Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease... The EMBO Journal vol. 1 1 no. 10 pp.3577 - 3583, 1992 Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc (BoNT, seven different serotypes) which cause the flaccid Schiavo, Bernard Poulain', Giampietro paralysis of botulism by inhibiting ACh release at the Ornella Rossetto, Fabio Benfenati2, neuromuscular junction (Simpson, 1989). Ladislav Tauc1 and Cesare Montecucco Recently the cDNA-deduced amino acid sequences of five Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, BoNT serotypes have been determined and aligned with Universita di Padova, Via Trieste 75, 35121 Padova, Italy, that of TeTx (Niemann, 1991). Despite the very similar 'Laboratoire de Neurobiologie Cellulaire et Moleculaire, Centre mechanism of action, these neurotoxins show an overall National de la Recherche Scientifique, Gif-sur-Yvette, 91198 France low degree of homology, having a few segments of close 2Istituto di Universita di Modena, Via Campi and Fisiologia Umana, similarity. The most conserved segment is located in the 287, 41100 Modena, Italy central region of the L chain and the common elements Communicated by J.Meldolesi of this peptide are reported in Figure lB. We noticed that this segment includes the HExxH zinc binding motif of Tetanus and botulinum neurotoxins are the most potent metalloendopeptidases. In these enzymes the zinc ion is toxins known. bind to nerve cells, penetrate the They tetra-coordinated and it is essential for their catalytic activity release. cytosol and block neurotransmitter Comparison 1988; Vallee and Auld, 1990). The two histidines (Matthews, reveals a of their predicted amino acid sequences highly of interact with the zinc ion while the glutamic the motif conserved that contains the HExxH zinc binding segment residues a catalytic role. The third ligand is another play motif of metalloendopeptidases. The metal content of glutamic residue whose position in the sequence has been and it was found that tetanus toxin was then measured identified in bacterial neutral zinc endopeptidases (Vallee and zinc is to the chain of tetanus one atom of bound light Auld, 1990), but it is still unidentified in matrix metallo- be removed incubation toxin. Zinc could reversibly by proteases and it is also not evident in sequences of TeTx is coordinated two with heavy metal chelators. Zn2+ by and BoNTs. The fourth ligand is water. with no involvement in cysteines, suggesting histidines In this work we demonstrate that TeTx does indeed contain that it a rather than a structural role. plays catalytic a zinc atom coordinated by two histidines and that the zinc was found to be essential for the tetanus Bound Zn + ion is essential for TeTx inhibition of neurotransmitter release toxin inhibition of neurotransmitter release in Aplysia in neurons. We also report that highly purified Aplysia with the light chain. The intracellular neurons injected preparations of the L chain of TeTx show a proteolytic toxin was blocked a activity of the by phosphoramidon, activity specific for synaptobrevin, an integral membrane Purified very specific inhibitor of zinc endopeptidases. protein of small synaptic vesicles, and that its effect on preparations of light chain showed highly specific neurotransmitter release is inhibited by phosphoramidon, a an proteolytic activity against synaptobrevin, integral inhibitor of zinc endopeptidases. highly specific vesicles. The membrane of small synaptic present protein that tetanus and also the indicate toxin, possibly findings are and that C botulinum neurotoxins, metalloproteases release via this they block neurotransmitter protease H chain activity. release/ Key words: metalloprotease/neurotransmitter N C L chain TI toxin/zinc synaptobrevin/tetanus Introduction toxin is the for the Tetanus (TeTx) agent responsible of an often fatal due development tetanus, spastic paralysis block of neurotransmitter release of the cord to the spinal DPhLH.HLuHxY h uHu uG h hHEh interneurons It is inhibitory (Simpson, 1989). produced by as a strains of Clostridium tetani toxigenic single chain, motifs of tetanus and 1. Schematic structure and zinc Fig. binding at an is cleaved which readily by proteases exposed loop botulinum neurotoxins and of matrix metalloendoproteases. 100 kDa the active two-chain to generate protein (H chain, of the clostridial neurotoxins of tetanus and Schematic structure (A) in IA et and L, kDa), depicted Figure (Krieglstein al., of closest between the six botulism. (B) The segment similarity is involved in cell BoNT D and The H chain neurospecific binding clostridial neurotoxins (TeTx, serotypes A, B, Cl, E) 1990). are known is located in the central is for the whose sequences (Niemann, 1991) while the L chain and responsible penetration to the six clostridial of the L chain. The features common portion of the of a inactivation key component neuroexocytosis amino acid one letter code and with h are in the neurotoxins reported machinery (Montecucco, 1986; Simpson, 1989; Niemann, x for the zinc u for and for hydrophobic, uncharged any residue; and et zinc and is 1991; Schiavo al., 1991). Structurally functionally motif of matrix Auld, 1990) binding endopeptidases (Vallee below. the botulinum neurotoxins aligned just related to TeTx are closely © Press Oxford University G.Schiavo et a/. Vallee and Auld, 1990; Thayer et al., 1991). Results 1988; Chemical modification of TeTx with diethylpyrocarbonate Metal content of TeTx a histidine et al., 1967; (DEPC), modifying reagent (Ovadi To determine the nature and amount of metals bound to Miles, 1977; Papini et al., 1989), indicates that also in TeTx TeTx, the toxin and its L chain were isolated by chromato- are involved in zinc coordination. In fact an histidines graphic methods followed by high performance size of 1.8 histidine out of the six were average residues, present, et Ozutsumi exclusion liquid chromatography (Lin al., 1985; L while this number modified with DEPC in the native chain, et was determined et al., 1985; Schiavo al., 1990). Toxicity varied in the Zn2'-depleted L chain of TeTx according to < 1 and was estimated to be in mice ng/kg) purity (MLD50 An of 1.5 additional the level of residual Zn2+. average >99% by densitometric scanning of SDS -PAGE gels DEPC in L chain preparations histidines were modified by blue. As shown in atomic stained with Coomassie Figure 2, of different of TeTx absorption analysis preparations gave zinc content between 0.70 and 0.85 atoms a range of per and 1.0 zinc atoms TeTx molecule and between 0.9 per the H chain contains no zinc molecule of L chain, while (not below an value is shown). A metal content slightly integer 4,~~~~~~~~~~~~~~~~~~4 with due to metal loss a common finding metalloproteins These values hence indicate the during protein purification. of zinc TeTx molecule and that presence of one atom per 0.06 SzE0aS is associated with the L chain. all the zinc content of TeTx that BoNT Preliminary experiments indicate serotypes A, AS S ig 3.Hsiiemdfcto ihDP fntv n toxin molecule B and E also contain one zinc atom per idepleted et data). Co, Cu, Fe, Mn (G.Schiavo al., unpublished and Ni were also measured and found to be below the limits of detection. +Zn -Zn Zinc removal with heavy metal chelators incubation with the toxin released most of its Upon EDTA, Histidine modification with DEPC of native and Fig. zinc-depleted content bars in This occurred Zn2+ (filled Figure 2). L chain of TeTx. (A) The increase in absorbance at 243 nm is without protein denaturation because Zn2+ could be indicative of the formation of N-carbethoxyhistidine. (B) The number of histidines modified as determined absorbance change incubation in a Zn2+- con- by DEPC, by reassociated with the toxin upon at 243 bars are the SD of three different on L nm; experiments bars in 2 and Zn2+ taining medium (hatched Figures 4). an residual zinc content of No chain preparations with average 19%. could also be removed treatment with other partially by nm of absorbance change was detected at 278 indicative no heavy metal chelating agents such as o-phenanthroline (OP), modification of tyrosine residues. acid dipicolinic (DPA), tetrakis-(2-pyridylmethyl)ethylene- and diamine anhydride (TPEN) diethylenetriaminepenta- acetic anhydride (DTPA). histidines and Chemical modification of cysteines In the zinc two histidines are involved in endopeptidases, zinc coordination et et (Matthews al., 1972; Pauptit al., L chain TeTx 1.0- E- C Q5- 0-i- N EDTA R OP TPEN N EDTA R Df UrTPA Fig. 4. Dot blot assay of [65Zn2+] binding to various samples of L Fig. 2. Zinc content, measured by atomic absorption, of TeTx and of chain and of the Leu230-Gly244 peptide of TeTx. The