Mechanism of substrate recognition by botulinum neurotoxin serotype A

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting neurotransmitter-carrying vesicle fusion to the plasma membrane of peripheral neurons. Unlike other zinc proteases, BoNTs recognize extended regions of SNAP25 for cleavage; however, the molecular basis for this extended substrate recognition is unclear. Here, we define a multistep mechanism for recognition and cleavage of SNAP25 by BoNT/A. SNAP25 initially binds along the belt region of BoNT/A, which aligns the P5 residue to the S5 pocket at the periphery of the active site. Although the exact order of each step of recognition of SNAP25 by BoNT/A at the active site is not clear, the initial binding could subsequently orient the P4'-residue of SNAP25 to form a salt bridge with the S4'-residue, which opens the active site allowing the P1'-residue access to the S1'-pocket. Subsequent hydrophobic interactions between the P3 residue of SNAP25 and the S3 pocket optimize alignment of the scissile bond for cleavage. This explains how the BoNTs recognize and cleave specific coiled SNARE substrates and provides insight into the development of inhibitors to prevent botulism.     

Crystal structure of botulinum neurotoxin type G light chain: serotype divergence in substrate recognition

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) block neurotransmitter release through their specific proteolysis of one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) complex. BoNTs have stringent substrate specificities that are unique for metalloprotease in that they require exceptionally long substrates (1). To understand the molecular reasons for the unique specificities of the BoNTs, we determined the crystal structure of the catalytic light chain (LC) of Clostridium botulinum neurotoxin type G (BoNT/G-LC) at 2.35 A resolution. The structure of BoNT/G-LC reveals a C-terminal beta-sheet that is critical for LC oligomerization and is unlike that seen in the other LC structures. Its structural comparison with thermolysin and the available pool of LC structures reveals important serotype differences that are likely to be involved in substrate recognition of the P1' residue. In addition, structural and sequence analyses have identified a potential exosite of BoNT/G-LC that recognizes a SNARE recognition motif of VAMP.     

Structural analysis of botulinum neurotoxin serotype F light chain: implications on substrate binding and inhibitor design

The seven serologically distinct Clostridium botulinum neurotoxins (BoNTs A-G) are zinc endopeptidases which block the neurotransmitter release by cleaving one of the three proteins of the soluble N-ethylmaleimide-sensitive-factor attachment protein receptor complex (SNARE complex) essential for the fusion of vesicles containing neurotransmitters with target membranes. These metallopeptidases exhibit unique specificity for the substrates and peptide bonds they cleave. Development of countermeasures and therapeutics for BoNTs is a priority because of their extreme toxicity and potential misuse as biowarfare agents. Though they share sequence homology and structural similarity, the structural information on each one of them is required to understand the mechanism of action of all of them because of their specificity. Unraveling the mechanism will help in the ultimate goal of developing inhibitors as antibotulinum drugs for the toxins. Here, we report the high-resolution structure of active BoNT/F catalytic domain in two crystal forms. The structure was exploited for modeling the substrate binding and identifying the S1' subsite and the putative exosites which are different from BoNT/A or BoNT/B. The orientation of docking of the substrate at the active site is consistent with the experimental BoNT/A-LC:SNAP-25 peptide model and our proposed model for BoNT/E-LC:SNAP-25.     

Multiple pocket recognition of SNAP25 by botulinum neurotoxin serotype E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. The molecular basis for SNAP25 recognition and cleavage by BoNT serotype E is currently unclear. Here we define the multiple pocket recognition of SNAP25 by LC/E. The initial recognition of SNAP25 is mediated by the binding of the B region of SNAP25 to the substrate-binding (B) region of LC/E comprising Leu166, Arg167, Asp127, Ala128, Ser129, and Ala130. The mutations at these residues affected substrate binding and catalysis. Three additional residues participate in scissile bond cleavage of SNAP25 by LC/E. The P3 site residues, Ile178, of SNAP25 interacted with the S3 pocket in LC/E through hydrophobic interactions. The S3 pocket included Ile47, Ile164, and Ile182 and appeared to align the P1' and P2 residues of SNAP25 with the S1' and S2 pockets of LC/E. The S1' pocket of LC/E included three residues, Phe191, Thr159, and Thr208, which contribute hydrophobic and steric interactions with the SNAP25 P1' residue Ile181. The S2 pocket residue of LC/E, Lys224, binds the P2 residue of SNAP25, Asp179, through ionic interactions. Deletion mapping indicates that main chain interaction(s) of residues 182-186 of SNAP25 contribute to substrate recognition by LC/E. Understanding the mechanism for substrate specificity provides insight for the development of inhibitors against the botulinum neurotoxins.     

