The secondary structure of staphylococcal enterotoxins A, B and C.

The circular dichroism (CD) of staphylococcal enterotoxins A, B and C was measured. The CD of enterotoxins B and C were almost identical from 250 to 320 nm, but differed from the CD of enterotoxin A. The spectrum of enterotoxin A in this wavelength region contained the same bands with respect to both location and sign, but with significant differences in intensity. The CD spectra of enterotoxins B and C were also much more alike from 190 to 250 nm. Although all three enterotoxins had a major negative extremum at 215--218 nm, its magnitude was equal in enterotoxins B and C, but was substantially decreased in enterotoxin A. The secondary structure of the enterotoxins contained little alpha-helix as analyzed with CD models. A secondary structure of entertoxin B compured from a scheme based on a joint prediction histogram of five separate methods, placed 29 residues in alpha-helices, 71 in beta-pleated sheets, 88 in beta-turns and 55 in aperiodic conformation.

Immune recognition of botulinum neurotoxin type A: regions recognized by T cells and antibodies against the protective H(C) fragment (residues 855-1296) of the toxin.

Botulism toxicity is caused by botulinum neurotoxins (BoNTs), a group of protein neurotoxins produced by Clostridium botulinum. Recent studies have shown that immunization with a C-terminal fragment [H(C), residues 855-1296] of BoNT type A (BoNT/A) affords excellent protection against BoNT/A toxicity. The present work was carried out in order to map the molecular and cellular immunological recognition of H(C). We have previously described the synthesis of 31 overlapping peptides encompassing the entire H(C)-fragment of BoNT/A. These peptides were employed in this study to localize the continuous regions recognized by T cells and by antibodies (Abs) generated in two mouse strains against H(C). T cells from SJL that had been primed with H(C) gave a strong proliferative response to challenge in vitro with each of the six peptides spanning residues 897-985 and a lower response to peptide 1051- 1069. While H(C)-primed T cells of BALB/c recognized three regions residing within residues 939-957, 1009-1027 and 1135-1153 (strong). Recognition regions by Abs in SJL or BALB/c anti-H(C) antisera essentially overlapped. However, the level of Abs bound to each region differed between the two strains. These common or similar recognition regions by the two strains were: 855-915 (SJL) or 855-901 (BALB/c); 939-957; 967-1013 (BALB/c) or 981-1013 (SJL); 1051-1069; 1079-1111 (BALB/c) or 1093-1125 (SJL); 1177-1195; and 1275-1296. In addition, BALB/c recognized region 1135-1153. Some of these regions show considerable sequence similarity in BoNT types B and E and, therefore, H(C) of these two BoNTs might offer protection against the correlate clostridial toxins.

Physicochemical properties of glucocorticoid receptors from mouse fibroblasts.

Glucocorticoid-bound receptor macromolecules were prepared in three forms from mouse fibroblast cells: the cytosol receptor, the nuclear extractable receptor, and the nuclear residual form obtained by DNase digestion of chromatin samples. These receptor complexes were studied with respect to gel filtration properties, sedimentation velocities in various salt concentrations, partial specific volumes, isoelectric points, and thermal stability properties. The results indicate that the three forms of the receptor differ in their molecular properties, and nuclear translocation and binding ofthe receptor complex is associated with conformational and physical changes consistent with a reduction in apparent molecular weight.

Antibodies and T cells against synthetic peptides of the C-terminal domain (Hc) of botulinum neurotoxin type A and their cross-reaction with Hc.

