Transdermal delivery of botulinum toxin-A through phosphatidylcholine/cholesterol nanoliposomes for treatment of post-acne scarring.

As an acne sequela, post-acne scarring (PSA) has huge negative impact on sufferers' quality of life because of aesthetical embarrassment. Transdermal delivery of botulinum toxin-A (BTXA) is a promising strategy for PAS treatment, but currently reported approaches are far from satisfactory. In this work, phosphatidylcholine/cholesterol (PC/Chol) nanoliposomes were utilized for encapsulation and transdermal delivery of BTXA. The composition, structure, morphology, size, size distribution, etc. of as-prepared BTXA@liposome nanoparticles were investigated in detail. Simulated transdermal delivery assay indicated that the diffusion depth of the BXTA@liposome nanoparticles was nearly 8 times that of pure BTXA and reached 380 µm. 12 facial PSA patients were recruited to evaluate the curative effect of the BTXA@liposome nanoparticles on PSA. Through ECCA (échelle d'évaluation clinique des cicatrices d'acné) scoring and self-evaluation of patients, the resultant data indicated that compared to hyaluronic acid (HA) hydrogel treatment the BTXA@liposome/HA hydrogel treatment could better relieve PSA to some extent but didn't show significant advantage. Further work is needed to verify the feasibility and curative effect of this method in PSA treatment in the future.

Characterization of a membrane binding loop leads to engineering botulinum neurotoxin B with improved therapeutic efficacy.

Botulinum neurotoxins (BoNTs) are a family of bacterial toxins with seven major serotypes (BoNT/A-G). The ability of these toxins to target and bind to motor nerve terminals is a key factor determining their potency and efficacy. Among these toxins, BoNT/B is one of the two types approved for medical and cosmetic uses. Besides binding to well-established receptors, an extended loop in the C-terminal receptor-binding domain (HC) of BoNT/B (HC/B) has been proposed to also contribute to toxin binding to neurons by interacting with lipid membranes (termed lipid-binding loop [LBL]). Analogous loops exist in the HCs of BoNT/C, D, G, and a chimeric toxin DC. However, it has been challenging to detect and characterize binding of LBLs to lipid membranes. Here, using the nanodisc system and biolayer interferometry assays, we find that HC/DC, C, and G, but not HC/B and HC/D, are capable of binding to receptor-free lipids directly, with HC/DC having the highest level of binding. Mutagenesis studies demonstrate the critical role of consecutive aromatic residues at the tip of the LBL for binding of HC/DC to lipid membranes. Taking advantage of this insight, we then create a "gain-of-function" mutant HC/B by replacing two nonaromatic residues at the tip of its LBL with tryptophan. Cocrystallization studies confirm that these two tryptophan residues do not alter the structure of HC/B or the interactions with its receptors. Such a mutated HC/B gains the ability to bind receptor-free lipid membranes and shows enhanced binding to cultured neurons. Finally, full-length BoNT/B containing two tryptophan mutations in its LBL, together with two additional mutations (E1191M/S1199Y) that increase binding to human receptors, is produced and evaluated in mice in vivo using Digit Abduction Score assays. This mutant toxin shows enhanced efficacy in paralyzing local muscles at the injection site and lower systemic diffusion, thus extending both safety range and duration of paralysis compared with the control BoNT/B. These findings establish a mechanistic understanding of LBL-lipid interactions and create a modified BoNT/B with improved therapeutic efficacy.

Biology activity and characterization of the functional L-HN fragment derivative of botulinum neurotoxin serotype E.

