Influence of the Carboxy Terminus of Serum Amyloid A on Protein Oligomerization, Misfolding, and Fibril Formation

The fibrillar deposition of serum amyloid A (SAA) has been linked to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform, SAA2.2, from the CE/J mouse strain, as a model system to explore the inherent structural and biophysical properties of SAA. Despite its nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in vitro, suggesting that SAA proteins are inherently amyloidogenic. However, whereas the importance of the amino terminus of SAA for fibril formation has been well documented, the influence of the proline-rich and presumably disordered carboxy terminus remains poorly understood. To clarify the inherent role of the carboxy terminus in the oligomerization and fibrillation of SAA, we truncated the proline-rich final 13 residues of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal truncated SAA2.2 (SAA2.2ΔC) did not oligomerize to a hexamer or octamer, but formed a high molecular weight soluble aggregate. Moreover, SAA2.2ΔC also exhibited a pronounced decrease in the rate of fibril formation. Intriguingly, when equimolar amounts of denatured SAA2.2 and SAA2.2ΔC were mixed and allowed to refold together, the mixture formed an octamer and exhibited rapid fibrillation kinetics, similar to those for full-length SAA2.2. These results suggest that the carboxy terminus of SAA, which is highly conserved among SAA sequences in all vertebrates, might play important structural roles, including modulating the folding, oligomerization, misfolding, and fibrillation of SAA

Characterization of hSAA1.1 and MetSAA1.1 by SDS-PAGE, SEC, far UV-CD, tryptophan fluorescence, and thermal denaturation studies.

<p>(A) SDS-PAGE gel (lanes: 1, protein ladder; 2, hSAA1.1; 3, MetSAA1.1 (B) SEC elution profiles of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line); (C) far UV-CD spectra of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line); (D) Thermal denaturation profiles of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line) (E) Tryptophan emission spectra of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line); (F) Tryptophan fluorescence-based thermal denaturation profiles of MetSAA1.1 (red solid line) and hSAA1.1 (blue solid line). The concentration of protein used in all the experiments was 20 µM. All experiments were performed at 4°C.</p

Characterization of aggregation of MetSAA1.1 and hSAA1.1 by the ThT Fluorescence assay, Congo red binding assay, far UV CD, and solubility assay.

<p>(A) ThT fluorescence intensity profile for MetSAA1.1 (black bars) and hSAA1.1 (red bars); (B) Congo red absorbance spectra for Congo red only (blue solid line); Congo red plus MetSAA1.1 sample (red solid line); MetSAA1.1 difference spectra (red dash line); Congo red plus hSAA1.1 sample (black solid line); hSAA1.1 difference spectra (black dash line); (C) Far UV CD spectra of MetSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (D) Far UV CD spectra of hSAA1.1 samples incubated at 37°C for 6 h (red solid line), 24 h (blue solid line), and 72 h (green solid line); (E) solubility profile for MetSAA1.1 (black solid line) and hSAA1.1 (red solid line). The starting concentration of protein was 20 µM. All assays were performed after the proteins were allowed to aggregate at 37°C.</p

Characterization of “seeding” properties of MetSAA1.1 and hSAA1.1 by ThT fluorescence assay.

<p>(A) ThT fluorescence intensity profile for freshly refolded MetSAA1.1 only (black bars) and MetSAA1.1+ MetSAA1.1 “seed” (gray bars); (B) ThT fluorescence intensity profile for freshly refolded hSAA1.1 only (black bars) and hSAA1.1+ hSAA1.1 “seed” (gray bars). The concentration of protein was 20 µM. ThT fluorescence intensities were recorded by incubating the samples at 37°C.</p