Ammodytoxin A, a highly lethal phospholipase A2 from Vipera ammodytes ammodytes venom.

The amino acid sequence of ammodytoxin A, the most toxic presynaptically active phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined. The primary structure was deduced from peptides obtained by Staphylococcus aureus proteinase and trypsin digestion of reduced and carboxymethylated protein and from the automated Edman degradation of the N-terminal part of the non-reduced molecule. According to the sequence, the enzyme classifies to the subgroup IIA of the phospholipase A2 family of enzymes. The location of basic residues believed to be responsible for the toxic activity of presynaptically active phospholipases differs substantially from those in the highly toxic enzymes of other subgroups. Comparison of the sequence with sequences of other snake venom enzymes indicates that the toxic site(s) may not be the same in all subgroups of presynaptically active phospholipases.

The role of tryptophan in structural and functional properties of equinatoxin II.

A pore-forming, cytolytic and lethal polypeptide, equinatoxin II, from the sea anemone Actinia equina, was subjected to oxidation with N-bromosuccinimide to study the role of five present tryptophan residues in structure-function relationships. In the folded toxin molecule, 1-2 tryptophan residues were readily susceptible to oxidation with N-bromosuccinimide, whereas modification of a single residue resulted in complete impairment of the toxin lethal and hemolytic activities as well as the ability of an oxidized toxin to precipitate with serum lipoproteins. CD and fluorescence spectra indicated a slight alteration of a toxin secondary structure following N-bromosuccinimide treatment. Incubation with sphingomyelin of the toxin prior to oxidation did not prevent subsequent modification with N-bromosuccinimide and loss of its activities, indicating that the modified tryptophan residue is not directly involved in toxin binding and insertion into lipid membranes. It was concluded that the modified tryptophan residue is essential for the structure of equinatoxin II.

The primary structure of Vipera ammodytes venom trypsin inhibitor I.

The primary structure of Vipera ammodytes venom trypsin inhibitor I consists of 61 amino acid residues [sequence in text]. The N-terminal group of the inhibitor is pyrrolidonecarboxylic acid. The sequential data were obtained by analysis of peptides isolated from tryptic and chymotryptic digests and by analysis of peptides derived from the hydrolysis of the aspartyl-prolyl bond of the carboxymethylated inhibitor. The primary structure of trypsin inhibitor I presented shows approximately 80% sequence homology with chymotrypsin inhibitor isolated from the venom of the same snake, and nearly 50% homology with bovine basic pancreatic trypsin inhibitor. It belongs to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.

Primary structure of ammodytoxin C further reveals the toxic site of ammodytoxin.

The sequence of ammodytoxin C, a presynaptically toxic, basic phospholipase A2 of Vipera ammodytes ammodytes venom was determined. The toxin differs only in two amino acid residues from the most toxic isotoxin ammodytoxin A and is 18-times less lethal. Ammodytoxin B which is 30-times less lethal than ammodytoxin A differs from it only in three amino acid residues. From the three-dimensional model of ammodytoxin A, it can be seen that mutated regions of ammodytoxin B and ammodytoxin C are on the surface, and relatively distant from each other. The observed decrease in toxicity of ammodytoxin C could be a consequence of changed charge in position 128 where a Lys is exchanged for Glu. The resulting change in electrostatic properties of the molecule which influences the orientation of the molecule during the approach to the charged nerve-terminal membrane might be responsible for the observed decrease in toxicity.

Isolation, partial characterization and complete amino acid sequence of the toxic phospholipase A2 from the venom of the common viper, Vipera berus berus.

A basic, toxic phospholipase A2 was purified from the venom of Vipera berus berus (Vbb) by a single purification step, using hydrophobic chromatography. The primary structure of isolated protein was established from peptides generated by Gly-specific papaya proteinase IV, beta-trypsin, CNBr and mild acid hydrolysis. The enzyme consists of a single chain of 122 amino acid residues with 14 Cys in positions characteristic for the phospholipase A2 subgroup IIA. As far as we know, this is the first complete Vipera berus phospholipase A2 amino acid sequence reported.

Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L., and its association with lipid vesicles.

The complete amino acid sequence of equinatoxin II, a potent pore-forming toxin with hemolytic, cytotoxic and cardiotoxic activity from the venom of the sea anemone, Actinia equina L., is reported. In addition, circular dicroism was used to estimate the secondary structure of this toxin either in the water-soluble or in the membrane-anchored form. Equinatoxin II when in water was found to contain about 29-33% of alpha-helical structure, 53-58% of beta-strand+beta-turn and 10-16% of random structure. Upon association with phospholipids, in particular with sphingomyelin, a rearrangement of the secondary structure occurs resulting in an increase of the alpha-helix content. An amphiphilic alpha-helical segment is predicted at the N-terminus, which shares structural homology with membrane active peptides like melittin and viral fusion peptides. In analogy to the behaviour of these peptides we propose that at least part of the alpha-helix content increase of equinatoxin II is due to the insertion of its N-terminus into the lipid bilayer. As in the case of melittin, association of 3-4 equinatoxin molecules is necessary to induce membrane permeabilisation.