OBJECTIVES: The mature botulinum neurotoxin (BoNT) is a long peptide chain consisting of a light chain (L) and a heavy chain (H) linked by a disulfide bond, where the heavy chain is divided into a translocation domain and an acceptor binding domain (Hc). In this study, we further explored the biology activity and characteristics of recombinant L-HN fragment (EL-HN) composed of the L and HN domains of BoNT/E in vivo and in vitro. METHODS: Neurotoxicity of L-HN fragments from botulinum neurotoxins was assessed in mice. Cleavage of dichain EL-HN in vitro and in neuro-2a cells was assessed and compared with that of single chain EL-HN. Interaction of HN domain and the receptor synaptic vesicle glycoprotein 2C (SV2C) was explored in vitro and in neuro-2a cells only expressing SV2C. RESULTS: We found that the 50% mouse lethal dose of the nicked dichain EL-HN fragment (EL-HN-DC) was 0.5 µg and its neurotoxicity was the highest among the L-HN's of the four serotypes of BoNT (A/B/E/F). The cleavage efficiency of EL-HN-DC toward synaptosome associated protein 25 (SNAP25) in vitro was 3-fold higher than that of the single chain at the cellular level, and showed 200-fold higher animal toxicity. The EL-HN-DC fragment might enter neuro-2a cells via binding to SV2C to efficiently cleave SNAP25. CONCLUSIONS: The EL-HN fragment showed good biological activities in vivo and in vitro, and could be used as a drug screening model and to further explore the molecular mechanism of its transmembrane transport.

Elucidation of critical pH-dependent structural changes in Botulinum Neurotoxin E.

Botulinum Neurotoxins (BoNT) are the most potent toxins currently known. However, they also have therapeutic applications for an increasing number of motor related conditions due to their specificity, and low diffusion into the system. Although the start- and end- points for the BoNT mechanism of action are well-studied, a critical step remains poorly understood. It is theorised that BoNTs undergo a pH-triggered conformational shift, activating the neurotoxin by priming it to form a transmembrane (TM) channel. To test this hypothesis, we combined molecular dynamics (MD) simulations and small-angle x-ray scattering (SAXS), revealing a new conformation of serotype E (BoNT/E). This conformation was exclusively observed in simulations below pH 5.5, as determined by principal component analysis (PCA), and its theoretical SAXS profile matched an experimental SAXS profile obtained at pH 4. Additionally, a localised secondary structural change was observed in MD simulations below pH 5.5, in a region previously identified as instrumental for membrane insertion for serotype A (BoNT/A). These changes were found at a critical pH value for BoNTs in vivo, and may be relevant for their therapeutic use.

Crystal structure of the catalytic domain of botulinum neurotoxin subtype A3.

Botulinum neurotoxins (BoNTs) are among the most widely used therapeutic proteins; however, only two subtypes within the seven serotypes, BoNT/A1 and BoNT/B1, are currently used for medical and cosmetic applications. Distinct catalytic properties, substrate specificities, and duration of enzymatic activities potentially make other subtypes very attractive candidates to outperform conventional BoNTs in particular therapeutic applications. For example, BoNT/A3 has a significantly shorter duration of action than other BoNT/A subtypes. Notably, BoNT/A3 is the subtype with the least conserved catalytic domain among BoNT/A subtypes. This suggests that the sequence differences, many of which concern the α-exosite, contribute to the observed functional differences in toxin persistence by affecting the binding of the substrate SNAP-25 and/or the stability of the catalytic domain fold. To identify the molecular determinants accounting for the differences in the persistence observed for BoNT/A subtypes, we determined the crystal structure of the catalytic domain of BoNT/A3 (LC/A3). The structure of LC/A3 was found to be very similar to that of LC/A1, suggesting that the overall mode of SNAP-25 binding is common between these two proteins. However, circular dichroism (CD) thermal unfolding experiments demonstrated that LC/A3 is significantly less stable than LC/A1, implying that this might contribute to the reduced toxin persistence of BoNT/A3. These findings could be of interest in developing next-generation therapeutic toxins.

Gangliosides interact with synaptotagmin to form the high-affinity receptor complex for botulinum neurotoxin B.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b-synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with a model in which a preassembled GT1b-synaptotagmin complex constitutes the high-affinity BoNT/B receptor.

Crystal Structures of the Clostridium botulinum Neurotoxin A6 Cell Binding Domain Alone and in Complex with GD1a Reveal Significant Conformational Flexibility.

