Small Molecule Receptor Binding Inhibitors with In Vivo Efficacy against Botulinum Neurotoxin Serotypes A and E.

Botulinum neurotoxins (BoNTs) are the most poisonous substances in nature. Currently, the only therapy for botulism is antitoxin. This therapy suffers from several limitations and hence new therapeutic strategies are desired. One of the limitations in discovering BoNT inhibitors is the absence of an in vitro assay that correlates with toxin neutralization in vivo. In this work, a high-throughput screening assay for receptor-binding inhibitors against BoNT/A was developed. The assay is composed of two chimeric proteins: a receptor-simulating protein, consisting of the fourth luminal loop of synaptic vesicle protein 2C fused to glutathione-S-transferase, and a toxin-simulating protein, consisting of the receptor-binding domain of BoNT/A fused to beta-galactosidase. The assay was applied to screen the LOPAC1280 compound library. Seven selected compounds were evaluated in mice exposed to a lethal dose of BoNT/A. The compound aurintricarboxylic acid (ATA) conferred 92% protection, whereas significant delayed time to death (p < 0.005) was observed for three additional compounds. Remarkably, ATA was also fully protective in mice challenged with a lethal dose of BoNT/E, which also uses the SV2 receptor. This study demonstrates that receptor-binding inhibitors have the potential to serve as next generation therapeutics for botulism, and therefore the assay developed may facilitate discovery of new anti-BoNT countermeasures.

Special Delivery: Potential Mechanisms of Botulinum Neurotoxin Uptake and Trafficking within Motor Nerve Terminals.

Botulinum neurotoxins (BoNTs) are highly potent, neuroparalytic protein toxins that block the release of acetylcholine from motor neurons and autonomic synapses. The unparalleled toxicity of BoNTs results from the highly specific and localized cleavage of presynaptic proteins required for nerve transmission. Currently, the only pharmacotherapy for botulism is prophylaxis with antitoxin, which becomes progressively less effective as symptoms develop. Treatment for symptomatic botulism is limited to supportive care and artificial ventilation until respiratory function spontaneously recovers, which can take weeks or longer. Mechanistic insights into intracellular toxin behavior have progressed significantly since it was shown that toxins exploit synaptic endocytosis for entry into the nerve terminal, but fundamental questions about host-toxin interactions remain unanswered. Chief among these are mechanisms by which BoNT is internalized into neurons and trafficked to sites of molecular toxicity. Elucidating how receptor-bound toxin is internalized and conditions under which the toxin light chain engages with target SNARE proteins is critical for understanding the dynamics of intoxication and identifying novel therapeutics. Here, we discuss the implications of newly discovered modes of synaptic vesicle recycling on BoNT uptake and intraneuronal trafficking.

Uptake of Clostridial Neurotoxins into Cells and Dissemination.

Clostridial neurotoxins, botulinum neurotoxins (BoNT) and tetanus neurotoxin (TeNT), are potent toxins, which are responsible for severe neurological diseases in man and animals. BoNTs induce a flaccid paralysis (botulism) by inhibiting acetylcholine release at the neuromuscular junctions, whereas TeNT causes a spastic paralysis (tetanus) by blocking the neurotransmitter release (glycine, GABA) in inhibitory interneurons within the central nervous system. Clostridial neurotoxins recognize specific receptor(s) on the target neuronal cells and enter via a receptor-mediated endocytosis. They transit through an acidic compartment which allows the translocation of the catalytic chain into the cytosol, a prerequisite step for the intracellular activity of the neurotoxins. TeNT migrates to the central nervous system by using a motor neuron as transport cell. TeNT enters a neutral pH compartment and undergoes a retrograde axonal transport to the spinal cord or brain, where the whole undissociated toxin is delivered and interacts with target neurons. Botulism most often results from ingestion of food contaminated with BoNT. Thus, BoNT passes through the intestinal epithelial barrier mainly via a transcytotic mechanism and then diffuses or is transported to the neuromuscular junctions by the lymph or blood circulation. Indeed, clostridial neurotoxins are specific neurotoxins which transit through a transport cell to gain access to the target neuron, and use distinct trafficking pathways in both cell types.