samples were its L chain treated with various chelators. Amount of zinc, in atoms onto nitrocellulose paper and overlaid with 50 nM 65ZnC12 for applied per protein molecule SD of four different toxin preparations, bound 60 min. After washings and drying, the nitrocellulose paper strips to TeTx and to its L chain before (N, open bars) or after EDTA were exposed at -80°C to a Kodak X-OMAT film. Samples are as treatment (EDTA, filled bars) or after EDTA incubation, dialysis and follows: L+Zn, native L chain; L+Zn+DEPC, native L chain treated further incubation with 50 ZnC12 (R, hatched bars). The effect of with a 30-fold molar excess of DEPC; L-Zn, L chain treated with ,M the following heavy metal chelators is also shown: o-phenanthroline 10 mM EDTA and dialysed; L-Zn+DEPC, L chain depleted of (OP), dipicolinic acid (DPA), tetrakis-(2-pyridylmethyl)ethylenediamine incubation Zn2+ by with EDTA and treated with a 30-fold molar anhydride (TPEN) and diethylenetriaminepentaacetic anhydride excess of DEPC; DT, diphtheria toxin; Peptide, Leu230-Gly244 (DTPA). Co, Cu, Fe, Mn and Ni were also assayed and were found peptide; Peptide+DEPC, peptide treated with a Leu230-Gly244 to be below the limits of detection. Experimental details are in 30-fold molar excess of DEPC. Other experimental details are in Materials and methods. Materials and methods. 3578 Tetanus toxin and zinc endopeptidase activity with an average residual zinc content of 19%, with no [65Zn2"] binding to TeTx light chain and to concomitant formation of o-carbethoxytyrosine (not shown). Leu230- Gly244 fragment Taken together, the results of Figure 3 indicate that two To gain further evidence for the involvement of the in Zn2+ coordination and that bound histidines are involved conserved segment shown in Figure lB in zinc coordination, Zn2+ protects them from reacting with DEPC. the peptide Leu230-Gly244 of TeTx was chemically In many zinc proteins, cysteine residues are involved in synthesized. Figure 4 shows that this peptide binds [65Zn2+] zinc coordination (Vallee and Auld, 1990). However, the in a dot blot test and that this binding is abolished by DEPC same number of sulfhydryl groups (1.3 0.2 per L chain treatment. It would be expected that the peptide binds less molecule) were titrated with Ellman's reagent (5,5'-dithiobis- [65Zn2+] than the TeTx L chain because the peptide lacks 2-nitrobenzoic acid) on the L chain before and after Zn2+ the third zinc ligand present in the native protein. Figure depletion. Hence cysteines are not involved in Zn2+ 4 also shows that the zinc atom of the L chain of TeTx is coordination of TeTx unlike in alcohol dehydrogenase, exchangeable and that, after DEPC modification, the aspartate transcarbamoylase or in the zinc finger of DNA- Zn2+-depleted L chain is unable to reincorporate the zinc binding proteins (Vallee and Auld, 1990). The absence of ion, while a parallel DEPC treatment of the native L chain cysteine coordination emphasizes the similarity of TeTx to does not affect its ability to exchange its zinc atom. This zinc endoproteinases and suggests that the Zn2+ ion of result provides further support for the involvement of TeTx plays a catalytic, rather than a structural role, as coordination. The dot blot histidines in Zn2+ assay in all zinc enzymes with histidine coordination (Vallee used here is very specific since diphtheria toxin, which and Auld, 1990). histidines interspaced by three residues also has three + / TPEN DTPA A0~~~#toA a a 100- LAA04 .0 &O4 % "5 A.-^..' A A . .^s A~ *I, &Ae t0 0%& &db t s OSOZ& i0 ° r~~~~~~~-0)o 4 50- 'IO '9 ' Zn++ depleted 0:.0 -W ~0. 4, V,. native *1 0 60 120 -60 time (min) h. 0 0: 100- 0 o1 i, o'0 0neS*)) Zn " depleted 0 00io0o % , eo 'Po 0 * 0 a DEPC *0 4- 0 IPo e ,+ 4A ',ocjb 0.50 *0 @0 native DEPC treated>\V. f I -80 0 60 120 time (min) release induced intracellular of the L chain of TeTx. Identified of neurons identified 5. Inhibition of by application couples making Fig. acetylcholine in the buccal of were used for the of modified L chain of the TeTx as detailed in ganglion Aplysia califomica assay chemically cholinergic synapses at time zero were: native L chain and its form (19% methods. The L chain A, Zn2'-depleted Materials and samples, injected (arrows) panel DEPC-treated native chain histidines and DEPC-treated L chain (18% residual in this Zn2+ residual Zn2+); panel B, (1.8 modified) Zn-depleted In order to remove ions from both extracellular and intracellular medium, in one series of (panel 3.3 histidines experiments sample, modified). Zn2+ for 100 in the of 50 DTPA and 30 TPEN; DTPA was also was min jtM mixed filled the buccal pre-incubated presence AM A, triangles), ganglion L chain before to a final intracellular concentration of 200 Control showed that, while DTPA and into the injection AM. experiments Zn2'-depleted TPEN are inefficient in zinc removal as shown in zinc the L chain. Figure 2, they prevent re-uptake by 3579 G.Schiavo et al. does not show of zinc endopeptidases (Komiyama et al., 1975; Weaver et al., (His484xxxHisxxxHis492), any sign 1977; Lennarz and Strittmatter, 1991). When [65Zn2 ] binding. phosphor- amidon was injected into Aplysia neurons, as shown in Figure 6, it completely blocked the L chain effect TeTx inhibition of neurotransmitter release in Aplysia on neurons is zinc dependent neurotransmitter release, while by itself the drug was for the intracellular ineffective. The small inhibition of neurotransmitter release The best characterized system testing of clostridial neurotoxins is the identified detectable long after toxin injection is expected because of activity couples in the buccal of diffusion of phosphoramidon out of the nerve cell and of neurons making synapses ganglion Aplysia of toxin et dissociation the -inhibitor complex. Given the high californica (Poulain al., 1988, 1990, 1991; Mochida In this cellular it could be specificity of phosphoramidon inhibition, this result strongly et al., 1990). system directly that the L chain of TeTx is the supports the proposal that TeTx is a zinc endopeptidase. demonstrated intracellularly active portion of the toxin (Mochida et al., 1990; Poulain Protease activity of the light chain of TeTx et al., 1990, 1991). Figure 5A shows that both the native The zinc ion plays a central role in the peptide L chain and the Zn2+-depleted L chain of TeTx were bond neurotransmitter hydrolysis catalysed by zinc proteinases (Matthews, effective in inhibiting the evoked release 1988; level of inhibition. eventually reaching the same Vallee and Auld, 1990). The experiments described above However, the L chain indicate that two histidines are involved in Zn2+ co- the kinetics of inhibition of Zn2+-depleted were if the L chain ordination in TeTx. The putative zinc binding site of TeTx much slower. On the contrary, Zn2+-depleted into a neuron with the is suggested by secondary structure prediction methods to was injected presynaptic pre-treated et no be a-helical with the two histidines on the same face of heavy metal chelators (Arslan al., 1985), inhibitory the for least 3 h ae-helix as is the case for the three zinc proteases whose activity was recorded at (filled triangles in 5B that DEPC treatment of the three-dimensional structure Figure 5A). Figure shows has been resolved (Matthews its with et al., 1972; Pauptit et al., 1988; Thayer et al., native L chain does not modify inhibitory activity 1991). Together with the phosphoramidon inhibition of the intra- respect to the untreated control (Figure 5A). On the contrary, L treated with cellular toxin activity, these similarities suggest that TeTx the chain DEPC after Zn2+ depletion release residual may be a zinc-dependent protease. inhibited neurotransmitter very poorly; As a first could be accounted for the residual approach, we incubated TeTx with a large activity by Zn2+- L chain. number of chromophoric protease model substrates with no containing that the L for These experiments suggest Zn2+-depleted positive findings except a weak protease activity on the its zinc ion from the chain is slowly able to regain general protease substrate casein. As a possible candidate and DEPC for the toxin action cytoplasmic pool, while a Zn2+-depleted we next assayed small synaptic vesicles Zn2+ is for (SSVs), purified from rat cerebral cortex (Huttner et al., modified one is not, and that bound necessary L chain inhibition of neurotransmitter release. 1983; Benfenati et al., 1989). Figure 7 shows that the L the chain of TeTx does not change the SDS -PAGE protein blocks the TeTx inhibition of pattern of highly purified SSVs, except for the disappearance Phosphoramidon in neurons of a single band of 19 kDa with the concomitant appearance neurotransmitter release Aplysia of a 7 kDa and a 12 kDa fragment; the latter peptide overlaps Phosphoramidon [(N-a-L-rhamnopyranosyl-hydroxyphos- of with an SSV component of the same apparent molecular phinyl)-L-leucyl-L-tryptophan] is a very specific inhibitor phosphoramidon 6e, a a , ° W - treated 100- * ^) 0 s.