Article |
Address correspondence to E.R. Chapman, Dept. of Physiology, SMI 129, University of Wisconsin, Madison, 1300 University Ave., Madison, WI 53706. Tel.: (608) 263-1762. Fax: (608) 265-5512. email: chapman{at}physiology.wisc.edu
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Abstract |
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Key Words: synaptotagmin; synaptobrevin; clostridial neurotoxin; neurotoxin receptor; gangliosides
Abbreviations used in this paper: BoNT, botulinum neurotoxin; CNT, clostridial neurotoxin; syb, synaptobrevin; syt, synaptotagmin; TeNT, tetanus neurotoxin.
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Introduction |
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The first step in the action of CNTs involves binding to receptors on the surface of neurons. Current evidence suggests that the receptors are composed of gangliosides and proteins that cooperate to form high affinity toxin-binding sites. Alternatively, gangliosides may constitute relatively low affinity toxin-binding sites that serve to capture CNTs to facilitate interactions with cell surface receptor proteins (Montecucco, 1986; Nishiki et al., 1996a). Gangliosides are ubiquitous glycosphingolipids in the outer leaflet of plasma membranes. They are classified according to the number and position of sialic acids present in their head groups. Polysialiogangliosides, which are present almost exclusively in neurons and neuroendocrine cells, bind to CNTs with the greatest avidity (Halpern and Neale, 1995). Although a protein component is also clearly involved in toxincell recognition, at present, a protein that mediates toxin entry has not been identified (Schiavo et al., 2000).
Biochemical experiments have led to the identification of a handful of CNT-binding proteins. In most cases, these binding proteins do not appear to function as receptors that mediate entry of the toxins. For example, BoNT/A, B, E, and TeNT were reported to bind synapsin I and adducin, respectively (Schengrund et al., 1992, 1993, 1996). Because neither of these proteins are exposed to the outside surface of cells, they are unlikely to function as cell surface receptors. TeNT was reported to bind Thy-1, a GPI-anchored plasma membrane protein. However, neurons from mice lacking Thy-1 are still sensitive to TeNT, suggesting that Thy-1 is not essential for TeNT entry into cells (Herreros et al., 2001).
At present, the only remaining candidate receptor proteins for BoNT/B are synaptotagmins (syts) I and II (Nishiki et al., 1994). These are homologous synaptic vesicle membrane proteins thought to function as Ca2+ sensors for exocytosis (Schiavo et al., 1998; Chapman, 2002). Syts I and II were reported to bind BoNT/B in the presence of gangliosides; the dissociation constant for the syt IBoNT/B complex was 2.3 nM and the dissociation constant for syt IIBoNT/B was 0.23 nM (Nishiki et al., 1996a). High affinity binding of BoNT/B to fibroblasts was reconstituted by expression of syt II and incorporation of exogenous gangliosides into surface membranes. However, binding did not result in the cleavage of the BoNT/B target protein, syb II, that had been coexpressed with syt II, indicating that the toxin was not internalized (Nishiki et al., 1996b). Although biochemical experiments clearly established that syt binds to BoNT/B, evidence that binding mediates entry into cells is lacking. Thus, whether this interaction has any functional role remains unknown. More recently, BoNT/A and E have also been reported to bind syt I, albeit in a ganglioside-independent manner (Li and Singh, 1998).
In the current work, we characterized the interactions between BoNT/B, A, and E with syts I and II, and have investigated whether syts I and II function as receptors for BoNT/B. Our data demonstrate that syts I and II mediate BoNT/B entry into PC12 cells and motor nerve terminals. Moreover, the action of BoNT/B on these model systems in vitro and in mice in vivo can be neutralized by syt II fragments that contain the toxin-binding domain.
