Properties of a protease-sensitive acceptor component in mouse brain synaptosomes for Clostridium botulinum type B neurotoxin

To characterize an acceptor for Clostridium botulinum type B neurotoxin, its binding kinetics were examined with mouse brain synaptosomes treated with various enzymes. The amount of 125I-labelled neurotoxin bound to synaptosomes decreased upon treatment with lysyl endopeptidase, neuraminidase, or phospholipase C. The binding of the neurotoxin was partially recovered by incubation of neuraminidase-treated synaptosomes with ganglioside GT1b or GD1a. Gangliosides incorporated into untreated, lysyl endopeptidase-treated, and phospholipase C-treated synaptosomes had no effect on the binding of the neurotoxin. These results may suggest that type B neurotoxin binds to gangliosides in cooperation with a certain protease-sensitive substance on the neural membranes.     

Lipid interaction of tetanus neurotoxin. A calorimetric and fluorescence spectroscopy study.

The interaction of Tetanus toxin with phospholipid vesicles containing gangliosides (GD1a, GD1b or GT1b) or phosphatidic acid has been investigated at neutral or acidic pH. Change in the thermotropic properties of the vesicles occurred only after addition of the toxin at acidic pH, and led to surface binding or membrane insertion of the protein, dependent on the physical state of the membrane. Most remarkably, toxin addition at acidic pH to dipalmitoyl-phosphatidylcholine vesicles containing GT1b ganglioside, caused formation of ganglioside microdomains on the vesicle surface.     

Dalargin-binding proteins of synaptic membranes from the rat brain: isolation and comparison of their properties with opiate receptor properties]

The proteins isolated from rat brain synaptic membranes were studied by affinity chromatography on dalargin-omega-aminohexyl-Sepharose 4B and specific elution with DAGO (Tyr-D-Ala-Gly-N-Me-Gly-ol). These proteins were shown to bind specifically 3H-naloxone (Kd = 6.6 nM; Bmax = 690 pmol/mg of protein). SDS electrophoresis of the dalargin-binding proteins termed as DBPDAGO revealed one major protein band with M(r) of 42 kDa and two minor bands with M(r) of 29 and 67 kDa. The glycoprotein component was found in DBPDAGO; their isoelectric properties were established (pI 5.4). The close similarity of DBP properties with those of isolated brain opiate receptors suggest them to be opiate receptor components.     

Solubilization and various properties of NAD-binding receptor protein from rat brain synaptic membranes

The NAD-binding receptor protein has been solubilized from the synaptic membranes of the rat brain by different detergents. Digitanin proved to be the most effective detergent which exerted no action on the specific binding of [14C]NAD with the solubilized receptor protein. Kinetic parameters of the soluble ligand-receptor complex were studied. The affinity of the solubilized receptor protein to NAD did not change as compared to the protein of native membranes. The specific binding of [14C]NAD was saturated at Kd = 0.53 microM, Bmax = 0.011 nmol per 1 mg of protein. The molecular weight of the soluble NAD-receptor complex determined under native conditions was equal to 115 kDa.     

Effect of tetanus toxin and colchicine on synaptic membranes of rat cerebral cortex]

It was shown that purified tetanus toxin did not influence the activity of the Na, K-ATP-ase fractions of the synaptic membranes of the rat cerebral cortex, it had no effect on the inhibition of Na, K,-ATP-ase under electrical stimulation of the synaptic membrane suspension, or the GABA--3H binding by the synaptosomes in vitro. Tetanus toxin (400--4000 DLM) and colchicine (1 mM) induced a decrease of osmotic sensitivity of the nerve endings. Colchicine in low concentrations (10(-5)-10(-3) M) failed to influence Mg-and Na, K-ATP-ases, but considerably inhibited both ATP-ases at higher concentrations.     

Botulinum neurotoxin type B. Its purification, radioiodination and interaction with rat-brain synaptosomal membranes

Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein. 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially. Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites. No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction. Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity. Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively). These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors. Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin. Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity. These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.     

