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Botulinum neurotoxins (BoNTs) cause botulism by cleaving proteins necessary for nerve transmission. There are seven serotypes of BoNT, A-G, characterized by their response to antisera. Many serotypes are further distinguished into differing subtypes based on amino acid sequence some of which result in functional differences. Our laboratory previously reported that all tested subtypes within each serotype have the same site of enzymatic activity. Recently, three new subtypes of BoNT/F; /F3, /F4, and /F5, were reported. Here, we report that BoNT/F5 cleaves substrate synaptobrevin-2 in a different location than the other BoNT/F subtypes, between 54L and 55E. This is the first report of cleavage of synaptobrevin-2 in this location.
Botulism is a disease which can be fatal if untreated and is caused by exposure to any one of the highly toxic proteins known as botulinum neurotoxins (BoNTs). BoNT are composed of a heavy chain, which binds to receptors on the neuron, and a light chain that is a protease. In vivo, the BoNT light chain cleaves proteins necessary for nerve signal transmission. This enzymatic cleavage leads to flaccid paralysis. Botulinum neurotoxins are currently classified into seven serotypes, labeled A-G. BoNT/A, /C, and /E cleave SNAP-25 (synaptosomal-associated protein) [1–6]whereas BoNT/B, /D, /F, and /G cleave synaptobrevin-2 (also known as VAMP-2) [7–11].
Our laboratory previously reported on BoNT’s ability to cleave a peptide substrate that mimics its natural target, with peptide cleavage detected by mass spectrometry [12,13]. BoNT/A through /F are known to exhibit genetic and amino acid variance within each serotype, and this has led to the designation of subtypes, some with identified antigenic differences [14–16]. However, it was uncertain if the amino acid variance would also result in differential enzymatic activity. Therefore, previous work in our laboratory focused on determining the cleavage location of all available subtypes of BoNT/A, /B, /E, and /F, the serotypes known to cause botulism in humans. Previously, 15 such subtypes were available in sufficient quantities for testing, and all BoNT subtypes within the same serotype cleaved their respective substrate in the same location . At the time that this work was reported, only four subtypes of BoNT/F were known.
In 2010, three new subtypes of BoNT/F; /F3, /F4, and /F5, were reported based on phylogenetic analysis of botulinum neurotoxin type F genes . All three subtypes were defined as BoNT/F based on their ability to cause signs of botulism in mice which could be prevented by preincubation of toxin with F antitoxins, although it was reported that the BoNT/F5 toxin required 20 times more type F antiserum to neutralize an equivalent amount of BoNT/F1 toxin . Additionally, it was noted that BoNT/F5 was highly different in its gene and amino acid sequences compared to the other BoNT/F subtypes . For instance, Table 1 shows that the whole toxin (holotoxin) of BoNT/F1 and BoNT/F2 have similar (83.5% identical) amino acid sequences. However, BoNT/F1 and /F5 holotoxins are dissimilar (69.9% identical). The amino acid dissimilarity (47.3% identity) observed in the light chain (the enzymatic domain) between BoNT/F5 and BoNT/F1 is even more pronounced. Until the identification of BoNT/F5, this level of variation in the light chain amino acid sequences within a serotype had not been reported. Indeed, an amino acid identity of only 47% is much less than the light chain identity between differing serotypes which have different sites of enzymatic activity, such as 56% between BoNT/E and /F and 61% between BoNT/B and /G, based on amino acid comparison.
The second-most divergent BoNT/F subtype, F7, was reported to have different peptide substrate recognition requirements . BoNT/F7, produced by Clostridium baratii, is 73.7% identical to BoNT/F1 at the holotoxin level and 63.3% identical at the enzymatic domain level. BoNT/F7 was reported to cleave both synaptobrevin-2 and peptides based on synaptobrevin-2 in the same location as the other BoNT/F subtypes, between 58Q and 59K. However, BoNT/F7 did not cleave a peptide substrate cleaved by other available BoNT/F subtypes based on the sequence of synaptobrevin-2, and would only cleave TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDD RADAL, a lengthened peptide substrate . A similar phenomenon was reported with two neurotoxins which also cleave synpatobrevin-2, BoNT/B and tetanus toxin . This was due to differential binding of BoNT/F7 with synaptobrevin-2 as compared to other BoNT/F. Because the amino acid sequence of the BoNT/F5 light chain is even more divergent than BoNT/F7, it was theorized that BoNT/F5 may have a different molecular reaction with its substrate. In this work, we describe the discovery of a novel enzymatic cleavage site on synaptobrevin-2 by BoNT/F5.
