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Functional Assay for Botulinum Neurotoxin Type A Utilizing the Neuronal Receptor Protein SV2c.

Christian, T.; Rummel, A.; Shine, N.

Botulinum toxin progresses through a three step process leading to the interruption of synaptic transmission. The first step is binding of toxin to neuronal cells and incorporation of the toxin into an endosome; the second step is translocation of the enzymatic light chain out of the endosome and into the cytosol of the neuron; and the final step is cleavage of synaptosomal proteins to prevent vesicle docking and neurotransmitter release. Recent biochemical and molecular genetic studies have established synaptic vesicle glycoprotein 2C (SV2c) as the protein receptor for Botulinum Neurotoxin Type A (BTA). The luminal domain loop of SV2c, between transmembrane domains 7 and 8, has been shown to be the location of BoNT/A binding (Mahrhold et al FEBS Lett. 2006). Utilizing recombinant GST-SV2c, we have shown through pull-down experiments that we can detect both the binding of BTA to SV2c and enzymatic activity using the FRET peptide SNAPtide. We describe the reaction conditions and the detection limits using multiple FRET substrates. With this assay we can examine two of the three steps of toxin activity, binding and cleavage. In the future, we plan to establish this as a functional assay to monitor binding and enzymatic activity of Botulinum Neurotoxin Type A in order to demonstrate a direct correlation to the gold standard, the mouse bioassay.