I. INTRODUCTION

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With the general aim of increasing their chance of survival, many thousands of living species produce toxins that are used to modify the physiology of other species. Toxins can be of any chemical complexity from very simple molecules, such as the formic acid used by ants, to the multimillion-dalton protein toxins produced by several bacteria. Some toxins are rather unspecific, but many of them are specific for a selected target molecule. It is conceivable that the specificity of certain toxins has been progressively refined during the course of evolution to alter the function of a selected target molecule, thus attaining specific goals within the strategy of survival of the toxin producer. Although most plant toxins are used for defense, animal toxins can be used for defense or predation of other animals or for both roles. Some bacterial toxins are directed against competing bacteria, whereas other toxins alter the physiology of the animal host to increase multiplication and/or diffusion of toxigenic bacteria. Being the product of a long-term coevolution of the toxin-producing species with the target species, a toxin has very frequently been shaped around the target; hence, the study of its mechanism of action can reveal important features of host physiology (495).

In this light, it is not surprising that most known toxins are selective for molecules of the nervous tissue. All the most poisonous toxins are neurotoxins. Given the essential role of the nervous system in animal physiology, even a minor biochemical modification of a few neurons may result in a profound modification of behavior. Neurotoxins have played, and will play without doubt, a major role in unravelling nerve physiology (245, 257, 620).

In general, neurotoxins block in one way or another the transmission of the nerve impulse. A variety of animal neurotoxins act postsynaptically. They bind the acetylcholine receptor, the acetylcholinesterase or ion channels thereby blocking or altering their function. The majority of neurotoxins act presynaptically by binding specifically to ion channels, and the ensuing strong alteration of the permeability of the neuronal plasma membrane to selected ions results in an indirect inhibition of neuroexocytosis and in the blockade of the transmission of nerve signals. Neurotoxins that act simply by binding to neuronal molecules, thereby altering their physiological activity, are not dealt with in the present review, which focuses on the structure and mechanism of action of three groups of presysnaptic neurotoxins that interfere directly and specifically with neurotransmitter release. Previous reviews have analyzed structural and functional aspects of these neurotoxins (149, 245, 419, 440, 509, 527, 620) as well as electrophysiological, ultrastructural, and molecular aspects of neuroexocytosis (48, 125, 590, 620). This review aims to provide an analysis of the mode of action of neurotoxins directly altering neuroexocytosis, in relation to the recent knowledge acquired on the synaptic vesicle exocytosis and endocytosis cycle.

	II. EXO-ENDOCYTOSIS OF SYNAPTIC VESICLES

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Transmission of a nerve muscle impulse follows a presynaptic depolarization that causes the opening of voltage-gated Ca²⁺ channels. This leads to a very rapid local increase of Ca²⁺, up to 200 mM, which triggers, within 200-300 ms, the fusion of small synaptic vesicles (SSV) bound to specialized "active" zones of the presynaptic membrane (12, 180, 239, 291, 350, 493, 620). The synchronous release of these ACh quanta causes a large postsynaptic depolarization, termed end-plate potential (EPP). The resting neuromuscular junction (NMJ) spontaneously releases quanta of ACh, each of which is contained in a single small synaptic vesicle having a diameter of 40-50 nm (Fig. 1). This release causes a postsynaptic depolarization, termed miniature end-plate potential (MEPP) (291). Occasionally, giant MEPP can be observed. They account for 1-3% of the total number of synaptic events and correspond to a large Ca²⁺-independent discharge of ACh, since the amount released is sufficient to activate the muscle fiber (296, 345, 597, 599). It has been suggested that giant MEPP derive from the release of ACh contained in endosomal compartments precursors of the SSV (35) or as a result of repair processes at damaged neuronal terminals (484, 599). After release, the SSV undergo rapid reuptake in a dynamin-dependent process and are refilled with neurotransmitter by proton-driven neurotransmitter transporters (Fig. 1) (48, 125, 138, 284, 590).

View larger version (27K): [in this window] [in a new window]   Fig. 1. The exocytosis-endocytosis cycle of synaptic vesicles at nerve terminals. Neurotransmitters (NT) are accumulated in the lumen of synaptic vesicles via specific vesicular transporters in a process driven by the pH gradient generated by the vacuolar ATPase proton pump (top). Most synaptic vesicles present in a typical synaptic terminal are bound to the actin cytoskeleton via interactions regulated by phosphorylation of proteins such as the synapsins (black comma on left). A small proportion of synaptic vesicles binds to the cytosolic face of the presynaptic membrane at active zones, via protein-protein interactions. Biochemical steps of this process have not been clarified, and the following part of the scheme is hypothetical and based on work performed mainly with systems not strictly related to the synapse such as the granule exocytosis in chromaffin cells and mast cells and the homotypic membrane fusion of yeast vacuoles. The binding process may involve a first phase of tethering, which implicates rab proteins and may be followed by priming catalyzed by cytosolic proteins, including N-ethylmaleimide-sensitive factor (NSF) and synaptosomal-associated proteins (SNAP) and the hydrolysis of ATP. Completion of the priming step leads to stabilization of the binding by additional protein-protein interactions involving a set of SNARE (docking), which form a trans-SNARE complex between the vesicle-associated memberane protein (VAMP) of the docked vesicle and 25-kDa SNAP (SNAP-25) and syntaxin, present on the cytosolic face of the presynaptic membrane. Docked vesicles may then become ready to bind Ca2+ and to fuse with the plasmalemma in a maturation reaction. Fusion is very rapidly triggered by the local increase of Ca2+ concentration that follows the opening of Ca2+ channels, located within the active zone. At the neuromuscular junction, the release of the ACh, contained inside one vesicle, causes a miniature end-plate potential, whereas the release of several vesicles corresponds to an end-plate potential. Exocytosis is rapidly (<1 s) followed by endocytosis in a process dependent on the formation of a clathrin coat and of a GTP-dependent action of dynamin. After pinching off the membrane, the coated vesicle uncoats and another cycle starts again.

An extraordinary amount of research with the convergence of all experimental approaches presently available has focused on the identification and on the structural and functional characterization of the proteins involved in the SSV life cycle. This has led to the understanding that a very similar set of proteins and lipids is involved in all cellular events involving membrane fusion between a vesicular compartment and its target membrane (216, 346, 437, 514). Furthermore, these studies demonstrate that additional proteins are essential to sustain the unique features of neuroexocytosis, which is the most tightly regulated of such membrane trafficking events (48). In section IIIK, we limit the discussion to an introduction of the family of proteins, known as SNARE (514), which are the target of the action of clostridial neurotoxins (CNT), and we refer the reader to recent reviews (48, 125, 587, 662) for synaptic proteins not included here.

