Convenient nomenclature of cysteine-rich polypeptide toxins from sea anemones

Polypeptide toxins are the main constituents of natural venoms. Considerable progress in the study of these molecules has resulted in the determination of a large number of structurally related sequences. To classify newly discovered molecules, a rational nomenclature for naming peptide toxins was developed, which takes into account toxin biological activity, the species name, and structural peculiarities of the polypeptide. Herein, we suggest modifications to this nomenclature for cysteine-rich polypeptide toxins from sea anemones and describe 11 novel polypeptide structures deduced after common database revision.     

Molecular mechanisms of neurotoxin action on voltage-gated sodium channels

Voltage-gated sodium channels are the molecular targets for a broad range of neurotoxins that act at six or more distinct receptor sites on the channel protein. These toxins fall into three groups. Both hydrophilic low molecular mass toxins and larger polypeptide toxins physically block the pore and prevent sodium conductance. Alkaloid toxins and related lipid-soluble toxins alter voltage-dependent gating of sodium channels via an allosteric mechanism through binding to intramembranous receptor sites. In contrast, polypeptide toxins alter channel gating by voltage sensor trapping through binding to extracellular receptor sites. The results of recent studies that define the receptor sites and mechanisms of action of these diverse toxins are reviewed here.     

Polypeptide neurotoxins from spider venoms.

Spider venoms contain a variety of toxic components. The polypeptide toxins are divided into low and high molecular mass types. Small polypeptide toxins interacting with cation channels display spatial structure homology. They can affect the functioning of calcium, sodium, or potassium channels. A family of high molecular mass toxic proteins was found in the venom of the spider genus Latrodectus. These neurotoxins, latrotoxins, cause a massive transmitter release from a diversity of nerve endings. The latrotoxins are proteins of about 1000 amino acid residues and share a high level of structure identity. The structural and functional properties of spider polypeptide toxins are reviewed in this paper.     

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.     

Toxins A and B of Clostridium difficile

Abstract: The toxins produced by Clostridium difficile share several functional properties with other bacterial toxins, like the heat-labile enterotoxin of Escherichia coli and cholera toxin. However, functional and structural differences also exist. Like cholera toxin, their main target is the disruption of the microfilaments in the cell. However, since these effects are not reversible, as found with cholera toxin, additional mechanisms add to the cytotoxic potential of these toxins. Unlike most bacterial toxins, which are built from two structurally and functionally different small polypeptide chains, the functional and binding properties of the toxins of C. difficile are confined within one large polypeptide chain, making them the largest bacterial toxins known so far.     

Toxin entry: how bacterial proteins get into mammalian cells

Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation — the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Characterization of the C-terminal domain essential for toxic activity of adenylate cyclase toxin

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartate-rich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca²⁺, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat region and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca²⁺ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca²⁺ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca²⁺ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca²⁺ binding repeats of the short C-terminal polypeptide and the Ca²⁺ binding repeats of the CyaA mutant lacking block A.     

Processing and secretion of a virally encoded antifungal toxin in transgenic tobacco plants: evidence for a Kex2p pathway in plants.

Ustilago maydis is a fungal pathogen of maize. Some strains of U. maydis encode secreted polypeptide toxins capable of killing other susceptible strains of U. maydis. We show here that one of these toxins, the KP6 killer toxin, is synthesized by transgenic tobacco plants containing the viral toxin cDNA under the control of a cauliflower mosaic virus promoter. The two components of the KP6 toxin, designated alpha and beta, with activity and specificity identical to those found in toxin secreted by U. maydis cells, were isolated from the intercellular fluid of the transgenic tobacco plants. The beta polypeptide from tobacco was identical in size and N-terminal sequence to the U. maydis KP6 beta polypeptide. Processing of the KP6 preprotoxin in U. maydis requires a subtilisin-like processing protease, Kex2p, which is present in both animal and fungal cells and is required for processing of (among other things) small secreted polypeptide hormones and secreted toxins. Our findings present evidence for Kex2p-like processing activity in plants. The systemic production of this viral killer toxin in crop plants may provide a new method of engineering biological control of fungal pathogens in crop plants.     