Seventeen peptides containing T cell and/or antibody (Ab) epitopes previously localized on Hc of botulinum neurotoxin type A were used in SJL and BALB/c mice as immunogens either individually or as an equimolar mixture of groups that contained epitopes of T cells, Abs or both, to determine their abilities to generate T cells and/or Abs that recognize intact Hc. In SJL, peptide 897-915 which included both T cell and Ab epitopes, elicited Abs that cross-reacted very strongly with Hc. In BALB/c, peptides 869-887, 883-901, 981-999 and 1275-1296 which contained Ab epitopes generated Abs that cross-reacted strongly with Hc. A mixture of peptides that contained T cell and Ab epitopes was effective in both strains in eliciting T cells and Abs that cross-reacted with Hc. This mixture form gave a quicker rise (after two injections) in cross-reactive (with Hc) Ab titer as compared to other peptide mixtures or the individual peptides, and sustained in BALB/c a high Ab titer upon further booster injections. Some of the regions that elicited crossreactive immunity to Hc have sequence similarity to other clostridial toxins, suggesting that one or more of these synthetic peptides might provide cross-protection against those toxins.

Specific association of T-2 toxin with mammalian cells.

The binding of radiolabeled T-2 toxin to a mammalian cell line derived from a Chinese hamster ovary (CHO) was studied. The toxin bound to, or was taken up by, cells in a time-, temperature- and concentration-dependent manner. The binding was saturable, of high affinity (Kd approximately 0.1 to 1 nM), reversible at 37 degrees (half-time approximately 2 hr), and specific. The kinetics of T-2-cell association and the rate of toxin-induced inhibition of protein synthesis closely paralleled one another. Likewise, the concentration-response for inhibition of protein synthesis and the toxin binding isotherm were similar. A synthetically derived epimer of T-2 bound less tightly to cells, but apparently to the same site as authentic T-2. The epimer was also less potent at inducing inhibition of protein synthesis. Two other trichothecene toxins, one more and one less toxic than T-2, blocked labeled T-2 binding to cells in a manner reflective of their protein synthesis inhibitory potencies. We conclude that the binding we defined is an accurate measure of the toxin responsible for inhibition of protein synthesis in CHO cells. The data also suggested that, at equilibrium, the interaction of T-2 with cells is not static, but is the sum of a continuous uptake and release process.

Binding of T-2 toxin to eukaryotic cell ribosomes.

The binding of radiolabeled T-2 to eukaryotic ribosomes was studied. The toxin bound to ribosomes in a time-, temperature- and concentration-dependent manner. The binding was saturable (0.3 nM), reversible at 37 degrees (half-time approximately 2.5 hr) and specific. The stoichiometry was one toxin molecule bound per ribosome. Binding of T-2 appeared to stablize the toxin recognition site to thermal degradation. A synthetically derived epimer of T-2 bound to the same ribosomal site as authentic T-2, but apparently with lower affinity. Two other trichothecene toxins tested blocked the binding of T-2 to ribosomes in a manner reflecting their protein synthesis inhibitory potencies. Anisomycin blocked the binding of T-2 to both isolated ribosomes and cells, whereas emetine blocked binding only to cells. Our data, together with that in the accompanying paper (Middlebrook JL and Leatherman DL, Biochem Pharmacol 38: 3093-3102, 1989), suggest that T-2 interaction with CHO cells is best viewed as a free, bidirectional movement of toxin across the plasma membrane and specific high-affinity binding to ribosomes.

Antibodies having markedly different effects on enzymatic activity and induction of acetylcholine release by two presynaptically-acting phospholipase A2 neurotoxins.

The enzymatic and acetylcholine-releasing activities of two presynaptically-acting phospholipase A2 neurotoxins (pseudexin B and scutoxin) were studied in a synaptosomal fraction. Scutoxin (100 nM) induced greater [14C]acetylcholine release than did pseudexin B (100 nM). Both toxins caused fatty acid production in the synaptosomal fraction, although pseudexin B was more active than scutoxin. One monoclonal antibody raised against pseudexin B (#4) had no effect on the enzymatic activity of either pseudexin B or scutoxin. Two other monoclonal antibodies (#3 and #7), also raised against pseudexin B, antagonized the enzymatic activity of pseudexin B and scutoxin. Monoclonal antibody #3 was more effective than #7 in reducing the amount of acetylcholine released by the toxins, whereas #7 was more effective than #3 in reducing fatty acid production. Although antibody #3 caused complete inhibition of phospholipase A2 activity of pseudexin B on purified substrates, it only reduced phospholipase A2 activity by 35% in synaptosomes. These findings support the hypothesis that gross phospholipase A2 activity does not play a role in stimulation of acetylcholine release by the presynaptically-acting phospholipase A2 neurotoxins.