Entry of Botulinum Neurotoxin Subtypes A1 and A2 into Neurons.

Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans but also are common therapies for neurological diseases. BoNTs are dichain toxins, comprising an N-terminal catalytic domain (LC) disulfide bond linked to a C-terminal heavy chain (HC) which includes a translocation domain (HN) and a receptor binding domain (HC). Recently, the BoNT serotype A (BoNT/A) subtypes A1 and A2 were reported to possess similar potencies but different rates of cellular intoxication and pathology in a mouse model of botulism. The current study measured HCA1 and HCA2 entry into rat primary neurons and cultured Neuro2A cells. We found that there were two sequential steps during the association of BoNT/A with neurons. The initial step was ganglioside dependent, while the subsequent step involved association with synaptic vesicles. HCA1 and HCA2 entered the same population of synaptic vesicles and entered cells at similar rates. The primary difference was that HCA2 had a higher degree of receptor occupancy for cells and neurons than HcA1. Thus, HCA2 and HCA1 share receptors and entry pathway but differ in their affinity for receptor. The initial interaction of HCA1 and HCA2 with neurons may contribute to the unique pathologies of BoNT/A1 and BoNT/A2 in mouse models.

Characterization of the functional activity of botulinum neurotoxin subtype B6.

Clostridium botulinum produces the highly potent neurotoxin, botulinum neurotoxin (BoNT), which is classified into seven serotypes (A-G); the subtype classification is confirmed by the diversity of amino acid sequences among the serotypes. BoNT from the Osaka05 strain is associated with type B infant botulism and has been classified as BoNT/B subtype B6 (BoNT/B6) by phylogenetic analysis and the antigenicity of its C-terminal heavy chain (HC ) domain. However, the molecular bases for its properties, including its potency, are poorly understood. In this study, BoNT/B6 holotoxin was purified and the biological activity and receptor binding activity of BoNT/B6 compared with those of the previously-characterized BoNT/B1 and BoNT/B2 subtypes. The derivative BoNT/B6 was found to be already nicked and in an activated form, indicating that endogenous protease production may be higher in this strain than in the other two strains. BoNT/B1 exhibited the greatest lethal activity in mice, followed by BoNT/B6, which is consistent with the sensitivity of PC12 cells. No significant differences were seen in the enzymatic activities of the BoNT/Bs against their substrate. HC /B1 and HC /B6 exhibited similar binding affinities to synaptotagmin II (SytII), which is a specific protein receptor for BoNT/B. Binding to the SytII/ganglioside complex is functionally related to the toxic action; however, the receptor recognition sites are conserved. These results suggest that the distinct characteristics and differences in biological sensitivity of BoNT/B6 may be attributable to the function of its Hc .domain.

Myasthenia gravis and related disorders: Pathology and molecular pathogenesis.

Disorders affecting the presynaptic, synaptic, and postsynaptic portions of the neuromuscular junction arise from various mechanisms in children and adults, including acquired autoimmune or toxic processes as well as genetic mutations. Disorders include autoimmune myasthenia gravis associated with acetylcholine receptor, muscle specific kinase or Lrp4 antibodies, Lambert-Eaton myasthenic syndrome, nerve terminal hyperexcitability syndromes, Guillain Barré syndrome, botulism, organophosphate poisoning and a number of congenital myasthenic syndromes. This review focuses on the various molecular and pathophysiological mechanisms of these disorders, characterization of which has been crucial to the development of treatment strategies specific for each pathogenic mechanism. In the future, further understanding of the underlying processes may lead to more effective and targeted therapies of these disorders. This article is part of a Special Issue entitled: Neuromuscular Diseases: Pathology and Molecular Pathogenesis.

