The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn2+ from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn2+. In contrast to soluble chelators, immobilized chelators have no effect on bound Zn2+, nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn2+ from its coordination site in botulinum toxin is relatively slow. When exogenous Zn2+ is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn2+ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn2+ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn2+ or exogenous Zn2+ means that productive internalization does not produce conformational changes that block rebinding of cation. Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn2+ from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn2+. In contrast to soluble chelators, immobilized chelators have no effect on bound Zn2+, nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn2+ from its coordination site in botulinum toxin is relatively slow. When exogenous Zn2+ is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn2+ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn2+ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn2+ or exogenous Zn2+ means that productive internalization does not produce conformational changes that block rebinding of cation. tetrakis-(2-pyridylmethyl) ethylenediamine glutathioneS-transferase synaptosomal protein of 25 kDa botulinum neurotoxin serotype A Botulinum toxin poisons vulnerable cells by proceeding through a sequence of three major steps: binding, productive internalization, and intracellular expression of catalytic activity (1Humeau Y. Doussau F. Grant N.J. Poulain B. Biochimie (Paris). 2000; 85: 427-446Crossref Scopus (388) Google Scholar). Each of the three major steps in toxin action can be further subdivided into two or more events. Thus, the binding step reportedly involves two sequential processes. During the first, toxin associates with low affinity receptors on the cell surface, and during the second, the low affinity complex shuttles the toxin to a high affinity receptor (2Montecucco C. Trends Biochem. Sci. 1986; 11: 315-317Abstract Full Text PDF Scopus (349) Google Scholar). Productive internalization also involves a number of events (see “Discussion”), with the two major ones being receptor-mediated endocytosis across the plasma membrane and pH-dependent translocation across the endosome membrane. During the final step, botulinum toxin acts as a zinc-dependent endoprotease to cleave polypeptides that govern exocytosis (3Montecucco C. Schiavo G. Q. Rev. Biophys. 1995; 28: 423-472Crossref PubMed Scopus (409) Google Scholar). Enzymatic cleavage of these polypeptides must also involve multiple events (viz. substrate binding, substrate cleavage), although this has not been studied extensively. During the interval since toxin action was first described as a sequence of three steps (4Simpson L.L. J. Pharmacol. Exp. Ther. 1980; 212: 16-21PubMed Google Scholar, 5Simpson L.L. Pharmacol. Rev. 1981; 33: 155-188PubMed Google Scholar), investigators have tended to examine these steps as though they are separate and independent. However, recent and apparently contradictory findings may necessitate a change in perspective. In 1992, Schiavo et al. (6Schiavo G. Benfanti F. Poulain B. Rossetto O. Polverino de Laureto P. DasGupta B. Montecucco C. Nature. 1992; 359: 832-835Crossref PubMed Scopus (1495) Google Scholar) and Linket al. (7Link E. Edelmann L. Chou J.H. Binz T. Yamasaki S. Eisel U. Baumert M. Sudhof T.C. Niemann H. Jahn R. Biochem. Biophys. Res. Commun. 1992; 189: 1017-1023Crossref PubMed Scopus (268) Google Scholar) reported that botulinum toxin type B and tetanus toxin were zinc-dependent endoproteases. In 1993, Simpsonet al. (8Simpson L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Ther. 1993; 267: 720-727PubMed Google Scholar) reported that all seven botulinum toxin serotypes (A–G) were likely to be zinc-dependent endoproteases, because the toxicity of all seven serotypes was diminished by zinc chelation. However, the work with Zn2+ chelators revealed an unexpected outcome. It was observed that removing Zn2+from toxin was necessary but not sufficient to cause a loss of toxicity against intact cells. Toxin stripped of Zn2+ could still produce blockade of exocytosis, apparently because the toxin could replenish its divalent cation from tissue stores. In keeping with this idea, the chelation of toxin as well as tissue was necessary to produce marked reductions in toxin activity. More recently, Fu et al. (9Fu F.