Limits...
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.

Show MeSH

Related in: MedlinePlus

Light scattering intensities measured on-line by in situ DLS.(A) 1 g/L light chain solution in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–), NaH2PO4 (…), Na2SO4 (―) (runs 3, 4, 6 and 9 in Table 1); (B) The same as (A) but salts are compared at similar ionic strength (see text): 0.45 M NaCl (–), 0.49 M NaH2PO4 (…), 0.15 M Na2SO4 (―) (runs 4, 6 and 8 in Table 1); (C) The same as (A) but for a solution in 20 mM HCl buffer at pH 2.0, with 0.45 M NaCl (―) and KCl (…) (runs 4 and 10 in Table 1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3303827&req=5

pone-0033372-g003: Light scattering intensities measured on-line by in situ DLS.(A) 1 g/L light chain solution in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–), NaH2PO4 (…), Na2SO4 (―) (runs 3, 4, 6 and 9 in Table 1); (B) The same as (A) but salts are compared at similar ionic strength (see text): 0.45 M NaCl (–), 0.49 M NaH2PO4 (…), 0.15 M Na2SO4 (―) (runs 4, 6 and 8 in Table 1); (C) The same as (A) but for a solution in 20 mM HCl buffer at pH 2.0, with 0.45 M NaCl (―) and KCl (…) (runs 4 and 10 in Table 1).

Mentions: The situation changed when the concentrations of NaCl and NaH2PO4 were increased to 0.45 M and 0.49 M, respectively (run 4 and 6 in Table 1) or when Na2SO4 was added (runs 7 to 9 in Table 1). The stability behavior for the different salts and different salt concentrations, followed by DLS, is shown in Figure 3. In Figure 3A, the results obtained using different salts but at the same constant concentration of 0.15 M are compared, while in Figure 3B salts are compared at similar ionic strength I, being I defined as , where ci is the concentration of ionic species and zi the corresponding valence (I is equal to 0.45 M for a 0.45 M NaCl and 0.49 M NaH2PO4 solution, and equal to 0.35 M for a 0.15 M Na2SO4 solution). In all cases where instability occurred, the scattered intensity increased almost linearly from the beginning of the incubation, without the lag phase typically encountered in amyloidogenic systems [22], indicating that the aggregate formation started immediately after incubation without a nucleation process. NaH2PO4 followed a behavior similar to NaCl, i.e., aggregation was observed only at sufficiently large concentration values, but aggregation was slower with NaH2PO4 than with NaCl. Instead, Na2SO4 showed a peculiar effect in accelerating the aggregation at low pH, which occurred even at low salt concentration (64 mM, corresponding to an ionic strength of 0.15 M).


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 scattering intensities measured on-line by in situ DLS.(A) 1 g/L light chain solution in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–), NaH2PO4 (…), Na2SO4 (―) (runs 3, 4, 6 and 9 in Table 1); (B) The same as (A) but salts are compared at similar ionic strength (see text): 0.45 M NaCl (–), 0.49 M NaH2PO4 (…), 0.15 M Na2SO4 (―) (runs 4, 6 and 8 in Table 1); (C) The same as (A) but for a solution in 20 mM HCl buffer at pH 2.0, with 0.45 M NaCl (―) and KCl (…) (runs 4 and 10 in Table 1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0033372-g003: Light scattering intensities measured on-line by in situ DLS.(A) 1 g/L light chain solution in 20 mM HCl buffer at pH 2.0 without salt (▵), with 0.15 M NaCl (–), NaH2PO4 (…), Na2SO4 (―) (runs 3, 4, 6 and 9 in Table 1); (B) The same as (A) but salts are compared at similar ionic strength (see text): 0.45 M NaCl (–), 0.49 M NaH2PO4 (…), 0.15 M Na2SO4 (―) (runs 4, 6 and 8 in Table 1); (C) The same as (A) but for a solution in 20 mM HCl buffer at pH 2.0, with 0.45 M NaCl (―) and KCl (…) (runs 4 and 10 in Table 1).
Mentions: The situation changed when the concentrations of NaCl and NaH2PO4 were increased to 0.45 M and 0.49 M, respectively (run 4 and 6 in Table 1) or when Na2SO4 was added (runs 7 to 9 in Table 1). The stability behavior for the different salts and different salt concentrations, followed by DLS, is shown in Figure 3. In Figure 3A, the results obtained using different salts but at the same constant concentration of 0.15 M are compared, while in Figure 3B salts are compared at similar ionic strength I, being I defined as , where ci is the concentration of ionic species and zi the corresponding valence (I is equal to 0.45 M for a 0.45 M NaCl and 0.49 M NaH2PO4 solution, and equal to 0.35 M for a 0.15 M Na2SO4 solution). In all cases where instability occurred, the scattered intensity increased almost linearly from the beginning of the incubation, without the lag phase typically encountered in amyloidogenic systems [22], indicating that the aggregate formation started immediately after incubation without a nucleation process. NaH2PO4 followed a behavior similar to NaCl, i.e., aggregation was observed only at sufficiently large concentration values, but aggregation was slower with NaH2PO4 than with NaCl. Instead, Na2SO4 showed a peculiar effect in accelerating the aggregation at low pH, which occurred even at low salt concentration (64 mM, corresponding to an ionic strength of 0.15 M).

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