Artikel Tetanus

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Molecular Microbiology (1994) 13(1), 1-8 MicroReview Mechanism of action of tetanus and botulinum neurotoxins Cesare Montecucco* and Giampietro Schiavo Centro CNR Biomembrane and Dipartimento di Scienze Biomediche, Universita di Padova, via Trieste 75, 35121 Padova,  Italy. Summary The clostridiai neurotoxins responsibie for tetanus and botuiism are metalio-proteases that enter nerve cells and biock neurotransmitter release via zinc- dependent cleavage ot protein components o1 the neuroexocytosis apparatus. Tetanus neurotoxin (TeNT) binds to the presynaptic membrane of the neuromuscular Junction and is internaiized and trans- ported retroaxonally to the spinai cord. Whilst TeNT causes spastic paralysis by acting on the spinai inhibi- tory interneurons, the seven serotypes of botulinum neurotoxins (BoNT) induce a fiaccid paralysis because they intoxicate the neuromuscular junction. TeNT and BoNT serotypes B, D, F and G specificaily cleave VAMP/synaptobrevIn, a membrane protein of smaii synaptic vestcies, at different single peptide bonds. Proteins of the presynaptic membrane are specificaily attacked by the other BoNTs: serotypes A and E cleave SNAP-25 at two different sites located within the carboxyi terminus, whereas the specific target of serotype C is syntaxin. Introduction About one century has elapsed since protein neurotoxins produced by clostridia were identified as the cause of the paralytic syndromes of tetanus and botuiism (Carle and Rattone, 1884; Faber, 1890; Tizzoni and Cattani, 1890; van Ermengem, 1887). As in the case of diphtheria, this led to disease prevention by injection of formaldehyde- treated toxins: completely atoxic and highly immunogenic derivatives capable of eliciting very elevated antitoxin titres (van l-leyningen, 1968; Simpson, 1989). The effec- tiveness of these vaccines has lowered the interest of clinical microbiologists, and the subject has been taken Received 25 January, 1994; revised 24 March, 1994; accepted 28 March, 1994. 'For correspondence. Tei. (49) 8286556; Fax (49) 8286576. over by molecular and cell biologists. This new era was opened with the demonstration that botulinum neurotoxins (BoNTs) block the release of acetylcholine at the neuro- muscular junction (Burgen  etal. 1948). A spectacular con- sequence of this finding is that BoNTs are now currently used as therapeutic agents in the treatment of a variety of dystonias (Jankovic and Hallett, 1994). In contrast to BoNTs, tetanus neurotoxin (TeNT) blocks neurotrans- mitter release in the inhibitory intemeurons of the spina cord, which results in a spastic paralysis (van Heyningen 1968). Hence, despite the opposite clinical symptoms o tetanus and botulism, their aetiological agents intoxicate neuronal cells in the same way (Simpson, 1986; 1989) This has unified research on these neurotoxins, and has led to the finding that they have a very similar structura organization. More recently their intracellular catalytic activity was discovered, and was shown to be identica for TeNT and BoNTs. They are zinc-endopeptidases acting specifically on protein components of the same cellular machine: the neuroexocytosis apparatus (Schiavo etal. 1993d; Montecucco and Schiavo, 1993). Structurai features All toxigenic strains of  Clostridium tetani  produce only one type of TeNT, while different species of clostridia (botulinum, barati and butyricum)  produce seven differen serotypes of BoNT (A, B, C, D, E, F, and G). These neuro- toxins are synthesized as a single inactive polypeptide chain of 150kDa and released by bacterial lysis. Bacteria or tissue proteases cleave them within an exposed loop and generate the active di-chain neurotoxins composed of a heavy chain (H, 100 kDa) and  a  light chain (L, 50kDa bridged by a single interchain disulphide bond (Fig. 1). Structural and biochemical data (Robinson  et ai,  1988 Simpson, 1989; Wellhoner, 1992; Montecucco and Schiavo, 1993; Schiavo  etal. 1993d) and some similarities with other toxins of known three-dimensional stnjcture, such as diphtheria toxin and  Pseudomonas aeruginosa exotoxin A (Choe  etal. 1992; Allured  etal. 1986), indicate that clostridiai neurotoxins are folded into three functionally distinct domains, which play different roles in cell intoxica- tion (Fig. 1). Domain L is responsible for the blockade of neuroexocytosis, domain  HN,  the 50 kDa amino-temiina

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Transcript of Artikel Tetanus

  • Molecular Microbiology (1994) 13(1), 1 -8

    MicroReviewMechanism of action of tetanus and botulinumneurotoxins

    Cesare Montecucco* and Giampietro SchiavoCentro CNR Biomembrane and Dipartimento diScienze Biomediche, Universita di Padova, viaTrieste 75, 35121 Padova, Italy.