Structure and heterologous expression of the Ustilago maydis viral toxin KP4

Killer toxins are polypeptides secreted by some fungal species that kill sensitive cells of the same or related species, in the best-characterized cases, they function by creating new pores in the ceil membrane and disrupting ion fluxes. Immunity or resistance to the toxins is conferred by the preprotoxins (or products thereof) or by nuclear resistance genes. In several cases, the toxins are encoded by one or more genomic segments of resident double-stranded RNA viruses. The known toxins are composed of one to three polypeptides, usually present as multimers. We have further characterized the KP4 killer toxin from the maize smut fungus Ustilago maydis. This toxin is also encoded by a single viral double-stranded RNA but differs from other known killer toxins in several respects: it has no N-linked glycosylation either in the precursor or in the mature polypeptide, it is the first killer toxin demonstrated to be a single polypeptide, and h Is not processed by any of the known secretory protelnases (other than the signal peptidase). It is efficiently expressed in a heterologous fungal system.     

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

Summary The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (P _o ) increased with depolarization;P _o was 0.5 at –40 to –50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1nm at –45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in theP _o vs. V _m relation such that the channels were more likely to be open at potentials <40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.     

Dimethyl sulphoxide protects cells against polypeptide toxins and poliovirus.

The effect of dimethyl sulphoxide and other cryoprotective compounds on the sensitivity of cells to polypeptide toxins and to poliovirus was tested. In the presence of these compounds, which all affect membrane fluidity, the cells were protected against the toxic proteins and against poliovirus. The large protection obtained was not due to reduced binding and endocytosis of the toxins. Apparently, the cryoprotective compounds interfere with the entry of toxins and of the poliovirus genome across the cell membrane.     

Calitoxin, a neurotoxic peptide from the sea anemone Calliactis parasitica: amino acid sequence and electrophysiological properties

We have isolated a new toxin, calitoxin (CLX), from the sea anemone Calliactis parasitica whose amino acid sequence differs greatly from that of other sea anemone toxins. The polypeptide chain contains 46 amino acid residues, with a molecular mass of 4886 Da and an isoelectric point at pH 5.4. The amino acid sequence determined by Edman degradation of the reduced, S-carboxymethylated polypeptide chain and tryptic and chymotryptic peptides is Ile-Glu-Cys-Lys-Cys-Glu-Gly-Asp-Ala-Pro-Asp-Leu-Ser-His-Met-Thr-Gly-Thr- Val-Tyr - Phe-Ser-Cys-Lys-Gly-Gly-Asp-Gly-Ser-Trp-Ser-Lys-Cys-Asn-Thr-Tyr-Thr-Ala- Val-Ala - Asp-Cys-Cys-His-Glu-Ala. No cysteine residues were present in the peptide. Similarly to other sea anemone toxins, calitoxin interacts, in crustacean nerve muscle preparations, with axonal and not with muscle membranes, inducing a massive release of neurotransmitter that causes a strong muscle contraction. The low homology of CLX with RP II and ATX II toxins has implications regarding the role played by particular amino acid residues.     

omega-Aga-I: a presynaptic calcium channel antagonist from venom of the funnel web spider, Agelenopsis aperta

Spider venoms are proving to be important sources of specific ion channel toxins. Venom of Agelenopsis aperta, a funnel web spider, contains a class of polypeptide toxins which blocks neuromuscular synapses at nanomolar concentrations. Detailed physiological analyses of block caused by one of these toxins, omega-Aga-I, show that it suppresses transmitter release at insect and frog neuromuscular junctions and blocks calcium spikes in insect neuronal cell bodies. omega-Aga-I may define a binding site on neuronal calcium channels which is common to both vertebrates and invertebrates.     

Amino-acid sequence of toxin I from Anemonia sulcata.

Toxin I from Anemonia sulcata, a major component of the sea anemone venom, consists of 46 amino acid residues which are linked by three disulfide bridges. The [14C]carboxymethylated polypeptide was sequenced to position 29 by automated Edman degradation. The remaining sequence was determined from cyanogen bromide peptides and from tryptic peptides of the citraconylated [14C]carboxymethylated toxin. Toxin I is homologous to toxin II from Anemonia sulcata and to anthopleurin A, a toxin from the sea anemone Anthopleura xanthogrammica. These toxins constitute a new class of polypeptide toxins. No significant homologies exist with toxin III from Anemonia sulcata nor with known sequences of neurotoxins or cardiotoxins of various origin.     

