VHHs as model proteins to investigate amyloid fibril formation

The term "amyloidosis" covers up a group of diseases associated with deposition in different organs of protein aggregates organized into amyloid fibrils. About twenty-five amyloidosis are known so far, amongst which Alzheimer's disease, type II diabetes and immunoglobulin amyloidosis [1].

Although the mechanism of amyloid fibrils formation at the molecular level is not yet completely understood, it has been shown that the capacity to form amyloid fibrils in vitro is an intrinsic property of all polypeptide chains [1]. The choice of model proteins to investigate the aggregation process in vitro is therefore no more restrained to proteins involved in amyloidosis but can be settled on a wide variety of proteins.

In this study, we have chosen two variable domains of camelid heavy-chain antibodies (referred to as VHHs or nanobodies), cAb-HuL6 and cAb-BcII10, and this choice was motivated by the following reasons:
- First, they are small monomeric domains (~14 kDa) presenting high stability and high solubility [2], which permits their expression with a high yield (20-40 mg.L-1).
- Second, a wide range of stable mutants of these two VHHs is available. Mutations located at the disulfide bond [3,4], the CDRs [3] and the framework have been introduced. Characterisation of the aggregating properties of these mutants will allow the investigation of the impact of these structural elements on the process of fibril formation.

In order to determine conditions in which cAb-HuL6 and cAb-BcII10 are more susceptible to form intermediates and thus amyloid fibrils, heat induced infolding experiments at pHs comprised in a range from 2,5 to 9,5 have been monitored by intrinsic fluorescence, ANS binding and circular dichroism. Then, aggregation experiments have been performed in the selected conditions and the presence of amyloid fibrils has been acknowledged by thioflavineT fluorescence experiments and electronic microscopy.

[1] Chiti, F. and Dobson, C. M., Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem., 75, 2006, 333-366.
[2] Dumoulin, M., Conrath, K., Van Meirhaeghe, A., Meersman, F., Heremans, K., Frenken, L. G., Muyldermans, S., Wyns, L. & Matagne, A., Single-domain antibody fragments with high conformational stability, Protein Sci., 11, 2002, 500-515.
[3] Saerens, D., Pellis, M., Loris, R., Pardon, E., Dumoulin, M., Matagne, A., Wyns, L., Muyldermans, S., Conrath, K., Identification of a universal VHH framework to graft non-canonical antigen-binding loops of camel single-domain antibodies, J. Mol. Biol., 352, 2005, 597-607.
[4] Saerens D., Conrath K., Govaert J., Muyldermans S., Disulfide bond introduction for general stabilization of immunoglobulin heavy-chain variable domains, J Mol Biol., 377, 2008, 478-488.