RESUMEN
A new specific voltage-sensitive calcium channel (VSCC) blocker has been isolated from the venom of the fish-hunting cone snail Conus consors. This peptide, named omega-Ctx CNVIIA, consists of 27 amino acid residues folded by 3 disulfide bridges. Interestingly, loop 4, which is supposed to be crucial for selectivity, shows an unusual sequence (SSSKGR). The synthesis of the linear peptide was performed using the Fmoc strategy, and the correct folding was achieved in the presence of guanidinium chloride, potassium buffer, and reduced/oxidized glutathione at 4 degrees C for 3 days. Both synthetic and native toxin caused an intense shaking activity, characteristic of omega-conotoxins targeting N-type VSCC when injected intracerebroventricularly to mice. Binding studies on rat brain synaptosomes revealed that the radioiodinated omega-Ctx CNVIIA specifically and reversibly binds to high-affinity sites with a K(d) of 36.3 pM. Its binding is competitive with omega-Ctx MVIIA at low concentration (K(i) = 2 pM). Moreover, omega-Ctx CNVIIA exhibits a clear selectivity for N-type VSCCs versus P/Q-type VSCCs targeted respectively by radioiodinated omega-Ctx GVIA and omega-Ctx MVIIC. Although omega-Ctx CNVIIA clearly blocked N-type Ca(2+) current in chromaffin cells, this toxin did not inhibit acetylcholine release evoked by nerve stimuli at the frog neuromuscular junction, in marked contrast to omega-Ctx GVIA. omega-Ctx CNVIIA thus represents a new selective tool for blocking N-type VSCC that displays a unique pharmacological profile and highlights the diversity of voltage-sensitive Ca(2+) channels in the animal kingdom.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , omega-Conotoxinas/farmacología , Secuencia de Aminoácidos , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/síntesis química , Bloqueadores de los Canales de Calcio/aislamiento & purificación , Canales de Calcio Tipo N/metabolismo , Células Cromafines/fisiología , Ciprinodontiformes , Relación Dosis-Respuesta a Droga , Inyecciones Intraventriculares , Masculino , Potenciales de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Músculo Liso/fisiología , Técnicas de Placa-Clamp , Unión Proteica , Ensayo de Unión Radioligante , Rana esculenta , Ratas , Homología de Secuencia de Aminoácido , Caracoles/química , Relación Estructura-Actividad , omega-Conotoxinas/síntesis química , omega-Conotoxinas/aislamiento & purificaciónRESUMEN
The alpha-ImI conotoxin, a selective potent inhibitor of the mammalian neuronal alpha7 nicotinic acetylcholine receptor (n-AchR), was shown by point mutation or by L-alanine scanning to display two regions essential for bioactivity: the active site Asp5-Pro6-Arg7 in the first loop and Trp10 in the second loop. The deletion of the Cys3,Cys12 disulfide bond in the alpha-ImI scaffold, e.g. peptide II, had no effect on its binding affinity. CD spectra, NMR studies and structure calculations were carried out on the wild type alpha-ImI, the weakest analog (R7A) and peptide II (equipotent to alpha-ImI) in order to point out the conformational differences between these compounds. Then, an attempt to correlate the conformational data and the affinity results was proposed. CD and NMR data were identical for the R7A analog and alpha-ImI, revealing the crucial functional role of the Arg7 side chain. On the other hand, the scaffold of the first loop in peptide II was shown by NMR to represent the minimal conformation for the optimal interaction of the toxin with the neuronal alpha7 n-AchR. Last, the beta-turn forming property of the 6th residue (Pro) in the active site of the alpha-ImI can be correlated with its affinity.