Monoclonal antibodies to human stefin B and determination of their epitopes.

Monoclonal antibodies (MAbs) to human stefin B were developed and three of them were characterised. Stefin B was cleaved into four peptides which were later subfragmented to smaller peptides. Only two peptides, of 24 and 30 amino acids, could bind to MAbs. In one instance, two peptides that were not consecutive in the sequence were recognised by the same antibody, proving that the epitope was discontinuous. Location of the epitopes was further narrowed by measuring the binding of MAbs to the complex of stefin B with papain. A sandwich ELISA (enzyme-linked immunosorbent assay), which measures the concentration of free inhibitor, was developed. It confirms that two out of three MAbs bind to different sites of stefin B. On the basis of the crystal structure of complex of stefin B with papain, the surface, accessible to a sphere with a radius of 5 A which simulates the accessibility of variable regions of antibody was determined. From the difference between accessibilities of free stefin B and stefin B in complex, the epitope location was determined more accurately.

Phylogenomic analysis of chromoviruses.

Genome sequences of model organisms provide a unique opportunity to obtain insight into the complete diversity of any transposable element (TE) group. A limited number of chromoviruses, the chromodomain containing genus of Metaviridae, is known from plant, fungal and vertebrate genomes. By searching diverse eukaryotic genome databases, we have found a surprisingly large number of new, structurally intact and highly conserved chromoviral elements, greatly exceeding the number of previously known chromoviruses. In this study, we examined the diversity, origin and evolution of chromoviruses in Eukaryota. Chromoviral diversity in plants, fungi and vertebrates, as shown by phylogenetic analyses, was found to be much greater than previously expected. A novel centromere-specific chromoviral lineage was found to be widespread and highly conserved in all seed plants. The age of chromoviruses has been significantly extended by finding their representatives in the most basal plant lineages (green and red algae), in Heterokonta (oomycetes) and in Cercozoa (plasmodiophorids). The evolutionary origin of chromoviruses has been found to be no earlier than in Cercozoa, since none can be found in the basal eukaryotic lineages, despite the extensive genome data. The evolutionary dynamics of chromoviruses can be explained by a strict vertical transmission in plants and fungi, while in Metazoa it is more complex. The currently available genome data clearly show that chromoviruses are the most widespread and one of the oldest Metaviridae clade.

Adaptive evolution of animal toxin multigene families.

Animal toxins comprise a diverse array of proteins that have a variety of biochemical and pharmacological functions. A large number of animal toxins are encoded by multigene families. From studies of several toxin multigene families at the gene level the picture is emerging that most have been functionally diversified by gene duplication and adaptive evolution. The number of pharmacological activities in most toxin multigene families results from their adaptive evolution. The molecular evolution of animal toxins has been analysed in some multigene families, at both the intraspecies and interspecies levels. In most toxin multigene families, the rate of non-synonymous to synonymous substitutions (dN/dS) is higher than one. Thus natural selection has acted to diversify coding sequences and consequently the toxin functions. The selection pressure for the rapid adaptive evolution of animal toxins is the need for quick immobilization of the prey in classical predator and prey interactions. Currently available evidence for adaptive evolution in animal toxin multigene families will be considered in this review.

Molecular evolution of Bov-B LINEs in vertebrates.

Since their discovery in family Bovidae (bovids), Bov-B LINEs, believed to be order-specific SINEs, have been found in all ruminants and recently also in Viperidae snakes. The distribution and the evolutionary relationships of Bov-B LINEs provide an indication of their origin and evolutionary dynamics in different species. The evolutionary origin of Bov-B LINE elements has been shown unequivocally to be in Squamata (squamates). The horizontal transfer of Bov-B LINE elements in vertebrates has been confirmed by their discontinuous phylogenetic distribution in Squamata (Serpentes and two lizard infra-orders) as well as in Ruminantia, by the high level of nucleotide identity, and by their phylogenetic relationships. The direction of horizontal transfer from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution of Bov-B LINE elements. The ancestor of Colubroidea snakes has been recognized as a possible donor of Bov-B LINE elements to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINE elements in Ruminantia and the fossil data of Ruminantia to be 40-50 My ago. The phylogenetic relationships of Bov-B LINE elements from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINE elements have been stably maintained by vertical transmission since the origin of Squamata in the Mesozoic era.

