The Kinetic Stability of a Full-Length Antibody Light Chain Dimer Determines whether Endoproteolysis Can Release Amyloidogenic Variable Domains

Abstract

Light chain amyloidosis (AL amyloidosis) appears to be caused by the aggregation of an antibody light chain (LC) or fragment thereof, and is fatal if untreated. LCs are secreted from clonally expanded plasma cells as disulfide-linked dimers, with each monomer comprising one constant and one variable domain. The energetic contribution of each domain and the role of endoproteolysis in AL amyloidosis remain unclear. To investigate why only some LCs form amyloid and cause organ toxicity, we measured the aggregation propensity and kinetic stability of LC dimers and associated variable domains from AL amyloidosis patients and non-patients. All the variable domains studied readily form amyloid fibrils, whereas none of the full-length LC dimers, even those from AL amyloidosis patients, are amyloidogenic. Kinetic stability — that is, the free energy difference between the native state and the unfolding transition state — dictates the LC’s unfolding rate. Full-length LC dimers derived from AL amyloidosis patients unfold more rapidly than other full-length LC dimers and can be readily cleaved into their component domains by proteases, whereas non-amyloidogenic LC dimers are more kinetically stable and resistant to endoproteolysis. Our data suggest that amyloidogenic LC dimers are kinetically unstable (unfold faster) and thus are susceptible to endoproteolysis that results in the release amyloidogenic LC fragments, whereas other LCs are not as amenable to unfolding and endoproteolysis and are therefore aggregation resistant. Pharmacologic kinetic stabilization of the full-length LC dimer could be a useful strategy to treat AL amyloidosis.

Stabilization of amyloidogenic immunoglobulin light chains by small molecules

“…Sequence also seems to play a role, as not all destabilized FL LCs aggregate in patients (4-8). How aggregation occurs in patients is not known, but several processes have been described in vitro, including destabilization-dependent endoproteolysis that releases amyloidogenic LC fragments (4,9,10). LC fragments including V domains are observed in patient deposits alongside FL LCs (11-13).Since we do not understand the structure-proteotoxicity relationships driving AL, a conservative strategy is to block FL LC misfolding at its origin by stabilizing the FL LC native state.…”

“…However, since amyloidogenic full-length (FL) LCs are generally less stable than nonamyloidogenic FL LCs, they can misfold, or misfold and misassemble, into nonnative species including cross-β-sheet amyloid fibrils, which are a hallmark of AL (4)(5)(6)(7)(8). Sequence also seems to play a role, as not all destabilized FL LCs aggregate in patients (4)(5)(6)(7)(8). How aggregation occurs in patients is not known, but several processes have been described in vitro, including destabilization-dependent endoproteolysis that releases amyloidogenic LC fragments (4,9,10).…”

In vitro co-expression of human amyloidogenic immunoglobulin light and heavy chain proteins: a relevant cell-based model of AL amyloidosis

“…In addition, the intact Ig generated by our cell system can be used to determine the role of thermodynamic and kinetic structural stability in protein misfolding. Previous studies by our group and others have established the kinetic stability of LC alone and its likely role in protein misfolding in AL [2,23]. The model system described in this report enables us to extend these studies to intact Ig, and thereby determine the potential role of kinetic barriers in the misfolding of both LC and HC in AL amyloidosis.…”

Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic Protein

“…This reengagement of destabilized ALLC with 'pro-folding' factors similarly prevents targeting to degradation pathways, resulting in the ER retention observed following ATF6 activation (15). In contrast, energetically normal LCs such as JTO can efficiently fold following release from chaperoning pathways in the ATF6remodeled ER environment due to its increased stability relative to ALLC (15,49). This allows JTO to adopt a trafficking-competent conformation that can then be secreted to the extracellular space.…”