Synaptobrevin-2, SNAP-25, and synatxin recombinant proteins were purchased from GenWay Biotech, Inc. (San Diego, CA). Monoclonal antibody 6F5 was obtained from Dr. James Marks at the University of California at San Francisco. Dynabeads® (M-280/Streptavidin) 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 LQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL and TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL were synthesized by Biopeptide (San Diego, CA). Commercially purified BoNT/F1 (strain Langeland) was purchased from Metabiologics (Madison, WI).
An existing BoNT/F1 LC ORF was PCR amplified using primers to add 5’ BamHI and 3’ EcoRI sites. The PCR reaction product was run on a 1% TAE gel and the amplified band at approximately 1400 bp was excised, purified, ligated into the homologous restriction enzymes sites of pGEX6P-1, and transformed into the DH5-strain of E. coli. A single recombinant colony was used to inoculate 5 ml of LB-50 mg/L ampicillin and grown overnight at 37°C. Plasmid DNA was recovered, completely sequenced to insure accuracy, mobilized into E. coli strain BL21 DE3, and spread onto LB plates with 50 mg/L ampicillin. A single recombinant colony was used to inoculate a LB-50 mg/L ampicillin, grown overnight at 37°C. The overnight culture was used to inoculate a fresh flask of 1L of LB-50 mg/L ampicillin and grown to an OD of 0.7, cooled to 18°C, IPTG was added to a final concentration of 1 mM, and the induction was allowed to proceed at 18°C overnight. The same procedure was followed for BoNT/F5, with the exception that the BoNT/F5 light chain ORF (representing nucleotides 1–438 from GU213211.1) was synthesized de novo using a generalized E. coli K12 codon bias and ligated into the BamHI/EcoRI sites of bacterial expression vector pGEX6P-1.
Both proteins bound to glutathione sepharose resin and were eluted in the presence of 10 mM reduced glutathione; however, the protein was not purified at this stage and required further chromatography steps. Both proteins were further separated with cation exchange chromatography after a pH change from ~8.0 to ~5.7. After cation exchange chromatography the purified BoNT/F5 LC-GST was approximately 100 g/ml, while the BoNT/F1 LC-GST was approximately 40 g /ml.
CDC54075, originally isolated in March 1978 from soil in a cornfield in Mendoza, Argentina at latitude 34° 98' and longitude 67° 59', was inoculated into 10 mls of Trypticase-Peptone-Glucose-Yeast Extract (TPGY) broth (Remel, Inc. Lexana, KS) at 35°C for 5 days in an anaerobe chamber system (Coy Laboratory Products, Inc. Grass Lake, MI) utilizing 10% Hydrogen, 5% CO2, and 85% Nitrogen. The culture was tested by PCR for the presence of neurotoxin genes as previously reported . The culture was centrifuged at 4,000 × g for 10 minutes and filtered using a 0.45µm syringe filter (Whatman #6876-2504) to remove viable cells. The presence of botulinum toxin in the culture supernatant was confirmed by ELISA and mouse bioassay .
The BoNT fusion proteins were serially diluted in water to achieve three different concentrations of each protein. For F1, concentrations of 40 g/mL (52.7 pmol/mL), 4 g/mL (5.27 pmol/mL), and 2 g/mL (2.64 pmol/mL) were used; whereas concentrations of 100 g/mL (1.32 nmol/mL), 10 g/uL (132 pmol/mL), and 5 g/mL (66 pmol/mL) were used for F5. 2 L of each concentration of fusion protein was added to 16 L of reaction buffer consisting of 0.05 M Hepes (pH 7.3), 25 mM dithiothreitol, and 20 M ZnCl2. Finally, 2 L of recombinant synaptobrevin-2 or peptide substrate (LQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL or TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL) was added to achieve a final concentration of 250 ng/ L (18 pmol/ L) for synaptobrevin-2 and 50 pmol/ L for the peptide reactions. All samples then were incubated at 37°C for 4 hrs with no agitation.
