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1.
C. Lecomte G. Ferrat Z. Fajloun J. Van Rietschoten H. Rochat M.-F. Martin-Eauclaire H. Darbon J.-M. Sabatier 《Chemical biology & drug design》1999,54(5):369-376
Abstract: Tityus kappa (Ts κ), a novel toxin from the venom of the scorpion Tityus serrulatus, is a 35-residue polypeptide cross-linked by three disulphide bridges and acts on small-conductance calcium-activated potassium channels (SK channels). Ts κ was chemically synthesized using the solid-phase method and characterized. The synthetic product, sTs κ, was indistinguishable from the natural toxin when tested in vitro in competition assay with radiolabelled apamin for binding to rat brain synaptosomes (IC50 = 3 nm ). The sTs κ was further tested in vivo for lethal activity to mice following intracerebroventricular inoculation (LD50 = 70 ng per mouse). The half-cystine pairings were formerly established by enzyme-based cleavage of sTs κ; they were between Cys7–Cys28, Cys13–Cys33 and Cys17–Cys35, which is a disulphide bridge pattern similar to that of other short scorpion toxins. According to previous studies on SK channel-acting toxins, the putative influence of certain basic residues of Ts κ (i.e. Arg6, Arg9, Lys18, Lys19) in its pharmacological activity was investigated using synthetic point-mutated analogues of the toxin with an Ala substitution at these positions. Data from binding assay, together with conformational analysis of the synthetic analogues by 1H-NMR, suggest that Arg6, and to a lesser extent Arg9, are important residues for an high-affinity interaction of this toxin with SK channels; interestingly these residues are located outside the α-helical structure, whereas the pharmacologically important basic residues from other SK channel-specific toxins had been located inside the α-helix. 相似文献
2.
The amino acid sequences of neurotoxins I and II, which are active on mammals, purified from the venom of Buthus occitanus tunetanus have been determined using standard methods, including mainly automatic phenylisothiocyanate degradation of S-carboxymethylated derivatives of the two proteins and peptides derived by enzymatic hydrolyses. Both toxins are made of sixty-five amino acid residues cross-linked with four disulfide bridges. For toxin II, the complete covalent structure, including the positions of the four disulfide bridges was determined: the positions are similar to those previously found in toxin II of another scorpion from Africa, Androctonus australis Hector. This finding is in favor of a similar structure for all of the scorpion neurotoxins active on mammals. 相似文献
3.
V. CALABRO J.M. SABATIER E. BLANC C. LECOMTE J. VAN RIETSCHOTEN H. DARBON 《Chemical biology & drug design》1997,50(1):39-47
Leiurotoxin I is a neurotoxin, blocker of Ca2+-activated apamin-sensitive K+ channel, purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. It is a 31-residue polypeptide reticulated by three disulfide bridges, i.e. Cys3-Cys21, Cys8-Cys26 and Cys12-Cys28. To investigate the role of these disulfide bridges in the folding of this toxin, analogs lacking one disulfide bridge were synthesized. The structures of two analogs in which two half-cystines were replaced by α-aminobutyrate residues to suppress one disulfide bridge, were analyzed by 1H NMR. The NMR studies reveal a three-dimensional structure identical with the native toxin for the analog lacking disulfide bridge Cys3-Cys21 and a loss of organized structure for another analog lacking disulfide bridge Cys12-Cys28. These analogs are, respectively, fully active and only weakly active (2% of the residual activity) when tested in vitro for their ability to interact with their receptor channel and in vivo for their neurotoxic activity in mice. This suggests that disulfide bridge Cys12-Cys28 is essential for the folding process. In contrast, the lack of disulfide bridge Cys3-Cys21 does not affect the folding and the maintenance of bioactive conformation of Leiurotoxin I. 相似文献
4.
