A novel peptide mimicking the interaction of α-neurotoxins with acetylcholine receptor

A peptide corresponding to residues 26–41 of α-bungarotoxin, and closed by a disulfide bond between two cysteine residues at the amino and C terminal ends of the peptide, was synthesized and the monomeric form was purified. The peptide, which represents the exposed part of the long central loop of the toxin molecule, was examined for binding to acetylcholine receptor. The peptide was shown by radiometric titrations to bind radiolabeled receptor, and radiolabeled peptide was bound by receptor. The specificity of the binding was confirmed by inhibition with the parent toxin. A synthetic analog of the peptide in which Trp-28 was replaced by glycine had very little (10%) of the original activity. Succinylation of the amino groups of the peptide resulted in virtually complete (98%) loss of the binding activity. These results indicate that a shortened loop peptide corresponding to the region 26–41 of α-bungarotoxin exhibits binding activities mimicking those of the parent molecule. In this region, Trp-28, and one or both of Lys-26 and Lys-38, are essential contact residues in the binding to receptor.     

Mapping by synthetic peptides of the binding sites for acetylcholine receptor on alpha-bungarotoxin

A set of seven peptides constituting the various loops and most of the surface areas of -bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind¹²⁵I-labeled AChR and, conversely,¹²⁵I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47).     

Variability among the sites by which curaremimetic toxins bind to torpedo acetylcholine receptor, as revealed by identification of the functional residues of alpha-cobratoxin

alpha-Cobratoxin, a long chain curaremimetic toxin from Naja kaouthia venom, was produced recombinantly (ralpha-Cbtx) from Escherichia coli. It was indistinguishable from the snake toxin. Mutations at 8 of the 29 explored toxin positions resulted in affinity decreases for Torpedo receptor with DeltaDeltaG higher than 1.1 kcal/mol. These are R33E > K49E > D27R > K23E > F29A >/= W25A > R36A >/= F65A. These positions cover a homogeneous surface of approximately 880 A(2) and mostly belong to the second toxin loop, except Lys-49 and Phe-65 which are, respectively, on the third loop and C-terminal tail. The mutations K23E and K49E, and perhaps R33E, induced discriminative interactions at the two toxin-binding sites. When compared with the short toxin erabutoxin a (Ea), a number of structurally equivalent residues are commonly implicated in binding to muscular-type nicotinic acetylcholine receptor. These are Lys-23/Lys-27, Asp-27/Asp-31, Arg-33/Arg-33, Lys-49/Lys-47, and to a lesser and variable extent Trp-25/Trp-29 and Phe-29/Phe-32. In addition, however, the short and long toxins display three major differences. First, Asp-38 is important in Ea in contrast to the homologous Glu-38 in alpha-Cbtx. Second, all of the first loop is insensitive to mutation in alpha-Cbtx, whereas its tip is functionally critical in Ea. Third, the C-terminal tail may be specifically critical in alpha-Cbtx. Therefore, the functional sites of long and short curaremimetic toxins are not identical, but they share common features and marked differences that might reflect an evolutionary pressure associated with a great diversity of prey receptors.     

On the molecular mechanisms of neutralization of a cobra neurotoxin by specific antibodies

Examination of 76 homologous neurotoxin sequences suggested that the "toxic" domain of these compounds consists of twelve highly conserved residues. Five of these, namely Lys-27, Trp-29, Asp-31, Arg-33 and Glu-38, together with a variant residue at position 36 are organized into a pattern which resembles that of d-tubocurarine. Two lines of experimental evidence are in agreement with the proposed topology of the "toxic" site in Naja nigricollis toxin alpha--Three highly conserved residues (Lys-27, Trp-29 and Lys-47) have been modified individually in toxin alpha. These modifications induce a decrease in binding affinity of toxin alpha for its target, the nicotinic acetylcholine receptor. In contrast, modifications of three residues (Leu-1, Lys-15 and Lys-51) excluded from the "toxic" domain, do not alter the binding properties of toxin alpha.--Five toxin derivatives carrying a nitroxide group at residues 1, 15, 27, 47 or 51 have been prepared. ESR spectra have been recorded for each derivative in both the free state and bound to the receptor. Mobility of the probes of the residues excluded from the "toxic" site is not altered upon receptor binding. In contrast mobility of the nitroxide of the presumed "toxic" Lys-47 becomes markedly reduced after toxin receptor complex formation. Lys-27 nitroxide is immobilized in both the free and bound state. The antigenic structure of N. nigricollis toxin alpha has been partially clarified using two different approaches. --Fifteen antigenically important residues of toxin alpha have been identified by analyzing cross-reactions between toxin alpha and eleven homologous neurotoxins, using polyclonal antibodies.--- One monoclonal antibody (M alpha 1) specific for toxin alpha has been prepared. Competition experiments, made with (3H) toxin alpha, six mono modified toxin derivatives or alpha three homologous neurotoxins, showed that the binding site of (M alpha 1) comprises the N-terminal group, Lys-15, Pro-18 and probably Thr-16. This site is topographically different from the "toxic" domain. (M alpha 1) inhibits the toxicity of toxin alpha under both in vivo and in vitro conditions. In addition, (M alpha 1) is capable of "removing" toxin molecules bound to the receptor, allowing a rapid recovery of the functional properties of the receptor.     