-N. Lomneth R.B. Cai S. Singh B.R. Biochemistry. 1998; 37: 5267-5278Crossref PubMed Scopus (64) Google Scholar) have published findings that seem to contradict those just described. They found that the use of a chelator to remove Zn2+ from toxin led to irreversible changes in tertiary structure as measured by various light-scattering techniques. They also found that removal of the cation led to irreversible losses in toxicity as measured by norepinephrine release from permeabilized PC-12 cells. Close inspection of these contradictory findings on Zn2+reveals that there could be implications that extend beyond the final or catalytic step in toxin action. These data may also have an impact on proposed models for productive internalization and particularly on models for toxin penetration of the endosome membrane (see “Discussion”). Thus, any model that calls for a significant relaxation of the toxin molecule, which in turn could cause the loss of Zn2+ binding, would not be plausible unless the toxin could subsequently regain Zn2+ and biological activity. Conversely, any model that calls for toxin to remain tightly bound to Zn2+ during translocation would not be viable unless the translocation process could accommodate a somewhat bulky protein that retains at least some of its secondary and tertiary structure. In the work that follows, both experimental studies and deduction have been used to gauge the likelihood that removal of Zn2+produces irreversible losses in botulinum toxin activity. This work then has been used to assess whether events that occur during the internalization step can impact events during the catalytic step. 65ZnCl2 (6,146.68 MBq/mg) was purchased from PerkinElmer Life Sciences. Sephadex G-25 gel filtration columns were obtained from Amersham Pharmacia Biotech. Chelex® 100 Resin was purchased from Bio-Rad. EGTA and EDTA disodium salts were from Sigma, and TPEN1 was purchased from Molecular Probes. Rabbit anti-C-terminal SNAP-25 polyclonal antibody was purchased from StressGen Biotechnologies Corp. (Victoria, B.C., Canada), and donkey anti-rabbit IgG horseradish peroxidase was fromAmersham Pharmacia Biotech. All other reagents were from Sigma or Fisher Scientific (Malvern, PA). Botulinum neurotoxin type A was purified to homogeneity by procedures that have been described previously (10Sakaguchi G. Pharmacol. Ther. 1982; 19: 165-194Crossref PubMed Scopus (300) Google Scholar, 11DasGupta B.R. Sathyamoorthy V. Toxicon. 1984; 22: 415-424Crossref PubMed Scopus (127) Google Scholar, 12Simpson L.L. Schmidt J.J. Middlebrook J.L. Methods Enzymol. 1988; 165: 76-85Crossref PubMed Scopus (23) Google Scholar). Recombinant GST-SNAP-25 (∼50 kDa) was constructed and expressed according to published techniques. Murine SNAP-25 cDNA, pSNAP8.52, which was derived from a BALB/c strain brain Lambda cDNA library (13Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billingsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (704) Google Scholar) kindly provided by Dr. Michael C. Wilson (Scripps Clinic, La Jolla, CA), was used as a polymerase chain reaction template. The polymerase chain reaction was carried out with primers 5′-TCT TGG ATC CGC CGA AGA CGC AGA CAT GC-3′ and 5′-TCT TGG ATC CTT AAC CAC TTC CCA GCA TCT T-3′. A product of ∼880 base pairs was digested with BamHI and ligated into pGEX-KG vector Biochem. PubMed Scopus Google Scholar). The was to that SNAP-25 cDNA been into pGEX-KG The expression of in and of GST-SNAP-25 protein were as described T. Y. G. Biol. 1998; PubMed Scopus Google Scholar). All were that were with to remove of as Zn2+. on that of Chelex® 100 of remove all from The used in these were with a of for at and this was by filtration through a to remove the to all and were three with all experimental described with toxin was Sephadex G-25 by were by with a and experimental of toxin were through Sephadex G-25 and were The were added to of and the of in the was by toxin at and the in the was to the of with the toxin Zn2+ of toxin was carried out in two toxin was added to EDTA and EGTA and at for the toxin was at of two against this EDTA and the toxin was into that been with Chelex® 100 for Stripped toxin was at in that been three with and it was used a of Zn2+ final and final were in a reaction in Chelex® and at for Zn2+ final final were in a reaction in Chelex® and at for and with the toxin was as described final was with final for at in reaction The with toxin was by the to and subsequently of Chelex® 100 EDTA final or final to reaction used toxin added were at for of (see which the was final was with final as described The with toxin was by to and the reaction to a of or by the of the of at were at for the with the toxin was tissue was from and and in and The was with a at a of for The were at for The was at for and The was at for The was in The protein of was a protein was for endoprotease activity either GST-SNAP-25 or synaptosomal In both an anti-C-terminal SNAP-25 antibody was used as a in the of activity M. J. P. C. M. M. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). When substrate was of GST-SNAP-25 was in reaction when were of protein was added to In the latter was added to the reaction to and were by with at for in cleavage When the were with stripped toxin or chelators, Zn2+ was not added to the In substrate was added to toxin, and the reaction was to proceed for at Enzymatic cleavage were by an of and The were by at with and then in according to Nature. PubMed Scopus Google Scholar). SNAP-25 was from to and Fisher and with antibody at anti-rabbit IgG with horseradish peroxidase was used as a secondary antibody at a of The antibody reaction was by Amersham Pharmacia were from as described previously J.A. Bakry N. J. L.L. J. Pharmacol. Exp. Ther. Google Scholar, N. L.L. PubMed Google Scholar). The were in solution that was with and and at the solution the and The were with as an protein to of were and was was measured as a in to were used to to soluble In the first, toxin was for of to a of EDTA and EGTA to strip Zn2+ (see In the second, toxin was with for at the both endoprotease activity was substrate or substrate in toxin cleavage of substrate in toxin that been with and in and toxin that been stripped of Zn2+ in were free of activity. the of exogenous Zn2+ the of chelation. of whether toxin been to in or stripped of Zn2+ and in the of of endoprotease activity. of toxin that been stripped of Zn2+ with EDTA and EGTA or been to were for to produce neuromuscular blockade of In both toxin to be in blocking exocytosis. This was obtained though toxin was not to exogenous Zn2+ its to were with an immobilized chelator that to Zn2+ its coordination site in the toxin molecule but can free Zn2+ in The toxin was with chelator as described for at which the was to separate the toxin was then for biological activity. toxin to Chelex® activity and neuromuscular blocking activity with those of toxin These results were observed the of exogenous not were to the of spontaneous of zinc at the active site of botulinum this of were with a of were for at the of exogenous Zn2+ that Zn2+ was in there was of Zn2+. was at and was more at and These results are with the previously reported finding that the for Zn2+ at its binding site is G. Rossetto O. DasGupta B.R. Montecucco C. J. Biol. 1992; 267: Full Text PDF PubMed Google Scholar). In a of the of Zn2+ was This was at of which is on the of the in The results that a for was and the for was This means that when studied in at Zn2+ can with Zn2+. The of this is as to it for botulinum toxin to at least a of Zn2+ during and release by and by and binding, internalization, and of neuromuscular The results presented and in that stripped toxin can regain Zn2+ and activity. the in toxicity with that stripped toxin can Zn2+ rapidly were to the at which Zn2+ can be toxin was stripped of Zn2+ with a of EDTA and EGTA as described Stripped toxin was then for various of and the of of exogenous zinc was The which are in that the for was This finding two the of Zn2+ by stripped toxin is the of in toxin, and the of Zn2+ by stripped toxin can for the of this to exogenous Zn2+ and block neuromuscular for toxin activity in substrate and in blocking neuromuscular have that a soluble chelator as EDTA toxicity an immobilized chelator as Chelex® 100 does not (see This that the of an EDTA Zn2+, but Chelex® 100 does were to this Botulinum toxin was stripped of Zn2+ as described and then This was then with EDTA or at which the with the toxin was The data from three that Chelex® no to strip Zn2+ from the other EDTA more of bound Zn2+, and of the bound cation. These findings that soluble chelators rapidly to remove Zn2+ from the results that soluble chelators do more Zn2+. In all they the active site and bound Zn2+. the data that Chelex® is not in stripping Zn2+ for the finding that Chelex® is not in toxicity (see Botulinum toxin is not likely to a chelator when it acts on neuromuscular to However, there are ambient by the toxin that could