    Summary

    The clostridiai neurotoxins responsibie for tetanusand botuiism are metalio-proteases that enter nervecells and biock neurotransmitter release via zinc-dependent cleavage ot protein components o1 theneuroexocytosis apparatus. Tetanus neurotoxin(TeNT) binds to the presynaptic membrane of theneuromuscular Junction and is internaiized and trans-ported retroaxonally to the spinai cord. Whilst TeNTcauses spastic paralysis by acting on the spinai inhibi-tory interneurons, the seven serotypes of botulinumneurotoxins (BoNT) induce a fiaccid paralysisbecause they intoxicate the neuromuscular junction.TeNT and BoNT serotypes B, D, F and G specificailycleave VAMP/synaptobrevIn, a membrane protein ofsmaii synaptic vestcies, at different single peptidebonds. Proteins of the presynaptic membrane arespecificaily attacked by the other BoNTs: serotypesA and E cleave SNAP-25 at two different sites locatedwithin the carboxyi terminus, whereas the specifictarget of serotype C is syntaxin.

    Introduction

    About one century has elapsed since protein neurotoxinsproduced by clostridia were identified as the cause of theparalytic syndromes of tetanus and botuiism (Carle andRattone, 1884; Faber, 1890; Tizzoni and Cattani, 1890;van Ermengem, 1887). As in the case of diphtheria, thisled to disease prevention by injection of formaldehyde-treated toxins: completely atoxic and highly immunogenicderivatives capable of eliciting very elevated antitoxintitres (van l-leyningen, 1968; Simpson, 1989). The effec-tiveness of these vaccines has lowered the interest ofclinical microbiologists, and the subject has been taken

    Received 25 January, 1994; revised 24 March, 1994; accepted 28March, 1994. 'For correspondence. Tei. (49) 8286556; Fax (49)8286576.

    over by molecular and cell biologists. This new era wasopened with the demonstration that botulinum neurotoxins(BoNTs) block the release of acetylcholine at the neuro-muscular junction (Burgen etal., 1948). A spectacular con-sequence of this finding is that BoNTs are now currentlyused as therapeutic agents in the treatment of a varietyof dystonias (Jankovic and Hallett, 1994). In contrast toBoNTs, tetanus neurotoxin (TeNT) blocks neurotrans-mitter release in the inhibitory intemeurons of the spinalcord, which results in a spastic paralysis (van Heyningen,1968). Hence, despite the opposite clinical symptoms oitetanus and botulism, their aetiological agents intoxicateneuronal cells in the same way (Simpson, 1986; 1989).This has unified research on these neurotoxins, and hasled to the finding that they have a very similar structuralorganization. More recently their intracellular catalyticactivity was discovered, and was shown to be identicalfor TeNT and BoNTs. They are zinc-endopeptidasesacting specifically on protein components of the samecellular machine: the neuroexocytosis apparatus (Schiavoetal., 1993d; Montecucco and Schiavo, 1993).

    Structurai features

    All toxigenic strains of Clostridium tetani produce onlyone type of TeNT, while different species of clostridia(botulinum, barati and butyricum) produce seven differentserotypes of BoNT (A, B, C, D, E, F, and G). These neuro-toxins are synthesized as a single inactive polypeptidechain of 150kDa and released by bacterial lysis. Bacterialor tissue proteases cleave them within an exposed loopand generate the active di-chain neurotoxins composedof a heavy chain (H, 100 kDa) and a light chain (L, 50kDa)bridged by a single interchain disulphide bond (Fig. 1).Structural and biochemical data (Robinson et ai, 1988;Simpson, 1989; Wellhoner, 1992; Montecucco andSchiavo, 1993; Schiavo etal., 1993d) and some similaritieswith other toxins of known three-dimensional stnjcture,such as diphtheria toxin and Pseudomonas aeruginosaexotoxin A (Choe etal., 1992; Allured etal., 1986), indicatethat clostridiai neurotoxins are folded into three functionallydistinct domains, which play different roles in cell intoxica-tion (Fig. 1). Domain L is responsible for the blockade ofneuroexocytosis, domain HN, the 50 kDa amino-temiinal

