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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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Protein characterization.(A) SEC chromatogram of 1 g/L light chain solution in 25 mM PBS buffer at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–). Insertion: calibration curve obtained with bovine serum albumin (60 kDa), chymotrypsinogen A (25 kDa) and lysozyme (14.5 kDa); (B) and (C) Mass spectroscopy analysis in 25 mM PBS buffer at pH 7.4 (B) and in 20 mM HCl at pH 2.0 (C).
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pone-0033372-g001: Protein characterization.(A) SEC chromatogram of 1 g/L light chain solution in 25 mM PBS buffer at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–). Insertion: calibration curve obtained with bovine serum albumin (60 kDa), chymotrypsinogen A (25 kDa) and lysozyme (14.5 kDa); (B) and (C) Mass spectroscopy analysis in 25 mM PBS buffer at pH 7.4 (B) and in 20 mM HCl at pH 2.0 (C).

Mentions: Many domains (both single variable and complete constant- variable domains) have been found to form dimers in physiological and stable conditions [16]. Particularly, amyloidogenic light chains are commonly found to be in equilibrium between the dimer and the monomeric form in patients' urine [16]. After the production and purification step, the protein has been characterized in terms of monomer-dimer equilibrium at physiological and low pH. The SEC chromatograms reported in Figure 1A show a single, symmetric peak under both conditions. According to the column calibration curve, the elution volume of such a peak corresponds to a molecular weight of 42 kDa, indicating the presence of a dimer consisting of both variable and constant domain [1]. The linearity between the elution volume and the natural logarithm of the molecular weight applies well only for globular proteins and non-spherical macromolecules can deviate from such relationship [19]. Therefore, to get a more accurate evaluation of the molecular weight, mass spectroscopy (MS) analysis was performed. The results in Figure 1B and 1C show one single peak with molecular weight equal to 45.7 kDa for both conditions, in agreement with SEC analysis. It is worth noting that the results of SEC and MS are inconsistent with the western Blot and SDS-PAGE analysis (data not shown), which revealed two bands, at around 50 kDa and 25 kDa. This may arise from the denaturating conditions used in the electrophoresis analysis, which can induce breakage of the dimer disulphide-bond. Indeed, when SDS-Page was performed in the presence of a reducing agent, only the 25 kDa monomer band was detected. We can conclude that the produced light chains are composed of both constant and variable part and associate into dimers through covalent disulphide bonds, which can be broken under reducing 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)

Protein characterization.(A) SEC chromatogram of 1 g/L light chain solution in 25 mM PBS buffer at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–). Insertion: calibration curve obtained with bovine serum albumin (60 kDa), chymotrypsinogen A (25 kDa) and lysozyme (14.5 kDa); (B) and (C) Mass spectroscopy analysis in 25 mM PBS buffer at pH 7.4 (B) and in 20 mM HCl at pH 2.0 (C).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033372-g001: Protein characterization.(A) SEC chromatogram of 1 g/L light chain solution in 25 mM PBS buffer at pH 7.4 (―) and in 20 mM HCl at pH 2.0 (–). Insertion: calibration curve obtained with bovine serum albumin (60 kDa), chymotrypsinogen A (25 kDa) and lysozyme (14.5 kDa); (B) and (C) Mass spectroscopy analysis in 25 mM PBS buffer at pH 7.4 (B) and in 20 mM HCl at pH 2.0 (C).
Mentions: Many domains (both single variable and complete constant- variable domains) have been found to form dimers in physiological and stable conditions [16]. Particularly, amyloidogenic light chains are commonly found to be in equilibrium between the dimer and the monomeric form in patients' urine [16]. After the production and purification step, the protein has been characterized in terms of monomer-dimer equilibrium at physiological and low pH. The SEC chromatograms reported in Figure 1A show a single, symmetric peak under both conditions. According to the column calibration curve, the elution volume of such a peak corresponds to a molecular weight of 42 kDa, indicating the presence of a dimer consisting of both variable and constant domain [1]. The linearity between the elution volume and the natural logarithm of the molecular weight applies well only for globular proteins and non-spherical macromolecules can deviate from such relationship [19]. Therefore, to get a more accurate evaluation of the molecular weight, mass spectroscopy (MS) analysis was performed. The results in Figure 1B and 1C show one single peak with molecular weight equal to 45.7 kDa for both conditions, in agreement with SEC analysis. It is worth noting that the results of SEC and MS are inconsistent with the western Blot and SDS-PAGE analysis (data not shown), which revealed two bands, at around 50 kDa and 25 kDa. This may arise from the denaturating conditions used in the electrophoresis analysis, which can induce breakage of the dimer disulphide-bond. Indeed, when SDS-Page was performed in the presence of a reducing agent, only the 25 kDa monomer band was detected. We can conclude that the produced light chains are composed of both constant and variable part and associate into dimers through covalent disulphide bonds, which can be broken under reducing 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