Asunto(s)
Conotoxinas , Venenos de Moluscos/metabolismo , Oligopéptidos/química , Oligopéptidos/metabolismo , Conformación Proteica , Receptores Nicotínicos/metabolismo , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutación , Mutación Puntual , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Two novel alpha-conotoxins were purified and characterized from the venom of the fish-hunting cone snail Conus consors. These peptides were identified by screening HPLC fractions of the crude venom and by binding experiments with Torpedo nicotinic acetylcholine receptor. The toxins named alpha-CnIA and alpha-CnIB exhibited sequences of 14 and 12 amino acids, respectively. The alpha-CnIA represents the main alpha-conotoxin contained in the venom, whereas alpha-CnIB is present in a relatively small amount. Chemical synthesis of alpha-CnIA was carried out using the Fmoc methodology by selective disulfide bond formation. The biological activity of the toxin was assessed in fish and mice. The alpha-CnIA inhibited the fixation of iodinated alpha-bungarotoxin to Torpedo nicotinic acetylcholine receptors with an IC50 of 0.19 microM which can be compared to the IC50 of 0.31 microM found for the previously characterized alpha-MI isolated from the piscivorous Conus magus. The synthetic alpha-CnIA blocked spontaneous and evoked synaptic potentials in frog and mouse isolated neuromuscular preparations at sub-micromolar concentrations. Solution NMR of this toxin indicated a conformational heterogeneity with the existence of different conformers in solution, at slow and intermediate exchange rates relative to the NMR chemical shift time scale, similar to that reported for alpha-GI and alpha-MI. NMR structures were calculated for the major NMR signals representing more than 80% of the population at 5 degrees C.
Asunto(s)
Conotoxinas , Venenos de Moluscos/química , Oligopéptidos/química , Péptidos/química , Secuencia de Aminoácidos , Animales , Bioensayo , Bungarotoxinas/farmacología , Radioisótopos de Yodo , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Moluscos/química , Antagonistas Nicotínicos/farmacología , Oligopéptidos/aislamiento & purificación , Oligopéptidos/farmacología , Péptidos/aislamiento & purificación , Péptidos/farmacología , Péptidos Cíclicos/química , Conformación Proteica , Receptores Nicotínicos/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
kappa-Conotoxin PVIIA from the venom of Conus purpurascens is the first cone snail toxin that was described to block potassium channels. We synthesized chemically this toxin and showed that its disulfide bridge pattern is similar to those of omega- and delta-conotoxins. kappa-conotoxin competes with radioactive alpha-dendrotoxin for binding to rat brain synaptosomes, confirming its capacity to bind to potassium channels; however, it behaves as a weak competitor. The three-dimensional structure of kappa-conotoxin PVIIA, as elucidated by NMR spectroscopy and molecular modeling, comprises two large parallel loops stabilized by a triple-stranded antiparallel beta-sheet and three disulfide bridges. The overall fold of kappa-conotoxin is similar to that of calcium channel-blocking omega-conotoxins but differs from those of potassium channel-blocking toxins from sea anemones, scorpions, and snakes. Local topographies of kappa-conotoxin PVIIA that might account for its capacity to recognize Kv1-type potassium channels are discussed.
Asunto(s)
Conotoxinas , Venenos de Moluscos/química , Neurotoxinas/química , Bloqueadores de los Canales de Potasio , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Disulfuros/química , Modelos Moleculares , Datos de Secuencia Molecular , Venenos de Moluscos/síntesis química , Venenos de Moluscos/metabolismo , Neurotoxinas/síntesis química , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Ratas , Ratas Sprague-Dawley , Homología de Secuencia de Aminoácido , Caracoles , Sinaptosomas/metabolismoRESUMEN
During the time course of disulfide bond formation by iodine oxidation (in a methanolic and hydrochloric acid solution) of a cysteinyl(S-acetamidomethyl)-glutaminyl tridecapeptide, we observed by ESI, FAB mass spectrometry (pseudo-molecular ion and ion-fragments) and 1H-NMR a side reaction due to a shift of the Acm leaving group from cysteine to the carboxamide side chain of glutamine. This type of Acm-shift at low level was described previously by L.W. Mendelson et al. (Int. J. Pept. Protein Res. 35:249-257) for an aspariginyl-cysteinyl(S-acetamidomethyl) peptide in an anhydrous hydrochloric solution. We report here the efficiency of glutamine as a scavenger to suppress the S-->N shift of the acetamidomethyl group during S-acetamidomethyl cleavage and sulfhydryl oxidation with iodine, as the folded tridecapeptide was obtained with the expected molecular weight.