C. Lecomte R. Ben Khalifa M.‐F. Martin‐eauclaire R. Kharrat M. El Ayeb H. Darbon H. Rochat M. Crest J.‐M. Sabatier 《Chemical biology & drug design》2000,55(3):246-254
Abstract: Maurotoxin (MTX) is a 34‐amino acid polypeptide cross‐linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half‐cystine pairings at Cys3–Cys24, Cys9–Cys29, Cys13–Cys19 and Cys31–Cys34. This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C‐terminal ‘14‐membered disulfide ring’ (i.e. cyclic domain 31–34), We therefore studied structure–activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C‐terminus {i.e. MTX (1–29), [Abu31,34]‐MTX and [Cys31,34, Tyr32]d ‐MTX} or mimicking the cyclic C–terminal domain [i.e. MTX (31–34)]. Unexpectedly, the absence of a disulfide bridge Cys31–Cys34 in [Abu 31,34]‐MTX and MTX (1–29) resulted in MTX‐unrelated half‐cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX (31–34) was also biologically inactive. [Cys31,34, Tyr32]d ‐MTX, which had a ‘native’, MTX‐related, disulfide bridge organization, but a d ‐residue‐induced reorientation of the C–terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide‐induced Kv1.1 blockage was voltage‐dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on‐rate changes in ligand binding. Thus, the cyclic C–terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel. 相似文献
5.
Nobuto Matsushita Masahiro Miyashita Atsushi Sakai Yoshiaki Nakagawa Hisashi Miyagawa 《Toxicon》2007,50(6):861-867
Scorpion venoms contain a variety of peptides toxic to mammals, insects and crustaceans. Most of the scorpion toxins have been isolated from the venoms of scorpions in the family Buthidae, but little interest has been paid to non-Buthidae scorpions. In this study, we isolated a short-chain insecticidal toxin (LaIT1) from the venom of the scorpion Liocheles australasiae belonging to the Hemiscorpiidae family. This toxin showed insect toxicity against crickets at a dose of 1.0 microg/insect, but no toxicity was observed against mice even after injection of 1.0 microg of LaIT1 via the intracerebroventricular route, suggesting that the effect of the toxin is insect-selective. Edman sequencing and mass spectrometric analysis revealed that the toxin is composed of 36 amino acid residues and cross-linked by only two disulfide bridges. The pattern of the disulfide bridges was assigned by LC/MS analysis after enzymatic digestion. LaIT1 shows no sequence homology to any other known toxins, suggesting that this toxin represents a novel structural motif class. 相似文献
6.
The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins. 相似文献
7.
The primary structure of TsTX-IV, a neurotoxin isolated from Tityrus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K+ channels. TsTx-IV was able to block Ca2+-activated K+ channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced. 相似文献
8.
The effects of the crude venom of the scorpion Androctonus australis Hector and its insect toxin, mammal toxin II and the ‘crustacean fraction’ were tested on the crayfish stretch receptor organ. The ‘crustacean fraction’ was able to mimic the excitatory and blocking action of the crude venom, while the insect and mammal toxins were inactive. It is suggested that the specificity in the action of the ‘crustacean fraction’ is due to a specific affinity to a crustacean neural system. 相似文献
9.
The venom of South African scorpion Parabuthus transvaalicus contains a novel group of peptide toxins. These peptides resemble the long chain neurotoxins (LCN) of 60-70 residues with four disulfide bridges; however they are 58 residues long and have only three disulfide bridges constituting a new family of peptide toxins. Here we report the isolation and characterization of three new members of this mammal specific group of toxins. Dortoxin is a lethal peptide, bestoxin causes writhing in mice and altitoxin is a highly depressant peptide. Binding ability of these peptides to rat brain synaptosomes is tested. While the crude venom of P. transvaalicus enhances the binding of [(3)H] BTX to rat brain synaptosomes none of these individual toxins had a positive effect on binding. Although the primary structures of these toxins are very similar to birtoxin, their 3D models indicate significant differences. Dortoxin, bestoxin and altitoxin cumulatively constitute at least 20% of the peptide contained in the venom of P. transvaalicus and contribute very significantly to the toxicity of the venom of this medically important scorpion species. Therefore the amino acid sequences presented here can be used to make more specific and effective antivenins. Possible approaches to a systematic nomenclature of toxins are suggested. 相似文献
10.
11.
