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In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

Arosio P, Owczarz M, Müller-Späth T, Rognoni P, Beeg M, Wu H, Salmona M, Morbidelli M - PLoS ONE (2012)

Bottom Line: Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT.A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation.It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

ABSTRACT
Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m) at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

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Aggregates morphology.AFM pictures of light chain aggregates obtained after one month incubation in 20 mM HCl at pH 2.0 with 0.45 M NaCl (A), with 0.15 M Na2SO4 (B) or with 0.49 M NaH2PO4 (C) and after two hours incubation in 25 mM PBS at pH 7.4, T = 55°C (D) (runs 4, 8, 6 and 11 in Table 1).
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pone-0033372-g006: Aggregates morphology.AFM pictures of light chain aggregates obtained after one month incubation in 20 mM HCl at pH 2.0 with 0.45 M NaCl (A), with 0.15 M Na2SO4 (B) or with 0.49 M NaH2PO4 (C) and after two hours incubation in 25 mM PBS at pH 7.4, T = 55°C (D) (runs 4, 8, 6 and 11 in Table 1).

Mentions: The aggregate morphology was further studied by AFM microscopy and hydrated thin film ATR-FTIR. In Figure 6 the pictures of samples taken after one month incubation in the conditions of runs 4, 6, 8 and 11 in Table 1 are shown. It can be seen that only spherical aggregates are visible for all the investigated conditions.


In vitro aggregation behavior of a non-amyloidogenic λ light chain dimer deriving from U266 multiple myeloma cells.

Arosio P, Owczarz M, Müller-Späth T, Rognoni P, Beeg M, Wu H, Salmona M, Morbidelli M - PLoS ONE (2012)

Aggregates morphology.AFM pictures of light chain aggregates obtained after one month incubation in 20 mM HCl at pH 2.0 with 0.45 M NaCl (A), with 0.15 M Na2SO4 (B) or with 0.49 M NaH2PO4 (C) and after two hours incubation in 25 mM PBS at pH 7.4, T = 55°C (D) (runs 4, 8, 6 and 11 in Table 1).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303827&req=5

pone-0033372-g006: Aggregates morphology.AFM pictures of light chain aggregates obtained after one month incubation in 20 mM HCl at pH 2.0 with 0.45 M NaCl (A), with 0.15 M Na2SO4 (B) or with 0.49 M NaH2PO4 (C) and after two hours incubation in 25 mM PBS at pH 7.4, T = 55°C (D) (runs 4, 8, 6 and 11 in Table 1).
Mentions: The aggregate morphology was further studied by AFM microscopy and hydrated thin film ATR-FTIR. In Figure 6 the pictures of samples taken after one month incubation in the conditions of runs 4, 6, 8 and 11 in Table 1 are shown. It can be seen that only spherical aggregates are visible for all the investigated conditions.

Bottom Line: Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT.A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation.It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

ABSTRACT
Excessive production of monoclonal light chains due to multiple myeloma can induce aggregation-related disorders, such as light chain amyloidosis (AL) and light chain deposition diseases (LCDD). In this work, we produce a non-amyloidogenic IgE λ light chain dimer from human mammalian cells U266, which originated from a patient suffering from multiple myeloma, and we investigate the effect of several physicochemical parameters on the in vitro stability of this protein. The dimer is stable in physiological conditions and aggregation is observed only when strong denaturating conditions are applied (acidic pH with salt at large concentration or heating at melting temperature T(m) at pH 7.4). The produced aggregates are spherical, amorphous oligomers. Despite the larger β-sheet content of such oligomers with respect to the native state, they do not bind Congo Red or ThT. The impossibility to obtain fibrils from the light chain dimer suggests that the occurrence of amyloidosis in patients requires the presence of the light chain fragment in the monomer form, while dimer can form only amorphous oligomers or amorphous deposits. No aggregation is observed after denaturant addition at pH 7.4 or at pH 2.0 with low salt concentration, indicating that not a generic unfolding but specific conformational changes are necessary to trigger aggregation. A specific anion effect in increasing the aggregation rate at pH 2.0 is observed according to the following order: SO(4)(-)≫Cl(-)>H(2)PO(4)(-), confirming the peculiar role of sulfate in promoting protein aggregation. It is found that, at least for the investigated case, the mechanism of the sulfate effect is related to protein secondary structure changes induced by anion binding.

Show MeSH
Related in: MedlinePlus