Small molecule non-peptide inhibitors of botulinum neurotoxin serotype E: Structure-activity relationship and a pharmacophore model.

Botulinum neurotoxins (BoNTs) are the most poisonous biological substance known to humans. They cause flaccid paralysis by blocking the release of acetylcholine at the neuromuscular junction. Here, we report a number of small molecule non-peptide inhibitors of BoNT serotype E. The structure-activity relationship and a pharmacophore model are presented. Although non-peptidic in nature, these inhibitors mimic key features of the uncleavable substrate peptide Arg-Ile-Met-Glu (RIME) of the SNAP-25 protein. Among the compounds tested, most of the potent inhibitors bear a zinc-chelating moiety connected to a hydrophobic and aromatic moiety through a carboxyl or amide linker. All of them show low micromolar IC50 values.

Synchronized chaperone function of botulinum neurotoxin domains mediates light chain translocation into neurons.

Clostridium botulinum neurotoxin (BoNT) is a multidomain protein in which the individual modules work in synchronized cooperative action in order to enter into neurons and inhibit synaptic transmission. The di-chain protein is made up of the ~50 kD light chain and the ~100 kD heavy chain. The HC can be further subdivided into the N-terminal translocation domain (H(N)) and the C-terminal Receptor Binding Domain (H(C)). BoNT entry into neurons requires the toxin to utilize the host cell's endocytosis pathway where it exploits the acidic environment of the endosome. Within the endosome the H(C) triggers the H(N) to change conformation from a soluble protein to a membrane inserted protein-conducting channel in precise timing with LC refolding. The LC must partially unfold to a translocation competent conformation in order to be translocated by the H(N) channel in an N to C terminal direction. Upon completion of translocation, the LC is released from the HC and allowed to interact with its substrate SNARE protein. This article discusses the individual functions of each module as well as the mechanisms by which each domain serves as a chaperone for the others, working in concert to achieve productive intoxication.

Persistence of Botulinum neurotoxin inactivation of nerve function.

The extraordinary persistence of intoxication occurring after exposure to some Botulinum neurotoxin (BoNT) serotypes is both a therapeutic marvel and a biodefense nightmare. Understanding the mechanisms underlying BoNT persistence will offer new strategies for improving the efficacy and extending the applications of BoNT therapeutic agents as well as for treating the symptoms of botulism. Research indicates that the persistence of BoNT intoxication can be influenced both by the ability of the toxin protease or its cleaved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein substrate to resist turnover. Protease turnover seems to be mediated in part by the ubiquitin-proteasome system (UPS) and efforts to manipulate the UPS may prove to be an effective strategy for improving therapeutic utility of BoNT products and in the development of botulism antidotes.

Beltless translocation domain of botulinum neurotoxin A embodies a minimum ion-conductive channel.

Botulinum neurotoxin, the causative agent of the paralytic disease botulism, is an endopeptidase composed of a catalytic domain (or light chain (LC)) and a heavy chain (HC) encompassing the translocation domain (TD) and receptor-binding domain. Upon receptor-mediated endocytosis, the LC and TD are proposed to undergo conformational changes in the acidic endocytic environment resulting in the formation of an LC protein-conducting TD channel. The mechanism of channel formation and the conformational changes in the toxin upon acidification are important but less well understood aspects of botulinum neurotoxin intoxication. Here, we have identified a minimum channel-forming truncation of the TD, the "beltless" TD, that forms transmembrane channels with ion conduction properties similar to those of the full-length TD. At variance with the holotoxin and the HC, channel formation for both the TD and the beltless TD occurs independent of a transmembrane pH gradient. Furthermore, acidification in solution induces moderate secondary structure changes. The subtle nature of the conformational changes evoked by acidification on the TD suggests that, in the context of the holotoxin, larger structural rearrangements and LC unfolding occur preceding or concurrent to channel formation. This notion is consistent with the hypothesis that although each domain of the holotoxin functions individually, each domain serves as a chaperone for the others.