~~~~~~~~~~~~~~ 0og~0Oe-s 0 I~* ~ ~ O OzC ~~~~~~~~ 50- .U *. -0 native L chain ^.- 0* 0% 0W .0 -60 0 60 120 180 time (min) Fig. 6. Effect of phosphoramidon on the intracellular Aplysia neuron activity of the L chain of TeTx. L chain of TeTx (48 pgI/ml) was incubated with 10.7 mM phosphoramidon (Serva) or with buffer for 30 at room temperature. Two presynaptic Aplysia neurons afferent to the same min postsynaptic cell were used. Neuron 1 (open circles) were made 0.37 mM in phosphoramidon and the 1 h recording before time 0 shows that the inhibitor does not by itself affect neurotransmitter release. At time 0 (arrow) neuron 1 was injected with the phosphoramidon-treated L chain while the control neuron 2 (filled circles) was injected with L chain (final concentration 10 nM). The slow recovery of activity of the toxin in neuron 1 with prolonged incubation is to be attributed to diffusion of phosphoramidon in the inhibitor free extracellular medium. 3580 zinc Tetanus toxin and endopeptidase activity 3 of Figure 7). The electrophoretic mobility The lack of a cell line highly sensitive to TeTx, similar weight (lane of the 19 kDa band and its immunoblot staining with an to Vero cells for diphtheria toxin (Sandvig and Olsnes, anti-synaptobrevin monoclonal antibody (Figure 7, lane 1) 1981), represents a major obstacle to the direct demonstration indicate that this protein is synaptobrevin. The H chain did of a proteolytic activity of TeTx and to the identification of not cause any change to the SSV protein profile (not shown). its intracellular target(s). The high specificity of TeTx in vivo The synaptobrevin specific cleavage by the L chain was and the lack of proteolysis in vitro (35 different synthetic completely blocked by EDTA, as expected for a zinc peptide substrates of various proteases assayed) suggested protease (lane 4 of Figure 7), and was only partially that the activity of TeTx should be very specific. inhibited by phosphoramidon (not shown). This latter result Possible targets of TeTx proteolytic activity are at the is probably due to the fact that the very strict binding nerve terminal level. A protein of SSVs, the organelles that requirements of phosphoramidon were not met in the in vitro store and release neurotransmitters, appeared to be a good assay (Weaver et al., 1977; Lennarz and Strittmatter, 1991). candidate. TeTx has been shown to act on the rat brain causing an epileptiform syndrome (Mellanby et al., 1985; Bagetta et al., 1991). Using SSVs isolated from rat cerebral Discussion cortex in a highly purified form (Huttner et al., 1983. Bacteria produce a whole range of protein toxins that are Benfenati et al., 1989), we were able to show that TeTx is their (Alouf and Freer, 1991). Here a very specific zinc endopeptidase since it cleaves only essential to pathogenicity we provide direct evidence that the toxin responsible for all synaptobrevin, out of the many components of the vesicles. the symptoms of tetanus contains one atom of zinc This protein is also expressed in PC12 and adrenal medulla clinical active L chain. No metal is bound which are both sensitive to bound to its intracellularly cells (Baumert et al., 1989), of the toxin responsible for to the H chain, the portion TeTx (Niemann, 1991). membranes (Schiavo et al., 1991) and binding to neuronal Our observations suggest that TeTx acts inside neuronal The between TeTx and for cell penetration. similarity cells by specifically cleaving synaptobrevin. The role of to the amino acid sequence synaptobrevin in neuroexocytosis is still obscure (De Camilli metalloproteases is not confined of zinc atom. Here we have also and Jahn, 1990), but the present findings suggest that this and to the presence the zinc is coordinated via two histidines and that protein plays a key role in the process of neurotransmitter shown that Leu230-Gly244, which includes the zinc release. In this respect, it is noteworthy that all bacterial the TeTx peptide motif of binds zinc. The protein toxins with intracellular targets act on key elements binding metalloendopeptidases, zinc is indicative of a of regulation such as G proteins (for cholera, histidine involvement in binding cell physiology of toxin bound metal and this is in or (for catalytic role the pertussis and botulinum C3 toxins) protein synthesis in vivo. In when and Pizza, 1991; with the data obtained fact, only diphtheria and Shiga toxins) (Rappuoli agreement L chain able to inhibit Boquet and Gill, 1991). zinc is bound to the toxin is the in neurons, as clearly shown or activation of important biological neurotransmitter release Aplysia Regulation the experiments of Figure 5. phenomena via highly selective proteolytic cleavage is by not uncommon, e.g. blood coagulation or complement activation. The protease activity of TeTx, described here, is consistent with its known specificity and potency. An z -3 intracellular catalytic activity of TeTx accounts for its -I.. extreme because one molecule of toxin would be potency _ Tm`_ A&: sufficient to inactivate a large number of synaptobrevin- _i containing vesicles. *It Our findings provide the first explanation at the molecular level of a high incidence neuroparalytic disease such as tetanus and it new for the development opens perspectives 4. 4* of based on inhibitors of the zinc therapeutic protocols Moreover, our data may become of protease activity. -..4 for the dissection of the molecular paramount importance of neurotransmitter release. events underlying the process methods Materials and SYB:> Toxin and fragments were isolated from culture filtrates of Clostridium TeTx and the L chain Ozutsumi et Schiavo before et 1985; tetani, as detailed (Lin al., 1985; al., in 10 mM 50 mM sodium and stored at -800C HEPES-Na, et al., 1990), H chain was as described Matsuda 7.2. The by chloride, pH prepared and Yoneda (1975). of metal content Determination with with a were rinsed water, All materials Milli-Q grade previously is cleaved the L chain of Fig. Synaptobrevin proteolytically by were with chemicals of the > 10 Buffers MQ prepared highest conductibility blue stained SDS-PAGE of Lanes 2-4 show Coomassie TeTx.- the of metals and with to available respect presence heavy pre-treated incubated alone or in the purity SSV 2) of (lane samples purified (35 Ag) Before metal the MB-3 determination, with Amberlite (Sigma). protein L chain or with L chain with of TeTx (lane 3) pretreated presence at 150 mM Tris-Cl, were samples extensively dialysed 4°C against pH Lane refers to a of SSV at 1 EDTA for 15 min 37°C (lane 4). sample boiled 100 mM sodium 7.4 or 10 mM chloride, pH 7.0, using HEPES-Na, monoclonal with the immunoblotted antibody IgGi anti-synaptobrevin were for Zn, Co, Cu, dialysis tubing (Spectrum, TX). Samples analysed et Cl 10.1 1989). (Baumert al., 3581 et al. G.Schiavo Fe, Mn and Ni with a Perkin -Elmer 4000 atomic absorption flame of TeTx Proteolytic activity with impact bed loading. Metal contents were measured from rat cerebral cortex et spectrophotometer 35 of SSVs purified (Huttner al., 1983; jig after standardization in the linear range of concentration for each ion (0-0.5 Benfenati et al., 1989) were incubated with 1 of TeTx L chain in 270 Ag Al p.p.m. for Zn, Ni and Co; 0-1 p.p.m. for Cu; 0-5 p.p.m. for Fe and Mn). M 7.4. contained of 5 mM HEPES-Na, 0.3 glycine, pH Some samples 10 mM EDTA. After 30 min at 370C, 30 50% TCA were added and, Zinc removal were dissolved in after 30 min at room temperature, centrifuged. Pellets L chain were diluted to 0.5-1.0 mg/ml with 150 mM Tris-Cl, electrophoresis sample buffer 5 mM EDTA and 5 TeTx or the containing % 2-mercapto- 7.4, and incubated in the presence of 10 mM EDTA for 60 min at 37°C. ethanol and boiled for 2 min. were in 12% pH Samples electrophoresed were dialysed extensively against the same buffer at 4°C and their was carried out Samples polyacrylamide gels (Laemmli, 1970). Immunoblotting zinc content was determined as above. Concentrations of other chelators according to Towbin et al. (1979), using the IgGi anti-synaptobrevin used are as follows: 10 mM o-phenanthroline (Erba, Milan), 50 mM monoclonal antibody Cl 10.1 (Baumert et al., 1989) and revealed by 30 tetrakis-(2-pyridylmethyl)ethylenediamine antibodies. dipicolinic acid (Fluka), uM anti-mouse alkaline phosphatase coupled secondary Probes, Eugene, OR), 200 anhydride (Molecular diethylenetriamine- lsM pentaacetic anhydride (Molecular Probes). Toxicity tests The toxicity of TeTx was determined by injecting intraperitoneally serial dilutions of the toxin with phosphate-buffered saline containing 0.1% BSA. Peptide synthesis to the sequence LLMHELIHVLH- Time before death was recorded TeTx segment 230-244 corresponding and mouse lethal doses were calculated Dr A.Santucci (University of Siena) by solid as described (Weller GLYG was kindly prepared by et al., 1988). 