Botulinum toxin serotype A (BoNT/A)

From CFGparadigms

Revision as of 14:16, 3 October 2011

The clostridial neurotoxins are the most lethal protein toxins for humans and recently have been utilized as therapeutic agents to treat numerous human neurological inflictions. The Botulinum neurotoxins produced by Clostridium botulinum and Tetanus toxin produced by C. tetani are members of the family of clostridial neurotoxins. The clostridial neurotoxins are di-chain toxins with the N-terminal catalytic domains (Light Chain, LC) possessing metalloprotease activity that is disulfide linked to the C-terminal domain (Heavy Chain, HC). The neurological toxicity and therapeutic utility of the clostridial neurotoxins is due to the HC’s tropism for neuronal receptors and the LC’s cleavage of neuron-specific target proteins, termed SNARE proteins. SNARE proteins are responsible for the fusion of neurotransmitter vesicles with the plasma membrane. There are seven serotypes of the BoNTs that share primary and ternary structure-function properties. Each BoNT serotype utilizes dual receptors for entry into neurons and each cleaves a specific SNARE protein or a unique site on a specific SNARE protein.

The Botulinum neurotoxins elicit flaccid paralysis, while tetanus toxin elicits spastic paralysis. The differential toxicity is due to the unique trafficking of these toxins in motor neurons. Botulinum neurotoxin HC binds to dual host receptors on the surface of motor neurons to deliver the BoNT to acidified vesicles where the LC is translocated into the cytoplasm of the neuron. Upon entry into the cytoplasm, LC cleaves a SNARE protein which inhibits fusion of neurotransmitter vesicles to the plasma membrane, inhibiting the release of neurotransmitter molecules. In contrast, TeNT HC binds to dual host receptors on the surface of motor neurons to deliver the TeNT to neutral vesicles for retrograde trafficking to the central nervous system. Following this transcytosis, Tetanus toxin binds dual receptors on inhibitory neurons to deliver TeNT to acidified endosomes where the LC is translocated into the cytoplasm of the neuron. TeNT LC cleaves the SNARE proteins, VAMP-2, which inhibits the release of neurotransmitters from inhibitory motor neurons. The inability to release neurotransmitter from inhibitory motor neurons yields spastic paralysis.

Botulinum toxin serotype A (BoNT/A) was chosen as the paradigm for the clostridial neurotoxins, since the dual host receptors for BoNT/A have been determined and the basis for recognizing cleavage of the SNARES substrate SNAP25 has been characterized. In addition, BoNT/A is the most common serotype used in clinical therapies. It is anticipated that understanding BoNT/A action will provide new information relevant to the entire family of clostridial neurotoxins. These studies will enhance the understanding of the unique properties of each BoNT serotype and Tetanus toxin to extend their utility in human inflictions. The CFG has been used to facilitate the characterization of the ganglioside binding pocket of these neurotoxins. Prior studies utilized low throughput analyses that provided limited insight into interactions between these neurotoxins and gangliosides, and but high throughput analysis of the CFG core provided a better understanding of the biochemical and structural interactions of the neurotoxins with glycans. For example, array analysis showed that the dual receptors for Tetanus toxin recognized unique components of gangliosides.

CFG Participating Investigators contributing to the understanding of this paradigm

CFG Participating Investigators (PIs) contributing to the understanding of BoNT/A include: Joseph Barbieri, Edwin Chapman, Minoru Fukuda, Raymond Stevens, Willie Vann

Progress toward understanding this GBP paradigm

Carbohydrate ligands

Botulinum neurotoxins bind to two co-receptors on neuronal cell surfaces: common glycolipid receptors and different protein receptors that target them to specific cell types. The common ganglioside co-receptor is GT1b^[1].^[2]

Cellular expression of GBP and ligands

Botulinum toxins are produced by Clostridium botulinum Their target glycolipid and protein receptors are found on the surface of motorneurons.

Biosynthesis of ligands

Ganglioside GT1b is synthesized stepwise from the lipid ceramide by the action of the following seven glycosyltransferases [genes]:

ceramide glucosyltransferase [UGCG]

beta-1,4-galactosyltransferase 6 [B4GALT6]

lactosylceramide a-2,3-sialyltransferase [ST3GAL5]

alpha-2,8-sialyltransferase [ST8SIA1]

N-acetylgalactosaminyltransferase [B4GALNT1]

beta-1,3-galactosyltransferase [B3GALT4]

alpha-2,3-sialyltransferase [ST3GAL2, ST3GAL3]

Evidence for the action of each of the above genes in this pathway is supported by either in vitro enzyme assays, genetic studies, or both. Other (redundant) enzymes may also act in this pathway.

Structure

The crystal structure of BoNT/A has been solved both alone and bound to its ganglioside co-receptor (see entry in Panthema NIAID Bioinformatics Resource Center).

Biological roles of GBP-ligand interaction

Botulinum toxins cause neurotoxicity by cleaving SNARE proteins, which normally allow neurotransmitter-containing vesicles to fuse with the neuronal plasma membrane.

CFG resources used in investigations

The best examples of CFG contributions to this paradigm are described below, with links to specific data sets. For a complete list of CFG data and resources relating to this paradigm, see the CFG database search results for botulinum.

Glycan profiling

Glycogene microarray

BoNT/A is not represented on the CFG microarrays, which only contain probes for mouse and human glycogenes.

Knockout mouse lines

Not applicable.

Glycan array

The CFG synthesized ganglioside derivates that have been used in co-crystallization studies with the clostridial neurotoxins. The CFG glycan array was used to identify the ganglioside binding specificity to the clostridial neurotoxins (BoNT/C, BoNT/D, BoNT/F).

Related GBPs

Botulinum neurotoxins serotypes B-G (CFG data: BoNT/B, BoNT/C,BoNT/D,BoNT/E,BoNT/F,BoNT/G), Tetanus toxin (CFG data)

References

1. ↑ Yowler BC, Schengrund CL. Botulinum neurotoxin A changes conformation upon binding to ganglioside GT1b. "Biochemistry" 43, 9725-9731 (2004)
2. ↑ Stenmark. P, Dupuy1, J, Imamura, A, Kiso, M, and Stevens, RC. Crystal Structure of Botulinum Neurotoxin Type A in Complex with the Cell Surface Co-Receptor GT1b—Insight into the Toxin–Neuron Interaction “PLOS Pathogens” 4, e1000129 (2008)

• C. Chen, Z. Fu, J-J. P. Kim, J.T. Barbieri, and M. R. Baldwin. 2009. Gangliosides as High Affinity Receptors for Tetanus Neurotoxin. J Biol Chem. 284: 26569-77. PMC2785345
• M. Dong, W. H. Tepp, H. Liu, E. A. Johnson, and E. R. Chapman. 2007. Mechanism of botulinum neurotoxin B and G entry into hippocampal neurons. J. Cell Biol. 179: 1511-1522. PMC2373501

Acknowledgements

The CFG is grateful to the following PIs for their contributions to this wiki page: Joseph Barbieri, James Paton