Franc Gubenšek

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.

Adaptive evolution in the snake venom Kunitz/BPTI protein family

Snake venoms are rich sources of serine proteinase inhibitors that are members of the Kunitz/BPTI (bovine pancreatic trypsin inhibitor) family. However, only a few of their gene sequences have been determined from snakes. We therefore cloned the cDNAs for the trypsin and chymotrypsin inhibitors from a Vipera ammodytes venom gland cDNA library. Phylogenetic analysis of these and other snake Kunitz/BPTI homologs shows the presence of three clusters, where sequences cluster by functional role. Analysis of the nucleotide sequences from the snake Kunitz/BPTI family shows that positive Darwinian selection was operating on the highly conserved BPTI fold, indicating that this family evolved by gene duplication and rapid diversification.

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.

Equinatoxins, pore-forming proteins from the sea anemone Actinia equina, belong to a multigene family.

The multigene family of equinatoxins, pore-forming proteins from sea anemone Actinia equina, has been studied at the protein and gene levels. We report the cDNA sequence of a new, sphingomyelin inhibited equinatoxin, EqtIV. The N-terminal sequences of natural Eqt I and III were also determined, confirming two isoforms of EqtI, differing at position 13. The number of Eqt genes determined by Southern blot hybridization was found to be more than five, indicating that Eqts belong to a multigene family.

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.

Fractionation of Vipera ammodytes venom and seasonal variation of its composition

Abstract Crude venom was fractionated on CM- and DEAE-cellulose and on Sephadex G-50. Lethality, hemorrhagic and proteolytic activity and arterial blood pressure depressing activities were tested in the various fractions. Composition of fractions was monitored by disc electrophoresis. Lethal basic proteins of low molecular weight were separated from hemorrhagic proteins. Proteolytic and hemorrhagic activities could not be separated. Complete disappearance of two basic low molecular weight proteins was observed in the venoms milked during the winter season. Some smaller changes in other protein constituents were also evident.

Identification and Purification of a Novel Receptor for Secretory Phospholipase A2 in Porcine Cerebral Cortex

A specific phospholipase A2receptor from porcine cerebral cortex has been characterized (K d = 145 nm,B max = 0.4 pmol/mg membrane protein) by using a radioiodinated derivative of ammodytoxin C (AtxC), a snake venom presynaptically neurotoxic group IIA phospholipase A2. After the receptor was solubilized in a ligand-binding form, it was approximately 14,000-fold enriched by chromatography on wheat germ lectin-Sepharose and AtxC-Affi-Gel 10. The receptor is a single chain glycoprotein with an apparent molecular mass of 180 kDa and binds toxic and non-toxic phospholipases A2 of either group I or II. It also recognizes conjugates of bovine serum albumin with mannose,N-acetylglucosamine, and galactose. In its molecular mass and pharmacological profile, the AtxC receptor resembles the M-type receptor for secretory phospholipases A2 from rabbit skeletal muscle (a C-type multilectin, homologous to macrophage mannose receptor), yet in terms of relative abundance in brain and antigenicity, these two receptors are completely different. A further AtxC receptor of approximately 200 kDa discovered in porcine liver was, however, recognized by anti-rabbit M-type phospholipase A2receptor antibodies. There are, therefore, two immunologically distinct secretory phospholipase A2 receptors of about 200 kDa in the same species. Although the liver receptor is related to the M-type secretory phospholipase A2 receptors, the brain receptor is not and belongs to a novel group of secretory phospholipase A2 receptors.

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.

The neurotoxic phospholipase A2 associates, through a non-phosphorylated binding motif, with 14-3-3 protein γ and ε isoforms

Two novel acceptors for ammodytoxin C, a presynaptically neurotoxic phospholipase A2 from snake venom, have been purified from porcine cerebral cortex by a toxin-affinity-based procedure. Usingtandem mass spectrometry, the isolated acceptors were identified as 14-3-3c and e isoforms, highly conserved cytoplasmic proteins involved in the regulation of numerous physiological processes. The interaction between ammodytoxin C and 14-3-3 proteins is direct and not mediated by calmodulin, a high-affinity acceptor for both ammodytoxin C and 14-3-3 proteins, as demonstrated in pull-down experiments and by surface plasmon resonance. The latter technique gave an apparent dissociation constant of 1:0 � 0:2lM for the interaction between chip-immobilized 143-3 and ammodytoxin C. 14-3-3 usually interacts with proteins through specific phospho-Ser/Thr motifs. Ammodytoxin C is not a phospho-protein, therefore the interaction must occur through a non-phosphorylated binding site, most probably the KEESEK sequence at its C-terminal end. The interaction we describe suggests an explanation for the pathophysiological effects evoked by some secreted phospholipases A2, such as the inhibition of protein phosphorylation, of terminal ion currents, and of neurotransmission, as well as the initiation of neuronal cell death, all processes regulated by 14-3-3 proteins. 2003 Elsevier Science (USA). All rights reserved.

Purification and characterisation of two hemorrhagic metalloproteinases from the venom of the long-nosed viper, Vipera ammodytes ammodytes

Two hemorrhagic proteins, VaH1 and VaH2, have been purified from Vipera ammodytes ammodytes venom. They are monomeric glycoproteins of an apparent molecular mass of 70kDa and multiple isoelectric points around pH 5.5. Both molecules are proteolytically active against azocasein as substrate. VaH1, which was characterised in detail, showed maximum activity at pH 7.5. Ethylenediaminetetraacetic acid eliminated the proteolytic as well as the hemorrhagic activity of VaH1 while iodoacetamide, phenylmethylsulfonyl fluoride and pepstatin A, inhibitors of cysteine, serine and aspartic proteinases respectively, had no effect. VaH1 is therefore a metalloproteinase whose hemorrhagic activity is very likely the result of its proteolytic activity. VaH1 is a fibrinogenase, hydrolysing exclusively the Aalpha-chain of fibrinogen. In the B-chain of insulin it cleaved with a high preference the bond between Ala(14) and Leu(15). Based on its molecular mass, VaH1 (as well as VaH2) is a Class P-III metalloproteinase. Partial amino acid sequences of its CNBr fragments demonstrated a high level of identity with the reprolysin subfamily of zinc-metalloproteinases.