Stopped-flow fluorescence studies on binding of snake neurotoxins to acetylcholine receptor

The stopped-flow technique has been applied to observe the time dependence of a tryptophanyl fluorescence change upon binding of postsynaptic snake neurotoxins to nicotinic acetylcholine receptor (Narke japonica). Examination of the kinetics of the fluorescence change reflecting a conformational change in the receptor in the process of binding of 28 short neurotoxins and 8 long neurotoxins to the receptor has revealed the following. Short neurotoxins associate with the receptor more rapidly than do long neurotoxins. A positive charge on the side chains of residues 27 and 30 and the overall net charge of the toxin molecule governs the magnitude of the binding rates of toxins to the receptor. The invariant residue Asp-31 is important for neurotoxicity, but is not critical for binding ability with the receptor.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Mass spectrometry analysis of the phospholipase A2 activity of snake pre-synaptic neurotoxins in cultured neurons

Snake pre-synaptic phospholipase A₂ neurotoxins paralyse the neuromuscular junction by releasing phospholipid hydrolysis products that alter curvature and permeability of the pre-synaptic membrane. Here, we report results deriving from the first chemical analysis of the action of these neurotoxic phospholipases in neurons, made possible by the use of high sensitivity mass spectrometry. The time–course of the phospholipase A₂ activity (PLA₂) hydrolysis of notexin, β-bungarotoxin, taipoxin and textilotoxin acting in cultured neurons was determined. At variance from their enzymatic activities in vitro , these neurotoxins display comparable kinetics of lysophospholipid release in neurons, reconciling the large discrepancy between their in vivo toxicities and their in vitro enzymatic activities. The ratios of the lyso derivatives of phosphatidyl choline, ethanolamine and serine obtained here together with the known distribution of these phospholipids among cell membranes, suggest that most PLA₂ hydrolysis takes place on the cell surface. Although these toxins were recently shown to enter neurons, their intracellular hydrolytic action and the activation of intracellular PLA₂s appear to contribute little, if any, to the phospholipid hydrolysis measured here.     

Comparison of the pH-induced conformational change of different clostridial neurotoxins

Clostridial neurotoxins are internalized inside acidic compartments, wherefrom the catalytic chain translocates across the membrane into the cytosol in a low pH-driven process, reaching its proteolytic substrates. The pH range in which the structural rearrangement of clostridial neurotoxins takes place was determined by 8-anilinonaphthalene-1-sulfonate and tryptophan fluorescence measurements. Half conformational change was attained at pH 4.55, 4.50, 4.40, 4.60, 4.40, and 4.40 for tetanus neurotoxin and botulinum neurotoxin serotypes /A, /B, /C, /E, and /F, respectively. This similarity indicates the key residues for the conformation transition are strongly conserved. Acidic liposomes support the conformational rearrangement shifting the effect versus higher pH values, whereas zwitterionic liposomes do not. The disulfide bridge linking the light and the heavy chains together needs to be oxidized to allow toxin membrane insertion, indicating that in vivo its reduction follows exposure to the cytosol after penetration of the endosomal membrane.     

Actions of snake neurotoxins on an insect nicotinic cholinergic synapse

Here we examine the actions of six snake neurotoxins (α-cobratoxin from Naja naja siamensis, erabutoxin-a and b from Laticauda semifasciata; CM12 from N. haje annulifera, toxin III 4 from Notechis scutatus and a long toxin from N. haje) on nicotinic acetylcholine receptors in the cercal afferent, giant interneurone 2 synapse of the cockroach, Periplaneta americana. All toxins tested reduced responses to directly-applied ACh as well as EPSPs evoked by electrical stimulation of nerve XI with similar time courses, suggesting that their action is postsynaptic. Thus, these nicotinic receptors in a well-characterized insect synapse are senstive to both long and short chain neurotoxins. This considerably expands the range of snake toxins that block insect nicotinic acetylcholine receptors and may enable further pharmacological distinctions between nAChR subtypes.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

Electric dipole reorientation in the interaction of botulinum neurotoxins with neuronal membranes

Botulinum neurotoxins are highly potent toxins capable of rapid and specific interaction with the presynaptic membrane. We have hypothesised that: (1) these neurotoxins possess an electric dipole with the positive pole on receptor binding domain Hc-C and that (2) on approaching the negatively charged presynaptic membrane, they reorient themselves and hit the membrane surface with Hc-C; this electrostatic effect would contribute efficient binding. Electrostatic calculations confirm these hypotheses and strongly indicate that electrostatics effects can play an important role in the unique presynaptic membrane binding properties of these neurotoxins and generally on the interaction of other plasma membrane protein ligands.     