Substrates and controls for the quantitative detection of active botulinum neurotoxin in protease-containing samples.

Botulinum neurotoxins (BoNTs) are used in a wide variety of medical applications, but there is limited pharmacokinetic data on active BoNT. Monitoring BoNT activity in the circulation is challenging because BoNTs are highly toxic and are rapidly taken up by neurons and removed from the bloodstream. Previously we reported a sensitive BoNT "Assay with a Large Immunosorbent Surface Area" that uses conversion of fluorogenic peptide substrates to measure the intrinsic endopeptidase activity of bead-captured BoNT. However, in complex biological samples, protease contaminants can also cleave the substrates, reducing sensitivity and specificity of the assay. Here, we present a novel set of fluorogenic peptides that serve as BoNT-specific substrates and protease-sensitive controls. BoNT-cleavable substrates contain a C-terminal Nle, while BoNT-noncleavable controls contain its isomer ε-Ahx. The substrates are cleaved by BoNT subtypes A1-A3 and A5. Substrates and control peptides can be cleaved by non-BoNT proteases (e.g., trypsin, proteinase K, and thermolysin) while obeying Michaelis-Menten kinetics. Using this novel substrate/control set, we studied BoNT/A1 activity in two mouse models of botulism. We detected BoNT/A serum activities ranging from ~3600 to 10 amol/L in blood of mice that had been intravenously injected 1 h prior with BoNT/A1 complex (100 to 4 pg/mouse). We also detected the endopeptidase activity of orally administered BoNT/A1 complex (1 µg) in blood 5 h after administration; activity was greatest 7 h after administration. Redistribution and elevation rates for active toxin were measured and are comparable to those reported for inactive toxin.

Improved detection of botulinum neurotoxin serotype A by Endopep-MS through peptide substrate modification.

Botulinum neurotoxins (BoNTs) are a family of seven toxin serotypes that are the most toxic substances known to humans. Intoxication with BoNT causes flaccid paralysis and can lead to death if untreated with serotype-specific antibodies. Supportive care, including ventilation, may be necessary. Rapid and sensitive detection of BoNT is necessary for timely clinical confirmation of clinical botulism. Previously, our laboratory developed a fast and sensitive mass spectrometry (MS) method termed the Endopep-MS assay. The BoNT serotypes are rapidly detected and differentiated by extracting the toxin with serotype-specific antibodies and detecting the unique and serotype-specific cleavage products of peptide substrates that mimic the sequence of the BoNT native targets. To further improve the sensitivity of the Endopep-MS assay, we report here the optimization of the substrate peptide for the detection of BoNT/A. Modifications on the terminal groups of the original peptide substrate with acetylation and amidation significantly improved the detection of BoNT/A cleavage products. The replacement of some internal amino acid residues with single or multiple substitutions led to further improvement. An optimized peptide increased assay sensitivity 5-fold with toxin spiked into buffer solution or different biological matrices.

Dynamin inhibition blocks botulinum neurotoxin type A endocytosis in neurons and delays botulism.

The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.

Molecular diversity of the two sugar-binding sites of the ß-trefoil lectin HA33/C (HA1) from Clostridium botulinum type C neurotoxin.

