Biomedical subjects

F Gubensek

Publications and source records attributed to F Gubensek.

At least 37 records · Page 2Linked to original sources

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

Horizontal transfer of non-LTR retrotransposons 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 ancestral snake lineage (Boidae) 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 mya. 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.

Animals↗

PubMed1999

Cysteine-scanning mutagenesis of an eukaryotic pore-forming toxin from sea anemone: topology in lipid membranes.

Equinatoxin II is a cysteineless pore-forming protein from the sea anemone Actinia equina. It readily creates pores in membranes containing sphingomyelin. Its topology when bound in lipid membranes has been studied using cysteine-scanning mutagenesis. At approximately every tenth residue, a cysteine was introduced. Nineteen single cysteine mutants were produced in Escherichia coli and purified. The accessibility of the thiol groups in lipid-embedded cysteine mutants was studied by reaction with biotin maleimide. Most of the mutants were modified, except those with cysteines at positions 105 and 114. Mutants R144C and S160C were modified only at high concentrations of the probe. Similar results were obtained if membrane-bound biotinylated mutants were tested for avidin binding, but in this case three more mutants gave a negative result: S1C, S13C and K43C. Furthermore, mutants S1C, S13C, K20C, K43C and S95C reacted with biotin only after insertion into the lipid, suggesting that they were involved in major conformational changes occurring upon membrane binding. These results were further confirmed by labeling the mutants with acrylodan, a polarity-sensitive fluorescent probe. When labeled mutants were combined with vesicles, the following mutants exhibited blue-shifts, indicating the transfer of acrylodan into a hydrophobic environment: S13C, K20C, S105C, S114C, R120C, R144C and S160C. The overall results suggest that at least two regions are embedded within the lipid membrane: the N-terminal 13-20 region, probably forming an amphiphilic helix, and the tryptophan-rich 105-120 region. Arg144, Ser160 and residues nearby could be involved in making contacts with lipid headgroups. The association with the membrane appears to be unique and different from that of bacterial pore-forming proteins and therefore equinatoxin II may serve as a model for eukaryotic channel-forming toxins.

2-Naphthylamine↗

PubMed1999

Mutational analysis of two stefin A epitopes.

Stefin A, an intracellular inhibitor of cysteine proteinases, is expressed most abundantly in epithelial cells and in cells of lymphatic origin. In order to study its role in normal and pathological conditions we have prepared and characterized monoclonal antibodies against recombinant stefin A. Two high affinity monoclonal antibodies (mAbs) (A22 and C52) were tested for binding to free and papain-complexed stefin A and to a chimeric inhibitor, consisting of 61 amino acid residues of stefin A and 37 carboxy-terminal residues of stefin B. mAb A22 recognized not only free stefin A but also stefin A in complex with papain. The mAbs were further tested for their cross-reactivity against stefin A and B isolated from different mammalian species. On the basis of sequence similarity and tertiary structure of human stefin A we have prepared three mutants - Glu33Lys, Asp61Gly and Asn62Tyr and their reactivity with the mAbs was tested. The binding affinities of mAb A22 for the Asp61Gly and Asn62Tyr mutants were significantly lower, indicating thatthe two amino acids are part of the stefin A epitope recognized by A22. The binding of both mAbs to the mutants Gly4Arg and Gly4Glu was comparable to wild-type stefin A.

Amino Acid Sequence↗

PubMed1998

Positive Darwinian selection in Vipera palaestinae phospholipase A2 genes is unexpectedly limited to the third exon.

The venom of Vipera palaestinae contains a two-component toxin, consisting of an acidic phospholipase A2 (PLA2) and a basic protein. Here we report the cloning and sequence analysis of the complete V. palaestinae PLA2 genes. Since in all Viperidae PLA2 multigene families the 5' and 3' flanking regions are highly conserved, we designed oligonucleotide primers that allow amplification of the whole PLA2 multigene family in a single step. The structural organization of both genes is the same as in the Vipera ammodytes PLA2 multigene family, there being five exons separated by four introns. Comparison of V. palaestinae PLA2 genes with other Viperidae PLA2 genes has shown that the structural organization of the genes and the nucleotide sequence of all introns and flanking regions are highly conserved, whereas the third exon clearly shows a higher number of amino acid replacements, an indication of positive Darwinian selection. The positive Darwinian selection is surprisingly limited to the third exon, in contrast to other Viperidae PLA2 genes, where it is present in all mature protein coding exons.