Structural and biochemical studies of botulinum neurotoxin serotype C1 light chain protease: implications for dual substrate specificity

Clostridial neurotoxins are the causative agents of the neuroparalytic disease botulism and tetanus. They block neurotransmitter release through specific proteolysis of one of the three soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs) SNAP-25, syntaxin, and synaptobrevin, which constitute part of the synaptic vesicle fusion machinery. The catalytic component of the clostridial neurotoxins is their light chain (LC), a Zn2+ endopeptidase. There are seven structurally and functionally related botulinum neurotoxins (BoNTs), termed serotype A to G, and tetanus neurotoxin (TeNT). Each of them exhibits unique specificity for their target SNAREs and peptide bond(s) they cleave. The mechanisms of action for substrate recognition and target cleavage are largely unknown. Here, we report structural and biochemical studies of BoNT/C1-LC, which is unique among BoNTs in that it exhibits dual specificity toward both syntaxin and SNAP-25. A distinct pocket (S1') near the active site likely achieves the correct register for the cleavage site by only allowing Ala as the P1' residue for both SNAP-25 and syntaxin. Mutations of this SNAP-25 residue dramatically reduce enzymatic activity. The remote alpha-exosite that was previously identified in the complex of BoNT/A-LC and SNAP-25 is structurally conserved in BoNT/C1. However, mutagenesis experiments show that the alpha-exosite of BoNT/C1 plays a less stringent role in substrate discrimination in comparison to that of BoNT/A, which could account for its dual substrate specificity.     

Botulinum neurotoxin serotype F: identification of substrate recognition requirements and development of inhibitors with low nanomolar affinity

Botulinum neurotoxins (BoNTs A-G) are zinc metalloendoproteases that exhibit extraordinary specificities for proteins involved in neurotransmitter release. In view of the extreme toxicities of these molecules, their applications in human medicine, and potential for misuse, it is of considerable importance to elucidate the mechanisms underlying substrate recognition and to develop inhibitors, with the ultimate goal of obtaining anti-botulinum drugs. We synthesized peptides based on vesicle-associated membrane protein (VAMP) to investigate the substrate requirements of BoNT F, which cleaves VAMP between residues Q58 and K59. The minimum substrate was a peptide containing VAMP residues 32-65, which includes only one of the two VAMP structural motifs thought to be required for botulinum substrate recognition. BoNT F exhibited a strict requirement for residues D57 (P(2)), K59 (P(1)'), and L60 (P(2)'), but peptides containing substitutions for R56 (P(3)), Q58 (P(1)), and S61 (P(3)') were cleaved. Therefore, the P(2), P(1)', and P(2)' residues of VAMP are of paramount importance for BoNT F substrate recognition near the scissile bond. K(i) values of uncleavable analogues were similar to K(m) values of the substrate, suggesting that substrate discrimination occurs at the cleavage step, not at the initial binding step. We then synthesized inhibitors of BoNT F that incorporated d-cysteine in place of glutamine 58, exhibited K(i) values of 1-2 nM, and required binding groups on the N-terminal but not the C-terminal side of the zinc ligand. The latter characteristic distinguishes BoNT F from other zinc metalloendoproteases, including BoNTs A and B.     

Time-dependent botulinum neurotoxin serotype A metalloprotease inhibitors

Botulinum neurotoxins (BoNTs) are the most lethal of biological substances, and are categorized as class A biothreat agents by the Centers for Disease Control and Prevention. There are currently no drugs to treat the deadly flaccid paralysis resulting from BoNT intoxication. Among the seven BoNT serotypes, the development of therapeutics to counter BoNT/A is a priority (due to its long half-life in the neuronal cytosol and its ease of production). In this regard, the BoNT/A enzyme light chain (LC) component, a zinc metalloprotease responsible for the intracellular cleavage of synaptosomal-associated protein of 25 kDa, is a desirable target for developing post-BoNT/A intoxication rescue therapeutics. In an earlier study, we reported the high throughput screening of a library containing 70,000 compounds, and uncovered a novel class of benzimidazole acrylonitrile-based BoNT/A LC inhibitors. Herein, we present both structure–activity relationships and a proposed mechanism of action for this novel inhibitor chemotype.     

