Formation of Calprotectin Inhibits Amyloid Aggregation of S100A8 and S100A9 Proteins

Abstract

Calcium-binding S100A8 and S100A9 proteins play a significant role in various disorders due to their pro-inflammatory functions. Substantially, they are also relevant in neurodegenerative disorders via the delivery of signals for the immune response. However, at the same time, they can aggregate and accelerate the progression of diseases. Natively, S100A8 and S100A9 exist as homo- and heterodimers, but upon aggregation, they form amyloid-like oligomers, fibrils, or amorphous aggregates. In this study, we aimed to elucidate the aggregation propensities of S100A8, S100A9, and their heterodimer calprotectin by investigating aggregation kinetics, secondary structures, and morphologies of the aggregates. For the first time, we followed the in vitro aggregation of S100A8, which formed spherical aggregates, unlike the fibrillar structures of S100A9 under the same conditions. The aggregates were sensitive to amyloid-specific ThT and ThS dyes and had a secondary structure composed of β-sheets. Similarly to S100A9, S100A8 protein was stabilized by calcium ions, resulting in aggregation inhibition. Finally, the formation of S100A8 and S100A9 heterodimers stabilized the proteins in the absence of calcium ions and prevented their aggregation.

Keywords: S100; aggregation; amyloid; inflammation.

Figure 1

S100A8 and S100A9 play roles in the neurodegeneration. S100A9 was detected in Tau neurofibrillary tangles and coaggregated with amyloid-β 40 (Aβ40). Both proteins are upregulated in microglial cells in response to amyloid plaques.

Figure 2

S100A8 and S100A9 amino acid sequence and secondary structure depiction with possible aggregation sites predicted by PASTA 2.0 (A). The aggregation-prone residues identified by Aggrescan3D using available protein structures (S100A8–1MR8; S100A9–1IRJ, CP-1XK4) (B). The positive scores (red, sticks) indicate high-potential aggregation sites, and negative scores (blue) show more stable residues.

Figure 3

Characterization of the CP formation. Size exclusion chromatography chromatogram of S100A8, S100A9, and CP (A). The first derivative of the normalized DSF fluorescence signal of S100A8 (B) and S100A9 (C) in the absence and presence of calcium ions (0, 50, 100, 200, 400, and 800 μM Ca²⁺). Calculated melting temperatures (using the first derivative) of each protein (D, E).

Figure 4

Aggregation kinetics of S100A8 (A), S100A9 (B), and CP (C) followed by ThT fluorescence intensity. The inflection points (t_i) of S100A8 aggregation curves (D) and half-time values (t₅₀) of S100A9 aggregation kinetics (E). ThT fluorescence intensity after 70 h of aggregation of S100A8 (F) and S100A9 (G) in the presence of calcium ions (0, 25, 50, 100, 200, 400, and 800 μM Ca²⁺).

Figure 5

FTIR (A) and CD spectra (B) of native S100A8, S100A9, and CP proteins and after 65 h of aggregation at 37 °C.

Figure 6

AFM images (A) of S100A8, S100A9, and CP after 65 h of aggregation at 37 °C (scale bar, 500 nm). Aggregates height distribution (B) with box plots indicating the interquartile range and error bars are for one standard deviation (sample size, 50). Fluorescence microscopy images (C) of S100A8, S100A9, CP aggregates, and buffer stained with ThS (scale bar, 10 μm). Transmission electron microscopy image (D) of S100A8 aggregates stained with uranyl acetate (scale bar, 500 nm).