BOTULINUM TOXIN
Introduction
Toxins are biomolecules which are produced by bacteria, fungi, insects, plants, vertebrate and invertebrate animals. Their main function is to harm other organisms as a form of protection.
There are various classifications of BoNTs, with approximately 40 different BoNT types and subtypes. Botulinum toxin (BoNT) is considered as a lethal biological toxin. It is produced by obligate anaerobic gram positive bacteria, Clostridium botulinum and can have many adverse effects on the nervous system, giving rise to what is known as botulism. In addition to this, it has an important role in cosmetics and also has a positive effect when treating various neurological, movement and gastrointestinal disorders.
Throughout this page, we will review information from scientific papers about the structure and function of botulinum neurotoxin, the mechanism of action on human cells, the extraction process to be used in cosmetics as Botox® injections, the role of botulinum toxin in disease, and finally the uses of this protein.
Structure and Function
BoNT is classified as a zinc dependent metalloprotease (this means that its mechanism of action depends on zinc). There are eight different exotoxins (toxins secreted by bacteria) classified as BoNTs, which are diverse from each other and all bring about neurological damage.These are: A, B, C1, C2, D, E, F and G. (Whitemarsh, et al. 2013)
In addition to this, it can act on the following body sites:
neuromuscular junction
autonomic ganglia
postganglionic parasympathetic nerve endings
postganglionic sympathetic nerve endings. (Pirazzini et al., 2017)
These 150 kDa large neurotoxic proteins consist of a 50 kDa light chain and a 100 kDa heavy chain connected by a disulfide bond.
A structural representation of botulinum can be observed in Figure 1 as well as a short pymol animation representing the botulinum protein can be seen below.
Figure 1
Here we see the structure of the protein being divided in a heavy chain and a light chain linked by a disulfide bond. The catalytic domain is responsible for the catalytic activity of the protein, and it is located within the light chain. Also, within the light chain the HEXXH motif is located, this is responsible for binding Zm. These structural domains are important to mediate protein functioning.
The role of the heavy chain is to help BoNT attach to neuronal membranes at neuromuscular junctions. The heavy chain is further divided into 2 regions:
N-terminal translocation domain, which has a free amine group on the first alpha Carbon and 6 parallel alpha-helices (2 long and 4 short). It allows the uptake of the toxin in the neural cells.
C-terminal receptor binding domain, which has a free carboxylic group on the last alpha Carbon. It mediates interactions with nerve cells (Lam & Jin., 2015).
The light chain is dependent on the zinc ion to bring about its function. It also contains the catalytic site of the protein which is characterised by the HEXXH motif, where the H refers to histidine, E to glutamate and X to any amino acid.This part of the protein is the domain which possesses metalloprotease activity. (Rossetto et al., 2014)
Mechanism of Action
Different BoNT serotypes bind to different target proteins. After BoNT is injected into the target tissue in the body, its heavy chain is able to specifically bind to the cholinergic nerve terminals’ glycoprotein structures. After binding takes place, the glycoprotein structures are internalised into the vesicles. When this process is complete, BoNTs light chain also binds to the SNARE protein complex with high specificity. The light chain is proteolytically cleaved, which avoids any docking of the acetylcholine vesicle on the cellular membranes’ inner surface. As a result, vesicle fusion is blocked.
The effect of the blockage depends on the target tissues it occurs in. For instance in the muscle, chemical denervation gives rise to paresis (weakened muscle movement) whilst glandular secretion is halted in the exocrine glands. Exocytosis of acetylcholine is inhibited; such inhibition is terminated by restoration of the SNARE protein complex turnover (Dressler & Saberi 2005; Padda & Tadi, 2021)
Figure 2 and 3 provide a depiction of the mechanism of action of botulinum toxin: When an action potential in a neuron is released, the axon terminal of neurons becomes depolarised. Acetylcholine is released by SNARE from the cytosol into the synaptic cleft.
Figure 2
Normal Transmitter Release:
Synaptobrevin, syntaxin and SNAP 25 are SNARE proteins. Synaptobrevin is found attached to the synaptic vesicle while the syntaxin and the SNAP 25 are present on the cell membrane of the neuron.
The SNARE proteins bind together and bring the synaptic vesicle closer to the membrane.
The vesicle binds to the neuron cell membrane and the acetylcholine is released into the synaptic cleft.
