Clostridial neurotoxins (e.g., 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. This fusion has been replaced with a hexahistidine fusion which produces Hc fragment in greater yields and purity. Homology modeling was performed using available crystallographic data in order to identify putative ganglioside-binding domains on the toxin. Based on these analyses, two regions in tetanus toxin were identified which shared structural homology with the binding domains of other sialic acid-binding proteins. These two regions of the toxin were hypothesized as potential binding sites for gangliosides. Amino acids within these regions were targeted for site-directed mutagenesis, and the binding properties of the mutant proteins were assessed using surface plasmon resonance. Of the mutagenized amino acids, one residue was found to be critical for binding of the toxin Hc fragment to ganglioside GT1b. Circular dichroism analysis confirmed that the integrity of the mutant toxin fragment was not compromised by the amino acid change, thus this mutation is involved in ganglioside binding. Further efforts to map the binding site have identified two additional residues in the same region that are also important for binding of tetanus toxin Hc fragment to ganglioside GT1b. While efforts continue to define the binding site, significant progress has been made toward the mapping of the ganglioside-binding domain of tetanus toxin.