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Results |
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If syts I and II are physiologically relevant receptors for BoNT/B, binding must be mediated by the region of syt that is exposed outside of cells (i.e., the luminal domain) during cycles of exocytosis and endocytosis. To clarify how BoNT/B binds to syt II, we first used syt II and syt IX chimeras. Swapping the luminal domains of these proteins was sufficient to transfer the BoNT/B-binding activity from syt II to syt IX (Fig. 1 B), indicating that BoNT/B binding is mediated by the luminal domain of syt II. Consistent with this finding, a shorter fragment of syt II, composed of only the luminal and transmembrane domain (residues 187), mediated stoichiometric binding of the toxin (Fig. 1 C).
Truncation analysis was used to further map the toxin-binding site of syts I and II. Within the luminal domain of syt II, residues 4060, which are adjacent to the transmembrane domain, are critical for toxin binding (Fig. 2 A). We note that fragment 61267 of syt II can bind gangliosides via its transmembrane domain (residues 6187; Kozaki et al., 1998), yet this fragment fails to bind BoNT/B in the presence of gangliosides (Fig. 2 A, middle). These data suggest that gangliosides do not directly mediate toxin binding under our assay conditions, but rather cooperate with the luminal domain to form high affinity BoNT/B-binding sites. The analogous membrane proximal region of syt I (residues 3252) was also critical for binding of BoNT/B (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200305098/DC1). This segment is highly conserved between syts I and II (Fig. 2 B); minor sequence differences may account for the differences in affinity for BoNT/B (Nishiki et al., 1996a). We note that the isolated luminal domain of syt II (residues 161) but not syt I (residues 153), bound to BoNT/B (Fig. 2 A and Fig. S1). This result is likely due to the strong ganglioside requirements for syt IBoNT/B interactions; deletion of the transmembrane domain of syt I abolishes ganglioside binding (Kozaki et al., 1998), and thereby decreases BoNT/B-binding activity.
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Syt I mediates ganglioside-dependent binding and entry of BoNT/B into PC12 cells
The experiments described above demonstrate that syts I and II bind to BoNT/B through a conserved region in their ectodomains. However, the key question is whether syt I or syt II mediate entry of the toxin; namely, are they functional protein receptors for BoNT/B? To address this question, we first used PC12 cells, a neuroendocrine cell line that serves as a model system to study Ca2+-triggered exocytosis. These cells express the substrates for all of the CNTs, but are resistant to entry of BoNT/B, probably due to lack of functional toxin receptors (Lomneth et al., 1991). PC12 cells express syt I and IX and trace levels of syt IV (Zhang et al., 2002). Because syts IX and IV do not bind BoNT/B and syt I binds only in the presence of gangliosides (Fig. 1 A, middle), toxin resistance could be due to the fact that these cells contain low levels of gangliosides as compared with neurons (Walton et al., 1988). We tested this idea by preloading exogenous gangliosides into the plasma membrane of wild-type PC12 cells. As shown in Fig. 3 A, detectable toxin binding was observed only when cells were loaded with gangliosides. Then, we determined whether the toxin can enter ganglioside-treated cells. To monitor entry, we assayed for cleavage of the cytoplasmic substrate of BoNT/B, syb II (Schiavo et al., 1992), by immunoblot analysis using anti-syb II antibodies. Cleavage of syb II by BoNT/B occurred only when cells were first preloaded with gangliosides (Fig. 3 B). These data are consistent with a model in which syt I and gangliosides cooperate to mediate the binding and entry of BoNT/B, and are in agreement with biochemical data showing that the toxin binds to syt I only in the presence of gangliosides. To further test this model, we took advantage of a PC12 cell line that lacks syt I (Syt I-; Shoji-Kasai et al., 1992). This cell line is still capable of Ca2+-triggered exocytosis, presumably via the redundant action of syt IX (Fukuda et al., 2001; Zhang et al., 2002). As shown in Fig. 3 C, BoNT/B failed to cleave syb II in ganglioside-loaded syt I- PC12 cells. These data indicate that gangliosides plus syt I are both needed for toxin entry.