Amino Acid Sequence↗

PubMed1998

Identification of a new high-affinity binding protein for neurotoxic phospholipases A2.

Ammodytoxin C is a neurotoxic phospholipase A2 which blocks the release of neurotransmitter from the nerve terminal. Using a radioiodinated derivative of the toxin, we located its specific high-affinity binding site in the demyelinated P2 fraction of porcine cerebral cortex (Kd = 15 nM; Bmax = 1.5 pmol/mg membrane protein). In cross-linking experiments on a membrane preparation, 125I-ammodytoxin C labeled a protein of 25 kDa. The formation of a specific adduct was not inhibited by nontoxic phospholipases A2 or even by neurotoxic phospholipases A2 which have practically identical pathophysiological activities to ammodytoxin C: agkistrodotoxin, Oxyuranus scutellatus 2 phospholipase A2, taipoxin, beta-bungarotoxin, notexin, and crotoxin. 125I-ammodytoxin C specific cross-linking was inhibited, however, by mannosylated BSA, suggesting the presence of a carbohydrate-recognition domain in the acceptor structure. According to the pharmacological and structural properties, the ammodytoxin acceptor from porcine cerebral cortex differs from other so far identified as phospholipase A2 acceptors and represents a new type of a high-affinity binding protein for neurotoxic phospholipases A2.

Affinity Labels↗

PubMed1998

Unusual horizontal transfer of a long interspersed nuclear element between distant vertebrate classes.

We have shown previously by Southern blot analysis that Bov-B long interspersed nuclear elements (LINEs) are present in different Viperidae snake species. To address the question as to whether Bov-B LINEs really have been transmitted horizontally between vertebrate classes, the analysis has been extended to a larger number of vertebrate, invertebrate, and plant species. In this paper, the evolutionary origin of Bov-B LINEs is shown unequivocally to be in Squamata. The previously proposed horizontal transfer of Bov-B LINEs 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 horizontal transfer of Bov-B LINEs from Squamata to the ancestor of Ruminantia is evident from the genetic distances and discontinuous phylogenetic distribution. The ancestor of Colubroidea snakes is a possible donor of Bov-B LINEs to Ruminantia. The timing of horizontal transfer has been estimated from the distribution of Bov-B LINEs in Ruminantia and the fossil data of Ruminantia to be 40-50 My ago. The phylogenetic relationships of Bov-B LINEs from the various Squamata species agrees with that of the species phylogeny, suggesting that Bov-B LINEs have been maintained stably by vertical transmission since the origin of Squamata in the Mesozoic era.

Animals↗

PubMed1998

Ammodytin L, an inactive phospholipase A2 homologue with myotoxicity in mice, binds to the presynaptic acceptor of the beta-neurotoxic ammodytoxin C in Torpedo: an indication for a phospholipase A2 activity-independent mechanism of action of beta-neurotoxins in fish?

A Ser48 phospholipase A2-homologue, ammodytin L, which is myotoxic in mammals and devoid of any phospholipase A2 activity, completely inhibits the specific binding of the neurotoxic phospholipase A2, ammodytoxin C, to fish presynaptic membranes from Torpedo marmorata electric organ. In cross-linking experiments, 125I-ammodytin L labels the same membrane proteins as 125I-ammodytoxin C (70, 38.5-57.4 and 19.7 kDa). The formation of these adducts is completely prevented by the presence of ammodytoxin C but not of a non-toxic phospholipase A2, ammodytin I2. A chimeric phospholipase A2, constructed by associating the N-terminal half of ammodytoxin to the C-terminal half of ammodytin L, possesses a low, but significant phospholipase A2 activity, however it is not toxic to mice, probably due to abolition of the specific neuronal acceptor binding in mammals. Nevertheless, the chimeric phospholipase A2 is able to interact with the ammodytoxin acceptor in Torpedo marmorata electric organ. The existence of neuronal acceptors for ammodytin L and for the chimeric phospholipase A2 suggests that they may act as neurotoxins in fish. As ammodytin L does not possess any enzymatic activity it, therefore, appears to be an excellent tool to investigate the mechanism of action of beta-neurotoxins independently of their phospholipase A2 activity.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1997

Bov-B long interspersed repeated DNA (LINE) sequences are present in Vipera ammodytes phospholipase A2 genes and in genomes of Viperidae snakes.