	III. NEUROTOXINS WITH METALLOPROTEASE ACTIVITY (CLOSTRIDIAL NEUROTOXINS)

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Eight neurotoxins endowed with a metalloprotease activity have been characterized so far, and the consequences of the activity of one of them (tetanus neurotoxin) have been known since the very beginning of medical literature. In fact, it was Hippocrates who described 25 centuries ago the symptoms of a paralyzed patient with hypercontracted skeletal muscles (358). He termed such a spastic paralysis tetanus (tetanos in greek means contraction). Tetanus is often fatal. Death follows body exhaustion and occurs by respiratory failure or heart failure (70). Tetanus still takes hundreds of thousands of lives per year, concentrated in those parts of the world where antitetanus vaccination is not compulsory (198). This disease was thought to be of nervous origin until it was shown to be caused by a bacterium (97), which was isolated, characterized, and termed Clostridium tetani by Kitasato (301). This name derives from the elongated shape of the bacterium, which frequently harbors a subterminal spore, thus resembling a drum stick (clostridium in latin). Clostridium tetani is strictly anaerobic because it does not possess the redox enzymes necessary to reduce oxygen. Thus, in the presence of oxygen, radicals accumulate and lead eventually to bacterial death. Clostridium tetani is widespread in nature in the form of spores, which germinate under appropriate conditions of very low oxygen tension, slight acidity, and availability of nutrients (481). Such conditions may be present in anaerobic wounds or skin ruptures or abrasions (even minor ones such as those caused by piercing or tattooing), where spores can germinate and produce a protein toxin that fills the bacterial cytosol and is released by autolysis. The toxin, termed tetanus neurotoxin (TeNT), is responsible for all of the symptoms of tetanus (169, 301, 602, 603).

Adult botulism was first recognized and described much later than tetanus (294), and infant botulism was described only 20 yr ago (17, 400, 473). This later recognition of botulism is to be attributed to the much less evident symptoms with respect to those of tetanus. In fact, botulism is characterized by a generalized muscular weakness, which first affects ocular and throat muscles and extends later to the whole skeleton (249, 522, 576). Botulism is likely to be much more frequent than can be deduced from the number of officially recorded outbreaks, because a partial muscular flaccidity may be considered not relevant enough to be reported (576, 621). In the more severe forms, a generalized flaccid paralysis accompanied by impairment of respiration and of autonomic functions develops, and death may result from respiratory failure (249, 576). Massive fatal outbreaks of botulism are not infrequent in animals, particularly among birds and fishes, both in the wild and on farms (576).

Botulism is caused by intoxication with one of the seven neurotoxins produced under anaerobic conditions by toxigenic strains of Clostridium botulinum (621) or Clostridium barati and Clostridium butirycum (22, 235). The seven serotypically distinct botulinum neurotoxins (BoNT) are indicated with letters from A to G. The spores of the different toxigenic Clostridia sp. germinate under different conditions, and the bacteria differ for nutrient and temperature requirements (481). Such differences in growth conditions explain why, contrary to tetanus, botulism very rarely follows wound infection with spores of C. botulinum (wound botulism) (249). Usually, a BoNT is introduced by eating foods contaminated by spores of C. botulinum and preserved under anaerobic conditions that favor germination, proliferation, and toxin production (249, 522, 576). Similarly to most known proteins, BoNT are sensitive to the proteolytic and denaturating conditions found in the stomach lumen. It is believed that, to overcome this difficulty, they are produced as complexes with other nontoxic proteins (280, 402), which enable a proportion of BoNT to reach the intestine undamaged. Here, the slightly alkaline pH causes dissociation of the toxin complexes. BoNT could then reach general circulation by transcytosis from the apical to the basolateral side of intestinal epithelial cells (359) or by uptake from the M cells. There is evidence that in humans this process may be inefficient: as many as 10⁶ mouse LD₅₀ of BoNT/A per milliliter of stool have been found in children showing moderate botulism symptoms during a recent outbreak of botulism in Italy (23) (P. Aureli, personal communication).

At the present time, we cannot exclude that part of the toxic complex is adsorbed as such in the oral cavity and/or in the esophagus and/or the stomach and that dissociation of the BoNT from the nontoxic proteins takes place in the circulating fluids. Alternatively, the complex may dissociate at early stages, and BoNT can be adsorbed in the first portions of the alimentary tract. In this case, one could hypothesize that the nontoxic proteins are made to preserve the BoNT from proteolytic attack in the bacterial culture medium.

As a consequence of the fact that a single protein is responsible for all the clinical symptoms of tetanus and botulism, these diseases can be completely prevented by antitoxin specific antibodies (198, 398, 399). Toxin-neutralizing antibodies can be acquired passively by injection of immunoglobulins isolated from immunized donors or, actively, as a result of vaccination with tetanus toxoid. The toxoid is obtained by treating TeNT or BoNT with paraformaldheyde (198, 494). Tetanus toxoid is very immunogenic and is used as a standard immunogen in a variety of immunonological studies (123). More recently, antitetanus and antibotulism vaccines have been developed by genetic engineering techniques employing the COOH-terminal third of the TeNT or BoNT molecules (399). The general population is not vaccinated against botulism, since the disease is rather rare in developed countries, but vaccination may be performed on people involved in manipulation of toxigenic Clostridia or of large quantities of BoNT. Vaccination does not appear necessary for scientists working with BoNT; the avoidance of using sharp objects, such as needles, and the availability in the laboratory of antisera anti-BoNT appear to be sufficient safety measures. The only known case of laboratory intoxication with BoNT occurred in workers attempting to administer an aerosol of BoNT/A to animals (268).

The mouse LD₅₀ values of TeNT and BoNT are between 0.1 and 1 ng toxin/kg body wt. Thus they are the most toxic substances known. Such values are expected to be even lower in the wild, where even a very small deficit in mobility may be sufficient to impair survival. Different animal species show a great range of sensitivity to TeNT and to the different BoNT. Humans and horses are at least as sensitive to these neurotoxins as mice, whereas rats, birds, snakes, and amphibians are rather resistant to TeNT, and turtles are insensitive (211, 460). The recent, ever-growing, use of BoNT/A as a therapeutic agent for a variety of dystonias and other diseases has uncovered significant variations in the response of patients to the same dose of BoNT/A, with some individuals being unresponsive. The absolute neurospecificity of TeNT and BoNT and their catalytic activity (see below) are at the basis of such high toxicity. The time of onset of paralysis of animals injected with these neurotoxins is variable depending on species, dose, and route of injection. However, a lag phase, ranging from several hours to days, is always present between the time of injection and the appearance of symptoms. Of course, the lag phase is much longer when the disease is caused by contamination of wounds with spores of toxigenic Clostridia because in this case the time of germination and bacterial proliferation is to be included. The lag phase of tetanus in humans may be longer than 1 mo.

After entering the general circulation, CNT bind very specifically to the presynaptic membrane of motoneuron nerve endings. TeNT also binds to sensory and adrenergic neurons. Presynaptic receptor(s) have not been identified, but CNT are expected to bind rapidly and with high affinity to account for the limited spreading around the site of injection, experienced in clinical treatments, and for the low LD₅₀ values. After binding to the presynaptic membrane, the BoNT enter the neuronal cytosol and block the release of ACh, thus causing a flaccid paralysis (87, 387, 570). TeNT also binds to the motoneuron presynaptic membrane, but its peripheral action is zero or very limited, unless very high doses are injected (370). Contrary to BoNT, TeNT is transported retrogradely inside the motoneuron axon, in a microtubule-dependent movement, up to the spinal cord, where it accumulates in the ventral horn of the gray matter (85, 167, 186, 231, 238, 490, 588, 615). An intra-axonal ascent transport rate of 7.5 mm/h has been estimated (588). Neuromuscular stimulation enhances the extent of uptake of CNT (233, 270, 319, 479, 644). Within the spinal cord, TeNT migrates trans-synaptically from the dendrites of peripheral motoneurons into coupled inhibitory interneurons across the synaptic cleft (547, 548), and it blocks the release of inhibitory neurotransmitters (40, 47, 82, 83, 129). Excitatory synapses appear not to be affected at early stages (46, 47, 82, 83, 390, 643, 646, 654), but they may be inhibited at later stages (596). This specificity of TeNT for inhibitory versus excitatory synapses is maintained when TeNT is applied to hippocampal slices (92) or injected into the hippocampus (389, 393). Such specificity for inhibitory synapses of the central nervous system (CNS) also accounts for the neurodegenerative and epileptogenic effects of TeNT, which mainly result from unopposed release of glutamate from excitatory synapses (24-26, 339). The selective action of TeNT on inhibitory synapses within the spinal cord may be at least in part due to the anatomical organization of the tissue because it is not preserved in spinal cord neurons in culture (47, 654). During trans-synaptic migration, TeNT can be neutralized by antitoxin antibodies injected in the spinal fluid (166).

