Mapping of the Synaptosome-Binding Regions on the Heavy Chain of Botulinum Neurotoxin A By Synthetic Overlapping Peptides Encompassing the Entire Chain

The purpose of this work was to map, on the heavy (H) chain of botulinum neurotoxin A (BoNT/A), the regions that bind to mouse brain synaptosomes (snps). We prepared 60 synthetic overlapping peptides that had uniform size and overlaps and encompassed the entire H chain (residues 499 to 1296) of BoNT/A. The ability of each peptide to inhibit the binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was studied. The binding of ¹²⁵I-labeled BoNT/A to mouse brain snps was completely inhibited by free unlabeled BoNT/A, but not by unrelated proteins, indicating that the binding of BoNT/A to mouse brain snps was a specific event. Inhibition studies with the individual peptides showed that, on the H_N domain, inhibitory activities greater than 10% were exhibited, in decreasing order, by peptides 799–817, 659–677, 729–747, 533–551, 701–719, and 757–775. Lower inhibitory activities (between 5.6% and 8.7%) were exhibited by five other peptides, 463–481, 505–523, 519–537, 603–621 and 645–663. The remaining 18 H_N peptides had little or no inhibitory activity. In the H_C domain, peptides 1065–1083, 1163–1181 and 1275–1296 had the highest inhibitory activities (between 25% and 29%), followed (10–12% inhibitory activity) by peptides 1107–1125, 1191–1209 and 1233–1251. Two other peptides, 1079–1097 and 1177–1195, had very low (5.8% and 4.9 %) inhibitory activities. The remaining 23 H_C peptides had no inhibitory activity. Inhibition with mixtures of equimolar quantities of the most active 6 peptides of H_N, 5 of H_C or all 11 of H_N and H_C revealed that the peptides contain independent non-competing binding regions. Comparison of the locations of the snp-binding regions on the H-subunit with the regions that bind blocking mouse anti-BoNT/A Abs helped explain the protecting ability of these Abs. In the three-dimensional structure of BoNT/A, the snp-binding regions that completely coincide or significantly overlap with the antigenic regions occupy surface locations and most of them reside in the last half of the H_C domain. But some of the regions reside in the H_N domain and might play a role in the translocation event.     

Synaptotagmin II and Gangliosides Bind Independently with Botulinum Neurotoxin B but Each Restrains the Other

Botulinum neurotoxin type B (BoNT/B) initiates its toxicity by binding to synaptotagmin II (SytII) and gangliosides GD1a and GT1b on the neural membrane. We synthesized two 27-residue peptides that carry the BoNT/B binding sites on mouse SytII (mSytII 37–63) or human SytII (hSytII 34–60). BoNT/B bound to these peptides, but showed substantially higher binding to mSytII peptide than to hSytII peptide. The mSytII peptide inhibited almost completely BoNT/B binding to synaptosomes (snps) and displayed a high affinity. BoNT/B bound strongly to mSytII peptide and binding was inhibited by the peptide. Binding of BoNT/B to snps was also inhibited (~80 %) by a larger excess of gangliosides GD1a or GT1b. The mSytII peptide inhibited very strongly (at least 80 %) the toxin binding to snps, while the two gangliosides were much less efficient inhibitors requiring much larger excess to achieve similar inhibition levels. Furthermore, gangliosides GD1a or GT1b inhibited BoNT/B binding to mSytII peptide at a much larger excess than the inhibition by mSytII peptide. Conversely, BoNT/B bound well to each ganglioside and binding could be inhibited by the correlate ganglioside and much less efficiently by the mSytII peptide. There was no apparent collaboration between mSytII peptide and either ganglioside. mSytII peptide displayed some protective activity in vivo in mice against a lethal BoNT/B dose. We concluded that SytII peptide and gangliosides bind independently but, with their binding sites on BoNT/B being spatially close, each can influence BoNT/B binding to the other due to regional conformational perturbations or steric interference or both. Ganglioside involvement in BoNT/B binding might help in toxin translocation and endocytosis.     

