Introduction
Botulinum toxins (BT) are the most active protein toxins among bacterial, animal, plant and chemical agents. These toxins cause botulism [1]. At the same time, BT-based drugs have great prospects in the treatment of various diseases. Until now, research of many issues of BT pharmacology is being continued. These studies are very important to create BT-based drugs and anti-botulinum agents.
This review is devoted to molecular mechanisms underlying pharmacodynamics of BT type A (BTA) and possible factors influencing sensitivity to BT.
Structure of toxin complex and BT molecule
BT is a 150 kDa protein dimer with chemical formula C6760H10447N1743O2010S32. BT consists of 2 chains (light (L-chain) and heavy (H-chain) ones). L-chain is about 1/3 of molecular weight and linked with H-chain by a single disulfide bond [2].
L-chain is a protease destroying intracellular transport proteins. This chain forms catalytic domain of BT molecule. H-chain contains 2 domains. Binding domain binds to receptors on the surface of the target cell. Translocation domain ensures L-chain translocation via transmembrane channel. BT molecule is a dipole. It has an electric charge decreasing from the binding domain to the catalytic one [3]. This feature is essential for orienting the molecule near cellular membrane and binding to receptors.
In natural environment, bacteria synthesize BTs in a complex with several proteins: one non-toxic non-hemagglutinin (NTNHA) and several hemagglutinins [1].
NTNHA has a molecular weight of 130 kDa and very high amino acid sequence homology with BT. In contrast to BT molecule, there is no only protease aspect in NTNHA. Non-toxic non-hemagglutinin protects BT molecule from aggressive environmental influences including proteolytic enzymes of digestive tract [4].
There are 3 classes of hemagglutinins (molecular weight 33–35 kDA, 15–18 kDA and 70 kDA) [5]. Hemagglutinins do not directly contact with BT molecule but bind to NTNHA and ensure adhesion during absorption of toxin complex.
Non-toxic hemagglutinin and hemagglutinins can form various complexes with BT. Each complex contains only one BT molecule, which can be released under environmental pH changes [2].
BT absorption and distribution
BT can enter the body through both damaged and intact tissues. For medical purposes, BTA drugs are mainly administered via injection in maximum proximity to the target cells. Topical transdermal forms of BTA have been already developed. These forms do not imply integumentary tissue damage. However, these forms have not yet passed clinical trials phase III [6, 7].
In natural conditions, systemic effect of BT causes botulism after entering through the intact membranes.
There are several forms of botulism depending on toxin gateway: foodborne (intake of food contaminated with botulinum toxin), infant (intake of food contaminated with bacterial spores), inhalation (inhaling BT-containing aerosols), wound (drug injections in most cases), iatrogenic [8].
In natural conditions, BT should penetrate epithelial barriers and achieve systemic circulation to get to target cells. This process is called absorption.
BT penetration through intestinal or pulmonary epithelium is possible by 2 mechanisms (via the cell and intercellular contacts).
In case of transcytosis (penetration through the epithelial cell), BT binds to ganglioside receptors on the epithelial cell surface and undergoes endocytosis. Transport vesicles carry the toxin through the entire cell and release it into systemic circulation. Neither toxin structure change, nor chain release into cellular cytosol occur during transcytosis. This feature distinguishes BT binding with epithelial cells and neuronal cells [9, 10].
Paracellular pathway (through intercellular junctions) proceeds with or without hemagglutinins. Hemagglutinins are able to bind to E-cadherin of epithelial intercellular junctions, destroy these junctions and ensure BT release into systemic circulation [4]. However, BT molecule can destroy epithelial barriers even without these proteins. A. Maksymowych et al. [11] and F. Al-Saleem et al. [12] experimentally confirmed equivalent BT titers in systemic circulation with the same toxicity and effectiveness after injection of equimolar amounts of free BTA and BTA complex. However, it is assumed that hemagglutinins increase the rate of BT transport across the epithelium.
BT can bind to cholinergic and serotonergic neurons of intestinal nerve system during transport through the enteric wall. These neurons are located in submucosal layer and regulate motor and secretory activity of the bowel. Blockade of these neurons explains intestinal motility impairment (constipation) as one of the early symptoms of foodborne and infant botulism [13].
As soon as BT enters systemic circulation, it distributed in extracellular fluid in the whole body with the exception of central nervous system.
