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At least 19 recordsLinked to original sources
PubMed1990

Kappa 2-bungarotoxin and kappa 3-bungarotoxin: two new neuronal nicotinic receptor antagonists isolated from the venom of Bungarus multicinctus.

Neuronal nicotinic acetylcholine receptors are recognized with high affinity by two snake venom kappa-neurotoxins, kappa-bungarotoxin and kappa-flavitoxin. Native and radiolabeled kappa-neurotoxins have been used to localize and quantitate neuronal nicotinic receptors in a variety of species. We now report the identification of two new kappa-neurotoxins. kappa 2-Bungarotoxin and kappa 3-bungarotoxin were purified from the venom of Bungarus multicinctus collected in the province of Guangdong, China. kappa-Bungarotoxin has as yet not been found in this venom, although it is the only kappa-neurotoxin to be isolated thus far from Taiwanese Bungarus multicinctus. The geographical separation of Guangdong and Taiwan might account for this evolutionary divergence within the species. Both of the new kappa-neurotoxins are potent antagonists of nicotinic transmission in the chick ciliary ganglion. kappa 3-Bungarotoxin, the least potent of the kappa-neurotoxins, produces a complete blockage of nicotinic transmission in 60 min at 250 nM. Protection experiments using the short-acting nicotinic antagonists dihydro-beta-erythroidine and (+)-tubocurarine demonstrate that kappa 2-bungarotoxin blocks transmission by binding to the acetylcholine recognition sites of neuronal nicotinic receptors. The isoelectric point of kappa 2-bungarotoxin (pI = 8.9) is similar to that of kappa-bungarotoxin and kappa-flavitoxin, but kappa 3-bungarotoxin is considerably more basic, with pI greater than 11. Partial amino acid sequences are reported for both kappa 2-bungarotoxin and kappa 3-bungarotoxin. These sequences show a high degree of homology (approximately 80%) with other kappa-neurotoxins, and allow the determination of the critical differences between the kappa-neurotoxins and the structurally related alpha-neurotoxins. For example, all 4 kappa-neurotoxins lack a tryptophanyl residue which is invariant and important for function in the alpha-neurotoxins. The kappa-neurotoxins also differ from the alpha-neurotoxins by having an invariant prolinyl residue at a critical sequence position. Heterodimers were detected consisting of one subunit each of kappa 2-bungarotoxin and kappa 3-bungarotoxin. These heterodimers, which form between any combination of two kappa-neurotoxins, appear to be physiologically active and confirm that a further distinction between kappa-neurotoxins and alpha-neurotoxins is the strong tendency of the former to self-associate in solution. The present results help to establish the definition of 'kappa-neurotoxin'. These snake toxins are now being used by a number of laboratories in physiological and biochemical experiments on neuronal nicotinic receptors from a variety of species.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed1992

Localization of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin binding to nicotinic sites in the chicken forebrain and midbrain.

We have previously localized cholinergic cell bodies and fibers within the midbrain of the chicken with choline acetyltransferase immunohistochemistry. In a continuing effort to characterize the central cholinergic system, the present study examines the distribution of various nicotinic acetylcholine receptors in the forebrain and midbrain of the chicken. The binding of 3H-nicotine, 125I-kappa-bungarotoxin, and 125I-alpha-bungarotoxin was localized by film autoradiography in adjacent sections of the adult chicken brain, allowing a comparison of the distribution of different classes of nicotinic binding sites within the brain. Although all three ligands were often co-localized, there were areas that bound 3H-nicotine but not the 125I-neurotoxins, or vice versa. Very high densities of all three ligands were found in the hyperstriatum ventrale; the nucleus geniculatus lateralis, pars ventralis; the griseum tectale; the nucleus dorsolateralis anterior thalami; the nucleus lentiformis mesencephali, pars lateralis and pars medialis; the periventricular organ; and the stratum griseum et fibrosum superficiale, layer f of the optic tectum. The nucleus spiriformis lateralis had the highest levels of 3H-nicotine binding in the chicken brain, but it did not bind either of the two snake neurotoxins. On the other hand, high levels of both 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding were found in the nucleus semilunaris and the nucleus ovoidalis, but these areas contained little or no 3H-nicotine binding. No unique 125I-kappa-bungarotoxin sites, unrecognized by 125I-alpha-bungarotoxin, were identified by the low resolution autoradiography performed in this study. In general, nicotinic receptors were found in areas that have been reported to contain cholinergic cell bodies or fibers. Comparison of our results with the expression of neuronal nicotinic receptor subunits, as determined by in situ hybridization, suggests that many of the high affinity 3H-nicotine sites are localized presynaptically, as, for example, in the retinorecipient nuclei and the nucleus interpeduncularis. The lack of 125I-kappa-bungarotoxin binding in the presence of alpha-bungarotoxin indicates that the chicken brain has only very low levels of a unique kappa-bungarotoxin site. This is in marked contrast to chicken, frog, and rat autonomic ganglia, where a unique kappa-neurotoxin-sensitive receptor has been identified and shown to mediate nicotinic neurotransmission.

