Induction with a synthetic peptide of antibodies to HLA class I C-terminal intracytoplasmic region.

Site-specific antibodies to HLA class I molecules have been raised in rabbits immunized with a synthetic peptide with the same amino acid sequence as HLA residues 328-338, which corresponds to the highly conserved intracytoplasmic region. Antibodies were detected by radioimmunoassay and were able to recognize isolated HLA heavy chains blotted onto nitrocellulose as well as the biosynthetically labeled HLA-beta 2 microglobulin complexes solubilized by non-ionic detergents. The intracellular localization of the determinants recognized by the antibodies was shown by indirect immunofluorescence labeling and the specificity of the reaction confirmed by its inhibition with the synthetic peptide. No cross-reaction was seen with H-2 antigens on murine cells. These antibodies will be important for further characterization of HLA antigens and detection of their expression in mouse cells transfected with human genes.

Evidence for a new class of scorpion toxins active against K+ channels.

cDNAs encoding novel long-chain scorpion toxins (64 amino acid residues, including only six cysteines) were isolated from cDNA libraries produced from the venom glands of the scorpions Androctonus australis from Old World and Tityus serrulatus from New World. The encoded peptides were very similar to a recently identified toxin from T. serrulatus, which is active against the voltage-sensitive 'delayed-rectifier' potassium channel, but they were completely different from the long-chain and short-chain scorpion toxins already characterised. However, there was some sequence similarity (42%) between these new toxins, Aa TX Kbeta and Ts TX Kbeta, and scorpion defensins purified from the hemolymph of Buthidae scorpions Leiurus quinquestriatus and A. australis. Thus, according to a multiple sequence alignment using CLUSTAL, these new toxins seem to be related to the scorpion defensins.

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus.

A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.

Biochemical, pharmacological and genomic characterisation of Ts IV, an alpha-toxin from the venom of the South American scorpion Tityus serrulatus.

The venom of the scorpion, Tityus serrulatus, was fractionated to investigate the chemical and pharmacological properties of its alpha-toxin content. Three alpha-toxins (Ts III, Ts IV and Ts V) were purified by conventional chromatography (gel filtration and ion-exchange chromatography), followed by immunoaffinity chromatography. Competition experiments using reference alpha- and beta-toxins suggested that these alpha-toxins were contaminated with around 0.01% of beta-toxin. The sequence of the first 30 amino acids of Ts IV was established. Using an oligonucleotide probe, a cDNA encoding its precursor was cloned from a venom gland cDNA library. The primary structure deduced from the cDNA nucleotide sequence provides possible explanations for the polymorphism of these three molecules.

Antibodies raised against a peptide corresponding to a Gs alpha domain reveal a vimentin-associated protein.

In an attempt to localize the guanine nucleotide binding protein Gs alpha by immunocytochemistry, we synthetized peptides corresponding to several stretches of residues deduced from the published cDNA sequence of Gs alpha and raised antibodies against them. Among the peptides, one corresponding to residues 367-381 elicited antibodies that immunocytochemically detected, at the optical level, what appeared to be vimentin in several cells and tissues. Studies at the ultrastructural level confirmed this observation and also showed weak staining of some areas of plasma membranes of glial and nerve cells. Analysis by Western blots of rat brain subcellular fractions indicated that: (a) the protein stained by the anti-peptide antibodies was associated with the cytoskeleton; and (b) it was not vimentin but a protein of higher molecular weight, 65 KD. We accordingly suggest that the Gs alpha-derived peptide elicited two types of antibodies, one recognizing Gs alpha in fixed tissues, the other recognizing an epitope located in a vimentin-associated protein. This study emphasizes the caution that is needed when conclusions are drawn on the basis of immunocytochemical studies using antipeptide antibodies.

Accessibility of the highly conserved amino- and carboxy-terminal regions from HIV-1 external envelope glycoproteins.

Amino- and carboxy-terminal extremities of the envelope external glycoproteins are regions that have remained highly conserved between human immunodeficiency viruses HIV-1 and HIV-2. The corresponding peptides have been synthesized and their structure and function analyzed. Circular dichroism spectra showed evidence of alpha helical conformation when the peptides were dissolved in the nonpolar solvent trifuoroethanol. These two regions are indeed exposed on the molecule because they were accessible to their respective specific antibodies on the native gp160 precursor or processed gp120 glycoproteins of HIV-1. Neither the peptides nor rabbit or human antibodies directed against the N- and C-terminal peptides interfered with the interaction between HIV-1 external glycoprotein gp120 and its CD4 cellular receptor. Taken together, these results indicate that N- and C-terminal regions of gp120 are accessible on the quaternary structure of the virion as well as on the soluble form of gp120 and that these regions are not directly or indirectly involved in the binding of gp120 to CD4.