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Syt II is sufficient to mediate entry of BoNT/B into PC12 cells
To determine directly whether syt II can function as a receptor for BoNT/B, we took advantage of the observation that this syt isoform is able to bind BoNT/B to some extent in the absence of gangliosides (Fig. 1 A). We generated PC12 cell lines that stably express syt II (Syt II+; Fig. 4 A), and observed that they bind BoNT/B without preloading cells with exogenous gangliosides (Fig. 4 B). A key finding was that expression of syt II was sufficient to reconstitute toxin entry into the transfected cells, as shown by the cleavage of syb II (Fig. 4 C). The efficiency of cleavage was proportional to the level of syt II expression, and cleavage was not observed in cells lacking syt II (Fig. 4 C, right). These findings demonstrate that syt II can function as a receptor for BoNT/B without preloading cells with exogenous gangliosides.
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Activity-dependent entry of BoNT/B into vesicles that contain syt I
The data above suggest a model in which BoNT/B gains entry into PC12 cells by binding to the luminal domain of syt I or syt II. This model predicts that BoNT/B will follow the internalization of syts I and II from the cell surface and that internalization will be activity dependent. To answer this question, we first demonstrated that an anti-syt I luminal domain antibody (-syt IN) and BoNT/B can bind to syt I simultaneously (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200305098/DC1). This is the expected result because the antibody recognizes the first 12 amino acids at the NH2 terminus of syt I, whereas the BoNT/B-binding site lies at the COOH-terminal end of the luminal domain.
We took advantage of this finding and determined whether the antibody and toxin are taken up into the same compartment in response to stimulation. PC12 cells were preloaded with gangliosides and depolarized with high [K+] to induce exocytosis of secretory vesicles in the presence of -syt IN antibodies and BoNT/B. Exocytosis and endocytosis were allowed to proceed for 10 min, followed by extensive washes to remove surface-bound antibody and toxin. As shown in a confocal section (Fig. 6), both
-syt IN antibodies and BoNT/B were internalized into the same compartment. Depolarization of cells significantly increased the internalization of both the antibody and BoNT/B; only low levels of internalization, due to spontaneous exocytosis and recycling, were observed in the control (Fig. 6 A).
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Similar results were obtained using syt II+ PC12 cells; BoNT/B entered syt I containing vesicles in an activity-dependent manner (unpublished data). We have been unable to localize syt II in the syt II+ cell lines using antibodies currently available. However, syt II is colocalized with syt I on secretory vesicles in brain, and is likely to be targeted to syt I-containing organelles in PC12 cells (Osborne et al., 1999).
Activity-dependent uptake of BoNT/B into motor nerve terminals
The cause of death from BoNT/B intoxication is asphyxiation due to blockade of neurotransmission at the diaphragm. Therefore, we extended our analyses to explore the mechanism of toxin entry into neurons in this tissue. As shown in Fig. 7 A, stimulation with KCl results in a dramatic increase in the levels of BoNT/B associated with the neuromuscular junction (5.7-fold; Fig. 7 B), and a concomitant loss of syb II immunoreactivity (3.2-fold; Fig. 7 C). The increase in binding of BoNT/B and the loss of syb II were virtually abolished by incubation with the syt II fragment 1267/gangliosides, but not by a mixture of syt II 61267/gangliosides (Fig. 7, AC). These data demonstrate that uptake of BoNT/B is activity dependent at its natural target. Moreover, binding and entry of the toxin can be completely prevented by syt II fragments that contain the toxin-binding site, whereas syt fragments lacking the toxin-binding site have no effect.
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Syt II 61267 plus gangliosides did not neutralize the toxin (Fig. 8 B), further establishing the essential role of the luminal domain of syt II for toxin entry in vivo. The potencies of syt II 1267 and 187 were determined (Fig. 8 C); both fragments yielded dose-dependent protection at sub-µM concentrations. Finally, prior i.v. injection with syt II 1267 or 187, mixed with gangliosides, neutralized 7080% of BoNT/B that was injected 1 min later (Fig. 8 D), indicating that animals can be protected before exposure to toxin. Together, these results support the idea that the physiological receptor for BoNT/B is composed of syt II and gangliosides.