Elisabeth F. Schwartz Carlos A. Schwartz Froylan Gmez-Lagunas Fernando Z. Zamudio Lourival D. Possani 《Toxicon》2006,48(8):1046-1053
A novel toxin was identified, purified and characterized from the venom of the Mexican scorpion Hadrurus gertschi (abbreviated HgeTx1). It has a molecular mass of 3950 atomic mass units (a.m.u.) and contains 36 amino acids with four disulfide bridges established between Cys1–Cys5, Cys2–Cys6, Cys3–Cys7 and Cys4–Cys8. It blocks reversibly the Shaker B K+-channels with a Kd of 52 nM. HgeTx1 shares 60%, 45% and 40% sequence identity, respectively, with Heterometrus spinnifer toxin1 (HsTX1), Scorpio maurus K+-toxin (maurotoxin) and Pandinus imperator toxin1 (Pi1), all four-disulfide bridged toxins. It is 57–58% identical with the other scorpion K+-channel toxins that contain only three disulfide bridges. Sequence comparison, chain length and number of disulfide bridges analysis classify HgeTx1 into subfamily 6 of the -KTx scorpion toxins (systematic name: -KTx 6.14). 相似文献
12.
The ‘insect’, ‘crustacean’ and ‘mammal’ I and II toxins previously purufied from the venom of the scorpion Androctonus australis Hector according to their fly larvae paralysis, isopods paralysis and mice lethality, respectively, were applied on the guinea pig ileum smooth muscle preparation and it has been found that: (1) the ‘crustacean’ toxin induced a sustained-prolonged contraction in contrast to the rhythmic spasmodic ileal behaviour caused by the crude venom and the ‘mammal’ toxins. (2) The excitatory effect of these substances was due to a postganglionic presynaptic stimulation resulting in the release of a cholinergic transmitter. (3) Prolonged application of the ‘mammal’ as well as ‘crustacean’ toxins, caused a depression of postsynaptic reactivity as observed in a reduced ileal response to several agonists. (4) The ‘insect’ toxin, was inactive in the ileum smooth muscle preparation 相似文献
13.
The crystal and molecular structure of a toxin from the scorpion Centruroides sculpturatus has been solved by standard x-ray crystallographic methods at 3 Å resolution. Subsequently, the 3 Å model has been refined and the resolution has been extended to 1.8 Å using the gradient-curvature method. The final reliability index is 0.17The structure has two and a half turns of α-helix, a three-strand stretch of antiparallel β-sheet and several β-turns. Three of the four disulfide bridges are found in close interaction with the α-helix and β-sheet structures in what constitutes a very rigid part of the molecule.Examination of available scorpion toxin sequences reveals several sections containing invariant and/or semiinvariant amino acids. Many of these residues are found clustered on a rather large flat surface which is also clearly more hydrophobic than other areas on the molecule. These observations suggest that this surface may play a role in the biological action of scorpion toxins.Secondary structure predictions calculated using the method of Dufton and Hider agree well with the x-ray structure. This is also true for other scorpion toxins and reinforces the idea that scorpion toxins are a family of structurally related proteins. 相似文献
14.
A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects. 相似文献
15.
Y.‐M. Sun W. Liu R.‐H. Zhu D.‐C. Wang C. Goudet J. Tytgat 《Chemical biology & drug design》2002,60(5):247-256
Abstract: The unique fold of scorpion toxins is a natural scaffold for protein engineering, in which multiple disulfide bonds are crucial structural elements. To understand the respective roles of these disulfide bridges, a mutagenesis analysis for the four disulfide bonds, 12–63, 16–36, 22–46 and 26–48, of a representative toxin BmK M1 from the scorpion Buthus martensii Karsch was carried out. All cysteines were replaced by serine with double mutations. The recombinant mutants were expressed in the Saccharomyces cerevisiae S‐78 system. Toxic activities of the expressed mutants were tested on ICR mice in vivo and on neuronal Na+ channels (rNav1.2) by electrophysiological analysis. Recombinant variants M1 (C22S,C46S) and M1 (C26S,C48S) were not expressed at all; M1 (C16S,C36S) could be expressed at trace levels but was extremely unstable. Variant M1 (C12S,C63S) could be expressed in an amount comparable with that of unmodified rBmK M1, but had no detectable bioactivities. The results indicated that among the four disulfide bonds for long‐chain scorpion toxins, loss of either bridge C22?C46 or C26?C48 is fatal for the general folding of the molecule. Bridge C16?C36 mainly contributes to the global stability of the folded scaffold, and bridge C12?C63 plays an essential role in the functional performance of scorpion toxins. 相似文献
16.
Abstract: Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of α-conotoxin SI, a tridecapeptide amide from marine cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two ‘interlocking’ disulfide bridges connecting Cys2–Cys7 and Cys3–Cys13 (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys2–Cys13 and Cys3–Cys7 (2/13 & 3/7, ‘nested’) and Cys2–Cys3 and Cys7–Cys13 (2/3 & 7/13, ‘discrete’), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation ofS-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers. 相似文献
17.
