Elongation of the C-terminal domain of an anti-amyloid β single-chain variable fragment increases its thermodynamic stability and decreases its aggregation tendency.
Amyloid β (Aβ) immunotherapy is considered a promising approach to Alzheimer disease treatment. In contrast to the use of complete antibodies, administration of single-chain variable fragments (scFv) has not been associated with either meningoencephalitis or cerebral hemorrhage. ScFv-h3D6 is known to preclude cytotoxicity of the Aβ 1-42 peptide by removing its oligomers from the amyloid pathway. As is the case for other scFv molecules, the recombinant production of scFv-h3D6 is limited by its folding and stability properties. Here, we show that its urea-induced unfolding pathway is characterized by the presence of an intermediate state composed of the unfolded VL domain and the folded VH domain, which suggests the VL domain as a target for thermodynamic stability redesign. The modeling of the 3D structure revealed that the VL domain, located at the C-terminal of the molecule, was ending before its latest β-strand was completed. Three elongation mutants, beyond VL-K107, showed increased thermodynamic stability and lower aggregation tendency, as determined from urea denaturation experiments and Fourier-transform infrared spectroscopy, respectively. Because the mutants maintained the capability of removing Aβ-oligomers from the amyloid pathway, we expect these traits to increase the half-life of scFv-h3D6 in vivo and, consequently, to decrease the effective doses. Our results led to the improvement of a potential Alzheimer disease treatment and may be extrapolated to other class-I scFv molecules of therapeutic interest.
Protein Folding and Stability Group; Departament de Bioquímica i Biologia Molecular; Unitat de Biociències; Universitat Autònoma de Barcelona; Barcelona, Spain.
Geovanny
G
Rivera-Hernández
Marta
M
Marin-Argany
Bernat
B
Blasco-Moreno
Jaume
J
Bonet
Baldo
B
Oliva
Sandra
S
Villegas
D016229:Amyloid beta-Peptides / Q000276:immunology*; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D009154:Mutation; D011489:Protein Denaturation; D055550:Protein Stability; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D057127:Single-Chain Antibodies / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
D016229:Amyloid beta-Peptides; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D057127:Single-Chain Antibodies; D014508:Urea
Alzheimer disease; CD; FTIR; aggregation; immunotherapy; scFv; stability