The acetylcholine binds to the acetylcholine receptors and enters the muscle cell. Figure created with BioRender.com
Figure 3
Mechanism of Action of Botulinum Toxin:
The botulinum toxin is present in the synaptic cleft.
The cell membrane of the nerve terminus forms a vesicle around the botulinum, allowing it to enter the nerve cell.
The botulinum heavy (green) and light (purple) chains dissociate from each other. The light chain exits the vesicle.
The free light chain attaches to the synaptobrevin and the SNAP 25 proteins on the acetylcholine vesicles and the nerve terminal membrane respectively. This binding of the light chain prevents the SNARE proteins from forming and thus prevents the fusion of the acetylcholine vesicle to the nerve terminal membrane. Figure created with BioRender.com
Extraction and Purification Process
In the cosmetics industry, BONT-A is used in botox injections. C. botulinum, specifically Hall strain, is grown in media composed of casein hydrolysate, yeast extract and glucose. Within the bacterium, the BoNT-A protein is found associated with other proteins, including hemagglutinin (HA) and nontoxic-non hemagglutinin (NTNH). These proteins become associated and form complexes. Following bacterial fermentation, these proteins are isolated and the BoNT-A protein is purified through a series of techniques including acid precipitation and column chromatography (Wortzman & Pickett, 2009). Acid precipitation is a technique by which addition of an acid to a solution causes the soluble protein to become positively charged and dissociate as a cation. Column chromatography is a separating technique by which a single, specific compound is separated from a mixture. These techniques ensure that the eluted protein is the purified BoNT-A of interest.
Following purification, taxological testing is carried out, then the product is diluted, filled into vials in a sterile manner and freeze-dried. The extraction procedure is standard, regardless of the company or brand name of the product. However, the formulation of the product varies (Wortzman & Pickett, 2009). For example, the product sold under the brand name Botox® Cosmetic consists of 100 units (U) of BoNT-A, 0.5 mg of albumin protein and 0.9 mg of sterile saline solution which then is further diluted prior to intramuscular injection.
Role in Disease
BoNT is responsible for botulism which nowadays is a rare disease. However, in the 18th and 19th centuries, several botulism outbreaks took place, and BoNT became a public health hazard in the USA (Patil et al., 2016). World War II saw this toxin being employed as a biological weapon, where prostitutes stealthily administered botulinum capsules to Japanese officials.
This disease has different ways how it can manifest - all having similar clinical symptoms. Initially, there can be cranial nerve palsies, which might be followed by paralysis of certain voluntary muscles. These symptoms can progress to compromise the respiratory tract, and lead to death, as explained in the “Mechanism of Action'' section above. (Sobel, 2005). Such symptoms affect the peripheral nerve terminals due to the metalloprotease activity of BoNTs.
BoNT could either be:
Inhaled: this does not occur naturally. It is mostly associated with intentional events where this toxin is either weaponised or spilled accidentally.
Produced in a wound: if C.botulinum is present in the environment, its spores can infect open wounds and germinate in the human body to produce BoNT.
Produced in the intestines: this occurs in adults due to either bowel abnormalities or antimicrobial ingestion for a period of time. In infants, bacterial colonies can also grow in the intestines, leading to infant botulism.
Ingested through canned food: fortunately this is not an issue nowadays since canned food goes through a process designed specifically to kill the bacterial spores (Palma et al., 2019).
Once a patient is diagnosed with this disease, intensive care is required alongside treatment using botulinum antitoxin. The extent of paralysis could be limited through the administration of the botulinum antitoxin. This anti-toxin works by binding to the toxin molecules in the body, which have not yet bonded to the nerves. By doing so, the antitoxin will neutralise the toxin molecule, hence preventing the toxin from attaching to the neuromuscular junction (Sobel, 2005).
An overview of the dominant pathological effects linked with botulinum can be seen in Figure 4.
Figure 4
Pathological effects of botulinum toxin in the human body.
Figure created with BioRender.com
Uses and Applications: Cosmetic and Therapeutic
Within the past decade, BoNT has not only become a standard in cosmetic procedures but is also used as a therapeutic agent (Patil et al., 2016).
