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Botulinum Neurotoxin B: The Biochemical Blade
Nathan Hale High School SMART Team: Nicholas Dolan, Nick Goldner, Sam Hall, Brenna Hanley, Katie Milos, Rebecca Ruechel, Ajay Sreekanth
Teachers: Anne Xiong and Susan Getzel
Clostridium
Mentor: Joseph T. Barbieri, Ph.D., Medical College of Wisconsin, Milwaukee, WI
botulinum
Abstract
www.foodpoisonblog.com
Botulinum neurotoxins (BoNTs) are highly toxic proteins that cause the fatal
neuroparalytic illness called botulism. BoNTs are produced by the anaerobic
bacteria Clostridium botulinum. Although most prominently contracted from
contaminated food, botulism can also be contracted from the soil, through the
air, or from an open wound. BoNTs are AB toxins composed of three
domains. The first domain, the A domain of BoNT, is the catalytic
component, a Zinc-dependent protease. The second and third domain, or the
translocation and receptor-binding domain respectively, comprise the B
domain of the toxin, the binding component. Botulinum neurotoxin serotype
B (BoNT/B) is a specific type of neurotoxin that binds to the neurons. The
membrane of a neuron depolarizes which then stimulates the transport of
calcium ions into the neuron. Proteins on the transmitter vesicle bind the
calcium and the vesicles are then transported to the plasma membrane by
SNARE proteins to release the neurotransmitter acetylcholine. BoNT/B
enters the lumen of the neurotransmitter vesicle and binds to the luminal loop
of synaptotagmin, causing the loop to change from a coil to an alpha helix.
This represents the high affinity binding of BoNT/B to neurons. Inside the
neuron, the A domain of the toxin is translocated across the vesicle
membrane by the translocation domain in a pH-dependent mechanism and
cleaves the vesicle associated membrane protein (VAMP). This prevents
neurotransmitter vesicles from fusing to the plasma membrane and inhibits
further release of acetylcholine. Since acetylcholine is important in
movement and memory, a lack of acetylcholine causes the nervous system to
slow down and causes flaccid paralysis of the muscles, otherwise known as
botulism.
The Structure and Binding of BoNT/B
Nathan Hale
The Action of Botulinum Neurotoxin B
Arnon, S. S. et al. JAMA 2001;285:1059-1070. Copyright © 2001 American Medical Association. All rights reserved.
Normal Vesicle Function
Chai Q, et al. Nature 2006.
• The A domain of the toxin translocates across the vesicle membrane
through the translocation domain
• Once in the neuron with a neutral pH level, the A domain refolds and
cleaves the VAMP (vesicle associated membrane protein)
• Without VAMP, exocytosis is prevented, ultimately inhibiting the release of
acetylcholine.
•Synaptotagmin protein on ACh-filled vesicle detects
incoming calcium
•Three SNARE proteins coil together, brining the vesicle to
the membrane: SNAP-25, Syntaxin, VAMP
•Through exocytosis, ACh is released and the vesicle is
then recycled back into the neuron.
Botulinum Neurotoxin Therapies
• First used as a therapeutic agent in the 1960’s
• Used to treat spastic muscle disorders such as dystonia (a movement
disorder where the muscles contract involuntarily) and blepharospasms
(a disability due to muscle spasms in eyelids which causes visual
impairment, resulting in functional blindness)
• Treated by injecting minute dosages of the toxin directly to the muscles
in the eyes, which will temporarily paralyze them by blocking the release
of acetylcholine
Arnon, S. S. et al. JAMA 2001;285:1059-1070. Copyright © 2001 American Medical Association. All rights reserved.
Impaired Vesicle Function
2nm1.pdb
Above and right: Botulinum Neurotoxin B (shown above in
gray) interacting with the colorful synaptotagmin peptide
located in the axon terminal membrane.
Ganglioside Binding Assay
www.egeneralmedical.com/info-botox.htm
Arnon, S. S. et al. JAMA 2001;285:1059-1070. Copyright © 2001 American Medical Association. All rights reserved.
Absorbance (at 450nM)
Inhalation Botulism
Symptoms of inhaling the neurotoxin consist of difficulty moving eyes,
slurred speech, and severe muscle weakness. Other symptoms include
sluggishly reactive pupils, dry mouths, and hyperventilation. In Picture A,
patient is at rest; one can see his drooping eyelids, dilated pupils, and
muscle symmetry. In Picture B, when asked to smile, the patient exhibits
unsymmetrical muscle movement. The patient also demonstrates an
absence of smile creases.
Because BoNT/B binds
BoNT binding to co‐receptor Gt1b
simultaneously to the protein
synaptotagmin as well as a
Hcr A1 + Gt1b
3
lipid ganglioside, an
Hcr A1 ‐Gt1b
2.5
experiment was designed to
2
discover the amount of Hcr
1.5
A1(Heavy Chain Receptor
1
subtype A) needed to reach
0.5
the optimal level of
0
ganglioside (Gt1b) binding.
1
4
12
37
111 333 1000
‐0.5 0.45
Hcr A1 (nM)
Bovine albumin was added to
block all other receptors
except the ganglioside; then Hcr A1 was added into each well of the assay
incrementally. The data show that more concentrated amounts of Hcr A1 bind
greater amounts of Gt1b, resulting in a higher absorbency. The 50% binding was
observed at ~ 300 nM HCR A1. This knowledge helps scientists in further
research towards the discovery of vaccines.
A SMART Team project supported by the National Institutes of Health (NIH) – National Center for Research Resources Science Education Partnership Award (NCRR-SEPA)
•BoNT/B sits on exterior of membrane bound with high
affinity
•When the luminal domain of the vesicle is exposed,
BoNT/B binds to the synaptotagmin
•A signal transduction pathway is initiated and vesicle is
recycled back into the neuron with the toxin
•The toxin’s ganglioside receptors also bind to the lumen of
the vesicle
•Through a proton pump, H+ ions enter the vesicle, and the
pH of the lumen decreases
Conclusion
The BONT/B ultimately prevents the release of acetylcholine
from the axon terminal membrane, causing flaccid paralysis
of the skeletal muscles. Knowing about this toxin and the
way it binds allows us to develop therapies that can help
cure people with spastic muscle disorders.
References
Jin R, Rummel A, Binz T, Brunger AT Botulinum neurotoxin B recognizes its protein receptor
with high affinity and specificity. Nature. 2006 Dec 21;444(7122):1092-5. Epub 2006 Dec 13.
"Dystonias Fact Sheet." National Institute of Neurological Disorders and Stroke (NINDS). 04
Dec. 2008. National Institutes of Health. 26 Feb.
2009<http://www.ninds.nih.gov/disorders/dystonias/detail_dystonias.htm>.
"Botox for Migraines." MAGNUM: The National Migraine Association. Home Page. 2009. The
National Migraine Association. 26 Feb. 2009
<http://www.migraines.org/treatment/probotox.htm>.