produce a loss of Zn2+. The of these is the in that is when the toxin from the of the endosome to the cytosol. The in could produce a loss of low to of that Zn2+ binding, low conformational changes that Zn2+ binding, or were to Zn2+ at that the a for the chain of in these also the for those that conformational changes with toxin Botulinum toxin was stripped of Zn2+ as described and with This was then with of for at When the of were the results that there was a significant loss of cation at a of was an loss of cation with further of The at which toxin is to from the endosome to the cytosol has not been the of is in the of and the of is M. Y. Cell Biol. 2000; PubMed Scopus Google Scholar). the fact that there is a significant loss of Zn2+ at at least some cation could be during This could be by of change in or reported of toxin with a soluble chelator is not sufficient to cause the molecule to neuromuscular blocking activity L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Ther. 1993; 267: 720-727PubMed Google Stripped toxin can regain Zn2+ from tissue and regain catalytic and neuromuscular blocking activity. A loss of activity is observed both toxin and tissue are with the the results of an in which toxin and toxin were added either to or to These data show that toxin and stripped toxin were in to block neuromuscular in They also show that toxin nor stripped toxin when added to in and In a of toxin and stripped toxin were added to with an to that necessary for toxin to a of Zn2+ was added to It is that there was of toxin action that in neuromuscular and there was no significant toxin and stripped toxin in of of of The results in that was by toxin that productive internalization. To this were to that for in that were to the of Zn2+. Thus, there was no toxin in solution for binding and internalization. When were free of toxin and then to Zn2+, there was a of toxin action there was no that were to toxin and those that were to remove toxin The data presented show that but not chelators can strip Zn2+ from Stripped toxin has to cleave substrate or to block neuromuscular However, toxin that regains Zn2+ from tissue or from regains biological activity. The data in and particularly those on toxin added to a The fact that toxin is when added to in that a soluble chelator can strip the toxin in that for the toxin to the and This was by the of loss of Zn2+. of toxin were stripped and with as described This was with for of which zinc was in a significant loss of zinc and the loss to occur for at least These data for the that can strip Zn2+ during the interval when toxin is to its site of action. the sequence of events in the tissue is likely to be more that by the Zn2+ stripping the of chelator to toxin was the neuromuscular blocking the was the of Zn2+ stripping the toxin is to its site of action is likely to be that in Each of the three major steps in toxin binding, productive internalization, and intracellular involves some of (see the However, productive internalization may be the complex of the This step is to involve at least seven penetration of the plasma membrane by the change in toxin structure (viz. of an of the of the chain into the endosome of the that the and of the and translocation of the chain from the of the endosome to the presumably a (viz. or and of chain structure with from an to a more In productive internalization may on events that occur during the or binding, step. The changes in structure and of chain into the endosome membrane may not occur or may occur with low the toxin is not with its In other a toxin molecule that is free in solution may have into the endosome membrane and the process of Thus, productive internalization may on toxin with the receptor during the binding step. a recent of apparently contradictory findings may to of step in toxin intracellular on a step, productive internalization. Fu et al. (9Fu F.-N. Lomneth R.B. Cai S. Singh B.R. Biochemistry. 1998; 37: 5267-5278Crossref PubMed Scopus (64) Google Scholar) have reported that a loss of Zn2+ by botulinum toxin both irreversible loss of catalytic activity and irreversible changes in structure. these are they two botulinum toxin must not bound Zn2+ during productive internalization. it the the catalytic chain could not to block exocytosis. the spontaneous loss of Zn2+ by botulinum toxin in the and the must be slow. the toxin would its to were to the that stripping toxin of Zn2+ irreversible loss of catalytic activity. This the use of soluble and immobilized of the soluble chelators, was the as that used by Fu et al. (9Fu F.