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Alan L. Harvey - One of the best experts on this subject based on the ideXlab platform.
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Chemical synthesis of Dendrotoxin-I: Revision of the reported structure
The journal of peptide research : official journal of the American Peptide Society, 2009Co-Authors: H. Nishio, Alan L. Harvey, Tatsuya Inui, Y. Nishiuchi, C. L. C. De Medeiros, Edward G. Rowan, E. Katoh, T. YamazakiAbstract:Dendrotoxin I (DTX-I) is a 60-residue peptide from the venom of the black mamba snake Dendroaspis polylepis, which binds to neuronal K+ channels. The structure reported previously for DTX-I was synthesized for the first time by a solution procedure. The synthetic product was confirmed to have the correct primary and disulfide structure determined by peptide mapping, sequence analysis and mass measurements. Comparison of synthetic DTX-I with the natural one by high-performance liquid chromatography and capillary zone electrophoresis, as well as by sequence analysis, revealed that the Asn residue at position 12 in the synthetic peptide was Asp in the natural product. Synthesis of DTX-I with Asp at position 12 gave a peptide identical with the natural product in all aspects. NMR analysis of synthetic [Asn12]- and [Asp12]-DTX-I also supported our findings that the Asn residue at position 12 in the DTX-I molecule should be revised as Asp. [Asn12]- and [Asp12]-DTX-I had very similar binding affinities when tested against radiolabeled Dendrotoxin binding to rat brain synaptosomal membranes.
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Dendrotoxins: structure-activity relationships and effects on potassium ion channels
Current Medicinal Chemistry, 2004Co-Authors: Alan L. Harvey, Boyd RobertsonAbstract:Dendrotoxins are small proteins isolated from mamba (Dendroaspis) snakes. The original Dendrotoxin was found in venom of the Eastern green mamba, Dendroaspis angusticeps, and related proteins were subsequently found in other mamba venoms. The Dendrotoxins contain 57-60 amino acid residues crosslinked by three disulphide bridges, and they are homologous to Kunitz-type serine protease inhibitors, such as aprotinin (BPTI). The Dendrotoxins have little or no anti-protease activity, but they block particular subtypes of voltage-dependent potassium channels of the Kv1 subfamily in neurones. alpha-Dendrotoxin from green mamba Dendroaspis angusticeps and toxin I from the black mamba Dendroaspis polylepis block cloned Kv1.1, Kv1.2 and Kv1.6 channels in the low nanomolar range; toxin K, also from the black mamba Dendroaspis polylepis, preferentially blocks Kv1.1 channels and is active at picomolar concentrations. Structural modifications and mutations to Dendrotoxins have helped to define the molecular recognition properties of different types of K+ channels, although more work is needed to characterise the chemical features of the toxins that underlie their selectivity and potency at particular subtypes of channels. Dendrotoxins have been useful markers of subtypes of K+ channels in vivo, and Dendrotoxins have become widely used as probes for studying the function of K+ channels in physiology and pathophysiology. With some pathological conditions being associated with voltage-gated K+ channels, analogues of Dendrotoxins might have therapeutic potential.
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Twenty years of Dendrotoxins.