350 automatic synthesizer (Zynsser Analytic, phase synthesis with a SMPS The product was detached from Frankfurt) employing F-moc chemistry. Acknowledgements the resin with 93% TFA, purified by HPLC on a Vydac column and C18 on an Applied Biosystems controlled by automatic Edman degradation We thank V.Albergoni and F.Cattalini, Department of Biology, University 475A microsequencer. of Padua, for the atomic absorption measurements, S.Fabbiani of SCLAVO Spa for samples of culture filtrates of Clostridium tetani, P.De Camilli, DEPC modification Department of Cell Biology, Yale University, for generously supplying the 50 mM disaerated sodium phosphate The L chain of TeTx was diluted with anti-synaptobrevin monoclonal antibody Cl 10.1, T.Pozzan for advice on were filtered 50 mM, pH 7.8 to a final concentration of 7.0 uM. The samples the use of TPEN and DTPA, and A.Fontana, E.Papini and T.Pozzan for through a 0.22 filter (Anotec, Oxford, UK) and DEPC from a freshly ltm critically reading the manuscript. This work was supported by grants The DEPC/L chain ratio prepared solution in absolute ethanol was added. from CNR Target Project 'Biotechnology and Bioinstrumentation', from 30 mol/mol L chain. The reaction was followed at 25°C never exceeded Association Francaise contre les Myopathies, and from Direction des the differential absorbance at 243 nm and at by simultaneously recording Recherches et Etudes Techniques (granted to L.T.) and it is in partial 1977; Papini et al., 1989) in a 278 nm as previously described (Miles, fulfilment of the doctorate degree of the University of Padua in 'Molecular Modified residues were Perkin-Elmer Lambda 5 spectrophotometer. and Cellular Biology and Pathology' of O.R. coefficients of 3200 M- l cm-1 at estimated with differential extinction nm and 1310 M-1 cm-' at 278 nm for 243 for N-carbethoxyhistidine o-carbethoxytyrosine. References thiol groups Titration of free Alouf,J.E. and Freer,J.H. (eds) (1991) A Sourcebook ofBacterial Protein or L chain were diluted to 1 yM with 50 mM Native zinc-depleted samples Toxins. Academic Press, London. 7.8. Subsequently 10 of DTNB Arslan,P., Di Virgilio,F., Beltrame,M., Tsien,R.Y. and Pozzan,T. (1985) disareated sodium phosphate, pH 1z1 J. Biol. Chem., 260, in 50 mM sodium pH 8.0; final concentration 0.4 mM) 2719-2727. (5 mg/mi phosphate, Bagetta,P., and Bowery,N.G. (1991) Trends Pharmacol. Nistic6,G. Sci., at to the and the absorbance at 410 nm was were added 25°C samples 12, 285-289. content was calculated determined against a blank without protein. Sulfhydryl Baumert,M., Maycox,P.R., Navone,F., Camilli,P. De and Jahn,R. (1989) on the basis of molar absorptivity of 13 600 M-l cm-1 at 412 nm for EMBO J., 8, - 379 384. 2-nitro-5-thio-benzoate anion and expressed as moles of free thiol groups Benfenati,F., Bahler,M., Jahn,R. and Greengard,P. (1989) J. Cell Biol., mole of L chain (Ellman, 1959; Schiavo et al., 1990). per 108, 1863-1872. Boquet,P. and Gill,D.M. (1991) In Alouf,J.E. and Freer,J.H. (eds), Sourcebook of Bacterial Protein Toxins. Academic Press, London, pp. Determination of [65Zn2+] binding 23-44. L chains in their native or zinc-depleted forms, treated or not with 1:30 De Camilli,P. and Jahn,R. (1990) Annu. Rev. Physiol., 52, 625-645. molar excess of DEPC as above, or diphtheria toxin (2-32 pmol) or the Ellman,G.L. (1959) Arch. Biochem. Biophys., 82, 70-77. zinc binding peptide (25-400 pmol) were dot-blotted onto putative Huttner,W.B., Schiebler,W., Greengard,P. and De Camilli,P. (1983) J. nitrocellulose strips (0.22 Hoefer, CA). The strips were rinsed paper 96, 1374-1378. ltm; Cell Biol., for 5 in 25 mM 100 mM sodium chloride, pH 7.5 and min Tris-C1, Komiyama,T., Suda,H., Aoyagi,T., Takeuchi,T. and Umezawa,H. (1975) successively immersed for 60 mmn at room temperature into 50 nM 65ZnC12 Arch. Biochem. Biophys., 171, 727-731. (Amersham, UK; specific activity 575 mCi/mg zinc) in the same buffer. Krieglstein,K.G., Henschen,A.H., Weller,U. and Habermann,E. (1990) Eur. J. Biochem., 188, 39-45. Excess was removed by the paper strips six times with [65Zn2+] washing Laemmli,U.K. (1970) Nature, 227, 680-685. the same buffer. The amount of [65Zn +] bound was determined by Lennarz,W.J. and Strittmatter,W.J. (1981) Biochim. Biophys. Acta, 1071, exposing the dried paper strips to Kodak X-OMAT films at -800C. 149-158. Lin,C.S., Habig,W.H. and Hardegree,M.C. (1985) Infect. lmmun., 49, Tissue preparations and electrophysiological recordings 111-115. Experiments were performed on buccal ganglia of the sea slug Aplysia Matsuda,M. and Yoneda,M. (1975) Infect. Immun., 12, 1147-1153. californica as detailed before (Poulain et al., 1991). Neuronal somata Matthews,B.W. (1988) Acc. Chem. Res., 21, 333-340. (200-300 diameter) were impaled with two glass microelectrodes (3 M Matthews,B.W., Jansonius,J.N., Colman,P.M., Schoenborn,B.P. and ltm 1-5 Mg ). Postsynaptic responses due to ACh release Dupourque,D. (1972) Nature, 238, 37-41. potassium chloride; were induced once a minute by a presynaptic action potential and recorded Mellanby,J., Mellanmy,H, Hawkins,C.A, Rawlins,J.N.P. and Impey,M.E. every 2 min as current changes by conventional two electrode voltage-clamp (1985) J. Physiol. (Paris), 79, 207-215. techniques. By taking into account the reversal potential of the response, Miles,E.W. (1977) Methods Enzymol., 47, 431-442. current values were subsequently expressed as membrane conductance (nS). Mochida,S., Poulain,B., Eisel,U., Binz,T., Kurazono,H., Niemann,H. and At this kind of synapse, ACh release per impulse remains stable for several Tauc,L. (1990) Proc. Natl. Acad. Sci. USA, 87, 7844-7848. hours, even in the absence of superfusion (Poulain et al., 1986). Prior to Montecucco,C. (1986) Trends Biochem. Sci., 11, 314-317. intracellular administration, TeTx L chain samples in 10 mM Tris-HCI, Niemann,H. (1981) In Alouf,J.E. and Freer,J.H. (eds), A Sourcebook of pH 7.8 were mixed with a solution of Fast Green FCF (20% v/v, Sigma) Bacterial Protein Toxins. Academic Press, London, pp. 303-348. to a final L chain concentration of 1 In all experiments, after control Ovadi,J., Libor,S. and Elodi,P. (1967) Acta Biochim. Biophys. Acad. Sci. tiM. were the L chain Hung., 2, 455-458. were in the made, recordings samples injected presynaptic neuron under visual and Final intrasomatic Ozutsumi,K., Sugimoto,N. and Matsuda,M. (1985) Appl. Environ. electrophysiological monitoring. concentration of L chain was - 10 nM. Microbiol., 49, 939-943. 3582 Tetanus toxin and zinc endopeptidase activity Papini,E., Schiavo,G., Sandona,D., Rappuoli,R. and Montecucco,C. (1989) J. Biol. Chem., 264, 12385-12388. Picot,D., Jenkins,J.A., Niklaus-Reimer,A.S. Pauptit,R.A., Karlsson,R., 525-537. and Jansonius,J.N. (1988) J. Mol. Biol., 199, Poulain,B., Baux,G. and Tauc,L. (1986) Proc. Natl. Acad. Sci. USA, 83, 170-173. Poulain,B., Tauc,L., Maisey,E.A., Wadsworth,J.D.F., Mohan,P.M. and Acad. Sci. 4090-4094. Dolly,J.O. (1988) Proc. Natl. USA, 85, Poulain,B., Mochida,M., Wadsworth,J.D.F., Weller,U., Habermann,E., (Paris), 84, 247-261. Dolly,J.O. and Tauc,L. (1990) J. Physiol. Hogy,B., Habermann,E., Wadsworth, Poulain,B., Mochida,S., Weller,U., J. Biol. Chem., 266, J.D.F., Dolly,J.O., Shone,C.C. and Tauc,L. (1991) 1-6. Rappuoli,R. and Pizza,M.G. (1991) In Alouf,J.E. and Freer,J.H. (eds), Academic A Sourcebook ofBacterial Protein Toxins. Press, London, pp. 1-21. Sandvig,K. and Olsnes,S. (1981) J. Biol. Chem., 256, 9068-9076. Schiavo,G., Papini,E., Genna,G. and Montecucco,C. (1990) Infect. Immun., 58, 4136-4141. and Schiavo,G., Ferrari,G., Rossetto,O. Montecucco,C. (1991) FEBSLett., 290, 227-230. Botulinum Neurotoxin and Tetanus Toxin. Simpson,L.L. (ed.) (1989) San Academic Press, Diego. Thayer,M.M., Flaherty,K.M. and McKay,D.B. (1991) J. Biol. Chem., 266, 2864-2871. Towbin,H., Staehelin,T. and Gordon,J. (1979) Proc. Natl. Acad. Sci. USA, 4350-4354. 76, and Auld,D.S. (1990) Biochemistry, 29, 5647-5659. Vallee,B.L. Kester,W.R. and Matthews,B.W. (1977) J. Mol. Biol., 114, Weaver,L.H., 119-132. Weller,U., Mauler,F. and Habermann,E. (1988) Naunyn-Schmiedeberg's Arch. Pharmacol., 338, 99-106. on revised on June 12, 1992 Received April 10, 1992; http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc.

Download PDF
7 pages

Loading next page...