The blockade of inhibitory synapses brought about by TeNT at the spinal cord impairs the neuronal circuit that ensures balanced voluntary muscle contraction, thus causing the spastic paralysis characteristic of tetanus (390, 570, 643, 646). The half-life of ¹²⁵I-TeNT in the rat spinal cord and in cells in culture is several days (231, 234, 368). Such a figure compares well with the documented fact that tetanus symptoms may develop more than a month after wound infection, when the wound may have already healed. The amount of toxin that reaches the CNS, after uptake at the parasympathetic nervous system, is clearly an important parameter that determines the severity of the disease and may partly account for the different toxicity of TeNT in different vertebrates (460). Hence, the opposite clinical symptoms of tetanus and botulism result from different sites of action of TeNT and BoNT, rather than from a different mechanism of action (see also sect. IIIL). This neat distinction between the central site of activity of TeNT and the peripheral sites of action of the BoNT exists only at subpicomolar concentrations. To rapidly obtain consistent effects, hundreds of mouse lethal doses are frequently used in the laboratory, particularly when insensitive animals such as birds or fishes are studied or in vitro with cultured cells or isolated hemidiaphragm muscle preparations. Under such conditions, TeNT also inhibits peripheral synapses causing a botulism-like flaccid paralysis (370). In any case, CNT only act presynaptically causing a persistent inhibition of the exocytosis of a variety of neurotransmitters (reviewed in Ref. 643).

The action of TeNT and BoNT can be extended to a variety of nonneuronal cells by microinjection or addition to permeabilized cells: these neurotoxins then inhibit many, but not all, exocytotic events in a wide range of cells (6-8, 10, 29, 53, 57-59, 77, 108, 136, 137, 196, 197, 212, 226, 266, 278, 306, 307, 335, 377, 405, 454, 468, 500, 520, 584, 586, 592, 664).

Contrary to what has been seen with the animal neurotoxins described in sections IV and V, morphological examinations of synapses intoxicated in vivo or in vitro with CNT does not reveal major alterations of structure (Fig. 2). Synapses are not swollen; mitochondria, SSV, and large electron-dense vesicles are well preserved in terms of number, size, and intraterminal distribution. The only consistent change is an increase in the number of synaptic vesicles close to the cytosolic face of the presynaptic membrane (155, 156, 271, 320, 388, 434, 450, 462, 489). The BoNT/A poisoning of the frog NMJ causes the disappearance of the small membrane invagination normally detectable close to the active zones, which are believed to represent SSV fusion events (491).

View larger version (94K): [in this window] [in a new window]   Fig. 2. The nerve terminal poisoned by tetanus neurotoxin (TeNT). Electron micrographs of control (left) and TeNT-treated dissociated spinal cord neurons (100 ng/ml for 10 h) (right) are shown. Notice the increase of synaptic vesicles juxtaposed to the presynaptic membrane of the intoxicated and electrically silent synapse. (Photos courtesy of Dr. E. A. Neale, National Institutes of Health, Bethesda, MD.)

The first electrophysiological investigation of the effect of a BoNT on a NMJ was conducted by Burgen et al. (87) on the rat hemidiaphragm preparation. Following this seminal study, the consequences of CNT poisoning have been studied on different synaptic terminals, but only for the vertebrate NMJ is a large set of data available to compare the effects of TeNT and of BoNT. Most of these studies have been recently reviewed (96, 419, 484, 620). They can be summarized here as follows. 1) Clostridial neurotoxins cause a large and persistent blockade of EPP, responsible for the impaired synaptic transmission at intoxicated synaptic terminals; in vitro, on isolated neuronal cells in culture, they were shown to be effective on any synapse tested. 2) These neurotoxins greatly reduce the frequency, but not the amplitude, of evoked MEPP. Hence, CNT lower the number of vesicles capable of undergoing fusion and release, without affecting the ACh quantum. 3) CNT do not interfere with the processes of neurotransmitter synthesis, uptake, and storage (reviewed in Ref. 223). 4) TeNT and BoNT affect neither the propagation of the nerve impulse nor Ca²⁺ homeostasis at the synaptic terminal (152, 224, 361, 410). 5) The frequency of spontaneous MEPP is reduced, but not abolished, at poisoned terminals, and the neurotransmitter released during such residual events tends to be less and delivered more slowly. 6) The frequency of giant MEPP is not altered or even increases at the intoxicated NMJ (296, 408, 554, 555, 597). The effect of BoNT/C has been investigated only at CNS synapses with results comparable to those obtained with BoNT/A (96), whereas no report is available for BoNT/G. Clearly, more studies are necessary to provide a solid scientific basis for the clinical use of these neurotoxins. Meanwhile, BoNT/C has been proven to be as valuable as BoNT/A in the therapeutic treatment of human dystonias (165).

Based on the available data, CNT can be divided into two groups. BoNT/A and /E poison the NMJ in such a way that the quantal release of ACh evoked by nerve stimulation remains synchronous. On the other hand, TeNT, BoNT/B, /D, and /F cause a desynchronization of the quanta released after depolarization (51, 154, 202, 241, 410). Aminopyridines, by inhibiting potassium channels, indirectly cause an increase in the Ca²⁺ level of the synapse and synchronize evoked neurotransmitter release in BoNT/A and /E poisoned terminals, leaving the release largely asynchronous in NMJ treated with TeNT and BoNT/B, /D, and /F (408, 484, 553, 643). Similar conclusions were reached with Ca²⁺ ionophores. An increased Ca²⁺ concentration within the synaptic terminal partially reverses the effect of BoNT/A and BoNT/C, is poorly effective on BoNT/E, and has no effect on TeNT-treated preparations (18, 29, 96, 128). A difference in the mechanism of action of these two groups of neurotoxins is also suggested by experiments of double poisoning of the NMJ with a CNT, followed by -latrotoxin (LTX), which causes a massive release of SSV (see sect. V). -Latrotoxin counteracts the action of BoNT/A, but not that of TeNT or BoNT/B (202).

These extensive electrophysiological studies led to several clear conclusions with which recent molecular data have to be compared: 1) CNT hit on synaptic terminal components playing essential roles within the neuroexocytosis machinery; 2) the CNT fall into two groups having different targets within the presynaptic terminal. On one side there are TeNT, BoNT/B, BoNT/D, and BoNT/F, and on the other side there are BoNT/A, /C, and /E; this conclusion is now fully substantiated by the identification of the molecular targets of each CNT (see sect. IIIL). 3) The neurotoxin-impaired neuroexocytosis apparatus can still mediate some spontaneous residual synaptic activity, but with reduced efficiency with respect to the amount of neurotransmitter released and the rate of the overall process. 4) Giant MEPP occur via a mechanism not involving the CNT targets. Giant MEPP may be considered as indicators of immature or pathological states of the synapse, such as those occurring after tetanic stimulation or -LTX-induced stimulation. It has been proposed that they result from a constitutive, rather than highly regulated, type of exocytosis of ACh-containing endosomal compartments precursors of the SSV (35, 554).