Mapping by synthetic peptides of the binding sites for acetylcholine receptor on α-bungarotoxin

A set of seven peptides constituting the various loops and most of the surface areas of α-bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind¹²⁵I-labeled AChR and, conversely,¹²⁵I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47).     

Subunit Structure of α-Bungarotoxin Binding Component in Mouse Brain

Abstract: The α-bungarotoxin binding component in mouse brain was purified by affinity chromatography with toxin-Sepharose, gel-chromatography on Sepharose 6B, and ion-exchange chromatography with DE52 resin. The iodinated product of the last step produced one major and one minor band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the minor peak was twice as large as that of the major one. The iodinated product could bind α-bungarotoxin, and this binding was inhibited by a nicotinic antagonist, d -tubocurarine, which demonstrated that the iodinated product was a true α-bungarotoxin binding component. The molecular structure of the product was analysed by cross-linking followed by SDS-PAGE. The results fitted the model for an α-bungarotoxin binding component in the mouse brain composed of six identical or very similar subunits of 51,000-52,000. One subunit carrying the binding site for toxin bound one molecule of toxin. This subunit structure of an α-bungarotoxin binding component in the brain is discussed in comparison with that of a nicotinic acetylcholine receptor in the electric organ.     

State of functionally essential Trp-29 in snake venom neurotoxins: A proton nuclear magnetic resonance study

Proton nuclear magnetic resonance (NMR) spectra have been recorded of various neurotoxins from snake venoms.pH dependence of the chemical shifts and resonance intensity has been followed for the functionally essential Trp-29. The indole N-1 proton of Trp-29 in -bungarotoxin, toxin B, and cobrotoxin exhibits appreciably large upfield shifts as thepH is lowered and the suppressed exchange with the solvent hydrogen atpH 3–4, but not inNaja haje annulifera 10 where Asp-31 is replaced with Gly-31. This observation strongly suggests the presence of a hydrogen bond between Trp-29 and Asp-31 that is probably important in stabilizing the arrangement of the functionally essential residues to form a distinct binding region for the receptor.     

Structure-function relationships of curaremimetic neurotoxin loop 2 and of a structurally similar segment of rabies virus glycoprotein in their interaction with the nicotinic acetylcholine receptor

Peptides corresponding to portions of curaremimetic neurotoxin loop 2 and to a structurally similar segment of rabies virus glycoprotein were synthetically modified in order to gain information on structure-function relationships of neurotoxin loop 2 interactions with the acetylcholine receptor. Binding of synthetic peptides to the acetylcholine receptor of Torpedo electric organ membranes was assessed by measuring their ability to inhibit the binding of 125I-alpha-bungarotoxin to the receptor. The peptides showing the highest affinity for the receptor were a peptide corresponding to the sequence of loop 2 (residues 25-44) of Ophiophagus hannah (king cobra) toxin b (IC50 = 5.7 x 10(-6) M) and the structurally similar segment (residues 173-203) of CVS rabies virus glycoprotein (IC50 = 2.6 x 10(-6) M). These affinities were comparable to those of d-tubocurarine (IC50 = 3.4 x 10(-6) M) and suberyldicholine (IC50 = 2.5 x 10(-6) M). These results demonstrate the importance of loop 2 in the neurotoxin interaction with the receptor. N- and C-terminal deletions of the loop 2 peptides and substitution of residues invariant or highly conserved among neurotoxins were performed in order to determine the role of individual residues in binding. Residues 25-40 are the most crucial in the interaction with the acetylcholine receptor. Modifications involving Lys-27, Trp-29, Phe-33, Arg-37, and Gly-38 reduced affinity of binding. R37D and F33T modifications reduced the affinity of alpha-bungarotoxin residues 28-40 by an order of magnitude. Arg-37 may correspond to the positively charged quaternary ammonium group and Phe-33 to the hydrophobic acetyl methyl group of acetylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Peptide binding characteristics of the coeliac disease-associated DQ(α1*0501, β1*0201) molecule

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of theHLA-DQ2 (α1^*0501, β1^*0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several sizevariants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClα46–63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClα46–63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides. Substitutions at P1 and P4 were most critical, suggesting a more prominent role as anchor residues. Structural features of the DQ2 molecule that may relate to the binding motif and to gluten sensitivity are discussed.