[Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue]. / Synthèse chimique de la lactococcine B et évaluation fonctionnelle de son extrémité N-terminale grâce à un analogue synthétique tronqué.

The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

Electron transfer in an acidophilic bacterium: interaction between a diheme cytochrome and a cupredoxin.

Acidithiobacillus ferrooxidans, a chemolithoautotrophic Gram-negative bacterium, has a remarkable ability to obtain energy from ferrous iron oxidation at pH 2. Several metalloproteins have been described as being involved in this respiratory chain coupling iron oxidation with oxygen reduction. However, their properties and physiological functions remain largely unknown, preventing a clear understanding of the global mechanism. In this work, we focus on two metalloproteins of this respiratory pathway, a diheme cytochrome c4 (Cyt c4) and a green copper protein (AcoP) of unknown function. We first demonstrate the formation of a complex between these two purified proteins, which allows homogeneous intermolecular electron-transfer in solution. We then mimic the physiological interaction between the two partners by replacing one at a time with electrodes displaying different chemical functionalities. From the electrochemical behavior of individual proteins, we show that, while electron transfer on AcoP requires weak electrostatic interaction, electron transfer on Cyt c4 tolerates different charge and hydrophobicity conditions, suggesting a pivotal role of this protein in the metabolic chain. The electrochemical study of the proteins incubated together demonstrates an intermolecular electron transfer involving the protein complex, in which AcoP is reduced through the high potential heme of Cyt c4. Modelling of the electrochemical signals at different scan rates allows us to estimate the rate constant of this intermolecular electron transfer in the range of a few s-1. Possible routes for electron transfer in the acidophilic bacterium are deduced.

Fine molecular analysis of the antigenicity of the Androctonus australis hector scorpion neurotoxin II: a new antigenic epitope disclosed by the Pepscan method.

A set of 58 overlapping rod-bound peptides was used to map the antigenic reactivity pattern of a 64-residue neurotoxin (AaH II) from the venom of the scorpion Androctonus australis hector. Five anti-toxin rabbit antisera were assayed serially for their capacity to bind to each peptide in the set. Six regions of antigenic reactivity were thus identified (sequences: 1-8, 4-12, 27-35, 39-45, 52-58 and 55-61). When positioned on a 3-D model of the toxin, these regions appeared to correspond to either beta-turn or extended parts of the molecule. The antigenic regions revealed by this technique agree fairly well with those previously mapped on the same toxin by different methods. One discrepancy was, however, that the present study shows the N-terminus to be strongly reactive with anti-toxin antibodies. The antigenicity of this region was confirmed, since rabbit antibodies raised against a synthetic peptide mimicking the sequence 1-8 of the toxin were found to bind the toxin with high efficiency. A fine analysis of the recognition of this region was performed. Alanine-containing analogs of the sequence 1-7 and peptides mimicking the N-terminal of the four main toxins of AaH were probed with anti-toxin and anti-peptide antibodies. Lysine 2, aspartic acid 3 and glycine 4 were shown to be key residues in the recognition of the N-terminal region of the AaH II toxin by anti-toxin antibodies. In contrast, a loose specificity of recognition was shown by one anti-peptide serum which was, in addition, able to recognize the N-termini of all four AaH toxins.

A novel immunoglobulin superfamily junctional molecule expressed by antigen presenting cells, endothelial cells and platelets.

The architecture of lymphoid microenvironments depends upon complex interactions between several stromal cell types. We describe in this report the cloning of a cDNA which encodes a novel membrane molecule containing two external Ig-like domains. It is expressed at the junction between endothelial cells including HEV. It is also expressed by platelets and MHC class II+ antigen presenting cells in thymic medulla and T-cell areas in peripheral lymphoid organs. These cells which lack in RelB-deficient mice include tissue-derived dendritic, epithelial cells and macrophages. Thus, this molecule might contribute to the organization of cell junctions in different microenvironments.

Characterization and ultrastructural localization of annexin VI from mitochondria.

Annexin VI, a member of a family of related intracellular proteins that associate reversibly with membrane phospholipids in a Ca(2+)-dependent manner, has been purified from bovine liver mitochondria and characterized. Moreover, biochemical and immunocytochemical lines of evidence are presented which strongly suggest that annexin VI is closely associated with the cristae in the inner membrane of mitochondria. These findings are consistent with a calcium channel activity of annexin VI in mitochondria.

Novel anti-platelet aggregation polypeptides from Vipera lebetina venom: isolation and characterization.