 
/lp/springer-journals/tetanus-toxin-is-a-zinc-protein-and-its-inhibition-of-neurotransmitter-ekf7VTKCth

References (2)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 1992
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1002/j.1460-2075.1992.tb05441.x
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal vol. 1 1 no. 10 pp.3577 - 3583, 1992 Tetanus toxin is a zinc protein and its inhibition of neurotransmitter release and protease activity depend on zinc (BoNT, seven different serotypes) which cause the flaccid Schiavo, Bernard Poulain', Giampietro paralysis of botulism by inhibiting ACh release at the Ornella Rossetto, Fabio Benfenati2, neuromuscular junction (Simpson, 1989). Ladislav Tauc1 and Cesare Montecucco Recently the cDNA-deduced amino acid sequences of five Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, BoNT serotypes have been determined and aligned with Universita di Padova, Via Trieste 75, 35121 Padova, Italy, that of TeTx (Niemann, 1991). Despite the very similar 'Laboratoire de Neurobiologie Cellulaire et Moleculaire, Centre mechanism of action, these neurotoxins show an overall National de la Recherche Scientifique, Gif-sur-Yvette, 91198 France low degree of homology, having a few segments of close 2Istituto di Universita di Modena, Via Campi and Fisiologia Umana, similarity. The most conserved segment is located in the 287, 41100 Modena, Italy central region of the L chain and the common elements Communicated by J.Meldolesi of this peptide are reported in Figure lB. We noticed that this segment includes the HExxH zinc binding motif of Tetanus and botulinum neurotoxins are the most potent metalloendopeptidases. In these enzymes the zinc ion is toxins known. bind to nerve cells, penetrate the They tetra-coordinated and it is essential for their catalytic activity release. cytosol and block neurotransmitter Comparison 1988; Vallee and Auld, 1990). The two histidines (Matthews, reveals a of their predicted amino acid sequences highly of interact with the zinc ion while the glutamic the motif conserved that contains the HExxH zinc binding segment residues a catalytic role. The third ligand is another play motif of metalloendopeptidases. The metal content of glutamic residue whose position in the sequence has been and it was found that tetanus toxin was then measured identified in bacterial neutral zinc endopeptidases (Vallee and zinc is to the chain of tetanus one atom of bound light Auld, 1990), but it is still unidentified in matrix metallo- be removed incubation toxin. Zinc could reversibly by proteases and it is also not evident in sequences of TeTx is coordinated two with heavy metal chelators. Zn2+ by and BoNTs. The fourth ligand is water. with no involvement in cysteines, suggesting histidines In this work we demonstrate that TeTx does indeed contain that it a rather than a structural role. plays catalytic a zinc atom coordinated by two histidines and that the zinc was found to be essential for the tetanus Bound Zn + ion is essential for TeTx inhibition of neurotransmitter release toxin inhibition of neurotransmitter release in Aplysia in neurons. We also report that highly purified Aplysia with the light chain. The intracellular neurons injected preparations of the L chain of TeTx show a proteolytic toxin was blocked a activity of the by phosphoramidon, activity specific for synaptobrevin, an integral membrane Purified very specific inhibitor of zinc endopeptidases. protein of small synaptic vesicles, and that its effect on preparations of light chain showed highly specific neurotransmitter release is inhibited by phosphoramidon, a an proteolytic activity against synaptobrevin, integral inhibitor of zinc endopeptidases. highly specific vesicles. The membrane of small synaptic present protein that tetanus and also the indicate toxin, possibly findings are and that C botulinum neurotoxins, metalloproteases release via this they block neurotransmitter protease H chain activity. release/ Key words: metalloprotease/neurotransmitter N C L chain TI toxin/zinc synaptobrevin/tetanus Introduction toxin is the for the Tetanus (TeTx) agent responsible of an often fatal due development tetanus, spastic paralysis block of neurotransmitter release of the cord to the spinal DPhLH.HLuHxY h uHu uG h hHEh interneurons It is inhibitory (Simpson, 1989). produced by as a strains of Clostridium tetani toxigenic single chain, motifs of tetanus and 1. Schematic structure and zinc Fig. binding at an is cleaved which readily by proteases exposed loop botulinum neurotoxins and of matrix metalloendoproteases. 100 kDa the active two-chain to generate protein (H chain, of the clostridial neurotoxins of tetanus and Schematic structure (A) in IA et and L, kDa), depicted Figure (Krieglstein al., of closest between the six botulism. (B) The segment similarity is involved in cell BoNT D and The H chain neurospecific binding clostridial neurotoxins (TeTx, serotypes A, B, Cl, E) 1990). are known is located in the central is for the whose sequences (Niemann, 1991) while the L chain and responsible penetration to the six clostridial of the L chain. The features common portion of the of a inactivation key component neuroexocytosis amino acid one letter code and with h are in the neurotoxins reported machinery (Montecucco, 1986; Simpson, 1989; Niemann, x for the zinc u for and for hydrophobic, uncharged any residue; and et zinc and is 1991; Schiavo al., 1991). Structurally functionally motif of matrix Auld, 1990) binding endopeptidases (Vallee below. the botulinum neurotoxins aligned just related to TeTx are closely © Press Oxford University G.Schiavo et a/. Vallee and Auld, 1990; Thayer et al., 1991). Results 1988; Chemical modification of TeTx with diethylpyrocarbonate Metal content of TeTx a histidine et al., 1967; (DEPC), modifying reagent (Ovadi To determine the nature and amount of metals bound to Miles, 1977; Papini et al., 1989), indicates that also in TeTx TeTx, the toxin and its L chain were isolated by chromato- are involved in zinc coordination. In fact an histidines graphic methods followed by high performance size of 1.8 histidine out of the six were average residues, present, et Ozutsumi exclusion liquid chromatography (Lin al., 1985; L while this number modified with DEPC in the native chain, et was determined et al., 1985; Schiavo al., 1990). Toxicity varied in the Zn2'-depleted L chain of TeTx according to < 1 and was estimated to be in mice ng/kg) purity (MLD50 An of 1.5 additional the level of residual Zn2+. average >99% by densitometric scanning of SDS -PAGE gels DEPC in L chain preparations histidines were modified by blue. As shown in atomic stained with Coomassie Figure 2, of different of TeTx absorption analysis preparations gave zinc content between 0.70 and 0.85 atoms a range of per and 1.0 zinc atoms TeTx molecule and between 0.9 per the H chain contains no zinc molecule of L chain, while (not below an value is shown). A metal content slightly integer 4,~~~~~~~~~~~~~~~~~~4 with due to metal loss a common finding metalloproteins These values hence indicate the during protein purification. of zinc TeTx molecule and that presence of one atom per 0.06 SzE0aS is associated with the L chain. all the zinc content of TeTx that BoNT Preliminary experiments indicate serotypes A, AS S ig 3.Hsiiemdfcto ihDP fntv n toxin molecule B and E also contain one zinc atom per idepleted et data). Co, Cu, Fe, Mn (G.Schiavo al., unpublished and Ni were also measured and found to be below the limits of detection. +Zn -Zn Zinc removal with heavy metal chelators incubation with the toxin released most of its Upon EDTA, Histidine modification with DEPC of native and Fig. zinc-depleted content bars in This occurred Zn2+ (filled Figure 2). L chain of TeTx. (A) The increase in absorbance at 243 nm is without protein denaturation because Zn2+ could be indicative of the formation of N-carbethoxyhistidine. (B) The number of histidines modified as determined absorbance change incubation in a Zn2+- con- by DEPC, by reassociated with the toxin upon at 243 bars are the SD of three different on L nm; experiments bars in 2 and Zn2+ taining medium (hatched Figures 4). an residual zinc content of No chain preparations with average 19%. could also be removed treatment with other partially by nm of absorbance change was detected at 278 indicative no heavy metal chelating agents such as o-phenanthroline (OP), modification of tyrosine residues. acid dipicolinic (DPA), tetrakis-(2-pyridylmethyl)ethylene- and diamine anhydride (TPEN) diethylenetriaminepenta- acetic anhydride (DTPA). histidines and Chemical modification of cysteines In the zinc two histidines are involved in endopeptidases, zinc coordination et et (Matthews al., 1972; Pauptit al., L chain TeTx 1.0- E- C Q5- 0-i- N