T. V. Andreeva

Naturally Occurring Disulfide-bound Dimers of Three-fingered Toxins A PARADIGM FOR BIOLOGICAL ACTIVITY DIVERSIFICATION

Disulfide-bound dimers of three-fingered toxins have been discovered in the Naja kaouthia cobra venom; that is, the homodimer of α-cobratoxin (a long-chain α-neurotoxin) and heterodimers formed by α-cobratoxin with different cytotoxins. According to circular dichroism measurements, toxins in dimers retain in general their three-fingered folding. The functionally important disulfide 26–30 in polypeptide loop II of α-cobratoxin moiety remains intact in both types of dimers. Biological activity studies showed that cytotoxins within dimers completely lose their cytotoxicity. However, the dimers retain most of the α-cobratoxin capacity to compete with α-bungarotoxin for binding to Torpedo and α7 nicotinic acetylcholine receptors (nAChRs) as well as to Lymnea stagnalis acetylcholine-binding protein. Electrophysiological experiments on neuronal nAChRs expressed in Xenopus oocytes have shown that α-cobratoxin dimer not only interacts with α7 nAChR but, in contrast to α-cobratoxin monomer, also blocks α3β2 nAChR. In the latter activity it resembles κ-bungarotoxin, a dimer with no disulfides between monomers. These results demonstrate that dimerization is essential for the interaction of three-fingered neurotoxins with heteromeric α3β2 nAChRs.

Azemiopsin from Azemiops feae Viper Venom, a Novel Polypeptide Ligand of Nicotinic Acetylcholine Receptor

Background: Venoms from rare snake species may contain toxins of new structural or/and pharmacological types. Results: Amino acid sequence of the new polypeptide azemiopsin isolated from Azemiops feae viper venom was established, and its biological activity was determined. Conclusion: Azemiopsin is the first natural toxin that blocks nicotinic acetylcholine receptors and does not contain disulfide bridges. Significance: Azemiopsin is the first member of a new toxin group. Azemiopsin, a novel polypeptide, was isolated from the Azemiops feae viper venom by combination of gel filtration and reverse-phase HPLC. Its amino acid sequence (DNWWPKPPHQGPRPPRPRPKP) was determined by means of Edman degradation and mass spectrometry. It consists of 21 residues and, unlike similar venom isolates, does not contain cysteine residues. According to circular dichroism measurements, this peptide adopts a β-structure. Peptide synthesis was used to verify the determined sequence and to prepare peptide in sufficient amounts to study its biological activity. Azemiopsin efficiently competed with α-bungarotoxin for binding to Torpedo nicotinic acetylcholine receptor (nAChR) (IC50 0.18 ± 0.03 μm) and with lower efficiency to human α7 nAChR (IC50 22 ± 2 μm). It dose-dependently blocked acetylcholine-induced currents in Xenopus oocytes heterologously expressing human muscle-type nAChR and was more potent against the adult form (α1β1ϵδ) than the fetal form (α1β1γδ), EC50 being 0.44 ± 0.1 μm and 1.56 ± 0.37 μm, respectively. The peptide had no effect on GABAA (α1β3γ2 or α2β3γ2) receptors at a concentration up to 100 μm or on 5-HT3 receptors at a concentration up to 10 μm. Ala scanning showed that amino acid residues at positions 3–6, 8–11, and 13–14 are essential for binding to Torpedo nAChR. In biological activity azemiopsin resembles waglerin, a disulfide-containing peptide from the Tropidechis wagleri venom, shares with it a homologous C-terminal hexapeptide, but is the first natural toxin that blocks nAChRs and does not possess disulfide bridges.

An unusual phospholipase A2 from puff adder Bitis arietans venom – a novel blocker of nicotinic acetylcholine receptors

