Clostridial neurotoxins (botulinum and tetanus toxins) are large proteins organized into three functional domains, an amino terminal proteolytic domain, central translocation domain, and a carboxyl terminal receptor binding region. The neurotoxic effects of clostridial neurotoxins results from binding to cell surface receptors, translocation into the neural cell, and the proteolytic cleavage of proteins essential for synaptic vesicle docking/fusion events (SNARE proteins) by the enzymatically active amino terminal domain. The subsequent block of neurotransmitter release at the neuromuscular junction by botulinum toxins or block of inhibitory neurotransmitter release within the central nervous system by tetanus toxin leads to flaccid or spasmodic paralysis of the victim, respectively. The focus of this project has been the development of an in vitro assay for toxin activity and the characterization of the receptor binding domain of tetanus C-fragment. An invitro assay for tetanus toxin has been developed based on its endopeptidase activty. An essential component of the assayis a recombinant substrate that is cleaved by tetanus neurotoxin and has features that allow detection of the extent of digestion. The SNARE protein VAMP was fused to S-tag at the amino terminus to allow detection and appended to a hexahistidine at the carboxyl terminus to facilitate purification and binding to the microtiter plate solid phase of the assay. The extent of digestion is measured as the intact VAMP chimera remaining and is detected by a affinity interaction ribonuclease A conjugated to alkaline phosphatase. The assay correlates with digestion detected by densitometry of SDS-PAGE gels. The Hc toxin fragment has been successfully expressed in Escherichia coli and purified as a maltose binding protein fusion through affinity chromatography. Specific mutations in the wild-type Hc fragment have been made based on homology to other known oligosaccharide binding domains. Each mutated plasmid has been sequenced in the mutagenesis region to confirm the introduction of the desired mutation. Mutant Hc fragments will be expressed and then analyzed for the ability to bind to ganglioside receptors. Several in vitro qualitative ELISA assays designed to assess binding to gangliosides were unsuccessful, so we have explored a quantitative assay to assess binding of Hc fragment to ganglioside receptors. Using the Biacore instrument, an optics-based system which can measure binding at a surface, we have successfully detected strong, rapid binding of wild-type tetanus toxin Hc fragment to known ganglioside receptors present in a liposomal membrane. Thus, the Biacore instrument will be used to assess binding between mutant Hc fragments and gangliosides. The binding data from each mutant will be used to direct the subsequent mutagenesis steps. By continually making different amino acid substitutions or deletions, then studying the binding of these mutants, we expect to be able to develop a model for the binding of tetanus toxin Hc fragment with ganglioside receptors.