Animals↗
PubMed1988

Structural studies of alpha-bungarotoxin. 3. Corrections in the primary sequence and X-ray structure and characterization of an isotoxic alpha-bungarotoxin.

The most plausible set of chemical shift assignments for alpha-bungarotoxin as deduced from the combined use of two-dimensional J-correlated and two-dimensional nuclear Overhauser effect 1H nuclear magnetic resonance (NMR) spectroscopy was in conflict with the accepted amino acid sequence between residues 8 and 12 and residues 66 and 70 [Basus, V. J., Billeter, M., Love, R. A., Stroud, R. M., & Kuntz, I. D. (1988) Biochemistry (first paper of three in this issue]). Furthermore, NMR spectra of alpha-bungarotoxin, purified by conventional methods, evidenced a second species at the level of approximately 10% total protein. The minor component was separated from alpha-bungarotoxin by Mono-S (cationic) chromatography. Sequencing of Mono-S-purified alpha-bungarotoxin and one of its tryptic peptides showed that the correct sequence for alpha-bungarotoxin is Ser-Pro-Ile at positions 9-11 and Pro-His-Pro at positions 67-69. The electron density map of alpha-bungarotoxin [Love, R. A., & Stroud, R. M. (1986) Protein Eng. 1, 37] was refined with the new sequence data. Improvements in the structure were found primarily for residues 9-11. Sequence analysis of two overlapping tryptic peptides proved that the minor species differed from alpha-bungarotoxin by replacement of a valine for an alanine at position 31. This new toxin, alpha-bungarotoxin(Val-31), binds to the acetylcholine receptor with an affinity that is comparable to that of alpha-bungarotoxin.

Amino Acid Sequence↗
PubMed1988

Kappa-bungarotoxin blocks an alpha-bungarotoxin-sensitive nicotinic receptor in the insect central nervous system.

Snake venom kappa-neurotoxins are selective antagonists of nicotinic acetylcholine responses in avian, murine and bovine neurons, and have been used as probes for functionally defined vertebrate neuronal nicotinic receptors. The actions of kappa-bungarotoxin, a kappa-neurotoxin, have now been examined at a central invertebrate nicotinic receptor. kappa-Bungarotoxin is a potent antagonist (IC50 = 100 nM) of nicotinic responses, producing a long-lasting blockade of insect nicotinic acetylcholine receptors. The blockade appears to be competitive, and voltage-clamp experiments on an identified cockroach motorneuron indicate that the actions of kappa-bungarotoxin are not dependent on membrane potential. alpha-Bungarotoxin is also a potent antagonist at the cockroach central nicotinic receptor, and binds (Kd = 4.3 nM) to a nicotinic site in cockroach nervous tissue. kappa-Bungarotoxin recognizes this invertebrate nicotinic site with high affinity (Ki = 27 nM). A comparison of the pharmacological properties of insect nicotinic receptors with those of functionally defined receptors identified by kappa-neurotoxins in avian autonomic ganglia reveals several similarities. However, a striking exception is alpha-bungarotoxin, which is the most potent antagonist examined at cockroach nicotinic receptors, but fails to recognize functional autonomic ganglia nicotinic receptors even at very high concentrations. It is concluded that kappa-neurotoxins can be used as selective probes for neuronal nicotinic receptors in both vertebrates and invertebrates. Although invertebrates diverged from vertebrates over 600 million years ago, the results indicate that the neuronal nicotinic receptors found in species as diverse as cockroach and chick retain considerable structural similarity, and thus neuronal nicotinic receptors appear to be highly conserved membrane proteins.