Asunto(s)
Acetilcisteína/análogos & derivados , Disulfuros/química , Glutamina/química , Yodo/química , Péptidos/química , Acetilcisteína/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Peso Molecular , Oxidación-ReducciónRESUMEN
Sarafotoxins (SRTXs) form a family of toxic and potent vasoconstrictor peptides of 21 amino acids and two disulfide bonds. They are present in the venom of the burrowing asp Atractaspis engaddensis. We have made two derivatives of the amino acid sequence of SRTX-b, one of the most potent isotoxins, in the solid phase. First, we replaced Ser2 by Thr, to investigate whether, as previously postulated, this change is responsible for the weak activities of SRTXs c and d. Secondly, we replaced Ser2, Asp18 and Val19 respectively by Thr, Gly and Ile, with a view to generating SRTX-e whose amino acid sequence was deduced from cDNA. Solid-phase peptide synthesis (SPPS) was performed according to the tert-butyloxycarbonyl strategy and the disulfides were paired sequentially using a selective chemistry. The disulfide 1-15 was formed by oxidation of cysteines1,15 with ferricyanide, whereas disulfide 3-11 was made by iodine oxidation of Acm-blocked cysteines3,11. By plasma desorption mass spectrometry (PDMS), we monitored all possible side reactions that occurred during the synthesis. We thus observed a benzyl shift in mass spectra when aspartic and glutamic acid side chains were protected by a benzyl group during the SPPS. This could be circumvented by using instead, a cyclohexyl protecting group. We also noted the oxidation of the methionine and the tryptophan side chain (formation of methionine sulfoxide and oxindole ring of tryptophan) to a small extent during the cleavage peptide/solid phase oxidation of the methionine side chain during the formation of the disulfide 1-15 by ferricyanide.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Vasoconstrictores/química , Venenos de Víboras/química , Secuencia de Aminoácidos , Derivados del Benceno/química , Cromatografía Líquida de Alta Presión , Espectrometría de Masas , Metionina/química , Datos de Secuencia Molecular , Peso Molecular , Oxidación-Reducción , Triptófano/química , Vasoconstrictores/aislamiento & purificación , Venenos de Víboras/aislamiento & purificaciónRESUMEN
The 21 amino acid sarafotoxins (SRTX) c and d/e as well as endothelin-3 (ET-3) are known to be less toxic and weaker pharmacologically than the other isopeptides SRTX-a, SRTX-b and ET-1. Since SRTX-c, SRTX-d/e and ET-3 possess a Thr instead of a Ser at position 2, we investigated the possibility that this mutation could be responsible for the observed biological differences. Here we show that the synthetic [Thr2]SRTX-b has indeed a lower vasoconstriction efficacy (approximately 35%) in the rabbit aorta, but it is nearly as potent as SRTX-b in toxicity tests and in influencing contraction of the rat uterus. Using monoclonal antibodies directed against the structurally related endothelin-1, we also show that the antigenicity of the analogue is comparable to that of SRTX-b, suggesting that the overall structure of the two peptides is similar, despite the substitution at position 2. We suggest that the Thr2 substitution contributes to the lower activity of the 'weak' peptides in some systems; however, additional substitutions found in the 'weak' peptides of the ET/SRTX family most probably contribute to their low pharmacological activity.
Asunto(s)
Músculo Liso Vascular/efectos de los fármacos , Contracción Uterina/efectos de los fármacos , Vasoconstrictores/toxicidad , Venenos de Víboras/toxicidad , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Aorta/efectos de los fármacos , Unión Competitiva , Cromatografía Líquida de Alta Presión , Electrocardiografía/efectos de los fármacos , Endotelinas/química , Endotelinas/inmunología , Endotelinas/toxicidad , Femenino , Técnicas para Inmunoenzimas , Dosificación Letal Mediana , Ratones , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Conejos , Ratas , Relación Estructura-Actividad , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/química , Venenos de Víboras/química , Venenos de Víboras/clasificaciónRESUMEN
In a previous paper, the systematic epitope screening of a snake curaremimetic toxin or toxin a was described by this group using a panel of synthetic octadecapeptides. The disulphide cyclized peptide (Cys-23,40)(23-40) corresponding to loop II of the native toxin was found to elicit, with no linkage to a carrier, neutralizing antisera against the toxin. We have now undertaken the conformational study of this immunogenic disulphide cyclized peptide by CD and FTIR. The CD study of the peptide was carried in aqueous solution under various conditions (pH, temperature, presence of micelles) and in trifluoroethanol solution. Low temperature, SDS micelles and trifluoroethanol were found to induce a beta-sheet formation (16 to 39%). FTIR spectra of the peptide in the solid state (dry film) and in D2O solution or deuterated-TFE solution (hydrated film) displayed some characteristic bands indicating the presence of beta-sheet (1623 cm-1) and beta-turn (1637 cm-1; 1694 cm-1). These studies indicate that the immunogenic disulphide cyclized peptide (23-40) can adopt in solution an ordered structure.