The similar effect of the eight CNT at nerve terminals is the result of a closely related protein structure. They are synthesized in the bacterial cytosol without a leader sequence, which is in keeping with the fact that they are released in the culture medium only after bacterial lysis. No protein is associated with TeNT, whereas BoNT are released in the form of multimeric complexes, with a set of nontoxic proteins coded for by genes adjacent to the neurotoxin gene: these complexes are termed progenitor toxins (280, 402). Some BoNT-associated proteins have hemagglutinating activity (HA): HA of 17 kDa (HA17), HA of 34 kDa (HA34), and HA of 71 kDa (HA71). In addition, a large nontoxic nonhemagglutinating protein of 139 kDa (NTNH), coded for by a gene upstream to the BoNT gene, is always present. The NTNH produced by the various neurotoxigenic strains of Clostridia are more conserved than the corresponding BoNT themselves. Moreover, a remarkable feature uncovered by gene sequencing is that the 100-amino acid-long NH₂-terminal region of NTNH is homologous to the corresponding region of BoNT (402). The significance of such an homology is unclear at the present time. It is tempting to suggest that the NH₂-terminal regions of BoNT and NTNH are independent domains with a strong tendency to dimerize. As such, they nest the formation of the BoNT-NTNH complex which may then, or may not, progress to the formation of larger complexes. In fact, three forms of progenitor toxins have been characterized: extra-large size (LL sediments at 19S, ~900 kDa), large size (L sediments at 16S, 500 kDa), and medium size (M sediments at 12 S, 300 kDa). An electron-density projection map of the 19S complex of BoNT/A shows a triangularly shaped protein complex with six lobes (89).

The molecular genetics of CNT are currently under investigation, and several remarkable features are becoming apparent. They are beyond the scope of this review, and the reader is referred to recent reviews (402, 481). One general point has been firmly established: the neurotoxin genes are mobile, and nontoxigenic strains cocultivated with toxigenic strains can become toxigenic by gene transfer mediated by phages or plasmids or conjugation transposons. Such processes are believed to occur during the enormous proliferation of anaerobic bacteria that takes place on animal cadavers converted by death into effective anaerobic fermentors. As a result of such genetic mobility, C. botulinum may harbor more than one toxin gene (275), and strains producing mosaic BoNT with type C and type D mixed elements have been recently characterized (428, 429).

Botulism neurotoxin in the form of progenitor toxins is more stable than isolated BoNT to proteolysis and denaturation induced by temperature, solvent removal, or acid pH (110, 522). Progenitor toxins that survive the harsh conditions of the stomach reach the intestine, where the slightly alkaline pH induces their dissociation and releases the BoNT, which is then transcytosed to the mucosal side of the intestinal epithelium (359). The inactive single-chain 150-kDa neurotoxins are activated by specific proteolysis within a surface-exposed loop subtended by a highly conserved disulfide bridge (Fig. 6). Several bacterial and tissue proteinases are able to generate the active dichain neurotoxin (130, 131, 318, 641). The heavy chain (H, 100 kDa) and the light chain (L, 50 kDa) remain associated via noncovalent protein-protein interactions and via the conserved interchain S-S bond, whose integrity is essential for neurotoxicity (144, 537).

The length of the polypeptide chains of CNT varies from the 1,251 amino acid residues of Clostridium butyricum BoNT/E to the 1,297 residues of BoNT/G and the 1,315 residues of TeNT (402, 439). The exact length of the L and H chains depends on the site of proteolytic cleavage within the exposed loop. The L chains range in size from the 419 amino acid residues of BoNT/E to the 449 residues of TeNT. The H chains vary in size from the 829 amino acid residues of BoNT/E to the 857 residues of TeNT. As more and more amino acid sequences of BoNT are determined, it appears that their subdivision into seven serotypically distinct types is not adequate to describe their diversity. Very relevant sequence variations are present within the same BoNT serotype, and type C and type D hybrid toxin have been described (428, 429).

These polypeptide chains present homologous segments separated by regions of little or no similarity. The most conserved portions of the L chains are the NH₂-terminal 100 residues, mentioned above, and the central regions (residues 216-244, numbering of TeNT). Eight NH₂-terminal residues and 65 COOH-terminal residues can be deleted from TeNT without loss of activity (322). The 216-244 region contains the His-Glu-Xaa-Xaa-His binding motif of zinc-endopeptidases (286, 287, 322, 538, 540, 614, 663). This observation led to the demonstration that CNT are zinc proteins (535, 538-540, 542, 543, 668). One atom of zinc is bound to the L chain TeNT, BoNT/A, /B, and /F (538, 540, 543) with a dissociation constant (K_d) value in the 50-100 nM range, at the lower limit of the known range of affinities among metalloproteases. Flow dialysis also showed multiple zinc binding sites with lower affinity (540, 663). Heavy metal chelators remove bound zinc and generate inactive aponeurotoxins (265, 538, 571), without appreciable changes in L chain secondary structure (139). The active site metal atom can be reacquired upon incubation in zinc-containing buffers to reform the active holotoxin (265, 535, 538, 540, 542, 543, 571). With the same procedure, the active site zinc atom can be exchanged with other divalent transition metal ions forming active metal-substituted toxins (604).

The crystallographic structures of BoNT/A and the COOH-terminal TeNT domain (H_C) have been recently determined at 3- and 1.61-Å resolution, respectively (304, 326, 611). The toxin structure reveals three distinct functional domains, a unique hybrid of previously characterized structural motifs, and new insight into this protein's mechanism of toxicity (Fig. 3). There is complete agreement with the three-domain structural model of CNT previously proposed to account for the available biochemical data (418). BoNT/A consists of three ~50-kDa domains: an NH₂-terminal domain endowed with zinc-endopeptidase activity; a membrane translocation domain characterized by the presence of two 10-nm-long -helices, which are reminiscent of similar elements present in colicin and in the influenza virus hemagglutinin; and a binding domain composed of two unique subdomains similar to the legume lectins and Kunitz inhibitor (326).

View larger version (31K): [in this window] [in a new window]   Fig. 3. Three domain structure of clostridial neurotoxins. These neurotoxins consist of 3 domains of similar size (50 kDa). NH2-terminal domain (left) is a zinc endopeptidase, which is inactive when disulfide bonded to the rest of the molecule; its activity is expressed after reduction of the interchain disulfide bond. The active site zinc atom is coordinated by 2 histidine residues, a water molecule bound to a conserved glutamate residue and by the carboxylate group of another glutamate, with the likely participation of a conserved tyrosine (residue numbering corresponds to TeNT). HN, the central domain, is responsible for the membrane translocation of the L chain into the neuronal cytosol. The COOH-terminal Hc domain (right) consists of two equally sized subdomains. The NH2-terminal subdomain has a structure similar to that of sugar binding proteins. The COOH-terminal subdomain folds similarly to proteins known to be involved in protein-protein binding functions such as the K+ channel specific dendrotoxin. Such structure is consistent with the toxin binding to the presynaptic membrane via a double interaction, most likely with two different molecules of the nerve terminal.

Such structural organization is functionally related to the fact that CNT intoxicate neurons via a four-step mechanism consisting of 1) binding, 2) internalization, 3) membrane translocation, and 4) enzymatic target modification (417, 419). The L chain is responsible for the intracellular catalytic activity (10, 57, 58, 407, 468, 486, 640). The NH₂-terminal 50-kDa domain of the H chain (H_N) is implicated in membrane translocation (69, 151, 201, 264, 412, 561), whereas the COOH-terminal part (H_C) is mainly responsible for the neurospecific binding (61, 238, 430, 642).