Acetylcholine↗
PubMed1978

beta-Bungarotoxin. The binding of [3H]pyridoxylated beta-bungarotoxin to a high-molecular-weight protein receptor.

beta-Bungarotoxin was labelled with pyridoxal 5'-phosphate (incorporating 3H). The kinetics of beta-bungarotoxin binding to several tissue subfragments of nervous tissue was studied. The dissociation constant of 3H-pyridoxylated beta-bungarotoxin in this reaction was 0.21-0.37 micron and that of unlabelled beta-bungarotoxin was 25 nM. Hill [(1910) J. Physiol. (London) 40, iv-vii] and Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672] analyses demonstrated no co-operativity of binding and only a single class of receptor sites, consistent with a bimolecular association of beta-bungarotoxin and its receptor. The iodinated toxin was physiologically inactive. Toxin was bound in non-specific unsaturable fashion by glass and/or plastic. This low-affinity binding was corrected by addition of bovine serum albumin to a final concentration of 30 mg/ml. A soluble protein receptor of beta-bungarotoxin was isolated and the mol.wt. is approx. 200000.

Animals↗
PubMed2003

Identification of alpha-bungarotoxin (A31) as the major postsynaptic neurotoxin, and complete nucleotide identity of a genomic DNA of Bungarus candidus from Java with exons of the Bungarus multicinctus alpha-bungarotoxin (A31) gene.

The Malayan krait (Bungarus candidus) is one of the most medically significant snake species in Southeast Asia. No specific antivenom exists to treat envenoming by this species. Death within 30 min after its bite has been reported from Java, suggesting the presence of highly lethal postsynaptic neurotoxins in the venom of these snakes. We purified and identified the major postsynaptic toxin in the venom of B. candidus from Java. The toxin was indistinguishable from alpha-bungarotoxin (A31), a toxin originally isolated from Bungarus multicinctus, in its mass (7983.75 Da), LD50 (0.23 microg/g in mice i.p.), affinity to nicotinic acetylcholine receptors, and by its 40 N-terminal amino acid residues as determined by Edman degradation. Identity with alpha-bungarotoxin was confirmed by cloning and sequencing a genomic DNA from B. candidus which encodes the 74 amino acid sequence of alpha-bungarotoxin (A31) and part of its signal peptide, revealing complete identity to the alpha-bungarotoxin (A31) gene in exon and 98.9% identity in intron sequences. The entire mitochondrial cytochrome b gene of the krait species B. candidus from Java and B. multicinctus from Taiwan was sequenced for comparison, suggesting that these snakes are phylogenetically closely related. alpha-Bungarotoxin appears to be widely present and conserved in Southeast and East Asian black-and-white kraits across populations and taxa.

Amino Acid Sequence↗
PubMed1978

Interactions of neuromuscular effects of edrophonium, alpha-bungarotoxin and beta-bungarotoxin.

Interactions of neuromuscular effects of edrophonium, alpha-bungarotoxin, and beta-bungarotoxin were studied in 12 chickens using the sciatic-gastrocnemius nerve-muscle preparation to elucidate the mechanism of action of each drug. Modification by the toxins of neuromuscular effects of edrophonium depended on the level of block pre-established by the toxins. Edrophonium-induced augmentation of muscle twitch ("facilitation") was decreased by both toxins. As the block reached 50 per cent, the facilitation was nearly abolished. Edrophonium-induced contracture of the muscle was blocked by alpha-bungarotoxin only. At 25 per cent block, it was no longer observable in five of six preparations. Beta-bungarotoxin enhanced the contracture. At complete block, the contracture reached 156 (SE 11, n = 6) per cent of control. The authors conclude that edrophonium facilitates neuromuscular transmission by a prejunctional mechanism and causes contracture of the chicken muscle by a post-junctional activation. The beta-bungarotoxin-blocked nerve-muscle preparation of the chicken is a model of acute denervation potentially useful for the study of drug effects on the postjunctional membrane.

Animals↗
PubMed1995

alpha-Bungarotoxin, kappa-bungarotoxin, alpha-cobratoxin and erabutoxin-b do not affect [3H]acetylcholine release from the rat isolated left hemidiaphragm.