Asunto(s)
Proteínas Neurotóxicas de Elápidos/química , Curare/química , Disulfuros/química , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Proteínas Neurotóxicas de Elápidos/inmunología , Curare/inmunología , Análisis de Fourier , Datos de Secuencia Molecular , Pruebas de Neutralización , Conformación Proteica , Soluciones , TemperaturaRESUMEN
The acetamidomethyl (Acm) group is a widely used protecting group for the thiol of cysteine during the SPPS process. We prepared the amino terminal loop of the snake alpha-neurotoxin, [Cys3,Cys23, Ser17](1-24) amide, from the linear peptide [Cys(Acm)3,23,Ser17](1-24) amide obtained by SPPS. Three different methods of deprotection of Cys(Acm) and disulfide bond formation were used: iodine, thallium(III) trifluoroacetate and mercuric acetate/potassium ferricyanide. The iodine method failed to yield the expected peptide, and gave instead the mono-iodinated tyrosine analog. The disulfide cyclized peptide obtained by thallium (III) or Hg(II) procedures displayed a MW value observed by mass spectrometry that was higher than the calculated value. The difference (MWobs-MWcalc) corresponded to a multiple of the Acm moiety, which is shifted intra- and/or intermoleculary. Furthermore, we observed, in addition to the Acm shift in the disulfide cyclized decapeptide with a highSer and Thr content (model peptide II), the dimerization phenomenon in the Tl(TFA)3 process. Therefore we conclude that a side reaction, a S--O(Ser,Thr) Acm shift, occurred during the Cys(Acm) deprotection. This shift was supported by the demonstration of Ser(O-Acm) formation in the reaction of Boc-(L)-Cys(Acm) with Tl(TFA)3 in the presence of an equimolar amount of (L)Ser. We report here the efficiency of a trivalent alcohol, glycerol, as scavenger in the both Tl(TFA)3 and mercuric/ferricyanide methods, in an attempt to circumvent this side-reaction during the disulfide bond formation step starting from a bis-Cys(Acm) peptide with a high Ser and Thr content, such as the N-terminal loop of neurotoxin, model peptide II or a similar peptide.
Asunto(s)
Acetilcisteína/análogos & derivados , Proteínas Neurotóxicas de Elápidos/química , Cistina/química , Fragmentos de Péptidos/química , Serina/química , Acetilcisteína/química , Secuencia de Aminoácidos , Animales , Química Orgánica/métodos , Ferricianuros/química , Fluoroacetatos , Yodo/química , Mercurio/química , Datos de Secuencia Molecular , Serpientes , Talio , Treonina/química , Ácido Trifluoroacético/químicaRESUMEN
We have synthesized a free peptide capable of eliciting antibodies that neutralize toxin alpha from Naja nigricollis, a protein that binds specifically to the acetylcholine nicotinic receptor. Of the five tested fragments that encompassed the whole toxin sequence, only fragment 24-41 stimulated T cells from BALB/c mice primed with the whole toxin and conversely, only T cells from mice primed with fragment 24-41 could be stimulated by both the toxin and priming peptide. No other peptides had such properties, indicating that only fragment 24-41 possessed T determinant(s) in BALB/c mice (H-2d haplotype). In agreement with the current view that B cell proliferation requires specific T cell stimulation, only fragment 24-41 elicited an antibody response. However, the antipeptide antisera failed to bind to the native toxin and thereby to neutralize it. Instead, it recognized an unfolded form of the toxin. The peptide 24-41 was then made cyclic. A circular dichroism analysis revealed that, in organic solvent, this peptide had a tendency to adopt a beta-sheet structure, as in the folded toxin, whereas the linear peptide adopted an helical structure. The cyclic peptide not only remained T stimulating but elicited antisera that recognized and neutralized the native toxin. Furthermore, the antisera cross-reacted with several toxin variants. Our data show, therefore, that it is possible to give an appropriate B cell specificity directly to a T cell-stimulating peptide, an approach that may be of value for the design of synthetic vaccines.