Toxicon : official journal of the International Society on Toxinology, 2001Co-Authors: Alan L. HarveyAbstract:Dendrotoxins are small proteins that were isolated 20 years ago from mamba (Dendroaspis) snake venoms (Harvey, A.L., Karlsson, E., 1980. Dendrotoxin from the venom of the green mamba, Dendroaspis angusticeps: a neurotoxin that enhances acetylcholine release at neuromuscular junctions. Naunyn-Schmiedebergs Arch. Pharmacol. 312, 1-6.). Subsequently, a family of related proteins was found in mamba venoms and shown to be homologous to Kunitz-type serine protease inhibitors, such as aprotinin. The Dendrotoxins contain 57-60 amino acid residues cross-linked by three disulphide bridges. The Dendrotoxins have little or no anti-protease activity, but they were demonstrated to block particular subtypes of voltage-dependent potassium channels in neurons. Studies with cloned K(+) channels indicate that alpha-Dendrotoxin from green mamba Dendroaspis angusticeps blocks Kv1.1, Kv1.2 and Kv1.6 channels in the nanomolar range, whereas toxin K from the black mamba Dendroaspis polylepis preferentially blocks Kv1.1 channels. Structural analogues of Dendrotoxins have helped to define the molecular recognition properties of different types of K(+) channels, and radiolabelled Dendrotoxins have also been useful in helping to discover toxins from other sources that bind to K(+) channels. Because Dendrotoxins are useful markers of subtypes of K(+) channels in vivo, Dendrotoxins have become widely used as probes for studying the function of K(+) channels in physiology and pathophysiology.
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delineation of the functional site of alpha Dendrotoxin the functional topographies of Dendrotoxins are different but share a conserved core with those of other kv1 potassium channel blocking toxins
Journal of Biological Chemistry, 1998Co-Authors: Sylvaine Gasparini, Suzanne Pinkasfeld, Alan L. Harvey, E G Rowan, Jeanmarc Danse, Alain Lecoq, Sophie Zinnjustin, Louise C Young, Cleane C L De Medeiros, Andre MenezAbstract:Abstract We identified the residues that are important for the binding of α-Dendrotoxin (αDTX) to Kv1 potassium channels on rat brain synaptosomal membranes, using a mutational approach based on site-directed mutagenesis and chemical synthesis. Twenty-six of its 59 residues were individually substituted by alanine. Substitutions of Lys5 and Leu9 decreased affinity more than 1000-fold, and substitutions of Arg3, Arg4, Leu6, and Ile8 by 5–30-fold. Substitution of Lys5 by norleucine or ornithine also greatly altered the binding properties of αDTX. All of these analogs displayed similar circular dichroism spectra as compared with the wild-type αDTX, indicating that none of these substitutions affect the overall conformation of the toxin. Substitutions of Ser38 and Arg46 also reduced the affinity of the toxin but, in addition, modified its dichroic properties, suggesting that these two residues play a structural role. The other residues were excluded from the recognition site because their substitutions caused no significant affinity change. Thus, the functional site of αDTX includes six major binding residues, all located in its N-terminal region, with Lys5 and Leu9 being the most important. Comparison of the functional site of αDTX with that of DTX-K, another Dendrotoxin (Smith, L. A., Reid, P. F., Wang, F. C., Parcej, D. N., Schmidt, J. J., Olson, M. A., and Dolly, J. O. (1997)Biochemistry 36, 7690–7696), reveals that they only share the predominant lysine and probably a leucine residue; the additional functional residues differ from one toxin to the other. Comparison of the functional site of αDTX with those of structurally unrelated potassium channel-blocking toxins from venomous invertebrates revealed the common presence of a protruding key lysine with a close important hydrophobic residue (Leu, Tyr, or Phe) and few additional residues. Therefore, irrespective of their phylogenetic origin, all of these toxins may have undergone a functional convergence. The functional site of αDTX is topographically unrelated to the “antiprotease site” of the structurally analogous bovine pancreatic trypsin inhibitor.
