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Appl Environ Microbiol. Feb 2011; 77(4): 1301–1308.
Published online Dec 17, 2010. doi:  10.1128/AEM.01662-10
PMCID: PMC3067225
Different Substrate Recognition Requirements for Cleavage of Synaptobrevin-2 by Clostridium baratii and Clostridium botulinum Type F Neurotoxins[down-pointing small open triangle]
Suzanne R. Kalb,1 Jakub Baudys,2 Christina Egan,3 Theresa J. Smith,4 Leonard A. Smith,4 James L. Pirkle,1 and John R. Barr1*
Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy. N.E., Atlanta, Georgia 30341,1 Battelle Memorial Institute at the Centers for Disease Control and Prevention, Atlanta, Georgia,2 Wadsworth Center, New York State Department of Health, Albany, New York 12208,3 Integrated Toxicology, United States Army Medical Research Institute of Infectious Diseases (USAMRIID), Ft. Detrick, Maryland 217024
*Corresponding author. Mailing address: Centers for Disease Control and Prevention, National Center for Environmental Health, Division of Laboratory Sciences, 4770 Buford Hwy. N.E., Atlanta, GA 30341. Phone: (770) 488-7848. Fax: (770) 488-0509. E-mail: jbarr/at/cdc.gov
Received July 12, 2010; Accepted December 4, 2010.
Botulinum neurotoxins (BoNTs) cause botulism, which can be fatal if it is untreated. BoNTs cleave proteins necessary for nerve transmission, resulting in paralysis. The in vivo protein target has been reported for all seven serotypes of BoNT, i.e., serotypes A to G. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target. Most serotypes of BoNT can be subdivided into subtypes, and previously, we demonstrated that three of the currently known subtypes of BoNT/F cleave a peptide substrate, a shortened version of synaptobrevin-2, between Q58 and K59. However, our research indicated that Clostridium baratii type F toxin did not cleave this peptide. In this study, we detail experiments demonstrating that Clostridium baratii type F toxin cleaves recombinant synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. In addition, we demonstrate that Clostridium baratii type F toxin can cleave a peptide substrate based on the sequence of synaptobrevin-2. This peptide substrate is an N-terminal extension of the original peptide substrate used for detection of other BoNT/F toxins and can be used to detect four of the currently known BoNT/F subtypes by mass spectrometry.
Botulism is caused by intoxication with a potent neurotoxin known as botulinum neurotoxin (BoNT) and is a disease that can be fatal if it is untreated. Botulism can be contracted by ingestion of food containing the toxin (11, 33), by colonization of bacteria in the gastrointestinal tract in infants or susceptible individuals, by inhalation of the toxin, or by contact of bacteria with a wound (11). Treatment of botulism involves administering a therapeutic immunoglobulin product and is most effective when performed within 24 h of exposure (11). Due to the extreme toxicity, availability, and ease of preparation of BoNT, it is considered a likely agent for bioterrorism (4).
The neurotoxin molecule is composed of three functional domains—the receptor-binding domain, the translocation domain, and the enzymatic domain. The receptor-binding domain interacts with ectoacceptors on target cell surfaces (25). The toxin is taken up into endosomes, where a conformational change in the translocation domain allows for the formation of pores (24). These pores enable the enzymatic domain to enter the cytoplasm of target neuronal cells, where docking proteins (SNARE proteins) necessary for nerve transmission are cleaved. This inhibits the nerve impulse, resulting in a flaccid paralysis that can affect the lungs and may necessitate ventilator support.