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Discussion |
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First, we confirmed that BoNT/B binds to syts I and II. This interaction is stoichiometric, highly specific, facilitated by gangliosides, and is mediated by a region of syt that is transiently exposed outside of cells during exocytosis. Using PC12 cells, which are resistant to BoNT/B, we performed experiments to reconstitute entry. These cells have low levels of gangliosides, but express syt I. We found that BoNT/B is able to enter PC12 cells if their plasma membranes are preloaded with sufficient levels of gangliosides. Moreover, entry into ganglioside-loaded cells was strictly dependent on the expression of syt I; entry was abolished when syt I expression was disrupted. Next, we took advantage of our observation that, in contrast to syt I, syt II exhibits significant levels of toxin-binding activity in the absence of gangliosides. Expression of syt II in PC12 cells resulted in the binding and entry of the toxin into cells and overcame the need for preloading the cells with gangliosides. In summary, both loss-of-function (syt I-) and gain-of-function experiments (syt II+) are consistent with a model in which BoNT/B enters cells via interactions with syt I or syt II.
If syt mediates binding and entry of BoNT/B, then syt ectodomains, which contain the toxin-binding site, should block the action of the toxin. Using syt II+ PC12 cells, we observed that was indeed the case; fragments of syt II effectively inhibited the binding of BoNT/B to the surface of cells, and prevented the cleavage of syb II.
We studied the pathway of BoNT/B internalization into PC12 cells using an antibody directed against the NH2 terminus of syt I. We found that BoNT/B was co-internalized with the antibody in an activity-dependent manner. These results further support our conclusion that syts I and II are functional receptors that mediate BoNT/B entry through recycling of secretory vesicles. This is also likely to be the route of entry of BoNT/B into neurons. In neurons, syts I and II reside on synaptic vesicles where their luminal domains are exposed during cycles of exo-endocytosis (Matteoli et al., 1992; Juzans et al., 1996), providing a means of entry. It is known that cholinergic neurons at the neuromuscular junction express syt II and are the major physiological targets of BoNT/B (Dolly et al., 1984; Juzans et al., 1996). We explored this issue using motor neurons that innervate the diaphragm. Our data unambiguously demonstrate that binding and uptake of BoNT/B is activity dependent and can be blocked by syt fragments in conjunction with gangliosides at the neuromuscular junction (Fig. 7). These results indicate that the mechanism of toxin entry is similar between PC12 cells and motor neurons. Finally, these observations were extended to a whole animal study where we found that syt fragment/ganglioside mixtures neutralized the toxin in mice.
Our model also indicates that syt I is a less effective receptor, but at high local ganglioside concentrations, syt I may also mediate entry of BoNT/B into neurons that lack syt II. Therefore, the sensitivity of a particular neuron to BoNT/B might depend on the local levels of gangliosides and whether it expresses syt I or syt II.
All of our BoNT/B data are consistent with the double-receptor model for CNTs, as proposed more than 15 years ago (Montecucco, 1986). First, gangliosides facilitate sytBoNT/B interactions (Fig. 1 A; Nishiki et al., 1996a; Kozaki et al., 1998); second, preloading cells with gangliosides is required for syt I-mediated toxin entry into wild-type PC12 cells (Fig. 3 B); third, preloading cells with gangliosides can facilitate toxin entry that is mediated by syt II (unpublished data); and fourth, gangliosides are required for syt ectodomains to protect mice from BoNT/B (Fig. 8 B). This hypothesis is further supported by the finding that disruption of ganglioside synthesis in mice reduces their sensitivity to CNTs (Kitamura et al., 1999; Bullens et al., 2002).
The simplest explanation for the fact that syt IIganglioside complexes efficiently block BoNT/B action in PC12 cells, intact hemi-diaphragm preparations, and mice is that syt II plus gangliosides functions as the toxin receptor in vivo. The in vivo experiments establish the physiological significance of our analyses and provide a proof-of-principle that receptor ectodomains might provide a novel means to antagonize the action of bacterial toxins in animals.