Inhibition of Botulinum Neurotoxin A Toxic Action In Vivo by Synthetic Synaptosome- and Blocking Antibody-Binding Regions

In previous studies, we showed that certain peptides of the H_N and H_C domains of the H-chain of BoNT/A bind to mouse brain synaptosomes (snps). There was either complete correspondence or overlap between peptides that bind snps and those that bind human or mouse blocking antibodies (Abs). An equimolar mixture of the overlapping peptides N5/N6/N7/N8 (residues 505–523/519–537/533–551/547–565) extended the survival time of the mice to 74 h (20%) relative to controls, which had a 50% survival time of 60 h. On the other hand, peptide N26 (residues 799–817) provided no protection (50% survival time, 58 h), but the overlapping peptide N25 (785–803) almost doubled the 50% survival time to 119 h. A mixture of the overlap N25/N26 provided an unexpected level of protection permitting 40% of the mice to survive a lethal BoNT/A dose. In the H_C domain, the overlap C23/C24 (1163–1181/1177–1195) provided no protection. Peptide C31 (1275–1296) also provided no significant protection. But an equimolar mixture of peptides C15/C16 (1051–1069/1065–1083) or peptides C18/C19/C20 (1093–1111/1107–1125/1121–1139) extended the 50% survival time by 41% (to 85 h) over controls (60 h) and was able to fully protect 20% of the mice which eventually recovered. Surprisingly, the mixture of the peptides C15/C16 and C18/C19/C20, which gave a 50% survival time of 75 h, was less protective than either peptides C15/C16 or peptides C18/C19/C20. The in vivo inhibitory activity of these peptides is discussed in relation to their location in the 3-dimensional structure of the toxin molecule and their membrane receptor binding.     

A peptide-based immunoassay for antibodies against botulinum neurotoxin A

Cervical dystonia (CD) is due to neck-muscle spasms that cause pain and involuntary contractions resulting in abnormal neck movements and posture. Symptoms can be relieved by injecting the affected muscle with a botulinum neurotoxin (BoNT, usually type A or type B). The therapeutic benefits are impermanent and toxin injections need to be repeated every 3-6 months. In a very small percentage of patients (less with BoNT/A than with BoNT/B) the treatment elicits blocking anti-toxin antibodies (Abs), which reduce or terminate the patient's responsiveness to further treatment. We have recently mapped (Dolimbek et al., 2006) the CD sera Ab-binding profile using a panel of 60, 19-residue peptides that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by 5 residues. Abs in CD sera bound to one or more of the peptides N25, C10, C15, C20, and C31. This suggested the possibility that binding to these peptides could be used for assay of Abs in CD sera. Data analysis reported here found that Ab binding to these regions showed very significant deviations from the control responses. Of these four peptides, C10 showed the most significant level of separation between patient and control groups (p = 5 x 10(-7)) and the theoretical resolution (i.e., ability to distinguish CD patients from control, see full definition under 'Statistical analysis' in Methods), 84%, was about 4% higher than the least resolved response, C31 (p = 6 x 10(-6), resolution 80%). Since the amounts of Abs bound to a given peptide varied with the patient and not all the patients necessarily recognized all four peptides, there was the possibility that binding to combinations of two or more peptides might give a better discriminatory capability. Using two peptides, C10 plus C31, the resolution improved to 87% (p = 4 x 10(-8)). These two peptides appeared to compliment each other and negate the lower resolution of C31. Combination of three peptides gave resolutions that ranged from 85 (N25 + C15 + C31; p = 2 x 10(-7)) to 88% (C10 + C15 + C31; p = 1 x 10(-8)). Finally, using the data of all four peptides, N25 + C10 + C15 + C31, gave a resolution of 86% (p = 1 x 10(-7)). Although these levels of resolution are somewhat lower than that obtained with whole BoNT/A (resolution 97%; p = 6 x 10(-12)), it may be concluded that the two-peptide combination C10 + C31, or the three-peptide combination C10 + C15 + C31 (affording resolutions of 87 and 88%, respectively) provide a good diagnostic, toxin-free procedure for assay of total specific anti-toxin Abs in BoNT/A-treated CD patients.     