BoNTs are produced by some species of the genus Clostridium, in particular Clostridium botulinum, C. butyricum, C. baratii, and C. argentinense. These toxins are divided into serotypes A through G based on their serological properties. C. botulinum strains produce toxins of types A through F, and C. argentinense strains produce type G toxin. Some strains of C. butyricum produce type E toxin, and some C. baratii strains produce type F toxin. Four BoNT serotypes (A, B, E, and F) are commonly associated with human botulism. Each serotype can be divided further into four to six toxin subtypes. Within BoNT/F strains, proteolytic (23, 26, 44) and nonproteolytic (12, 13, 26) BoNT/F strains and bivalent BoNT/Af toxin-producing strains (15, 17) have been isolated from environmental samples and food-borne botulism cases. To date, all cases associated with bivalent Bf strains have been in infants (5). BoNT/F-expressing Clostridium baratii strains have been responsible for both infant and, presumably, food-borne botulism (18, 19). However, with C. baratii botulism, there is only tenuous evidence, if any at all, linking cases with causative foods, and while multiple members of a household may have consumed common foodstuffs, only targeted members became ill (18). This information indicates that the immune status of the patient may be a major factor in susceptibility to botulism due to C. baratii.
Different toxin serotypes have different enzymatic targets. BoNT/A, -C, and -E cleave synaptosome-associated protein (SNAP-25) (7, 8, 16, 28, 29, 43), whereas BoNT/B, -D, -F, and -G cleave synaptobrevin-2 (VAMP-2) (27, 31, 45, 46). Only BoNT/C is known to cleave more than one protein, i.e., SNAP-25 and syntaxin (9, 16, 32). Each toxin serotype cleaves its target at a unique place in the molecule. Knowledge of the cleavage sites has led to the development of several assays to detect BoNT based on its ability to cleave a peptide substrate derived from its in vivo protein target (3, 6, 10, 14, 20, 34-36, 41, 42). Previously, our laboratory reported the development of an assay for BoNT detection and serotype differentiation, termed the Endopep-MS method (6, 10). This method detects all BoNT serotypes, BoNT/A through BoNT/G, and involves incubating BoNT with a peptide substrate that mimics BoNT's natural, in vivo target. Each BoNT cleaves its peptide substrate in a specific location, and that location is different for each BoNT serotype. The reaction mixture is then introduced into a mass spectrometer, which detects and accurately reports the masses of any peptides within the mixture. Detection of peptide cleavage products corresponding to the specific toxin-dependent location indicates the presence of a particular BoNT serotype.
Proteolytic BoNT/F (protBoNT/F) cleaves synaptobrevin-2 between Q58 and K59 (30). The BoNT/F subtypes contain substantial amino acid sequence differences from protBoNT/F, which may affect receptor binding and the cleavage of target SNARE proteins. In previous work, we demonstrated that the enzymatic activities of most BoNT/A, -B, -E, and -F subtypes were equivalent in both location and potency (22). As part of that study, we confirmed that protBoNT/F, nonproteolytic BoNT/F (npBoNT/F), and several bivalent BoNT/F (bvBoNT/F) subtypes all cleaved their peptide substrate in the same location (22). However, our research indicated that BoNT/F from C. baratii strains (BoNT/FC. baratii) did not cleave this peptide.
In the current study, we detail experiments which demonstrate that Clostridium baratii type F toxin cleaves synaptobrevin-2 in the same location as that cleaved by proteolytic F toxin. Failure to cleave our standard synaptobrevin-2 peptide was found to be due to amino acid differences in and near multiple exosites between protBoNT/F and BoNT/FC. baratii. Extension of the N terminus of the synaptobrevin-2 peptide enabled cleavage of the substrate target by BoNT/FC. baratii subtypes. Use of this extended synaptobrevin-2 peptide allows detection of four of the currently known BoNT/F subtypes by mass spectrometry.
Materials.
Botulinum neurotoxin is very toxic and therefore requires appropriate safety measures. All neurotoxins were handled in a level 2 biosafety cabinet equipped with HEPA filters. Commercially purified BoNT/B and protBoNT/F were purchased from Metabiologics (Madison, WI). Synaptobrevin-2 recombinant protein was purchased from GenWay Biotech, Inc. (San Diego, CA). Monoclonal antibody 4E17.1 was obtained from James Marks at the University of California at San Francisco. Protein G Dynabeads were purchased from Invitrogen (Carlsbad, CA) at 1.3 g/cm3 in phosphate-buffered saline (PBS), pH 7.4, containing 0.1% Tween 20 and 0.02% sodium azide. All chemicals were from Sigma-Aldrich (St. Louis, MO), except where indicated. Peptide substrates were synthesized by Los Alamos National Laboratory (Los Alamos, NM) (Fig. (Fig.11).
