Structure and action of heteronemertine polypeptide toxins: Inactivation of Cerebratulus lacteus toxin B-IV by tyrosine nitration

Reaction of Cerebratulus lacteus toxin B-IV with tetranitromethane in the presence of low concentrations of urea results in essentially complete loss of toxicity as measured by a sensitive quantal bioassay. Amino acid analysis and speetrophotometric studies both indicate the primary effect of reaction to be nitration of a single tyrosine residue per molecule of toxin. The nitrated residue has been identified as tyrosine-9 by automated Edman degradation of the modified protein. Since the secondary structure of toxin B-IV is not detectably altered by nitration, it is concluded that tyrosine-9 is directly involved in the interaction of this polypeptide with its axonal receptor, proposed to be involved in the inactivation of voltage-sensitive Na⁺ channels in crustacean nerves.     

Structure and action of heteronemertine polypeptide toxins: importance of amphipathic helix for activity of Cerebratulus lacteus toxin A-III

The marine heteronemertine Cerebratulus lacteus produces a family of protein cytolysins designated as A-toxins. Limited subtilisin digests of the most abundant homolog, toxin A-III, yield two major products which may be purified by high-performance liquid chromatography. One product is shown to represent residues 1-86 and the other contains the entire toxin sequence (1-95). Both polypeptides are shown to lack internal protease nicks. The 1-95 polypeptide retains full cytolytic activity in comparison to native toxin, whereas 1-86 has an activity that is approximately four times lower. Extensive treatment of A-III with carboxypeptidase Y yields a polypeptide containing residues 1-75 which is totally devoid of hemolytic activity. Residues 63-95 of native A-III have been predicted to form a relatively hydrophobic alpha-helix which is potentially important for activity. The circular dichroism spectrum of 1-95 is in excellent agreement with both experimental and Chou-Fasman-predicted secondary structures of native A-III, while the spectra of 1-86 and 1-75 indicate a loss of helicity quantitatively consistent with the removal of residues 87-95 and 76-95, respectively. Combined with our earlier data on bilayer penetration by N-terminal sequences (K. M. Blumenthal (1982) Biochemistry 21, 4229-4233], the current results indicate a direct involvement of both ends of A-III in lytic activity. The C-terminal region may function by contributing a membrane binding site in the form of an amphipathic helix.     

Structure and action of heteronemertine polypeptide toxins Binding of cerebratulus lacteus toxin B-IV to axon membrane vesicles

The binding of the crustacean selective protein neurotoxin, toxin B-IV, from the nemertine Cerebratulus lacteus to lobster axonal vesicles has been studied. A highly radioactive, pharmacologically active derivative of toxin B-IV has been prepared by reaction with Bolton-Hunter reagent. Saturation binding and competition of ¹²⁵I-labeled toxin B-IV by native toxin B-IV have shown specific binding of ¹²⁵I-labeled toxin B-IV to a single class of binding sites with a dissociation constant of 5–20 nM and a binding site capacity, corrected for vesicle sidedness, of 6–9 pmol per mg membrane protein. This compares to a value of 3.8 pmol [³H]saxitoxin bound per mg in the same tissue. Analysis of the kinetics of toxin B-IV association ($\text{k}{}_{\mathrm{+1}}\text{=7.3·10}{}^5\text{M}{}^{\mathrm{?1}}\text{·s}{}^{\mathrm{?1}}$) and dissociation ($\text{k}{}_{\mathrm{?\; 1}}\text{=2·10}{}^{\mathrm{?3}}\text{s}{}^{\mathrm{?1}}$) shows a nearly identical $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of about 3 nM. There is no competition of toxin B-IV binding by purified toxin from Leiurus quinquestriatus venom while Centruroides sculpturatus Ewing toxin I appears to cause a small enhancement of toxin B-IV binding.     

