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Alpha-latrotoxin (alpha-ltx), a component of the venom of black widow spiders (BWSV), binds to higher vertebrates presynaptic nerve terminals, stimulating massive neurotransmitter release. This neurotoxic protein is responsible for most of the symptoms elicited in men by the bite of black widow spider (BWS), i.e. a neurological syndrome named latrodectism. By reasoning that targeting this single component would abrogate most of the effect of BWS envenomation, we took advantage of the antibody phage display technology to generate monoclonal Fab fragments able to bind and neutralize the alpha-ltx. To this aim, we immunized Balb/c mice with purified toxin and cloned their antibody repertoire in the pCombIII phage display vector. By combining a high-stringency affinity selection with a sensitive 45Ca(2+) uptake assay, we isolated a Fab fragment (FM1) able to bind the alpha-ltx in the low nM range and neutralize its ionophore activity, in vitro and in vivo. After the onset of overt symptomatology, administration of FM1 to experimentally envenomed mice induced remission of symptoms and prevented lethality. Since alpha-ltx is the only molecule responsible for the great toxicity of BWS bites in mammals, the FM1 Fab, highly effective in neutralizing the toxin in vivo, represents a promising immunotherapy reagent for treating latrodectic patients.

A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from alpha-latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern-blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C-terminal sequence of latrodectin detected cross-reactive proteins in the venom fluid, venom gland extracts, and in purified alpha-latrotoxin, suggesting that latrodectin is intimately associated with alpha-latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.

When Xenopus oocytes are injected with rat brain mRNA, they acquire the ability to respond to bath applied α-latrotoxin. This spider venom toxin is normally highly selective for nerve endings, where its binding is associated with a high-frequency, quantal discharge of neurotransmitter. By ‘transplanting’ toxin acceptor sites to Xenopus oocytes, we have observed both a toxin-mediated rise in cellular ionized Ca along with the triggering of a calcium-dependent chloride channel in these cells. This approach may contribute both to a better understanding of the mechanism of action of this toxin and to efforts to clone the cDNA for this binding site.

Monoclonal antibodies, raised against alpha-latrotoxin, have been used on serial sections of the venom glands with localization by indirect immunofluorescence. Colloidal gold immunovisualization and electron microscopy were used in order to localize the structure of the cells that synthesize and store the toxin. The antibodies were able to recognize the native toxin revealing its presence mainly in the 'replacement cell' layer, a layer of cells close to the muscular sheath of the gland. These cells apparently replace the disintegrated epithelial cells at the end of the holocrine cycle. Gold grains were predominantly observed around the free ribosomes and the secretion droplets of the 'replacement cells'.

Regeneration of soleus motor nerve terminals after focal destruction by black widow spider venom (BWSV) or its active factor alpha-latrotoxin (LTx) was compared in young and old CBF-1 mice. The object was to determine whether previously reported delayed regeneration after nerve injury in old rodents was due to altered removal of debris, or delay or aberrancy in structural or functional restoration of the neuromuscular junction. In addition, the use of a new fluorescent technique permitted for the first time quantitation of the accuracy of early nerve terminal regeneration in mammalian muscle. Immunohistochemical and electron micrographic studies showed no age difference in destruction of terminals and removal of debris 2 days after toxin application. The indirect twitch and structural reinnervation (measured with flourescent techniques) returned to an equal extent in young and old mice beginning at 3 days after LTx treatment. BWSV (as opposed to LTx) delayed regeneration 1 day in young but not in old mice. On the first day of reinnervation, there was perisynaptic outgrowth in both young and old mice, although in the latter, there was a higher incidence of aberrant outgrowth. The relation between return of twitch strength and recovery of nerve terminal area (measured in teased zinc iodide-stained preparations) showed no age dependency. We conclude that factors cited to explain altered reactive sprouting in the aging CNS do not apply to regeneration of peripheral motor nerve terminals. However, it is possible that the aberrant regrowth observed at the neuromuscular junction in old mice will pertain to the aging CNS. Altered axonal rather than nerve terminal regeneration is the likely source of delayed peripheral nerve regeneration in old animals.