EDTA R OP TPEN N EDTA R Df UrTPA Fig. 4. Dot blot assay of [65Zn2+] binding to various samples of L Fig. 2. Zinc content, measured by atomic absorption, of TeTx and of chain and of the Leu230-Gly244 peptide of TeTx. The samples were its L chain treated with various chelators. Amount of zinc, in atoms onto nitrocellulose paper and overlaid with 50 nM 65ZnC12 for applied per protein molecule SD of four different toxin preparations, bound 60 min. After washings and drying, the nitrocellulose paper strips to TeTx and to its L chain before (N, open bars) or after EDTA were exposed at -80°C to a Kodak X-OMAT film. Samples are as treatment (EDTA, filled bars) or after EDTA incubation, dialysis and follows: L+Zn, native L chain; L+Zn+DEPC, native L chain treated further incubation with 50 ZnC12 (R, hatched bars). The effect of with a 30-fold molar excess of DEPC; L-Zn, L chain treated with ,M the following heavy metal chelators is also shown: o-phenanthroline 10 mM EDTA and dialysed; L-Zn+DEPC, L chain depleted of (OP), dipicolinic acid (DPA), tetrakis-(2-pyridylmethyl)ethylenediamine incubation Zn2+ by with EDTA and treated with a 30-fold molar anhydride (TPEN) and diethylenetriaminepentaacetic anhydride excess of DEPC; DT, diphtheria toxin; Peptide, Leu230-Gly244 (DTPA). Co, Cu, Fe, Mn and Ni were also assayed and were found peptide; Peptide+DEPC, peptide treated with a Leu230-Gly244 to be below the limits of detection. Experimental details are in 30-fold molar excess of DEPC. Other experimental details are in Materials and methods. Materials and methods. 3578 Tetanus toxin and zinc endopeptidase activity with an average residual zinc content of 19%, with no [65Zn2"] binding to TeTx light chain and to concomitant formation of o-carbethoxytyrosine (not shown). Leu230- Gly244 fragment Taken together, the results of Figure 3 indicate that two To gain further evidence for the involvement of the in Zn2+ coordination and that bound histidines are involved conserved segment shown in Figure lB in zinc coordination, Zn2+ protects them from reacting with DEPC. the peptide Leu230-Gly244 of TeTx was chemically In many zinc proteins, cysteine residues are involved in synthesized. Figure 4 shows that this peptide binds [65Zn2+] zinc coordination (Vallee and Auld, 1990). However, the in a dot blot test and that this binding is abolished by DEPC same number of sulfhydryl groups (1.3 0.2 per L chain treatment. It would be expected that the peptide binds less molecule) were titrated with Ellman's reagent (5,5'-dithiobis- [65Zn2+] than the TeTx L chain because the peptide lacks 2-nitrobenzoic acid) on the L chain before and after Zn2+ the third zinc ligand present in the native protein. Figure depletion. Hence cysteines are not involved in Zn2+ 4 also shows that the zinc atom of the L chain of TeTx is coordination of TeTx unlike in alcohol dehydrogenase, exchangeable and that, after DEPC modification, the aspartate transcarbamoylase or in the zinc finger of DNA- Zn2+-depleted L chain is unable to reincorporate the zinc binding proteins (Vallee and Auld, 1990). The absence of ion, while a parallel DEPC treatment of the native L chain cysteine coordination emphasizes the similarity of TeTx to does not affect its ability to exchange its zinc atom. This zinc endoproteinases and suggests that the Zn2+ ion of result provides further support for the involvement of TeTx plays a catalytic, rather than a structural role, as coordination. The dot blot histidines in Zn2+ assay in all zinc enzymes with histidine coordination (Vallee used here is very specific since diphtheria toxin, which and Auld, 1990). histidines interspaced by three residues also has three + / TPEN DTPA A0~~~#toA a a 100- LAA04 .0 &O4 % "5 A.-^..' A A . .^s A~ *I, &Ae t0 0%& &db t s OSOZ& i0 ° r~~~~~~~-0)o 4 50- 'IO '9 ' Zn++ depleted 0:.0 -W ~0. 4, V,. native *1 0 60 120 -60 time (min) h. 0 0: 100- 0 o1 i, o'0 0neS*)) Zn " depleted 0 00io0o % , eo 'Po 0 * 0 a DEPC *0 4- 0 IPo e ,+ 4A ',ocjb 0.50 *0 @0 native DEPC treated>\V. f I -80 0 60 120 time (min) release induced intracellular of the L chain of TeTx. Identified of neurons identified 5. Inhibition of by application couples making Fig. acetylcholine in the buccal of were used for the of modified L chain of the TeTx as detailed in ganglion Aplysia califomica assay chemically cholinergic synapses at time zero were: native L chain and its form (19% methods. The L chain A, Zn2'-depleted Materials and samples, injected (arrows) panel DEPC-treated native chain histidines and DEPC-treated L chain (18% residual in this Zn2+ residual Zn2+); panel B, (1.8 modified) Zn-depleted In order to remove ions from both extracellular and intracellular medium, in one series of (panel 3.3 histidines experiments sample, modified). Zn2+ for 100 in the of 50 DTPA and 30 TPEN; DTPA was also was min jtM mixed filled the buccal pre-incubated presence AM A, triangles), ganglion L chain before to a final intracellular concentration of 200 Control showed that, while DTPA and into the injection AM. experiments Zn2'-depleted TPEN are inefficient in zinc removal as shown in zinc the L chain. Figure 2, they prevent re-uptake by 3579 G.Schiavo et al. does not show of zinc endopeptidases (Komiyama et al., 1975; Weaver et al., (His484xxxHisxxxHis492), any sign 1977; Lennarz and Strittmatter, 1991). When [65Zn2 ] binding. phosphor- amidon was injected into Aplysia neurons, as shown in Figure 6, it completely blocked the L chain effect TeTx inhibition of neurotransmitter release in Aplysia on neurons is zinc dependent neurotransmitter release, while by itself the drug was for the intracellular ineffective. The small inhibition of neurotransmitter release The best characterized system testing of clostridial neurotoxins is the identified detectable long after toxin injection is expected because of activity couples in the buccal of diffusion of phosphoramidon out of the nerve cell and of neurons making synapses ganglion Aplysia of toxin et dissociation the -inhibitor complex. Given the high californica (Poulain al., 1988, 1990, 1991; Mochida In this cellular it could be specificity of phosphoramidon inhibition, this result strongly et al., 1990). system directly that the L chain of TeTx is the supports the proposal that TeTx is a zinc endopeptidase. demonstrated intracellularly active portion of the toxin (Mochida et al., 1990; Poulain Protease activity of the light chain of TeTx et al., 1990, 1991). Figure 5A shows that both the native The zinc ion plays a central role in the peptide L chain and the Zn2+-depleted L chain of TeTx were bond neurotransmitter hydrolysis catalysed by zinc proteinases (Matthews, effective in inhibiting the evoked release 1988; level of inhibition. eventually reaching the same Vallee and Auld, 1990). The experiments described above However, the L chain indicate that two histidines are involved in Zn2+ co- the kinetics of inhibition of Zn2+-depleted were if the L chain ordination in TeTx. The putative zinc binding site of TeTx much slower. On the contrary, Zn2+-depleted into a neuron with the is suggested by secondary structure prediction methods to was injected presynaptic pre-treated et no be a-helical with the two histidines on the same face of heavy metal chelators (Arslan al., 1985), inhibitory the for least 3 h ae-helix as is the case for the three zinc proteases whose activity was recorded at (filled triangles in 5B that DEPC treatment of the three-dimensional structure Figure 5A). Figure shows has been resolved (Matthews its with et al., 1972; Pauptit et al., 1988; Thayer et al., native L chain does not modify inhibitory activity 1991). Together with the phosphoramidon inhibition of the intra- respect to the untreated control (Figure 5A). On the contrary, L treated with cellular toxin activity, these similarities suggest that TeTx the chain DEPC after Zn2+ depletion release residual may be a zinc-dependent protease. inhibited neurotransmitter very poorly; As a first could be accounted for the residual approach, we incubated TeTx with a large activity by Zn2+- L chain. number of chromophoric protease model substrates with no containing that the L for These experiments suggest Zn2+-depleted positive findings except a weak protease activity on the its zinc ion from the chain is slowly able to regain general protease substrate casein. As a possible candidate and DEPC for the toxin action cytoplasmic pool, while a Zn2+-depleted we next assayed small synaptic vesicles Zn2+ is for (SSVs), purified from rat cerebral cortex (Huttner et al., modified one is not, and that bound necessary L chain inhibition of neurotransmitter release. 