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Materials and methods |
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Antibodies
mAbs directed against syb II (69.1), syt I (-syt IN, 604.4;
-syt IC, 41.1),
/ß-SNAP (77.1), and SNAP-25 (71.2) were provided by R. Jahn and S. Engers (Max Planck Institute for Biophysical Chemistry, Gottingen, Germany). Rabbit pAbs directed against syt II were provided by M. Fukuda (RIKEN, Saitama, Japan; Fukuda and Mikoshiba, 2000).
Anti-BoNT/A, B, and E antibodies were generated by immunizing rabbits with formalin-treated purified neurotoxin; antibodies were affinity purified using immobilized neurotoxin.
cDNA and recombinant proteins
cDNA encoding rat syt I (Perin et al., 1990), mouse syt II and IX (Fukuda and Mikoshiba, 2000), and rat syt IV (Vician et al., 1995) were provided by T.C. Sudhof (University of Texas Southwestern Medical Center, Dallas, TX), M. Fukuda, and H. Herschman (University of California, Los Angeles, CA), respectively. Full-length syb II was generated as a GST fusion protein as described previously (Lewis et al., 2001) using a cDNA provided by R. Scheller (Stanford University, Stanford, CA).
To screen for toxin-binding activity, we generated truncated versions of syt I, II, IV, and IX that lacked the C2B domain, but contained all other domains. A number of additional constructs (truncations and chimeras, as indicated in the figures) were also generated by PCR, subcloned into pGEX-2T, and expressed and purified as described previously (Chapman et al., 1996; Lewis et al., 2001). Syt II 1267 and 61267 were also subcloned into pTrcHis and purified as NH2-terminal tagged His6 fusion proteins as described previously (Chapman et al., 1996).
Pull-down assays
Recombinant proteins were immobilized as GST fusion proteins bound to glutathione-Sepharose beads. Unless otherwise indicated, 10 µg immobilized protein was mixed with the indicated concentrations of BoNT/B, A, or E either with (+; 25 µg/ml) or without (-) gangliosides in 100 µl TBS (20 mM Tris and 150 mM NaCl, pH 7.4) plus 0.5% Triton X-100 for 1 h at 4°C. Beads were washed three times, bound proteins were solubilized by boiling in SDS sample buffer and subjected to SDS-PAGE, and were visualized by staining with Coomassie blue or by immunoblot analysis using anti-toxin antibodies. In all blots, the immunoreactivity for the toxin heavy chain is shown.
A peptide corresponding to residues 4060 of mouse syt II, P21, and a scrambled version of this peptide, P21S (IKMNDAEFFGKSNFQEKLEKEC), were synthesized (Biotech Center, University of Wisconsin, Madison, Madison, WI) with an added COOH-terminal cys that was used to conjugate them to agarose beads (at 1 mg/ml) using a Sulfolink Kit (Pierce Chemical Co.). 50 µl of the conjugated agarose gel was used in the pull-down assays.
PC12 cell lines and immunoblot analysis
To generate cells that express syt II (syt II+), full-length mouse syt II was subcloned into pCDNA3.1(-) (CLONTECH Laboratories, Inc.) and transfected into PC12 cells via electroporation. Transfected cells were selected with 1 mg/ml G418, and several independent monoclonal cell lines were established. Cells were harvested in PBS plus 0.5% Triton X-100, 0.05% SDS, and 5 mM PMSF, and were incubated for 30 min at 4°C on a shaker. Samples were centrifuged at 21,000 g for 10 min, and the concentration of protein in the supernatant was determined using bicinchoninic acid (Pierce Chemical Co.). Samples were subjected to SDS-PAGE and immunoblot analysis; blots were developed using ECL (Pierce Chemical Co.).
