Botulinum Internalization

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). 

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... 

Verderio, C., Coco, S., Rossetto, O., Montecucco, C., Matteoli, M. (1999). Internalization and proteolytic action of botulinum toxins in CNS neurons and astrocytes. J. Neurochem. 7 372-9. 

Muscle mass affects the toxin s response. More toxin is needed locally to produce a desired effect in areas of increased muscle mass. Histologic examination of orbicularis oculi musculature after treatment with botulinum toxin shows no evidence of alteration of muscle fiber diameter, disruption of internal muscle architecture, or pathologic changes in the motor end plates. 

Anal fissures are often intensely painful due to sphincter spasm. Anaesthetic ointments and stool softening agents have been widely used, with surgery (lateral internal sphinterotomy) for severely affected cases, but this procedure can cause incontinence from loss of sphincter control. An alternative is topical application of nitrate which heals two-thirds of fissures. Preparations should be diluted to 0.2% as such use may be complicated by headache tolerance can develop. Intrasphincteric injection of botulinum toxin has also been shown to be effective. 

Ohishi I, Yanagimoto A (1992) Visualizations of binding and internalization of two nonlinked protein components of botulinum C2 toxin in tissue culture cells. In Infect. Immun. 60 4648-55... 

The two components of botulinum C2 toxin ore functionally different proteins component II is a binding molecule, whereas component I is an ADP-hbosyltransferase, of which substrate is cytoplasmic actin monomers (Ohishi and Tuyama, 1986 Ohishi, 1986 Ohishi et al., 1990). This indicates that the toxin binds to the cell surface and enters the cytoplasm. These steps, the binding of the two nonlinked components of C2 toxin to the cells and the endocytotic vesicles containing the components, can be visualized either directly by incubating the cells with the two differently fluorescently labeled components (Ohishi, 1992), or indirectly by immunofluorescence labeling of the two proteins with their specific antibodies. This endocytotic incorporation of the protein is not a specific feature of this toxin, but is common to all the proteins that enter cells by receptor-mediated endocytosis. However, the internalization of the toxin may be of an interest to those readers, who would like use this characteristic toxin as a tool to analyze cellular responses, especially those who would like to compare the incorporation processes of the two non-linked components with... 

Ohishi I, Hama Y (1992) Purification and characterization of heterologous component lls of botulinum C2 toxin. In Microbiol Immunol. 36 221 -9 Ohishi I, Iwasaki M, Sdkaguchi G. (1980) Purification and characterization of two components of botulinum 02 toxin. In Infect Immun. 30 668-73 Ohishi I, Miyake M (1985) Binding of the two components of 02 toxin to epithelial cells and brush borders of mouse intestine. In Infect Immun. 48 769-75 Ohishi I, Tsuyama S (1986) ADP-ribosylation of nonmuscle actin with component I of 02 toxin. In Biochem Biophys Res Comm. 136 802-6 Ohishi I, Yanagimoto A (1992) Visualizations of binding and internalization of two nonlinked protein components of botulinum 02 toxin in tissue culture cells. In Infect Immun. 60 4648-55... 

They bind initially to ganglioside in the neuromuscular jimction, one subunit then being internalized as with the diphtheria toxin (Box 29-A). Botulinum toxins specifically enter motor neurons, while tetanus toxin is taken up via synaptic vesicle endocytosis by both peripheral and central neurons. Retrograde axonal transport carries the toxin into the central nervous system and across synaptic clefts into cholinergic intemeurons, which are poisoned. 

McNally RE, Morrison MB, Berndt JE, et al. Effectiveness of Medical Defense Interventions Against Predicted Battlefield Levels of Botulinum Toxin A. Vol 1. Joppa, Md Science Applications International Corporation 1994 3. 

Sheean G, Lannin NA, Turner-Stokes L, Rawicki B, Snow BJ. Botulinum toxin assessment, intervention and after-care for upper limb hypertonicity in adults international consensus statement. Eur J Neurol 2010 17(Suppl. 2) 74-93. 

A meta-analysis to compare the efficacy of posterior internal anal sphincter (IAS) myectomy with intrasphinc-teric botulinum toxin injection for treatmenf of IAS achalasia involving 16 prospective and retrospective studies in a total of 395 patienfs [18 ] demonstrated no significant difference in continued use of laxatives or rectal... 

Baird-Parker, A. C. and M. A. H. Baillie. 1974. The inhibition of Clostridium botulinum by nitrite and sodium chloride. In Proceedings of the International Symposium on Nitrite in Meat Products, eds. Tinbergen, B. J., and B. Krol, p. 77. Zeist, The Netherlands PUDOC, Wageningen. 

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. 

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