Tryptophan modification of phospholipase A2 enzymes and presynaptic neurotoxins from snake venoms

Three phospholipases A₂ purified from cobra venoms and two presynaptically acting neurotoxins that exhibit phospholipase A₂ activity were subjected to tryptophan modification with 2-hydroxy-5-nitrobenzyl bromide. Associated with the modification of an increasing number of Trp residues were marked decreases in enzymatic activity and lethality, whereas antigenicity remained unchanged. The degree of exposure of tryptophanyl groups as determined by acrylamide quenching was consistent with the relative reactivity toward 2-hydroxy-5-nitrobenzyl bromide, except for Hemachatushaemachatus phospholipase A₂, which showed unusually high reactivity due to its characteristic dimeric conformation. Difference spectra of Trp-modified derivatives differed from those of their native enzymes by the presence of a new positive perturbation between 350 and 500 nm, with a maximum at 415 nm. Scatchard plots revealed only one type of binding site for Ca²⁺, and the binding abilities of the modified enzymes were not impaired. At pH 8.0, all native enzymes enhanced the emission intensity of 8-anilinonaphthalene sulfonate (ANS) dramatically, and the emission intensity of the ANS-enzyme complex increased or decreased in parallel with increasing concentration of Ca²⁺ for the respective enzyme. The Trp-modified derivatives did not enhance the emission intensity of ANS at all either in the presence or absence of Ca²⁺. By means of tryptophan modification, we were able to infer that the tryptophan residues are in the vicinity of the Ca²⁺ binding site and are directly involved in the binding with ANS. This, together with the suggestion that the hydrophobic pocket that interacts with ANS might be the site of binding of the phospholipase A₂ enzyme with the substrate, suggests that the Trp residues in phospholipase A₂ enzymes and presynaptic toxins are involved in substrate binding.     

B型肉毒毒素重链抗原表位的预测及表位合成肽抗原性的检测

目的:预测B型肉毒毒素蛋白抗原表位,研究表位合成肽的生物学活性。方法:利用生物信息学,结合Anthwine分析软件及网络平台,预测B型肉毒毒素蛋白重链的B细胞抗原表位。人工合成4条(P1,P2,P3,P4)表位多肽,与抗B型肉毒毒素类毒素的马血清反应;采用腹腔注射的方式用合成肽免疫BALB/c小鼠,检测免疫血清与合成肽的结合;对免疫小鼠腹腔注射B型肉毒毒素。结果:合成的多肽能与抗B型肉毒毒素的类毒素的马血清结合;多肽免疫小鼠后能产生针对多肽的抗体,其中P1、P3、P4产生的抗体对受毒素攻击的小鼠具有保护作用。结论:成功预测了抗原表位,合成的表位多肽具有较好的抗原性。     

Tetanus relaxation. Temperature effects and Arrhenius analysis

Time constants (τ) have been accurately measured for the exponentially falling, latter 60–70% of the relaxation phase of maximal isometric tetani of the mouse extensor digitorum longus muscle over the range 15–35°C. Corresponding to the τ values, the rate constants (k = 25.0?189 s^-1) are assumed to describe a temperature-sensitive, first-order, rate-limiting reaction underlying, and determining the kinetics of, muscle relaxation. The mean Arrhenius plot for the k values of 6 muscles consists of 2 linear segments with a 25°C transition temperature. The activation energies at the relatively lower and higher temperatures are 22.9 and 12.6 kcal/mol, respectively. These values are qualitatively, and to some extent quantitatively, similar to corresponding known Arrhenius results of the Ca²⁺ active transport mechanism and the physical properties of the membrane of isolated sarcoplasmic reticulum. Thus, the present findings strongly indicate that relaxation of living muscle critically involves the ‘relaxing factor’ activity of Ca²⁺ uptake, as previously inferred from research on isolated sarcoplasmic reticulum. Using transition-state theory, the Arrhenius results indicate that $\text{ΔG}{}^3$ of the assumed rate-limiting reaction is 14.8–15.0 kcal/mol at all temperatures studied, and $\text{ΔS}{}^3$ is about 25 and ?10 cal/degree per mol at temperatures below and above the transition temperature, respectively. These also correspond, at least qualitatively, to the values of the activation thermodynamic parameters of isolated sarcoplasmic reticulum. The negative $\text{ΔS}{}^3$ at the higher temperature range, denoting an increase in order associated with the assumed activation process of the Ca²⁺ transport system, requires clarification.     

