Exchange of the H CC domain mediating double receptor recognition improves the pharmacodynamic properties of botulinum neurotoxin

Abstract

The four-domain structure of botulinum neurotoxins (BoNTs) reflects their multistep intoxication process. The high toxicity of BoNTs primarily results from specific binding and uptake into neurons mediated by their 50-kDa cell-binding fragment (H C). X-ray crystallography data have revealed that the H C fragment consists of two domains of equal size, named the 25-kDa N-terminal half of H C (H CN) and the 25-kDa C-terminal half of H C (H CC). In recent years, the ganglioside-binding sites of all seven BoNT serotypes have been allocated to the H CC domain. For BoNT/A, BoNT/B and BoNT/G, the protein receptor-binding site has been also been localized to the H CC domain. Here, we demonstrate that the H CC serotype can modulate the affinity of the H C fragment for neuronal membranes as well as the potency of full-length BoNT by replacing the BoNT/A H CC domain with the BoNT/B H CC, BoNT/C H CC and BoNT/E H CC domains, which exhibit higher affinity for synaptosomes. Indeed, the hybrids H CAB and H CAC display a higher affinity than wild-type H CA. Furthermore, the potency of a BoNT/A-based full-length hybrid containing the H CCB domain (AAAB; letters represent the serotype origin of the four domains) was quadrupled as compared with wild-type BoNT/A. Analogously, exchange of the H C fragment (AABB) yielded a neurotoxin with four-fold higher potency. As BoNT/A and BoNT/B are extensively used to treat neurological disorders, thereby facing the problem of BoNT neutralizing antibody formation, a BoNT with increased potency would lower the repeatedly administered protein dosage while maintaining the clinical benefit. Such a lowered protein load will delay the onset of neurotoxin antibody formation in patients. The high structural homology of botulinum neurotoxin serotypes paves the way to exchange domains to alter pharmacodynamic characteristics of BoNTs without impairing structural integrity e.g.: the exchange of the H CC domain exhibiting the ganglioside and protein receptor binding sites in BoNT/A yielded a hybrid neurotoxin with increased receptor binding affinity and potency which leads towards an improved peripheral muscle relaxant © 2011 The Authors Journal compilation © 2011 FEBS.