Nucleotide sequence of the gene coding for non-proteolyticClostridium botulinum type B neurotoxin: Comparison with other clostridial neurotoxins

The neurotoxin gene of non-proteolyticClostridium botulinum type B (strain Eklund 17B) was cloned as a series of overlapping polymerase chain reaction (PCR) fragments generated with primers designed to conserved regions of published botulinal toxin (BoNT) sequences. The 3 end of the gene was obtained by using primers designed to the determined sequence of non-proteolytic BoNT/B and a published downstream region of BoNT/B gene from a proteolytic strain. Translation of the nucleotide sequence derived from cloned PCR fragments demonstrated the toxin gene encodes a protein of 1291 amino acid residues. Comparative alignment of the derived BoNT/B sequence with those of other published botulinal neurotoxins revealed highest sequence relatedness with BoNT/B of proteolyticC. botulinum. The sequence identity between non-proteolytic and proteolytic BoNT/B was 97.7% for the light chain (corresponding to 10 amino acid changes) and 90.2% for the heavy chain (corresponding to 81 amino acid changes), with most differences occurring at the C-terminal end. A genealogical tree constructed from all known botulinal neurotoxin sequences revealed marked topological differences with a phylogenetic tree ofC. botulinum types based upon small-subunit (16S) ribosomal RNA sequences.     

Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display

Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses that are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and has implications for vaccine development. Here, we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast-displayed BoNT/A binding domain (H(C)) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of H(C), with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of H(C), in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may explain, in part, the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.     

Evaluation of the therapeutic usefulness of botulinum neurotoxin B,C1, E,and F compared with the long lasting type A. Basis for distinct durations of inhibition of exocytosis in central neurons

Seven types (A-G) of botulinum neurotoxin (BoNT) target peripheral cholinergic neurons where they selectively proteolyze SNAP-25 (BoNT/A, BoNT/C1, and BoNT/E), syntaxin1 (BoNT/C1), and synaptobrevin (BoNT/B, BoNT/D, BoNT/F, and BoNT/G), SNARE proteins responsible for transmitter release, to cause neuromuscular paralysis but of different durations. BoNT/A paralysis lasts longest (4-6 months) in humans, hence its widespread clinical use for the treatment of dystonias. Molecular mechanisms underlying these distinct inhibitory patterns were deciphered in rat cerebellar neurons by quantifying the half-life of the effect of each toxin, the speed of replenishment of their substrates, and the degradation of the cleaved products, experiments not readily feasible at motor nerve endings. Correlation of target cleavage with blockade of transmitter release yielded half-lives of inhibition for BoNT/A, BoNT/C1, BoNT/B, BoNT/F, and BoNT/E (31, 25, approximately 10, approximately 2, and approximately 0.8 days, respectively), equivalent to the neuromuscular paralysis times found in mice, with recovery of release coinciding with reappearance of the intact SNAREs. A limiting factor for the short neuroparalytic durations of BoNT/F and BoNT/E is the replenishment of synaptobrevin or SNAP-25, whereas pulse labeling revealed that extended inhibition by BoNT/A, BoNT/B, or BoNT/C1 results from longevity of each protease. These novel findings could aid development of new toxin therapies for patients resistant to BoNT/A and effective treatments for human botulism.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Molecular cloning of the Clostridium botulinum structural gene encoding the type B neurotoxin and determination of its entire nucleotide sequence.

DNA fragments derived from the Clostridium botulinum type A neurotoxin (BoNT/A) gene (botA) were used in DNA-DNA hybridization reactions to derive a restriction map of the region of the C. botulinum type B strain Danish chromosome encoding botB. As the one probe encoded part of the BoNT/A heavy (H) chain and the other encoded part of the light (L) chain, the position and orientation of botB relative to this map were established. The temperature at which hybridization occurred indicated that a higher degree of DNA homology occurred between the two genes in the H-chain-encoding region. By using the derived restriction map data, a 2.1-kb BglII-XbaI fragment encoding the entire BoNT/B L chain and 108 amino acids of the H chain was cloned and characterized by nucleotide sequencing. A contiguous 1.8-kb XbaI fragment encoding a further 623 amino acids of the H chain was also cloned. The 3' end of the gene was obtained by cloning a 1.6-kb fragment amplified from genomic DNA by inverse polymerase chain reaction. Translation of the nucleotide sequence derived from all three clones demonstrated that BoNT/B was composed of 1,291 amino acids. Comparative alignment of its sequence with all currently characterized BoNTs (A, C, D, and E) and tetanus toxin (TeTx) showed that a wide variation in percent homology occurred dependent on which component of the dichain was compared. Thus, the L chain of BoNT/B exhibits the greatest degree of homology (50% identity) with the TeTx L chain, whereas its H chain is most homologous (48% identity) with the BoNT/A H chain. Overall, the six neurotoxins were shown to be composed of highly conserved amino acid domains interceded with amino acid tracts exhibiting little overall similarity. In total, 68 amino acids of an average of 442 are absolutely conserved between L chains and 110 of 845 amino acids are conserved between H chains. Conservation of Trp residues (one in the L chain and nine in the H chain) was particularly striking. The most divergent region corresponds to the extreme carboxy terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.     