The interaction of neurotoxin derivatives with either acetylcholine receptor or a monoclonal antibody. An electron-spin-resonance study

Five derivatives of Naja nigricollis toxin alpha, spin-labeled on a single amino group, were prepared. The toxin derivatives were purified to homogeneity by ion-exchange and high-pressure liquid chromatographies. The modified amino groups are localized at residue 1 and lysines 15, 27, 47 and 51. Competition data show that incorporation of spin label at residues 27 or 47 reduces the affinity of the toxin for the nicotinic acetylcholine receptor (AcChR), while incorporation at residues 1 or 15 diminishes toxin affinity for a monoclonal toxin-specific immunoglobulin (M alpha 1). Classical and/or saturation transfer electron spin resonance (ESR) analysis was carried out on each derivative, either in the free state or bound to AcChR or M alpha 1. The data obtained give the following indications. In the free state, the nitroxides incorporated at residues 1, 15, 47 and 51 have their own rapid motion, while that at residue 27 had no residual mobility and reflects the toxin rotation. Binding of AcChR to the toxin reduces the motion of the nitroxide bound to Lys47. Binding of M alpha 1 to the toxin immobilizes the two nitroxides fixed on residues 1 and 15. ESR spectra show that Lys27-bound nitroxide remains immobilized upon binding of either AcChR or M alpha 1. The change in nitroxide immobilization observed upon AcChR or M alpha 1 binding correlates well with the variation of nitroxide accessibility to a water-soluble paramagnetic N2+i ion. Binding of the labeled Lys47 toxin derivative to AcChR yields a complex ESR signal, disclosing the existence of a physical difference between the two toxin binding sites on AcChR. All the data indicate that AcChR and M alpha 1 bind at two topographically distinct sites on the toxin surface.     

Association of botulinum neurotoxin serotypes a and B with synaptic vesicle protein complexes

Botulinum neurotoxins (BoNTs) elicit flaccid paralysis through cleavage of SNARE proteins within peripheral neurons. There are seven serotypes of the BoNTs, termed A-G, which differ in the SNARE protein and/or site that is cleaved. BoNTs are single-chain toxins that comprise an N-terminal zinc metalloprotease domain that is disulfide linked to the C-terminal translocation/receptor binding domain. SV2 and synaptotagmin have been identified as receptors for BoNT serotypes A and B, respectively. Using affinity chromatography, BoNTs A and B were observed to bind synaptic vesicle protein complexes in synaptosome lysates. Tandem LC-MS/MS identified SV2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2 (VAMP2), and the vacuolar proton pump as components of the BoNT-receptor complex. Density gradient analysis showed that BoNT serotypes A and B exhibited unique interactions with the synaptic vesicle protein complexes. The association of BoNT serotypes A and B with synaptic vesicle protein complexes implicates a physiological role for protein complexes in synaptic vesicle biology and provides insight into the interactions of BoNT and neuronal receptors.     

Identification of the synaptic vesicle glycoprotein 2 receptor binding site in botulinum neurotoxin A

Botulinum neurotoxins (BoNTs) inhibit neurotransmitter release by hydrolysing SNARE proteins. The most important serotype BoNT/A employs the synaptic vesicle glycoprotein 2 (SV2) isoforms A-C as neuronal receptors. Here, we identified their binding site by blocking SV2 interaction using monoclonal antibodies with characterised epitopes within the cell binding domain (H_C). The site is located on the backside of the conserved ganglioside binding pocket at the interface of the H_CC and H_CN subdomains. The dimension of the binding pocket was characterised in detail by site directed mutagenesis allowing the development of potent inhibitors as well as modifying receptor binding properties.     

Electrokinetic properties of synaptic vesicles and synaptosomal membranes.