The H_C domains of TeNT and BoNT/A are very similar with an overall elongated shape (Fig. 4), and preliminary data on the crystallographic structure of BoNT/E reveal a closely similar organization (R. C. Stevens, personal communication). The H_C domains of the two BoNT appear to be very flexible with respect to the H_N domain. The binding domains of these three CNT consist of two distinct subdomains, the NH₂-terminal half (H_C-N) and the C-terminal half (H_C-C), with little protein-protein contacts among them. H_C-N has two seven-stranded -strands arranged in a jelly-roll motif closely similar to that of legume lectins, which are carbohydrate binding proteins. The amino acid sequence of this subdomain is highly conserved among CNT, suggesting that it has a closely similar three-dimensional structure in all the CNT. The H_C-C contains a modified -trefoil folding motif present in several proteins involved in recognition and binding functions such as interleukin-1, fibroblast growth factor, and Kunitz-type trypsin inhibitors. Its sequence is poorly conserved among CNT. Removal of H_C-N from H_C does not reduce H_C nerve membrane binding, whereas deletion of only 10 residues from the COOH terminus abolishes its binding to spinal cord neurons (237). The critical importance of the last 34 residues of H_C-C, and in particular of His-1293 of TeNT, for binding the oligosaccharide portion of polysialogangliosides was recently demonstrated by photoaffinity labeling (556). These data are supportive of a double receptor model of binding of CNT to the presynaptic membrane (413) (see sect. IIIG for a discussion) with H_C-N binding to a glycoprotein, different for the different CNT, and H_C-C binding to a polysialoganglioside, whose nature may be similar for the various CNT. Additional toxin-membrane protein interactions cannot be excluded. The major difference between H_C-C of TeNT and BoNT/A resides in the structure of the loops, thus suggesting that these external segments may be responsible for the binding to the different protein receptors.

View larger version (39K): [in this window] [in a new window]   Fig. 4. Stereo-pair view of the receptor binding domain of tetanus neurotoxin. COOH- and NH2-terminal ends of the fragment Hc of TeNT are labeled, and these correspond to residues Glu-875 and Asp-1315, respectively, of the intact neurotoxin. The first 11 residues at the NH2 terminus were disordered in the crystal structure and hence are not displayed here. Residues Glu-875 to Ser-1110 form a subdomain possessing a jelly-roll folding motif, and this fold is similar to that displayed by the legume lectins (top). Residues Ile-1111 to Asp-1315 form the second subdomain of the Hc fragment, containing the -trefoil motif, similar to Kunitz-type protease inhibitors and to dendrotoxin (bottom). For further explanations, see text. (Photo courtesy of Dr. T. C. Umland.)

The H_N portions are highly homologous among the various CNT (402), and their predicted secondary structure is also highly similar (337). The membrane translocation domain of BoNT/A has a cylindrical shape determined by the presence of a pair of unusually long and twisted 10-nm-long -helices, corresponding to segment 685-827, reminiscent of the -helices hairpin of colicin (652). At both ends of the pair there is a shorter -helix that lies parallel to the main helices and, in addition, several strands pack along the two core helices. It is difficult to identify the residues and segments involved in the formation of ion channel at low pH, but the overall structure of H_N resembles that of some viral proteins that undergo an acid-driven conformational change (86, 639). A remarkable feature of BoNT/A is an extended loop that wraps around the catalytic L chain and makes it difficult to account for the fact that a short incubation of BoNT/A with dithiothreitol is sufficient to release the L chain.

The catalytic metalloprotease domain, 55 Å × 55 Å × 62 Å, contains both -helix and -strand secondary structures and has little similarity with related enzymes of known structure, apart from the -helix including the zinc binding motif (326). In addition to the imidazole rings of the two histidines of the motif and a water molecule bound to the glutamic acid of the motif, the zinc atom of BoNT/A is coordinated by Glu-261 and the phenolic ring of Tyr-365 points versus the metal atom, but remains ~5 Å away from it. This type of zinc coordination resembles that of thermolysin, but sequence differences as well as the unique properties of metal substituted TeNT (604) and of the differences found in the multiple scattering analysis of the X-ray absorption spectra of TeNT in comparison with metalloproteases of known three-dimensional structure (425) clearly indicate that these neurotoxins have an active site of unique architecture. A recent biophysical analysis compared TeNT with astacin and thermolysin and suggested that the phenolic ring of the active site Tyr residue of the isolated L chain of TeNT may be closer to the zinc atom than that indicated by the crystallographic structure of BoNT/A (394). Another characteritic of the active site of BoNT/A is that it is 20-24 Å deep in the protein and that it is accessible via an anionic channel, not accessible in the intact molecule because it is shielded by H_N and its wrapping belt (326). This accounts for the lack of enzymatic activity of dichain CNT. This active site channel becomes accessible to the substrate upon reduction of the interchain disulfide bridge and appears to be capable of accommodating 16 amino acid residues.

From the site of production or absorption, BoNT and TeNT diffuse in body fluids and reach and bind to the presynaptic membrane of cholinergic terminals. Tetanus neurotoxin may also bind to sympathetic and adrenergic fibers (reviewed in Refs. 238, 643). The introduction of radiolabeling methods allowing the production of active CNT at highly specific activity has made possible binding studies with unprecedented and still unsurpassed sensitivity. For technical reasons, almost invariably, the binding of CNT to CNS acceptors present in particulate brain matter, isolated lipid preparations, or synaptosomes has been studied. Only more recently, isolated neuronal cells in culture have been considered. These studies are hampered by the fact that all CNT lose some activity upon radiolabeling and that the range of concentration of clinical significance are undetectable with radioiodinated CNT. These extensive studies have not provided definitive answers with respect to affinity and number of binding sites of the individual CNT but have revealed heterogeneous binding of these neurotoxins to the presynaptic membrane with subnanomolar and nanomolar binding sites. These studies have been carefully reviewed before (27, 232, 238, 643) and are not dealt with here since the conclusion presented in Habermann and Dreyer (232) is still largely valid. [There is no doubt that specific binding sites do exist for botulinum neurotoxins (and tetanus neurotoxin) and probably even for the individual types with a varying degree of cross-reactivity. However, only a very small percentage of total binding is expected to display sufficient affinity, which is a prerequisite for the extreme potency of this toxin in vivo and on isolated organs.] Radiolabeled BoNT can be monitored also by autoradiographic methods, and it was thus possible to demonstrate that they bind to the unmyelinated zone of motorneuron terminals in a temperature-independent mode (150). Hundreds of binding sites for BoNT/A and /B per square millimeter appear to be present in the rat NMJ (63), whereas the number of TeNT receptors in a neuroblastoma-glioma cell line is ~450/cell (645). BoNT/A and BoNT/B also bind to cholinergic nerve terminals of rat brain, without apparent binding to other terminals (65).

Available evidence indicates that the H_C domain plays a major role in neurospecific binding (61, 115, 255, 312, 328, 430, 560, 642). However, it appears that additional regions of CNT are involved in binding because H_C shows only a partial protection from intoxication with the intact CNT molecule, and the H_C fragment of TeNT does not prevent retroaxonal transport of the holotoxin (61, 191, 407, 483, 487, 488, 595, 640). If binding is carried out above 20°C, the radiolabeled and fluorescent, or gold-tagged, CNT are internalized inside intracellular compartments of different nature after an energy-dependent endocytosis step (63, 126, 150, 375, 457, 458, 482).

