Project
BoNT C - Licence to enter
The BoNTs belong to the family of AB toxins and can be divided into the serotypes A-H. As an AB-toxin, BoNT consists of two protein chains, the heavy chain (HC) and light chain (LC), which are connected by a highly conserved disulfide bond and multiple noncovalent interactions. The mechanism underlying the cell specific infiltration by BoNT is partly understood, yet many questions remain: For cell recognition, BoNT uses gangliosides in the presynaptic membrane to select for neuronal cells (Benson et al 2011). Besides the gangliosides themselves, which are enriched in neuronal cells, binding of BoNT requires a linkage between gangliosides and N-acetylneuraminic acid (Sia5) (Strotmeier et al 2011). The subsequent infiltration process is performed by the HC binding to the membrane and further unknown receptors. HC and LC then undergo endocytosis until only the LC is released into the cytosol. Lastly, the LC, being a zinc metalloprotease, cleaves Snap-25 and other proteins of the SNARE complex depending on the toxin’s serotype. This leads to prevention of neurotransmitter release into the synaptic cleft and therefore muscle contraction at the neuromuscular end plate.
Since BoNT is so highly specific for neuronal cells, its potency and therefore toxicity is among the highest for peptide toxins in the world. For BoNT serotype C the lethal concentration (LD50) in humans is approximately 1ng/kg (parenteral application) and 1µg/kg N (oral application). The high toxicity would therefore prevent from any usage of the specific shuttle mechanism. In 2017 three missense mutations in the active site of the LC were reported that conserve the overall structure and binding affinity of BoNTC, however reduce the activity and overall toxicity by the factor 106. (Vazquez-Cintron et al 2017) This option of an efficient detoxification provides us with a very powerful tool that might be used to transport a variety of molecules into neuronal cells with a high specitivity: Synthetic drugs, therapeutic peptides and labeling proteins for fundamental research imported this way might revolutionize neuronal targeting strategies to increase our overall knowledge about the complexity of the brain and the chances to treat severe neuronal diseases.
Toolbox Description
Linker Methods
GS-Linker
Click Chemistry
Sortase-mediated Linking
Planned Fusion Partners
Cre
Omomyc
pHluorin2
Eslicarbazepine
Fluor488
Nano-Glo® HiBit Extracellular Detection System
Molecular Biology
Protein Expression
Cell Culture
Chemistry