Using the technique of electrophoretic light scattering, we have measured the electrophoretic mobilities of synaptic vesicles and synaptosomal plasma membranes isolated from guinea-pig cerebral cortex. The electrophoretic mobility of synaptic vesicles is slightly greater than that of synaptosomal plasma membranes. Ca+2 and Mg+2 reduced the mobility of both species to the same extent at physiologically relevant concentrations (0-1 mM) and near-physiologic ionic strength. The extent of the reduction was not large (approximately 6% for synaptic vesicles in the presence of 100 mM KCl) at 1 mM divalent cation concentrations. At concentrations of approximately 2 mM and higher, Ca+2 reduced the mobility of synaptic vesicles more than did Mg/2. A similar but much smaller effect was observed in the case of synaptosomal plasma membranes. The addition of 1 mM Mg+2-ATP had no effect upon synaptic vesicle mobility either in the presence or absence of the ionophores nigericin or valinomycin. These data, together with earlier work (Siegel et al., 1978, Biophys. J. 22:341-346), demonstrate that substantial reduction of the average electrostatic surface charge density is not the most important role of divalent cations in promoting close approach of secretory granules and secretory cell membranes, and that it is certainly not the Ca+2-specific step in exocytosis.     

Muscle surface membranes: preparative methods affect apparent chemical properties and neurotoxin binding.

Considerable disagreement exists between results reported by various authors for lipid composition and enzyme activity in purified muscle membrane fractions presumed to be sarcolemma, although an explanation for these discrepancies has not been presented. We have prepared muscle light surface membrane fractions of comparable density (1.050--1.120) by a low-salt sucrose method and by an LiBr-KCl extraction procedure and compared them for density profile, total lipid and cholesterol content, protein composition and ATPase activity. In addition, sodium channels characteristic of excitable membranes have been quantitated in each preparation using [3H]saxitoxin binding assays, and the density of acetylcholine receptors determined in fractions from control and denervated muscle using alpha-[125I]bungarotoxin. Although both fractions contain predominantly surface membrane, the LiBr fraction consistently shows the higher specific activity of p-nitrophenylphosphatase, higher free cholesterol content, and higher density of sodium channels and acetylcholine receptors. The density distribution of sodium channels appears uniform throughout both fractions. Quantitative differences were seen between sodium dodecyl sulfate polyacrylamide gel electrophoresis patterns of membrane proteins from the two preparations although most bands are represented in both. A majority of the low-salt sucrose light membrane proteins were accessible in varying degrees to labelling with diazotized diiodosulfanylic acid in intact muscle. These results suggest that light surface membrane fractions may be mixtures of sarcolemma and T-tubular membranes. Using our preparative methods, the LiBr fraction may contain predominantly sarcolemma while low-salt sucrose light membranes may be enriched in T-tubular elements.     

Losartan's molecular basis of interaction with membranes and AT1 receptor

Physicochemical methods were used to study the thermal and dynamic changes caused by losartan in the membrane bilayers. In addition, molecular modeling was implemented to explore its topography both in membranes and AT(1) receptor. Its incorporation resulted in the modification of thermal profile of dipalmitoyl phosphatidylcholine (DPPC) bilayers in a concentration dependent way up to 20mol% as it is depicted from the combination of differential scanning calorimetry (DSC) and MAS data. In particular, the presence of losartan caused lowering of the phase transition temperature and abolishment of the pretransition. T(1) experiments revealed the location of the drug into the membrane bilayers. The use of a combination of biophysical methods along with docking experiments brought out a possible two-step mechanism which involves incorporation of losartan at the interface of membrane bilayers and diffusion in the upper parts of AT(1) receptor helices IV-VII.     

Spin-labeled extracellular loop from a seven-transmembrane helix receptor: studies in solution and interaction with model membranes

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.     

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.     

Characterization of tetanus toxin binding to rat brain membranes. Evidence for a high-affinity proteinase-sensitive receptor.