The venoms of snakes from Viperidae family mainly influence the function of various blood components. However, the published data indicate that these venoms contain also neuroactive components, the most studied being neurotoxic phospholipases A₂ (PLA₂s). Earlier we have shown (Gorbacheva et al., 2008) that several Viperidae venoms blocked nicotinic acetylcholine receptors (nAChRs) and voltage-gated Ca²+ channels in isolated identified neurons of the fresh-water snail Lymnaea stagnalis. In this paper, we report on isolation from puff adder Bitis arietans venom and characterization of a novel protein bitanarin that reversibly blocks nAChRs. To isolate the protein, the venom of B. arietans was fractionated by gel-filtration, ion-exchange and reversed phase chromatography and fractions obtained were screened for capability to block nAChRs. The isolated protein competed with [¹²⁵I]iodinated α-bungarotoxin for binding to human α7 and Torpedo californica nAChRs, as well as to acetylcholine-binding protein from L. stagnalis, the IC₅₀ being 20 ± 1.5, 4.3 ± 0.2, and 10.6 ± 0.6 μM, respectively. It also blocked reversibly acetylcholine-elicited current in isolated L. stagnalis neurons with IC₅₀ of 11.4 μM. Mass-spectrometry analysis determined the molecular mass of 27.4 kDa and the presence of 28 cysteine residues forming 14 disulphide bonds. Edman degradation of the protein and tryptic fragments showed its similarity to PLA₂s from snake venoms. Indeed, the protein possessed high PLA₂ activity, which was 1.95 mmol/min/μmol. Bitanarin is the first described PLA₂ that contains 14 disulphide bonds and the first nAChR blocker possessing PLA₂ activity.

Dimeric α-Cobratoxin X-ray Structure LOCALIZATION OF INTERMOLECULAR DISULFIDES AND POSSIBLE MODE OF BINDING TO NICOTINIC ACETYLCHOLINE RECEPTORS

Background: α-Cobratoxin (αCT) dimer (αCT-αCT) has recently been discovered and found to bind both with α7 and α3β2 nicotinic receptors (nAChR). Results: αCT-αCT x-ray structure and intermolecular disulfides were established, intramolecular disulfides in central loops II were reduced, and interaction with distinct nAChRs was analyzed. Conclusion: Loop II disulfide is necessary for αCT-αCT binding to α7 but not α3β2 nAChR. Significance: Dimeric α-neurotoxins provide new means for distinguishing distinct nAChRs. In Naja kaouthia cobra venom, we have earlier discovered a covalent dimeric form of α-cobratoxin (αCT-αCT) with two intermolecular disulfides, but we could not determine their positions. Here, we report the αCT-αCT crystal structure at 1.94 Å where intermolecular disulfides are identified between Cys3 in one protomer and Cys20 of the second, and vice versa. All remaining intramolecular disulfides, including the additional bridge between Cys26 and Cys30 in the central loops II, have the same positions as in monomeric α-cobratoxin. The three-finger fold is essentially preserved in each protomer, but the arrangement of the αCT-αCT dimer differs from those of noncovalent crystallographic dimers of three-finger toxins (TFT) or from the κ-bungarotoxin solution structure. Selective reduction of Cys26-Cys30 in one protomer does not affect the activity against the α7 nicotinic acetylcholine receptor (nAChR), whereas its reduction in both protomers almost prevents α7 nAChR recognition. On the contrary, reduction of one or both Cys26-Cys30 disulfides in αCT-αCT considerably potentiates inhibition of the α3β2 nAChR by the toxin. The heteromeric dimer of α-cobratoxin and cytotoxin has an activity similar to that of αCT-αCT against the α7 nAChR and is more active against α3β2 nAChRs. Our results demonstrate that at least one Cys26-Cys30 disulfide in covalent TFT dimers, similar to the monomeric TFTs, is essential for their recognition by α7 nAChR, although it is less important for interaction of covalent TFT dimers with the α3β2 nAChR.

Inhibition of nicotinic acetylcholine receptors, a novel facet in the pleiotropic activities of snake venom phospholipases A2.

Phospholipases A2 represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A2, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A2 from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A2 content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A2, which add a new target to the numerous activities of these enzymes.

Phospholipases a2 from Viperidae snakes: Differences in membranotropic activity between enzymatically active toxin and its inactive isoforms

We describe the interaction of various phospholipases A2 (PLA2) from snake venoms of the family Viperidae (Macrovipera lebetina obtusa, Vipera ursinii renardi, Bothrops asper) with giant unilamellar vesicles (GUVs) composed of natural brain phospholipids mixture, visualized through fluorescence microscopy. The membrane fluorescent probes 8-anilino-1-naphthalenesulfonicacid (ANS), LAUDRAN and PRODAN were used to assess the state of the membrane and specifically mark the lipid packing and membrane fluidity. Our results have shown that the three PLA2s which contain either of aspartic acid, serine, or lysine residues at position 49 in the catalytic center, have different effects on the vesicles. The PLA2 with aspartic acid at this position causes the oval deformation of the vesicles, while serine and lysine-containing enzymes lead to an appreciable increase of fluorescence intensity in the vesicles membrane, wherein the shape and dimensions of GUVs have not changed, but in this case GUV aggregation occurs. LAURDAN and PRODAN detect the extent of water penetration into the bilayer surface. We calculated generalized polarization function (GP), showing that for all cases (D49 PLA2, S49 PLA2 and K49 PLA2) both LAUDRAN and PRODAN GP values decrease. A higher LAURDAN GP is indicative of low water penetration in the lipid bilayer in case of K49 PLA2 compared with D49 PLA2, whereas the PRODAN mainly gives information when lipid is in liquid crystalline phase.

