Fluorigenic Substrates for the Protease Activities of Botulinum Neurotoxins, Serotypes A, B, and F

The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P₁ and P₃′ residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a K_i value of 4 μM. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.     

Use of a Recombinant Fluorescent Substrate with Cleavage Sites for All Botulinum Neurotoxins in High-Throughput Screening of Natural Product Extracts for Inhibitors of Serotypes A, B, and E

The seven serotypes of botulinum neurotoxin (BoNTs) are zinc metalloproteases that cleave and inactivate proteins critical for neurotransmission. The synaptosomal protein of 25 kDa (SNAP-25) is cleaved by BoNTs A, C, and E, while vesicle-associated membrane protein (VAMP) is the substrate for BoNTs B, D, F, and G. BoNTs not only are medically useful drugs but also are potential bioterrorist and biowarfare threat agents. Because BoNT protease activity is required for toxicity, inhibitors of that activity might be effective for antibotulinum therapy. To expedite inhibitor discovery, we constructed a hybrid gene encoding (from the N terminus to the C terminus, with respect to the expressed product) green fluorescent protein, then a SNAP-25 fragment encompassing residues Met-127 to Gly-206, and then VAMP residues Met-1 to Lys-94. Cysteine was added as the C terminus. The expressed product, which contained the protease cleavage sites for all seven botulinum serotypes, was purified and coupled covalently through the C-terminal sulfhydryl group to maleimide-activated 96-well plates. The substrate was readily cleaved by BoNTs A, B, D, E, and F. Using this assay and an automated 96-well pipettor, we screened 528 natural product extracts for inhibitors of BoNT A, B, and E protease activities. Serotype-specific inhibition was found in 30 extracts, while 5 others inhibited two serotypes.     

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.     

A label-free biosensor assay for botulinum neurotoxin B in food and human serum

Botulinum neurotoxins (BoNTs) are among the most toxic substances known. Surveillance and diagnostics require methods for rapid detection of BoNTs in complex media such as foodstuffs and human serum. We have developed in vitro assays to specifically detect the protease activity of botulinum neurotoxin B (BoNT/B) on a time scale of minutes. Cleavage of the BoNT/B substrate VAMP2, a membrane SNARE protein associated with synaptic vesicles, was monitored using real-time surface plasmon resonance to measure vesicle capture by specific antibodies coupled to microchips. The assay is functional in low-ionic-strength buffers and stable over a wide range of pH values (5.5–9.0). Endoproteolytic cleavage of VAMP2 was detected in 10 min with 2 pM native BoNT/B holotoxin. Contamination of liquid food products such as carrot juice, apple juice, and milk with low picomolar amounts of BoNT/B was revealed within 3 h. BoNT/B activity was detected in sera from patients with type B botulism but not in healthy controls or patients with other neurological diseases. This robust, sensitive, and rapid protein chip assay is appropriate for monitoring BoNT/B in food products and diagnostic tests for type B botulism and could replace the current in vivo mouse bioassay.     

肉毒神经毒素抑制剂的研究进展

肉毒神经毒素(botulinum neurotoxins,BoNTs)是由梭状芽孢杆菌属分泌的外毒素,是目前已知毒性最强的生物类毒素.BoNTs共分为7种血清型(A-G),其中A型导致的肉毒中毒最为常见.由于肉毒毒素的强毒性及易于制备,其已被列为A类生物恐怖制剂.目前,针对肉毒中毒的有效治疗手段为早期注射抗毒素血清.但...     

Development of a fusion protein SNVP as substrate for assaying multi-serotype botulinum neurotoxins

The SNARE super family has three core members, namely SNAP-25, VAMP-2, and syntaxin. SNAP-25 is cleaved by botulinum toxins (BoNTs)/A, /C, and /E, whereas VAMP-2 is the substrate for proteolytic BoNTs/B, /D, /F, and /G. In this study, we constructed a hybrid gene encoding the fusion protein SNVP that encompasses SNAP-25 residues Met1 to Gly206 and VAMP-2 residues Met1 to Lys94. The hybrid gene was cloned in a prokaryotic vector carrying an N-terminal pelB signal sequence and overexpressed in Escherichia coli BL21(DE3) Rosetta. To easily purify the protein, 6× His double-affinity tags were designed as the linker and C terminus of the fusion protein. SNVP was purified to homogeneity by affinity chromatography on a HisTrap FF column and determined to be more than 97% pure by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. N-terminal sequencing of the purified protein showed that signal peptide was successfully removed. The fusion protein SNVP contained the protease cleavage sites of all seven serotypes of BoNTs. SNVP was also proved to be recognized and cleaved by the endopeptidase of BoNTs (BoNT/A–LC, BoNT/B–LC, BoNT/E–LC, and BoNT/G–LC). The novel fusion substrate SNVP exhibited high biological activity under the optimal conditions, suggesting its potential use as a reagent for BoNT assay.     