Clostridium botulinum neurotoxin A (BoNT/A) targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, by cleaving synaptosomal-associated protein of 25 kDa size (SNAP-25). Cleavage of SNAP-25 results in flaccid paralysis due to repression of synaptic transmission at the neuromuscular junction. This activity has been exploited to treat a range of diseases associated with hypersecretion of neurotransmitters, with formulations of BoNT/A commercially available as therapeutics. Generally, BoNT activity is facilitated by three essential domains within the molecule, the cell binding domain (HC), the translocation domain (HN), and the catalytic domain (LC). The HC, which consists of an N-terminal (HCN) and a C-terminal (HCC) subdomain, is responsible for BoNT's high target specificity where it forms a dual-receptor complex with synaptic vesicle protein 2 (SV2) and a ganglioside receptor on the surface of motor neurons. In this study, we have determined the crystal structure of botulinum neurotoxin A6 cell binding domain (HC/A6) in complex with GD1a and describe the interactions involved in ganglioside binding. We also present a new crystal form of wild type HC/A6 (crystal form II) where a large 'hinge motion' between the HCN and HCC subdomains is observed. These structures, along with a comparison to the previously determined wild type crystal structure of HC/A6 (crystal form I), reveals the degree of conformational flexibility exhibited by HC/A6.

Synaptotagmin Binding to Botulinum Neurotoxins.

Botulinum neurotoxins (BoNTs) are exceptionally toxic proteins that cause paralysis but are also extensively used as treatment for various medical conditions. Most BoNTs bind two receptors on neuronal cells, namely, a ganglioside and a protein receptor. Differences in the sequence between the protein receptors from different species can impact the binding affinity and toxicity of the BoNTs. Here we have investigated how BoNT/B, /DC, and /G, all three toxins that utilize synaptotagmin I and II (Syt-I and Syt-II, respectively) as their protein receptors, bind to Syt-I and -II of mouse/rat, bovine, and human origin by isothermal titration calorimetry analysis. BoNT/G had the highest affinity for human Syt-I, and BoNT/DC had the highest affinity for bovine Syt-II. As expected, BoNT/B, /DC, and /G showed very low levels of binding to human Syt-II. Furthermore, we carried out saturation transfer difference (STD) and STD-TOCSY NMR experiments that revealed the region of the Syt peptide in direct contact with BoNT/G, which demonstrate that BoNT/G recognizes the Syt peptide in a model similar to that in the established BoNT/B-Syt-II complex. Our analyses also revealed that regions outside the Syt peptide's toxin-binding region are important for the helicity of the peptide and, therefore, the binding affinity.

Strategies to Counteract Botulinum Neurotoxin A: Nature's Deadliest Biomolecule.

