title: 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(C) AB and H(C) AC display a higher affinity than wild-type H(C) A. Furthermore, the potency of a BoNT/A-based full-length hybrid containing the H(CC) B 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.
authors.affiliation: Institut für Toxikologie, Medizinische Hochschule Hannover, Germany. rummel.andreas@mh-hannover.de
authors.forename: Andreas
authors.initials: A
authors.lastname: Rummel
authors.forename: Stefan
authors.initials: S
authors.lastname: Mahrhold
authors.forename: Hans
authors.initials: H
authors.lastname: Bigalke
authors.forename: Thomas
authors.initials: T
authors.lastname: Binz
mesh_terms: D000818:Animals; D001665:Binding Sites; D001905:Botulinum Toxins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D018360:Crystallography, X-Ray; D005732:Gangliosides / Q000737:chemistry / Q000378:metabolism; D066298:In Vitro Techniques; D051379:Mice; D009474:Neurons / Q000378:metabolism; D009498:Neurotoxins / Q000737:chemistry / Q000378:metabolism; D010791:Phrenic Nerve / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D013574:Synaptosomes / Q000378:metabolism
chemical_list: D005732:Gangliosides; D009498:Neurotoxins; D011994:Recombinant Proteins; D001905:Botulinum Toxins