Expression of fully active ammodytoxin A, a potent presynaptically neurotoxic phospholipase A2, in Escherichia coli.

A cDNA encoding the most presynaptically neurotoxic phospholipase A2, ammodytoxin A, from the venom of the long-nosed viper (Vipera ammodytes ammodytes) has been expressed in Escherichia coli. Ammodytoxin A was produced as a fusion protein with the 81 N-terminal residues of adenylate kinase followed by the tetrapeptide recognition site for factor Xa (IEGR) just preceding the first amino acid residue of the toxin. The fusion protein was expressed under the control of tac promoter without IPTG induction in the form of insoluble inclusion bodies. It was dissolved in guanidine hydrochloride, S-sulfonated and refolded in a reoxidation mixture including a reduced/oxidized glutathione redox couple. Ammodytoxin A was fully activated by limited hydrolysis with trypsin that preferentially cleaves the fusion protein at the factor Xa recognition site and purified by cation-exchange chromatography. The correct N-terminus was confirmed by protein sequencing. Recombinant ammodytoxin A has been proved to be indistinguishable from the native toxin in its enzymatic activity and toxicity.

Immunological studies of the toxic site in ammodytoxin A.

Two monoclonal antibodies against the native ammodytoxin A and four site-directed polyclonal antibodies against synthetic peptides derived from the primary structure of the toxin were prepared in order to estimate the localization of its toxic site. Some of the antibodies neutralized the lethal toxicity of the toxin, thus indicating an approximate position of the toxic or receptor binding site on the molecule that is different from those predicted by comparison with a number of known sequences.

Molecular cloning of a putative homolog of proline/arginine-rich antibacterial peptides from porcine bone marrow.

Screening of a porcine bone marrow cDNA library with a PCR-derived probe from rabbit LPS-binding protein CAP18 led to the discovery of two closely related clones. The longer, full-length cDNA clone encodes a 228 amino acid residue protein similar to the family of antibacterial/LPS-binding cationic peptides. In contrast to other hitherto discovered precursors of Pro/Arg-rich peptides from this family, they have a novel, unique structure of the C-terminal region of 100 amino acid residues with a repeating sequence of ten residues (FPPPNXPGPR, where X = V or F). These precursors could represent a part of the antibacterial peptide repertoire of porcine bone marrow.

Neuronal receptors for phospholipases A(2 )and beta-neurotoxicity.

Some phospholipases A(2) interrupt neuromuscular communication by blocking the release of neurotransmitter into the synaptic cleft. Despite numerous studies, the molecular mechanism of their action is still largely obscure. In this review the best-characterized receptors for beta-neurotoxins are presented. We propose a model which could be useful in investigating the apparent inconsistency between the observed heterogeneity in the neuronal binding of beta-neurotoxins and the very similar pathomorphological and electrophysiological effects which they produce in the intoxicated tissue. We assume that beta-neurotoxins enter the nerve ending to exert their toxic effect. The model involves different pathways for phospholipase A(2) neurotoxins to reach the site of action inside the neuron, their respective extra- and intracellular neuronal receptors being key features of the pathway. Once in the nerve cell, beta-neurotoxins impair the function of the synaptic vesicles by phospholipid hydrolysis of the inner leaflet of the vesicle bilayer. The proportion of the products of the phospholipid hydrolysis, lysophospholipids and phospholipids in the membrane, has been demonstrated to be very important for the shaping of the membrane, affecting its fusogenic properties. Due to the same final step in the action of beta-neurotoxins, phospholipid hydrolysis, the consequences of their poisoning are practically identical.

The Bov-B lines found in Vipera ammodytes toxic PLA2 genes are widespread in snake genomes.

In the fourth intron of two toxic Vipera ammodytes PLA2 genes a Ruminantia specific 5'-truncated Bov-B LINE element was identified. Southern blot analysis of Bov-B LINE distribution in vertebrates shows that, apart from the Ruminantia, it is limited to Viperidae snakes (V. ammodytes, Vipera palaestinae, Echis coloratus, Bothrops alternatus, Trimeresurus flavoviridis and Trimeresurus gramineus). The copy number of the 3' end of Bov-B LINE in the V. ammodytes genome is between 62,000 and 75,000. At orthologous positions in other snake PLA2 genes the Bov-B LINE element is absent, indicating that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 Myr ago. The amplification of Bov-B LINEs in snakes may have occurred before the divergence of the Viperinae and Crotalinae subfamilies. Due to its wide distribution in Viperidae snakes it should be a valuable phylogenetic marker. The neighbour-joining phylogenetic tree shows two clusters of truncated Bov-B LINE, a Bovidae and a snake cluster, indicating an early horizontal transfer of this transposable element.