A critical role in internalizing the Clostridium botulinum neurotoxin into gastrointestinal cells is played by nontoxic components complexed with the toxin. One of the components, a ß-trefoil lectin has been known as HA33 or HA1. The HA33 from C. botulinum type A (HA33/A) has been predicted to have a single sugar-binding site, while type C HA33 (HA33/C) has two sites. Here we constructed HA33/C mutants and evaluated the binding capacities of the individual sites through mucin-assay and isothermal titration calorimetry. The mutant W176A (site I knockout) had a K(d) value of 31.5mM for galactose (Gal) and 61.3mM for N-acetylgalactosamine (GalNAc), while the K(d) value for N-acetylneuraminic acid (Neu5Ac) was too high to be determined. In contrast, the double mutant N278A/Q279A (site II knockout) had a K(d) value of 11.8mM for Neu5Ac. We also determined the crystal structures of wild-type and the F179I mutant in complex with GalNAc at site II. The results suggest that site I of HA33/C is quite unique in that it mainly recognizes Neu5Ac, and site II seems less important for the lectin specificity. The architectures and the properties of the sugar-binding sites of HA33/C and HA33/A were shown to be drastically different.

Neuronal functions associated with endo- and exocytotic events-cum-molecular trafficking may be cell maturation-dependent: lessons learned from studies on botulism.

The passion in the scientific endeavors of Marshall Warren Nirenberg had been his quest for knowledge regarding the storage, retrieval, and processing of information in the cell. After deciphering the genetic code for which he shared the Nobel Prize in Physiology and Medicine in 1968, Nirenberg devoted his attention to unraveling the mysteries in the most complex cellular organization in the body, i.e., the nervous system, especially those governing neuronal development, plasticity, and synaptogenesis. During the tenure of the primary author (RR) as a postdoctoral Staff Fellow in the Nirenberg laboratory in the late seventies to early eighties, he had the opportunity of working on projects related to what Nirenberg used to broadly define as the "synaptic code." The major aspects of these projects dealt with the functional macromolecules relevant to neuronal growth, organization, lineage, selectivity, stabilization, synaptogenesis, and functions such as neuroexocytosis. This author's emphasis was particularly on voltage-gated calcium channels that regulate stimulus-induced neurotransmitter release. One central as well as crucial theme in these studies was the fact that the neurons had to be mature and differentiated in order to study these entities (Science 222: 794-799, 1983; Cold Spring Harb Symp Quant Biol 48: 707-715, 1983). In this communication, we illustrate how did this basic knowledge, i.e., cell maturation-dependent properties being essential for neuronal functions, led to a successful experimental design and demonstration of the validity of the targeted neurologic therapeutic delivery approach based on recombinant botulinum toxin serotype A (BoNT/A) heavy chain (rHC) serving as a neuron-specific targeting molecule (BMC Pharmacol 9: 12, 2009).

Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies.

BACKGROUND: Botulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical. RESULTS: In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs. CONCLUSIONS: In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.

Formulating a new basis for the treatment against botulinum neurotoxin intoxication: 3,4-Diaminopyridine prodrug design and characterization.

Botulism is a disease characterized by neuromuscular paralysis and is produced from botulinum neurotoxins (BoNTs) found within the Gram positive bacterium Clostridium botulinum. This bacteria produces the most deadliest toxin known, with lethal doses as low as 1 ng/kg. Due to the relative ease of production and transport, the use of these agents as potential bioterrorist weapons has become of utmost concern. No small molecule therapies against BoNT intoxication have been approved to date. However, 3,4-diaminopyridine (3,4-DAP), a potent reversible inhibitor of voltage-gated potassium channels, is an effective cholinergic agonist used in the treatment of neuromuscular degenerative disorders that require cholinergic enhancement. 3,4-DAP has also been shown to facilitate recovery of neuromuscular action potential post botulinum intoxication by blocking K(+) channels. Unfortunately, 3,4-DAP displays toxicity largely due to blood-brain-barrier (BBB) penetration. As a dual-action prodrug approach to cholinergic enhancement we have designed carbamate and amide conjugates of 3,4-DAP. The carbamate prodrug is intended to be a slowly reversible inhibitor of acetylcholinesterase (AChE) along the lines of the stigmines thereby allowing increased persistence of released acetylcholine within the synaptic cleft. As a secondary activity, cleavage of the carbamate prodrug by AChE will afford the localized release of 3,4-DAP, which in turn, will enhance the pre-synaptic release of additional acetylcholine. Being a competitive inhibitor with respect to acetylcholine, the activity of the prodrug will be greatest at the synaptic junctions most depleted of acetylcholine. Here we report upon the synthesis and biochemical characterization of three new classes of prodrugs intended to limit previously reported stability and toxicity issues. Of the prodrugs examined, compound 32, demonstrated the most clinically relevant half-life of 2.76 h, while selectively inhibiting AChE over butyrylcholinesterase--a plasma-based high activity esterase. Future in vivo studies could provide validation of prodrug 32 as a potential treatment against BoNT intoxication as well as other neuromuscular disorders.