Vietnamese Heterometrus laoticus scorpion venom: Evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel

The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K⁺-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (K(i) 59 nM) to Kv1.3 potassium channel.

Biological basis for amyloidogenesis in Alzheimer’S disease

Certain cellular proteins normally soluble in the living organism under certain conditions form aggregates with a specific cross-β sheet structure called amyloid. These intraor extracellular insoluble aggregates (fibers or plaques) are hallmarks of many neurodegenerative pathologies including Alzheimer’s disease (AD), Huntington’s disease, Parkinson’s disease, prion disease, and other progressive neurological diseases that develop in the aging human central nervous system. Amyloid diseases (amyloidoses) are widespread in the elderly human population, a rapidly expanding demographic in many global populations. Increasing age is the most significant risk factor for neurodegenerative diseases associated with amyloid plaques. To date, nearly three dozen different misfolded proteins targeting brain and other organs have been identified in amyloid diseases and AD, the most prevalent neurodegenerative amyloid disease affecting over 15 million people worldwide. Here we (i) highlight the latest data on mechanisms of amyloid formation and further discuss a hypothesis on the amyloid cascade as a primary mechanism of AD pathogenesis and (ii) review the evolutionary aspects of amyloidosis, which allow new insight on human-specific mechanisms of dementia development.

Whole exome sequencing links dental tumor to an autosomal-dominant mutation in ANO5 gene associated with gnathodiaphyseal dysplasia and muscle dystrophies

Tumors of the jaws may represent different human disorders and frequently associate with pathologic bone fractures. In this report, we analyzed two affected siblings from a family of Russian origin, with a history of dental tumors of the jaws, in correspondence to original clinical diagnosis of cementoma consistent with gigantiform cementoma (GC, OMIM: 137575). Whole exome sequencing revealed the heterozygous missense mutation c.1067G > A (p.Cys356Tyr) in ANO5 gene in these patients. To date, autosomal-dominant mutations have been described in the ANO5 gene for gnathodiaphyseal dysplasia (GDD, OMIM: 166260), and multiple recessive mutations have been described in the gene for muscle dystrophies (OMIM: 613319, 611307); the same amino acid (Cys) at the position 356 is mutated in GDD. These genetic data and similar clinical phenotypes demonstrate that the GC and GDD likely represent the same type of bone pathology. Our data illustrate the significance of mutations in single amino-acid position for particular bone tissue pathology. Modifying role of genetic variations in another gene on the severity of the monogenic trait pathology is also suggested. Finally, we propose the model explaining the tissue-specific manifestation of clinically distant bone and muscle diseases linked to mutations in one gene.

Venoms of kraits Bungarus multicinctus and Bungarus fasciatus contain anticoagulant proteins

53 Kraits (genus Bungarus) belong to the family Elap idae and are spread in South and Southeast Asia. Ven oms of kraits have primarily neurotoxic effects and have been studied in sufficient detail. In particular, different neurotoxins have been identified in them, including α neurotoxins (the most well known of which is α bungarotoxin), κ bungarotoxin, and β bungaro toxin [1]. The first two types of toxins belong to the family of three finger toxins, whereas the latter is a member of the family of heterodimeric phospholi pases A2. In addition, a number of proteins possessing different types of enzymatic activity were isolated. However, data on the effect of venoms of kraits or pro teins from these venoms on the hemostatic system are almost missing. Only when this manuscript was in preparation, a paper reporting the anticoagulant activ ity of β bungarotoxin from the venom of Bungarus fas ciatus was published [2]. To detect other proteins with anticoagulant activity in the venoms of kraits Bungarus fasciatus and Bungarus multicinctus, we analyzed the influence of these venoms on the blood coagulation system using the standard tests.