FIG. 1.
FIG. 1.
Amino acid sequence of the recombinant synaptobrevin-2 substrate utilized in and produced by cleavage of BoNT/F and -B. The original BoNT/F substrate is in red, with the new substrate in red and purple. The observed m/z of each peptide or protein is also (more ...)
Production and characterization of Clostridium baratii type F culture supernatants.
Clostridium baratii type F organisms from the Wadsworth Center Culture Collection were cultured in 5 ml of Trypticase-peptone-glucose-yeast extract (TPGY) broth prepared in-house at 35°C for approximately 3 days. Anaerobic conditions were created by using an Oxoid AnaeroGen atmosphere generation system (Basingstoke, United Kingdom). The supernatants were then filtered by using an Acrodisc 0.22-μm syringe filter purchased from Pall Corporation (Port Washington, NY). Fifty microliters of filtered supernatant was cultured on sheep blood agar plates prepared in-house and incubated under the conditions described above to ensure that there were no viable organisms present in the supernatants. The culture supernatants were tested at the Wadsworth Center by using a mouse bioassay to determine the presence of type F toxin (38).
BoNT extraction.
Monoclonal antibody 4E17.1 was immobilized and cross-linked to protein G Dynabeads as previously described (21, 22), by using 40 μg of antibody diluted in 500 μl of PBS for every 100 μl of protein G Dynabeads. Cross-linked IgG-coated Dynabeads were made fresh daily. An aliquot of 20 μl of antibody-coated beads was mixed for 1 h with a solution of 500 μl of culture supernatant mixed with 50 μl of 10× phosphate-buffered saline with 0.01% Tween 20 (PBST), with 500 mouse 50% lethal doses (mLD50) of protBoNT/F mixed with 500 μl of PBST, or with 24,000 mLD50 of BoNT/B mixed with 500 μl of PBST. After being mixed for 1 h with constant agitation at room temperature, the beads were washed twice with 1 ml (each time) of PBST and then once with 100 μl of water. Negative controls consisted of PBST or blank culture supernatant medium with no spiked toxin. The remainder of the extraction protocol was as described above. The commercially purified toxins did not require antibody extraction, but antibody extraction was performed with all samples to ensure that all toxins were exposed to the same procedures to eliminate differential procedures as a source of inaccurate results. Levels of the commercially purified toxins were chosen in order to achieve a reaction in which the cleavage products and unmodified substrate were clearly visible. The level of the C. baratii toxin in the culture used is not known.
Incubation of BoNT with peptide or protein target.
The incubation of BoNT with a target was performed as previously described (21), with a few modifications. In all cases, a final reaction volume of 20 μl was added to the beads; the final concentration of the reaction buffer was 0.05 M HEPES (pH 7.3), 25 mM dithiothreitol, and 20 μM ZnCl2. For peptide reactions, the final concentration of the peptide substrate was 50 pmol/μl, and the peptide sequences are listed in Fig. Fig.1.1. For recombinant synaptobrevin-2 reactions, the final concentration of synaptobrevin-2 was 250 ng/μl. All samples were incubated at 37°C for 4 h.
Peptide mass spectrometric detection.
A 2-μl aliquot of each reaction supernatant was mixed with 18 μl of matrix solution consisting of alpha-cyano-4-hydroxycinnamic acid (CHCA) at 5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA), and 1 mM ammonium citrate. A 0.5-μl aliquot of this mixture was pipetted onto each spot of a 192-spot matrix-assisted laser desorption ionization (MALDI) plate (Applied Biosystems, Framingham, MA). Mass spectra for each spot were obtained by scanning from 1,100 to 4,800 m/z in mass spectrometry (MS) positive-ion reflector mode on an Applied Biosystems 4800 proteomic analyzer (Framingham, MA). The instrument uses an Nd-YAG laser at 355 nm, and each spectrum is an average of 2,400 laser shots.