Monoclonal antibody 6F5, a clonal relative of mAb E17.1  engineered toward higher affinity for BoNT/F subtypes, was immobilized to streptavidin Dynabeads® after being rinsed three times with HBS-EP buffer (GE Healthcare; Piscataway, NJ.). A 2 g aliquot of antibody was used per 100 L of beads. A standard orbital shaker was used to bind antibody onto the beads for 1.5 hours. An aliquot of 20 L of antibody-coated beads was added to a solution of 400 uL of HBS-EP buffer and 100 L of culture supernatant containing BoNT/F5. After mixing for 1 hr with constant agitation at room temperature, the beads were washed twice in 1 mL each of HBS-EP buffer and then once in 100 L of water. Negative controls consisted of blank culture supernatant medium with no toxin, with the remainder of the extraction protocol as above. Positive controls consisted of blank culture supernatant medium spiked with 20 mLD50 of BoNT/F1 complex. The level of BoNT/F5 toxin in the culture used was not known.
The beads were reconstituted in 18 L of reaction buffer consisting of 0.05 M Hepes (pH 7.3), 25 mM dithiothreitol, and 20 M ZnCl2 and 2 L of recombinant synaptobrevin-2 or peptide substrate (LQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL or TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL). The final concentration of substrate was 250 ng/ L (18 pmol/ L) for synaptobrevin-2 and 50 pmol/ L for the peptide reactions. All samples then were incubated at 37°C for 4 hrs with no agitation.
A 2- L aliquot of each reaction supernatant was mixed with 18 L of matrix solution consisting of alpha-cyano-4-hydroxy cinnamic acid (CHCA) at 5 mg/mL in 50% acetonitrile, 0.1% trifluoroacetic acid (TFA), and 1 mM ammonium citrate. A 0.5- L aliquot of this mixture was pipeted onto each spot of a 192-spot matrix-assisted laser desorption/ionization (MALDI) plate (Applied Biosystems, Framingham, MA). Mass spectra of each spot were obtained by scanning from 1100 to 4800 m/z in MS-positive ion reflector mode on an Applied Biosystems 4800 Proteomics Analyzer (Framingham, MA). The instrument uses an Nd-YAG laser at 355 nm, and each spectrum is an average of 2400 laser shots.
All reactions were first separated by using a nano-ACQUITY 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 × 5 mm (Dionex, Sunnyvale, CA), and washed for 4 min at a flow rate of 7.5 L/min with 99% mobile phase A. Intact synaptobrevin-2 and cleavage products were eluted and separated by using a 70 min RP gradient at 750 nL/min (1–50% mobile phase B over 35 minutes) on a Pepswift PS-DVB monolithic 100 µm × 5 cm nanoscale LC column (Dionex). The column temperature was set to 60°C.
A NanoMate TriVersa (Ithaca, NY) was used for infusion and on-line 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 QTOF (Waters); the instrument was calibrated for a mass range of 550– 4550 m/z with Cesium Iodide through direct infusion. The sampling and extraction cone voltage were optimized at 40V and 4V, 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 to transmit masses from 800–3500 Da. Trap and transfer collision energies were set to 6V and 2V, respectively, for maximum transmission of the most abundant synaptobrevin-2 charge states. The data were acquired in TOF V-mode at a mass range of 800–3000 m/z and a 2 scan/s acquisition time. All data were processed by using the Waters MassLynx MaxEnt 1 software to obtain the deconvoluted mass at a range of 700 to 15000 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.