Endplate preparations of the rat left hemidiaphragm were incubated with [3H]choline to label neuronal transmitter stores. Nerve evoked release of newly-synthesized [3H]acetylcholine was measured in the absence of cholinesterase inhibitors to investigate whether snake venom neurotoxins by blocking presynaptic nicotinic autoreceptors affect evoked transmitter release. Contractions of the indirectly stimulated hemidiaphragm were recorded to characterize the blocking effect of alpha-neurotoxins at the post-synaptic nicotinic receptors. Neither the long chain neurotoxins alpha-cobratoxin (1 microgram ml-1) and alpha-bungarotoxin (5 microgram ml-1) nor the short chain neurotoxin erabutoxin-b (0.1, 1 and 10 micrograms ml-1) affected the nerve-evoked release of [3H]acetylcholine. kappa-Bungarotoxin (1 and 5 micrograms ml-1), a toxin preferentially blocking neuronal nicotinic receptors, did also not affect evoked [3H]acetylcholine release, whereas (+)-tubocurarine (1 microM) under identical conditions reduced the release by about 50%. alpha-Bungarotoxin, alpha-cobratoxin and erabutoxin-b concentration-dependently (0.01-0.6 micrograms ml-1) inhibited nerve-evoked contractions of the hemidiaphragm. All neurotoxins except erabutoxin-b enhanced the basal tritium efflux immediately when applied to the endplate preparation or to a non-innervated muscle strip labelled with [3H]choline. This effect was attributed to an enhanced efflux of [3H]phosphorylcholine, whereas the efflux of [3H]choline and [3H]acetylcholine was not affected. It is concluded that the alpha-neurotoxins and kappa-bungarotoxin do not block presynaptic nicotinic receptors of motor nerves. These nicotinic autoreceptors differ from nicotinic receptors localized at the muscle membrane and at autonomic ganglia.

Acetylcholine↗
PubMed1992

Neurons of the chick brain and retina expressing both alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors: an immunohistochemical analysis.

Immunohistochemical methods were used to study the possible co-localization of two alpha-bungarotoxin-sensitive (alpha 7 and alpha 8) and two alpha-bungarotoxin-insensitive (beta 2 and alpha 3) subunits of the nicotinic acetylcholine receptors in neurons of the chick brain and retina. Several structures contained neurons that were doubly-labeled with antibodies against the alpha 7 subunit and the beta 2 subunit. These structures included, for example, the interpeduncular nucleus, nucleus spiriformis lateralis, optic tectum, pretectal visual nuclei, and the lateral hypothalamus. Double-labeling with antibodies against the alpha 7 and alpha 8 subunits was also seen in several regions, which included the interpeduncular nucleus, visual pretectum, lateral hypothalamus, dorsal thalamus, and the habenular complex. In the retina, many cells in the inner nuclear layer were observed to contain alpha 8 and alpha 3 subunits, whereas neurons in the ganglion cell layer were seen to contain alpha 7 and alpha 8 or, less frequently, alpha 7 and alpha 3 subunits. These results indicate that alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive subunits of the nicotinic receptors are co-expressed by neurons of the chick brain and retina.

Animals↗
PubMed1994

The sensitivity of nicotinic synapses in bullfrog sympathetic ganglia to alpha-bungarotoxin and neuronal-bungarotoxin.

1. The sensitivity of nicotinic synapses to alpha-bungarotoxin (alpha-Bgt) and neuronal-bungarotoxin (n-Bgt) was measured in the B and C cell systems of bullfrog paravertebral sympathetic ganglia 9 and 10 by recording extracellular compound postganglionic action potentials from the rami communicantes. 2. High concentrations (10 microM) of alpha-Bgt applied for up to 8 h had no effect upon synaptic transmission in either the B or C cell system. Ganglia pretreated with collagenase were also insensitive to alpha-Bgt. In control experiments on isolated sartorius muscle preparations, nerve-evoked twitches were fully blocked by 30-100 nM alpha-Bgt. 3. Nicotinic transmission in the B and C cell systems was reversibly blocked by 30-300 nM n-Bgt. Block appeared within 25-45 min of exposure to toxin and reversed fully with a half-time of 40-80 min. This was indistinguishable from washout times after block by 100 microM (+)-tubocurarine. 4. The results demonstrate close parallels between the bungarotoxin sensitivity of neuronal nicotinic receptors mediating ganglionic transmission in functional subclasses of bullfrog sympathetic neurones and the bungarotoxin sensitivity which has been reported for autonomic in avian and mammalian preparations.

Animals↗
PubMed1982

Amino acid sequences of three beta-bungarotoxins (beta 3-, beta 4-, and beta 5- bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains.

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.

Amino Acid Sequence↗
PubMed1994

Differential development of alpha-bungarotoxin-sensitive and alpha-bungarotoxin-insensitive nicotinic acetylcholine receptors in the chick retina.

