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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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Light chain structural changes induced by pH, denaturant addition and temperature.(A) CD spectra for the 0.3 g/L light chain solution in 25 mM PBS at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–); (B) Fraction of unfolded protein as a function of guanidinium hydrochloride (GuHCl) concentration evaluated by intrinsic tryptophan fluorescence measurements (see Materials and Methods). The continuous line represents the interpolation of experimental data according to Eq. 1; (C) Fraction of unfolded protein as a function of temperature evaluated by CD measurements (see Materials and Methods). The continuous line corresponds to the interpolation of experimental data according to Eq.1.
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pone-0033372-g002: Light chain structural changes induced by pH, denaturant addition and temperature.(A) CD spectra for the 0.3 g/L light chain solution in 25 mM PBS at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–); (B) Fraction of unfolded protein as a function of guanidinium hydrochloride (GuHCl) concentration evaluated by intrinsic tryptophan fluorescence measurements (see Materials and Methods). The continuous line represents the interpolation of experimental data according to Eq. 1; (C) Fraction of unfolded protein as a function of temperature evaluated by CD measurements (see Materials and Methods). The continuous line corresponds to the interpolation of experimental data according to Eq.1.

Mentions: In Figure 2A the far-UV CD spectra of the protein at pH 7.4 and pH 2.0 are reported. The spectrum at pH 7.4 shows a minimum at 220 nm characteristic of the Greek key β-barrel folding of the immunoglobulin fragment [20], [21]. The β-sheet structure content increases as the pH value decreases from 7.4 to 2.0 and the minimum shifts from 220 to 218 nm.


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)

Light chain structural changes induced by pH, denaturant addition and temperature.(A) CD spectra for the 0.3 g/L light chain solution in 25 mM PBS at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–); (B) Fraction of unfolded protein as a function of guanidinium hydrochloride (GuHCl) concentration evaluated by intrinsic tryptophan fluorescence measurements (see Materials and Methods). The continuous line represents the interpolation of experimental data according to Eq. 1; (C) Fraction of unfolded protein as a function of temperature evaluated by CD measurements (see Materials and Methods). The continuous line corresponds to the interpolation of experimental data according to Eq.1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033372-g002: Light chain structural changes induced by pH, denaturant addition and temperature.(A) CD spectra for the 0.3 g/L light chain solution in 25 mM PBS at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–); (B) Fraction of unfolded protein as a function of guanidinium hydrochloride (GuHCl) concentration evaluated by intrinsic tryptophan fluorescence measurements (see Materials and Methods). The continuous line represents the interpolation of experimental data according to Eq. 1; (C) Fraction of unfolded protein as a function of temperature evaluated by CD measurements (see Materials and Methods). The continuous line corresponds to the interpolation of experimental data according to Eq.1.
Mentions: In Figure 2A the far-UV CD spectra of the protein at pH 7.4 and pH 2.0 are reported. The spectrum at pH 7.4 shows a minimum at 220 nm characteristic of the Greek key β-barrel folding of the immunoglobulin fragment [20], [21]. The β-sheet structure content increases as the pH value decreases from 7.4 to 2.0 and the minimum shifts from 220 to 218 nm.

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