K. Eisele et al. [14] experimentally showed BT complex dissociation into active BT and hemagglutinins under pH values close to neutral (arterial blood pH 7.37–7.43 [15, 16]). Half-life period is less than a minute. Hemagglutinins cease to be of clinical significance after toxin complex dissociation.
F. Al-Saleem et al. [17] experimentally proved that BT reaches systemic circulation without obvious changes in structure and biological activity. Systemic circulation acts as a storage compartment for BT until toxin reaches the target cells. BT undergoes slight biotransformation in systemic circulation, does not accumulate in blood cells and remains mainly in a free active state. The concept of “systemic circulation — BT storage compartment” is confirmed by various studies. R. Fagan et al. [18] described serum active BTA in 11 days after ingestion of contaminated food, A. Sheth et al. [19] — after 25 days, L. Delbrassinne et al. [20] — after 29 days.
From intravascular liquid compartment, BT enters extravascular compartment and then extracellular fluid. In case of local injection for medical indications, BT immediately enters extravascular (or intravascular) compartment near the target cells without absorption. From extracellular fluid, BT should get to the target structure (peripheral cholinergic nerve) and bind to receptors.
One should understand normal neurotransmission in a synapse for comprehending the mechanism of BT binding to receptors.
Normal neurotransmission in a synapse
Neurotransmitters are synthesized in the cytosol of neurons and stored in the presynaptic nerve endings inside the vesicles. A proton pump (vesicular ATPase) in the membrane of synaptic vesicles increases the concentration of protons inside the vesicle [8]. Electrochemical proton gradient determines the entry of mediator from the cytosol into the vesicle and accumulation inside. Vesicles with a transmitter are located in the neuron cytoplasm and bind to the specialized domains of presynaptic membrane (active zones [21]). This process is known as docking [22]. Docking occurs only in active zones and is regulated by a large number of transport proteins [23].
Nerve impulse is followed by depolarization of presynaptic membrane of the axon, calcium channel opening and inwards Ca2+ current [24]. In response to Ca2+ current, vesicle with neurotransmitter fuses with presynaptic membrane within the active zone. This stage is called priming and regulated by 2 integral membrane proteins of synaptic vesicle (synaptobrevin and synaptotagmin), as well as 2 proteins in presynaptic membrane (SNAP25 and syntaxin) and cytosolic proteins, including complexin [25–28].
Fast conformational changes of the vesicle ensured by regulatory proteins results complete fusion of synaptic vesicle with presynaptic membrane and formation of a pore for neurotransmitter release into synaptic cleft [29].
Neurotransmitter diffuses from axon and binds to postsynaptic receptor. This process triggers signaling pathways in postsynaptic cell. In case of neuromuscular junction, acetylcholine binds to receptor on the membrane of myocyte and results depolarization of its membrane. Inward Ca2+ current and muscle contraction follow depolarization.
Synaptic vesicle temporarily opens outward into synaptic cleft during neurotransmitter release. Later, the vesicle is internalized into axon during endocytosis. After endocytosis, the vesicle is again filled with neurotransmitter, and the next cycle of neurotransmission begins [30].
BTA binding to the target cell
BTA binding to the target cells is realized through the membrane receptors [31]. Binding to presynaptic membrane requires interaction of BTA molecule with a combination of high- and low-affinity receptors [32]. Currently, 3 receptors and several co-receptors have been described in this combination.
Endocytosis of active toxin molecule and its further conformational changes are possible only after binding to the entire combination of axonal receptors [5]. Binding to only one receptor will not internalize the toxin. This multiple-stage mechanism of BTA binding to receptors compensates low environmental concentration of BTA, high extracellular fluid flow velocity around the cells and small surface area of the axons.
The first receptor is polysialicganglioside
The first receptor for BTA on the axon surface is polysialic ganglioside GT1b (PSG).
Gangliosides are glycosylated lipids of cell membranes. Although gangliosides are present in all vertebrate tissues, they are more abundant in neuronal membranes [33]. Gangliosides are involved in synthesis of myelin, axon-myelin interactions, peripheral and central axonal stability [32].
Density of PSG on presynaptic membrane is high. PSGs are organized into microdomains near the active zones of presynaptic membrane [34]. Arrangement of PSG receptors in these zones is of great importance for BT binding with other receptors.
Oligosaccharide (BT-binding part) of PSG protrudes into synaptic cleft far beyond the membrane and has a negative electrical charge [8]. BTA molecule is a dipole with positive charge of binding domain [3]. Different charges of BTA binding domain and PSG receptors (and other anionic lipids of axon membrane) ensure BTA molecule reorientation and higher likelihood of binding to the receptor.