Clinically, BoNT injections were first administered to help treat dystonia. This neurological disorder manifests itself in involuntary and repetitive muscle contractions and movements. BoNT therapy can also be employed to help with other movement disorders, including tremors, spasticity such as stroke and cerebral palsy, gastrointestinal disorders such as dysphagia, as well as neurodegenerative disorders (Jankovic, 2004). More specifically, BoNT can help patients with Parkinson's disease, by easing symptoms like cervical dystonia, involuntary eyelid contractions, urinary incontinence and drooling (Wagle Shukla & Malaty, 2017).
BoNT Type A injections are used for cosmetic purposes, providing a minimally invasive treatment with predictable results to treat cosmetic imperfections such as facial wrinkles, most commonly being frown and forehead lines. BoNT was only approved for cosmetic use by the U.S. Food and Drug Administration (FDA) in 2002 and today is one of the most frequently performed cosmetic procedures with 4.4 million procedures done in the U.S. alone in 2020 (American Society of Plastic Surgeons, 2020).
Conclusion
To conclude, a number of applications have been associated with BoNT. Amongst its varied uses, BoNTs have been applied in the cosmetics industry to reduce the effect of wrinkles, and have been applied in medicine to help with movement disorders such as focal dystonia and cerebral palsy. On the other hand, although rare, it can also give rise to botulism. Currently, research is being performed with aims of optimising dosages as well as finding new applications for this molecule (Kivi et al., 2020).
Check out the video linked below for more information on Botulinum toxin!
References:
Allergan Pharmaceuticals (Ireland) Ltd. (2002). BOTOX® COSMETIC (Botulinum Toxin Type A) Purified Neurotoxin Complex. Allergan Inc.
American Society of Plastic Surgeons. (2020). Plastic Surgery Statistics Report. https://www.plasticsurgery.org/documents/News/Statistics/2020/plastic-surgery-statistics-full-report-2020.pdf
Dressler, D., & Adib Saberi, F. (2005). Botulinum toxin: Mechanisms of action. European Neurology, 53(1), 3-9. https://doi.org/10.1159/000083259
Jankovic, J. (2004). Botulinum toxin in clinical practice. J Neurol Neurosurg Psychiatry, 75(7), 951. 10.1136/jnnp.2003.034702
Kivi, A., Ri, S., & Wissel, J. (2020). What clinicians and patients want: The past, the presence, and the future of the botulinum toxins. Toxicon, 177, 46-51. https://doi.org/https://doi.org/10.1016/j.toxicon.2020.02.004
Lam, K. H., & Jin, R. (2015). Architecture of the botulinum neurotoxin complex: a molecular machine for protection and delivery. Current opinion in structural biology, 31, 89–95. https://doi.org/10.1016/j.sbi.2015.03.013
Padda, I., & Tadi, P. (2021). Botulinum toxin.
Palma, N. Z., da Cruz, M., Fagundes, V., & Pires, L. (2019). Foodborne Botulism: Neglected Diagnosis. European Journal of Case Reports in Internal Medicine, 6(5)10.12890/2019_001122
Patil, S., Willett, O., Thompkins, T., Hermann, R., Ramanathan, S., Cornett, E. M., Fox, C. J., & Kaye, A. D. (2016). Botulinum Toxin: Pharmacology and Therapeutic Roles in Pain States. Current Pain and Headache Reports, 20(3), 15. 10.1007/s11916-016-0545-0
Pirazzini, M., Rossetto, O., Eleopra, R., & Montecucco, C. (2017). Botulinum Neurotoxins: Biology, Pharmacology, and Toxicology. Pharmacological reviews, 69(2), 200–235. https://doi.org/10.1124/pr.116.012658
Rossetto, O., Pirazzini, M. & Montecucco, C. Botulinum neurotoxins: genetic, structural and mechanistic insights. Nat Rev Microbiol 12, 535–549 (2014). https://doi.org/10.1038/nrmicro3295
Sobel, J. (2005). Botulism. Clinical Infectious Diseases, 41(8), 1167-1173. 10.1086/444507
Wagle Shukla, A., & Malaty, I. A. (2017). Botulinum Toxin Therapy for Parkinson's Disease. Semin Neurol, 37(02), 193-204.
Wortzman, M. S., & Pickett, A. (2009). The Science and Manufacturing Behind Botulinum Neurotoxin Type A-ABO in Clinical Use. Aesthetic Surgery Journal, 29(6), S34-S42. https://doi.org/10.1016/j.asj.2009.09.014