  • 2 C. Montecucco and G. Sctiiavo

    Pig. 1. Structure-function reiationships ofclostridial neurotoxins. The 50i(Dacartx>xy-terminal domain of the H chain (He)of dostrldial neurotoxins is mainly responsiblefor neurospeclfic binding to their respectivereceptors locaiized on the surface of thepresynaptic membrane (top-left panel).Productive binding is followed by theintemaiization of the toxin-receptor complexinside vesicles that are different for tetanusand ix)tulinum neurotoxins. BoNT-containingvesicles remain within the neuromuscuiarjunction, while TeNT-containing vesicles movebackwards along the motor neuron axon tothe soma. The amino-terminal domain (HN) issuggested to be Involved in the membranetranslocation of the L chain into the nen/e-ceiiCytoplasm (top-right panel). This process Istriggered by the acidification of the vesiclelumen by a proton-pumping ATPase whichleads to a conformational change of the toxin.In its acid conformation, the toxin Inserts Intothe llpid biiayer and H N promotes thetranslocation of the L chain across thevesicular membrane. The L chain, set free inthe cytosol by reduction of the interchaindisulphide bond, can display itszinc-endopeptidase activity by the seiectlvecieavage of proteins of the neuroexocytosismachinery (lower panel).

    haif of the H chain, appears to govern cell penetration,and domain He, the 50 kDa cartx>xyl-terminal half of the Hchain, seems to be mainly responsible for the neuro-specific binding.

    The mechanism of cell intoxication with clostridial neuro-toxins has been proposed to consist of three steps: cellbinding, intemaiization, and cytosoiic activity (Simpson,1986). However, on the basis of recent findings on the cellpenetration of other toxins (Ogata et al., 1990; Sandvigetal., 1992; Paplnie/a/., 1993) and of some aspects of theintracellular trafficking of TeNT (see below), we proposethat the cell intoxication of TeNT and BoNTs is more appro-priately described by a four-step process: (i) cell binding,(ii) intemaiization, (iii) membrane translocation, and (iv)target modification in the cytosol.

    Cell binding

    Clostridial neurotoxins bind specifically to nerve cells.Because of the central importance of the nervous systemin the physiology and behaviour of vertebrates, thisabsolute tissue specificity is the basis of their enormouspotency. Mouse LD50 (lethal dose, 50%) ranges between0.1 and 1 ng kg ~' for the various neurotoxins, but It shouldbe pointed out that the amount of toxin needed to cause

    death in the wild is probably much lower. Indeed, evena small decrease in fitness, such as diplopia, an earlyclinical symptom of botulism, is sufficient to precludesurvival of the intoxicated animai outside the laboratory.

    The lack of neurotoxin-sensitive cell lines with largenumbers of high-affinity toxin receptors has preventedthe identification of the molecular entities involved inTeNT and BoNT binding. From available data the follow-ing main points emerge: (i) TeNT and BoNT receptorsare located on the motor neuron plasmalemma at theneuromuscuiar junction (NMJ) (Wemig etal., 1977; Dollyet al., 1984); (ii) these receptors must display highaffinities if they are to bind the minute concentrations(sub-picoMolar) of neurotoxins sufficient to cause death;(iii) polysialogangliosides, the first suggested receptors(van Heyningen, 1968), are unlikely to be solely respon-sible for neurospecific binding, as discussed in detail else-where (Mellanby and Green, 1981; Montecucco, 1986);(iv) different receptors must be implicated in the binding ofTeNT and BoNT to account for the different intracellularrouting of these toxins inside motor neurons (see below)and for the different sensitivity of animal species to thedifferent BoNTs (Payling-Wright, 1955). Indeed, Blackand Dolly (1986a) found that NMJ receptors for BoNT/Aand BoNT/B are different.

