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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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Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1.AFM analysis of (A) MetSAA1.1, 3 h, 37°C; (B) MetSAA1.1, 72 h, 37°C; (C) hSAA1.1, 3 h, 37°C; (D) hSAA1.1, 200 h, 37°C; Immunoblot analysis of aggregates formed by MetSAA1.1 using (E) A11 antibody and (F) OC antibody; Immunoblot analysis of aggregates formed by hSAA1.1 using (G) A11 antibody and (H) OC antibody. All scale bars for AFM images represent 1 µm.
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pone-0064974-g003: Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1.AFM analysis of (A) MetSAA1.1, 3 h, 37°C; (B) MetSAA1.1, 72 h, 37°C; (C) hSAA1.1, 3 h, 37°C; (D) hSAA1.1, 200 h, 37°C; Immunoblot analysis of aggregates formed by MetSAA1.1 using (E) A11 antibody and (F) OC antibody; Immunoblot analysis of aggregates formed by hSAA1.1 using (G) A11 antibody and (H) OC antibody. All scale bars for AFM images represent 1 µm.

Mentions: Intrigued by the observation that both versions of hSAA1.1 exhibited similar aggregation kinetics, but differed in the solubilities of their aggregates, we decided to further characterize the species formed during aggregation. We performed AFM studies for morphological characterization of the protein aggregates. We incubated the MetSAA1.1 “native’-like” oligomers (see AFM in Fig. S5A in File S1) at 37°C and performed AFM on protein samples obtained after different time intervals. As shown in Figure 3A, “spherical” aggregates with heights in the range of ∼ 4–6 nm were predominantly observed following the incubation of MetSAA1.1 at 37°C for 3 h. Further incubation up to 6 h at 37°C resulted in the formation of short fibrils with “rod-like” architecture (Fig. S6A in File S1and inset) with heights ranging from 4–6 nm and lengths ranging from 10–15 nm. Small “spherical” prefibrillar aggregates, similar to the ones observed after 3 h, were observed along with these short fibrils. Incubating MetSAA1.1 at 37°C for 24 h resulted in the formation of large “rod-like” fibrils (Fig. S6B in File S1). By 72 h, MetSAA1.1 formed 15–20 µm long full-length mature amyloid fibrils (Fig. 3B). Although the heights of most of these fibrils were ∼ 4–6 nm, taller fibrils with heights of ∼ 10–12 nm were also observed. This observation would be consistent with the length-wise stacking of full-length fibrils over each other. Collectively, the AFM studies and solubility assay results for MetSAA1.1 suggest that the spherical aggregates formed by this protein might represent the population of soluble oligomers while the “rod-like” fibrillar structures might represent the population of insoluble aggregates.


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)

Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1.AFM analysis of (A) MetSAA1.1, 3 h, 37°C; (B) MetSAA1.1, 72 h, 37°C; (C) hSAA1.1, 3 h, 37°C; (D) hSAA1.1, 200 h, 37°C; Immunoblot analysis of aggregates formed by MetSAA1.1 using (E) A11 antibody and (F) OC antibody; Immunoblot analysis of aggregates formed by hSAA1.1 using (G) A11 antibody and (H) OC antibody. All scale bars for AFM images represent 1 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3672174&req=5

pone-0064974-g003: Biophysical characterization of aggregates formed by MetSAA1.1 and hSAA1.1.AFM analysis of (A) MetSAA1.1, 3 h, 37°C; (B) MetSAA1.1, 72 h, 37°C; (C) hSAA1.1, 3 h, 37°C; (D) hSAA1.1, 200 h, 37°C; Immunoblot analysis of aggregates formed by MetSAA1.1 using (E) A11 antibody and (F) OC antibody; Immunoblot analysis of aggregates formed by hSAA1.1 using (G) A11 antibody and (H) OC antibody. All scale bars for AFM images represent 1 µm.
Mentions: Intrigued by the observation that both versions of hSAA1.1 exhibited similar aggregation kinetics, but differed in the solubilities of their aggregates, we decided to further characterize the species formed during aggregation. We performed AFM studies for morphological characterization of the protein aggregates. We incubated the MetSAA1.1 “native’-like” oligomers (see AFM in Fig. S5A in File S1) at 37°C and performed AFM on protein samples obtained after different time intervals. As shown in Figure 3A, “spherical” aggregates with heights in the range of ∼ 4–6 nm were predominantly observed following the incubation of MetSAA1.1 at 37°C for 3 h. Further incubation up to 6 h at 37°C resulted in the formation of short fibrils with “rod-like” architecture (Fig. S6A in File S1and inset) with heights ranging from 4–6 nm and lengths ranging from 10–15 nm. Small “spherical” prefibrillar aggregates, similar to the ones observed after 3 h, were observed along with these short fibrils. Incubating MetSAA1.1 at 37°C for 24 h resulted in the formation of large “rod-like” fibrils (Fig. S6B in File S1). By 72 h, MetSAA1.1 formed 15–20 µm long full-length mature amyloid fibrils (Fig. 3B). Although the heights of most of these fibrils were ∼ 4–6 nm, taller fibrils with heights of ∼ 10–12 nm were also observed. This observation would be consistent with the length-wise stacking of full-length fibrils over each other. Collectively, the AFM studies and solubility assay results for MetSAA1.1 suggest that the spherical aggregates formed by this protein might represent the population of soluble oligomers while the “rod-like” fibrillar structures might represent the population of insoluble aggregates.

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