Interaction of botulinum toxin with the epithelial barrier.

Botulinum neurotoxin (BoNT) is a protein toxin (approximately 150 kDa), which possesses a metalloprotease activity. Food-borne botulism is manifested when BoNT is absorbed from the digestive tract to the blood stream and enters the peripheral nerves, where the toxin cleaves core proteins of the neuroexocytosis apparatus and elicits the inhibition of neurotransmitter release. The initial obstacle to orally ingested BoNT entering the body is the epithelial barrier of the digestive tract. Recent cell biology and molecular biology studies are beginning to elucidate the mechanism by which this large protein toxin crosses the epithelial barrier. In this review, we provide an overview of the structural features of botulinum toxins (BoNT and BoNT complex) and the interaction of these toxins with the epithelial barrier.

Characterization of immune response induced against catalytic domain of botulinum neurotoxin type E.

Botulinum neurotoxins (BoNTs) represent a family of bacterial toxins responsible for neuroparalytic disease 'botulism' in human and animals. Their potential use as biological weapon led to their classification in category 'A' biowarfare agent by Centers for Disease Control and Prevention (CDC), USA. In present study, gene encoding full length catalytic domain of BoNT/E-LC was cloned, expressed and protein was purified using Ni-NTA chromatography. Humoral immune response was confirmed by Ig isotyping and cell-mediated immunity by cytokine profiling and intracellular staining for enumeration of IFN-γ secreting CD4+ and CD8+ T cells. Increased antibody titer with the predominance of IgG subtype was observed. An interaction between antibodies produced against rBoNT/E-LC was established that showed the specificity against BoNT/E in SPR assay. Animal protection with rBoNT/E-LC was conferred through both humoral and cellular immune responses. These findings were supported by cytokine profiling and flow cytometric analysis. Splenocytes stimulated with rBoNT/E-LC showed a 3.27 and 2.8 times increase in the IFN-γ secreting CD4+ and CD8+ T cells, respectively; in immunized group (P < 0.05). Protection against BoNT/E challenge tended to relate with increase in the percentage of rBoNT/E-LC specific IL-2 in the splenocytes supernatant (P = 0.034) and with IFN-γ-producing CD4+ T cell responses (P = 0.045). We have immunologically evaluated catalytically active rBoNT/E-LC. Our results provide valuable investigational report for immunoprophylactic role of catalytic domain of BoNT/E.

Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells.

Botulinum neurotoxins (BoNTs) cause botulism by entering neurons and cleaving proteins that mediate neurotransmitter release; disruption of exocytosis results in paralysis and death. The receptors for BoNTs are thought to be composed of both proteins and gangliosides; however, protein components that mediate toxin entry have not been identified. Using gain-of-function and loss-of-function approaches, we report here that the secretory vesicle proteins, synaptotagmins (syts) I and II, mediate the entry of BoNT/B (but not BoNT/A or E) into PC12 cells. Further, we demonstrate that BoNT/B entry into PC12 cells and rat diaphragm motor nerve terminals was activity dependent and can be blocked using fragments of syt II that contain the BoNT/B-binding domain. Finally, we show that syt II fragments, in conjunction with gangliosides, neutralized BoNT/B in intact mice. These findings establish that syts I and II can function as protein receptors for BoNT/B.