  • Metalloprotease activity of dostridiai neurotoxins 3

    Recently, TeNT was found to interact specificaily, via theHe domain, with a 20 l

  • 4 C. Montecucco and G. Schiavo

    Table 1. Comparison of the dostridiai neuro-toxin ion channels in planar iipid biiayers withthose of a protein-oorKlucting channei of theendopiasmic reticulum. Property

    Conductance (pS)Ion selectivitySize of

    permeant ionpH dependenceVoltage dependence

    of conductance

    TeNT*

    45X*>Glucosamine

    4.0-5.0+

    BoNT/B"

    15X*NO

    4.0-5.0+

    BoNT/C"

    J5X*>Glucosamlne

    6.1

    Endoplasmicreticulumchannel'

    220Non-selective>Glucosamine

    ND-

    a. Hoch etal. (1985).b. Donovan and Middlebrook (1986).c. Simon and Blobei (1991).

    its properties are different from those of the channelinvolved in the translocation of nascent polypeptide chainacross the endoplasmic reticulum membrane (Simon andBlobei, 1991). Hence, at this stage, it must be concludedthat either the neurotoxin ion channel is an epipheno-menon not involved in membrane translocation of theL chain or that it does participate in the process andwhat is seen in model membranes is just a remnantof the channel that has driven the L chain throughthe membrane. This point has been discussed indetail elsewhere, with particular emphasis on diphtheriatoxin (the prototype of bacterial toxins entering cellsvia a low-pH intracellular compartment), for whichmore data are available (Montecucco etal., 1991; 1992).As for diphtheria toxin (Papini etal, 1993), the interchaindisulphide bond of TeNT and BoNTs plays a crucial rolein cell penetration (Schiavo et a/., 1990; de Paiva et al.,1993a).

    Zinc-endopeptidase activity and biocking ofneurotransmltter release

    The intracellular activity of TeNT and BoNTs is due to theirL domain (Ahnert-Hilger et al, 1989; Bittner et al, 1989;Mochida etal, 1989). Within a few years of the publicationof the sequence of TeNT (Eisel et al, 1986; Fairweatherand Lyness, 1986), the nucleotide sequences of allBoNT serotypes became available (Binz et ai, 1990a,b;Hauser et al, 1990; Thompson et al, 1990; 1993; Eastetal., 1992; Poulet etal, 1992; Wheian etal, 1992a,b;Campbell etal, 1993; Willems etal, 1993). The L chainsshow a low degree of amino acid sequence similaritylimited to segments scattered along the chain. The longestof these segments is in the middle of the L chain(Fig. 2) and contains the zinc-binding motif of zinc-endopeptidases His-Glu-X-X-His (Vallee and Auld,1990; Bode et al, 1993). Indeed, physico-chemicalmeasurements demonstrated that clostridial neurotoxinscontain one atom of zinc per molecule of toxin, bound tothe L chain with affinities in the 50-100 nM range.

    comparable to those of known zinc-proteinases (Schiavoetal, 1992a,c; 1993a).

    Zinc-endopeptidases can be divided into two groupsdepending on the involvement of two or three histidinesin zinc co-ordination, and on the presence of Gly andMet tums (Bode et al., 1993). In all cases, one waterrrralecule is the additional zinc ligand responsible for thehydrolysis of the peptide bond. Chemical modification,site-directed mutagenesis and spectroscopic investi-gations (Schiavo et al., 1992a,c; Dayanithi et al, 1994;Hohne-Zell ef a/., 1993; our unpublished results) indicatethat clostridial neurotoxins bind zinc via two histidinesand a glutamic acid, which is similar to thermolysin.

    The intracellular activity of clostridial rmiroXoxms wasrecently identified as a zinc-dependent specific proteoly-sis of components of the neuroexocytosis apparatus(Schiavo et al, 1992a,b; 1993a,b,c,d; Blasi et al,1993a,b; Montecucco and Schiavo, 1993; Hohne-Zellet al, 1993). The activity of TeNT was shown to dependstrictly on the zinc atom, but this metal could be sub-stituted by Ni^* and Co^* (Schiavo et al, 1992a;