E. Carlier S. Geib M. De Waard V. Avdonin T. Hoshi Z. Fajloun H. Rochat J.‐M. Sabatier R. Kharrat 《Chemical biology & drug design》2000,55(6):419-427
Abstract: Maurotoxin is a 34‐residue toxin isolated from the venom of the Tunisian chactoid scorpion Scorpio maurus palmatus and contains four disulfide bridges that are normally found in long‐chain toxins of 60–70 amino acid residues, which affect voltage‐gated sodium channels. However, despite the unconventional disulfide‐bridge pattern of maurotoxin, the conformation of this toxin remains similar to that of other toxins acting on potassium channels. Here, we analyzed the effects of synthetic maurotoxin on voltage‐gated Shaker potassium channels (ShB) expressed in Xenopus oocytes. Maurotoxin produces a strong, but reversible, inhibition of the ShB K+ current with an IC50 of 2 nm . Increasing concentrations of the toxin induce a progressively higher block at saturating concentrations. At nonsaturating concentrations of the toxin (5–20 nm ), the channel block appears slightly more pronounced at threshold potentials suggesting that the toxin may have a higher affinity for the closed state of the channel. At the single channel level, the toxin does not modify the unitary current amplitude, but decreases ensemble currents by increasing the number of depolarizing epochs that failed to elicit any opening. A point mutation of Lys23 to alanine in maurotoxin produces a 1000‐fold reduction in the IC50 of block by the toxin suggesting the importance of this charged residue for the interaction with the channel. Maurotoxin does not affect K+ currents carried by Kir2.3 channels in oocytes or Na+ currents carried by the αIIa channel expressed in CHO cells. 相似文献
18.
19.
Yvonne M. M. Bishop Christine C. Katsioules Johannes Meienhofer 《Chemical biology & drug design》1974,6(4):231-244
A Fortran computer program to aid in disulfide bond analysis in proteins has been developed. The program evaluates amino acid analysis data from isolated cystine-containing peptides by comparing it with known sequences around pairs of half-cystine residues in the primary structure of a protein and recording all matches. The method involves presetting upper and lower bounds for each amino acid present in a cystine-containing peptide by considering both partial degradation during acid hydrolysis and contamination sources for certain amino acids. Initially, limiting path terminals for each half-cystine residue in the primary structure are determined by examining the surrounding sequences in both directions for “alien” amino acid residues, not present in the composition of a given cystine-containing peptide. Subsequently, pairwise examination proceeds through several cycles incorporating minimal path and upper bound routines with intermittent revision of path ends. Finally, all pairs of half-cystines whose surrounding sequences fall within the preset bounds for this peptide are listed. If too many possible pairs or none are found, the bounds are revised. The lists for all available cystine-containing peptides are graphed to facilitate final determination of disulfide bond positions in the native protein. The computer program provided finite disulfide bond assignments for ovine lactogenic hormone and human growth hormone. In the case of hen egg-white lysozyme, a considerable reduction was obtained from the large number of theoretically possible disulfide bond combinations to a few alternatives, and one of the four disulfide bonds was unequivocally determined. To assign the positions of the other three disulfide bridges, additional considerations, not included in the program, were applied, such as specificity of the enzymes used in digesting the protein and electrophoretic mobility of the ensuing cystine-containing peptides 相似文献
20.
T.J. Zamborelli W.S. Dodson B.J. Harding J. Zhang B.D. Bennett D.M. Lenz C.‐F. Liu T. Jones M.A. Jarosinski Y. Young M. Haniu 《Chemical biology & drug design》2000,55(5):359-371
Abstract: The 52‐residue α/β chimera of the epidermal growth factor‐like domain in neu differentiation factor (NDFeα/β) has been synthesized and folded to form a three disulfide bridge (Cys182–Cys196, Cys190–Cys210, Cys212–Cys221) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single‐step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one‐step approach may not always provide the desired disulfide pairing. Here, we compare the single‐step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys182 to Cys196, Cys190 to Cys210 and Cys212 to Cys221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP‐HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via I2 oxidation. Only the peptide having the Cys182–Cys196 pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys190–Cys210/Cys212–Cys221) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large‐scale syntheses of NDFeα/β and other novel EGF‐like peptides. 相似文献