1. Synaptic Vesicle Proteins - A Molecular Study 2. Dissection of the Secretory Machinery 3. Regulatory Roles for Lipids in Vesicle Trafficking and Secretion 4. Fusion Proteins and the Fusion Events 5. Chromaffin Cells as a Secretory System - The Use of Neurotoxins 6. Botulinum Neurotoxins and Their Substrates 7. Purification, Function and Selectivity in a-Latrotoxin 8. The Synapsins and Neurotransmission 9. Morphological Studies of the Secretory Machinery Using Neurotoxin Probes 10. Molecular Mechanisms of the Action of Clostridium botulinum Type B Neurotoxin 11. Neurotoxins and Safety-Latches of the Secretory Process

alpha-Latrotoxin, a protein toxin present in the venom of black widow spider, interacts with membrane receptors of neurons and other secretory cells to stimulate exocytosis. Two types of receptors have been identified and cloned. Our attention has been focused on the calcium independent receptor, a G-protein coupled receptor, named latrophilin to see whether alpha-latrotoxin interaction was capable to produce an ionotropic effect, in alternative to the metabotropic hypothesis. Expression of latrophilin receptor is sufficient for the alpha-latrotoxin effect to become manifest. By inducing the transient expression of latrophilin receptor in non-neuronal human embryonic cells, we made them susceptible to toxin action as demonstrated by the increase in 45Ca(2+) accumulation detected after toxin treatment. Since the presence of a monoclonal antibody against alpha-latrotoxin (4C4.1 mAb) was able to obliterate toxin-dependent effects, we further investigated the nature of toxin-antibody interaction by characterization of the binding epitope using phage display-peptide libraries. A conformational epitope was recognized and partially localized on a region of the peptide toxin whereby a tetrameric structure is formed and inserted into the membrane of target cells where it functions as a pore.

alpha-Latrotoxin (alpha-LTX) induces exocytosis of small synaptic vesicles (SSVs) in neuronal cells both by a calcium-independent mechanism and by opening cation-permeable pores. Since the basic molecular events regulating exocytosis in neurons and endocrine cells may be similar, we have used the exocytosis of insulin-containing large dense core vesicles (LDCVs) as a model system. In primary pancreatic beta-cells and in the derived cell lines INS-1 and MIN6, alpha-LTX increased insulin release in the absence of extracellular calcium, but the insulin-secreting cell lines HIT-T15 and RINm5F were unresponsive. alpha-LTX did not alter membrane potential or cytosolic calcium, and its stimulatory effect on exocytosis was still observed in pre-permeabilized INS-1 cells kept at 0.1 microM Ca2+. Consequently, pore formation or ion fluxes induced by alpha-LTX could be excluded. The Ca2+-independent alpha-LTX-binding protein, latrophilin, is a novel member of the secretin family of G protein-coupled receptors (GPCR). Sensitivity to alpha-LTX correlated with expression of latrophilin, but not with synaptotagmin I or neurexin Ialpha expression. Moreover, transient expression of latrophilin in HIT-T15 cells conferred alpha-LTX-induced exocytosis. Our results indicate that direct stimulation of exocytosis by a GPCR mediates the Ca2+-independent effects of alpha-LTX in the absence of altered ion fluxes. Therefore, direct regulation by receptor-activated heterotrimeric G proteins constitutes an important feature of the endocrine exocytosis of insulin-containing LDCVs and may also apply to SSV exocytosis in neurons.

A neurotoxin of the venom of the spider Latrodectus mactans tredecimguttatus, has been prepared in a homogeneous form and examined by a variety of techniques. The protein has a molecular weight of 130 000 and its toxicity in mice is about 49 000 LD50 mg pure protein/g body weight. The toxin releases norepinephrine from synaptosomes prepared from rat brain and shows most of the toxic effects of the crude venom preparation.

A purified neurotoxin from the venom of black widow spider Latrodectus mactans tredecimguttatus, when tested on synaptosomes by means of a perfusion technique, stimulates the release of previously accumulated radioactive transmitters. Black widow spider toxin has also been shown to be a potent inhibitor of the uptake systems for γ-aminobutyrate and N-epinephrine. The release from synaptosomes of radioactive N-epinephrine and γ-aminobutyrate was rapidly increased by the presence of a low concentration of toxin (∼ 1 nM). The toxin does not appear to cause either non-specific membrane damage or the release of a non-transmitter substance like γ-amino[14C]isobutyric acid. The venom-induced release of γ-aminobutyrate was shown not to be dependent on extracellular calcium concentration provided that sodium ions are present. By contrast, that of N-epinephrine appeared to be Ca-dependent. The release of γ-aminobutyrate induced by the toxin is not modified by the presence in the perfusing fluid of tetrodotoxin, dithioerythritol and strontium ions, or by pretreatment of synaptosomes with diaminobutyrate. The mechanism of toxin-induced release is different from that of a depolarization-induced release. By correlating previous electrophysiological observations with the results obtained in this biochemical study it may be suggested that the toxin can be used in studies aimed at the identification of components specific to the presynaptic terminal membrane involved in neurotransmitter release.