Lebetins 1 and Lebetins 2, two polypeptide groups that inhibit platelet aggregation, were isolated from Vipera lebetina venom by gel filtration and reverse phase chromatography. Amino acid sequencing indicated that the first group contains two major polypeptides of 13 and 12 residues; their molecular weight was determined by electrospray mass spectrometry. The second was composed of two peptides of 38 and 37 residues, each with one disulfide bond. Sequence analysis revealed neither RGD sequence nor homology with other proteins including known snake or tick polypeptides. Lebetins 1 were Pro and Lys rich peptides and their sequences were identical to the N-terminus of Lebetins 2. Lebetins inhibited platelet aggregation induced by thrombin, collagen and PAF-acether. The 50% concentration that inhibited human and rabbit platelet aggregation induced by thrombin was 590 nM and 125 nM for Lebetins 1 and 100 nM and 8 nM for Lebetins 2, respectively. Lebetins 1 and Lebetins 2 also inhibited fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets as well as in vivo collagen-induced thrombocytopenia in rats with half effective doses of 2 nmol/kg and 4.2 nmol/kg, respectively. Lebetins were not toxic after intravenous injection into mice and rats. These polypeptides form novel platelet inhibitors that could be used to delineate further the mechanisms of platelet aggregation and serve as a model for developing antithrombotic agents.

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus.

The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.

Characterization of a new leiurotoxin I-like scorpion toxin. PO5 from Androctonus mauretanicus mauretanicus.

Three novel peptide inhibitors of the SKCa channels were purified to homogeneity from the venom of the scorpion Androctonus mauretanicus mauretanicus using one step of RP-HPLC and competition assays with [125I]apamin to rat brain synaptosomes. PO1, PO2 and PO5 have K0.5 of 100, 100 and 0.02 nM, respectively, for the apamin binding site. The sequence of PO5 was established and compared to that of other scorpion toxins active on K+ channels: it contains 31 residues and has a free carboxyl end. it shares sequence similarity with apamin and leiurotoxin I.

Maurotoxin, a four disulfide bridge toxin from Scorpio maurus venom: purification, structure and action on potassium channels.

A new toxin acting on K+ channels, maurotoxin (MTX), has been purified to homogeneity from the venom of the chactoid scorpion Scorpio maurus. MTX is a basic single chain 34 amino acid residue polypeptide, amidated at its C terminal, and crosslinked by four disulfide bridges. It shows 29-68% sequence identity with other K+ channel toxins, and presents an original disulfide pattern, the last two half-cystine residues (31-34) being connected. Although the first three disulfide bonds have not been defined experimentally, modelling based on the structure of charybdotoxin favored two combinations out of six, one of which has two bridges (3-24 and 9-29) in common with the general motif of scorpion toxins. The last bridge would connect residues 13 and 19. MTX inhibits the binding to rat brain synaptosomal membranes of both [125I]apamin, a SK(Ca) channel blocker (IC50 5 nM), and [125I]kaliotoxin, a Kv channel blocker (IC50 30 pM). MTX blocks the Kv1.1, Kv1.2 and Kv1.3 currents expressed in Xenopus oocytes with IC50 of 45, 0.8 and 180 nM, respectively. MTX represents a member of a new class of short toxins with 4 disulfide bridges, active on voltage-dependent K+ channel and also competing with apamin for binding to its receptor.

Aah VI, a novel, N-glycosylated anti-insect toxin from Androctonus australis hector scorpion venom: isolation, characterisation, and glycan structure determination.

Aah VI was isolated from the venom of the North African scorpion, Androctonus australis hector. It is the first glycosylated neurotoxin from scorpion venom to be described. It was not toxic to mice, when injected intracerebroventricularly at a dose of 1.2 microg per animal. However, it had typical activity in Blatella germanica cockroaches resulting in gradual paralysis and very low toxicity (LD50 = 8.5 microg/g of animal). It consists of 66 amino acid residues and is heterogeneously N-glycosylated at a single site, on asparagine 9, of the Asn-Gly-Thr sequence. The potential N-glycosylation site was deduced from automatic Edman degradation and amino acid analysis, and glycan heterogeneity was evidenced by ESMS. Determination of the N-glycan structures (dHex, Hex and HexNAc) was assessed by nanoESMS/MS with picomolar amounts of sample. Current knowledge of N-glycan structure and composition suggests that the glycan structures are derived from a common core.

Disulfide bridge reorganization induced by proline mutations in maurotoxin.

Maurotoxin (MTX) is a 34-residue toxin that has been isolated from the venom of the chactidae scorpion Scorpio maurus palmatus, and characterized. Together with Pi1 and HsTx1, MTX belongs to a family of short-chain four-disulfide-bridged scorpion toxins acting on potassium channels. However, contrary to other members of this family, MTX exhibits an uncommon disulfide bridge organization of the type C1-C5, C2-C6, C3-C4 and C7-C8, versus C1-C5, C2-C6, C3-C7 and C4-C8 for both Pi1 and HsTx1. Here, we report that the substitution of MTX proline residues located at positions 12 and/or 20, adjacent to C3 (Cys(13)) and C4 (Cys(19)), results in conventional Pi1- and HsTx1-like arrangement of the half-cystine pairings. In this case, this novel disulfide bridge arrangement is without obvious incidence on the overall three-dimensional structure of the toxin. Pharmacological assays of this structural analog, [A(12),A(20)]MTX, reveal that the blocking activities on Shaker B and rat Kv1.2 channels remain potent whereas the peptide becomes inactive on rat Kv1.3. These data indicate, for the first time, that discrete point mutations in MTX can result in a marked reorganization of the half-cystine pairings, accompanied with a novel pharmacological profile for the analog.