It is assumed that PSG are initial binding sites that capture BTA from large 3D space of extracellular fluid and move it into 2D space of membrane [5]. It is necessary for toxin binding to subsequent receptors. On the one hand, binding to PSG is irreversible, since BTA is extracted from extracellular fluid and fixed on the axon membrane. On the other hand, toxin is still exposed and available for neutralizing antibodies at this stage.
PSG are membrane receptors for both BT and anti-ganglioside autoantibodies associated with human neuropathy. PSG autoantibody release in case of neuropathy may be important in impaired sensitivity and resistance to BT [35].
The second receptor is fibroblast growth factor receptor 3
HC subdomain of BTA has structural homology with basic fibroblast growth factor (FGF) [36]. This structural similarity enables BTA high-affinity binding to fibroblast growth factor receptor 3 (FGFR3b) on the axon surface [37].
However, FGFR3b is affine not only to BTA, but also to many fibroblast growth factors. Moreover, tropism of this receptor to growth factors is higher than to BT. Native ligands for FGFR3 (growth factors FGF1, FGF2 and FGF9) compete for binding with FGFR3 and can block BTA uptake by cells after binding to these receptors [8].
Moreover, several low-affinity cofactors regulate activity of FGFR3b receptors including heparan sulfate, neuropilin-1, anosmin, etc. [38]. FGFR3 non-specificity, competition between BTA and fibroblast growth factors and effect of cofactors on receptor activity can explain vulnerability of this receptor mechanism and variable sensitivity to BT. Moreover, defective FGFR3 can be expressed in some conditions associated with FGFR3 mutation (skeletal dysplasia, epidermal nevi, seborrheic keratosis, hyperinsulinemia) [39–43]. The effect of FGFR3 mutation on BT susceptibility is still unclear.
The third receptor is transmembrane vesicular receptor SV2C
SV2 is a protein receptor in the vesicle membrane [44] of all peripheral and central neurons, as well as secretory granules of endocrine cells [45]. SV2 is expressed by membranes of cellular vesicles accumulating not only acetylcholine, but also GABA, dopamine, glutamate, substance P and some other mediators [46].
In contrast to PSG receptor expressed in synaptic cleft, BTA-binding site of SV2 receptor protrudes into the lumen of synaptic vesicle. This site is inaccessible to toxin while the vesicle is in the axon cytosol [47]. SV2 becomes available for BTA after fusion of the vesicle with presynaptic membrane and exocytosis of acetylcholine [48].
Thus, BTA binding to the entire combination of receptors occurs in active zones only after fusion of synaptic vesicle with presynaptic membrane and opening of vesicle lumen into synaptic cleft. This process facilitates subsequent endocytosis of BTA. BT becomes inaccessible for neutralizing antibodies after binding to a combination of receptors and endocytosis.
Endocytosis
BTA molecule binding to the receptors results receptor-mediated endocytosis of receptors and toxin [49].
Vesicle lumen has a neutral pH immediately after endocytosis. Proton pump of vesicular ATPase controls reuptake of neurotransmitter [50] and pumps the protons into synaptic vesicle. This process results a gradual decrease of pH in the vesicle [51].
L-chain translocation
Acidification leads to irreversible conformational changes of H-chain and L-chain of BTA. As a result, H-chain connected with a vesicle membrane through receptors forms a transmembrane H-channel [52, 53]. L-chain leaves the vesicle through this channel [54]. After that, disulfide bond is destroyed.
L-chain translocation occurs in pH 4.5 — 6. pH decrease results protonation of carboxylate amino acid residues of H- and L-chains of BTA. Carboxylate residues are located on one side of toxin molecule, and their protonation results significant conformational changes of molecule [55]. Positively charged surface of BTA molecule interacts with anionic surface of the vesicle membrane and forms a protein-lipid complex [56]. It is assumed that L-chain becomes a “molten protein globule” with hydrophobic properties [8]. On the one hand, hydrophobicity of L-chain ensures its passage through the membrane channel formed by H-chain. On the other hand, pH decrease is followed by more hydrophobic properties of the molecule surface with disulfide bond. This ensures disulfide bond integrity until complete L-chain translocation.
To cross the vesicle membrane, the L-chain should be disulfide-linked to the H-chain throughout the translocation process [55]. Premature destruction of disulfide bond at any stage before L-chain release into cytosol interrupts translocation [57].