Botulinum neurotoxin serotype A (BoNT/A), marketed commercially as Botox, is the most toxic substance known to man with an estimated intravenous lethal dose (LD50) of 1-2 ng/kg in humans. Despite its widespread use in cosmetic and medicinal applications, no postexposure therapeutics are available for the reversal of intoxication in the event of medical malpractice or bioterrorism. Accordingly, the Centers for Disease Control and Prevention categorizes BoNT/A as a Category A pathogen, posing the highest risk to national security and public health as a result of the ease with which BoNT/A can be weaponized and disseminated. BoNT/A-mediated lethality results from neurons impeded from releasing acetylcholine, which ultimately causes muscle paralysis and possible death by asphyxiation with the loss of diaphragm function. Currently, the only available respite for BoNT/A poisoning is antibody-based therapy; however, this intervention is only effective within 12-24 h postexposure. Small molecule therapeutics remain the only opportunity to reverse BoNT/A intoxication after neuronal poisoning and are urgently needed. Nevertheless, no small molecule BoNT/A inhibitors have reached the clinic or even advanced to clinical trials. This Account highlights the accomplishments and existing challenges facing BoNT/A drug discovery today. Using the comprehensive body of work from our laboratory, we illustrate our nearly two-decade endeavor to discover a clinically relevant BoNT/A inhibitor. Specifically, a discussion on the identification and characterization of new chemical leads, the development of in vitro and in vivo assays, and pertinent discoveries in BoNT/A structural biology related to small molecule inhibition is presented. Lead discovery efforts in our laboratory have leveraged both in vitro high-throughput screening and rational design, and an array of mechanistic strategies for inhibiting BoNT/A has been discovered, including noncovalent inhibition, metal-binding active site inhibition, covalent inhibition, and α- and ß-exosite inhibition. We contrast the strengths and weaknesses of each of these mechanistic strategies and propose the most favorable approach for success. Finally, we discuss multiple serendipitous discoveries of antibotulism small molecules with alternative mechanisms of action. Remaining challenges facing clinically relevant BoNT/A inhibition are presented and analyzed, including the current inability to reconcile toxin half-life (months to greater than one year) in neurons with in vivo pharmaceutical lifetimes and reoccurring inconsistencies between in vitro, cellular, and in vivo translation. Our Account of BoNT/A chemical research emphasizes the present accomplishments and critically analyzes the remaining obstacles for drug discovery. Importantly, we call for an increased focus on the discovery of safe and effective covalent inhibitors of BoNT/A that compete with the inherent half-life of the toxin.

The role of the single interchains disulfide bond in tetanus and botulinum neurotoxins and the development of antitetanus and antibotulism drugs.

A large number of bacterial toxins consist of active and cell binding protomers linked by an interchain disulfide bridge. The largest family of such disulfide-bridged exotoxins is that of the clostridial neurotoxins that consist of two chains and comprise the tetanus neurotoxins causing tetanus and the botulinum neurotoxins causing botulism. Reduction of the interchain disulfide abolishes toxicity, and we discuss the experiments that revealed the role of this structural element in neuronal intoxication. The redox couple thioredoxin reductase-thioredoxin (TrxR-Trx) was identified as the responsible for reduction of this disulfide occurring on the cytosolic surface of synaptic vesicles. We then discuss the very relevant finding that drugs that inhibit TrxR-Trx also prevent botulism. On this basis, we propose that ebselen and PX-12, two TrxR-Trx specific drugs previously used in clinical trials in humans, satisfy all the requirements for clinical tests aiming at evaluating their capacity to effectively counteract human and animal botulism arising from intestinal toxaemias such as infant botulism.

Translocation domain of botulinum neurotoxin A subtype 2 potently induces entry into neuronal cells.

Botulinum neurotoxin (BoNT) is the causative agent of botulism in humans and animals. Only BoNT serotype A subtype 1 (BoNT/A1) is used clinically because of its high potency and long duration of action. BoNT/A1 and BoNT/A subtype 2 (BoNT/A2) have a high degree of amino acid sequence similarity in the light chain (LC) (96%), whereas their N-and C-terminal heavy chain (HN and HC ) differ by 13%. The LC acts as a zinc-dependent endopeptidase, HN as the translocation domain, and HC as the receptor-binding domain. BoNT/A2 and BoNT/A1 had similar potency in the mouse bioassay, but BoNT/A2 entered faster and more efficiently into neuronal cells. To identify the domains responsible for these characteristics, HN of BoNT/A1 and BoNT/A2 was exchanged to construct chimeric BoNT/A121 and BoNT/A212. After expression in Escherichia coli, chimeric and wild-type BoNT/As were purified as single-chain proteins and activated by conversion to disulfide-linked dichains. The toxicities of recombinant wild-type and chimeric BoNT/As were similar, but dropped to 60% compared with the values of native BoNT/As. The relative orders of SNAP-25 cleavage activity in neuronal cells and toxicity differed. BoNT/A121 and recombinant BoNT/A2 have similar SNAP-25 cleavage activity. BoNT/A2 HN is possibly responsible for the higher potency of BoNT/A2 than BoNT/A1.

Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A.

Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral ß-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open ß-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

Botulinum Toxin-Chitosan Nanoparticles Prevent Arrhythmia in Experimental Rat Models.

Several experimental studies have recently demonstrated that temporary autonomic block using botulinum toxin (BoNT/A1) might be a novel option for the treatment of atrial fibrillation. However, the assessment of antiarrhythmic properties of BoNT has so far been limited, relying exclusively on vagal stimulation and rapid atrial pacing models. The present study examined the antiarrhythmic effect of specially formulated BoNT/A1-chitosan nanoparticles (BTN) in calcium chloride-, barium chloride- and electrically induced arrhythmia rat models. BTN enhanced the effect of BoNT/A1. Subepicardial injection of BTN resulted in a significant antiarrhythmic effect in investigated rat models. BTN formulation antagonizes arrhythmia induced by the activation of Ca, K and Na channels.

An Integrated Pharmacophore/Docking/3D-QSAR Approach to Screening a Large Library of Products in Search of Future Botulinum Neurotoxin A Inhibitors.

Botulinum toxins are neurotoxins produced by Clostridium botulinum. This toxin can be lethal for humans as a cause of botulism; however, in small doses, the same toxin is used to treat different conditions. Even if the therapeutic doses are effective and safe, the adverse reactions could be local and could unmask a subclinical impairment of neuromuscular transmissions. There are not many cases of adverse events in the literature; however, it is possible that sometimes they do not occur as they are transient and, if they do occur, there is no possibility of a cure other than to wait for the pharmacological effect to end. Inhibition of botulinum neurotoxin type A (BoNT/A) effects is a strategy for treating botulism as it can provide an effective post-exposure remedy. In this paper, 13,592,287 compounds were screened through a pharmacophore filter, a 3D-QSAR model, and a virtual screening; then, the compounds with the best affinity were selected. Molecular dynamics simulation studies on the first four compounds predicted to be the most active were conducted to verify that the poses foreseen by the docking were stable. This approach allowed us to identify compounds with a calculated inhibitory activity in the range of 316-500 nM.

Development of Botulinum Toxin A-Coated Microneedles for Treating Palmar Hyperhidrosis.

Hyperhidrosis is a disorder that is characterized by the production of excess amounts of sweat. The botulinum neurotoxin A (BoNT/A) has been used to treat hyperhidrosis through multiple intradermal injections at the site of the condition. However, because of BoNT/A toxicity, it is important to precisely deliver the proper dose of the toxin to the target site. In addition, the use of a conventional hypodermic needle for multiple injections in the palm makes the approach undesirable and painful. Here, we designed a BoNT/A-coated microneedle (BoNT-MN) array and tested its efficacy as a substitute pain-free method to treat hyperhidrosis. BoNT-MNs were prepared by coating polylactic acid microneedles with a BoNT/A formulation and were found to successfully penetrate into a thick skin in vitro. The coating formulations were then tested for their stability at 4, 25, and 37 °C for 24 h. BoNT-MNs were found to be much more stable than BoNT/A in a liquid state. Additionally, we carried out in vivo experiments by treating the right paws of mice with BoNT-MNs and found that the treatment induced a significant reduction in the sweating response in the mouse foot pad. Thus, BoNT/A treatment using microneedles is beneficial and may be used as a more efficient and less painful approach to treat hyperhidrosis.

Do complexing proteins provide mechanical protection for botulinum neurotoxins?