Cloning and expression of a synthetic gene for Cerebratulus lacteus neurotoxin B-IV

A synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV has been designed, cloned, and expressed in Escherichia coli. Although expression of the toxin alone appears to be incompatible with host viability, large amounts could be synthesized as a fusion protein with either E. coli beta-galactosidase or the gene 9 protein of bacteriophage T7, the latter system being the more efficient. The fusion protein has been purified, and, after Factor Xa-catalyzed hydrolysis at a customized linker site, we have obtained the equivalent of 12 mg of pure toxin B-IV per liter of bacterial culture. The recombinant protein is identical with B-IV isolated from Cerebratulus with respect to high performance liquid chromatography mobility and secondary structure, and its amino acid composition differs only by the presence of an amino-terminal methionine residue and replacement of Hyp10 by Pro. Quantal bioassay indicates that the cloned protein is comparable to the natural toxin in specific toxicity. The small differences observed in comparing the activities of the two proteins are most likely due to the presence of the methionine extension at the amino terminus of the recombinant, although lack of hydroxylation of Pro10 may also contribute.     

Circular dichroism and laser Raman spectroscopic analysis of the secondary structure ofCerebratulus lacteus toxin B-IV

The secondary structure ofCerebratulus lacteus toxin B-IV, a neurotoxic polypeptide containing 55 amino acid residues and four disulfide bonds, was experimentally estimated by computer analyses of toxin circular dichroism (CD) and laser Raman spectra. The CD spectrum of the toxin displayed typical α-helical peaks at 191, 208, and 222 nm. At neutralpH, the α-helix estimates from CD varied between 49 and 55%, when nonrepresentative spectrum analytical methods were used. Analysis of the laser Raman spectrum obtained at a much higher toxin concentration yielded a 78% α-helix estimate. Both CD and Raman spectroscopic methods failed to detect any β-sheet structure. The spectroscopic analyses revealed significantly more α-helix and less β-sheet for toxin B-IV than was predicted from its sequence. To account for the difference between the 49–55% helix estimate from CD spectra and the 78% helix estimate from the Raman spectrum, we postulate that some terminal residues are unfolded at the low toxin concentrations used for CD measurements but form helix at the high toxin concentration used for Raman measurements. Our CD observations showing thatCerebatulus toxin B-IV helix content increases about 15% in trifluoroethanol or at highpH are consistent with this interpretation.     

Synthesis of sequences 1-16 and 63-95 of Cerebratulus lacteus toxin AIII. Hemolytic activity in a toxin fragment

Sequences 1-16 and 63-95 of C. lacteus toxin AIII were synthesized on benzhydrylamine and PAM resins respectively; the crude peptide products released by HF contained 90 and 33% of the target peptides. Fragment 63-95 was nearly a full agonist, producing greater than 90% of the hemolytic activity of the intact toxin molecule at 10(3)-fold higher concentrations. Fragment 1-16 had no activity.     

1H-NMR study of neurotoxin B-IV from the marine worm Cerebratulus lacteus. Solution properties, sequence-specific resonance assignments, secondary structure and global fold.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.     

Purification and characterization of the cytotoxic Cerebratulus A toxins

Mucus secreted from the skin of a marine worm, Cerebratulus lacteus, contains a family of polypeptide cytotoxins (A toxins) in addition to the previously reported polypeptide neurotoxins (B toxins). The A toxins were purified by Sephadex G-50 chromatography and then CM-cellulose gradient chromatography at pH 7.5 and pH 3.5. The three most abundant A toxins (designated according to their order of CM-cellulose elution) were homogeneous by gel electrophoreses, amino acid composition, and by NH2-terminal and COOH-terminal partial sequence analyses. Each of the three A toxins consists of a single basic polypeptide chain of 93 to 99 residues, cross-linked by three or four disulfide bonds, lacking reducing sugar and cysteinyl residues. The three A toxins rapidly lysed human red cells and Ehrlich ascites tumor cells at 1 to 10 microgram/ml concentrations. On a molar basis toxin A-III is about 4 times more active than melittin (bee venom lysin) and over 10 times more active than cardiotoxin (elapid snake lysin) upon human red cells. Purified A toxins lacked phospholipase A activity. The cytoxins as well as the neurotoxins were concentrated within the body wall integument.     