Protein mass spectrometric detection.
All reaction mixtures were first separated by using a nanoACQUITY UltraPerformance LC (UPLC) system (Waters, Milford, MA). Mobile phases were 0.04% TFA with 0.06% formic acid (FA) in water (mobile phase A) and 0.04% TFA and 0.06% FA in acetonitrile (mobile phase B). Synaptobrevin-2 and cleavage products were trapped at 500 ng on a Pepswift PS-DVB monolithic trapping column (200 μm by 5 mm; Dionex, Sunnyvale, CA) and then washed for 4 min at a flow rate of 7.5 μl/min with 99% mobile phase A. Intact synaptobrevin-2 and cleavage products were eluted and separated by using a 70-min reverse-phase (RP) gradient at 750 nl/min (1 to 50% mobile phase B over 35 min) on a Pepswift PS-DVB monolithic nanoscale LC column (100 μm by 5 cm; Dionex). The column temperature was set to 60°C.
A NanoMate TriVersa instrument (Ithaca, NY) was used for infusion and online LC coupling analysis of the samples at a capillary spray voltage of 1.82 kV. The mass spectral data were acquired on a Synapt HDMS quadrupole time of flight (QTOF) instrument (Waters); the instrument was calibrated for a mass range of 550 to 4,550 m/z with cesium iodide through direct infusion. The sampling and extraction cone voltages were optimized at 40 V and 4 V, respectively, for maximum intact synaptobrevin-2 sensitivity, by comparing on-column injections. Source temperature was set to 150°C. A quadrupole RF transmission profile was defined for transmit masses from 800 to 5,000 Da. Trap and transfer collision energies were set to 6 V and 2 V, respectively, for maximum transmission of the most abundant synaptobrevin-2 charge state. The data were acquired in TOF V mode at a mass range of 700 to 2,500 m/z and a 2-scan/s acquisition time. All data were processed by using Waters MassLynx MaxEnt 1 software to obtain the deconvoluted mass at a range of 3,000 to 20,000 Da, with a mass resolution of 0.5 Da. All spectra were processed with a uniform Gaussian damage model with an iterate to convergence option selected.
C. baratii type F does not cleave a peptide substrate recognized by other type F toxins.
Three of the currently known subtypes of BoNT/F cleave a peptide substrate based on the sequence of synaptobrevin-2 (22). This peptide substrate has the sequence LQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL, with cleavage by BoNT/F occurring between the bolded and underlined Q and K. These data (Fig. 2B to D) and a negative control (Fig. (Fig.2A)2A) are depicted by their mass spectra in Fig. Fig.2.2. Following toxin extraction and concentration of BoNT/FC. baratii, the peptide substrate remained unaltered as depicted in Fig. Fig.2E,2E, indicating that BoNT/FC. baratii does not cleave this peptide substrate.
FIG. 2.
FIG. 2.
MALDI mass spectra of Endopep-MS BoNT/F reactions for no toxin (A), protBoNT/F (B), npBoNT/F (C), bvBoNT/F (D), and BoNT/FC. baratii (E) extracted using 4E17.1 antibody-coated beads. The peptide cleavage products indicating that BoNT/F is present are (more ...)
C. baratii type F cleaves synaptobrevin-2 as other type F toxins do.
Because BoNT/FC. baratii did not cleave the peptide substrate as expected, its in vivo protein target was investigated. Schiavo and coauthors demonstrated that protBoNT/F cleaved the protein synaptobrevin-2 when BoNT/F was incubated with rat synaptic vesicles (30). Because other tested subtypes had the same cleavage site under in vitro conditions (22), we theorized that BoNT/FC. baratii had the same cleavage site as the other type F toxins under in vitro conditions. To test this theory, full-length recombinant synaptobrevin-2 needed to be detected by mass spectrometry. Following extraction using blank PBST buffer, antibody-coated beads were incubated with synaptobrevin-2 in the presence of reaction buffer. The supernatant of this reaction was analyzed by QTOF mass spectrometry. Figure Figure33 shows the deconvoluted mass spectrum of this reaction. The peak at mass 13,824 corresponds to intact full-length recombinant synaptobrevin-2.
