Mechanisms of clostridial neurotoxin binding and entry
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The botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) make up the clostridial neurotoxin (CNT) family. The CNTs are produced by Clostridium botulinum and Clostridum tetani respectively, and are the most potent human protein toxins. Eight CNT family members have been identified: seven botulinum neurotoxins (A-G) and tetanus neurotoxin (TeNT). Intoxication with BoNT is largely restricted to peripheral motor neurons, and results in flaccid paralysis. TeNT is sorted into a retrograde axonal trafficking pathway, transported to the central nervous system, and causes spastic paralysis. The CNTs are typical AB toxins. They are secreted as ~150 kDa single chain proteins that undergo processing to produce a disulfide linked, dichain active form. The “A” or active domain is ~50 kDa zinc dependent protease, also known as the light chain (LC), that inhibits synaptic vesicle fusion with the plasma membrane through cleavage of soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins. LCs cleave one of three neuronal SNARE proteins, preventing synaptic vesicle exocytosis. The “B” subunit is ~100 kDa, and contains a heavy chain translocation domain (HCT) and heavy chain receptor binding domain (HCR). Through an unclear mechanism, the HCT undergoes a conformational change upon acidification and forms pH dependent channels, facilitating transport of the LC into the neuronal cytosol. The HCR binds neuronal receptors on the presynaptic membrane of α-motor neurons. In order to explain the neuronal specificity of the CNTs, a dual receptor model was put forward. One co-receptor is ganglioside, a glycosphingolipid with a carbohydrate backbone decorated with sialic acids and a sphingolipid anchor. To satisfy the coreceptor model, the CNTs bind either a resident synaptic vesicle protein or a second ganglioside. The focus of this work was to examine mechanisms of CNT binding and translocation to better understand CNT pathogenesis. Special emphasis was placed on understanding the ganglioside binding interactions in retrograde axonal trafficking, BoNT/A1 and A2 subtype specific ganglioside interactions, and the role of receptor contributions in CNT translocation. Improving our understanding of basic mechanisms of CNT pathogenesis, including binding, entry, and translocation we can improve inhibitor designs, vaccine development, and further CNT platforms for pharmaceutical development.