-N. Lomneth R.B. Cai S. Singh B.R. Biochemistry. 1998; 37: 5267-5278Crossref PubMed Scopus (64) Google Scholar), but the results were In the of the both EDTA and loss of activity as measured by the cleavage of When a of Zn2+ was added to stripped there was and of activity. These findings are in with those obtained by other investigators in B. B. M. 1993; PubMed Scopus Google Scholar, F. Schiavo G. Montecucco C. Biochem. J. PubMed Scopus Google Scholar). They are also in keeping with the findings of and J. 2000; 19: PubMed Scopus Google Scholar), studied the loss and of Zn2+ by a chain derived from botulinum were also with an immobilized and the data were when with toxin at a the chelator no loss of activity the that The major the soluble chelator and the immobilized chelator is that the can Zn2+ its coordination site and strip from toxin, the latter from the coordination site and can that Zn2+ that has been by toxin and is free in The fact that Chelex® not cause a loss of activity that there must be a of spontaneous loss of cation. The of studies was to whether there is spontaneous of bound Zn2+, and the of this The data that the for spontaneous bound Zn2+ and Zn2+ in the was This of is with the for neuromuscular but this is not the when with the of a toxin molecule in its the and the of a The of a toxin molecule could the for bound Zn2+, and this in turn to of three the toxin is in a and irreversible process of biological because molecule that a Zn2+ catalytic toxin that is in with exogenous zinc biological because the of bound zinc and exogenous Zn2+ is with the of from an active to an when toxin is stripped of Zn2+, or the loss of Zn2+ does not cause irreversible toxin can through a of and Zn2+ and and catalytic activity. The data on toxin with EDTA or that the first is not when the toxin was stripped of Zn2+, it biological activity when with The data also that the has biological When the toxin was in a for a of that to stripped there was still a of activity. The data the as by the cell-free studies but also as by studies on intact neuromuscular To the of toxin and stripped toxin to described in more (8Simpson L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Ther. 1993; 267: 720-727PubMed Google Scholar), this is likely because of stripped toxin Zn2+ from tissue stores. However, the results of a may of a more When stripped toxin was added to in with there was no of in the of bound Zn2+ the toxin could not catalytic activity. When a of exogenous Zn2+ was added to these there was of This that the toxin can through a of and Zn2+ and and activity. The latter with and Zn2+ on neuromuscular The data in that exogenous Zn2+ to in that been free of This that exogenous Zn2+ was toxin that was in the cell This must that botulinum toxin can and be internalized in the of Zn2+. To to an binding and internalization are complex and of these events involve changes in into the endosome of and and translocation of chain to the Thus, the in which stripped toxin Zn2+ and catalytic activity the cytosol on the of The data that any conformational changes in the botulinum toxin molecule that from the removal of Zn2+ are not of a or sufficient to with conformational changes that are essential for productive internalization, and the conformational changes with productive internalization do not have an effect on the toxin molecule of a or sufficient to block the of Zn2+ in a previously stripped These two when that it is not essential for Zn2+ to remain bound during productive internalization. Zn2+ is from either or exogenous the catalytic and neuromuscular blocking of the toxin be is an and of the on toxicity and tissue of Zn2+. To be more this is the that the in the toxin molecule have been to have a with the of the it has been that botulinum toxin is as a relatively have a that the and it into an active structure. However, the toxin is not by the it may still be This is because when the molecule can be by in the and to the active The results with Zn2+ described in the to be a the toxin and the toxin molecule has a bound Zn2+ that is essential for activity. It has not been whether the toxin this bound Zn2+ through all of the steps with blockade of exocytosis. this does then the bound Zn2+ However, it does not the toxin can still be The stripped molecule can regain Zn2+ from tissue and in the process the structure and the activity for the blockade of exocytosis.

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