Previous attempts at identification of the presynaptic receptor(s) of CNT have been reviewed before (for reviews and references, see Refs. 238, 390, 413, 414, 643). Here we only mention results that are relevant with respect to the recent identification of a sugar-binding subdomain in BoNT/A and TeNT (326, 610). Beginning with the seminal work of van Heyningen (622-624), a large number of studies have established that polysialogangliosides are involved in binding CNT (54, 126, 168, 230, 237, 267, 289, 299, 300, 369, 392, 421, 422, 446, 530, 533, 567, 589, 628, 630, 653, 671, 677). The results of these studies are briefly summarized hereafter: 1) CNT bind to polysialogangliosides, particularly to G_D1b, G_T1b, and G_Q1b; 2) preincubation with polysialogangliosides partially prevents the BoNT poisoning of the NMJ and the retroaxonal transport of TeNT; 3) incubation of cultured cells with polysialogangliosides increases their sensitivity to TeNT and BoNT/A; and 4) treatment of membranes with neuraminidase, which removes sialic acid residues, decreases toxin binding. Binding to polysialogangliosides well accounts for an unsaturable low-affinity binding of the CNT to nerve cells and to nerve tissue membranes. However, as discussed in detail previously (390, 413), it is unlikely that polysialogangliosides are the sole receptors of these neurotoxins. Experiments carried out with cells in culture have indicated that proteins of the cell surface may be involved in toxin binding (458, 474, 534, 672). The sugar-binding and protein binding subdomains present in the H_C domain of TeNT and BoNT/A (326, 611) and the protection experiments mentioned above support the suggestion that CNT may bind strongly and specifically to the presynaptic membrane because they display multiple interactions with sugar and protein binding sites (413). Recent experiments provided strong evidence in favor of such a model by showing that BoNT/B binds strongly to the synaptic vesicle protein synaptotagmin II in the presence of polysialogangliosides and that Chinese hamster ovary cells transfected with the synaptotagmin II gene bind the toxin with low affinity and with a high affinity after membrane incorporation of gangliosides GT1b (441-443). More recently, BoNT/E was also reported to interact with synaptotagmin (343).

Generally, receptors for toxins and viruses are cell surface molecules essential for the life of the cell, and their study has led to important progresses in cell biology and neurosciences. The identification of the receptors of the CNT is particularly relevant for several theoretical and practical reasons. In fact, both TeNT and BoNT bind the presynaptic membrane of -motoneurons, but then they follow different intracellular trafficking paths. The electrophysiological studies discussed above have clearly shown that BoNT block neuroexocytosis at peripheral terminals, whereas TeNT causes the same effect on CNS synapses of the spinal cord. These different final destinations of TeNT and BoNT must be determined by specific receptors that drive them to different intracellular routes. The determination of the nature of the peripheral motoneuron TeNT receptor(s) will uncover an entry gateway leading from the peripheral to the central nervous system. This is expected to help in devising novel routes to deliver biologicals, including analgesic and anesthetic agents, into the spinal cord. The knowledge of the receptors for the various BoNT will also contribute to improving present therapeutic protocols and may explain the lack of effect of BoNT/A in a subset of patients that do not benefit from the current BoNT/A treatment.

To reach its final site of action, TeNT has to enter inside two different neurons: a peripheral motoneuron and an inhibitory interneuron of the spinal cord. Its binding to peripheral and central presynaptic terminals is different, as indicated by several pieces of evidence. 1) Cats and dogs are highly resistant to TeNT administered peripherally but very sensitive to the toxin injected directly in the spinal cord (564). 2) The L-H_N fragment of TeNT injected in the cat leg is not toxic, whereas it causes a spastic paralysis upon direct injection into the spinal cord (595). It is possible that the concentration of TeNT in the limited space of the synaptic cleft between peripheral motoneuron and inhibitory interneuron is significantly higher than that at the periphery, with the motoneuron acting as a sort of "toxin pump." If this is the case, even a low-affinity receptor could mediate the entry of TeNT in the latter cells, because of its anatomically restricted location within the intersynaptic space. Lipid monolayer studies have clearly documented the ability of 10⁸ M TeNT to interact with acidic lipids (533). Similar concentrations are routinely used with cells in culture and in hippocampal injections in vivo (391) or in experiments of induction of a flaccid paralysis in mice treated with 1,000 times the mouse LD₅₀ dose (370). On the other hand, in clinical tetanus and botulism, at the periphery, TeNT and BoNT act at subpicomolar concentrations. A possible scenario that reconciles the presently available data is summarized hereafter. Glycoprotein and glycolipid binding sites are implicated in the peripheral binding of CNT, which is characterized by high affinity and high specificity. The protein receptor of TeNT would be responsible for its inclusion in an endocytic vesicle that moves in a retrograde direction all along and inside the axon, whereas BoNT protein receptors would guide them inside vesicles that acidify within the NMJ. The TeNT-carrying vesicles reach the cell body and then move to dendritic terminals to release the toxin in the intersynaptic space. The TeNT equilibrates between pre- and postsynaptic membranes and then binds and enters the inhibitory interneurons via synaptic vesicle endocytosis.

Contrary to TeNT, there is no evidence that BoNT can reach the CNS in botulism patients. However, in rats injected with high doses of BoNT/A, a little fraction can reach the spinal cord (229, 650, 651), and cats in the lateral rectus muscle of the eye show some signs of central effects (427). On the other hand, BoNT/A does inhibit neuroexocytosis in isolated CNS preparations (reviewed in Ref. 643). Thus is possible that BoNT at high doses, like high doses of TeNT do, act on sites of the nervous system that are unaffected in clinical botulism.

Because the L chains of CNT block neuroexocytosis by acting in the cytosol, at least this toxin domain must reach the cell cytosol. All available evidence indicates that CNT do not enter the cell directly from the plasma membrane, rather are endocytosed inside acidic cellular compartments. Electron microscopic studies have shown that, after binding, CNT enter the lumen of vesicular structures in a temperature- and energy-dependent process (62, 63, 126, 150, 375, 457, 583). The H_C domains of TeNT and BoNT/A, /B, and /E appear to be sufficient for the internalization process in murine spinal cord neurons (328). Montesano et al. (424) found TeNT inside non-clathrin-coated vesicles, but their study was performed on liver cells exposed to very large concentrations of TeNT. Gold-labeled TeNT was internalized by spinal cord neurons inside a variety of vesicular structures, and only a minority of TeNT was in the lumen of SSV (457). In contrast, Matteoli et al. (375) found TeNT almost exclusively inside small synaptic vesicles of hippocampal neurons after a 5-min membrane depolarization. It was long known that nerve stimulation facilitates intoxication (233, 270, 319, 479, 644). A prominent neuroexocytosis correlates with a high rate of synaptic vesicle recycling via endocytosis and refilling with neurotransmitter, being the two processes tightly coupled (48, 125, 549, 590). The simplest way to account for the shorter onset of paralysis induced by CNT under conditions of nerve stimulation is that the neurotoxins enter the synaptic terminal via endocytosis inside the lumen of SSV. Hippocampal neurons are the best available test system for such an hypothesis because 1) TeNT is active on the hippocampus, causing an epileptic-like syndrome when injected in this brain area (78, 207); 2) antibodies specific for epitopes of SSV luminal proteins bind to them during neurotransmitter release and are taken up inside the terminals after SSV endocytosis (316, 373, 432); 3) SSV endocytosis can be followed accurately with dyes such as FM1-43 (49, 50); 4) a high rate of SSV exo-endocytosis can be induced at synaptic terminals simply by briefly incubating the cells in a Ca²⁺-containing, high-potassium medium, using as a control a Ca²⁺-free medium; and 5) during their development, growing axons are characterized by a high rate of spontaneous SSV recycling (316, 374). Tetanus neurotoxin was found to enter synaptic terminals of hippocampal neurons inside the lumen of SSV (375). The toxin was also found to be internalized inside SSV spontaneously recycling in growing axons of hippocampal neurons. Similarly, TeNT enters inside granular cells of the cerebellum (O. Rossetto, P. Caccin, and C. Montecucco, unpublished results). These studies indicate that TeNT uses SSV as "Trojan horses" to enter inside CNS neurons. Similar experiments on peripheral motoneurons would permit the evaluation of such a possibility for BoNT at peripheral synapse, but it is presently difficult to maintain these cells in culture and to perform similar experiments.

