1H, 13C and 15N assignments of a camelid nanobody directed against human α-synuclein

Abstract

Nanobodies are single chain antibodies that are uniquely produced in Camelidae, e.g. camels and llamas. They have the desirable features of small sizes (Mw < 14 kDa) and high affinities against antigens (Kd approximately nM), making them ideal as structural probes for biomedically relevant motifs both in vitro and in vivo. We have previously shown that nanobody binding to amyloidogenic human lysozyme variants can effectively inhibit their aggregation, the process that is at the origin of systemic amyloid disease. Here we report the NMR assignments of a new nanobody, termed NbSyn2, which recognises the C-terminus of the intrinsically disordered protein, human alpha-synuclein (aS), whose aberrant self-association is implicated in Parkinson's disease.

Nanobodies as novel agents for disease diagnosis and therapy

“…The affinities of Nbs are comparable or superior to those of conventional Ab derivatives; 15 Nbs with 100 pM affinity constants have been isolated. 35,50 The fast retrieval of high-affinity and target-specific Nbs provides a rich pool for intracellular signaling molecules, protein-protein interactions, and cancer biomarkers. Nbs can be fused with fluorescent proteins to produce chromobodies that may be utilized in single-molecule localization with super-resolution imaging techniques.…”

“…23 The hydrophilic content and the CDR3 loop folding confer thermal tolerance, aggregation resistance, and conformational stability at extreme pH values and in the presence of chaotropic agents. 28 Melting points are in the range of 67°C-78°C, 50 with thermal unfolding often shown to be fully reversible; functional activity can be retained up to 90°C. 21 The ruggedness of Nbs is suitable for harsh-condition engineering, 27,44 tumor targeting, 15 and surfactant-based pharmaceutical formulations.…”

“…Structure characterization with NMR. 50 Nbs expressed in the ER and in the cytoplasm of cells. 51 Nbs to target mitochondria.…”

“…63 The CDR3 elongations can extend into cavities on antigens, such as the active site crevice of enzymes; indeed, the immune system of camelids seems to possess an inherent tendency to form enzyme-blocking HCAbs. 50 However, an extended loop implies larger flexibility, and this is expected to be entropically counterproductive for binding. This is avoided in camelids by stabilizing their CDR3 loops with an additional disulfide bond toward either the CDR1 loop in camels or the CDR2 in llamas.…”

Intrabodies as Neuroprotective Therapeutics

“…Vuchelen et al [78] reported a camelid VHH nanobody, NbSyn2, which bound to the C-terminal of α-Syn. This nanobody, obtained by immunization followed by phage display, has a high affinity for monomeric α-Syn (Kd1 0 nM).…”