Analysis by high-performance liquid chromatography of Androctonus mauretanicus mauretanicus (black scorpion) venom.

The venom of the black scorpion, Androctonus mauretanicus mauretanicus, was obtained by means of manual stimulation and was analyzed using high-performance liquid chromatography. Starting from 20 mg of venom and using only two chromatographic steps, six toxins were purified to homogeneity. They have been characterized by their amino acid content and compared to those already isolated from a pool of venoms obtained using electric stimulation (Rosso and Rochat, Toxicon 23, 113-125, 1985). The toxins Amm I and Amm II were not found, suggesting either different levels of toxin expression or the existence of Androctonus mauretanicus mauretanicus subspecies. Using rat brain synaptosomes, it was demonstrated that the toxins Amm III, Amm IV and Amm V were alpha-toxins. The toxin Amm VI was neither alpha- or beta-toxin. Unexpectedly, the toxin Amm VII was found to be a beta-toxin, the first one identified in a north African scorpion venom. In addition, some toxins active on mammals exhibited different levels of specificity towards phylogenetically related groups of arthropods.

Purification of the main beta-toxin from Tityus serrulatus scorpion venom using high-performance liquid chromatography.

The venom of the Brazilian scorpion Tityus serrulatus was fractionated using high-performance liquid chromatography which allowed us to purify in two steps the main beta-type toxin of the venom. The toxin constituted about 15% of the absorbance at 280 nm and 50% of the toxicity of the venom. According to its amino acid content, its electrophoretic migration on Phast-Gel homogenous 20 and its biological properties both in vivo by intracerebroventricular injection to the mouse (LD50 = 30 ng/kg mouse) and in vitro by competition receptor assay on rat brain synaptosomes (K0.5 = 80 pM), the toxin was identified as toxin Ts VII already purified from the same venom using low-pressure liquid chromatography (BECHIS et al., 1984 Biochem. biophys. Res. Commun. 122, 1146). The high-performance liquid chromatographic technique used improved by a factor of four the amount of toxin purified.

Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus.

The full-length cDNA encoding the scorpion alpha-toxin Amm V was amplified from a cDNA library produced from the venom glands of the scorpion Androctonus mauretanicus mauretanicus from Morocco. We deduced the amino acid sequence of the encoded precursor protein and found that the mature toxin was similar to the previously characterised toxin. The genomic DNA sequence encoding the toxin was also amplified, subcloned and sequenced. This also led to the isolation of a new Amm V related-gene. Then, for the first time, we studied changes in the level of toxin mRNA synthesis over time.

Localization of the toxic site of Naja mossambica cardiotoxins: small synthetic peptides express an in vivo lethality.

Cardiotoxins are small basic proteins which cause heart failure when they are injected in vivo. In order to better understand their molecular mode of action, short peptides designed on the model of the first loop of the molecule of cardiotoxin IV from Naja mossambica mossambica venom have been synthetized by the solid-phase procedure of Merrifield. These peptides express lethality in mouse when they are injected intravenously. Taking into account the respective molecular weights, they are 3.5 to 5% as toxic as the cardiotoxin. Furthermore, the symptomatology they induce is undistinguishable from that induced by cardiotoxins. These results strongly support our previous hypothesis that the first loop of the molecule is the toxic site of cardiotoxins.

Structural analysis of the interaction of apamin with Ia and its recognition by Ad- or Ab-restricted mouse T cells.

