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Botulinum neurotoxin E (BoNT/E) is one of the major causes of human botulism and paradoxically also a promising therapeutic agent. Here we determined the co-crystal structures of the receptor-binding domain of BoNT/E (HCE) in complex with its neuronal receptor synaptic vesicle glycoprotein 2A (SV2A) and a nanobody that serves as a ganglioside surrogate. These structures reveal that the protein-protein interactions between HCE and SV2 provide the crucial location and specificity information for HCE to recognize SV2A and SV2B, but not the closely related SV2C. At the same time, HCE exploits a separated sialic acid-binding pocket to mediate recognition of an N-glycan of SV2. Structure-based mutagenesis and functional studies demonstrate that both the protein-protein and protein-glycan associations are essential for SV2A-mediated cell entry of BoNT/E and for its potent neurotoxicity. Our studies establish the structural basis to understand the receptor-specificity of BoNT/E and to engineer BoNT/E variants for new clinical applications.

The application of botulinum neurotoxins (BoNTs) for medical treatments necessitates a potency quantification of these lethal bacterial toxins, resulting in the use of a large number of test animals. Available alternative methods are limited in their relevance, as they are based on rodent cells or neuroblastoma cell lines or applicable for single toxin serotypes only. Here, human motor neurons (MNs), which are the physiological target of BoNTs, were generated from induced pluripotent stem cells (iPSCs) and compared to the neuroblastoma cell line SiMa, which is often used in cell-based assays for BoNT potency determination. In comparison with the mouse bioassay, human MNs exhibit a superior sensitivity to the BoNT serotypes A1 and B1 at levels that are reflective of human sensitivity. SiMa cells were able to detect BoNT/A1, but with much lower sensitivity than human MNs and appear unsuitable to detect any BoNT/B1 activity. The MNs used for these experiments were generated according to three differentiation protocols, which resulted in distinct sensitivity levels. Molecular parameters such as receptor protein concentration and electrical activity of the MNs were analyzed, but are not predictive for BoNT sensitivity. These results show that human MNs from several sources should be considered in BoNT testing and that human MNs are a physiologically relevant model, which could be used to optimize current BoNT potency testing.

Cesare Montecucco is a member of the board of various leading scientific journals. His research focus is on the regeneration of the peripheral nervous system and on the mechanisms of action of toxins and related diseases, including tetanus, botulism, anthrax and Helicobacter pylori-associated diseases. In this interview, we talked to Cesare Montecucco about his career, learning and culture.

Botulinum neurotoxins (BoNT) cause muscle paralysis by inhibiting the exocytosis at motor nerve terminals. Initially, they bind to abundant complex polysialogangliosides via a conserved pocket within the C-terminal half of their cell binding domain (Hcc-domain). Engineering of this binding site in BoNT/A leads to mutants with >3-fold increased potency, which can be exploited as new therapeutics with decreased dosage and thus might prolong the period until immune response in patients. Additionally, protein receptors are required for productive neurotoxin uptake. While BoNT/B and G share the synaptic vesicle proteins synaptotagmin (Syt) I and Syt-II as protein receptors, BoNT/A uniquely binds SV2. Recently, the interaction site of Syt I and Syt-II was located near the ganglioside binding pocket within the Hcc-domain of BoNT/B and G. Furthermore, the BoNT/B–Syt II interaction could be characterised at the atomic level. None of the so-far identified protein receptors interacts with BoNT/C, D, E, F and TeNT. Employing the ex vivo mouse phrenic nerve-hemidiaphragm as a physiological target, the type and number of unknown neurotoxin receptor candidates were narrowed down. The productive uptake of BoNT/C, D, E and F is stimulation dependent, which points towards receptors being part of synaptic vesicles. Moreover, the endocytosis of BoNT/C, E and F, is inhibited by addition of any Hc-fragment derived from BoNT/C, E and F, indicating a binding to the same receptor structures.

The main object of this work is to deliver the catalytically active portion of neurotoxins (light chain) into the cytosol of mast cells to inhibit the specific release of inflammatory mediators. Mast cells play a significant role in the pathophysiology of many different diseases. In most of these diseases there is increased recruitment of mast cells and sustained secretion of many proinflammatory mediators, including autacoids, cytokines and proteases. We use the human mast cell line 1 (HMC-1), a leukaemia line expressing several features of mature human mast cells that has proved a valuable model for studying human mast cell biology. In order to stimulate fusion among secretory granules, we use intracellular application of GTPgammaS and extracellular stimulation with concanavalin A. By patch-clamp technique, we determine the changes in cell membrane capacitance, thereby studying exocytosis. The whole-cell configuration of patch-clamp is used. By electroporation, the light chains of TeNT and BoNT/C & D will be delivered into the cytoplasm of mast cells to estimate their effects on mast cell activity. Cells treated with neurotoxins will be subjected to stimulation tests “in vitro”, recording of membrane capacitance by patch-clamp and detection of cytokine release to estimate effects on mast cell activity.