Ammodytin L is a myotoxic Ser49 phospholipase A2 (PLA2) homologue, which is tissue-specifically expressed in the venom glands of Vipera ammodytes. The complete DNA sequence of the gene and its 5' and 3' flanking regions has been determined. The gene consists of five exons separated by four introns. Comparative analysis of the ammodytin L and ammodytoxin C genes shows that all intron and flanking sequences are considerably more conserved (93-97%) than the mature protein-coding exons. The pattern of nucleotide substitutions in protein-coding exons is not random but occurs preferentially on the first and the second positions of codons, which suggests positive Darwinian evolution for a new function. An Ruminantia specific ART-2 retroposon, recently recognised as a 5'-truncated Bov-B long interspersed repeated DNA (LINE) sequence, was identified in the fourth intron of both genes. This result suggests that ammodytin L and ammodytoxin C genes are derived by duplication of a common ancestral gene. The phylogenetic distribution of Bov-B LINE among vertebrate classes shows that, besides the Ruminantia, it is limited to Viperidae snakes (Vipera 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 Vipera ammodytes genome is between 62,000 and 75,000. The absence of Bov-B LINE at orthologous positions in other snake PLA2 genes indicates that its retrotransposition in the V. ammodytes PLA2 gene locus has occurred quite recently, about 5 My 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 may be a valuable phylogenetic marker. The neighbor-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.

Animals↗

PubMed1997

Neurotoxic phospholipases A2 ammodytoxin and crotoxin bind to distinct high-affinity protein acceptors in Torpedo marmorata electric organ.

We studied the binding of radioiodinated ammodytoxin C, a monomeric phospholipase A2 neurotoxin from Vipera ammodytes, and of radioiodinated crotoxin, a dimeric phospholipase A2 neurotoxin from Crotalus durissus terrificus, to presynaptic membranes from the electric organ of Torpedo marmorata. In both cases, two different families of specific binding sites were identified and characterized. The high-affinity binding sites for both toxins have been shown to be proteins. The low-affinity binding sites were not affected by proteinases or heat, suggesting the involvement of certain lipid structures in this type of binding. By affinity-labeling, [125I]ammodytoxin C was shown to be associated predominantly with membrane proteins of apparent molecular masses of 70,000 and 20,000 Da and to a lesser extent with several proteins of apparent molecular masses ranging between 39,000 and 57,000 Da. [125I]crotoxin, on the other hand bound primarily to a 48,000 Da membrane protein. All phospholipases A2 tested, except beta-bungarotoxin, inhibited the low-affinity specific binding of ammodytoxin C, whereas only neurotoxic phospholipases A2 prevented the high-affinity binding and the cross-linking of ammodytoxin C and crotoxin. The inhibition profiles of high-affinity binding for [125I]crotoxin and for [125I]ammodytoxin C were quite different. Ammodytoxin C and crotoxin did not inhibit each other on their respective high-affinity binding sites. These observations indicate that at least high-affinity binding sites of these two toxins are different. In contrast with crotoxin, the isolated basic subunit CB of crotoxin was able to completely inhibit the high-affinity binding of [125I]ammodytoxin C. Therefore, the acidic subunit CA of crotoxin does not simply act as a chaperone for CB subunit, but it also confers a distinct binding specificity to the crotoxin.

Affinity Labels↗

PubMed1997

N-terminal truncation mutagenesis of equinatoxin II, a pore-forming protein from the sea anemone Actinia equina.

The role of the N-terminal segment 1-33 of equinatoxin II, a 20 kDa pore-forming protein from the sea anemone Actinia equina, was studied by N-truncation mutagenesis. A part of this segment was classified as being amphiphilic and membrane seeking. Wild-type equinatoxin II and its mutants lacking 5, 10 and 33 amino acid residues, respectively, were produced in Escherichia coli using T7 RNA polymerase-based expression vector. Soluble recombinant proteins were isolated from bacterial lysates and assayed for their inhibition by sphingomyelin, binding to red blood cells and hemolytic activity. The N-terminal deletion of 33 amino acids resulted in an insoluble protein, while mutants lacking 5 and 10 residues expressed increased relative avidity for sphingomyelin and red blood cell membranes. Their specific hemolytic activity was decreased, however, with increasing truncation. The results suggest that the N-terminus, which has been found to be conserved in sea anemone pore-forming toxins, contributes to the solubility of the equinatoxin II, but it is not essential for binding to lipid membranes. It is very likely that the N-terminus play a role in the formation of functional pores.