Preparation and characterization of monoclonal antibodies against pseudexin.

Fifteen hybridoma cell lines secreting monoclonal antibodies against pseudexin were developed. The cell lines were grown as ascites tumors and the resulting antibodies were purified by Protein A affinity-chromatography. Several of the antibodies exhibited extensive ELISA cross-reactions with different phospholipase A2 toxins from various snake venoms, while other of the antibodies reacted only with the pseudexins. Three of the antibodies neutralized pseudexin A and B, but none of the 10 other phospholipase A2 toxins tested. These same three antibodies inhibited the enzymatic activity of pseudexin A and B and also that of notexin. After each antibody was labeled with biotin, competition experiments were carried out to determine the binding relationships among the antibodies and the pseudexins. Competitions were frequently observed, with a low of zero to a high of eight out of the 14 possibilities. Competition experiments were also carried out with biotin-labeled rabbit IgG against the pseudexins. Some of the monoclonal antibodies had no effect on rabbit IgG binding to pseudexin, while others blocked up to 50% of the binding.

Cross-neutralizations of phospholipase A2 neurotoxins from snake venoms.

Rabbit antisera were produced against several purified phospholipase A2 neurotoxins from snake venoms. The neutralizing and cross-neutralizing capacities of these antisera were evaluated using mice. In all except one case, homologous antisera neutralized the lethal effects of the neurotoxins. In several instances, antisera that exhibited strong ELISA cross-reactivity (MIDDLEBROOK and KAISER, Toxicon, 27, 965-977, 1989) were also capable of cross-neutralizations. Homologous antisera neutralized elapid neurotoxins much more effectively than did heterologous antisera. With the crotalid neurotoxins, homologous and heterologous antisera had similar neutralization potentials. These observations further define the immunological relatedness of the phospholipase A2 neurotoxins and suggest that common neutralizing epitopes exist within subgroups of these toxins.

Effects of steroids on association of T-2 toxin with mammalian cells.

The effects of steroids on the association of T-2 toxin with cultured cells were evaluated. Preincubating cells with certain steroids led to a time- and concentration-related increase in total T-2-cell association. At maximally effective concentrations, the increase in association was 300-500%. This effect required a preincubation at 37 degrees C for a minimum of 10 min and was completely reversible after 20-30 min. Steroid treatment increased the rate of toxin-cell association and decreased the rate of dissociation. The effect was elicited by progesterone, estradiol, testosterone and diethylstilbestrol, but not by several other steroids tested. Binding of T-2 to isolated ribosomes was not altered by the steroids. We speculated that steroids somehow alter the state of ribosomal aggregation or assembly such that more toxin can bind after entering the cell.

Monoclonal antibodies to VRV-PL-VIIIa, a basic multitoxic phospholipase A2 from Vipera russelli venom.

VRV-PL-VIIIa, the most basic phospholipase A2 (PLA2) from the venom of Vipera russelli, induces multiple toxic effects, including neurotoxicity, myotoxicity, edema and hemorrhage. Rabbit polyclonal anti-serum was raised against VRV-PL-VIIIa. The antiserum cross-reacted in enzyme-linked immunosorbant assay (ELISA) with two other PLA2 from the same venom, VRV-PL-V and VRV-PL-VI, and with ammodytoxin A, caudoxin and crotoxin. Twenty-two hybridoma cell lines secreting monoclonal antibodies against VRV-PL-VIIIa were isolated. The monoclonal antibodies exhibited apparent binding affinities in ELISA with VRV-PL-VIIIa ranging over two orders of magnitude. Most of the monoclonal antibodies cross-reacted moderately with VRV-PL-V and weakly with VRV-PL-VI. None of the antibodies cross-reacted with ammodytoxin, caudoxin or crotoxin. Reducing the disulfide bonds of VRV-PL-VIIIa lowered the ELISA signals of each monoclonal antibody to nonspecific levels, suggesting that all the antibodies recognize conformational epitopes. Four of the 22 antibodies neutralized the enzymatic activity of VRV-PL-VIIIa. Interestingly, two of the four exhibited the lowest affinities of the monoclonal antibody library for VRV-PL-VIIIa in ELISA, while the other two exhibited the highest. Each of the monoclonal antibodies was biotinylated and spatial binding relationships were evaluated by competition ELISA.

