Botulinum Receptor

Bullens, R. W., O Hanlon, G. M., Wagner, E. etal. Complex gangliosides at the neuromuscular junction are membrane receptors for autoantibodies and botulinum neurotoxin but redundant for normal synaptic function. /. Neurosci. 22 6876-6884, 2002. 

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). 

C. botulinum toxins belong to the AB group of toxins, which also includes diphtheria toxin, pseudomonas exotoxin A, anthrax toxin, Shiga(like) toxin, cholera toxin, pertussis toxin, and plant toxins, e.g., ricin. Moiety A has an enzymatic activity and usually modified cellular-target entering cytosol. Moiety B consists of one or more components and binds the toxin to surface receptors, and is responsible for translocation of the A component into cells. AB toxins are produced in a non-active form and are activated by a split between two cysteine residues within a region (Falnes and Sandvig, 2000). 

B. Botulinum toxin has no ability to inhibit esterase (A) or inhibit enzymes involved in the acetylcholine synthetic pathways (C), nor does it possess muscarinic receptor blocking properties (D). 

Schematic illustration of a generalized cholinergic junction (not to scale). Choline is transported into the presynaptic nerve terminal by a sodium-dependent choline transporter (CHT). This transporter can be inhibited by hemicholinium drugs. In the cytoplasm, acetylcholine is synthesized from choline and acetyl -A (AcCoA) by the enzyme choline acetyltransferase (ChAT). Acetylcholine is then transported into the storage vesicle by a second carrier, the vesicle-associated transporter (VAT), which can be inhibited by vesamicol. Peptides (P), adenosine triphosphate (ATP), and proteoglycan are also stored in the vesicle. Release of transmitter occurs when voltage-sensitive calcium channels in the terminal membrane are opened, allowing an influx of calcium. The resulting increase in intracellular calcium causes fusion of vesicles with the surface membrane and exocytotic expulsion of acetylcholine and cotransmitters into the junctional cleft (see text). This step can he blocked by botulinum toxin. Acetylcholine s action is terminated by metabolism by the enzyme acetylcholinesterase. Receptors on the presynaptic nerve ending modulate transmitter release. SNAPs, synaptosome-associated proteins VAMPs, vesicle-associated membrane proteins.

Another important example is the nicotinic acetylcholine receptor, which is activated by the agonist nicotine causing muscular fibrillation and paralysis. Indirect effects can also occur. For example, organophosphates and other acetylcholinesterase inhibitors increase the amount of acetylcholine and thereby overstimulate the receptor, leading to effects in a number of sites (see chap. 7). Alternatively, botulinum toxin inhibits the release of acetylcholine and causes muscle paralysis because muscular contraction does not take place (see chap. 7). 

This type of response may be caused by several mechanisms. For instance, the muscle relaxation induced by succinylcholine, discussed in more detail in chapter 7, is due to blockade of neuromuscular transmission. Alternatively, acetylcholine antagonists such as tubocurarine may compete for the receptor site at the skeletal muscle end plate, leading to paralysis of the skeletal muscle. Botulinum toxin binds to nerve terminals and prevents the release of acetylcholine the muscle behaves as if denervated, and there is paralysis. This will be discussed in more detail in chapter 7. 

Botulinum toxin is a mixture of six large molecules, each of which consist of two components, a heavy (lOOkDa) and light (50kDa) polypeptide chain. The heavy chain binds to the walls of nerve cells, which then allows the whole toxin molecule to be transported into the cell inside a vesicle via receptor-mediated endocytosis. Once inside, the light chain translocates into the cytosol and acting as a peptidase, destroys a synaptosomal protein. 

This chapter deals with botulinum toxin type A (BOTOX) in the treatment of strabismus, blepharospasm, and related disorders. Botulinum toxin type A (BOTOX) has been used to treat strabismus, blepharospasm, Meige s syndrome, and spasmodic torticollis. By preventing acetylcholine release at me neuromuscular junction, botulinum toxin A usually causes a temporary paralysis of the locally injected muscles. The variability in duration of paralysis may be related to me rate of developing antibodies to me toxin, upregulation of nicotinic cholinergic postsynaptic receptors, and aberrant regeneration of motor nerve fibers at me neuromuscular junction. Complications related to this toxin include double vision (diplopia) and lid droop (ptosis). 

Remarkably, SV2 also appears to be the cellular receptor for botulinum neurotoxin A (Dong et al., 2006). In particular, a segment of the large lumenal loop binds to the toxin, and all of the SV2 isoforms appear capable of serving as receptors. Thus, levetiracetam, even if it is bound to the same site as the toxin, seems unlikely to protect against poisoning since it interacts only with SV2A. 

Kirkpatrick LL, Matzuk MM, Dodds DC, Perin MS (2000) Biochemical interactions of the neuronal pentraxins. Neuronal pentraxin (NP) receptor binds to taipoxin and taipoxin-associated calcium-binding protein 49 via NP1 and NP2. J Biol Chem 275 17786-92 Kitamura M, Igimi S, Furukawa K, Furukawa K (2005) Different response of the knockout mice lacking b-series gangliosides against botulinum and tetanus toxins. Biochim Biophys Acta 1741 1-3... 

LaUi G, Bohnert S, Deinhardt K, Verastegui C, Schiavo G (2003) The journey of tetanus and botulinum neurotoxins in neurons. Trends Microbiol 11 431-7 Lambeau G, Lazdunski M (1999) Receptors for a growing family of secreted phospholipases A2. Trends Pharmacol Sd 20 162-70... 

Montecucco C, Schiavo G, Tugnoli V, de Grandis D (1996) Botulinum neurotoxins mechanism of action and therapeutic applications. Mol Med Today 2 418-24 Montecucco C, Rossetto O, Schiavo G (2004) Presynaptic receptor arrays for clostridial neurotoxins. Trends Microbiol 12 442-6... 

Rummel A, Eichner T, Weil T, Karnath T, Gutcaits A et al. (2007) Identification of the protein receptor binding site of botulinum neurotoxins B and G proves the double-receptor concept. Proc Natl Acad Sci U S A 104 359-64... 

The effects of curare develop rapidly after it enters the body. Victims develop rapid weakness of voluntary muscles followed by paralysis, respiratory failure, and death. The cause is a blockade of nicotinic cholinergic receptors at the neuromuscular junctions in skeletal muscle. Unlike botulinum toxin, release of acetylcholine by the cholinergic nerve terminals is not affected. When curare is present, however, the acetylcholine that is released cannot bind to the receptors because they are reversibly occupied by the curare. As a consequence, nerve-muscle communication fails and paralysis ensues. 

Jin, R., Rummel, A., Binz, T., Brunger, A.T. (2006). Botulinum neurotoxin B recognizes its protein receptor with high affinity and specificity. Nature 444 1092-5. 

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