Entry of BoNTs into PC12 cells
In experiments that did not involve preloading, cells were grown to 70% confluence and incubated with BoNT for 48 h. For experiment in which cells were preloaded with gangliosides, cells were grown to 80% confluence followed by incubation in serum-free media plus 250 µg/ml gangliosides. 24 h later, the serum-free/ganglioside media was replaced with complete media, and the cells were incubated with toxin for 48 h. Cells were harvested and entry of CNTs was assayed via immunoblot analysis using antibodies directed against syb II or SNAP-25.
For blocking experiments, syt II 1267 and syt II 61267 were generated as His6 fusion proteins; syt II 187 was generated as a GST fusion protein that was eluted from beads using 10 mM glutathione plus 0.5% Triton X-100. Protein fragments or peptides were premixed with BoNT/B in 200 µl TBS for 1 h at 4°C before adding into 2 ml cell culture media (per well in a 6-well plate). In some cases, gangliosides were also added in the binding buffer (Fig. 5 B, bottom). The final concentration of BoNT/B was 30 nM, the final concentration of gangliosides was 25 µg/ml, and the final [syt fragment] is indicated in the figure legends.
Binding of BoNT/B to PC12 cells
Cells treated with toxin, plus or minus preincubation with syt fragments, were washed three times with PBS, fixed with 4% PFA (15 min at RT), permeabilized with 0.1% Triton X-100 (for 10 min at RT), and stained with a rabbit anti-BoNT/B primary antibody and an FITC-conjugated goat antirabbit secondary antibody (Jackson ImmunoResearch Laboratories). In the syt II fragment competition assays described in Fig. 5, syt II 1267 and 61267 fragments form aggregates bound to cells (Bai et al., 2000); these were visualized using a mouse anti-His6 primary antibody (QIAGEN) and a rhodamine-conjugated goat antimouse secondary antibody (Jackson ImmunoResearch Laboratories). The fluorescence images were obtained as described for the motor nerve terminal experiments. We note that for these experiments, free detergent was removed from the recombinant syt fragments by washing the immobilized proteins with detergent-free buffers before elution. However, in the case of syt II 187, low levels of Triton X-100 were needed to elute the protein from the beads; because of this, experiments using this fragment were performed within 6 h to avoid effects of detergent on the cells.
Antibody and toxin uptake experiments
Cells were treated with either control solution (15 mM Hepes, 145 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, 0.5 mM ascorbic acid, and 0.1% BSA, pH 7.4), or high [K+] solution (same as control solution but adjusted to 95 mM NaCl and 56 mM KCl), for 10 min at 37°C, in the presence of BoNT/B plus 10 µl mAb against the luminal (-syt IN; clone 604.4) or cytoplasmic domain of syt I (
-syt 1c; clone 41.1). Cells were washed with culture media, incubated for 30 min at 37°C, fixed, and permeabilized. A rabbit anti-BoNT/B primary antibody was used to stain BoNT/B; staining was visualized using an FITC-conjugated goat antirabbit secondary antibody. Rhodamine-conjugated goat antimouse secondary antibodies were used to visualize internalized syt I antibodies. Confocal images were collected with a confocal microscope (MRC 1000; Bio-Rad Laboratories; Keck Center for Biological Imaging, University of Wisconsin, Madison, Madison, WI) using a 100x oil immersion objective.
Co-immunoprecipitation
Recombinant syt I 1265 GST was purified as described earlier in the Materials and methods section and was and cleaved from the GST tag using thrombin. 5 µl of mAb -syt IN (604.4) was incubated with 300 nM BoNT/B, with or without 1.5 µM Syt I 1265, in 100 µl TBS plus 0.5% Triton X-100 and 25 µg/ml gangliosides, for 1 h at 4°C. 30 µl Protein G fast flow beads (Amersham Biosciences) was added, samples were mixed for 1 h, beads were washed three times in binding buffer, and bound material was analyzed by SDS-PAGE and immunoblotting using an anti-BoNT/B pAb and
syt IN (604.4).