Monoclonal antibodies to toxin II from the scorpion Androctonus australis Hector: further characterization of epitope specificities and neutralizing capacities.

The epitope specificities of two previously prepared monoclonal antibodies (mAb) to the toxin II from Androctonus australis Hector were characterized. Neither mAb 4C1 nor mAb 3C5 was able to recognize any of the 58 overlapping synthetic heptapeptides which cover the whole sequence of toxin II. Thus, both mAbs probably recognize conformation-dependent epitopes at the surface of the toxin. Experiments were designed to check whether or not the two mAbs, or their Fab fragments, were able to bind simultaneously to the toxin. The results indicated that the epitopes recognized by the two antibodies are probably close together at the surface of the toxin, thus preventing the simultaneous binding of both mAbs to a single toxin molecule. Given the proximity of the two epitopes and the fact that mAb 4C1 is known to be a neutralizing antibody, the capacity of mAb 3C5 to inhibit the toxic effects of the toxin was re-evaluated in C57BL/6 mice. A clear, but weak, neutralizing effect was found, consistent with the low affinity binding of the mAb in the proximity of a neutralizing site of the toxin.

KTX3, the kaliotoxin from Buthus occitanus tunetanus scorpion venom: one of an extensive family of peptidyl ligands of potassium channels.

A new ligand of the K+ channels sensitive to KTX was purified from the venom of Buthus occitanus tunetanus, using two steps of high-performance-liquid-chromatography and by following its ability to compete with [125I]-KTX for binding to the KTX receptor on rat brain synaptosomes. Amino-acid analysis, amino acid sequencing and mass spectroscopy defined this new ligand. KTX3, as a 37-amino acid peptide, with three disulfide bridges. Its sequence shares 76% identity with KTX. The main differences between the two peptides are in the N-terminal region and the residue position 34 located in the region involved in channel recognition. These differences may explain the 5-fold lower binding affinity of KTX3, IC50=50 pM, than KTX to rat brain synaptosomes. Specific antibodies raised against KTX (1-37) were not able to recognize KTX3.

Two neurotoxins (BmK I and BmK II) from the venom of the scorpion Buthus martensi Karsch: purification, amino acid sequences and assessment of specific activity.

Two neurotoxins, BmK I and BmK II, were purified from the venom of the Chinese scorpion Buthus martensi Karsch. The complete amino acid sequences of both toxins, each containing 64 amino acid residues, were determined by the automatic sequencing of reduced and S-carboxymethylated toxins and their peptides, obtained after cleavage with TPCK-treated trypsin and Staphylococcus aureus V8 protease, respectively. Toxicity as minimum lethal dose tested by i.c.v. injection in mice showed that BmK I was six times more potent than BmK II. Only two amino acid replacements were found: at position 59 Val in BmK I was replaced by Ile in BmK II, and at position 62 a basic Lys residue in BmK I was substituted by a neutral Asn residue in BmK II. These features suggest that the positively charged residue (Lys or Arg) in the C-terminal position 62 (or 61 or 63) may also play an important role in facilitating the interaction between scorpion neurotoxins and the receptor on sodium channels. The effects of BmK I on nerve excitability were examined with the crayfish axon using intracellular recording and voltage-clamp conditions. The results indicate that BmK I preferentially blocks the sodium channel inactivation process. Thus, functional and structural similarities suggest that BmK I and BmK II belong to group 3 of scorpion alpha-type toxins.

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom.

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects (LD50 to Blatella germanica = 110 ng/100 mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity (K0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin (125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na+ current. The contracturant toxin BotIT1 is highly toxic to B. germanica (LD50 = 60 ng/ 100 mg body weight) and barely toxic to mice (LD50 = 1 microgram/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na+ channels. Thus, BotIT1 belongs to the group of alpha insect-selective toxins and shows a strong sequence identity (> 90%) with Lqh alpha IT and LqqIII, two insect alpha-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus. respectively.

Maurotoxin, a four disulfide bridges scorpion toxin acting on K+ channels.