Structure of botulinum neurotoxin type D light chain at 1.65 A resolution: repercussions for VAMP-2 substrate specificity

The seven serotypes (A-G) of botulinum neurotoxins (BoNTs) function through their proteolytic cleavage of one of three proteins (SNAP-25, Syntaxin, and VAMP) that form the SNARE complex required for synaptic vesicle fusion. The different BoNTs have very specific protease recognition requirements, between 15 and 50 amino acids in length depending on the serotype. However, the structural details involved in substrate recognition remain largely unknown. Here is reported the 1.65 A resolution crystal structure of the catalytic domain of BoNT serotype D (BoNT/D-LC), providing insight into the protein-protein binding interaction and final proteolysis of VAMP-2. Structural analysis has identified a hydrophobic pocket potentially involved in substrate recognition of the P1' VAMP residue (Leu 60) and a second remote site for recognition of the V1 SNARE motif that is critical for activity. A structural comparison of BoNT/D-LC with BoNT/F-LC that also recognizes VAMP-2 one residue away from the BoNT/D-LC site provides additional molecular details about the unique serotype specific activities. In particular, BoNT/D prefers a hydrophobic interaction for the V1 motif of VAMP-2, while BoNT/F adopts a more hydrophilic strategy for recognition of the same V1 motif.     

A Simple,Rapid and Sensitive FRET Assay for Botulinum Neurotoxin Serotype B Detection

Botulinum neurotoxins (BoNTs), the most potent naturally-occurring neurotoxins known to humans, comprise seven distinct serotypes (BoNT/A-G), each of which exhibits unique substrate specificity. Many methods have been developed for BoNT detection, in particular for BoNT/A, with various complexity and sensitivity, while substrate based FRET assay is considered as the most widely used approach due to its simplicity and sensitivity. In this study, we designed a vesicle-associated membrane protein 2 (VAMP2) based FRET assay based on the understanding of the VAMP2 and light chain/B (LC/B) interactions in our previous studies. The current design constituted the shortest peptide, VAMP2 (63–85), with FRET dyes (EDAN and Dabcyl) labelled at position 76 and 85, respectively, which showed minimal effect on VAMP2 substrate catalysis by LC/B and therefore enhanced the sensitivity of the assay. The FRET peptide, designated as FVP-B, was specific to LC/B, with a detection sensitivity as low as ∼20 pM in 2 h. Importantly, FVP-B showed the potential to be scaled up and used in high throughput screening of LC/B inhibitor. The currently developed FRET assay is one of the most economic and rapid FRET assays for LC/B detection.     

A new peptide substrate for enhanced botulinum neurotoxin type B detection by endopeptidase–liquid chromatography–tandem mass spectrometry/multiple reaction monitoring assay

Botulinum neurotoxins (BoNTs) are the most toxic proteins in nature. Rapid and sensitive detection of BoNTs is achieved by the endopeptidase–mass spectrometry (Endopep–MS) assay. In this assay, BoNT cleaves a specific peptide substrate and the cleaved products are analyzed by MS. Here we describe the design of a new peptide substrate for improved detection of BoNT type B (BoNT/B) in the Endopep–MS assay. Our strategy was based on reported BoNT/B–substrate interactions integrated with analysis method efficiency considerations. Incorporation of the new peptide led to a 5-fold increased sensitivity of the assay both in buffer and in a clinically relevant human spiked serum.     

Monoclonal antibodies to type A, B, E and F botulinum neurotoxins

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.     

Unique substrate recognition by botulinum neurotoxins serotypes A and 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. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.     