    BoNT/ABoNT/A INFANTBoNT/BBoNT/B NPBoNT/CBoNT/DBoNT/EBoNT/E BUTYRICUMBoNT/FBONT/F BARATIBoNT/GTEHT

    ZN-CNOOPEPTIOASES

    F A TF A TYYF C

    F S DF S 0M D

    F C H DF I 0 D

    Q DA 0

    F IF IF IYY

    A DF A DF Q 0

    T L A HT L A H1 L H H

    L H HH HH H

    L T L M H

    A VA VA LA L II L I LV I A LA L T LAA I S L A HA I S L A H

    H HM H

    E L I H A G H R L Y 6E L I H A E H R L Y G

    L I H V L H G L Y 6L I H V L H G L Y 6

    EEE LEE

    N H A H H N L YL rL I

    S LS L

    H Q L Y 6H G L Y

    E L I H S L H G L YE LE L

    I H A L H G

    T LL L

    E L IE L I

    H G LH G L

    L YYH V L

    H V LH V L H G L Y G

    H E X X H

    Fig. 2. The histidine-rich segment of the central part of the L chainsof clostridial neurotoxins contairw the zinc-binding motif ofmetallo-endopeptidases. The most conserved segment of the sevenBoNT serotypes, plus BoNT/A associated with infant botuiism(BoNT/A in^tnt), BoNT/B produced from a non-proteoiytic strain ofC. botuiinum (BoNT/B NP), BoNT/E and BoNT/F produced fromC. butyricum and C. baratl. respectively, and TeNT are aligned. Allof them contain the structural motif of zinc-endopeptidases. Starsindicate residue identity and dots indicate conservation of residueproperties.

  • Table 2. Tetanus and botulism neurotoxins:gene location, target, and peptide bondspecificities.

    Metalloprotease activity of ciostridial neurotoxins 5

    Toxin

    TeNTBoNT/ABoNT/BBoNT/CBoNT/DBoNT/HBoNT/FBoNT/G

    Gene location

    PiasmidChromosomeChromosomeBacteriophageBacteriophageChromosomeChromosome/phagePiasmid

    Neuronal target

    VAMPSNAP-25VAMPSyntaxinVAMPSNAP-25VAMPVAMP

    Peptide Bond Cleaved

    P2-P,-P,-P2'

    Ser-Gln-Phe-GluAsn-Gln-Arg-AlaSer-Gln-Phe-Glu?GIn-Lys-Leu-SerAsp-Arg-lle-MetAsp-Gln-Lys-LeuSer-Ala-Ala-Lys

    This table summarizes data from: Ekiund etal. (1989); Schiavo etal. (1992a,b); (1993a.b,c); inpreparation; Blasi etal. (1993a,b).

    Hohne-Zell et al., 1993; our unpublished results). TeNTand BoNT/B, -/D, -/F and -/G share a common intra-neuronal target; they specifically recognize and cleaveVAMP/synaptobrevin (VAMP, vesicle-associated mem-brane protein). BoNT/A and -/E, on the other hand,cause the specific hydroiysis of SNAP-25 (synaptosomal-associated protein of 25 kDa), while serotype C cleavessyntaxin. With the exception of TeNT and BoNT/B,each of the different clostridial neurotoxins catalyses thehydrolysis of different peptide bonds, as summarized inTable 2.

    An important feature of the zinc-endopeptidase activityof clostridial neurotoxin is that they do not cleave shortpeptides containing the cleavage site of their targetproteins. Only long peptides are cleaved by BoNT/B(Shone et al.. 1993) thus suggesting that these proteasesrecognize other segments and/or that they only bind andcleave the folded target molecule.

    Together with their neurospecificity, the zinc-endopeptidase activity of TeNT and BoNT explains theirextraordinary potency. It has b>een estimated that, intoxin-injected Aplysia neurons, 4-10 molecules of Lchains are sufficient to cause the biockade of neuro-transmitter release with a ti/2 of 20-40 min at 20"C(B. Poulain, personal communication). In view of the longdelay between intoxication and appearance of the firstsymptoms of tetanus and botulism and the higher bodytemperature of mammals, it is conceivable that onemolecule of toxin is capable of cleaving all molecules ofsubstrate present in the intoxicated mammalian synapse.

    It should be noted that, in contrast to necrotizing toxinssuch as diphtheria or Shiga toxins, and similarly withcholera and pertussis toxins, the clostridial neurotoxinsdo not cause death of the neuron they have entered.They only cause the paralysis and degeneration of theintoxicated synapse (Borodic etal.. 1994) and the animaldies btecause of blocking of neurotransmission. Within afew weeks, the motor neuron sprouts around the deadsynapse and, in a period of two to four months, reforms afunctional NMJ (Borodic etal.. 1994). This is at the basis

    of the reversibility of the effect of BoNTs in dystonicpatients treated with these toxins. The beneficial effectsof BoNTs, because of paralysis of the muscles causingthe dystonia, last only for two to four months and thepatient needs to be reinjected.