Lack of cross-resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to cry toxins from Bacillus thuringiensis subsp. israelensis

Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp. jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with B. thuringiensis subsp. jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp. jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.     

Omega-agatoxins: novel calcium channel antagonists of two subtypes from funnel web spider (Agelenopsis aperta) venom

A new series of polypeptide presynaptic antagonists ("omega-agatoxins") was purified from venom of the funnel web spider Agelenopsis aperta. Physiological data indicate that all of these peptides are antagonists of voltage-sensitive calcium channels. Although all three omega-agatoxins (Aga) described here (omega-Aga-IA, omega-Aga-IB, and omega-Aga-IIA) block insect neuromuscular transmission presynaptically, biochemical data permit their subclassification as Type I and Type II toxins. Type I toxins (omega-Aga-IA and -IB) are 7.5 kDa, have closely related amino acid sequences, and exhibit characteristic tryptophan-like UV absorbance spectra. Complete Edman sequencing of omega-Aga-IA reveals it to be a 66-amino acid polypeptide containing 9 cysteines and 5 tryptophan residues. omega-Aga-IIA, a Type II toxin, is 11 kDa, shows limited amino acid sequence similarity to the Type I toxins, and exhibits mixed tryptophan- and tyrosine-like absorbance. Nanomolar concentrations of omega-Aga-IIA inhibit the specific binding of 125I-labeled omega-conotoxin GVIA to chick synaptosomal membranes while omega-Aga-IA and -IB have no effect under identical conditions. The omega-agatoxins thus are defined as two subtypes of neuronal calcium channel toxins with different structural characteristics and calcium channel binding specificities.     

Comparison of three classes of snake neurotoxins by homology modeling and computer simulation graphics

We present a systematic structure comparison of three major classes of postsynaptic snake toxins, which include short and long chain alpha-type neurotoxins plus one angusticeps-type toxin of black mamba snake family. Two novel alpha-type neurotoxins isolated from Taiwan cobra (Naja naja atra) possessing distinct primary sequences and different postsynaptic neurotoxicities were taken as exemplars for short and long chain neurotoxins and compared with the major lethal short-chain neurotoxin in the same venom, i.e., cobrotoxin, based on the derived three-dimensional structure of this toxin in solution by NMR spectroscopy. A structure comparison among these two alpha-neurotoxins and angusticeps-type toxin (denoted as FS2) was carried out by the secondary-structure prediction together with computer homology-modeling based on multiple sequence alignment of their primary sequences and established NMR structures of cobrotoxin and FS2. It is of interest to find that upon pairwise superpositions of these modeled three-dimensional polypeptide chains, distinct differences in the overall peptide flexibility and interior microenvironment between these toxins can be detected along the three constituting polypeptide loops, which may reflect some intrinsic differences in the surface hydrophobicity of several hydrophobic peptide segments present on the surface loops of these toxin molecules as revealed by hydropathy profiles. Construction of a phylogenetic tree for these structurally related and functionally distinct toxins corroborates that all long and short toxins present in diverse snake families are evolutionarily related to each other, supposedly derived from an ancestral polypeptide by gene duplication and subsequent mutational substitutions leading to divergence of multiple three-loop toxin peptides.     

ω-Aga-I: A presynaptic calcium channel antagonist from venom of the funnel web spider,Agelenopsis aperta

Spider venoms are proving to be important sources of specific ion channel toxins. Venom of Agelenopsis aperta, a funnel web spider, contains a class of polypeptide toxins which blocks neuromuscular synapses at nanomolar concentrations. Detailed physiological analyses of block caused by one of these toxins, ω-Aga-I show that it suppresses transmitter release at insect and frog neuromuscular junctions and blocks calcium spikes in insect neuronal cell bodies. ω-Aga-I may define a binding site on neuronal calcium channels which is common to both vertebrates and invertebrates.