Maurotoxin, a toxin from the venom of the Tunisian chactoid scorpion Scorpio maurus, has been purified to homogeneity by gel filtration/reversed-phase HPLC, and characterized. It is a basic and C-terminal amidated 34-residue polypeptide cross-linked by four disulfide bridges. From Edman sequencing results, only six different pairings between the first six half-cystines were retained whereas a disulfide bridge was predicted between the two half-cystines in positions 31 and 34. Modelling based on the structure of charybdotoxin favored two different pairings, one of which possessed two disulfides in common with the general motif of scorpion toxins. The solid-phase technique was used to obtain synthetic maurotoxin, sMTX. The half-cystine pairings of sMTX were determined by enzymatic cleavage and were found to be Cys3 Cys24, Cys9-Cys29, Cys13-Cys19, and Cys31-34, in agreement with experimental data obtained with natural maurotoxin. Both natural and synthetic maurotoxins were lethal to mice following intracerebroventricular injection (LD50, 80 ng/mouse). They blocked the Kv1.1, Kv1.2, and Kv1.3 channels expressed in Xenopus oocytes with almost identical half-effects (IC50) in the range of 40, 0.8 and 150 nM, respectively. They also competed with 125I-apamin (SKca channel blocker) and 125I-kaliotoxin (Kv channel blocker) for binding to rat brain synaptosomes with IC50 of about 5 and 0.03 nM. As the natural and synthetic maurotoxins exhibit indistinguishable physicochemical and pharmacological properties, they are likely to adopt the same half-cystine pairing pattern which is unique among known scorpion toxins. However, this disulfide organization is different from those reported for Pandinus imperator and Heterometrus spinnifer toxins 1 (Pi1 and HsTx1), two novel four-disulfide bridged K+ channel-acting scorpion toxin sharing about 50-70% sequence identity with maurotoxin.

Amino acid sequence of an excitatory insect-selective toxin (BmK IT) from venom of the scorpion Buthus martensi Karsch.

The insect-selective neurotoxin (BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective bioinsecticides.

Neurotoxins active on insects: amino acid sequences, chemical modifications, and secondary structure estimation by circular dichroism of toxins from the scorpion Androctonus australis Hector.

Two scorpion neurotoxins active only on insects, the insect toxins AaH IT1 and AaH IT2, were purified from the venom of scorpions Androctonus australis Hector collected in Tozeur (Tunisia) and characterized. AaH IT2 was sequenced and found to differ in four amino acid positions from AaH IT, the single previously sequenced insect toxin [Darbon, H., Zlotkin, E., Kopeyan, C., Van Rietschoten, J., & Rochat, H. (1982) Int. J. Pept. Protein Res. 20, 320-330] which possessed an equal potential for paralyzing fly larvae. The basic amino acid residues of AaH IT1, which differs from AaH IT by one amino acid residue, were selectively chemically modified. Six derivatives were characterized. Their toxicity toward fly larvae and cockroach was determined, and their affinity for the AaH IT1 synaptosomal receptor from cockroach nerve cord was measured. Modification of His-30, Lys-34, and Arg-60 showed no significant effect on biological activity. However, the modification of Lys-28 or Lys-51 demonstrated that these two amino acids are important for toxicity. Furthermore, simultaneous modifications of both Lys-28 and Lys-51 led to a cumulative decrease in biological activity. AaH IT1 and AaH IT2 show similar CD spectra. The secondary structures content of AaH IT2 was estimated from circular dichroism data. Results showed that this class of toxin should possess an additional alpha-helical region and a beta-sheet strand, not found in toxins active on mammals. Attempts to localize these secondary structural features in the amino acid sequence of AaH IT2 indicated that these two regions would be located within the last 20 C-terminal amino acid residues. From these studies on secondary structures, it is possible to consider that toxins active on insects are more structurally constrained than those active on mammals; a decreased molecular flexibility may be, at least partially, responsible for the observed specificity of these toxins for the insect sodium channel. Furthermore, the two alpha-helices found in insect toxins enclosed the two conserved Lys-28 and Lys-51 and might thus be implicated in the toxic site of insect toxins.

The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion alpha-toxins on their antigenic and toxic properties.

The complete amino acid sequence (64 residues) of the AaH IV toxin from the scorpion Androctonus australis Hector was determined by automated Edman degradation and was compared with the sequences of other Androctonus toxins. AaH IV was also tested by radioimmunoassay for binding to antisera raised against other toxins of the same species. The results indicated that AaH IV shares some of the antigenic properties of AaH I and AaH III toxins, but does not cross-react with anti-AaH II antibodies. The structural basis for the observed antigenic relationships can be found in the high degree of homology displayed by AaH IV with regard to AaH I and III, the changes in amino acid residues equally affecting regions included or excluded from the main predicted antigenic sites of AaH IV. The lower biological potency of AaH IV is presumably the result of some of the sequence differences. In particular, substitution affecting the charge and bulkiness of residue 61 could account for the poor receptor binding and consequential weak toxic properties of this molecule.