Tetanus toxin specifically binds to neuronal surfaces and interferes with the release of transmitters. The effect of tetanus toxin pretreatment of PC12 cell line, taken as a model of neuronal cells in culture, was studied and found that it depresses depolarization-dependent catecholamines secretion. This effect is limited to PC12 cells fully differentiated by the action of Nerve Growth Factor (NGF) and is indicative of the expression of specific binding sites for tetanus toxin during transition from the undifferentiated state. Specific binding of [125I] tetanus toxin to NGF-treated PC12 was demonstrable. The toxin has no effect on the 45Ca accumulation coupled with the depolarization dependent release of catecholamines.

A brain specific protein, 14-3-2, has been isolated from bovine brain by the use of ammonium sulfate precipitation, gel filtration on Sephadex G-150, and ion exchange chromatography on DEAE-cellulose and DEAE-Sephadex. It is an acidic protein, in agreement with the high content of glutamic and aspartic acid, that is composed of a single polypeptide chain of mol. wt. 50,000. Immunochemical tests using antiserum to purified 14-3-2 showed that the protein is present in at least 100-fold greater amounts in brain than in any other rat tissue. Furthermore, 14-3-2 was found in brains of a number of vertebrate species, although the antigen is apparently not entirely identical in all species tested. The protein 14-3-2 can be considered to be a species non-specific protein which is neuronal in origin.

Latrodectus venom is toxic to the crayfish Astacus astacus , its toxicity being in the same range as that for the guinea pig and housefly. Low doses of the venom affect the activity of the stretch receptor neuron of the crayfish modifying and blocking the impulse frequency. Picrotoxin does not prevent this effect.

14-3-2, a nervous system specific protein, has been studied in mouse neuroblastoma cells grown in culture by means of the microcomplement fixation assay. The experiments reported indicate that 14-3-2 protein is rapidly accumulated during the early phase of cell growth.

The level of several glycolytic intermediates, phosphate compounds, and reduced and oxidized pyridine nucleotides was determined in an isolated crustacean nerve cell preparation both at rest and after prolonged physiological stimulation. The glycogen decreased consistently with the lactate increase during impulse activity. ATP showed a continuous decrease during prolonged stimulation while ADP and Pi showed opposite patterns. After stimulation, the ratio TPN/TPNH fell by more than 50 per cent. This was due to a decrease of TPN. Similarly, the DPN/DPNH ratio fell by 50 per cent, but in this case it was due to a decrease of DPN and a simultaneous increase of DPNH. The results strongly suggest the involvement of glycolysis in the mechanism maintaining impulse activity in this neurone. Impulse activity in the crustacean nerve cell is linked to an energy requiring system. This energy is furnished by ATP splitting and glycolysis directly or through the arginine phosphate step supporting the ATP resynthesis.

The simplified purification protocol established for the isolation of alpha-latrotoxin from the venom of the spider Latrodectus tredecimguttatus, has been employed for the purification of toxic components present in the venom of the spider Steatoda paykulliana. The venom of this spider, frequently mistaken for L. tredecimguttatus, is by tradition considered to cause an envenomation potentially dangerous to man. The venom of S. paykulliana has little toxic effect on guinea-pigs but is extremely toxic to houseflies (Musca domestica). No proteolytic activity was detectable. Interaction of microgram/ml amounts of the venom extract with artificial lipid membranes produces an increase of membrane conductance through the formation of stable ion-permeable channels modulated by the direction and size of the electric potential differences across the membrane. Higher concentrations of this venom are able to stimulate the release of transmitters from neurosecretory cells in a fashion reminiscent of black widow spider venom. Antibodies against the whole L. tredecimguttatus venom gave a few positive cross-reactions in the immunodiffusion test with S. paykulliana venom gland extract indicating the presence of common molecular sequences in the two venoms. Polyclonal antibodies against alpha-latrotoxin did not cross-react in the immunodiffusion test with S. paykulliana venom extracts, nor in the immunofluorescence assay with its cephalothorax sections, thus suggesting that the venom glands do not contain alpha-latrotoxin. A partial characterization of S. paykulliana venom has been performed and a high molecular weight protein toxic to houseflies has been partially purified.

