final concentration = 100%) after 168 h of incubation. The thickness of aggregates in the presence of mouse and cat AEFs was much greater than that of camel, cattle, and goat AEFs. Three-dimensional white lines with the letters x, y, and z each indicates a scale of 50 μm. (B) Slice images of aggregates of each sample in panel (A). The morphology of each aggregate was different depending on the added AEF. Scale bar in the goat fluorescent micrograph indicates 100 μm.

Figure 5

Phylogenetic tree of SAA gene sequences from camel, cattle, goat, cat, mouse, and human. The numbers following the species’ names indicate the National Center for Biotechnology Information (NCBI) accession numbers. The red box indicates clustering of sequences from mouse, human, and cat isoforms with similar homology.

Figure 6

Monomeric structure of serum amyloid A (SAA) sequences from five animal species and human. Color description of SAA gene homology sequences: 100% similarity (blue); similarity > 75% and <100% (pink); similarity >5 0% and <75% (green); similarity <50% has no color. Red and blue arrows indicate the position of the 48th and 125th amino acid. Red boxes indicate clustering of sequences from mouse, human, and cat isoforms with similar homology.