Biomedical subjects

B Selisko

Publications and source records attributed to B Selisko.

8 recordsLinked to original sources

PubMed1999

Antibody BCF2 against scorpion toxin Cn2 from Centuroides noxius Hoffmann: primary structure and three-dimensional model as free Fv fragment and complexed with its antigen.

The antibody BCF2 generated against the mammal-specific toxin Cn2 of the scorpion Centuroides noxius Hoffmann neutralizes the effect of both the toxin and the venom. We cloned and sequenced the genes coding for the Fv fragment of BCF2. A three-dimensional (3D) model of the Fv fragment was generated using a knowledge-based approach. Furthermore, a 3D model of the complex Cn2-BCF2 was built using the nuclear magnetic resonance (NMR) structure of Cn2 and experimental results on a putative epitope region around the N and C termini. The initial complex conformations were submitted to a new refinement procedure of rigid-body energy minimization combined with flexible-side-chain molecular dynamics. The final complex, selected after an extensive evaluation, uses the loop 7-11 as the central part of the epitope. The generated complex allows the following conclusions: 1) the neutralizing capacity of BCF2 toward the venom of C. noxius might rather be caused by the high venom concentration and toxicity of Cn2 than by a broad specificity, 2) the region involved in the binding of Cn2 to the Na(+) channel, should overlap with the employed epitope region, and 3) contact residues SerL91, AsnL92, LeuH50, AspH56, TyrH95, and TyrH98 of BCF2 are candidates for mutations to broaden its specificity. Proteins 1999;37:130-143.

Algorithms↗

PubMed1999

Mapping of an epitope recognized by a neutralizing monoclonal antibody specific to toxin Cn2 from the scorpion Centruroides noxius, using discontinuous synthetic peptides.

The Na+-channel-affecting toxin Cn2 represents the major and one of the most toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown previously to be able to neutralize the toxic effect of Cn2 and of the whole venom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N- and C-terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three-dimensional model of Cn2. The study of peptides of varying length resulted in the identification of segments 5-14 and 56-65 containing residues essential for recognition by BCF2. The peptide (abbreviated SP7) with the highest affinity to BCF2 (IC50 = 5.1 microM) was a synthetic heterodimer comprising the amino acid sequence from position 3-15 (amidated) of Cn2, bridged by disulfide to peptide from position 54-66, acetylated and amidated. Similar affinity was found with peptide SP1 [heterodimer comprising residues 1-14 (amidated) of Cn2, bridged with synthetic peptide 52-66 (acetylated)]. SP1 and SP7 were used to induce anti-peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2 in vitro. The most efficient protection (8.3 microgram Cn2 neutralized per mL of serum) was induced by rabbit anti-SP1 serum.

Amino Acid Sequence↗

PubMed1998

Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium-channel-blocking scorpion toxins.

Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.

Amino Acid Sequence↗

PubMed1997

Isolation, characterization and comparison of a novel crustacean toxin with a mammalian toxin from the venom of the scorpion Centruroides noxius Hoffmann.

A novel crustacean-specific toxin, Cn5, containing 66 amino acid residues was isolated from the venom of the scorpion Centruroides noxius Hoffmann. It is stabilized by four disulfide bridges, formed between Cys12-Cys65, Cys16-Cys41, Cys25-Cys46 and Cys29-Cys48. Toxicity tests revealed that Cn5 is a toxin that affects arthropods but not mammals. However, at high concentrations, Cn5 does displace the mammal-specific toxin Cn2 from rat brain synaptosomes. The concentration of Cn5 that produces half-maximal inhibition (IC50) was estimated to be 100 microM. Sequence comparison of Cn5 with toxin Cn2, a mammal-specific toxin from the same scorpion, showed the presence of two sequence stretches, at positions 30 to 38 and 49 to 58, where the majority of the differences are concentrated. On the three-dimensional structure of Cn5 it is demonstrated that these two sequence stretches form a continuous surface region near the site thought to bind to the sodium channel. We assume that this region might be implicated in determining species specificity.

Amino Acid Sequence↗

PubMed1996

An insect-specific toxin from Centruroides noxius Hoffmann. cDNA, primary structure, three-dimensional model and electrostatic surface potentials in comparison with other toxin variants.

Scorpion toxins acting on sodium channels differ in their specificity. Toxic peptides specific towards mammals and arthropods (insects and/or crustaceans) have been described. Because of the similar three-dimensional fold of these peptides, the molecular base of their specificity is thought to reside in certain differences at the level of amino acid residues especially within or near the binding site of the toxin to the particular ion channel. The cDNA, amino acid sequence and biological activity of an insect-specific toxin, Cn10, from the scorpion Centruroides noxius Hoffmann is reported. The electrostatic potential surface around a three-dimensional model of Cn10 was calculated. It revealed that residues Tyr4, Lys13, Ile18, Leu19, Gly20, Lys43, Leu44, Thr57, Tyr58, Pro59, Thr64 and Cys65, situated at the side of the toxin proposed in the literature to bind to the sodium channel, constitute a positive surface region. Therefore, they may form the site that binds to the channel. Cn10 was included in a comparative analysis of two groups of natural variants, highly similar peptides of the genus Centruroides with specificities towards mammals or arthropods. A number of surface-accessible residues, consistently different between the two groups and situated near the putative binding site, may be of importance for the specificity of the analyzed toxins.

