Incomplete Refolding of Antibody Light Chains to Non-Native, Protease-Sensitive Conformations Leads to Aggregation: A Mechanism of Amyloidogenesis in Patients?

Genetic, biochemical, and pharmacologic evidence supports the hypothesis that conformationally altered or misfolded protein states enable aggregation and cytotoxicity in the systemic amyloid diseases. Reversible structural fluctuations of natively folded proteins are involved in the aggregation of many degenerative disease associated proteins. Herein, we use antibody light chains (LCs) that form amyloid fibrils in AL amyloidosis to consider an alternative hypothesis of amyloidogenesis: that transient unfolding and incomplete extracellular refolding of secreted proteins can lead to metastable, alternatively folded states that are more susceptible to aggregation or to endoproteolysis that can release aggregation-prone fragments. Refolding of full-length λ6a LC dimers comprising an interchain disulfide bond from heat- or chaotrope-denatured ensembles in buffers yields the native dimeric state as well as alternatively folded dimers and aggregates. LC variants lacking an interchain disulfide bond appear to refold fully reversibly to the native state. The conformation of a backbone peptidyl-proline amide in the LC constant domain, which is cis in the native state, may determine whether the LC refolds back to the native state. A proline to alanine (P147A) LC variant, which cannot form the native cis-amide conformation, forms amyloid fibrils from the alternatively folded ensemble, whereas all the full-length λ6a LCs we have studied to date do not form amyloid under analogous conditions. P147A LC variants are susceptible to endoproteolysis by thrombin, enabling amyloidogenesis of the fragments released. Thus, non-native LC structural ensembles containing a tyrosine 146-proline 147 trans-amide bond can initiate and propagate amyloid formation, either directly or after aberrant endoproteolysis.