Amino Acid Sequence↗

PubMed1996

Protein and cDNA structures of an acidic phospholipase A2, the enzymatic part of an unusual, two-component toxin from Vipera palaestinae.

In the venom of Vipera palaestinae an unusual, two-component toxin was found. The two components of the toxin are an acidic phospholipase A2 (VpaPLA2) and a basic protein, both with an apparent molecular mass of about 15 kDa. Each component alone is not toxic; however, their mixture is lethal. We have determined the amino acid and cDNA sequences of VpaPLA2. The protein primary structure was solved by sequencing the peptides generated by chemical cleavage of the molecule using CNBr, formic acid and hydroxylamine-hydrochloride and by enzymatic fragmentation with trypsin and chymotrypsin. VpaPLA2 consists of 122 amino acid residues and has all the structural characteristics of subgroup IIA PLA2s. It shows the highest amino acid similarity to a non-toxic phospholipase A2 from Eristocophis macmahoni (82%), whereas the most similar toxic phospholipases A2 share about 70% of residues with VpaPLA2. The substitution of His20 for a hydrophobic residue (Leu) in VpaPLA2 might be one of the reasons that its complex with the basic protein could not be observed.

Amino Acid Sequence↗

PubMed1996

Ammodytoxin C gene helps to elucidate the irregular structure of Crotalinae group II phospholipase A2 genes.

Ammodytoxin C is a presynaptically neurotoxic phospholipase A2 (PLA2) expressed in the venom glands of Vipera ammodytes (subfamily Viperinae). The gene spans more than 4 kb and consists of five exons and four introns characteristic of group II phospholipase A2 genes. The first exon encodes the 5' untranslated region, the second exon encodes most of the signal peptide, while exons 3-5 encode three parts of the mature protein. Comparison of the Crotalinae and Viperinae PLA2 genes has shown that Crotalinae PLA2 retain the first intron in their mRNAs. The apparent cause of this retention is a deletion of 40 bp in the first exon of PLA2 genes of the subfamily Crotalinae, which prevents splicing of the first intron. Analysis of the secondary structure of the pre-mRNA of the ammodytoxin C gene has shown that the first exon is able to form an intra-exon hairpin which is absent in Crotalinae PLA2 pre-mRNAs. Our results indicate that this intra-exon hairpin structure is essential for the splicing of the retained first intron. Contrary to the predictions of the neutral theory of molecular evolution, the introns of all known snake venom PLA2 genes are conserved up to 90%, that is considerably more than the exons. Consequently it is proposed that highly conserved introns, in multigene families, which evolve under positive Darwinian selection, may have an important role in enabling homologous recombination.

Amino Acid Sequence↗

PubMed1996

Cloning, sequencing, and expression of equinatoxin II.

Equinatoxin II (EqtII), a basic protein of 179 amino acids lacking cysteine residues, is the most abundant cytolysin isolated from the sea anemone Actinia equina. Its mode of action is still poorly understood. In order to initiate further structure-function studies by protein engineering, cDNA library was prepared from the whole animal and hybridized with a PCR-derived probe, deduced from the EqtII primary structure. The longest positive clone of 899 bp was shown to encode a 214 residue precursor of EqtII. The mature protein region was amplified by PCR, cloned into a T7 RNA polymerase-based expression vector and expressed in Escherichia coli. Recombinant toxin was isolated by a simple, two-step isolation procedure including separation on CM-cellulose and gel filtration using an FPLC system. Its biochemical properties and hemolytic activity were practically indistinguishable from those of native toxin.

Amino Acid Sequence↗

PubMed1996

Re-examination of crotoxin-membrane interactions.