Optimization of peptide substrates for botulinum neurotoxin E improves detection sensitivity in the Endopep–MS assay

Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are the most poisonous substances known to humankind. It is essential to have a simple, quick, and sensitive method for the detection and quantification of botulinum toxin in various media, including complex biological matrices. Our laboratory has developed a mass spectrometry-based Endopep–MS assay that is able to rapidly detect and differentiate all types of BoNTs by extracting the toxin with specific antibodies and detecting the unique cleavage products of peptide substrates. Botulinum neurotoxin type E (BoNT/E) is a member of a family of seven distinctive BoNT serotypes (A–G) and is the causative agent of botulism in both humans and animals. To improve the sensitivity of the Endopep–MS assay, we report here the development of novel peptide substrates for the detection of BoNT/E activity through systematic and comprehensive approaches. Our data demonstrate that several optimal peptides could accomplish 500-fold improvement in sensitivity compared with the current substrate for the detection of both not-trypsin-activated and trypsin-activated BoNT/E toxin complexes. A limit of detection of 0.1 mouse LD₅₀/ml was achieved using the novel peptide substrate in the assay to detect not-trypsin-activated BoNT/E complex spiked in serum, stool, and food samples.     

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.     

Improved detection of botulinum neurotoxin type A in stool by mass spectrometry

Botulinum neurotoxins (BoNTs) are the most toxic substances known to humankind. Rapid and sensitive detection of BoNTs is necessary for timely clinical confirmation of the disease state in botulism. BoNTs cleave proteins and peptide mimics at specific sites. A mass spectrometry (MS)-based method, Endopep–MS, can detect these cleavages and has detection limits of 0.05–0.5 mouse LD₅₀ (U) in serum, depending on the BoNT serotypes. In this method, the products generated from cleavage of peptide substrates using antibody affinity-purified toxins are detected by MS. Nonspecific bound endogenous proteases or peptidases in stool can coextract with the toxin, cleaving the peptide substrates and reducing the sensitivity of the method. Here we report a method to reduce nonspecific substrate cleavage by reducing stool protease coextraction in the Endopep–MS assay.     

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.     

A continuous fluorigenic assay for the measurement of the activity of endoplasmic reticulum aminopeptidase 1: Competition kinetics as a tool for enzyme specificity investigation

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a recently discovered enzyme that plays critical roles in antigen presentation and the immune response. Unlike other aminopeptidases, ERAP1 displays strong sequence preferences for residues distal to the peptide-substrate’s N terminus. This unusual substrate specificity necessitates the development of new assays that are appropriate for the study of such aminopeptidases. Here we describe a continuous fluorigenic assay suitable for the analysis of the enzymatic properties of ERAP1. In this assay, signal is generated by the excision of an internally quenched N-terminal tryptophan residue from a 10mer peptide by the aminopeptidase, resulting in the enhancement of tryptophan fluorescence in the solution. This method overcomes the limitations of previously used fluorigenic and high-performance liquid chromatography (HPLC)-based assays and is appropriate for small molecule inhibitor screening as well as for rapid substrate specificity analysis by kinetic competition experiments. Such efficient peptidic fluorigenic substrates like the ones described here should greatly simplify specificity analysis and inhibitor discovery for ERAP1 and similar aminopeptidases.     

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.     

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA.     

Association of botulinum neurotoxin serotypes a and B with synaptic vesicle protein complexes

Botulinum neurotoxins (BoNTs) elicit flaccid paralysis through cleavage of SNARE proteins within peripheral neurons. There are seven serotypes of the BoNTs, termed A-G, which differ in the SNARE protein and/or site that is cleaved. BoNTs are single-chain toxins that comprise an N-terminal zinc metalloprotease domain that is disulfide linked to the C-terminal translocation/receptor binding domain. SV2 and synaptotagmin have been identified as receptors for BoNT serotypes A and B, respectively. Using affinity chromatography, BoNTs A and B were observed to bind synaptic vesicle protein complexes in synaptosome lysates. Tandem LC-MS/MS identified SV2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2 (VAMP2), and the vacuolar proton pump as components of the BoNT-receptor complex. Density gradient analysis showed that BoNT serotypes A and B exhibited unique interactions with the synaptic vesicle protein complexes. The association of BoNT serotypes A and B with synaptic vesicle protein complexes implicates a physiological role for protein complexes in synaptic vesicle biology and provides insight into the interactions of BoNT and neuronal receptors.     

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.