Sequence of the gene for Clostridium botulinum type B neurotoxin associated with infant botulism,expression of the C-terminal half of heavy chain and its binding activity

Previously, we demonstrated that the neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a foodborne botulism-related strain, Okra. In this study, the neurotoxin genes of 111/NT and Okra/NT were amplified and the sequences determined. The nucleotide sequences of the genes for both neurotoxins possessed an open reading frame of 3873 bp that encoded 1291 amino acids. The identities of nucleotide sequences and amino acid sequences were 97.6% and 95.7%, respectively. The ratio of nonsynonymous to synonymous substitutions was 0.47. The amino acid substitutions between 111/NT and Okra/NT occurred mainly in the domain of the C-terminal half of heavy chain (H(C)) responsible for binding to its receptor complex of protein and ganglioside. To characterize the binding capability of the H(C), recombinant genes for the H(C) and two hybrid H(C) in which one half of Okra/NT was replaced by the homologous half of 111/NT were constructed and expressed in Escherichia coli. The binding activity of the recombinant H(C) of 111/NT to the protein receptor synaptotagmin II, in the presence of ganglioside GT1b, was 4.2-fold less than Okra/NT, consistent with the corresponding two NTs. The use of hybrid H(C) revealed that mutation of 23 residues in carboxy terminal half of H(C) (1029-1291) of Okra/NT could be attributed to the lower binding activity of 111/NT and thus the differences in binding affinity between the two BoNT/B.     

The sensitivity of catecholamine release to botulinum toxin C1 and E suggests selective targeting of vesicles set into the readily releasable pool

The impact of syntaxin and SNAP-25 cleavage on [3H]noradrenaline ([3H]NA) and [3H]dopamine ([3H]DA) exocytotic release evoked by different stimuli was studied in superfused rat synaptosomes. The external Ca2+-dependent K+-induced [3H]catecholamine overflows were almost totally abolished by botulinum toxin C1 (BoNT/C1), which hydrolyses syntaxin and SNAP-25, or by botulinum toxin E (BoNT/E), selective for SNAP-25. BoNT/C1 cleaved 25% of total syntaxin and 40% of SNAP-25; BoNT/E cleaved 40% of SNAP-25 but left syntaxin intact. The GABA uptake-induced releases of [3H]NA and [3H]DA were differentially affected: both toxins blocked the former, dependent on external Ca2+, but not the latter, internal Ca2+-dependent. BoNT/C1 or BoNT/E only slightly reduced the ionomycin-evoked [3H]catecholamine release. More precisely, [3H]NA exocytosis induced by ionomycin was sensitive to toxins in the early phase of release but not later. The Ca2+-independent [3H]NA exocytosis evoked by hypertonic sucrose, thought to release from the readily releasable pool (RRP) of vesicles, was significantly reduced by BoNT/C1. Pre-treating synaptosomes with phorbol-12-myristate-13-acetate, to increase the RRP, enhanced the sensitivity to BoNT/C1 of [3H]NA release elicited by sucrose or ionomycin. Accordingly, cleavage of syntaxin was augmented by the phorbol-ester. To conclude, our results suggest that clostridial toxins selectively target exocytosis involving vesicles set into the RRP.     

Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855–1296 with antitoxin antibodies from three host species

Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced byClostridium botluinum in seven known immunological serotypes. These are the most potent toxins and poisons known. BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release. Human botulism is most frequently caused by types A, B, and E. Recent studies have shown that immunization with a 43-kDa C-terminal fragment (H_C, residues 860–1296) of BoNT/A affords excellent protection against BoNT/A poisoning. We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice. Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the H_C of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera. Horse Abs against BoBT/A were bound by peptides 855–873, 939–957, 1079–1097/1093–1111 overlap, 1191–1209/1205–1223 overlap, 1261–1279 and 1275–1296. In addition, peptides 883–901, 911–929, 995–1013, 1023–1041/1037–1055 overlap, 1121–1139, and 1149–1167 gave low, but significant and reproducible, binding. With human antisera, high amounts of Abs were bound by peptides 869–887, 925–943, 981–999, 995–1013, 1051–1069, and 1177–1195. In addition, lower amounts of Abs were bound by peptides 911–929, 939–957, 967–985, and the overlaps 1121–1139/1135–1153 and 1247–1265/1261–1279/1275–1296. With outbred mouse antisera, high amounts of Abs were bound by peptides 869–887, 1051–1069, and 1177–1195, while peptides 939–957, 995–1013, 1093–1111, and 1275–1296 bound lower amounts of Abs. The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera. Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.     

Differentiation of Clostridium botulinum Serotype A Strains by Multiple-Locus Variable-Number Tandem-Repeat Analysis

Ten variable-number tandem-repeat (VNTR) regions identified within the complete genomic sequence of Clostridium botulinum strain ATCC 3502 were used to characterize 59 C. botulinum strains of the botulism neurotoxin A1 (BoNT/A1) to BoNT/A4 (BoNT/A1-A4) subtypes to determine their ability to discriminate among the serotype A strains. Two strains representing each of the C. botulinum serotypes B to G, including five bivalent strains, and two strains of the closely related species Clostridium sporogenes were also tested. Amplified fragment length polymorphism analyses revealed the genetic diversity among the serotypes and the high degree of similarity among many of the BoNT/A1 strains. The 10 VNTR markers amplified fragments within all of the serotype A strains but were less successful with strains of other serotypes. The composite multiple-locus VNTR analysis of the 59 BoNT/A1-A4 strains and 3 bivalent B strains identified 38 different genotypes. Thirty genotypes were identified among the 53 BoNT/A1 and BoNT/A1(B) strains, demonstrating discrimination below the subtype level. Contaminating DNA within crude toxin preparations of three BoNT/A subtypes (BoNT/A1 to BoNT/A3) also supported amplification of all of the VNTR regions. These markers provide clinical and forensics laboratories with a rapid, highly discriminatory tool to distinguish among C. botulinum BoNT/A1 strains for investigations of botulism outbreaks.     

Minimal essential domains specifying toxicity of the light chains of tetanus toxin and botulinum neurotoxin type A.

To define conserved domains within the light (L) chains of clostridial neurotoxins, we determined the sequence of botulinum neurotoxin type B (BoNT/B) and aligned it with those of tetanus toxin (TeTx) and BoNT/A, BoNT/C1, BoNT/D, and BoNT/E. The L chains of BoNT/B and TeTx share 51.6% identical amino acid residues whereas the degree of identity to other clostridial neurotoxins does not exceed 36.5%. Each of the L chains contains a conserved motif, HExxHxxH, characteristic for metalloproteases. We then generated specific 5'- and 3'-deletion mutants of the L chain genes of TeTx and BoNT/A and tested the biological properties of the gene products by microinjection of the corresponding mRNAs into identified presynaptic cholinergic neurons of the buccal ganglia of Aplysia californica. Toxicity was determined by measurement of neurotransmitter release, as detected by depression of postsynaptic responses to presynaptic stimuli (Mochida, S., Poulain, B., Eisel, U., Binz, T., Kurazono, H., Niemann, H., and Tauc, L. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7844-7848). Our studies allow the following conclusions. 1) Residues Cys439 of TeTx and Cys430 of BoNT/A, both of which participate in the interchain disulfide bond, play no role in the toxification reaction. 2) Derivatives of TeTx that lacked either 8 amino- or 65 carboxyl-terminal residues are still toxic, whereas those lacking 10 amino- or 68 carboxyl-terminal residues are nontoxic. 3) For BoNT/A, toxicity could be demonstrated only in the presence of added nontoxic heavy (H) chain. A deletion of 8 amino-terminal or 32 carboxyl-terminal residues from the L chain had no effect on toxicity, whereas a removal of 10 amino-terminal or 57 carboxyl-terminal amino acids abolished toxicity. 4) The synergistic effect mediated by the H chain is linked to the carboxyl-terminal portion of the H chain, as demonstrated by injection of HC-specific mRNA into neurons containing the L chain. This finding suggests that the HC domain of the H chain becomes exposed to the cytosol during or after the putative translocation step of the L chain.     