Preparation of a crotoxin neutralizing monoclonal antibody.

Crotoxin is a heterodimeric protein composed of an acidic and basic subunit from the venom of Crotalus durissus terrificus and is representative of a number of presynaptically acting neurotoxins found in the venom of rattlesnakes. Four different monoclonal antibodies, typed as IgG1 subclass, were raised against the basic subunit of this toxin. One was a potent neutralizing antibody of intact crotoxin, which could neutralize approximately 1.6 moles of purified crotoxin per mole of antibody. The monoclonal antibody enhanced the neutralizing ability of commercial polyvalent crotalid antivenom against the lethality of crude C. d. terrificus venom four-fold. Paradoxically, this monoclonal antibody by itself was ineffective against the lethality of crude C. d. terrificus venom. Using an enzyme-linked immunosorbent assay, we tested various proteins for competitive inhibition of binding of biotinylated-crotoxin to plates coated with the four individual monoclonal antibodies. Concolor toxin, vegrandis toxin, intact crotoxin, Mojave toxin, and the basic subunit of crotoxin showed increasing effectiveness as displacers of crotoxin from the neutralizing monoclonal antibody. None of the monoclonal antibodies reacted with purified phospholipase A2 enzymes from Crotalus atrox or Crotalus adamanteus, nor any of the components present in the crude venoms from four different elapids known to contain presynaptically acting neurotoxins, which show some sequence identity to crotoxin.

Effects of beta-bungarotoxin and Naja naja atra snake venom phospholipase A2 on acetylcholine release and choline uptake in synaptosomes.

Beta-bungarotoxin is a potent presynaptically acting snake venom toxin that exhibits phospholipase A2 activity. We compared the effects of beta-bungarotoxin and a less toxic snake venom phospholipase A2 on synaptosomal 3H-acetylcholine release and 3H-choline uptake. The purpose of these experiments was to study the mode by which beta-bungarotoxin inhibits 3H-acetylcholine release in this preparation. Under non-depolarizing conditions, both beta-bungarotoxin and Naja naja atra phospholipase A2 stimulated 3H-acetylcholine release from a synaptosomal fraction preloaded with 3H-choline. Beta-bungarotoxin was more potent, but less efficacious, than N. naja atra phospholipase A2. In contrast, both toxins inhibited 3H-acetylcholine release from the synaptosomal fraction incubated with 3H-choline after toxin exposure. In agreement with the results obtained by monitoring acetylcholine release, beta-bungarotoxin and N. naja atra phospholipase A2 appeared to block 3H-choline uptake into the synaptosomal fraction non-competitively. Although the toxins may cause the release of unlabeled choline from synaptosomes, the block of labeled choline uptake could not be explained by decreased specific activity of 3H-choline in the bathing medium. Therefore, beta-bungarotoxin and N. naja atra phospholipase A2 block 3H-acetylcholine release from synaptosomes indirectly by inhibiting the uptake of 3H-choline necessary for 3H-acetylcholine synthesis. In comparing these results using 3H-choline to those in the literature obtained with deuterated choline, there appears to be a difference in apparent toxin action that relates to the type of label (3H or 2H) attached to choline.

Effects of myonecrotic snake venom phospholipase A2 toxins on cultured muscle cells.