1983; Benfenati et al., 1989). Figure 7 shows that the L the chain of TeTx does not change the SDS -PAGE protein blocks the TeTx inhibition of pattern of highly purified SSVs, except for the disappearance Phosphoramidon in neurons of a single band of 19 kDa with the concomitant appearance neurotransmitter release Aplysia of a 7 kDa and a 12 kDa fragment; the latter peptide overlaps Phosphoramidon [(N-a-L-rhamnopyranosyl-hydroxyphos- of with an SSV component of the same apparent molecular phinyl)-L-leucyl-L-tryptophan] is a very specific inhibitor phosphoramidon 6e, a a , ° W - treated 100- * ^) 0 s.~~~~~~~~~~~~~~ 0og~0Oe-s 0 I~* ~ ~ O OzC ~~~~~~~~ 50- .U *. -0 native L chain ^.- 0* 0% 0W .0 -60 0 60 120 180 time (min) Fig. 6. Effect of phosphoramidon on the intracellular Aplysia neuron activity of the L chain of TeTx. L chain of TeTx (48 pgI/ml) was incubated with 10.7 mM phosphoramidon (Serva) or with buffer for 30 at room temperature. Two presynaptic Aplysia neurons afferent to the same min postsynaptic cell were used. Neuron 1 (open circles) were made 0.37 mM in phosphoramidon and the 1 h recording before time 0 shows that the inhibitor does not by itself affect neurotransmitter release. At time 0 (arrow) neuron 1 was injected with the phosphoramidon-treated L chain while the control neuron 2 (filled circles) was injected with L chain (final concentration 10 nM). The slow recovery of activity of the toxin in neuron 1 with prolonged incubation is to be attributed to diffusion of phosphoramidon in the inhibitor free extracellular medium. 3580 zinc Tetanus toxin and endopeptidase activity 3 of Figure 7). The electrophoretic mobility The lack of a cell line highly sensitive to TeTx, similar weight (lane of the 19 kDa band and its immunoblot staining with an to Vero cells for diphtheria toxin (Sandvig and Olsnes, anti-synaptobrevin monoclonal antibody (Figure 7, lane 1) 1981), represents a major obstacle to the direct demonstration indicate that this protein is synaptobrevin. The H chain did of a proteolytic activity of TeTx and to the identification of not cause any change to the SSV protein profile (not shown). its intracellular target(s). The high specificity of TeTx in vivo The synaptobrevin specific cleavage by the L chain was and the lack of proteolysis in vitro (35 different synthetic completely blocked by EDTA, as expected for a zinc peptide substrates of various proteases assayed) suggested protease (lane 4 of Figure 7), and was only partially that the activity of TeTx should be very specific. inhibited by phosphoramidon (not shown). This latter result Possible targets of TeTx proteolytic activity are at the is probably due to the fact that the very strict binding nerve terminal level. A protein of SSVs, the organelles that requirements of phosphoramidon were not met in the in vitro store and release neurotransmitters, appeared to be a good assay (Weaver et al., 1977; Lennarz and Strittmatter, 1991). candidate. TeTx has been shown to act on the rat brain causing an epileptiform syndrome (Mellanby et al., 1985; Bagetta et al., 1991). Using SSVs isolated from rat cerebral Discussion cortex in a highly purified form (Huttner et al., 1983. Bacteria produce a whole range of protein toxins that are Benfenati et al., 1989), we were able to show that TeTx is their (Alouf and Freer, 1991). Here a very specific zinc endopeptidase since it cleaves only essential to pathogenicity we provide direct evidence that the toxin responsible for all synaptobrevin, out of the many components of the vesicles. the symptoms of tetanus contains one atom of zinc This protein is also expressed in PC12 and adrenal medulla clinical active L chain. No metal is bound which are both sensitive to bound to its intracellularly cells (Baumert et al., 1989), of the toxin responsible for to the H chain, the portion TeTx (Niemann, 1991). membranes (Schiavo et al., 1991) and binding to neuronal Our observations suggest that TeTx acts inside neuronal The between TeTx and for cell penetration. similarity cells by specifically cleaving synaptobrevin. The role of to the amino acid sequence synaptobrevin in neuroexocytosis is still obscure (De Camilli metalloproteases is not confined of zinc atom. Here we have also and Jahn, 1990), but the present findings suggest that this and to the presence the zinc is coordinated via two histidines and that protein plays a key role in the process of neurotransmitter shown that Leu230-Gly244, which includes the zinc release. In this respect, it is noteworthy that all bacterial the TeTx peptide motif of binds zinc. The protein toxins with intracellular targets act on key elements binding metalloendopeptidases, zinc is indicative of a of regulation such as G proteins (for cholera, histidine involvement in binding cell physiology of toxin bound metal and this is in or (for catalytic role the pertussis and botulinum C3 toxins) protein synthesis in vivo. In when and Pizza, 1991; with the data obtained fact, only diphtheria and Shiga toxins) (Rappuoli agreement L chain able to inhibit Boquet and Gill, 1991). zinc is bound to the toxin is the in neurons, as clearly shown or activation of important biological neurotransmitter release Aplysia Regulation the experiments of Figure 5. phenomena via highly selective proteolytic cleavage is by not uncommon, e.g. blood coagulation or complement activation. The protease activity of TeTx, described here, is consistent with its known specificity and potency. An z -3 intracellular catalytic activity of TeTx accounts for its -I.. extreme because one molecule of toxin would be potency _ Tm`_ A&: sufficient to inactivate a large number of synaptobrevin- _i containing vesicles. *It Our findings provide the first explanation at the molecular level of a high incidence neuroparalytic disease such as tetanus and it new for the development opens perspectives 4. 4* of based on inhibitors of the zinc therapeutic protocols Moreover, our data may become of protease activity. -..4 for the dissection of the molecular paramount importance of neurotransmitter release. events underlying the process methods Materials and SYB:> Toxin and fragments were isolated from culture filtrates of Clostridium TeTx and the L chain Ozutsumi et Schiavo before et 1985; tetani, as detailed (Lin al., 1985; al., in 10 mM 50 mM sodium and stored at -800C HEPES-Na, et al., 1990), H chain was as described Matsuda 7.2. The by chloride, pH prepared and Yoneda (1975). of metal content Determination with with a were rinsed water, All materials Milli-Q grade previously is cleaved the L chain of Fig. Synaptobrevin proteolytically by were with chemicals of the > 10 Buffers MQ prepared highest conductibility blue stained SDS-PAGE of Lanes 2-4 show Coomassie TeTx.- the of metals and with to available respect presence heavy pre-treated incubated alone or in the purity SSV 2) of (lane samples purified (35 Ag) Before metal the MB-3 determination, with Amberlite (Sigma). protein L chain or with L chain with of TeTx (lane 3) pretreated presence at 150 mM Tris-Cl, were samples extensively dialysed 4°C against pH Lane refers to a of SSV at 1 EDTA for 15 min 37°C (lane 4). sample boiled 100 mM sodium 7.4 or 10 mM chloride, pH 7.0, using HEPES-Na, monoclonal with the immunoblotted antibody IgGi anti-synaptobrevin were for Zn, Co, Cu, dialysis tubing (Spectrum, TX). Samples analysed et Cl 10.1 1989). (Baumert al., 3581 et al. G.Schiavo Fe, Mn and Ni with a Perkin -Elmer 4000 atomic absorption flame of TeTx Proteolytic activity with impact bed loading. Metal contents were measured from rat cerebral cortex et spectrophotometer 35 of SSVs purified (Huttner al., 1983; jig after standardization in the linear range of concentration for each ion (0-0.5 Benfenati et al., 1989) were incubated with 1 of TeTx L chain in 270 Ag Al p.p.m. for Zn, Ni and Co; 0-1 p.p.m. for Cu; 0-5 p.p.m. for Fe and Mn). M 7.4. contained of 5 mM HEPES-Na, 0.3 glycine, pH Some samples 10 mM EDTA. After 30 min at 370C, 30 50% TCA were added and, Zinc removal were dissolved in after 30 min at room temperature, centrifuged. Pellets L chain were diluted to 0.5-1.0 mg/ml with 150 mM Tris-Cl, electrophoresis sample buffer 5 mM EDTA and 5 TeTx or the containing % 2-mercapto- 7.4, and incubated in the presence of 10 mM EDTA for 60 min at 37°C. ethanol and boiled for 2 min. were in 12% pH Samples electrophoresed were dialysed extensively against the same buffer at 4°C and their was carried out Samples polyacrylamide gels (Laemmli, 1970). Immunoblotting zinc content was determined as above. Concentrations of other chelators according to Towbin et al. (1979), using the IgGi anti-synaptobrevin used are as follows: 10 mM o-phenanthroline (Erba, Milan), 50 mM monoclonal antibody Cl 10.1 (Baumert et al., 1989) and revealed by 30 tetrakis-(2-pyridylmethyl)ethylenediamine antibodies. dipicolinic acid (Fluka), uM anti-mouse alkaline phosphatase coupled secondary Probes, Eugene, OR), 200 anhydride (Molecular diethylenetriamine- lsM pentaacetic anhydride (Molecular Probes). Toxicity tests The toxicity of TeTx was determined by injecting intraperitoneally serial dilutions of the toxin with phosphate-buffered saline containing 0.1% BSA. Peptide synthesis to the sequence LLMHELIHVLH- Time before death was recorded TeTx segment 230-244 corresponding and mouse lethal doses were calculated Dr A.Santucci (University of Siena) by solid as described (Weller GLYG was kindly prepared by et al., 1988). 