P19 embryonal carcinoma cells exhibit high sensitivity to botulinum type C and D/C mosaic neurotoxins

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release at peripheral nerve terminals. They are serologically classified from A to G, C/D and D/C mosaic neurotoxins forming further subtypes of serotypes C and D. Cultured primary neurons, as well as neuronal cell lines such as PC12 and Neuro-2a, are often utilized in cell-based experiments on the toxic action of botulinum toxins. However, there are very few reports of the use of neural cell lines for studying BoNTs/C and D. In addition, the differentiated P19 neuronal cell line, which possesses cholinergic properties, has yet to be tested for its susceptibility to BoNTs. Here, the responsiveness of differentiated P19 cells to BoNT/C and BoNT/DC is reported. Both BoNT/C and BoNT/DC were shown to effectively bind to, and be internalized by, neurons derived from P19 cells. Subsequently, the intracellular substrates for BoNT/C and BoNT/DC were cleaved by treatment of the cells with the toxins in a ganglioside-dependent manner. Moreover, P19 neurons exhibited high sensitivity to BoNT/C and BoNT/DC, to the same extent as cultured primary neurons. These findings suggest that differentiated P19 cells possess full sensitivity to BoNT/C and BoNT/DC, thus making them a novel susceptible cell line for research into BoNTs.     

Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes

Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes. To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours). This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors. Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A. Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner. The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM. Binding of [3H]imipramine and [3H]serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites. These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane.     

肉毒毒素结构及其重组疫苗的相关研究进展

肉毒毒素是肉毒梭状杆菌产生的外毒素,有7种血清型(A～G).肉毒毒素属神经强毒,是目前已知的毒性最强的细菌蛋白质.作为重要的生物战剂之一,对肉毒毒素的研究已经相当深入,基本明确了各型肉毒毒素的基因序列、同源性和三维结构及毒素作用的本质和机理.随着国际恐怖活动的日益猖獗,针对肉毒毒素的检测和预防也备受关注,对其疫苗的探索已成为研究的焦点.本扼要介绍了肉毒毒素的结构、作用机制及其疫苗的相关研究进展.     

Delineating the susceptibility of botulinum neurotoxins to denaturation through thermal effects

Botulinum neurotoxins (BoNT) are the etiological agents responsible for botulism and are acknowledged terrorist threat agents. Passive immunotherapy may provide one countermeasure. Importantly, in the virtually unlimited repertoire of antibody specificities, enzyme linked immunosorbent assays (ELISA) has become an indispensable method for antibody selection. We report that of the BoNTs, BoNT/E is highly susceptible to polystyrene induced denaturation. To further dissect this result and the potential susceptibility of other BoNTs to denaturation we selected a thermal platform, which could be readily quantified using surface plasmon resonance (SPR), a primary rat spinal cord cell-based assay and an animal lethality model.     

Botulism: the agent,mode of action of the botulinum neurotoxins,forms of acquisition,treatment and prevention

The botulinum neurotoxins are produced by anaerobic, spore-forming bacteria belonging to the Clostridium genus. They are synthesised as a single chain protein (150 kDa), which is not or weakly active. The active form results from a proteolysis cleaving the precursor in a light chain (about 50 kDa) and a heavy chain (about 100 kDa), which are linked by a disulfide bridge. The heavy chain is involved in the recognition of a specific neuronal surface receptor and mediates the internalization of the light chain into the cytosol. The light chain is responsible for the intracellular activity. It catalyses the proteolysis of SNARE proteins, which are involved in the exocytosis of synaptic vesicles containing acetylcholine. Hence, the release of acetylcholine at the neuromuscular junction is blocked, leading to a flaccid paralysis. Human botulism, usually type A, B or E, is associated with intoxination, ingestion of preformed toxin in food, with digestive toxi-infection, mainly in newborns (infant botulism), or with wound contamination (wound botulism). The treatment of botulism is usually symptomatic. The specific treatment is based on the serotherapy or on the use of purified specific antibodies. The vaccination against botulism is efficient. However, since the botulinum neurotoxins are widely used for the treatment of numerous dystonias, a generalised vaccination is not conceivable.     