Analysis of neurotoxin cluster genes in Clostridium botulinum strains producing botulinum neurotoxin serotype A subtypes

Neurotoxin cluster gene sequences and arrangements were elucidated for strains of Clostridium botulinum encoding botulinum neurotoxin (BoNT) subtypes A3, A4, and a unique A1-producing strain (HA(-) Orfx(+) A1). These sequences were compared to the known neurotoxin cluster sequences of C. botulinum strains that produce BoNT/A1 and BoNT/A2 and possess either a hemagglutinin (HA) or an Orfx cluster, respectively. The A3 and HA(-) Orfx(+) A1 strains demonstrated a neurotoxin cluster arrangement similar to that found in A2. The A4 strain analyzed possessed two sets of neurotoxin clusters that were similar to what has been found in the A(B) strains: an HA cluster associated with the BoNT/B gene and an Orfx cluster associated with the BoNT/A4 gene. The nucleotide and amino acid sequences of the neurotoxin cluster-specific genes were determined for each neurotoxin cluster and compared among strains. Additionally, the ntnh gene of each strain was compared on both the nucleotide and amino acid levels. The degree of similarity of the sequences of the ntnh genes and corresponding amino acid sequences correlated with the neurotoxin cluster type to which the ntnh gene was assigned.     

Benzoquinones as inhibitors of botulinum neurotoxin serotype A

Although botulinum neurotoxin serotype A (BoNT/A) is known for its use in cosmetics, it causes a potentially fatal illness, botulism, and can be used as a bioterror weapon. Many compounds have been developed that inhibit the BoNTA zinc-metalloprotease light chain (LC), however, none of these inhibitors have advanced to clinical trials. In this study, a fragment-based approach was implemented to develop novel covalent inhibitors of BoNT/A LC. First, electrophilic fragments were screened against BoNT/A LC, and benzoquinone (BQ) derivatives were found to be active. In kinetic studies, BQ compounds acted as irreversible inhibitors that presumably covalently modify cysteine 165 of BoNT/A LC. Although most BQ derivatives were highly reactive toward glutathione in vitro, a few compounds such as natural product naphthazarin displayed low thiol reactivity and good BoNT/A inhibition. In order to increase the potency of the BQ fragment, computational docking studies were employed to elucidate a scaffold that could bind to sites adjacent to Cys165 while positioning a BQ fragment at Cys165 for covalent modification; 2-amino-N-arylacetamides met these criteria and when linked to BQ displayed at least a 20-fold increase in activity to low μM IC₅₀ values. Unlike BQ alone, the linked-BQ compounds demonstrated only weak irreversible inhibition and therefore acted mainly as non-covalent inhibitors. Further kinetic studies revealed a mutual exclusivity of BQ covalent inactivation and competitive inhibitor binding to sites adjacent to Cys165, refuting the viability of the current strategy for developing more potent irreversible BoNT/A inhibitors. The highlights of this study include the discovery of BQ compounds as irreversible BoNT/A inhibitors and the rational design of low μM IC₅₀ competitive inhibitors that depend on the BQ moiety for activity.     

Substrate recognition of VAMP-2 by botulinum neurotoxin B and tetanus neurotoxin

Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60-87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40-87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1' residues of VAMP-2 had the greatest inhibition of LC/B cleavage (> or =32-fold), whereas mutations at P7, P4, P1', and P2' residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (> or =64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.     

Novel ganglioside-mediated entry of botulinum neurotoxin serotype D into neurons

Botulinum Neurotoxins (BoNTs) are organized into seven serotypes, A-G. Although several BoNT serotypes enter neurons through synaptic vesicle cycling utilizing dual receptors (a ganglioside and a synaptic vesicle-associated protein), the entry pathway of BoNT/D is less well understood. Although BoNT/D entry is ganglioside-dependent, alignment and structural studies show that BoNT/D lacks key residues within a conserved ganglioside binding pocket that are present in BoNT serotypes A, B, E, F, and G, which indicate that BoNT/D-ganglioside interactions may be unique. In this study BoNT/D is shown to have a unique association with ganglioside relative to the other BoNT serotypes, utilizing a ganglioside binding loop (GBL, residues Tyr-1235-Ala-1245) within the receptor binding domain of BoNT/D (HCR/D) via b-series gangliosides, including GT1b, GD1b, and GD2. HCR/D bound gangliosides and entered neurons dependent upon the aromatic ring of Phe-1240 within the GBL. This is the first BoNT-ganglioside interaction that is mediated by a phenylalanine. In contrast, Trp-1238, located near the N terminus of the ganglioside binding loop, was mostly solvent-inaccessible and appeared to contribute to maintaining the loop structure. BoNT/D entry and intoxication were enhanced by membrane depolarization via synaptic vesicle cycling, where HCR/D colocalized with synaptophysin, a synaptic vesicle marker, but immunoprecipitation experiments did not detect direct association with synaptic vesicle protein 2. Thus, BoNT/D utilizes unique associations with gangliosides and synaptic vesicles to enter neurons, which may facilitate new neurotoxin therapies.     