Botulinum toxin (BT) consists of botulinum neurotoxin and complexing proteins (CPs). CPs might provide mechanical protection for botulinum neurotoxin. As incobotulinumtoxinA (INCO, Xeomin®) does not contain CPs, we wanted to compare its mechanical stability to that of onabotulinumtoxinA (ONA, Botox®) containing CPs. For this, ONA and INCO were reconstituted without mechanical stress (NS) and with mechanical stress (WS) generated by a recently introduced stress test. Potencies were then measured by the paralysis times (PTs) in the mouse diaphragm assay. ONA-PT was 75.8 ± 10.3 min (n = 6) under NS and 116.7 ± 29.8 min (n = 6) under WS (two-tailed t test, p = 0.002). Mechanical stress increased the ONA-PT by 35.0% on the Growth Percentage Index. INCO-PT was 66.0 ± 7.0 min for NS and 76.0 ± 1.0 min for WS (t test, p = 0.129). Mechanical stress increased the INCO-PT by 13.2% on the Growth Percentage Index. Our data show that mechanical stress inactivates a CP-containing BT drug, but not a CP-free BT drug. We conclude that CPs do not provide protection against mechanical stress, supporting the view that CPs are not necessary for therapeutic purposes.

IncobotulinumtoxinA: A Highly Purified and Precisely Manufactured Botulinum Neurotoxin Type A

Aesthetic dermatologic applications of botulinum neurotoxin (BoNT), including treatment of glabellar lines, horizontal forehead lines, and crow's feet, were the most common non-surgical cosmetic procedures in the US in 2017, with high levels of subject satisfaction. Since the first BoNT type A (BoNT-A) formulation was approved in 1989, the number of formulations available on the world's commercial markets has increased and new approvals are expected. BoNT is produced by Clostridium botulinum in nature as part of a large protein complex. However, the unnecessary clostridial proteins, which dissociate from BoNT under physiological conditions with a half-life of <1 minute, have no role in clinical applications. Data demonstrate that BoNT administration can elicit an immunological response, leading to production of neutralizing antibodies that can be associated with reduced efficacy or treatment non-response. As repeat treatments are required to maintain efficacy, clinicians should be aware of the possibility of antibody development and choose a BoNT with the lowest risk of immunogenicity. IncobotulinumtoxinA is manufactured using advanced technology to precisely isolate the pure BoNT without unnecessary clostridial proteins, and with low immunogenicity and high specific activity. In incobotulinumtoxinA clinical studies, no previously BoNT-naïve subjects developed neutralizing antibodies, and there was no secondary non-response to incobotulinumtoxinA treatment. Here we review the role of unnecessary clostridial proteins in BoNT-A and discuss the unique incobotulinumtoxinA manufacturing and purification process with a focus on the implications for use in aesthetic medicine. J Drugs Dermatol. 2019;18(1):52-57.

Botulinum neurotoxins: new questions arising from structural biology.

Botulinum neurotoxins (BoNTs) are the most toxic substances known and cause botulism in vertebrates. They have also emerged as effective and powerful reagents for cosmetic and medical applications. One important prerequisite for understanding BoNT function in disease, and the further development of the toxins for cosmetic and medical applications, is a detailed knowledge of BoNT interactions with non-toxic neurotoxin-associated proteins and cell surface receptors. Based on the substantial recent progress in obtaining high-resolution crystal structures of key BoNT complexes, we summarize the major advances in understanding BoNT interactions and discuss the resulting potential implications, in particular those relating to BoNT serotype A.

High resolution crystal structures of Clostridium botulinum neurotoxin A3 and A4 binding domains.

Clostridium botulinum neurotoxins (BoNTs) cause the life-threatening condition, botulism. However, while they have the potential to cause serious harm, they are increasingly being utilised for therapeutic applications. BoNTs comprise of seven distinct serotypes termed BoNT/A through BoNT/G, with the most widely characterised being sub-serotype BoNT/A1. Each BoNT consists of three structurally distinct domains, a binding domain (HC), a translocation domain (HN), and a proteolytic domain (LC). The HC domain is responsible for the highly specific targeting of the neurotoxin to neuronal cell membranes. Here, we present two high-resolution structures of the binding domain of subtype BoNT/A3 (HC/A3) and BoNT/A4 (HC/A4) at 1.6 Šand 1.34 Šresolution, respectively. The structures of both proteins share a high degree of similarity to other known BoNT HC domains whilst containing some subtle differences, and are of benefit to research into therapeutic neurotoxins with novel characteristics.

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.

Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity.