Purification and molecular cloning of SE-cephalotoxin, a novel proteinaceous toxin from the posterior salivary gland of cuttlefish Sepia esculenta

Cephalopods contain toxins in their salivary glands, presumably to paralyze prey animals such as crabs and bivalves. Proteinaceous toxins (called cephalotoxins) with crab lethality have previously been purified from three species of octopodiform cephalopods (octopuses) but their detailed properties and primary structures have remained unknown. In this study, salivary glands of six species of decapodiform cephalopods were newly found to be toxic; three species of cuttlefish were lethal only to crabs and three species of squid to both mice and crabs. A proteinaceous toxin (named SE-cephalotoxin) in the salivary gland of cuttlefish Sepia esculenta was soluble only in high-salt solvents. This unique solubility enabled us to purify SE-cephalotoxin by gel filtration HPLC and hydroxyapatite HPLC. SE-cephalotoxin was shown to be a 100kDa monomeric glycoprotein with an LD(50) (against crabs) of 2mug/kg. Based on the determined partial amino acid sequence, a full-length cDNA (3402bp) coding for SE-cephalotoxin was cloned by RT-PCR and RACE. The SE-cephalotoxin precursor protein (1052 amino acid residues) is composed of a signal peptide (region 1-21), propeptide (region 22-29) and mature protein (region 30-1052). A database search failed to find any proteins sharing homology with SE-cephalotoxin.     

Complete amino acid sequences of two cardiotoxin-like analogues from Bungarus fasciatus (banded krait) snake venom

Three cardiotoxin-like proteins have been isolated from Bungarus fasciatus venom and the amino acid sequence of the major toxin (toxin VI) have been determined. The amino acid sequences of two other analogues (toxins V-2 and V-3) were investigated. The reduced and S-carboxymethylated toxins were digested with trypsin-TPCK and the resulting tryptic peptides were isolated by fingerprinting technique on paper. The amino acid compositions, N-terminal residues and partial amino acid sequences of some of the tryptic peptides were determined. Thirteen tryptic peptides were aligned by following the order of corresponding fragments of toxin VI. Toxins V-2 and V-3 contained 118 and 117 amino acid residues, respectively, in a single polypeptide chain cross-linked with six pairs of intramolecular disulphide bonds. There are only four (for toxin V-2) and five (for toxin V-3) places of differences in their primary structures when compared with that of the major toxin (toxin VI) of Bungarus fasciatus venom.     

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.     

Plancitoxins, lethal factors from the crown-of-thorns starfish Acanthaster planci, are deoxyribonucleases II.

Two lethal factors (named plancitoxins I and II for major and minor toxins, respectively) with the same LD50 (i.v. injection into mice) of 140 microg/kg were purified from spines of the crown-of-thorns starfish Acanthaster planci. Injection of a sublethal dose of plancitoxin I or II into mice remarkably elevated serum levels of glutamic oxaloacetic transaminase and glutamic pyruvic transaminase, demonstrating that both toxins are potently hepatotoxic. Analysis by SDS-PAGE revealed that both plancitoxins are composed of two subunits (alpha-subunit of 10 kDa and beta-subunit of 27 kDa) bridged by a disulfide bond. Based on the determined N-terminal amino acid sequences of alpha- and beta-subunits, the full-length cDNA (1820 bp) encoding plancitoxin I was cloned by RT-PCR, 3'-RACE and 5'-RACE. alpha-Subunit (92 amino acid residues) and beta-subunit (240 residues) are coded in this order by the same cDNA. Interestingly, the deduced amino acid sequence of plancitoxin I showed 40-42% homologies with mammalian deoxyribonucleases II (DNases II). In addition, plancitoxin I exhibited DNA degrading activity with an optimum pH of 7.2. Plancitoxin I is the first example of toxic DNases II whose structures have been elucidated.     

Toxins in the skin secretion of the oriental catfish (Plotosus lineatus): immunological properties and immunocytochemical identification of producing cells

Antiserum against toxin I, one of the lethal factors in the skin secretion from the oriental catfish (Plotosus lineatus), was used to examine immunological properties of the toxic factors and identify toxin-producing cells by an immunocytochemical technique. In immunodiffusion tests, the antiserum formed a precipitin line with toxin I while it formed no precipitin line either with another lethal factor (toxin II) or with a hemolysin. Lethal and edema-forming activities of toxin I were neutralized by the antiserum but lethal activity of toxin II and lytic activity of the hemolysin were not. These results suggested that toxin I can be antigenically distinguished from both toxin II and hemolysin. In immunocytochemical tests using the antiserum, club cells in the epidermis were positively stained, indicating that toxin I is produced in the club cells. Interestingly, venom glandular cells surrounding the dorsal and pectoral spines were also stained. The venom glandular cells appear to produce toxin I or a toxin with the same antigen determinants as toxin I.     

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.     

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

Three peptide toxins (gigantoxins I-III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD(50) (against crabs) of 70 and 120 microg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED(50) 215 microg/kg), although its lethality is very weak (LD(50)>1000 microg/kg). Interestingly, gigantoxin I has 31-33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.     

Huwentoxin-V, a novel insecticidal peptide toxin from the spider Selenocosmia huwena, and a natural mutant of the toxin: indicates the key amino acid residues related to the biological activity.

A neurotoxin peptide (named Huwentoxin-V) was purified from the venom of the Chinese bird spider Selenocosmia huwena by a combination of ion exchange chromatography and reverse phase HPLC. HWTX-V has 35 amino acid residues, and is in perfect agreement with the molecular mass 4111.4 Da identified by mass spectrometry. A natural mutant of the toxin (called mHuwentoxin-V) was also isolated from the venom. mHWTX-V was only truncated two amino acid residues from the C-terminus of HWTX-V, and its molecular weight is 3877.1 Da determined by mass spectrometry. The six cysteine residues in each sequence of the two peptides suggest three disulfide bridges, the present of which was demonstrated by mass spectrometry after dithiothreiotol reduce and S-carboxymethylation. The primary structure of the two toxins exhibits sequence identity with other spider toxins such as ProTx-I (64%), SGTx (57%), SNX-482 (55%), and Hanatoxin (54%). HWTX-V can reversibly paralyze locusts and cockroaches for several hours with a ED50 value as 16 +/- 5 microg/g to locusts, and a larger dose of the toxin can cause death. However, mHWTX-V shows no significant effect on locusts and cockroaches. The structure-activity relationship indicates that the residues Phe34 and Ser35 in the C-terminus of HWTX-V are the key residues of the biological activity.     

A simplified procedure for the fractionation of Tityus serrulatus venom: isolation and partial characterization of TsTX-IV, a new neurotoxin

Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX3, IX5, and X4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. LD50, in mice, of 826 +/- 156 micrograms/kg and an intracisternal LD50 of 11 +/- 9 micrograms/kg, compared to 375 +/- 45 and 4.9 +/- 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.     

Purification of three polypeptides with neuro- and cardiotoxic activity from the sea anemone Anemonia sulcata.

Three toxic polypeptides, Toxins I, II and III have been isolated from the sea anemone Anemonia sulcata Pennant (Actinaria). Toxin isolation was accomplished by alcoholic extraction of the homogenized sea anemones, batchwise adsorption on cation exchangers, gel filtration on Sephadex (G 50, G 25, G 10) and ion exchange chromatography on SP-Sephadex and QAE-Sephadex. It was demonstrated that the same toxins (Toxin I and Toxin II) are also present in the separated tentacles. Toxin I contains 45 amino acids, mol. wt 4702, Toxin II contains 44 amino acids, mol. wt 4197, Toxin III contains 24 amino acids, mol. wt 2678.The toxins were tested on the shore crab Carcinus maenas by intramuscular injection. For both Toxins I and II the lethal dose is less than 2 μg per kg Carcinus, whereas for Toxin III it is less than 50 μg per kg. Toxins I and II also have a strong paralysing effect on mammals and fishes.     

Primary structure and electrophysiological characterization of two almost identical isoforms of toxin from Isometrus vittatus (family: Buthidae) scorpion venom

Two almost identical proteins with 70 amino acid residues each, closely packed by four disufide bridges, and molecular masses of 7899.5 and 7884.7 were isolated and sequenced from the venom of the scorpion Isometrus vittatus from Pakistan. They differ by an acidic amino acid residue (glutamic or aspartic) at the same position 55 of the peptide chain, however, they exhibit the same length, the same charge and are undistinguishable when separated by C₁₈ reverse phase HPLC. The mixture of the two proteins called IsomTx1 depolarizes the cockroach isolated axon; artificial repolarization is followed by sustained repetitive activity, artificial hyperpolarization facilitates bursting activity observed as an answer to rapid depolarization to −60 mV. The depolarization is antagonized by TTX. In voltage-clamp experiments IsomTx1 increases axonal sodium permeability which has a particular importance between resting and threshold potentials and moderately slows down the fast inactivation. These characteristics closely resemble those of other anti-insect scorpion toxins classified as contractive toxins from Androctonus and Buthotus venoms.     

Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea.