We have attempted to establish a cell culture model suitable for molecular mechanism of action studies of necrotic phospholipases A2 (PLA2). Three myonecrotic PLA2 were purified, one basic PLA2 from Naja nigricollis venom and two basic PLA2 (VRV-PL-V and VRV-PL-VIIIa) from Vipera russelli venom. The effects of these PLA2 on several established muscle cell lines were evaluated. As judged by light microscopy, some, but not all, cell lines detached from the culture plate in a time- and concentration-related fashion. Naja nigricollis PLA2 was the most potent at eliciting this effect, followed by VRV-PL-V and VRV-PL-VIIIa. The two most sensitive cell lines, 1447 and 1456, were chosen for further study using N. nigricollis PLA2. Cellular protein and nucleic acid syntheses were inhibited by the toxin in a time- and dose-related manner. However, it appeared that most, if not all, of the inhibition was due to toxin-induced reduction of precursor uptake, suggesting effects at the plasma membrane level. The putative membrane effects were specific, in that uptake of calcium, choline or glucose was not inhibited by the toxin. Moreover, treating the cells with toxin failed to significantly increase lactate dehydrogenase release into the medium. Polyclonal antiserum prepared against N. nigricollis basic PLA2 neutralized the toxicity completely with 1456 cells, but only partially with the 1447 cell line. Both the 1447 and 1456 lines appear to be suitable as cell culture models for necrotizing PLA2 molecular mechanism of action studies.

Mosquito inoculation: an alternative bioassay for toxins.

Mosquitoes were evaluated as a bioassay host for several classes of biological toxins. Mosquitoes were sensitive to snake toxic or neurotoxic phospholipase A2 enzymes (but not to nontoxic phospholipase A2 enzymes), cobrotoxin, saxitoxin, microcystin and the scorpion insect sodium channel toxin. Mosquitoes were not sensitive to ricin, diphtheria toxin, anthrax toxin, botulinum toxin, tetanus toxin, conotoxin G or a scorpion sodium channel toxin toxic to mammals. Specific antisera neutralization tests with mosquitoes gave comparable results to those of a mouse assay. The mosquito is a suitable bioassay animal for many, but not all biological toxins, and offers a safer, more efficient and economical assay than mice.

Purification, sequencing and characterization of pseudexin phospholipases A2 from Pseudechis porphyriacus (Australian red-bellied black snake).

Pseudexin is the name given to a mixture of toxic phospholipase A2 isoenzymes isolated from the venom of the Australian red-bellied black snake, Pseudechis porphyriacus. We found that this mixture consists of three components: pseudexins A, B and C, which we individually purified by reverse phase chromatography or by hydrophobic interaction chromatography. Pseudexins A and B had relatively low specific toxicities in mice (i.p. LD50 of 1300 and 750 micrograms/kg, respectively), while C was non-toxic. All three had similar phospholipase A2 activities (43-53 muequiv H+ released/min/mg protein). The complete amino acid sequences of pseudexins A and B were determined. Amino acids were identical at 91 of the 117 residues. The first 28 residues of pseudexin C were determined, sufficient to show that C is structurally similar to A and B, but not identical with either. As judged by reactions with antisera against several other snake phospholipase A2 toxins, pseudexins A, B and C have very similar antigenic structures. We noted extensive homology with other phospholipases.

Cellular regulation of diphtheria toxin cell surface receptors.

The cellular regulation of diphtheria toxin cell surface receptors was studied. Treatment of Vero cells with cycloheximide reduced their diphtheria toxin binding capacity, while cells treated with actinomycin D did not lose their ability to bind diphtheria toxin. A non-toxic analogue of diphtheria toxin, CRM 197, produced a dose-related depletion of cell surface diphtheria toxin binding capacity that was reversible upon washing the cells. Vero cells depleted of toxin receptors by CRM 197 did not restore their ability to bind diphtheria toxin in the presence of cycloheximide. Phospholipase C treatment of Vero cells reduced their diphtheria toxin binding capacity in a dose-dependent manner. The loss of diphtheria toxin binding capacity was recovered within 2 hr after removal of the enzyme. Protein synthesis inhibition blocked this recovery while actinomycin D partially inhibited it. Receptors prebound with toxin were resistant to phospholipase C treatment, suggesting that the action of the enzyme was directly on the receptor. Inhibition of glycosylation with tunicamycin did not prevent reappearance of toxin receptors after CRM 197 or phospholipase C treatment. These data establish the requirement of a continuous protein synthesis for the maintenance of diphtheria toxin cell surface receptors and also suggest that these receptors do not recycle after binding ligand. A hypothesis is put forward that the diphtheria toxin receptor might be a lipid-linked cell surface protein.

