List Labs POSTER - Presented at the 6th International Conference on Basic and Therapeutic Aspects of Botulinum and Tetanus Toxins, June 2008 in Baveno
Recent biochemical and molecular genetic studies have established synaptic vesicle glycoprotein 2c (SV2c) as the protein receptor for Botulinum Neurotoxin Type A (BoNT/A). The luminal domain loop of SV2c, between transmembrane domains 7 and 8, has been shown to be the location of BoNT/A binding. Utilizing this information, we have shown through GST pull-down experiments that we can detect binding of BoNT/A to SV2c using an immunoassay. This can be done with either the rat or human forms of the SV2c protein. Upon specific binding of BoNT/A to its receptor protein, we can introduce our FRET peptide SNAPtide and detect enzymatic cleavage. We describe the reaction conditions and the detection limits using both rat and human forms of the SV2c binding region as well as multiple FRET substrates to determine the optimal conditions for this detection assay. There are three steps in the interruption of synaptic transmission by botulinum toxin. The first is binding to neuronal cells; the second is translocation of the enzymatic light chain out of the endosome; and finally cleavage of synaptosomal proteins to inhibit neurotransmitter release. With this assay we can monitor two of the three steps of toxin activity, binding and cleavage. In the future, we hope to establish this as a new functional assay for the detection and activity of Botulinum Neurotoxin Type A and demonstrate a direct correlation to the gold standard, the mouse bioassay.