As discussed above, TeNT and BoNT have to enter different vesicles at the NMJ to account for their different destiny inside peripheral motoneurons. Alternatively, they could enter inside the same vesicles with TeNT causing a vesicle modification/lesion such that the TeNT-containing vesicle is induced to bind to the microtubule-dependent motor involved in retroaxonal transport. In contrast, the BoNT-containing vesicles would remain within the motoneuron presynaptic terminal. In this respect, it is noteworthy that BoNT can intoxicate CNS neurons only when present at high concentrations. A high concentration of BoNT appears to enter hippocampal neurons via the aspecific process of fluid-phase endocytosis (C. Verderio, S. Coco, A. Bacci, O. Rossetto, P. De Camilli, C. Montecucco, and M. Matteoli, unpublished observations), but it is possible that this is not the case for cholinergic CNS neurons.

Whatever the nature of the vesicles containing the internalized neurotoxins, the L chains must cross the hydrophobic barrier of the vesicle membrane to reach the cytosol where they display their activity. The different trafficking of TeNT and BoNT at the NMJ clearly indicates that internalization is not necessarily linked to, and followed by, membrane translocation into the cytosol, i.e., internalization and membrane translocation are clearly distinct steps of the process of cell intoxication, as is the case for most intracellularly acting bacterial toxins (396, 417). There is indirect, but compelling, evidence that TeNT and BoNT have to be exposed to a low pH step for nerve intoxication to occur (3, 375, 568, 569, 572, 656). Acidic pH does not induce a direct activation of the toxin via a structural change, since the introduction of a non-acid-treated L chain in the cytosol is sufficient to block exocytosis (10, 57, 58, 407, 468, 486, 640). Hence, low pH is instrumental in the process of membrane translocation of the L chain from the vesicle lumen into the cytosol. In this respect, TeNT and BoNT appear to behave similarly to the other bacterial protein toxins characterized by a structure consisting of three distinct domains (417). Low pH induces TeNT and BoNT to undergo a conformational change from a water-soluble "neutral" form to an "acid" form with surface-exposed hydrophobic segments, which enable the penetration of both the H and L chains in the hydrocarbon core of the lipid bilayer (73, 74, 91, 395, 420, 421, 504, 532). After this low pH-induced membrane insertion, TeNT and BoNT form ion channels in planar lipid bilayers (69, 73, 151, 201, 264, 395, 496, 546, 561). These ion-conducting channels are cation-selective with conductances of a few tens of picoSiemens and are permeable to molecules smaller than 700 Da. There is evidence that these channels are formed by the oligomerization of the H_N domain (151, 395, 546, 561). The structure of the H_N domain of BoNT/A has elements of similarity with other membrane translocating toxins such as colicins and diphtheria toxin, which make channels (112, 456, 652) and with some viral proteins of viruses undergoing low pH-driven structural changes (174, 639). Site-directed mutagenesis coupled to electrophysiological investigations and biochemical studies of diphtheria toxin and colicins membrane insertion indicate that the hairpin pair of buried hydrophobic helices is the first part of the molecule that enters the lipid bilayer followed by other -helices of the same domain (reviewed in Refs. 124, 415). Peptides corresponding to segment 668-690 of TeNT (GVVLLLEYIPEITLPVIAALSIA) and segment 659-681 of BoNT/A (GAVILLEFIPEIAIPVLGTFALV), which are predicted to form amphipatic -helices, but are actually -stranded in the crystallographic structure neutral form (326), form channels with properties similar to those of the intact toxin molecule (412). On this basis, it was proposed that the channel is formed by a toxin tetramer that brings four amphipatic helices into proximity with the carboxylates of the two Glu residues of the segment pointing inside the channel (412). This is compatible with the three-dimensional image reconstruction of the channel formed by BoNT/B in phospholipid bilayers (546). Clostridial neurotoxin channel formation is not limited to model membranes, since TeNT forms ion channels in spinal cord neurons. They open with high frequency at pH 5.0, but not at neutral pH; are rather nonselective for Na⁺, K⁺, Ba²⁺, and Cl; and have a single-channel conductance of 45 pS (39).

There is a general consensus that these toxin channels are related to the process of translocation of the L domain across the vesicle membrane into the nerve cytosol. However, there is no agreement on how this process may take place. According to one hypothesis, the L chain unfolds at low pH and permeates through a transmembrane pore formed by H chain(s). After exposure to the neutral pH of the cytosol, the L chain refolds, and it is released from the vesicle by reduction of the interchain disulfide bond (73, 264). In this "tunnel" model, the formation of a transmembrane ion-conducting pore is a prerequisite for translocation. Two experimental results do not fit in this model: 1) the L chains of TeNT and BoNT/A, /B, and /E penetrate the lipid bilayer in such a way as to be exposed to the fatty acid chains of phospholipids, i.e., they are not shielded from lipids inside the H chain tunnel (420, 421); and 2) values of the order of a few tens of picoSiemens do not account for the dimensions expected for a protein channel that has to accommodate a polypeptide chain with lateral groups of different volume, charge, and hydrophilicity. The protein-conducting channels of the endoplasmic reticulum, of Escherichia coli and of mitochondrial membranes, characterized in planar lipid bilayers, have a conductance of 220 pS (181, 261, 321, 565, 566, 600). These channels are closed when plugged by a transversing polypeptide chain. Changing the size or polarity of the applied voltage does not influence their conductance or gating, whereas it does affect CNT channels.

A second model, advanced by Beise et al. (39), envisages that as the vesicle internal pH decreases after the operation of the vacuolar-type ATPase proton pump, CNT insert into the lipid bilayer, forming ion channels that grossly alter electrochemical gradients. Eventually, such permeability changes cause an osmotic lysis of the toxin-containing acidic vesicle, emphasized by possible toxin-induced destabilization of the lipid bilayer (91). The membrane barrier is broken, and the cargo of toxin molecules is released in the cytosol. Even though this model greatly simplifies the problems posed by membrane-translocating toxins, some experimental findings with diphtheria toxin, which provides the best-characterized system with respect to bacterial toxins entry into cells, do not support it. 1) Diphtheria toxin forms ion channels in the plasma membrane of living cells at low pH without causing cell lysis (11, 455, 526), and similarly, TeNT does not lyse the plasmalemma of neuronal cells at pH 5.0 (39). 2) Endosomes containing diphtheria toxin can be isolated from cells (38, 341). 3) A catalytically inactive diphtheria toxin mutant form alters the plasma membrane permeability to sodium and potassium without lysing the cell (455). 4) Diphtheria toxin that has not translocated in the cytosol moves further along the endosomal-lysosomal pathway to be eventually degraded (341, 453). This osmotic lysis model could be tested directly by determining if fluid-phase markers gain access to the cytosol in the presence of toxins.

