Internalization Botulinum neurotoxin

Dolly, J.O., Black, J., Williams, R.S. and Melling, J., Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization. Nature, 307, 457 60, 1984. 

Rossetto O, Seveso M, Caccin P, Schiavo G, Montecucco C (2001b) Tetanus and botulinum neurotoxins turning bad guys into good by research. Toxicon 39 27—41 Rossetto O, Morbiato L, Rossetto et al. 2006 Caccin P, Rigoni M, Montecucco C (2006) Presynaptic enzymatic neurotoxins. J Neurochem 97 1534—4 5 Roux S, Colasante C, Saint Clomcnt C, Barbier J, Curie T et al. (2005) Internalization of a GFP-tetanus toxin C-terminal fragment fusion protein at mature mouse neuromuscular junctions. Mol Cell Neurosci 30 572-82... 

Therefore, we developed a drug delivery vehicle (DDV) comprising the nontoxic recombinant heavy chain of BoNT-A coupled to a 10-kDa amino dextran via the heterobifunctional linker 3-(2-pyridylthio)-propionyl hydrazide. The heavy chain served to target botulinum neurotoxin-sensitive cells and promote internalization of the complex, while the dextran served as a platform to deliver model therapeutic molecules to the targeted cells. 

Botulinum neurotoxins (seven serotypes, A-G) are relatively large water soluble proteins (150 kDa) produced by the Clostridium botulinum. Each protein has two polypeptide chains (a 100 kDa heavy chain and a 50 kDa light chain) linked through a disulfide bond (Fig. 2). In the proposed mode of action of botulinum and tetanus neurotoxins (Simpson, 1986, 1989), the C-terminal half of the heavy chain binds to the nerve membrane leading to internalization of the neurotoxin in the nerve cell through endocytosis. Subsequently, the pH of the endosome is lowered causing the heavy chain to get integrated in the membrane... 

Figure 3. Schematic representation of the steps involved in the binding, internalization and intracellular activity of the botulinum neurotoxins. The steps with question mark are intensive areas of current research.

Some of the inherent difficulties associated with the use of this fluorescence-based immunosensor include fiber to fiber variability in the signal, cost of the instrument as well as fibers, and availability of portable commercial instrument. Still, further characterization of the instrument and the methods surrounding the immunoassay are necessary in order to increase its potential application as a sophisticated analytical technique. Relevant to the detection of botulinum neurotoxins, binding in a variety of biological and environmental matrices needs to be investigated. Also, to increase the applicability of the biosensor as a technique that offers reliable quantitative results, a method for internal standardization should be investigated. The fiberoptic immunosensor in its present state is best described as a technique that allows for the selective detection and monitoring of botulinum neurotoxins at concentrations at or above 300 pg/mL. However, this technique would provide only semi-quantitative results as far as the determination of unknown concentration of the toxin is concerned. 

Classical bacterial exotoxins, such as diphtheria toxin, cholera toxin, clostridial neurotoxins, and the anthrax toxins are enzymes that modify their substrates within the cytosol of mammalian cells. To reach the cytosol, these toxins must first bind to different cell-surface receptors and become subsequently internalized by the cells. To this end, many bacterial exotoxins contain two functionally different domains. The binding (B-) domain binds to a cellular receptor and mediates uptake of the enzymatically active (A-) domain into the cytosol, where the A-domain modifies its specific substrate (see Figure 1). Thus, three important properties characterize the mode of action for any AB-type toxin selectivity, specificity, and potency. Because of their selectivity toward certain cell types and their specificity for cellular substrate molecules, most of the individual exotoxins are associated with a distinct disease. Because of their enzymatic nature, placement of very few A-domain molecules in the cytosol will normally cause a cytopathic effect. Therefore, bacterial AB-type exotoxins which include the potent neurotoxins from Clostridium tetani and C. botulinum are the most toxic substances known today. However, the individual AB-type toxins can greatly vary in terms of subunit composition and enzyme activity (see Table 2). 

---