FIG. 3.
FIG. 3.
Deconvoluted mass spectrum of full-length recombinant synaptobrevin-2. The peak corresponding to synaptobrevin-2 is present at m/z 13,824.
ProtBoNT/F was then extracted with antibody-coated beads and incubated with full-length synaptobrevin-2. The mass spectra obtained from this reaction are shown in Fig. Fig.4.4. Figure Figure4A4A shows the deconvoluted mass spectrum produced on the QTOF instrument used to analyze larger-molecular-weight fragments. The peak at mass 13,823 corresponds to intact synaptobrevin-2, similar to that in Fig. Fig.3.3. However, Fig. Fig.4A4A shows a second peak of interest at mass 10,343, which corresponds to cleavage of synaptobrevin-2 by protBoNT/F.
FIG. 4.
FIG. 4.
Mass spectra of full-length recombinant synaptobrevin-2 cleaved by protBoNT/F. (A) Deconvoluted mass spectrum of the larger-molecular-weight pieces. (B) MALDI mass spectrum of the smaller-molecular-weight piece. The peak corresponding to intact synaptobrevin-2 (more ...)
After examination of the amino acid sequence of recombinant synaptobrevin-2 (Fig. (Fig.1),1), it was determined that the peak at mass 10,343 in Fig. Fig.4A4A corresponds to the N-terminal cleavage product from the 95th residue of recombinant synaptobrevin-2, or cleavage between glutamine 95 and lysine 96. This finding confirms the cleavage location for protBoNT/F reported by Schiavo and coauthors (30). Figure Figure4B4B shows the mass spectrum of protBoNT/F incubated with synaptobrevin-2. The peak at m/z 3,496.8 corresponds to the C-terminal cleavage product of synaptobrevin-2, also produced by cleavage with protBoNT/F.
In order to determine that the cleavage of full-length synaptobrevin-2 is indeed toxin dependent, BoNT/B, another toxin serotype capable of cleaving this protein, was extracted with antibody-coated beads and incubated with synaptobrevin-2. Following that reaction, the reaction supernatant that contained synaptobrevin-2 was analyzed by QTOF and MALDI-TOF mass spectrometry. Figure Figure55 shows the mass spectra obtained from that reaction, with Fig. Fig.5A5A showing the deconvoluted mass spectrum depicting the larger-molecular-weight fragments. As with Fig. Fig.33 and and4A,4A, Fig. Fig.5A5A shows a peak at mass 13,823.5 that corresponds to intact synaptobrevin-2. However, a peak is also found at mass 12,214 that corresponds to the cleavage of synaptobrevin-2 by BoNT/B.
FIG. 5.
FIG. 5.
Mass spectra of full-length recombinant synaptobrevin-2 cleaved by BoNT/B. (A) Deconvoluted mass spectrum of the larger-molecular-weight pieces. (B) MALDI mass spectrum of the smaller-molecular-weight piece. The peak corresponding to intact synaptobrevin-2 (more ...)
Upon examination of the amino acid sequence of recombinant synaptobrevin-2 (Fig. (Fig.1),1), we determined that this sequence corresponds to the N-terminal cleavage product from the 113th residue of recombinant synaptobrevin-2, or cleavage between a glutamine and a phenylalanine. This finding confirms the cleavage location for BoNT/B reported by Schiavo and coauthors (27). Furthermore, the C-terminal cleavage product was also visible by MALDI-TOF mass spectrometry. Figure Figure5B5B shows the MALDI-TOF mass spectrum of BoNT/B incubated with synaptobrevin-2. The peak at m/z 1,628.0 corresponds to the C-terminal cleavage product of synaptobrevin-2 produced by cleavage with BoNT/B. ProtBoNT/F and -B both cleave synaptobrevin-2, but each cleaves at a different site.
