Characterising and exploiting the binding of botulinum neurotoxins to their neuronal receptors

ARTICLE IN PRESS 4

Abstracts Toxins 2008 / Toxicon 51 (2008) 1–54

phosphorylation and to increase the viability of cultured cerebellar granular neurons upon challenge with high potassium. Keywords: Hc-TeTx; NGF; GD1b; GT1b; ERK 1/2 Akt 10.1016/j.toxicon.2008.04.010

9. Translocation of the light chain protease by the heavy chain protein-conducting channel Mauricio Montal University of California San Diego, La Jolla, USA

Clostridial botulinum neurotoxins (BoNTs) inhibit synaptic exocytosis; intoxication requires the di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol, colocalizing it with the substrate SNARE proteins. We investigate the dynamics of protein-translocation across membranes using a sensitive single-molecule assay to track translocation events with millisecond resolution on lipid bilayers and on membrane patches of Neuro 2A cells. Translocation of BoNT/A LC by the HC is observed in real time as changes of channel conductance: the channel is occluded by the light chain during transit, and open after completion of translocation and release of cargo, acting intriguingly similar to the protein-conducting/translocating channels of the endoplasmic reticulum, mitochondria, and chloroplasts. Our findings support the notion of an interdependent, tight interplay between the HC transmembrane chaperone and the LC cargo that prevents LC aggregation and dictates the productive passage of cargo through the channel and completion of translocation. Keywords: Channels; Chaperones; Protein translocation; Membranes; Modules 10.1016/j.toxicon.2008.04.011

10. Binding of botulinum neurotoxin A to the plasma membrane Lucia Muraro a, Serra Mauro Dalla b, Laura Morbiaro a, Ornella Rossetto a, Cesare Montecucco a a

Departement of Experimental Biomedical Sciences, University of Padua, Padua, Italy b FBK-CNR Institute of Biophysics, Unit at Trento, Povo (Trento), Italy

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most powerful clostridial toxins (CNTs). They are composed of a catalytic domain, the Light Chain (LC) and a Heavy chain (HC), which includes a translocation domain (HN) and a receptor binding domain (HC). HC is further composed of two subdomains, HCN and HCC. CNTs act at femtomolar concentrations; the high-affinity binding involved either membrane gangliosides or neuronal membrane proteins. Until now the whole plasma membrane binding capacity has been ascribed to HCC. The

aim of this project is to investigate whether HCN is also involved. The BoNT/A HCN coding sequence has been cloned as his-tag fusion protein, and fused to fluorescent probes. Observations by fluorescence microscopy have shown a plasma membrane staining enriched in bright spots in epithelial and differentiated neuronal cell lines. For TeNT a role of lipid rafts has been established, but for BoNTs the question seems to be still open. Our data show that the sphingomyelin binding toxin lysenin partially colocalized with HCN. From dot blot analysis it seems that HCN is able to directly interact with anionic lipids. A role for negative charged lipids in the binding of BoNTs and TeNT to lipid bilayers was already suggested, and these preliminary data indicate that the N-terminal portion of the binding domain is able to bind anionic lipids in the environment of membrane microdomains. Keywords: Membrane microdomains; Protein–lipid interaction; Toxin binding 10.1016/j.toxicon.2008.04.012

11. Characterising and exploiting the binding of botulinum neurotoxins to their neuronal receptors Andreas Rummel, Tino Karnath, Kirstin Ha¨fner, Stefan Mahrhold, Thomas Binz, Hans Bigalke Medizinische Hochschule Hannover, Toxikologie, Hannover, Germany

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 43-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. Keyword: Binding 10.1016/j.toxicon.2008.04.013