Summary Two aspects of the subunit structure of the bovine brain specific protein 14-3-2 have been examined. On the one hand, native 14-3-2 has been separated into two fractions by hydroxylapatite chromatography. One eluted at the same position when chromatographed on the same column, while the other redistributed into the same two fractions again. Amino acid analysis of these two forms of 14-3-2 gave results that were not significantly different under the condition of analysis. Furthermore, when each peak was subjected to cyanogen bromide cleavage, very similar elution profiles of the resultant fragments were obtained. The two pools also cross-reacted with antiserum to 14-3-2. Reaction of purified 14-3-2 with dimethylsuberimidate caused the formation of covalently bound protein units of 100,000 molecular weight when measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis, as opposed to the 50,000 minimal molecular weight normally detected. On the other hand, analysis of the soluble tryptic peptides of S-[14C]carboxymethyl 14-3-2 yielded only three distinct radioactive peptides, each with one residue of S-carboxymethylcysteine whereas 8 are expected on the basis of the amino acid composition of the 50,000 molecular weight polypeptide chains. Thermolysin digestion of a similarly modified 14-3-2 preparation yielded all of the radioactivity in 5 S-carboxymethylcysteine-containing peptides. The partial amino acid sequence of these peptides indicates that they represent 4 unique areas of the polypeptide chain. Since 8 such peptides were expected, that is, double the number found, the minimum structural unit of the protein must be of 25,000 molecular weight. The results of these experiments do not permit distinction between a duplication of the structure within a single polypeptide chain or the alternate possibility of two polypeptide chains bound by unusually strong non-covalent bonds. These results suggest that 14-3-2 is a covalently linked dimer of 25,000 mol.wt. units that can aggregate to form larger species of 100,000 mol. wt. and higher

The distribution of the 14-3-2 protein in rat brain was investigated by immunoelectron microscopy using antiserum to the protein conjugated with peroxidase. 14-3-2 was demonstrated in the nuclear membrane, the endoplasmic reticular membranes and in the plasma membrane of nerve cells. The protein was also localized to the presynaptic densities and to the pre- and postsynaptic membranes. It could not be demonstrated in the membranes of the Golgi complex, inner membrane of mitochondria or in the nucleoplasm of neurons. No 14-3-2 protein was found in astrocytes, oligodendrocytes or in non-neuroectodermal tissue elements.

A Na + , K + ATP-ase system is present in the cockroach ventral nerve cord. This system can be better differentiated from an Mg ++ ATP-ase in the “microsomial fraction”. Furthermore it fulfils the requirements described for a cation transport system mediated by Na + , K + ATP-ase. It is concluded that, in spite of the peculiar ion concentration of the haemolymph, the classical model of active transport can be considered operating insects nervous system.

Several polypeptide neurotoxins affect presynaptic functions by interfering with chemical neurotransmission. This group of toxins includes botulinum toxin, tetanus toxin, beta-bungaro-toxin and black widow spider toxin (BWSTx). While the effect of the first three toxins is mainly a rapid and severe block of neurotransmitter release, BWSTx affects transmission by a massive stimulation of mediator release. Despite various hypotheses put forward to explain the action of BWSTx at the level of nerve terminals, there is still a considerable degree of uncertainty as to the cation dependence of venom action. Study of the toxin mode of action at the biochamical level has been hampered by the complexity and cellular heterogeneity of the preparations used, neuromuscular junction or synaptosomes. PC12 cell line, derived from a rat phaeochromocytoma, seems to be an excellent model in view of its property of synthesising and storing noradrenaline, dopamine and acetylcholine, and releasing them in depolarising conditions. We have recently shown that highly purified BWSTx stimulates secretion from PC12 cells of previously taken up radioactive dopamine (DA) and noradrenaline (NA) (ref. 14 and manuscript in preparation). We report here that the earliest detectable event after toxin treatment of such cells is a massive increase of cytosolic calcium.