Apamin is a single-chain, disulfide-bonded, 18-amino acid peptide that elicits mouse T cell responses when presented by cells expressing syngeneic Ad or Ab class II MHC molecules. We previously showed that both the unfolding of this peptide by APC and the integrity of its N terminus segment were required for efficient apamin T cell recognition. To seek further information on the sites through which this peptide interacts with Ia and/or TCR, we used a panel of Ad- or Ab-restricted, apamin-specific THC to probe the antigenicity of a series of synthetic apamin analogs. These included peptides either truncated at the N terminus, or substituted by Ala at position 2, 4, 6, 7, 8, or 10. Analysis of THC responses to apamin analogs and use of the latter in competition assays for peptide presentation revealed the following: 1) optimal apamin T cell recognition critically involved Lys4, Ala5, Pro6, Glu7, and Leu10. The role of these residues in either "Ia or TCR binding regions" was found to depend upon the restricting Ia molecules at play. Thus, Lys4, Glu7, and Leu10 were TCR-binding residues in both Ad- and Ab-apamin complexes, whereas Lys4 participated in apamin/Ab but not, or to a marginal extent, in apamin/Ad interaction. Furthermore, Pro6 was associated either with an Ia contact region or a TCR interaction site when apamin was presented by Ab or Ad molecules, respectively. Unfolded apamin and the unrelated chicken OVA323-339 peptide were found to bind to the same, or closely related site(s) of Ad, as shown by their ability to compete reciprocally for recognition by appropriate Ad-restricted THC. Four distinct TCR V beta genes (V beta 2, V beta 4, V beta 6, and V beta 8) were found to be used in our panel of 16 apamin-specific THC. These data indicate that apamin interacts with Ad or TCR through a motif resembling other beta-sheeted, Ad-binding sequences; however, based on the spacing of the critical residues (i.e., 4, 7, and 10), the possibility exists that apamin processing permits the folding of this sequence into an alpha-helix.

Binding and toxicity of apamin. Characterization of the active site.

The structural features of apamin, a natural octadecapeptide from bee venom, enabling binding to its receptor and the expression of toxicity in mice, have been delineated by studying the effects on binding and toxicity of chemical modifications and amino acid substitutions in synthetic analogues. The results obtained indicate that the only hydrophobic residue, leucine at position 10, can be changed to alanine without a significant decrease in the specific activity. The need for a correct conformation has been established and also the importance of Gln-17 and the side chains of Arg-13 and Arg-14 (besides the charge effects). The interaction of apamin with its receptor, a calcium-activated potassium channel, is thus mediated by a precise topology around these three residues. Due to the ability to detect very low specific activities for some of the analogues, it has been shown that, individually, none of these interactions constitute an essential criteria for binding per se, but that their presence is necessary for the high specific activity of the toxin.

Use of fusion protein constructs to generate potent immunotherapy and protection against scorpion toxins.

We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal alpha-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I+II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1nM range. In vitro neutralisation assays indicated that 1ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD(50) of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD(50) of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.

The kaliotoxin family enlarged. Purification, characterization, and precursor nucleotide sequence of KTX2 from Androctonus australis venom.

Kaliotoxin (KTX) has been originally described as an inhibitor of the intermediate conductance Ca(2+)-activated K+ channel (Crest, M., Jacquet, G., Gola, M., Zerrouk, H., Benslimane, A., Rochat, H., Mansuelle, P., and Martin-Eauclaire, M.-F. (1992) J. Biol. Chem. 267, 1640-1647). However, the radioiodinated 125I-KTX-(1-37) was also able to bind to the dendrotoxin sensitive voltage-dependent K+ channel (Romi, R., Crest, M., Gola, M., Sampieri, F., Jacquet, G., Zerrouk, H., Mansuelle, P., Sorokine, O., Van Dorsselaer, A., Rochat, H., Martin-Eauclaire, M.-F., and Van Rietschoten, J. (1993) J. Biol. Chem. 268, 26302-26309). By following the ability to compete with 125I-KTX-(1-37) for binding to its receptor on rat brain synaptosomes, a new kaliotoxin-like peptide, KTX2, was isolated from Androctonus australis scorpion venom. It is a 37-amino acid residue peptide, and its sequence shares 76% identity with KTX. The differences between the two peptides concern the NH2-terminal region and the residues 31 and 34 located in the region involved in the channel recognition. These differences may explain the 5-fold decrease of the molluscan Ca(2+)-activated K+ channel blockage by KTX2 (kd = 135 nM) as well as of its binding affinity to rat brain synaptosomes (IC50 = 50 pM), compared with KTX. Specific antibodies raised against KTX-(1-37) were not able to recognize KTX2. Using degenerate primers, a 370-base pair cDNA encoding the KTX2 precursor was amplified by polymerase chain reaction from a cDNA library of A. australis venom glands. It encoded a presumed signal peptide of 22 residues followed by the sequence of the mature peptide.