    The finding that all of the clostridial neurotoxins respon-sible for tetanus and botulism are zinc-proteinases thatattack the same cellular apparatus suggests a possiblecommon evolutionary origin for these toxins. An ancestraiclostridial gene coding for a non-specific extracellular zinc-protease (Hase and Finkelstein, 1993) may have evolvedto allow selective cieavage of protein of the multi-subunitcomplex involved in neuroexocytosis, a fundamentalprocess of the physiology of higher eukaryotes. Thedifferent sites of attack of the same supramolecular struc-ture by the different neurotoxins mean that an animalspecies cannot become resistant to all ciostridial neuro-toxins by means of amino acid repiacements at thecleavage sites (see below).

    The neuroexocytosis multi-subunit complex as atarget of the clostridial neurotoxins

    Recently, Sollner etal. (1993a) have characterized a 20 Scomplex composed of VAMP, SNAP-25, syntaxin, NSF(/V-ethyl-maleimide-sensitive protein), ot/p SNAP (solubieNSF accessory protein) and y-SNAP. More recently,MUNC-18 and a cysteine-string protein have been added(Hata etal.. 1993; Zinsmaier etal.. 1994; MastrogiaconfK)et al., 1994). It has been proposed that every cellularevent of vesicle docking and fusion with a targetmembrane is mediated by such a complex (Sollner et al..1993a; Rothman and Warren, 1994). Vesicle dockingrequires both vesicle receptor(s) (v-SNARE) and targetmembrane receptor(s) (t-SNARE), and is followed by apriming of the complex and then fusion, triggered bycaicium in the case of neurotransmitter release.

    VAMP is a highly conserved protein anchored to themembrane of synaptic vesicles, the bulk of the moleculebeing exposed to the cytosoi (Bennett and Scheller,

  • 6 C. Montecucco and G. Schiavo

    1994). Three different isoforms have been identified;VAMP-1 and VAMP-2 present in the nervous and neuro-endocrine tissues (Trimble etal.. 1990) and cellubrevin,present in non-neuronal cells (McMahon et al.. 1993).These proteins are also expressed in different proportionsin other vertebrate tissues (O. Rossetto et al.. in prepara-tion), and they have been proposed to act as v-SNAREs(Sollner et al.. 1993a.b; Rothman and Warren, 1994).TeNT and BoNT/B, -ID, -/F and -/G attack VAMP,provided that the isoform possesses an appropriatecleavage site. VAMP-1 from chicken and rat brain is notcleaved by TeNT and BoNT/B because it carries a Vairesidue in place of the Gin residue carried by the otherVAMP isoforms, which are proteolysed specifically at aGin-Phe bond, as reported in Table 2. This single aminoacid replacement at the site of TeNT and BoNT/B cleavagehas been proposed to be associated with the resistanceof rats and chickens to tetanus and to type B botuiism,which contrasts with the sensitivity of humans and mice(Patamelloefa/., 1993).

    In addition, SNAP-25 and syntaxin are highly conservedand have various isoforms (Oyler et al.. 1989; Bennettetal.. 1993; Bennett and Scheller, 1994). Unlike VAMP,they are both located on the cytosolic surface of thenerve plasmalemma and were proposed to act ast-SNAREs. SNAP-25 is essential for axonal growth andregeneration (Osen-Sand etal.. 1993).

    a \ BoNT/F-VBONT/D

    Fig. 3. Target and site of cleavage of tetanus and botuiinumneurotoxins within the neuroexocytosis apparatus. The L chain is azinc-endopeptidase specific for components of the 20S multi-sub-unit vesicle docking/fusion complex. Tetanus and botuiinum B, 0, Fand G neurotoxins specifically cleave VAMP/synaptobrevin, aprotein of the neurotransmitter-containing vesicles. Botuiinumneurotoxins serotypes A and E recognize and cleave SNAP-25, apresynaptic membrane component, while syntaxin, also localized onthe plasma membrane, is specificaiiy proteolysed by serotype C.Arrows indicate the approximate positions of the cleavage sites.Empty circles refer to proteins that interact with the targets ofclostridial neurotoxins: MUNC-18, NSF. xJp and y-SNAP arecomponents of the 20S docking/fusion complex, while rab 3A andsynaptotagmin/p65 dissociate from syntaxin, VAMP and SNAP-25before complex assembly. SSV indicates neurotransmitter-containing small synaptic vesicles, although VAMP is also presenton the membrane of other exocytotic vesicles.