The interaction of crotoxin with synaptic membranes from Torpedo marmorata has been re-examined, using radioiodinated toxin. In competition experiments, the 'saturable binding' is usually calculated by subtracting the non-saturable binding, determined in the presence of an excess of unlabelled crotoxin, from total binding. Paradoxically, we observed a notable increase of the 'saturable binding' of 125I-crotoxin, defined in this manner in the presence of a high concentration of certain competitors (e.g. crotoxin subunit B, agkistrodotoxin, ammodytoxin and ammodytin I2). This potentiation effect was analysed by competition and cross-linking experiments. The dissociation of the basic crotoxin subunit CB, which differs markedly from crotoxin in its membrane-binding characteristics, was found to be the main reason for the observed phenomenon. 125I-CB could be released from 125I-crotoxin by exchange with a molecule having sufficient affinity towards CA (e.g. CB or agkistrodotoxin) or it could be dissociated from the specific crotoxin membrane-binding site(s) by a competitor molecule. Our results, therefore, suggest a reinterpretation of previous 125I-crotoxin binding studies.

Animals↗

PubMed1995

Autocatalytic acylation of phospholipase-like myotoxins.

Several snake venoms contain a phospholipase A2 in which position 49 in the active site is occupied by a lysine or a serine instead of the aspartate residue normally found. Although these proteins do not bind Ca2+ and are devoid of catalytic activity, they are still highly specific myotoxins and have recently been shown to induce membrane leakage by a new type of cytolytic mechanism. Three of these toxins, myotoxin II from Bothrops asper, ammodytin L from Vipera ammodytes, and the K49 protein from Agkistrodon piscivorus piscivorus, were examined for their interaction with fatty acids and were found to bind long-chain fatty acids covalently by a rapid, spontaneous, autocatalytic process. The fatty acids could be released by treatment with 1 M NH2OH or NaOH, but not with 1 M NaCl or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electron spin resonance studies using spin-labeled fatty acids showed that only the carboxyl headgroup of the fatty acid was linked to protein amino acid, the carbon chain had free mobility and did not bind tightly to the protein surface. Stearic acid methyl esters and short-chain fatty acids did not bind to the toxins. Acylated myotoxins bound to the surface of liposomes and isolated muscle membranes, with the fatty acid moiety inserted into the lipid bilayer and possibly acting as an anchor. The phospholipase-like myotoxins represent the first group of proteins able to undergo acylation by spontaneous reaction with free fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Acylation↗

PubMed1995

Ammodytoxin A acceptor in bovine brain synaptic membranes.

Ammodytoxin A, the presynaptic neurotoxin from Vipera ammodytes ammodytes venom, was found to bind specifically and with high affinity to bovine cortex synaptic membrane preparation. The detected ammodytoxin A high-affinity binding was characterized by equilibrium binding analysis which revealed a single high-affinity binding site with Kd 4.13 nM and Bmax 6.67 pmoles/mg of membrane protein. 125I-ammodytoxin A was covalently cross-linked to its neuronal acceptor using a chemical cross-linking technique. As revealed by subsequent SDS-PAGE analysis and autoradiography, 125I-ammodytoxin A specifically attached to membrane components with apparent mol. wts 53,000-56,000. Besides by the native ammodytoxin A, the binding of radioiodinated ammodytoxin A to the neuronal acceptor was highly attenuated, also by other two iso-neurotoxins from V. a. ammodytes venom, ammodytoxins B and C, and neurotoxin crotoxin B from the venom of the South American rattlesnake (Crotalus durissus terrificus). Vipera berus berus phospholipase A2 was a weaker inhibitor, whereas nontoxic phospholipase A2, ammodytoxin I2 and myotoxic phospholipase A2 homologue, ammodytin L, both from V. a. ammodytes venom as well, were very weak inhibitors. No inhibitory effect on 125I-ammodytoxin A specific binding at all was, however, obtained with alpha-dendrotoxin, beta-bungarotoxin and crotoxin A, respectively. Treatment of synaptic membranes with proteinase K and Staphylococcus aureus V-8 proteinase, a combination of PNGase F and neuroaminidase, heat or acid lowered the 125I-ammodytoxin A specific binding to various extents but never completely abolished it. The ammodytoxin A binding site in bovine synaptic membranes is thus most likely a combination of membrane glycoprotein acceptor and membrane phospholipids. As ammodytoxin A reduced the second negative component of the perineural waveform, measured on mouse triangularis sterni preparation, which is very likely a result of an inhibition of a fraction of the terminal K+ currents, the ammodytoxin A acceptor could well be connected with K+ channels.

Animals↗