The sequence of recombinant synaptobrevin-2 is MRGSHHHHHHGMASMTGGQQMGRDLYDDDDKDRWGSHMSATAATAPPAAPAGEGGPPAPPPNLTSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADALQAGASQFETSAAKLKRKYW, and BoNT/F1, /F2, /F6, and /F7 holotoxins have been reported to cleave it in the underlined location, between 58Q and 59K . The GST-BoNT/F1 light chain fusion protein was reacted with recombinant synaptobrevin-2, and Figure 1 shows that this resulted in two peaks in the mass spectrometer which correspond to cleavage of recombinant synaptobrevin-2 by the F1 fusion protein. The peak at mass 13824.0 in Figure 1A acquired by electrospray ionization mass spectrometry corresponds to intact recombinant synaptobrevin-2, whereas the peak at mass 10344.0 corresponds to the N-terminal cleavage product. The peak at m/z 3496.8 in Figure 1B acquired by MALDI-TOF/MS corresponds to the C-terminal cleavage product, and the peak at 1749.4 corresponds to the doubly-charged C-terminal cleavage product. Both cleavage products demonstrate that the GST-BoNT/F1 light chain fusion protein cleaves recombinant synaptobrevin-2 between 58Q and 59K, exactly where the BoNT/F1 holotoxin cleaves recombinant synaptobrevin-2.
Additionally, this fusion protein was tested with two additional peptide substrates, LQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL and TSNRRLQQTQAQVDEVVDIMRVNVDKVLERDQKLSELDDRADAL, whose sequences are based upon the sequence of synaptobrevin-2, and those results are in Figures 1C and 1D. The peak at m/z 1345.7 in Figures 1C and 1D corresponds to the C-terminal cleavage product of the substrates. Note that the C-terminal cleavage product is the same in both cases, as the two peptides are identical at the C-terminus. The peak at m/z 3168.6 in Figure 1C corresponds to the N-terminal cleavage product of the first peptide substrate, and the peak at m/z 3782.9 in Figure 1D corresponds to the N-terminal cleavage product of the second peptide substrate. In both cases, the GST-BoNT/F1 light chain fusion protein cleaved the peptide substrates in the above-underlined location, corresponding to a location between 58Q and 59K of synaptobrevin-2. These data demonstrate that the GST-BoNT/F1 light chain fusion protein has the same activity upon its substrates as BoNT/F1 holotoxin.
Next, GST-BoNT/F5 light chain fusion protein was reacted with recombinant synaptobrevin-2. Similar to the reaction with F1, this resulted in peaks in the mass spectrum corresponding to N-terminal and C-terminal cleavage products, as seen in Figure 2. However, the masses of these peaks did not correspond with cleavage between 58Q and 59K. The peaks at mass 9815.0 (Figure 2A) and m/z 4025.2 (Figure 2B) correspond to cleavage between 54L and 55E of synaptobrevin-2, with the peak at m/z 2013.6 corresponding to the doubly-charged C-terminal cleavage product. The GST-BoNT/F5 light chain fusion protein was also tested with the two peptide substrates used with BoNT/F1, and those results are in Figures 2C and 2D. The peak at m/z 1872.9 in Figures 2C and 2D corresponds to the C-terminal cleavage product of each of the peptides. The peak at m/z 2640.4 in Figure 2C corresponds to the N-terminal cleavage product of the first peptide substrate, and the peak at m/z 3254.6 in Figure 2D corresponds to the N-terminal cleavage product of the second peptide substrate. In all cases, the GST-BoNT/F5 light chain fusion protein cleaved the peptide substrates in a location which corresponds to between 54L and 55E of synaptobrevin-2.
Although the light chain of BoNT/F5 exhibits a different cleavage pattern than that of the other BoNT/F, it was unclear that this pattern would be repeated with the holotoxin or progenitor form of the toxin produced by BoNT/F5-encoding C. botulinum. Therefore, holotoxin BoNT/F5 was reacted with recombinant synaptobrevin-2 and the two peptide substrates listed above, and those results are in Figure 3. The peaks at mass 9815.0 (Figure 3A) and m/z 4025.2 (Figure 3B) correspond to cleavage between 54L and 55E of synaptobrevin-2, with the peak at m/z 2013.9 corresponding to the doubly-charged C-terminal cleavage product. BoNT/F5 was also tested with the two peptide substrates used with GST-BoNT/F5, and those results are in Figures 3C and 3D. The peak at m/z 1872.9 in Figures 3C and 3D corresponds to the C-terminal cleavage product of each of the peptides. The peak at m/z 2640.4 in Figure 3C corresponds to the N-terminal cleavage product of the first peptide substrate, and the peak at m/z 3254.6 in Figure 3D corresponds to the N-terminal cleavage product of the second peptide substrate. The results are identical to that of Figure 2, indicating that BoNT/F5 holotoxin cleaves synaptobrevin-2 between 54L and 55E, in the same location as the GST-BoNT/F5 light chain fusion protein.