The development of cells containing neuronal nicotinic receptors (nAChRs) in the chick retina was investigated by means of immunohistochemical techniques with antibodies directed against the alpha 3 and alpha 8 nAChR subunits. The alpha 3 subunit is one of the major alpha-bungarotoxin-insensitive nicotinic receptor subunits in the chick retina, whereas alpha 8 appears to be the most common alpha-bungarotoxin-sensitive subunit in the same structure, alpha 3-like immunoreactivity (alpha 3-LI) was first detected in cells of the vitreal margin, on the embryonic day 4.5 (E4.5). alpha 8-LI was first detected in the same type of cell almost a day later. However, the processes of alpha 8-LI cells developed much faster than those of alpha 3-LI cells, generating visible stained laminae in the prospective inner plexiform layer as early as E7. alpha 3-LI was only clearly seen in laminae of the inner plexiform layer by E12. By this date, both alpha 3 and alpha 8-LI were seen in the same types of cells as in the adult retina, i.e., amacrines, displaced ganglion cells, and cells of the ganglion cell layer for alpha 3-LI; and amacrines, bipolar cells, and cells of the ganglion cell layer for alpha 8-LI. These results reveal different patterns of development of cells containing the alpha 3 and alpha 8 nAChR subunits in the chick retina and indicate that those nAChR subunits are expressed in the chick retina before choline acetyltransferase-positive cells can be detected and well before synaptogenesis. These data also suggest that nAChRs may have a developmental function in the retina.

Animals↗
PubMed1996

Neuronal bungarotoxin displaces (125I) alpha-bungarotoxin binding at the neuromuscular junction as well as to the spinal cord during embryogenesis.

Alpha-Bungarotoxin (alpha BTX) administration in ovo prevents motoneuron apoptosis during development. This process may be mediated by alpha BTX-sensitive nicotinic cholinoceptors in the spinal cord, at the neuromuscular junction or at both sites. In order to differentiate between these possibilities, neuronal bungarotoxin binding (NBTX) binding to embryonic muscle and spinal cord was investigated in the chick.

Animals↗
PubMed1989

Neutralizing monoclonal antibody specific for alpha-bungarotoxin: preparation and characterization of the antibody, and localization of antigenic region of alpha-bungarotoxin.

We prepared an alpha-bungarotoxin-specific monoclonal antibody that neutralizes the biological activity of the toxin in vivo. The antigenic determinant combining specifically with this antibody was determined on the basis of cross-reaction experiments using three other long neurotoxins and peptide fragments of alpha-bungarotoxin. The antigenic determinant was located on the peptide fragment containing S34-S35-R36-G37-K38, which forms a part of the expected site that binds to the acetylcholine receptor proteins.

Amino Acid Sequence↗
PubMed1978

Characterization of phospholipase A activity of beta1-bungarotoxin from Bungarus multicinctus venom. II. Identification of the histidine residue of beta1-bungarotoxin modified by p-bromophenacyl bromide.

beta1-Bungarotoxin modified with p-bromophenacyl bromide (BPB) was reduced and carboxymethylated, and the resulting two constituent RCM-polypeptide chains (the RCM-A and B chains) were separated. The RCM-A chain was found to be modified by BPB by measuring its UV absorption spectrum and was shown to have lost one histidine residue by analyzing its amino acid composition. To determine the location of the modified histidine residue in the A chain of the toxin, the RCM-A chain was digested with TPCK-trypsin, and the resulting peptides were fractionated by gel filtration followed by DEAE-cellulose chromatography. The modified residue was finally identified as histidine-48 in the A chain by Edman degradation and from the amino acid composition of the BPB-modified peptide. The amino acid sequence around the modified histidine residue in the A chain is highly homologous with those of porcine pancreas phospholipase A2 and presynaptic toxin, notexin. We conclude that histidine-48 in the A chain participates in the phospholipase A activity of beta1-bungarotoxin.

Acetophenones↗
PubMed1993

Photoaffinity labeling of muscle-type nicotinic acetylcholine receptors and neuronal/nicotinic alpha-bungarotoxin binding sites with a derivative of alpha-bungarotoxin.