After determining that cleavage of full-length synaptobrevin-2 by either BoNT/B or protBoNT/F could be detected by mass spectrometry, BoNT/FC. baratii was extracted with antibody-coated beads and incubated with synaptobrevin-2. Following that reaction, the reaction supernatant that contained synaptobrevin-2 was analyzed by QTOF and MALDI-TOF mass spectrometry. Figure Figure66 shows the mass spectra obtained from that reaction, with Fig. Fig.6A6A showing the deconvoluted mass spectrum from the QTOF mass spectrometer. As with Fig. Fig.3,3, ,4A,4A, and and5A,5A, Fig. Fig.6A6A shows a peak at 13,824 Da that corresponds to intact synaptobrevin-2. However, there is also a peak at 10,344 Da, which corresponds to cleavage of synaptobrevin-2 by BoNT/FC. baratii. This peak is the same as that produced by cleavage of synaptobrevin-2 by protBoNT/F in Fig. Fig.4A4A and corresponds to the N-terminal cleavage product of synaptobrevin-2. The C-terminal cleavage product of this reaction is present in Fig. Fig.6B,6B, at m/z 3,496.8, and is identical to the C-terminal cleavage product of the reaction with protBoNT/F. Therefore, BoNT/FC. baratii cleaves synaptobrevin-2 between glutamine 95 and lysine 96, which is the same location as that cleaved by protBoNT/F.
FIG. 6.
FIG. 6.
Mass spectra of full-length recombinant synaptobrevin-2 cleaved by BoNT/FC. baratii. (A) Deconvoluted mass spectrum of the larger-molecular-weight pieces. (B) MALDI mass spectrum of the smaller-molecular-weight piece. The peak corresponding to intact (more ...)
C. baratii type F cleaves a lengthened peptide substrate for BoNT/F.
Because BoNT/FC. baratii cleaves synaptobrevin-2 yet does not cleave our original peptide based on a portion of the sequence of synaptobrevin-2, we decided to design a peptide substrate that could be recognized by BoNT/FC. baratii. Extension of the C terminus of our peptide did not result in cleavage of the peptide by BoNT/FC. baratii (data not shown). However, extending the N terminus of our peptide did result in cleavage by BoNT/FC. baratii. Figure Figure7A7A shows the mass spectrum of a negative control where no toxin was incubated with the peptide TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL, a 5-amino-acid N-terminal extension of the original peptide. The peak at 2,555.9 m/z corresponds to the doubly charged intact substrate. Figure Figure7B7B shows the mass spectrum of this peptide incubated with BoNT/FC. baratii. The peak at m/z 1,345.8 corresponds to the C-terminal cleavage product of the peptide substrate for BoNT/FC. baratii. This location corresponds to cleavage between Q37 and K38 of this peptide, or the same location as cleavage of full-length synaptobrevin-2 by BoNT/FC. baratii. The N-terminal cleavage product is also present at 3,783.2 m/z.
FIG. 7.
FIG. 7.
MALDI mass spectra of the updated peptide substrate in Endopep-MS BoNT/F reactions for no toxin (A), BoNT/FC. baratii (B), protBoNT/F (C), npBoNT/F (D), and bvBoNT/F (E) extracted using 4E17.1 antibody-coated beads. The peptide cleavage products indicating (more ...)
This peptide substrate was also tested with other known subtypes of BoNT/F to ensure that they also cleave this peptide substrate. Figure Figure7C7C shows the mass spectrum of the reaction of protBoNT/F with the peptide substrate. The peak at m/z 1,345.8 corresponds to the C-terminal cleavage product of the peptide substrate for protBoNT/F and is identical to the C-terminal cleavage product produced by BoNT/FC. baratii. This peak is also present in Fig. 7D and E, which correspond to the reactions of npBoNT/F and bvBoNT/F, respectively. Therefore, four of the currently known subtypes of BoNT/F cleave this new peptide substrate.