Mapping of the Antibody-Binding Regions on the HN-Domain (Residues 449–859) of Botulinum Neurotoxin A with Antitoxin Antibodies from Four Host Species. Full Profile of the Continuous Antigenic Regions of the H-Chain of Botulinum Neurotoxin A

Previously, we mapped the antibody (Ab) and T-cell recognition regions on the HC domain (residues 855-1296) of the 848-residue heavy (H) chain of botulinum neurotoxin A (BoNT/A). We have mapped here the HN-domain (residues 449-859) regions that bind protective anti-BoNT/A Abs raised in four different species. We synthesized, purified, and characterized 29 19-residue peptides that spanned the entire HN and overlapped consecutively by 5 residues, and also region L218-231 around the L-chain's substrate-binding site. Human, horse, mouse, and chicken anti-BoNT/A Abs did not bind to the L-peptide but recognized similar HN regions within peptides 519-537/533-551/547-565/561-579 (with slight left- or right-shifts), 743-761, 785-803, and 813-831/827-845 overlap. Recognition of other peptides that bound lower Ab levels showed similarities and also some differences. Peptide 463-481, strongly immunodominant with horse antisera, did not bind human, mouse, and chicken Abs. However, peptide 449-467 bound Abs in these three antisera, and the region may have shifted to the right (peptide 463-481) with horse Abs. The overlap 659-677/673-691 reacted strongly with human Abs whereas with mouse and chicken antisera, only peptide 673-691 showed low reactivity. Horse antisera had no detectable Ab binding to region(s) 659-691. The Ab-recognition regions on the H chain occupy surface locations in BoNT/A three-dimensional structure, but the great part of the surface is not immunogenic. Regions recognized by the protective antisera of the four different species are prime candidates for inclusion in synthetic vaccine designs.     

Crystal Structure of the Botulinum Neurotoxin Type G Binding Domain: Insight into Cell Surface Binding

Botulinum neurotoxins (BoNTs) typically bind the neuronal cell surface via dual interactions with both protein receptors and gangliosides. We present here the 1.9-Å X-ray structure of the BoNT serotype G (BoNT/G) receptor binding domain (residues 868-1297) and a detailed view of protein receptor and ganglioside binding regions. The ganglioside binding motif (SxWY) has a conserved structure compared to the corresponding regions in BoNT serotype A and BoNT serotype B (BoNT/B), but several features of interactions with the hydrophilic face of the ganglioside are absent at the opposite side of the motif in the BoNT/G ganglioside binding cleft. This may significantly reduce the affinity between BoNT/G and gangliosides. BoNT/G and BoNT/B share the protein receptor synaptotagmin (Syt) I/II. The Syt binding site has a conserved hydrophobic plateau located centrally in the proposed protein receptor binding interface (Tyr1189, Phe1202, Ala1204, Pro1205, and Phe1212). Interestingly, only 5 of 14 residues that are important for binding between Syt-II and BoNT/B are conserved in BoNT/G, suggesting that the means by which BoNT/G and BoNT/B bind Syt diverges more than previously appreciated. Indeed, substitution of Syt-II Phe47 and Phe55 with alanine residues had little effect on the binding of BoNT/G, but strongly reduced the binding of BoNT/B. Furthermore, an extended solvent-exposed hydrophobic loop, located between the Syt binding site and the ganglioside binding cleft, may serve as a third membrane association and binding element to contribute to high-affinity binding to the neuronal membrane. While BoNT/G and BoNT/B are homologous to each other and both utilize Syt-I/Syt-II as their protein receptor, the precise means by which these two toxin serotypes bind to Syt appears surprisingly divergent.     

Exchange of the H(CC) domain mediating double receptor recognition improves the pharmacodynamic properties of botulinum neurotoxin

The four-domain structure of botulinum neurotoxins (BoNTs) reflects their multistep intoxication process. The high toxicity of BoNTs primarily results from specific binding and uptake into neurons mediated by their 50-kDa cell-binding fragment (H(C) ). X-ray crystallography data have revealed that the H(C) fragment consists of two domains of equal size, named the 25-kDa N-terminal half of H(C) (H(CN) ) and the 25-kDa C-terminal half of H(C) (H(CC) ). In recent years, the ganglioside-binding sites of all seven BoNT serotypes have been allocated to the H(CC) domain. For BoNT/A, BoNT/B and BoNT/G, the protein receptor-binding site has been also been localized to the H(CC) domain. Here, we demonstrate that the H(CC) serotype can modulate the affinity of the H(C) fragment for neuronal membranes as well as the potency of full-length BoNT by replacing the BoNT/A H(CC) domain with the BoNT/B H(CC) , BoNT/C H(CC) and BoNT/E H(CC) domains, which exhibit higher affinity for synaptosomes. Indeed, the hybrids H(C) AB and H(C) AC display a higher affinity than wild-type H(C) A. Furthermore, the potency of a BoNT/A-based full-length hybrid containing the H(CC) B domain (AAAB; letters represent the serotype origin of the four domains) was quadrupled as compared with wild-type BoNT/A. Analogously, exchange of the H(C) fragment (AABB) yielded a neurotoxin with four-fold higher potency. As BoNT/A and BoNT/B are extensively used to treat neurological disorders, thereby facing the problem of BoNT neutralizing antibody formation, a BoNT with increased potency would lower the repeatedly administered protein dosage while maintaining the clinical benefit. Such a lowered protein load will delay the onset of neurotoxin antibody formation in patients.     

Analysis of the neurotoxin complex genes in Clostridium botulinum A1-A4 and B1 strains: BoNT/A3, /Ba4 and /B1 clusters are located within plasmids

Background

Clostridium botulinum and related clostridial species express extremely potent neurotoxins known as botulinum neurotoxins (BoNTs) that cause long-lasting, potentially fatal intoxications in humans and other mammals. The amino acid variation within the BoNT is used to categorize the species into seven immunologically distinct BoNT serotypes (A–G) which are further divided into subtypes. The BoNTs are located within two generally conserved gene arrangements known as botulinum progenitor complexes which encode toxin-associated proteins involved in toxin stability and expression.

Methodology/Principal Findings

Because serotype A and B strains are responsible for the vast majority of human botulism cases worldwide, the location, arrangement and sequences of genes from eight different toxin complexes representing four different BoNT/A subtypes (BoNT/A1-Ba4) and one BoNT/B1 strain were examined. The bivalent Ba4 strain contained both the BoNT/A4 and BoNT/bvB toxin clusters. The arrangements of the BoNT/A3 and BoNT/A4 subtypes differed from the BoNT/A1 strains and were similar to those of BoNT/A2. However, unlike the BoNT/A2 subtype, the toxin complex genes of BoNT/A3 and BoNT/A4 were found within large plasmids and not within the chromosome. In the Ba4 strain, both BoNT toxin clusters (A4 and bivalent B) were located within the same 270 kb plasmid, separated by 97 kb. Complete genomic sequencing of the BoNT/B1 strain also revealed that its toxin complex genes were located within a 149 kb plasmid and the BoNT/A3 complex is within a 267 kb plasmid.

Conclusions/Significance

Despite their size differences and the BoNT genes they contain, the three plasmids containing these toxin cluster genes share significant sequence identity. The presence of partial insertion sequence (IS) elements, evidence of recombination/gene duplication events, and the discovery of the BoNT/A3, BoNT/Ba4 and BoNT/B1 toxin complex genes within plasmids illustrate the different mechanisms by which these genes move among diverse genetic backgrounds of C. botulinum.     

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.