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changes to biological activity following acetylation of Dendrotoxin i from dendroaspis polylepis black mamba
Toxicon, 1997Co-Authors: Alan L. Harvey, Ake Engstrom, E G Rowan, Hossein Vatanpour, Bengt WesterlundAbstract:The potassium channel blocker Dendrotoxin I was acetylated with acetic anhydride. Mono-acetyl derivatives of all seven lysine residues (N-terminus blocked) and a di-derivative were isolated by chromatography on the cation-exchanger Bio-Rex 70 and reversed-phase high-performance liquid chromatography. The derivative acetyl-Lys 29 and the di-derivative of Tyr 24 and Lys 28 had more than 1000 times lower affinity than the native toxin as determined by inhibition of the 125I-Dendrotoxin binding to synaptosomal membranes from rat brain. Lys 29 is part of the triplet Lys-Lys-Lys (28-30) which also occurs in the homologous alpha-Dendrotoxin where the triplet is not in the functional site, as shown by site-directed mutagenesis. Acetylation of Lys 29 may have produced large structural perturbations that inactivated the toxin. Acetylation of Lys 28 alone had little effect, but the toxin became almost inactive when both Lys 28 and Tyr 24 were modified. Ten experiments were conducted under similar conditions, but a derivative of Tyr 24 was obtained only three times. In these cases the toxin apparently had a different structure, with Tyr 24 accessible to the reagent. This may depend on freeze-drying, which can alter the structure of proteins. The third derivative with low activity was acetyl-Lys 5, with affinity decreased 20-fold. Lys 5 has a protruding side-chain that does not interact with any other group in the toxin molecule. Therefore, Lys 5 is probably part of the functional site for Dendrotoxin's binding to the voltage-dependent K+ channels.
Alice M. Luther - One of the best experts on this subject based on the ideXlab platform.
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In vivo neutralization of Dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.
Nature communications, 2018Co-Authors: Andreas H. Laustsen, Aneesh Karatt-vellatt, Edward W. Masters, Ana Silvia Arias, Cecilie Knudsen, Saioa Oscoz, Peter Slavny, Daniel T. Griffiths, Urska Pus, Alice M. LutherAbstract:The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of Dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing Dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.
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In vivo neutralization of Dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies
Nature Publishing Group, 2018Co-Authors: Andreas H. Laustsen, Aneesh Karatt-vellatt, Edward W. Masters, Ana Silvia Arias, Cecilie Knudsen, Saioa Oscoz, Peter Slavny, Daniel T. Griffiths, Urska Pus, Alice M. LutherAbstract:Current anti-venoms against black mamba (Dendroaspis polylepis) bites are animal-derived and associated with several limitations. Here, Laustsen and colleagues develop an experimental recombinant anti-venom based on oligoclonal human IgG antibodies and establish its potential protective value in neutralizing Dendrotoxin-mediated neurotoxicity using venom challenge in vivo models
Andreas H. Laustsen - One of the best experts on this subject based on the ideXlab platform.
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In vivo neutralization of Dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies.
Nature communications, 2018Co-Authors: Andreas H. Laustsen, Aneesh Karatt-vellatt, Edward W. Masters, Ana Silvia Arias, Cecilie Knudsen, Saioa Oscoz, Peter Slavny, Daniel T. Griffiths, Urska Pus, Alice M. LutherAbstract:The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of Dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing Dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.
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In vivo neutralization of Dendrotoxin-mediated neurotoxicity of black mamba venom by oligoclonal human IgG antibodies
Nature Publishing Group, 2018Co-Authors: Andreas H. Laustsen, Aneesh Karatt-vellatt, Edward W. Masters, Ana Silvia Arias, Cecilie Knudsen, Saioa Oscoz, Peter Slavny, Daniel T. Griffiths, Urska Pus, Alice M. LutherAbstract:Current anti-venoms against black mamba (Dendroaspis polylepis) bites are animal-derived and associated with several limitations. Here, Laustsen and colleagues develop an experimental recombinant anti-venom based on oligoclonal human IgG antibodies and establish its potential protective value in neutralizing Dendrotoxin-mediated neurotoxicity using venom challenge in vivo models
Kurt Wüthrich - One of the best experts on this subject based on the ideXlab platform.
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Nuclear Magnetic Resonance Solution Structure of Dendrotoxin K from the Venom of Dendroaspis polylepis polylepis
Journal of molecular biology, 1993Co-Authors: Kurt D. Berndt, Peter Güntert, Kurt WüthrichAbstract:Nuclear-Magnetic-Resonance Solution Structure of Dendrotoxin-K from the Venom of Dendroaspis-Polylepis-Polylepis
Ake Engstrom - One of the best experts on this subject based on the ideXlab platform.