A thrombin-like enzyme from bushmaster (Lachesis muta stenophyrs) venom.

The clotting enzyme (Stenoxobin), from the venom of Lachesis muta stenophyrs, was purified by gel chromatography on Bio-gel P-100 followed by agmatine CH-Sepharose-4B and FPLC on Mono Q column. By SDS polyacrylamide gel electrophoresis the mol. wt was found to be 37,000. The enzyme is a glycoprotein with 1.6 moles of sialic acid per mole of protein and has an average content of 7.0% of neutral carbohydrates. The clotting and esterolytic (BAEE) activities were 843 NIH units/mg and 60.1 +/- 1.2 OD225 ml/min/mg, respectively, and could not be inhibited by heparin or hirudin. Amino acid analysis revealed a low content of tryptophan and a high content of acid residues. Stenoxobin acts upon human fibrinogen by releasing consecutively fibrinopeptides A and B from the alpha- and beta-chains of fibrinogen.

mRNA secondary structure can greatly affect production of recombinant phospholipase A(2) toxins in bacteria.

The neurotoxic activity of ammodytoxin A (AtxA), a phospholipase A(2) from Vipera ammodytes ammodytes venom, has been investigated by protein engineering. With the aim of obtaining AtxA as a non-fused protein in the bacterial cytoplasm and avoiding problems with incomplete cleavage in vivo of the initial Met preceding the first residue (Ser1), a double mutant (S1A/E4Q) was prepared and expressed in Escherichia coli. Immunoblotting of the bacterial lysate showed that the mutant was synthesized at a low level not exceeding 0.5% of total cell protein. Analysis of the potential secondary structure of the mutant mRNA in the translation initiation region suggested that the Ala1 (GCC) and Leu2 (CUG) codons used are likely to be involved in a hairpin structure with the Thr13 (ACG) and Gly14 (GGG) codons, hindering effective translation at the ribosome. To weaken this structure (by DeltaG of about 20 kJ/mol) the same double mutant was prepared using another mutagenic oligonucleotide with silent mutations in the Ala1 (GCU) and Leu2 (UUG) codons. The mutant was successfully produced at a level of approximately 15% of total protein, with the initial Met completely removed in the bacterial cell. Such an approach could be important in solving similar problems in bacterial production of other toxic proteins.

Coagulant proteinase from Bothrops colombiensis venom.

The venom of Bothrops colombiensis, like other Crotalidae venoms, contains thrombin-like activity. We purified a mixture of isoenzymes by chromatography of the crude venom on DEAE-Sephacel where coagulant proteinase were separated from other proteolytic enzymes. By subsequent chromatography on Sephadex G-100 we obtained coagulant proteinase as a single band on acrylamide gel electrophoresis at pH 7.5 which showed 4 major protein bands when subjected to flat gel isoelectric focusing. This heterogeneity is presumably due to carbohydrates present in this glycoprotein. The native molecular weight of the coagulant proteinase was found to be over 90000 by gel filtration. SDS electrophoresis showed, however, that the monomer molecular weight is around 67000. The specific coagulant activity of the purified enzyme was increased 13 fold by purification and was 231 NIH units/mg. The optimal pH for coagulation of bovine fibrinogen was at pH 7.0. The enzyme shows maximal stability in the pH range 5-6 when incubated for 1 hr at 37 degrees C. The intraperitoneal LD50 for white mice was 4.0 mg/kg. The enzyme is similar to other known coagulant proteinases from snake venoms and thus potentially useful as a therapeutic agent.

Characterization of a metallo-proteinase from Bothrops asper (terciopelo) snake venom.

Metalloproteinase from the venom of Bothrops asper (proteinase G) is a glycoprotein with 1% neutral hexose and 3.5 moles of sialic acid per mole of protein. It hydrolyses a number of protein substrates such as casein, hemoglobin, gelatin and fibrinogen, whose alpha chain is degraded preferentially. The pH optimum of hydrolysis of casein is approximately 9.5. The protease is devoid of hemorraghic, esterolytic and amidolytic activities. The proteolytic activity of the enzyme increases by about 20% in the presence of 0.2 mM Ca2+ and Mg2+. Among the other ions tested, only Cd2+ and Fe2+ markedly decreased its activity. EDTA and cysteine are also strong inhibitors. In the presence of Ca2+ and EDTA, Zn2+ ions restored 50% of the activity. The amino acid composition shows fewer acidic residues than in related proteinases from other snake venoms.