Plasmid encoded neurotoxin genes in Clostridium botulinum serotype A subtypes

Clostridium botulinum, an important pathogen of humans and animals, produces botulinum neurotoxin (BoNT), the most poisonous toxin known. We have determined by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations that the genes encoding BoNTs in strains Loch Maree (subtype A3) and 657Ba (type B and subtype A4) are located on large (approximately 280 kb) plasmids. This is the first demonstration of plasmid-borne neurotoxin genes in Clostridium botulinum serotypes A and B. The finding of BoNT type A and B genes on extrachromosomal elements has important implications for the evolution of neurotoxigenicity in clostridia including the origin, expression, and lateral transfer of botulinum neurotoxin genes.     

Structure- and substrate-based inhibitor design for Clostridium botulinum neurotoxin serotype A

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there is no post-exposure therapeutic intervention available. To develop inhibitors leading to drug design, it is imperative that critical interactions between the enzyme and the substrate near the active site are known. Although enzyme-substrate interactions at exosites away from the active site are mapped in detail for botulinum neurotoxin type A, information about the active site interactions is lacking. Here, we present the crystal structures of botulinum neurotoxin type A catalytic domain in complex with four inhibitory substrate analog tetrapeptides, viz. RRGC, RRGL, RRGI, and RRGM at resolutions of 1.6-1.8 A. These structures show for the first time the interactions between the substrate and enzyme at the active site and delineate residues important for substrate stabilization and catalytic activity. We show that OH of Tyr(366) and NH(2) of Arg(363) are hydrogen-bonded to carbonyl oxygens of P1 and P1' of the substrate analog and position it for catalytic activity. Most importantly, the nucleophilic water is replaced by the amino group of the N-terminal residue of the tetrapeptide. Furthermore, the S1' site is formed by Phe(194), Thr(215), Thr(220), Asp(370), and Arg(363). The K(i) of the best inhibitory tetrapeptide is 157 nm.     

A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than SNAP-25 substrate

Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.     

The receptor binding domain of botulinum neurotoxin serotype C binds phosphoinositides

Botulinum neurotoxins (BoNTs) are the most toxic proteins known for humans and animals with an extremely low LD₅₀ of ∼1 ng/kg. BoNTs generally require a protein and a ganglioside on the cell membrane surface for binding, which is known as a “dual receptor” mechanism for host intoxication. Recent studies have suggested that in addition to gangliosides, other membrane lipids such as phosphoinositides may be involved in the interactions with the receptor binding domain (HCR) of BoNTs for better membrane penetration. Using two independent lipid-binding assays, we tested the interactions of BoNT/C-HCR with lipids in vitro domain. BoNT/C-HCR was found to bind negatively charged phospholipids, preferentially phosphoinositides in both assays. Interactions with phosphoinositides may facilitate tighter binding between neuronal membranes and BoNT/C.     

A capillary electrophoresis method to assay catalytic activity of botulinum neurotoxin serotypes: implications for substrate specificity

The potent botulinum neurotoxin inhibits neurotransmitter release at cholinergic nerve terminals, causing a descending flaccid paralysis characteristic of the disease botulism. The currently expanding medical use of the neurotoxin to treat several disorders, as well as the potential misuse of the neurotoxin as an agent in biowarfare, has made understanding of the nature of the toxin's catalytic activity and development of inhibitors critical. To study the catalytic activity of botulinum neurotoxin more thoroughly and characterize potential inhibitors, we have developed a capillary electrophoresis method to measure catalytic activity of different serotypes of botulinum neurotoxin using peptides derived from the native substrates. This assay requires only a minute amount of sample (25 nl), is relatively rapid (15 min/sample), and allows the determination of enzyme kinetic constants for a more sophisticated characterization of inhibitors and neurotoxin catalytic activity. Using this method, we can measure activity of five of the seven serotypes of botulinum neurotoxin (A, B, E, F, and G) with two peptide substrates. Botulinum neurotoxin serotypes C and D did not cleave our peptides, lending insight into potential substrate requirements among the serotypes.     