Rat hemi-diaphragm experiments
Rat hemi-diaphragms were placed in an ice-cold ringer (in mM: NaCl 138.8, KCl 4, NaHCO3 12, KH2PO4 1, MgCl2 2, CaCl2 2, and glucose 11), gassed with 95% CO2/5% O2. Stimulation was performed with a similar solution where [KCl] was increased to 45 mM, and the NaCl appropriately reduced. Hemi-diaphragms were incubated with high potassium ringer containing 5 nM BoNT/B for 10 min at RT. In some experiments, the BoNT/B was premixed with either the syt II fragment 1267, or fragment 61267, both mixed with 25 µg/ml gangliosides. At the end of the stimulation/incubation period, the preparations were fixed (4% PFA), permeabilized (0.3% Triton X-100), and blocked in goat serum before immunolabeling with a rabbit anti-BoNT/B antibody and a monoclonal anti-Syb II antibody. Immunofluorescence was visualized using a FITC-conjugated antirabbit antibody, and a TRITC-conjugated antimouse antibody. A region of muscle adjacent to the site of nerve entry (where a large number of surface nerve terminals are to be found) was placed in a viewing chamber with a glass bottom comprising a single coverslip. Immunofluorescence images were obtained using a microscope (model TE300; Nikon), with a cooled CCD camera (MicroMAX; Princeton Instruments) controlled by MetaMorph® software (Universal Imaging Corp.). Fluorescence intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
Neutralization of BoNT/B activity in vivo
For each batch of BoNT/B, the LD50 value for mice (2022 g; Institute of Cancer Research strain; ICR, Philadelphia, PA) was determined using standard methods (Schantz and Kautter, 1978). The LD50 corresponds to the amount of toxin, introduced via i.p. injection, that results in 50% death after 4 d. Our preparations of BoNT/B had activities of 108 LD50/mg. For toxin neutralization experiments, we made use of the more rapid i.v. time-to-death assay (Boroff and Fleck, 1966). First, we generated a standard curve in which the relationship between time-to-death of mice injected, i.v., with 100 µl BoNT/B (expressed in min) is plotted versus the specific toxicity of BoNT/B that was determined using the standard method described earlier in the Materials and methods section (log [LD50/ml]). Within the linear range, 104106 LD50/ml, this plot was used to convert experimentally determined time-to-death, from i.v. injection of relatively large doses of toxin, to LD50/ml values. For the toxin neutralization experiments, BoNT/B was premixed with gangliosides alone (250 µg/ml) or gangliosides plus the indicated syt II fragments for 10 min at RT and then injected i.v. into mice. In all experiments, the total injection volume was always 100 µl. Neutralization of the toxin is indicated by an extension in the time-to-death of mice injected with toxin alone versus injection with toxin that had been pre-mixed wit syt fragments/gangliosides. The increase in the time-to-death was converted into a decrease in the apparent [LD50/ml] using the standard curve, and the percentage of neutralization was calculated using the expression: 1- [LD50/ml (+ syt II fragment)/LD50/ml (- syt II fragment)] x 100, where (+ syt II fragment) refers to samples that contain toxin, gangliosides, and recombinant proteins, and (- syt II fragment) samples were composed of toxin and gangliosides only.
Online supplemental material
Fig. S1 shows the mapping of the BoNT/B-binding site within the luminal domain of syt I. Fig. S2 demonstrates the simultaneous and specific internalization of syt I luminal domain antibodies and BoNT/B into PC12 cells. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200305098/DC1.
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Acknowledgments |
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This work was supported by grants from the National Institutes of Health (National Institute of General Medical Sciences grant GM 56827 and National Institute of Mental Health grant MH61876 to E.R. Chapman, and grant AI42226 to E.A. Johnson), the Milwaukee Foundation (to E.R. Chapman), and by industrial sponsors of the Food Research Institute (to E.A. Johnson). E.R. Chapman is a Pew Scholar in the Biomedical Sciences.
Submitted: 20 May 2003
Accepted: 4 August 2003
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