Amino Acid Sequence↗

PubMed1994

Quantitative analysis of polyethylene glycol (PEG) in PEG-modified proteins/cytokines by aqueous two-phase systems.

Covalent attachment of poly(ethylene glycol) (PEG) to proteins produces conjugates with altered/improved physicochemical and biological properties which depend upon the number of PEG chains linked. Quantification of the attached PEG is however not a trivial issue. The partition coefficient, K, of the PEG-protein conjugate in PEG/dextran two-phase systems provides a quantitative measure for the degree of modification. A linear relationship between log K and the number of PEG chains was observed in fractionated PEG-modified-granulocyte-macrophage colony stimulating factor conjugates having 1 to 3 substitutions. Furthermore, in mixtures of PEG-bovine-serum-albumin conjugates with increasing degrees of modification, a linear relationship was found between log K and n, the average substitution. The increment in log K per PEG chain added is protein specific and this suggests that the interactions between the PEG-protein conjugate and the polymers in the phase system are more complex than just a simple affinity of the PEG for the PEG-rich top phase. Increasing the polymer concentration in the phase system produces larger increments in log K per PEG molecule attached and the proportionality between log K and number of PEG molecules is only compromised for conjugates with high degree of substitution when partitioned in biphasic systems of high concentration of polymers.

Chemical Phenomena↗

PubMed1993

Entrapment of dextran in plant cell capsules by reversible change of cell wall permeability.

Vesicular packing material (VP) made of clusters of extracted higher plant cells with the intact framework of their cell wall was used so far for permeation chromatography (vesicle chromatography). The objective of this study was to devise a method to entrap dextran in the vesicles. This can provide a means to entrap biocatalysts and secondly, to create aqueous two-phase systems with a stationary dextran phase for liquid-liquid partition chromatography. Dextran of molecular sizes above the separation limit of the plant cell wall cannot permeate into the intracellular space in aqueous medium. However, in hydrophilic organic solvent/water mixtures, dextran molecules can diffuse into the capsules. The removal of the organic solvent leaves the dextran trapped inside. There was an inverse correlation between the percentage of dextran permeating through the cell wall (Pperm) and the concentration of solvent required for dextran precipitation. The increase of permeability is therefore considered to be caused, to a great extent, by the decrease of the effective size of dextran molecules due to decreased solvation. Pperm was inversely correlated to the dielectric constants and the polarities of the solvents and, in the case of protic solvents, the hydrogen-bond acidities. No correlation was found to the hydrogen-bond basicities.

Cell Membrane Permeability↗

PubMedDate not supplied

Isolation and structure-functional characterization of phage display library-derived mimotopes of noxiustoxin, a neurotoxin of the scorpion Centruroides noxius Hoffmann.

Noxiustoxin (NTX) is a short-chain toxin from the venom of the scorpion Centruroides noxius Hoffmann, whose molecular structure and physiological effects have been characterized in detail, whereas the antigenic properties of this and other K(+) channel-blocking toxins are poorly studied. A monoclonal antibody against NTX, BNTX18, able to inhibit the binding of NTX to rat brain synaptosomes, was used in the present study for selecting immunoreactive peptides, mimotopes, from a 12mer and a 7mer phage library. The peptides were characterized immunologically and used for mapping the epitope on NTX. In total, 75 phage clones carrying 43 different peptides were analyzed of which 42 clones carrying 17 different peptides, twelve 12mer and five 7mer peptides, presented a single consensus motif: Leu(Ile, Val)-Tyr(Phe, Trp, Leu)-Gly-Met(Ala). All but three of the peptides containing this motif were reactive with selected mAb BNTX18 in a dot-blot assay of which eight were clearly positive in ELISA and exhibited in competition-inhibition assay the antibody binding specificity of the NTX epitope recognized by BNTX18. The two most reactive mimotopes injected into mice showed the ability to induce antibodies reacting with NTX, thus, to mimic the epitope of NTX antigenically. Sequence comparison and the analysis of the three-dimensional structure of NTX led to the proposal that residues Glu19-Leu20-Tyr21-Gly22 and the hydrophobic part of the side chain of Lys18 form the C-terminal part of the epitope. Due to the frequent presence of residues Pro, Leu, Thr, Arg, and Gln in the N-terminal part of the mimotopes, corresponding homologous residues in the N-terminal proximity of the partial epitope may be part of an additional more hydrophilic epitope element.

Amino Acid Sequence↗