An alternative hypothesis, which explains all available experimental data, proposes that the L chain translocates across the vesicle membrane within a channel open laterally to lipids, rather than inside a proteinaceous pore (416, 417). The two toxin polypeptide chains are supposed to change conformation at low pH in a concerted fashion, in such a way that both of them expose hydrophobic surfaces and enter into contact with the hydrophobic core of the lipid bilayer. The toxin acid form may have the dynamic properties of a molten globule (90, 619). The H chain forms a transmembrane hydrophilic cleft that nests the passage of the partially unfolded L chain with its hydrophobic segments facing the lipids. The cytosolic neutral pH induces the L chain to refold and to regain its water-soluble neutral conformation, after reduction of the interchain disulfide. It is possible that cytosolic chaperones are involved in treadmilling the L chain out of the vesicle membrane and in assisting its cytosolic refolding, but as yet there is no supporting evidence. As the L chain is released from the vesicle membrane, the transmembrane hydrophilic cleft of the H chain is supposed to tighten up to reduce the amount of hydrophilic protein surface exposed to the membrane hydrophobic core. However, this leaves across the membrane a peculiarly shaped channel with two rigid protein walls and a flexible lipid seal on one side. This is proposed to be the structure responsible for the ion-conducting properties of TeNT and BoNT. In this "cleft" model, the ion channel is a consequence of membrane translocation rather than a prerequisite. Moreover, ion transport is mediated by a transmembrane structure that derives from the one involved in the L-chain translocation, but which is physically different.

The catalytic activity of these neurotoxins was discovered following the sequencing of the corresponding genes, which began with TeNT (160, 173) and, within a few years, was extended to all CNT (402). Sequence comparison revealed a highly conserved 20-residue-long segment, located in the middle of the L chain, containing the His-Glu-Xaa-Xaa-His zinc-binding motif of zinc-endopeptidases (322, 538, 540, 663). Building on this observation, investigators soon demonstrated that TeNT inhibited ACh release at synapses of the buccal ganglion of Aplysia californica via a zinc-dependent protease activity (538). Identification of the cytosolic substrates of such enzymic activity followed assays of proteolysis performed on SSV and on other synaptic proteins suggested as candidates for the neuroexocytosis apparatus by the characterization of a 7S brain complex (579).

The eight CNT are remarkably specific proteases; among the many proteins and synthetic substrates assayed so far, only three of them, the so-called SNARE proteins, have been identified (Figs. 5 and 6 and Table 1). TeNT and BoNT/B, /D, /F and /G cleave vesicle-associated membrane protein (VAMP)/synaptobrevin, but each at different sites (531, 535, 538, 539, 543, 666, 667); BoNT/A and /E cleave 25-kDa synaptosomal-associated protein (SNAP-25) at two different sites and BoNT/C cleaves both syntaxin and SNAP-25 (56, 67, 68, 189, 450, 539, 541, 542, 655). Strikingly, TeNT and BoNT/B cleave VAMP at the same peptide bond (Gln-76-Phe-77), yet when injected in the animal, they cause the opposite symptoms of tetanus and botulism, respectively (531). This observation clearly demonstrated that the different symptoms derive from different sites of intoxication rather than from a different molecular mechanism of action of the two neurotoxins.

View larger version (21K): [in this window] [in a new window]   Fig. 5. Schematic structure of syntaxin I and SNAP-25 with cleavage sites of clostridial neurotoxins. A: syntaxin is a type II membrane protein consisting of 4 parts: a NH2-terminal region (1-180) that folds in a bundle of -helices with a left-handed twist, followed by a region (180-262, gray box) that participates in SNARE complex formation via -helix coiling around complementary regions of VAMP and SNAP-25. Botulism neurotoxin (BoNT)/C cleaves within this second part of syntaxin and compromises the functional pairing of the vesicle with the presynaptic membrane, thus preventing the ensuing vesicle membrane fusion. The third part is a typical transmembrane segment (black box) followed by a short extracellular COOH-terminal segment. B: SNAP-25 lacks a classical transmembrane segment, and its membrane binding is mediated by the palmitoylation of a group of cysteines located in the middle of the polypeptide chain. Cleavage sites for BoNT/A, /C, and /E (arrows) and the 2 segments essential for the interaction with other SNARE (gray boxes) are indicated.

View larger version (24K): [in this window] [in a new window]   Fig. 6. Schematic structure of VAMP. VAMP/synaptobrevin is a type II membrane protein with a short COOH-terminal tail protruding in the vesicle lumen, a transmembrane segment (black box), followed by a 66-residue-long cytosolic part, which is highly conserved among isoforms and species (gray box). This central portion of VAMP coils around complementary regions of SNAP-25 and syntaxin in the SNARE complex and contains the site of interaction and cleavage by the clostridial neurotoxins. In contrast, the NH2-terminal part is poorly conserved and rich in prolines and remains outside the SNARE complex. It is likely to be involved in protein-protein interactions with other components of the neuroexocytosis apparatus and transport proteins. The cleavage sites for TeNT, BoNT/B, /D, /F, and /G are indicated by arrows. Rat VAMP-1 isoform is not cleaved by TeNT and BoNT/B, due to a sequence variation at the cleavage site, but this is not the case for other species, such as humans and mice.

Recombinant VAMP, SNAP-25, and syntaxin are cleaved at the same peptide bonds as the corresponding cellular proteins, thus indicating that no additional endogenous factors are involved in determining the specificity of the CNT. It was recently reported that CNT are phosphorylated inside the neuron and that this modification enhances the proteolytic activity of the toxins as well as their lifetime inside the cytosol (178). These findings have been exploited to develop in vitro assays of the metalloprotease activity of CNT (162, 163, 236, 536, 577). Particularly useful will be continuous assays based on the used of fluorescent substrates, whose fluorescence is internally quenched and is freed upon proteolysis of the peptide bond that keeps the two fluorophores close to each other (305) (F. Cornille and B. P. Roques, unpublished observations). The proteolytic activity of the CNT can be probed in cells and tissues with antibodies specific for epitopes present in the intact SNARE molecule, which are released into the cytosol following the action of the toxin. A highly sensitive single-cell assay can thus be performed by following the progressive loss of SNARE staining as its proteolysis progresses (375, 450, 492, 655). In parallel, the progressive block of SSV exo-endocytosis recycling consequent to substrate proteolysis can be monitored by assaying the internalization of antibodies specific for epitopes of SSV lumen (373).

Two groups of zinc-endopeptidase inhibitors are known: 1) zinc chelators and 2) molecules that bind with high affinity to the active site. Zinc chelators act either by complexing the free zinc that is in chemical equilibrium with the active site zinc, as EDTA does, or by actively removing the protein-bound metal atom, as ortho-phenantroline does (21). The latter type of chelators is much more rapid and should be used when a quick inactivation of a zinc-endopeptidase is needed. Some zinc chelators are plasma membrane permeable and can be used in intact cells (538). Although chelators are very effective on the CNT, none of the inhibitors active on the other classes of zinc-endopeptidases acts on CNT at low concentrations (120, 538) as a consequence of the different active site geometry of this novel group of zinc-endopeptidases. Specific inhibitors are badly needed not only for biochemical and cellular studies but also to evaluate them as potential drugs for the treatment of tetanus and botulism. Recently, a fluorescent coumarin derivative was found to inhibit BoNT/B (4), and a series of aminothiol derivatives of tripep