Investigation of serum amyloid a within animal species focusing on the 1-25 amino acid region

Abstract

AA amyloidosis, characterized by the misfolding of serum amyloid A (SAA) protein, is the most common amyloid protein disorder across multiple species. SAA is a positive-acute phase protein synthesized by the liver in response to inflammation or stress, and it normally associates with high-density lipoprotein at its N-terminus. In this study, we focused on the 1-25 amino acid (aa) region of the complete 104 aa SAA sequence to examine the aggregation propensity of AA amyloid. A library comprising eight peptides from different species was assembled for analysis. To access the aggregation propensity of each peptide region, a bioinformatic study was conducted using the algorithm TANGO. Congo red (CR) binding assays, Thioflavin T (ThT) assays, and transmission electron microscopy (TEM) were utilized to evaluate whether the synthesized peptides formed amyloid-like fibrils. All synthetic SAA 1-25 congeners resulted in amyloid-like fibrils formation (per CR and/or ThT staining and TEM detection) at the exception of the ferret SAA1-25 fragment, which generated plaque-like materials by TEM. Ten residues were preserved among SAA 1-25 congeners resulting in amyloid-like fibrils, i.e. F6, E9, A10, G13, D16, M17, A20, Y21, D23, and M24. Amino acid residues highlighted by this study may have a role in increasing the propensity for amyloid-like fibril formation. This study put an emphasis on region 1-25 in the mechanism of SAA1 misfolding.

Keywords: Animals; aggregation; amyloid-like fibrils; oligomers; serum amyloid A1; zoology.

Figure 1.

Representative visible spectra obtained from Congo red (CR) binding assays of the eight serum amyloid A1 synthetic peptides of the region 1-25 at 500 µM. Optical density (OD) data were plotted as function of wavelength (nm). The change in optical density (higher and right shift) is indicative of CR binding to β-plated sheets. Optical density measurements were recorded at 25 °C in 25 mM Tris buffer (pH 8) and 50% hexafluoroisopropanol (HFIP) after 5 days of incubation. ID# 1: human; ID# 2: common bottlenose dolphin; ID# 3: donkey; ID# 4: rhesus monkey; ID# 5: alpine ibex; ID# 6: Lesser-Egyptian jerboa; ID# 7: domestic ferret; ID# 8: chamois.

Figure 2.

Comparison of Thioflavin T (ThT) fluorescence intensity obtained at the end of fibrilization kinetics for synthetic SAA 1-25 fragments resourced from amino acid sequences of different animal species. The experiments were conducted using a concentration of 500 μM in 25 mM Tris buffer (pH 8) and 50% hexafluoroisopropanol (HFIP). Fragment peptides were monitored at 37 °C for 120 h. Average represents experimental triplicate. Fluorescence background signal was subtracted. Each numerical value in the figure corresponds with the respective organism as presented in Table 1. Specifically, ID# 1: human; ID# 2: common bottlenose dolphin; ID# 3: donkey; ID# 4: rhesus monkey; ID# 5: alpine ibex; ID# 6: Lesser-Egyptian jerboa; ID# 7: domestic ferret; ID# 8: chamois.

Figure 3.

Comparison of the Thioflavin T (ThT) fluorescence intensity. Graphs represent each species’ fibrilization intensity over 120 h (five days) in incubation at 37 °C. Three samples ran simultaneously were then averaged subtracting the background intensity from each prior to creating the graph. Error bars represent SEM. ID# 1: human; ID# 2: common bottlenose dolphin; ID# 3: donkey; ID# 4: rhesus monkey; ID# 5: alpine ibex; ID# 6: Lesser-Egyptian jerboa; ID# 7: domestic ferret; ID# 8: chamois.

Figure 4.

Transmission electron microscopy (TEM) was used to observe the different synthetic SAA fragment peptides solubilized at 500 µM in 25 mM Tris buffer (pH 8) and 50% hexafluoroisopropanol (HFIP), and subsequently incubated at 37 °C for seven days, at a magnification of 40K. Notation a corresponding to the human SAA1 peptide. Notation B corresponding to the common bottlenose dolphin SAA1 peptide. Notation C corresponding to the donkey SAA1 peptide. Notation D corresponding to the rhesus monkey SAA1 peptide. Notation E corresponding to the alpine ibex SAA1 peptide. Notation F corresponding to the Lesser-Egyptian jerboa SAA1 peptide. Notation G corresponding to the domestic ferret SAA1 peptide. Notation H corresponding to the chamois SAA1 peptide. Scale bar = 200 nm.

Figure 5.

Photomicrograph of the domestic ferret SAA1 peptide taken at low magnification (2000) by transmission electron microscopy. Experimental conditions are identical as in Figure 4.