Neuronal/nicotinic alpha-bungarotoxin binding sites (nBgtS) found in the nervous system are not well characterized. In this study, photolabile toxin derivatives have been used in affinity labeling protocols to investigate the subunit composition of nBgtS expressed by different neuron-like cell lines. Data obtained was compared to the known subunit composition of toxin-binding muscle-type nicotinic acetylcholine receptors (nAChR). Muscle-type nAChR-rich membranes prepared from Torpedo electroplax contain components with corrected apparent molecular sizes of 41, 46, 50, 62 and 66 kDa that are reactive with toxin. The photoaffinity labeling patterns for preparations derived from cells of the TE671 clone, which express muscle-type nAChR, are very similar to that of cells of the IMR-32 or SH-SY5Y clonal lines, which express nBgtS. There is consistent labeling of four polypeptides with corrected apparent molecular weights of 40, 43, 47 and 56 kDa. These results suggest that both mammalian muscle-type nAChR and mammalian nBgtS are similarly composed of at least four kinds of subunits.

Affinity Labels↗
PubMed1999

The functional role of positively charged amino acid side chains in alpha-bungarotoxin revealed by site-directed mutagenesis of a His-tagged recombinant alpha-bungarotoxin.

A polyhistidine tag was added to the N-terminus of alpha-bungarotoxin (Bgtx) recombinantly expressed in E. coli. The His-tagged Bgtx was identical to native, venom-derived Bgtx in its apparent affinity for the nicotinic acetylcholine receptor (nAChR) in Torpedo electric organ membranes. Furthermore, in a physiological assay involving mouse muscle nAChR expressed in Xenopus oocytes, the His-tagged Bgtx was as effective as authentic Bgtx at blocking acetylcholine-evoked currents. Ala-substitution mutagenesis of His-tagged Bgtx was used to evaluate the functional contribution of Arg36, a residue that is invariant among all alpha-neurotoxins. Replacement with Ala resulted in a 90-fold decrease in the apparent affinity for the Torpedo nAChR and a corresponding 150-fold increase in the IC50 for block of heterologously expressed mouse muscle nAChR, demonstrating the critical importance of this positive charge for the binding and functional activity of a long alpha-neurotoxin. The observed decrease in affinity corresponds to a DeltaDeltaG of 2.7 kcal/mol and indicates that Arg36 makes a major contribution to complex formation. This finding is consistent with the proposal that Arg36 mimics the positive charge found on acetylcholine and directs the toxin to interact with receptor sites normally involved in acetylcholine recognition. In comparison, Ala-substitution of the highly conserved Lys26 resulted in only a 9-fold decrease in apparent affinity. Truncation of the His-tagged Bgtx following residue 67 produces a toxin lacking the seven C-terminal residues including the two positively charged residues Lys70 and Arg72. Truncation leads to a 7-fold decrease in apparent binding affinity.

Amino Acid Substitution↗
PubMed1998

Neutralizing epitope mapping of six beta1-bungarotoxin monoclonal antibodies and its application in beta1-bungarotoxin peptide vaccine design.

Twenty three stable monoclonal antibodies (mAbs) against beta1-bungarotoxin (beta1-bgt) were prepared by the hybridoma technique. Seven of the 23 mAbs (mAbs 2, 6, 8, 11, 17, 21 and 22) could inhibit more than 70% of phospholipase A2 activity of beta1-bgt and neutralize the toxin. Six of these neutralizing mAbs (mAbs 2, 6, 8, 17, 21 and 22) recognized continuous epitopes on the A chain of beta1-bgt and the other one (mAb 11) recognized a conformational epitope on the toxin. The continuous epitopes of these six mAbs were mapped using synthetic peptide and proteolytic enzymes. Experimental results indicate that mAb 17 recognized the A-chain residues 31-37; mAbs 2 and 8 recognized residues 46-51; mAbs 21 and 22 recognized residues 91-98; and mAb 6 recognized residue 100-106. The competitive-antibody-binding inhibition experiments showed that the affinity of these neutralizing mAbs to the native beta1-bgt is compatible with synthetic peptides. Furthermore, mice immunized with BSA-conjugated A-chain-peptide sequences A(31-37), A(46-51), A(91-98) or A(100-106) were protected from a high-dose beta1-bgt challenge. Subsequently, the peptide-immunized sera were passively injected into Balb/c mice and a significantly protective effect was also observed. To our knowledge, this study is the first systematic demonstration of multiple neutralizing B-cell epitopes of beta1-bgt, and this study is also the first report of the protective synthetic-peptide vaccine against beta1-bgt challenge.

Amino Acid Sequence↗