Immunological relationships of phospholipase A2 neurotoxins from snake venoms.

Polyclonal rabbit antisera were raised against ten snake phospholipase A2 neurotoxins and one snake phospholipase A2 cytotoxin. Immunological cross-reactivities between these toxins, two other snake phospholipase A2 enzymes and pancreatic phospholipase A2 were studied using ELISA technology. All snake phospholipase A2 neurotoxins fell into two main antigenic classes. One antigenic class was composed of all the elapid toxins tested (textilotoxin, taipoxin, notexin, pseudexin and beta-bungarotoxin), the cytotoxic phospholipase A2 from Naja naja atra and pancreatic phospholipase A2. beta-Bungarotoxin seemed to be in an immunological subclass of its own compared to the rest of the elapid toxins. The second antigenic class was comprised of crotalid and viperid phospholipase A2 neurotoxins (crotoxin, concolor toxin, Mojave toxin, vegrandis toxin, ammodytoxin and caudoxin). Our data indicated that the viperid toxins, caudoxin and ammodytoxin, were an immunological subclass apart from the crotalid toxins.

Binding of myotoxin a to cultured muscle cells.

The binding of radiolabeled myotoxin a to various cultured cell lines was evaluated. One rat skeletal muscle-derived cell line, L8, bound substantially more myotoxin a than did all all other cell lines examined. Several biophysical parameters of myotoxin a-L8 binding were determined. Binding was saturable with a moderate binding affinity. Scatchard analysis and Hill plots indicated a single class of binding sites. The binding was reversible, as demonstrated by chase experiments. Radiolabeled myotoxin a bound to the cell surface at a site inaccessible to the general protease, pronase. Specificity and biological relevance of the binding was suggested by competition with unlabeled toxin and various peptides derived from the toxin. Biologically active peptides, corresponding to the N- and C-terminal sequence of myotoxin a, competed with radiolabeled toxin for L8 binding. It was concluded that the L8 system is a suitable cell model to study myotoxin a mechanism of action.

Primary sequence determination of the most basic myonecrotic phospholipase A2 from the venom of Vipera russelli.

The most basic phospholipase A2 (PLA2), VRV-PL-VIIIa, was purified from (Sri Lankan) Vipera russelli venom. It is a major component of the venom, contributing over 40% to the whole venom PLA2 activity. The purity of VRV-PL-VIIIa was ascertained by electrophoresis and by reverse phase high-pressure liquid-chromatography (RP-HPLC). VRV-PL-VIIIa had an apparent mol. wt of 13,000 and was a single polypeptide. The protein was reduced, pyridylethylated and subjected to sequence analysis. The N-terminal amino acid sequence was established up to the 39th residue. Pyridylethylated VRV-PL-VIIIa was digested with endoprotease Glu-C, and several peptides were purified by RP-HPLC; six purified peptides were sequenced. The sequence of the C-terminal was established by sequencing a CNBr-produced peptide purified by RP-HPLC. Several peptides were also generated by digestion with endoprotease Asp-N. Two peptides were sequenced to obtain overlapping regions. The complete structure was deduced from sequences of overlapping peptides and through homology with other group II PLA2 sequences. Sequence homology was greatest with ammodytoxin A: 99 amino acid residues out of 121 occurred in identical positions. Myotoxin III of Bothrops asper showed 73% homology, 89 out of 121 residues. In agreement with the sequence data, polyclonal antiserum against VRV-PL-VIIIa cross-reacted in ELISA with ammodytoxin A and, to a lesser extent, with caudoxin.