Regulation and the modification of axial properties in partial embryos of the nemertean, Cerebratulus lacteus

 Embryos acquire axial properties (e.g., the animal-vegetal, dorsoventral and bilateral axes) at various times over the course of their normal developmental programs. In the spiral-cleaving nemertean, Cerebratulus lacteus, lineage tracing studies have shown that the dorsoventral axis is set up prior to the first cleavage division; however, blastomeres isolated at the two-cell stage will regulate to form apparently perfect, miniature pilidium larvae. We have examined the nature of axial specification in this organism by determining whether partial embryos retain the original embryonic/larval axial properties of the intact embryo, or whether new axial relationships are generated as a consequence of the regulatory process. Single blastomeres in two-cell stage embryos were injected with lineage tracer, and were then bisected along the second cleavage plane at the four-cell stage. Thus, the relationship between the plane of the first cleavage division and various developmental axes could be followed throughout development in the ”half-embryos”. While some embryo fragments appear to retain their original animal-vegetal and dorsoventral axes, many fragments generate novel axial properties. These results indicate that axial properties set up and used during normal development in C. lacteus can be completely reorganized during the course of regulation. While certain embryonic axes, such as the animal-vegetal and dorsoventral axes, appear to be set up prior to first cleavage, these axes and associated cell fates are not irreversibly fixed until later stages of development in normal intact embryos. In C. lacteus, the process whereby these properties are ultimately determined is apparently controlled by complex sets of cell-cell interactions. Received: 11 October 1996 / Accepted: 21 February 1997     

Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin.

The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.     

Structure and mode of action of clostridial glucosylating toxins: the ABCD model

Toxins A and B, which are the major virulence factors of antibiotic-associated diarrhea and pseudomembranous colitis caused by Clostridium difficile, are the prototypes of the family of clostridial glucosylating toxins. The toxins inactivate Rho and Ras proteins by glucosylation. Recent findings on the autocatalytic processing of the toxins and analysis of the crystal structures of their domains have made a revision of the current model of their actions on the eukaryotic target cells necessary.     

Structure of tetanus toxin. I. Breakdown of the toxin molecule and discrimination between polypeptide fragments.

Tetanus toxin was digested with papain, yielding one major polypeptide (Fragment C) with a molecular weight corresponding to 47,000 +/- 5%, thus comprising about one-third of the toxin molecule. Fragment C was antigenically active, atoxic, and stimulated the formation of antibodies neutralizing the lethal action of tetanus toxin in vivo. Furthermore, a second split product (Fragment B) was isolated from the papain digest, containing two polypeptide chains linked together via a disulfide bond. Fragment B (Mr = 95,000 +/- 5%) was atoxic and showed a reaction of nonidentity with Fragment C on immunodiffusion analysis against tetanus antitoxin. The basic two-chain structure (heavy and light chain polypeptide, cf. Matsuda, M., and Yoneda, M. (1975) Infect. Immun. 12, 1147-1153) of tetanus toxin has been confirmed and the relationship between Fragments B and C within this framework has been established. Fragment C was distinguished from the light chain by electrophoresis in sodium dodecyl sulfate and by immunodiffusion analysis, indicating that this fragment constitutes a portion of the heavy chain polypeptide. Fragment B showed a reaction of partial identity with the light as well as the heavy chain from tetanus toxin. Reduction of Fragment B with dithiothreitol followed by gel chromatography yielded a fraction which was indistinguishable from the light chain portion of the toxin molecule. It is concluded that Fragment B comprises the complementary portion of the heavy chain (remaining after scission of the polypeptide bond(s) releasing Fragment C) linked to the light chain by a disulfide bond.     