Binding of 125I-labelled tetanus toxin to rat brain membranes in 25 mM-Tris/acetate, pH 6.0, was saturable and there was a single class of high-affinity site (KD 0.26-1.14 nM) present in high abundance (Bmax. 0.9-1.89 nmol/mg). The sites were largely resistant to proteolysis and heating but were markedly sensitive to neuraminidase. Trisialogangliosides were effective inhibitors of toxin binding (IC50 10 nM) and trisialogangliosides inserted into membranes lacking a toxin receptor were able to bind toxin with high affinity (KD 2.6 nM). The results are consistent with previous studies and the hypothesis that di- and trisialogangliosides act as the primary receptor for tetanus toxin under these conditions. In contrast, when toxin binding was assayed in Krebs-Ringer buffer, pH 7.4, binding was greatly reduced, was non-saturable and competition binding studies showed evidence for a small number of high-affinity sites (KD 0.42 nM, Bmax. 0.90 pmol/mg) and a larger number of low-affinity sites (KD 146 nM, Bmax. 179 pmol/mg). Treatment of membranes with proteinases, heat, and neuraminidase markedly reduced binding. Trisialogangliosides were poor inhibitors of toxin binding (IC50 11.0 microM), and trisialogangliosides inserted into membranes bound toxin with low affinity. The results suggest that in physiological buffers tetanus toxin binds with high affinity to a protein receptor, and that gangliosides represent only a low-affinity site.     

Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin

The type B neurotoxin (NT) isolated from Clostridium botulinum (strain 657) behaved as a mixture of single (unnicked) and dichain (nicked) proteins, both of Mr approximately 150 kDa. When the dichain NT was reduced by mercaptoethanol, the two chains migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as separate polypeptides of Mr approximately 100 and 50 kDa that appeared similar to the heavy and light chains of other serotypes of botulinum NT. The N-terminal amino acid sequences of the two chains were determined. They were as follows: light chain: Pro-Val-Thr-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asp-Asn-Asn-Asn-Ile- Ile-Met - Met-Glu-Pro-Pro-Phe-Ala-Arg-Gly-Met-Gly-Arg-Tyr-Tyr-Lys-Ala-Phe-Lys-Ile- Thr-Asp - Arg-Ile-Trp-Ile-; and heavy chain: Ala-Pro-Gly-Ile-X-Ile-Asp-Val-Asp-Asn-Glu-Asp-Leu-Phe-Phe-Ile-Ala-Asp-Ly s-Asn- Ser-Phe-Arg-Asp-Asp-Leu-. These two sequences matched exactly with those of the light and heavy chains of type B NT (strain Okra) of which only 16 and 18 residues were known (J. Biol. Chem. (1985) 260, 10461). The above sequences were different from those of type A NT. Immunoprecipitation reactions of type B NT isolated from strains 657 and Okra were indistinguishable against polyclonal anti-type B NT serum. These two preparations did not produce precipitin reactions with polyclonal anti-type A NT serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Targeted expression of tetanus neurotoxin interferes with behavioral responses to sensory input in Drosophila

Targeted inactivation of neurons by expression of toxic gene products is a useful tool to assign behavioral functions to specific neurons or brain structures. Of a variety of toxic gene products tested, tetanus neurotoxin light chain (TNT) has the least severe side effects and can completely block chemical synapses. By using the GAL4 system to drive TNT expression in a subset of chemo‐ and mechanosensory neurons, we detected walking and flight defects consistent with blocking of relevant sensory information. We also found, for the first time, an olfactory behavioral phenotype associated with blocking of a specific subset of antennal chemoreceptors. Similar behavioral experiments with GAL4 lines expressing in different subsets of antennal chemoreceptors should contribute to an understanding of olfactory coding in Drosophila. To increase the utility of the GAL4 system for such purposes, we have designed an inducible system that allows us to circumvent lethality caused by TNT expression during early development. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 221–233, 2002; DOI 10.1002/neu.10029