A proteomic investigation of botulinum toxin serotype A protein-protein interactions at the mammalian neuromuscular junction
Parrott, Tonia Marie
MetadataShow full item record
Botulinum toxin is one of the most toxic substances known to man. Ironically, it is the toxin’s specificity for cholinergic nerve terminals, its ubiquitous nature, and extremely low effective dose that has resulted in its use as a valuable therapeutic tool while at the same time earning it a place on the CDC’s list of biological weapons. While early studies have developed a model regarding the toxin’s mode of action, little is understood regarding intoxication involving cellular receptors at the physiological target, the mammalian neuromuscular junction. The first step toward identification of these receptors involves the ability to isolate a fraction rich in neuromuscular proteins. Earlier studies in our laboratory utilized a mouse diaphragm preparation to study substrate proteolysis. For receptor identification, it was important to determine whether this preparation would also serve as an adequate model of a “synaptosome.” After demonstrating that this preparation contained proteins common to both pre- and post-synaptic machinery, it was utilized in an affinity precipitation with beads coated with botulinum toxin serotype A. Proteins from this precipitation were identified utilizing tandem mass spectrometry. Most of the identified proteins were intracellular proteins that, while not serving as the toxin protein receptor, may be involved in intracellular toxin interactions. In the process, several peptides were also isolated that corresponded to conserved domains within growth factor receptors. Using antibodies to several growth factors, we were able to identify two growth factors in particular that demonstrated selective binding to toxin. These receptors were identified as Nogo-66 receptor isoform 2 (NGR2), and Fibroblast growth factor receptor III (FGFR3). Interestingly, both proteins have been shown to have roles in either neurogenesis or axonal development, which is a significant characteristic of botulinum toxin A intoxication. Both of these proteins have also been known to interact with complex gangliosides and to participate in lipid raft signaling. In these studies, we have identified two possible candidates in the binding of Botulinum toxin serotype A at the nerve cell terminal.