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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 salt addition monitored by CD (A) and ANS binding (B).Experiments were performed at 20°C for a 0.3 g/L protein solution in 25 mM PBS at pH 7.4 (…) and in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–),0.15 M Na2SO4 (―), 0.45 M NaCl (○), 0.49 M NaH2PO4 (□) and 0.5 M Na2SO4 (×).
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pone-0033372-g004: Light chain structural changes induced by salt addition monitored by CD (A) and ANS binding (B).Experiments were performed at 20°C for a 0.3 g/L protein solution in 25 mM PBS at pH 7.4 (…) and in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–),0.15 M Na2SO4 (―), 0.45 M NaCl (○), 0.49 M NaH2PO4 (□) and 0.5 M Na2SO4 (×).

Mentions: To investigate such peculiar effect, the initial light chain secondary structure in the presence of the sulfate and the chloride anion was characterized by CD spectroscopy and ANS binding, a method probing the solvent accessibility of hydrophobic patches. As shown in Figure 4A, with respect to the case in the absence of salt, in the presence of NaCl the minimum in the far-UV CD spectrum is decreased, indicating an increase of β-sheet structure; moreover, the minimum shifts from 218 to 217 nm. Such structural rearrangement is accompanied by a burying of hydrophobic patches, as indicated by the decrease of the maximum ANS fluorescence (Figure 4B) as a consequence of the reduced binding of the hydrophobic dye. In the presence of the sulfate anions, the light chain structure changes significantly: the far-UV CD spectrum shows a minimum at 204.6 nm, corresponding to a disordered structure (Figure 4A). In such a more open, disordered structure, with respect to the β-sheet, the hydrophobic patches expose more in the solvent. Indeed, the ANS binding in this case is larger than that in the presence of the chloride anion, as indicated by the increase in maximum ANS fluorescence (Figure 4B). It is likely that such significant structural change forms an intermediate more prone to aggregate. On the other hand, by increasing the salt concentration (by adding 0.45 M NaCl, 0.49 M NaH2PO4 and 0.5 M Na2SO4), the maximum ANS fluorescence decreases (Figure 4B), indicating less hydrophobic conformations. This is likely due to the additional effects induced at high salt concentration, which will be discussed in the Discussion section. Moreover, it is worth noticing that at 0.5 M Na2SO4 the fast aggregation rate affects the ANS measurements.


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 salt addition monitored by CD (A) and ANS binding (B).Experiments were performed at 20°C for a 0.3 g/L protein solution in 25 mM PBS at pH 7.4 (…) and in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–),0.15 M Na2SO4 (―), 0.45 M NaCl (○), 0.49 M NaH2PO4 (□) and 0.5 M Na2SO4 (×).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033372-g004: Light chain structural changes induced by salt addition monitored by CD (A) and ANS binding (B).Experiments were performed at 20°C for a 0.3 g/L protein solution in 25 mM PBS at pH 7.4 (…) and in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–),0.15 M Na2SO4 (―), 0.45 M NaCl (○), 0.49 M NaH2PO4 (□) and 0.5 M Na2SO4 (×).
Mentions: To investigate such peculiar effect, the initial light chain secondary structure in the presence of the sulfate and the chloride anion was characterized by CD spectroscopy and ANS binding, a method probing the solvent accessibility of hydrophobic patches. As shown in Figure 4A, with respect to the case in the absence of salt, in the presence of NaCl the minimum in the far-UV CD spectrum is decreased, indicating an increase of β-sheet structure; moreover, the minimum shifts from 218 to 217 nm. Such structural rearrangement is accompanied by a burying of hydrophobic patches, as indicated by the decrease of the maximum ANS fluorescence (Figure 4B) as a consequence of the reduced binding of the hydrophobic dye. In the presence of the sulfate anions, the light chain structure changes significantly: the far-UV CD spectrum shows a minimum at 204.6 nm, corresponding to a disordered structure (Figure 4A). In such a more open, disordered structure, with respect to the β-sheet, the hydrophobic patches expose more in the solvent. Indeed, the ANS binding in this case is larger than that in the presence of the chloride anion, as indicated by the increase in maximum ANS fluorescence (Figure 4B). It is likely that such significant structural change forms an intermediate more prone to aggregate. On the other hand, by increasing the salt concentration (by adding 0.45 M NaCl, 0.49 M NaH2PO4 and 0.5 M Na2SO4), the maximum ANS fluorescence decreases (Figure 4B), indicating less hydrophobic conformations. This is likely due to the additional effects induced at high salt concentration, which will be discussed in the Discussion section. Moreover, it is worth noticing that at 0.5 M Na2SO4 the fast aggregation rate affects the ANS measurements.

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