In previous work, our laboratory demonstrated that the activities of most noncommercially available subtypes on a peptide substrate were the same as those of the commercially available toxins (22). For instance, we determined that the activities of BoNT/A2, -A3, and -A4 were the same as that of BoNT/A1. This is important information, as the original experiments that discovered BoNT's target proteins and cleavage sites were performed with only one subtype of toxin per serotype, and it was not known if all subtypes would have the same target. The current experiments detail that four subtypes of BoNT/F do indeed have the same protein target and cleavage site, as those BoNT/F subtypes cleave synaptobrevin-2 in the same location. However, differences in peptide substrate recognition sites (exosites) exist among the various BoNT/F subtypes, and these differences translate to an inability to cleave our 39-amino-acid peptide.
Several publications have addressed binding of synaptobrevin-2 to protBoNT/F. James Schmidt et al. showed that a peptide including residues A37 to S75 of synaptobrevin-2 generated effective cleavage by protBoNT/F, between 58Q and 59K (35). Schmidt et al. later reported that one or more residues from L32 to Q36 of synaptobrevin-2 are important for substrate binding and cleavage by protBoNT/F (37) and also noted that residues R66 to S75 are not required for substrate recognition and cleavage by protBoNT/F. Therefore, the optimal substrate for recognition and cleavage by protBoNT/F was found to be residues L32 to D65 of synaptobrevin-2. A 39-amino-acid peptide substrate consisting of residues L32 to L70 of synaptobrevin-2 was tested in our laboratory and was recognized and cleaved by protBoNT/F, npBoNT/F, and bvBoNT/F but not by BoNT/FC. baratii.
Several publications have also analyzed the binding of synaptobrevin-2 to protBoNT/F through either structural analysis via X-ray crystallography (1, 2) or point mutation of synaptobrevin-2 (40). Point mutations indicated that 14 amino acids between residues T27 and Y88 are important for cleavage by protBoNT/F, with critical amino acids beginning at residue Q33. X-ray crystallography results indicated that synaptobrevin-2 makes contact with protBoNT/F mainly through residues E41 to D65 (1). Residues Q33 to E41 of synaptobrevin-2 also interact with protBoNT/F (2) but are not critical for binding, as point mutations in this region do not affect the activity of protBoNT/F upon the peptide substrate (40). Additional interactions with protBoNT/F are reported for residues N25 to N29 of synaptobrevin-2; however, these interactions are reported as weak, and including these residues has little effect on increasing the binding affinity (2).
It is clear from previously published studies and the experimental results presented here that the interaction of protBoNT/F with synaptobrevin-2 is different from that of BoNT/FC. baratii with synaptobrevin-2. Table Table11 illustrates that BoNT/F subtypes differ by as much as 31.4% in overall amino acid composition. BoNT/FC. baratii is the most divergent, with amino acid differences ranging from 26.3% to 31.4%, versus 7.6% to 16.6% among the other subtypes (Table (Table1).1). Examination of amino acid identities versus protBoNT/F by domain (Table (Table2)2) indicates that the translocation and receptor-binding domains are both fairly conserved, with amino acid identities of 78.9% or better. The highest degree of divergence between BoNT/FC. baratii and protBoNT/F is seen within the enzymatic domain, where more than one-third of all amino acids differ, compared with fewer than 20% with any other BoNT/F subtype (Table (Table2).2). BoNT subtypes typically do not display such differences, as other subtypes have enzymatic domain identities of >81% (data not shown). Indeed, an amino acid identity of only 63% is quite close to the overall identity between differing serotypes, such as 63% between BoNT/E and -F and 57% between BoNT/B and -G. With such a high degree of divergence in the enzymatic domain, it is not surprising that BoNT/FC. baratii interacts with its substrate in a different manner from that of protBoNT/F.