Structural analysis of the receptor binding domain of botulinum neurotoxin serotype D

Botulinum neurotoxins (BoNTs) are the most toxic proteins known. The mechanism for entry into neuronal cells for serotypes A, B, E, F, and G involves a well understood dual receptor (protein and ganglioside) process, however, the mechanism of entry for serotypes C and D remains unclear. To provide structural insights into how BoNT/D enters neuronal cells, the crystal structure of the receptor binding domain (S863-E1276) for this serotype (BoNT/D-HCR) was determined at 1.65 Å resolution. While BoNT/D-HCR adopts an overall fold similar to that observed in other known BoNT HCRs, several major structural differences are present. These structural differences are located at, or near, putative receptor binding sites and may be responsible for BoNT/D host preferences. Two loops, S1195-I1204 and K1236-N1244, located on both sides of the putative protein receptor binding pocket, are displaced >10 Å relative to the corresponding residues in the crystal structures of BoNT/B and G. Obvious clashes were observed in the putative protein receptor binding site when the BoNT/B protein receptor synaptotagmin II was modeled into the BoNT/D-HCR structure. Although a ganglioside binding site has never been unambiguously identified in BoNT/D-HCR, a shallow cavity in an analogous location to the other BoNT serotypes HCR domains is observed in BoNT/D-HCR that has features compatible with membrane binding. A portion of a loop near the putative receptor binding site, K1236-N1244, is hydrophobic and solvent-exposed and may directly bind membrane lipids. Liposome-binding experiments with BoNT/D-HCR demonstrate that this membrane lipid may be phosphatidylethanolamine.     

Production and purification of a chimeric monoclonal antibody against botulinum neurotoxin serotype A

Production of recombinant antibodies against botulinum neurotoxin is necessary for the development of a post-exposure treatment. CHO-DG44 cells were transfected with a plasmid encoding the light and heavy chains of a chimeric monoclonal antibody (S25) against botulism neurotoxin serotype A. Stable cell lines were obtained by dilution cloning and clones were shown to produce nearly equivalent levels of light and heavy chain antibody by an enzyme-linked immunosorbent assay (ELISA). In suspension culture, cells produced 35 μg/ml of chimeric antibody after 6 days, corresponding to a specific antibody productivity of 3.1 pg/cell/day. A method for the harvest and recovery of an antibody against botulism neurotoxin serotype A was investigated utilizing ethylenediamine-N,N′-tetra(methylphosphonic) acid (EDTPA) modified zirconia and MEP-hypercel, a hydrophobic charge interaction chromatography resin. Purification of the S25 antibody was compared to that achieved using rProtein A–Sepharose Fast Flow resin. After the direct load of culture supernatant, analysis by ELISA and gel electrophoresis showed that S25 antibody could be recovered at purities of 41 and 44%, from the EDTPA modified zirconia and MEP-hypercel columns, respectively. Although the purity obtained from each of these columns was low, the ability to withstand high column pressures and nearly 90% recovery of the antibody makes EDTPA modified zirconia well suited as an initial capture step. Combining the EDTPA modified zirconia and HCIC columns in series resulted in both purity and final product yield of 72%.     

Translocation of botulinum neurotoxin serotype A and associated proteins across the intestinal epithelia

Botulinum neurotoxins (BoNTs) are some of the most poisonous natural toxins. Botulinum neurotoxins associate with neurotoxin‐associated proteins (NAPs) forming large complexes that are protected from the harsh environment of the gastrointestinal tract. However, it is still unclear how BoNT complexes as large as 900 kDa traverse the epithelial barrier and what role NAPs play in toxin translocation. In this study, we examined the transit of BoNT serotype A (BoNT/A) holotoxin, complex and recombinantly purified NAP complex through cultured and polarized Caco‐2 cells and, for the first time, in the small mouse intestine. Botulinum neurotoxin serotype A and NAPs in the toxin complex were detectable inside intestinal cells beginning at 2 h post intoxication. Appearance of the BoNT/A holotoxin signal was slower, with detection starting at 4–6 h. This indicated that the holotoxin alone was sufficient for entry but the presence of NAPs enhanced the rate of entry. Botulinum neurotoxin serotype A detection peaked at approximately 6 and 8 h for complex and holotoxin, respectively, and thereafter began to disperse with some toxin remaining in the epithelia after 24 h. Purified HA complexes alone were also internalized and followed a similar time course to that of BoNT/A complex internalization. However, recombinant HA complexes did not enhance BoNT/A holotoxin entry in the absence of a physical link with BoNT/A. We propose a model for BoNT/A toxin complex translocation whereby toxin complex entry is facilitated by NAPs in a receptor‐mediated mechanism. Understanding the intestinal uptake of BoNT complexes will aid the development of new measures to prevent or treat oral intoxications.