At the end of translocation, disulfide bond is destroyed by thioredoxin reductase (TrxR) — thioredoxin (Trx) system for L-chain release into cytosol and expression of its catalytic activity [58].
TrxR — Trx system is a main cellular redox system. TrxR and Trx are proteins located on the cytosolic side of the vesicle membrane. Their inhibition can lock the BTA effect at the stage when the toxin is unavailable for neutralizing antibodies [59]. G. Zanetti et al. (2015) experimentally showed that TrxR — Trx enzyme pair inhibitors block protease activity of L-chain of all known BT serotypes in cultured neurons. In vivo, these inhibitors prevent toxin-induced paralysis in mice regardless the BT serotype [60].
Disulfide bond destruction means the end of intracellular existence of intact active BTA molecule (holotoxin). None of the chains per se could disrupt the cell function even if L-chain or H-chain would be exported from the cell. Only holotoxin is able to pass through many stages followed by blockade of neurotransmission [61]. On the other hand, conformational changes associated with pH-induced L-chain translocation, create a “trap”. The last one makes impossible inverse translocation into endosome and return of active toxin molecule to external environment [5].
L-chain-mediated cleavage of transport proteins
In neuron cytosol, L-chain functions as metalloprotease. L-chain enzymatically cleaves 9 amino acids from the C-terminus of V-soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein SNAP25 (SNAP25206). As a result, SNAP25197 protein occurs [62, 63]. Intact SNAP25 is required for attachment of a vesicle with mediator and subsequent neurotransmitter release. Moreover, this protein is directly involved in regulation of Ca-channels of presynaptic membrane [64]. SNAP25 cleavage disrupts exocytosis of mediators and causes blockade of neurotransmission [65].
Synaptic activity is very sensitive to minimal SNAP25 cleavage. It is hypothesized that cytosol SNAP25 in neurons exists in different pools and only a small amount of SNAP25 is actively involved in exocytosis and accessible for L-protease [66]. These data are experimentally confirmed by complete blockade of neurotransmitter release after cleavage of 10–15% of total intracellular pool of SNAP25 [67–69]. Cleavage of only 2–3% of SNAP25 pool by L-protease blocks miniature postsynaptic potentials of cells (weak depolarization of postsynaptic membranes in a state of neuromuscular resting) [70].
At the same time, SNAP25197 protein as a product of SNAP25 proteolysis independently inhibits exocytosis [71]. F. Meunier et al. [72] described long-term SNAP25197 persistence in cytosol as a part of unproductive SNARE complex and prolongation of BTA effect. At the same time, removal of several amino acids from SNAP25197 rapidly restores exocytosis.
Zinc metabolism during translocation
Zinc is essential for catalytic activity of the L-chain. One botulinum holotoxin molecule contains one zinc atom via reversible zinc-binding amino acid sequence of the L-chain [73].
Vesicle acidification causes protonation of zinc-binding sites of BT molecule. Translocation is associated with L-chain denaturation and loss of chelate site integrity. This results zinc dissociation and its release to the pool of cytosolic zinc.
L. Simpson et al. [74] experimentally found that removal of zinc from the active BT molecule was followed by loss of L-chain catalytic activity in acellular specimens. However, this activity was preserved in intact neuromuscular junctions because the internalized toxin bound cytosolic zinc. Thus, zinc retained by holotoxin (intact active molecule) is not the same zinc bound to catalytically active L-chain. L-chain binds cytosolic zinc.
Mediator release blockade
The main target of BTA is peripheral neurons. BT inhibits acetylcholine release in these cells [75].
Various authors reported BTA-mediated blockade of release of not only acetylcholine, but also other neurotransmitters accumulated in vesicles [32]. These are adrenaline, norepinephrine, dopamine [76, 77], glutamate [78], glycine [79], serotonin [80], substance P [81], etc. Thus, BT should be considered as a blocker of exocytosis of various mediators rather specific blocker of acetylcholine excretion. This feature makes BT perspective in the treatment and prevention of many diseases.
Conclusion
Further study of unique mechanisms of BT pharmacology opens up the prospects for opportunities to influence its action, i.e. prolong or reduce duration of effect, increase or decrease BT sensitivity in certain patients. Moreover, the protocols for optimal combination of BT with various aesthetic procedures could be developed using these data. Comprehensive understanding the multiple aspects of not only neuronal selectivity of BT, but also its interaction with non-neuronal cells will be valuable for therapeutic use of BT-based drugs in various fields of medicine.
The author declares no conflicts of interest.