350 automatic synthesizer (Zynsser Analytic, phase synthesis with a SMPS The product was detached from Frankfurt) employing F-moc chemistry. Acknowledgements the resin with 93% TFA, purified by HPLC on a Vydac column and C18 on an Applied Biosystems controlled by automatic Edman degradation We thank V.Albergoni and F.Cattalini, Department of Biology, University 475A microsequencer. of Padua, for the atomic absorption measurements, S.Fabbiani of SCLAVO Spa for samples of culture filtrates of Clostridium tetani, P.De Camilli, DEPC modification Department of Cell Biology, Yale University, for generously supplying the 50 mM disaerated sodium phosphate The L chain of TeTx was diluted with anti-synaptobrevin monoclonal antibody Cl 10.1, T.Pozzan for advice on were filtered 50 mM, pH 7.8 to a final concentration of 7.0 uM. The samples the use of TPEN and DTPA, and A.Fontana, E.Papini and T.Pozzan for through a 0.22 filter (Anotec, Oxford, UK) and DEPC from a freshly ltm critically reading the manuscript. This work was supported by grants The DEPC/L chain ratio prepared solution in absolute ethanol was added. from CNR Target Project 'Biotechnology and Bioinstrumentation', from 30 mol/mol L chain. The reaction was followed at 25°C never exceeded Association Francaise contre les Myopathies, and from Direction des the differential absorbance at 243 nm and at by simultaneously recording Recherches et Etudes Techniques (granted to L.T.) and it is in partial 1977; Papini et al., 1989) in a 278 nm as previously described (Miles, fulfilment of the doctorate degree of the University of Padua in 'Molecular Modified residues were Perkin-Elmer Lambda 5 spectrophotometer. and Cellular Biology and Pathology' of O.R. coefficients of 3200 M- l cm-1 at estimated with differential extinction nm and 1310 M-1 cm-' at 278 nm for 243 for N-carbethoxyhistidine o-carbethoxytyrosine. References thiol groups Titration of free Alouf,J.E. and Freer,J.H. (eds) (1991) A Sourcebook ofBacterial Protein or L chain were diluted to 1 yM with 50 mM Native zinc-depleted samples Toxins. Academic Press, London. 7.8. Subsequently 10 of DTNB Arslan,P., Di Virgilio,F., Beltrame,M., Tsien,R.Y. and Pozzan,T. (1985) disareated sodium phosphate, pH 1z1 J. Biol. Chem., 260, in 50 mM sodium pH 8.0; final concentration 0.4 mM) 2719-2727. (5 mg/mi phosphate, Bagetta,P., and Bowery,N.G. (1991) Trends Pharmacol. Nistic6,G. Sci., at to the and the absorbance at 410 nm was were added 25°C samples 12, 285-289. content was calculated determined against a blank without protein. Sulfhydryl Baumert,M., Maycox,P.R., Navone,F., Camilli,P. De and Jahn,R. (1989) on the basis of molar absorptivity of 13 600 M-l cm-1 at 412 nm for EMBO J., 8, - 379 384. 2-nitro-5-thio-benzoate anion and expressed as moles of free thiol groups Benfenati,F., Bahler,M., Jahn,R. and Greengard,P. (1989) J. Cell Biol., mole of L chain (Ellman, 1959; Schiavo et al., 1990). per 108, 1863-1872. Boquet,P. and Gill,D.M. (1991) In Alouf,J.E. and Freer,J.H. (eds), Sourcebook of Bacterial Protein Toxins. Academic Press, London, pp. Determination of [65Zn2+] binding 23-44. L chains in their native or zinc-depleted forms, treated or not with 1:30 De Camilli,P. and Jahn,R. (1990) Annu. Rev. Physiol., 52, 625-645. molar excess of DEPC as above, or diphtheria toxin (2-32 pmol) or the Ellman,G.L. (1959) Arch. Biochem. Biophys., 82, 70-77. zinc binding peptide (25-400 pmol) were dot-blotted onto putative Huttner,W.B., Schiebler,W., Greengard,P. and De Camilli,P. (1983) J. nitrocellulose strips (0.22 Hoefer, CA). The strips were rinsed paper 96, 1374-1378. ltm; Cell Biol., for 5 in 25 mM 100 mM sodium chloride, pH 7.5 and min Tris-C1, Komiyama,T., Suda,H., Aoyagi,T., Takeuchi,T. and Umezawa,H. (1975) successively immersed for 60 mmn at room temperature into 50 nM 65ZnC12 Arch. Biochem. Biophys., 171, 727-731. (Amersham, UK; specific activity 575 mCi/mg zinc) in the same buffer. Krieglstein,K.G., Henschen,A.H., Weller,U. and Habermann,E. (1990) Eur. J. Biochem., 188, 39-45. Excess was removed by the paper strips six times with [65Zn2+] washing Laemmli,U.K. (1970) Nature, 227, 680-685. the same buffer. The amount of [65Zn +] bound was determined by Lennarz,W.J. and Strittmatter,W.J. (1981) Biochim. Biophys. Acta, 1071, exposing the dried paper strips to Kodak X-OMAT films at -800C. 149-158. Lin,C.S., Habig,W.H. and Hardegree,M.C. (1985) Infect. lmmun., 49, Tissue preparations and electrophysiological recordings 111-115. Experiments were performed on buccal ganglia of the sea slug Aplysia Matsuda,M. and Yoneda,M. (1975) Infect. Immun., 12, 1147-1153. californica as detailed before (Poulain et al., 1991). Neuronal somata Matthews,B.W. (1988) Acc. Chem. Res., 21, 333-340. (200-300 diameter) were impaled with two glass microelectrodes (3 M Matthews,B.W., Jansonius,J.N., Colman,P.M., Schoenborn,B.P. and ltm 1-5 Mg ). Postsynaptic responses due to ACh release Dupourque,D. (1972) Nature, 238, 37-41. potassium chloride; were induced once a minute by a presynaptic action potential and recorded Mellanby,J., Mellanmy,H, Hawkins,C.A, Rawlins,J.N.P. and Impey,M.E. every 2 min as current changes by conventional two electrode voltage-clamp (1985) J. Physiol. (Paris), 79, 207-215. techniques. By taking into account the reversal potential of the response, Miles,E.W. (1977) Methods Enzymol., 47, 431-442. current values were subsequently expressed as membrane conductance (nS). Mochida,S., Poulain,B., Eisel,U., Binz,T., Kurazono,H., Niemann,H. and At this kind of synapse, ACh release per impulse remains stable for several Tauc,L. (1990) Proc. Natl. Acad. Sci. USA, 87, 7844-7848. hours, even in the absence of superfusion (Poulain et al., 1986). Prior to Montecucco,C. (1986) Trends Biochem. Sci., 11, 314-317. intracellular administration, TeTx L chain samples in 10 mM Tris-HCI, Niemann,H. (1981) In Alouf,J.E. and Freer,J.H. (eds), A Sourcebook of pH 7.8 were mixed with a solution of Fast Green FCF (20% v/v, Sigma) Bacterial Protein Toxins. Academic Press, London, pp. 303-348. to a final L chain concentration of 1 In all experiments, after control Ovadi,J., Libor,S. and Elodi,P. (1967) Acta Biochim. Biophys. Acad. Sci. tiM. were the L chain Hung., 2, 455-458. were in the made, recordings samples injected presynaptic neuron under visual and Final intrasomatic Ozutsumi,K., Sugimoto,N. and Matsuda,M. (1985) Appl. Environ. electrophysiological monitoring. concentration of L chain was - 10 nM. Microbiol., 49, 939-943. 3582 Tetanus toxin and zinc endopeptidase activity Papini,E., Schiavo,G., Sandona,D., Rappuoli,R. and Montecucco,C. (1989) J. Biol. Chem., 264, 12385-12388. Picot,D., Jenkins,J.A., Niklaus-Reimer,A.S. Pauptit,R.A., Karlsson,R., 525-537. and Jansonius,J.N. (1988) J. Mol. Biol., 199, Poulain,B., Baux,G. and Tauc,L. (1986) Proc. Natl. Acad. Sci. USA, 83, 170-173. Poulain,B., Tauc,L., Maisey,E.A., Wadsworth,J.D.F., Mohan,P.M. and Acad. Sci. 4090-4094. Dolly,J.O. (1988) Proc. Natl. USA, 85, Poulain,B., Mochida,M., Wadsworth,J.D.F., Weller,U., Habermann,E., (Paris), 84, 247-261. Dolly,J.O. and Tauc,L. (1990) J. Physiol. Hogy,B., Habermann,E., Wadsworth, Poulain,B., Mochida,S., Weller,U., J. Biol. Chem., 266, J.D.F., Dolly,J.O., Shone,C.C. and Tauc,L. (1991) 1-6. Rappuoli,R. and Pizza,M.G. (1991) In Alouf,J.E. and Freer,J.H. (eds), Academic A Sourcebook ofBacterial Protein Toxins. Press, London, pp. 1-21. Sandvig,K. and Olsnes,S. (1981) J. Biol. Chem., 256, 9068-9076. Schiavo,G., Papini,E., Genna,G. and Montecucco,C. (1990) Infect. Immun., 58, 4136-4141. and Schiavo,G., Ferrari,G., Rossetto,O. Montecucco,C. (1991) FEBSLett., 290, 227-230. Botulinum Neurotoxin and Tetanus Toxin. Simpson,L.L. (ed.) (1989) San Academic Press, Diego. Thayer,M.M., Flaherty,K.M. and McKay,D.B. (1991) J. Biol. Chem., 266, 2864-2871. Towbin,H., Staehelin,T. and Gordon,J. (1979) Proc. Natl. Acad. Sci. USA, 4350-4354. 76, and Auld,D.S. (1990) Biochemistry, 29, 5647-5659. Vallee,B.L. Kester,W.R. and Matthews,B.W. (1977) J. Mol. Biol., 114, Weaver,L.H., 119-132. Weller,U., Mauler,F. and Habermann,E. (1988) Naunyn-Schmiedeberg's Arch. Pharmacol., 338, 99-106. on revised on June 12, 1992 Received April 10, 1992;

Journal

The EMBO Journal – Springer Journals

Published: Oct 1, 1992

There are no references for this article.