In Vitro Aggregation Behavior of a Non-Amyloidogenic λ Light Chain Dimer Deriving from U266 Multiple Myeloma Cells

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 Tm 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: SO4 −≫Cl−>H2PO4 −, 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.

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

“…Whereas the denaturation of most LC was reversible, all -MM LC denatured irreversibly after heating. We found that -and -isotype LC had different stabilities in thermal denaturation, with an average T m of 53 Ϯ 5 and 59 Ϯ 2°C, respectively, which corresponds to previously published data (28,30,44,64). We did not find strong systematic differences in stability between AL-and MM-LC.…”

A Mechanistic Model for Amorphous Protein Aggregation of Immunoglobulin-like Domains

“…This has been seen several times before, especially for the amorphous aggregation of immunoglobulin-like domains. 12,20,32,39 The fact that I27 aggregation occurs from a specific, TFE-induced structure is reminiscent of recent work on the p53 core domain, which demonstrated that aggregation from the apoprotein is substantially faster than aggregation from the holo-protein. 40 It also agrees with observations that the stabilization of partly folded intermediate states causes increased aggregation in proteins such as transthyretin (TTR) and lysozyme.…”

Sulfate Anion Delays the Self-Assembly of Human Insulin by Modifying the Aggregation Pathway

“…In addition, the divalency of the anion confers peculiar properties in terms of charge screening, anion binding, and salt bridge formation. The overimposition of the different electrostatic effects can either promote (34)(35)(36) or inhibit (37) the protein aggregation propensity. In the case of an IgG2 monoclonal antibody, we have recently reported a maximum of the aggregation propensity as a function of sulfate concentration (22).…”