Structure and action of the binary C2 toxin from Clostridium botulinum

C2 toxin from Clostridium botulinum is composed of the enzyme component C2-I, which ADP-ribosylates actin, and the binding and translocation component C2-II, responsible for the interaction with eukaryotic cell receptors and the following endocytosis. Three C2-I crystal structures at resolutions of up to 1.75 A are presented together with a crystal structure of C2-II at an appreciably lower resolution and a model of the prepore formed by fragment C2-IIa. The C2-I structure was determined at pH 3.0 and at pH 6.1. The structural differences are small, indicating that C2-I does not unfold, even at a pH value as low as 3.0. The ADP-ribosyl transferase activity of C2-I was determined for alpha and beta/gamma-actin and related to that of Iota toxin and of mutant S361R of C2-I that introduced the arginine observed in Iota toxin. The substantial activity differences between alpha and beta/gamma-actin cannot be explained by the protein structures currently available. The structure of the transport component C2-II at pH 4.3 was established by molecular replacement using a model of the protective antigen of anthrax toxin at pH 6.0. The C-terminal receptor-binding domain of C2-II could not be located but was present in the crystals. It may be mobile. The relative orientation and positions of the four other domains of C2-II do not differ much from those of the protective antigen, indicating that no large conformational changes occur between pH 4.3 and pH 6.0. A model of the C2-IIa prepore structure was constructed based on the corresponding assembly of the protective antigen. It revealed a surprisingly large number of asparagine residues lining the pore. The interaction between C2-I and C2-IIa and the translocation of C2-I into the target cell are discussed.     

Transport of protein toxins into cells: pathways used by ricin,cholera toxin and Shiga toxin

Ricin, cholera, and Shiga toxin belong to a family of protein toxins that enter the cytosol to exert their action. Since all three toxins are routed from the cell surface through the Golgi apparatus and to the endoplasmic reticulum (ER) before translocation to the cytosol, the toxins are used to study different endocytic pathways as well as the retrograde transport to the Golgi and the ER. The toxins can also be used as vectors to carry other proteins into the cells. Studies with protein toxins reveal that there are more pathways along the plasma membrane to ER route than originally believed.     

Structure of presynaptic toxins: Crystallization and preliminary x-ray diffraction data on Notechis II-5, a presynaptic toxin phospholipase

The presynaptic neutrotoxin-phospholipase, Notechis II-5 from the venom of NotechisScutatus scutatus (Australian tiger snake) has been crystallized in a form suited for x-ray diffraction analysis. The crystals belong to the orthorhombic space group P2₁ 2₁,2₁, with unit cell dimensions, a=146.1,b =43.5 and c =39.0 A. There are two molecules of Notechis II-5 in the asymmetric unit. The molecular weight is about 13,500. Notechis II-5 is highly homologous to Notexin, another presynaptic toxin from the venom of the Australian tiger snake, to bovine and porcine pancreatic phospholipases A and other venom phospholipases.     

Human intoxication with paralytic shellfish toxins: clinical parameters and toxin analysis in plasma and urine

This study reports the data recorded from four patients intoxicated with shellfish during the summer 2002, after consuming ribbed mussels (Aulacomya ater) with paralytic shellfish toxin contents of 8,066 +/- 61.37 microg/100 gr of tissue. Data associated with clinical variables and paralytic shellfish toxins analysis in plasma and urine of the intoxicated patients are shown. For this purpose, the evolution of respiratory frequency, arterial blood pressure and heart rate of the poisoned patients were followed and recorded. The clinical treatment to reach a clinically stable condition and return to normal physiological parameters was a combination of hydration with saline solution supplemented with Dobutamine (vasoactive drug), Furosemide (diuretic) and Ranitidine (inhibitor of acid secretion). The physiological condition of patients began to improve after four hours of clinical treatment, and a stable condition was reached between 12 to 24 hours. The HPLC-FLD analysis showed only the GTX3/GTX2 epimers in the blood and urine samples. Also, these epimers were the only paralytic shellfish toxins found in the shellfish extract sample.