Heavy-chain antibody fragments as model proteins to investigate the molecular mechanism of formation of amyloid fibrils

Abstract

Amyloidoses are rather common and widespread debilitating and/or life-threatening diseases. They display structurally identical amyloid fibrils, and consequently appears to share a common, but still poorly understood, mechanism of fibrillogenesis. Variable domains of camelid heavy-chain antibodies, referred to as VHHs or nanobodies, were chosen to investigate the factors that favour amyloid fibrillogenesis and to get a better understanding of the mechanism of Ig fibrillogenesis. Our study focused on the contribution of protein regions, i.e. the framework (FR) and the complementary determining regions (CDRs) of VHHs, to the formation of amyloid fibrils. Three VHHs were selected: cAb HuL6, cAb BcII10 and cAb BcII10 H H H, a chimera bearing the FR of cAb BcII10 and the CDRs of cAb HuL6. We first established experimental conditions suitable for fibrillogenesis: even the especially stable and soluble VHHs can form amyloid fibrils in denaturing conditions. This comforts the proposal that amyloid conformation is a generic structural form that all proteins can adopt in the appropriate conditions. Amyloid fibrils were characterised by transmission electron microscopy (TEM), circular dichroism (CD), thioflavin T (ThT) fluorescence and X-ray fibre diffraction. The kinetics of fibril formation were monitored by turbidity measurements. We then determined conditions suitable for characterising self-seeding and cross-seeding kinetics, and ascertained that the three VHHs convert into amyloid fibrils according to a nucleation-dependent process. Fibril elongation rates in self-seeding and cross-seeding experiments suggest that amyloid fibril elongation occurs through sequential addition of protein monomers to preformed fibrils. The kinetics of aggregation and self-seeding of cAb-HuL6 and cAb-BcII10 differ significantly, and a remarkable specificity for cross-seeding was observed, with cAb-BcII10 being unable to cross-seed the formation of fibrils by the two other VHHs, while cAb-HuL6 and cAb BcII10 H-H-H mutually cross-seed each other’s fibril formation. With the help of three prediction algorithms (AGGRESCAN, Zyggregator and 3D-profile), we showed that various peptides of the VHHs had the ability to form fibrils by themselves. Our results strongly suggest that the sequences of the regions forming the core of the fibrils essentially correspond to the CDRs. Concurrently, we set up experimental conditions for interrupting the fibril formation process and we characterised the multimer and fibril species formed in the early stages of fibrillogenesis by analytical ultracentrifugation (AUC) and small angle neutron scattering (SANS) ex situ.Heavy-chain antibody fragments as model proteins to investigate the molecular mechanism of formation of amyloid fibril