TABLE 1.
TABLE 1.
BoNT/F subtype amino acid identities for four currently known subtypes
TABLE 2.
TABLE 2.
Amino acid identities of three BoNT/F subtypes to protBoNT/F for each domain of toxin, along with the strain identification for each subtype listed
These large amino acid differences in BoNT/FC. baratii account for its inability to bind a peptide substrate based on the sequence of synaptobrevin-2 from residues L32 to L70. Of the amino acids in protBoNT/F which are reported to be important for interactions with synaptobrevin-2, there are many which are mutated in BoNT/FC. baratii. Figure Figure88 lists the residues of synaptobrevin-2 from L32 to D57 and their reported contact with corresponding residues of protBoNT/F (2), with added sequence alignments of npBoNT/F, bvBoNT/F, and BoNT/FC. baratii. Of the 26 amino acids in BoNT/FC. baratii predicted to be contacted by residues L32 to D57 of synaptobrevin-2, half are significantly altered from those in protBoNT/F. Six of the amino acids from L32 to D57 of synaptobrevin-2—Q33, V39, E41, V43, L54, and D57—reduce the cleavability of synaptobrevin-2 by more than 66% when mutated, demonstrating that these residues are critical for binding of synaptobrevin-2 to protBoNT/F (40). Five of those six residues contact mutated residues on BoNT/FC. baratii, which could explain why residues L32 to L70 of synaptobrevin-2 cannot bind BoNT/FC. baratii effectively.
FIG. 8.
FIG. 8.
Amino acid contacts between residues 27 and 57 of synaptobrevin-2 and protBoNT/F. Sequence alignments with npBoNT/F, bvBoNT/F, and BoNT/FC. baratii are also included, with mutations shown in red and conserved mutations shown in green. The figure includes (more ...)
All currently known subtypes of BoNT/F cleave a peptide substrate based on the sequence of synaptobrevin-2 from residues T27 to L70, and residues T27 to R31 (TSNRR) are not necessary for binding to protBoNT/F, npBoNT/F, or bvBoNT/F. However, these residues are critical for binding to BoNT/FC. baratii. Residues T27 and N29 of TSNRR in synaptobrevin-2 may play important roles in the binding of synaptobrevin-2 to BoNT/FC. baratii. Residue T27 of synaptobrevin-2 is reported to contact Y319 and W322 of protBoNT/F (2). As indicated in Fig. Fig.8,8, W322 is conserved in BoNT/FC. baratii (W314), so T27 of synaptobrevin-2 could contact BoNT/FC. baratii via W314. In addition, N29 of synaptobrevin-2 is reported to contact Y316 of protBoNT/F (2). This residue is also conserved in BoNT/FC. baratii, as Y308, so N29 of synaptobrevin-2 could contact BoNT/FC. baratii via Y308. Residues R30 and R31 of synaptobrevin-2 could also play a role in binding to BoNT/FC. baratii. Both of these residues are disordered in the crystal structure and therefore could not be modeled (2).
The discovery that BoNT/FC. baratii requires a longer peptide substrate for cleavage than other BoNT/F subtypes is important to many assays that use activity to detect and differentiate BoNTs. Detection of BoNT via its activity upon a peptide substrate was first reported in 1994 (39), and many reports have followed, some of which report lower limits of detection than those of the mouse bioassay, in a shorter time. A substrate consisting of residues L32 to D65 of synaptobrevin-2 can be used to detect three of the currently known subtypes of BoNT/F. However, this peptide cannot be used to detect BoNT/FC. baratii. Therefore, it is possible to use the different responses of BoNT/FC. baratii toward the peptide substrates to identify the subtype of toxin as BoNT/FC. baratii based on an activity assay.
Acknowledgments
The opinions, interpretations, and recommendations are those of the authors and are not necessarily those of the Centers for Disease Control and Prevention or the U.S. Army.
Footnotes
[down-pointing small open triangle]Published ahead of print on 17 December 2010.
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