    VAMP, SNAP-25 and syntaxin have been shown to bethe targets of the clostridial neurotoxins, not only in vitrobut also in synaptosomes and, in the case of VAMP, ininjected Aplysia neurons (Link et al., 1992; Blasi et al.,1993a,b; Schiavo etal.. 1993c; Poulain etal.. 1993). Thefact that cleavage of these three proteins leads to a persis-tent and sustained blocking of neurotransmitter release isstrong evidence in favour of the actual involvement of the20S complex in neuroexocytosis. The cleavage of VAMPand of syntaxin by TeNT, BoNT/B, -/D, -/F, -/G and -/Cleads to the release of a substantial portion of themolecule in the cytosol (Fig. 3), and this is likely to impairthe assembly of the neuroexocytosis apparatus. In con-trast, BoNT/A only removes nine residues from theSNAP-25 carboxyl-terminus (Schiavo et al.. 1993c).Hence, this part of the molecule is essential for its foldingor, altematively, the cart>oxyl-terminal part of SNAP-25plays a direct role in the function of the neuroexocytosiscomplex.

    Apart from providing a moiecuiar understanding of thepathogenesis of tetanus and botulism, the discovery ofthe intraneuronal-specific zinc-endopeptidase activity ofthese toxins offers to cell biologists new and powerfultools for the study of the molecular mechanisms under-lying exocytosis and axonal growth and regeneration.

    Future perspectives

    The identification of the catalytic activity of TeNT andBoNTs widens the search for seiectlve inhibitors of theirzinc-endopeptidase activity for testing as therapeuticagents of tetanus and botuiism. This has already t>eendone with other zinc-endopeptidases such as theangiotensin-converting enzyme, which is specificallyinhibited by captopril (a widely used antihypertensiveagent; Vidt et al.. 1982). Captopril is only a weak inhibitorof the clostridial neurotoxins (Schiavo et al.. 1992b) andtherefore it cannot be useful in the management oftetanized patients. However, this finding ciearly indicatesthat specific inhibitors of the clostridial neurotoxinprotease activity can be identified. This will be of greatvalue for countries in which an appropriate anti-tetanusvaccination programme is not established and in whichtetanus is still responsible for the loss of several hundredsof thousands of lives per year.

    Although the specific zinc-endopeptidase activitydescribed above is sufficient to account for the blockingof neuroexocytosis brought about by TeNT and BoNTs, itcannot be excluded that these toxins may act on otherintracellular targets. In fact, if one runs a computer searchwith peptides spanning the cleavage sites of the varioustoxins, several potential intracellular substrates arefound. For example, processing of the amyloid pre-cursor protein occurs at two contiguous peptide bonds.

  • Metalloprotease activity ot clostridial neurotoxins 7

    Gin-Lys-Leu, identical to those cleaved by serotypes Dand F of BoNT (Sisodia, 1992), thus suggesting thepossibiiity that the endocellular protease involved in sucha process is similar to BoNT/D and BoNT/F.

    Moreover, Facchiano et al. (1993a,b) have recentlyshown that TeNT activates transglutaminase and thatsynapsin, a major cytoskeleton-interacting protein ofsmall synaptic vesicles, is an excellent transglutaminasesubstrate. Hence, TeNT could also affect neurotransmitterrelease by cross-linking the synaptic vesicies to the cyto-skeleton, thereby preventing their docking and fusionwith the presynaptic membrane (Facchiano et al..1993b). Moreover, Ray et al. (1993) have found thatBoNT/A interferes with the production of arachidonic acidassociated with neurotransmitter release in PC12 cells.These findings do not account for the fact that the neuro-exocytosis blocking activity of TeNT and BoNT is inhibitedby phosphoramidon and captopril, two well-characterizedzinc-endopeptidase inhibitors, in Aplysia neurons, in NMJpreparations, and in neurohypophysis (Schiavo et al..1992a; de Paiva etal.. 1993b; Dayanithi etal.. 1994; B.Poulain et al.. unpublished). Nevertheiess, the possiblerelevance of these additional biological activities andtargets of TeNT to its in vivo action remains to be testedin future experiments.

    Notes added in proofVery recently, Nishika etal. {JBid Chem ^^994) 269:10498-10503) have reported that the receptor of BoNT/B consists ofsynaptotagmin and polysialoganglioside providing the firstexperimental evidence for the mode of binding discussed inthis review. This result also indicates that BoNTs exploit there-uptake of small synaptic vesicles to penetrate neuronalcells.

    AcknowledgementsThe work described here and carried out in the authors'laboratory Is supported by CNR-Blotecnologie, Telethon-ltaliaGrant 473, and MURST.

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