A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.     

Characteristics of chitosanases from<Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> KB-1

Two chitosanases produced by Aspergillus fumigatus KB-1 were purified by ion exchange and size exclusion chromatographies. Molecular weights of chitosanases were 111.23 kDa (chitosanase I) and 23.38 kDa (chitosanase II). The N-terminal amino acid sequence of chitosanase II was determined as follows: YNLPNNLKQIYDKHKGKXSXVLAKGFTN. The optimum pH of the chitosanase I and II was 6.5 and 5.5, respectively. The optimum temperatures were 60 degrees C for chitosanase land 70 degrees C for chitosanase II. Hydrolysis products of two chitosanases were analyzed by HPLC and GPC. Chitosanase I hydrolyzed substrate to glucosamine. Chitosanase II produced chitooligosaccharides.     

Structure and Function of Clostridium Botulinum Progenitor Toxin

Clostridium botulinum strains produce seven immunologically distinct neurotoxins (NTX), type A to G. the NTXs associate with nontoxic components in cultures, and become large complexes with three forms (12S, 16S, and 19S) designated progenitor toxins. the 12S toxin consists of a NTX and a nontoxic component having no hemagglutinin (HA) activity (described here as non-toxic non-HA, NTNH), and the 16S and 19S toxins are formed by conjugation of the 12S toxin with HA. Based on the genetic-and protein chemical-analyses of the progenitor toxins it became clear that 1) the HA consists of four subcomponents namely HA1 (Mr. 33–35 kDa), HA2 (15–17 kDa), HA3a (19–23 kDa), and HA3b (52–53 kDa), 2) the genes coding for NTX (ntx), NTNH (ntnh), and HA (ha) occur as a cluster; ha lies just upstream of ntnh, and ntx lies just downstream of ntnh, 3) ha is in the opposite orientation from that of ntnh and ntx, 4) ha consists of three ORFs (ha1, ha2, and ha3), 5) the gene product (70 kDa) of ha3 is split into HA3a and HA3b after translation, 6) HA3a is cleaved at several different sites of its N-terminal region to form proteins with slightly different Mrs, 7) the 19S toxin is a dimer of 16S toxin crosslinked by HA1, 8) the NTNHs of type A to D 12S toxins have a cleavage(s) on their N-terminal regions. It also became clear that the HA plays an important role when the 16S (and 19S) toxin is absorbed from the small intestine.     

Structure and other chemical characterizations of gila toxin,a lethal toxin from lizard venom

The complete primary structure of a lethal toxin, horridum toxin, from the venom of the lizard, Heloderma horridum horridum, was determined by Edman degradation. The amino acid sequence was deduced by overlapping peptide fragments generated by chemical and enzymatic digestions. Horridum toxin causes hemorrhage in internal organs and particularly in the eye, leading to exophthalmia, an effect that has not been observed for other toxins. It is a glycoprotein with a total of 210 residues. Examination of the primary sequence revealed that horridum toxin has considerable homology to tissue-type kallikrein and trypsin. Furthermore, synthetic substrates for trypsin, such as tosyl-l -arginine methyl ester, benzoyl-l -arginine ethyl ester and other p-nitroanilide substrates, were hydrolyzed. The toxin released bradykinin upon hydrolysis of kininogen. This enzymatic behavior is similar to that of plasma kallikrein; however, the presence of a characteristic “kallikrein-like” loop at 91-100 (GTIYNCNYVN) in the primary structure and other features similar to tissue kallikrein suggest that horridum toxin is more like tissue kallikrein. This toxin degraded all three chains of fibrinogen but did not form a clot, which suggests that it is different from thrombin. Moreover, it differs from another lethal factor from H. horridum horridum, gila toxin, which has 245 amino acid residues and does not cause exophthalmia.     

Structure-activity relationships of scorpion alpha-neurotoxins: contribution of arginine residues

The role of arginine residues in the structure-activity relationships of alpha-scorpion neurotoxins was studied. Toxins I and II from Androctonus australis Hector (north African scorpion), containing respectively 2 and 3 arginines, were modified by phenylglyoxal or p-hydroxyphenylglyoxal. Modified derivatives were purified by reverse-phase HPLC and/or ion exchange HPLC. Subsequent bioassays showed that toxin I (AaH I) derivatives with single modifications on Arg 2 and Arg 60 had low activity (25 and 14% of residual activity, assessed in receptor binding experiments). Doubly modified (Arg 2, Arg 60) AaH I had 7% residual activity while further derivatization of the alpha-amino group led to an almost inactive derivative. These results agree with the involvement of arginines 2 and 60, as well as the alpha-amino group, of AaH I in the toxin/receptor interaction, probably via electrostatic interactions. Consistent with the role of N-terminal residues, the selective removal of the N-terminal dipeptide Val-Arg of toxin III from the same scorpion resulted in low activity (7% residual activity). The arginine residue in position 56 of toxin II was important for bioactivity since the derivative modified by phenylglyoxal on Arg 56 exhibited low residual activity (20%). Arg 62 and Arg 18, on the other hand, can be modified without any great effect on the pharmacological activity of AaH II. These results furnish a more precise picture of those residues involved in the "toxic region", which appears to be composed of residues belonging to the conserved hydrophobic surface and to the C-terminal and N-terminal sequences.     