As noted above, BoNT/F5 is the most divergent of the botulinum neurotoxins. Previously BoNT/F7, the second-most divergent of the botulinum neurotoxins, was shown to interact with its substrate, synaptobrevin-2, differently from most of the BoNT/F subtypes since F7 required a longer peptide substrate for cleavage by the toxin. However the actual cleavage site was identical to the other known BoNT/F subtypes In contrast, the previously unknown F5 subtype cleaves the substrate at a different site than all other BoNT/Fs tested. Of particular interest are several amino acid substitutions near the active site of BoNT/F5 as seen in Figure 4. The active site of the toxin, 228E, is conserved among all BoNT/F, and the region immediately surrounding the active site is also conserved among all BoNT/F, except for BoNT/F5. With BoNT/F5, three of the five amino acids immediately upstream of the active site differ from all other BoNT/F. This degree of variance so close to the active site is an important factor in the ability of this toxin to cleave synaptobrevin-2 in a different location.
BoNT/F5 has a large degree of amino acid variance, and this accounts for its ability to cleave synaptobrevin-2 in a different location from all other BoNT/F. These amino acid differences are located not only at the interaction of the active site of BoNT/F5 with synaptobrevin-2 as discussed above, but also at other interactions of BoNT/F5 with synaptobrevin-2. Of the amino acids in BoNT/F1 which are reported to be important for interactions with synaptobrevin-2, there are many which are mutated in BoNT/F5. Table 2 lists the residues of synaptobrevin-2 from 25N to 57D and their reported contact with corresponding residues of BoNT/F1 , with added sequence alignments of all other BoNT/F. Of the 28 amino acids in BoNT/F5 expected to be contacted by residues 25N to 57D of synaptobrevin-2, approximately three-quarters are significantly altered from that of BoNT/F1, with 20 of the 28 residues differing significantly. This is in contrast to the other BoNT/F, in which 0 to 14 residues differ. Six of the amino acids from 25Nto 57D of synaptobrevin-2—33Q, 39V, 41E, 43V, 54L, and 57D—reduce the cleavability of synaptobrevin-2 by more than 66% when mutated, demonstrating that these residues are critical for binding of synaptobrevin-2 to BoNT/F1 . Four of those six residues contact mutated residues on BoNT/F5, which could explain why residues 25N70L of synaptobrevin-2 bind BoNT/F5 differently, resulting in differential cleavage. It should be noted that BoNT/F5 was found to cleave only synaptobrevin-2 and not additional SNARE proteins such as SNAP-25 or a truncated version of syntaxin (data not shown).
Cleavage between 54L and 55E of synaptobrevin-2, has never been reported for any botulinum neurotoxin. BoNT/B, /D, and /G are also known to cleave snaptobrevin-2, but these toxins cleave between 76Q and 77F, 59K and 60L, and 81A and 82A of synaptobrevin-2, respectively [7,10,11]. Additionally, up to this point, all subtypes within a serotype have been reported to share the same cleavage site. Knowledge of a new cleavage site of BoNT will be of particular interest to scientists involved in developing medical countermeasures for botulism. Many scientists are investigating inhibitors such as peptide substrate blockers and mAb as next generation botulism therapeutics. Additionally, some botulism diagnostic methods are being developed which are based on activity towards a protein or peptide substrate. This report of a unique substrate cleavage site by BoNT/F5 subtype will alert scientists to potential functional variability within BoNT serotypes. Therefore, it is important to study the enzymatic activity of newly-emerging botulinum neurotoxins in order to design effective inhibitors of BoNTs and assays to detect all BoNTs.
This work was supported by NIAID IAA 120-B18. The opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Centers for Disease Control and Prevention, the U.S. Army, the National Institute of Allergy and Infectious Diseases, or the National Institutes of Health.
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Structured summary of protein interactions:
BoNT/F5 cleaves Synaptobrevin-2 by protease assay (View Interaction: 1, 2)
BoNT/F1 cleaves Synaptobrevin-2 by protease assay (View Interaction: 1, 2)