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changes to biological activity following acetylation of Dendrotoxin i from dendroaspis polylepis black mamba
Toxicon, 1997Co-Authors: Alan L. Harvey, Ake Engstrom, E G Rowan, Hossein Vatanpour, Bengt WesterlundAbstract:The potassium channel blocker Dendrotoxin I was acetylated with acetic anhydride. Mono-acetyl derivatives of all seven lysine residues (N-terminus blocked) and a di-derivative were isolated by chromatography on the cation-exchanger Bio-Rex 70 and reversed-phase high-performance liquid chromatography. The derivative acetyl-Lys 29 and the di-derivative of Tyr 24 and Lys 28 had more than 1000 times lower affinity than the native toxin as determined by inhibition of the 125I-Dendrotoxin binding to synaptosomal membranes from rat brain. Lys 29 is part of the triplet Lys-Lys-Lys (28-30) which also occurs in the homologous alpha-Dendrotoxin where the triplet is not in the functional site, as shown by site-directed mutagenesis. Acetylation of Lys 29 may have produced large structural perturbations that inactivated the toxin. Acetylation of Lys 28 alone had little effect, but the toxin became almost inactive when both Lys 28 and Tyr 24 were modified. Ten experiments were conducted under similar conditions, but a derivative of Tyr 24 was obtained only three times. In these cases the toxin apparently had a different structure, with Tyr 24 accessible to the reagent. This may depend on freeze-drying, which can alter the structure of proteins. The third derivative with low activity was acetyl-Lys 5, with affinity decreased 20-fold. Lys 5 has a protruding side-chain that does not interact with any other group in the toxin molecule. Therefore, Lys 5 is probably part of the functional site for Dendrotoxin's binding to the voltage-dependent K+ channels.
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characterization of a potassium channel toxin from the caribbean sea anemone stichodactyla helianthus
Toxicon, 1995Co-Authors: Olga Castaneda, Alan L. Harvey, Vivian Sotolongo, Ana Maria Amor, Reto Stocklin, A J Anderson, Ake Engstrom, Christer Wernstedt, Evert KarlssonAbstract:A peptide toxin, ShK, that blocks voltage-dependent potassium channels was isolated from the whole body extract of the Caribbean sea anemone Stichodactyla helianthus. It competes with Dendrotoxin I and alpha-Dendrotoxin for binding to synaptosomal membranes of rat brain, facilities acetylcholine release at an avian neuromuscular junction and suppresses K+ currents in rat dorsal root ganglion neurones in culture. Its amino acid sequence is R1SCIDTIPKS10RCTAFQCKHS20MKYRLSFCRK30TCGTC35. There is no homology with other K+ channel-blocking peptides, except for BgK from the sea anemone Bunodosoma granulifera. ShK and BgK appear to be in a different structural class from other toxins affecting K+ channels.
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a potassium channel toxin from the secretion of the sea anemone bunodosoma granulifera isolation amino acid sequence and biological activity
Biochimica et Biophysica Acta, 1993Co-Authors: Abel Aneiros, David L Marshall, Alan L. Harvey, A J Anderson, Ake Engstrom, Ileana Garcia, Joser Martinez, Ulf Hellman, Evert KarlssonAbstract:A peptide toxin affecting potassium channels was isolated from the sea anemone Bunodosoma granulifera. It facilitates acetylcholine release at avian neuromuscular junctions, competes with Dendrotoxin I, a probe for voltage-dependent potassium channels, for binding to synaptosomal membranes of rat brain with a Ki of 0.7 nM and suppresses K+ currents in rat dorsal root ganglion neurones in culture. It represents a new structural type of potassium channel toxin with the sequence V1RCDWFKETA10CRHAKSLGNC20RTSQKYRANC30AKTLQCC37 (M(r) 4275, three disulfides).