The Toxins Purified from Tityus Serrulatus (Lutz & Mello)Venom

Abstract

The toxicity of Tityus serrulatus venom is mainly due to a complex mixture of basic proteins of low molecular weight (MW< 8000 Da) which are active on the voltage-sensitive Na⁺ channel of excitable cells. One group of toxins, the α-toxins, delays inactivation of the Na⁺ channel. A second group, the β- toxins, produces a transient shift in the voltage-dependence of Na⁺ channel activation and increases the tendency of the cells to fire repetitively. The two groups bind specifically to two different binding sites, sites 3 and 4, of Na⁺ channels present in rat brain synaptosomes. The primary structure of the main toxins has been determined and consists of a single amino acid chain of 61 to 66 residues cross-linked by four disulfide bridges. Some secondary structural elements have also been determined. More recently, using molecular biological techniques, cDNAs encoding the precursors of α and β-toxins have been cloned from a cDNA library of Tityus serrulatus venom gland. The precursors contain a signal peptide of about 20 – 22 residues, the mature toxin and three additional Gly-Lys-Lys residues at the C-terminal that are not present in the mature toxins. The Lys residues are removed by a carboxypeptidase and the remaining Gly-extended peptides are converted into α-amidated C-terminal toxins. Toxins active at K⁺ channels have also been purified from Tityus serrulatus venom. At present, only two toxins have been characterized, namely a short polypeptide of 37 amino acid residues cross-linked by three disulfide bridges that competes for 125I-alpha DTX (dendrotoxin) binding sites in synaptic membranes, and a longer nonhomologous toxin of 8 016 Da, whose primary structure has only been determined at the level of its NH2 terminal.     

Characterization of a new family of toxin-like peptides from the venom of the scorpion Leiurus quinquestriatus hebraeus-NMR structure of leiuropeptide II

To extend our knowledge about the structural features of short scorpion toxins, the ion-exchange fractions obtained from Leiurus quinquestriatus hebraeus venom were investigated by plasma desorption mass spectrometry in order to select low molecular mass polypeptides. Three toxin-like peptides with molecular mass close to 3 kDa, named leiuropeptides I, II and III, were purified and found devoid of any significant toxicity against mammals and insects. Their amino acid sequences revealed a cysteine pattern analogous to that of short-chain scorpion toxins. The solution structure of leiuropeptide II was determined by 2D H-NMR spectroscopy and indicated the presence of a helix accomodating a proline, connected to a two-stranded β-sheet by three disulfide bonds. The overall fold of leiuropeptide II is found to be similar to that of leiurotoxin I, a 31-residue toxin present in the same scorpion venom which acts on K⁺ channels. In order to rationalize the absence of toxicity, the electrostatic potential of leiuropeptide II was compared to that of leiurotoxin I. The peptide is characterized by a large negative zone around Glu4, Asp5 and Asp8 residues, beginning in the neighbourhood of the β-turn and extending along the helix. In the same area, leiurotoxin I exhibits a positive surface, around Arg6 and Argl3 basic residues, which are essential for its receptor affinity. © Munksgaard 1997.     

Isolation and chemical characterization of a toxin isolated from the venom of the sea snake, Hydrophis torquatus aagardi

Sea snakes (family: Hydrophiidae) are serpents found in the coastal areas of the Indian and Pacific Oceans. There are two subfamilies in Hydrophiidae: Hydrophiinae and Laticaudinae. A toxin, aagardi toxin, was isolated from the venom of the Hydrophiinae snake, Hydrophis torquatus aagardi and its chemical properties such as molecular weight, isoelectric point, importance of disulfide bonds, lack of enzymatic activity and amino acid sequence were determined. The amino acid sequence indicated a close relationship to the primary structure of other Hydrophiinae toxins and a significant difference from Laticaudinae toxins, confirming that primary toxin structure is closely related to sea snake phylogenecity.