Husbandry of the little file snake,Acrochordus granulatus

Aquatic snakes of the family Acrochordidae are unusual in terms of appearance, biology, and natural history. In spite of many attractive and fascinating features, there are few zoological exhibits of acrochordid snakes, and as a result many aspects of their husbandry are poorly understood. The present paper summarizes aspects of acrochordid biology related to health and welfare of captive snakes, with emphasis on the little file snake, Acrochordus granulatus. Several key points emerge having crucial relevance to successful husbandry. (1) File snakes are sensitive to low temperatures and to rapid thermal change. Captive snakes do well when maintained at water temperatures of 27–30°C and will not thrive if water temperatures are below 25°C. (2) File snakes can be kept in either fresh water or seawater. Snakes in sea or brackish water dehydrate, however, and must be allowed to drink fresh water periodically. If snakes from marine populations are maintained in saline water, 60–70% seawater is recommended. In all cases, water should be filtered or changed periodically to maintain quality. (3) File snakes feed almost exclusively on fishes which are usually captured in body coils. Snakes are more inclined to feed well if live prey are offered in shallow water where they are more easily captured. (4) File snakes are nocturnal and prefer quiescent seclusion within darkened refugia during daylight hours. Providing snakes with refugia such as sections of PVC pipe (which simulate burrows) helps reduce stress and improves the chances of snakes feeding regularly. (5) Snakes tend to burrow, and they locomote by crawling as well as by swimming. Use of sharp or rough materials in aquaria should be avoided because of possible skin abrasion which increases permeability and provides sites for bacterial infection. © 1996 Wiley-Liss, Inc.     

Recombinant derivatives of clostridial neurotoxins as delivery vehicles for proteins and small organic molecules

Clostridial neurotoxins are the most powerful toxins known. Nevertheless, derivatives of these toxins may find broad applications both in science and medicine because of their unique abilities to recognize neurons and deliver small and large molecules into them. In this paper we describe the construction of two types of such derivatives. Proteins belonging to the first class were designed to allow direct conjugation with one or few molecules of interest. Proteins belonging to the second class contain biotin residue and therefore could be easily connected to streptavidin loaded with multiple molecules of interest. Only C-terminal regions of neurotoxin heavy chains were incorporated in the structure of recombinant proteins. Nevertheless, recombinant proteins were found to be able to recognize specific neuronal receptors and target model molecules to rat synaptosomes and human neuroblastoma cells.     

Ganglioside-Induced Adherence of Botulinum and Tetanus Neurotoxins to Adducin

Abstract: Preincubation of botulinum neurotoxin serotype A, B, or E with ganglioside GT1b was previously found to enhance adherence of botulinum neurotoxin to synapsin I and an ∼116-kDa bovine brain synaptosomal protein; in contrast, adherence to these two proteins by tetanus neurotoxin required preincubation with GT1b. We have now found that preincubation of the neurotoxins with ganglioside GD3 enhances their adherence to the ∼116-kDa protein more than that with GT1b. A purified preparation of the water-soluble ∼116-kDa protein was obtained from bovine brain synaptosomes by preparative column sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. N-Terminal amino acid sequences were obtained for two tryptic fragments of the ∼116-kDa protein. These sequences matched with the data bank sequences for β-adducin, a cytoskeletal protein. The carboxy-terminal tail region of adducin, but not the head region, was adhered to by the neurotoxins. Adherence of the neurotoxin to adducin and synapsin I may facilitate presentation of the neurotoxin to its specific substrate(s).     

Binding of Botulinum and Tetanus Neurotoxins to Ganglioside GT1b and Derivatives Thereof

The ability of fragments derived from botulinum neurotoxin (BTx) serotype A to bind to GT1b-coated plastic wells was investigated and compared with the binding characteristics of the parent approximately 150-kDa protein. Although the approximately 50-kDa light chain of BTxA had a marginal binding capacity, the predominant adherence to GT1b-coated wells was exhibited by the approximately 50-kDa carboxy-terminal half of the approximately 100-kDa heavy chain of BTxA; the amino-terminal half of the heavy chain lacked the ability to bind. Binding to GT1b by BTxA and its fragments was compared with that of tetanus neurotoxin (TTx) and the carboxy-terminal half of its heavy chain. Binding of BTxA and the C-terminal half of the heavy chain was optimal in buffers of low ionic strength (mu less than or equal to 0.04 and 0.06, respectively), whereas the heavy chain bound GT1b best at mu greater than or equal to 0.10. TTx and the approximately 50-kDa C-terminal half of its approximately 100-kDa heavy chain bound GT1b at ionic strengths similar to those of BTxA. Comparison of the binding of BTx serotypes A, B, and E to GT1b (using conditions that were found to be optimal for binding by BTxA) indicated differences in the interaction of the three serotypes with GT1b. Compared with BTxA, adherence to GT1b by serotypes B and E was reduced by approximately 60 and approximately 90%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel microsatellite loci in the grass snake (Natrix natrix) and cross-amplification in the dice snake (Natrix tessellata)

Six novel polymorphic microsatellite loci are presented for the grass snake (Natrix natrix), a species with declining populations in many regions. The number of alleles per locus ranged from two to seven. Four dice snake (Natrix tessellata) microsatellites were polymorphic in the grass snake with three to four alleles. At two loci, the expected heterozygosity differed significantly from observed heterozygosity. Cross-amplification of the grass snake markers in the dice snake showed two polymorphic microsatellites with two and four alleles.