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Characterization of the oligomerization and aggregation of human Serum Amyloid A.

Patke S, Srinivasan S, Maheshwari R, Srivastava SK, Aguilera JJ, Colón W, Kane RS - PLoS ONE (2013)

Bottom Line: We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C.Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway.A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA.

ABSTRACT
The fibrillation of Serum Amyloid A (SAA) - a major acute phase protein - is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA - human SAA1.1 (hSAA1.1) - using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C. Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

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Characterization of hSAA1.1 and MetSAA1.1 by SDS-PAGE, SEC, far UV-CD, tryptophan fluorescence, and thermal denaturation studies.(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.
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pone-0064974-g001: Characterization of hSAA1.1 and MetSAA1.1 by SDS-PAGE, SEC, far UV-CD, tryptophan fluorescence, and thermal denaturation studies.(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.

Mentions: Both versions of SAA were expressed, purified and refolded using the protocols described in the experimental procedures section (See Supporting Information for more details about the sequence, UniProtKD ID, estimated MW, and extinction coefficient values of the proteins). In order to have a consistent starting point for all the studies, the experiments were performed on freshly refolded protein. By doing so, we ensured that conformational changes associated with the storage of proteins for extended periods of time did not affect the consistency of the results [35]. Given the marginal stability of hSAA1.1, as discussed later in the text, refolding was performed at 4°C. Refolded protein was assessed for its purity by using SDS-PAGE (Fig. 1A) and ESI-MS (data not shown). As expected, MetSAA1.1 (MW ∼ 11.81 kDa) and hSAA1.1 (MW ∼ 11.68 kDa) showed a single band with MW between 6 and 16 kDa (Fig. 1A). Amino-terminal MS-MS analysis of the first 7 amino acids yielded MRSFFSF for MetSAA1.1 and RSFFSFL for hSAA1.1. These amino acid sequences are also consistent with the sequences of the protein available in the literature [8], [12], [18], [28].


Characterization of the oligomerization and aggregation of human Serum Amyloid A.

Patke S, Srinivasan S, Maheshwari R, Srivastava SK, Aguilera JJ, Colón W, Kane RS - PLoS ONE (2013)

Characterization of hSAA1.1 and MetSAA1.1 by SDS-PAGE, SEC, far UV-CD, tryptophan fluorescence, and thermal denaturation studies.(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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672174&req=5

pone-0064974-g001: Characterization of hSAA1.1 and MetSAA1.1 by SDS-PAGE, SEC, far UV-CD, tryptophan fluorescence, and thermal denaturation studies.(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.
Mentions: Both versions of SAA were expressed, purified and refolded using the protocols described in the experimental procedures section (See Supporting Information for more details about the sequence, UniProtKD ID, estimated MW, and extinction coefficient values of the proteins). In order to have a consistent starting point for all the studies, the experiments were performed on freshly refolded protein. By doing so, we ensured that conformational changes associated with the storage of proteins for extended periods of time did not affect the consistency of the results [35]. Given the marginal stability of hSAA1.1, as discussed later in the text, refolding was performed at 4°C. Refolded protein was assessed for its purity by using SDS-PAGE (Fig. 1A) and ESI-MS (data not shown). As expected, MetSAA1.1 (MW ∼ 11.81 kDa) and hSAA1.1 (MW ∼ 11.68 kDa) showed a single band with MW between 6 and 16 kDa (Fig. 1A). Amino-terminal MS-MS analysis of the first 7 amino acids yielded MRSFFSF for MetSAA1.1 and RSFFSFL for hSAA1.1. These amino acid sequences are also consistent with the sequences of the protein available in the literature [8], [12], [18], [28].

Bottom Line: We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C.Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway.A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA.

ABSTRACT
The fibrillation of Serum Amyloid A (SAA) - a major acute phase protein - is believed to play a role in the disease Amyloid A (AA) Amyloidosis. To better understand the amyloid formation pathway of SAA, we characterized the oligomerization, misfolding, and aggregation of a disease-associated isoform of human SAA - human SAA1.1 (hSAA1.1) - using techniques ranging from circular dichroism spectroscopy to atomic force microscopy, fluorescence spectroscopy, immunoblot studies, solubility measurements, and seeding experiments. We found that hSAA1.1 formed alpha helix-rich, marginally stable oligomers in vitro on refolding and cross-beta-rich aggregates following incubation at 37°C. Strikingly, while hSAA1.1 was not highly amyloidogenic in vitro, the addition of a single N-terminal methionine residue significantly enhanced the fibrillation propensity of hSAA1.1 and modulated its fibrillation pathway. A deeper understanding of the oligomerization and fibrillation pathway of hSAA1.1 may help elucidate its pathological role.

Show MeSH
Related in: MedlinePlus