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Venomous function is investigated in relation to innate immune function in two cases selected from scorpion venom and serpent venom. In the first case, structural analysis of scorpion toxins and defensins reveals a close interrelation between both functions (toxic and innate immune system function). In the second case, structural and functional studies of natural inhibitors of toxic snake venom phospholipases A2 reveal homology with components of the innate immune system, leading to a similar conclusion. Although there is a clear functional distinction between neurotoxins, which act by targeting membrane ion channels, and the circulating defensins which protect the organism from pathogens, the scorpion short toxins and defensins share a common protein folding scaffold with a conserved cysteine-stabilized alpha-beta motif of three disulfide bridges linking a short alpha helix and an antiparallel beta sheet. Genomic analysis suggests that these proteins share a common ancestor (long venom toxins were separated from an early gene family which gave rise to separate short toxin and defensin families). Furthermore, a scorpion toxin has been experimentally synthetized from an insect defensin, and an antibacterial scorpion peptide, androctonin (whose structure is similar to that of a cone snail venom toxin), was shown to have a similar high affinity for the postsynaptic acetylcholine receptor of Torpedo sp. Natural inhibitors of phospholipase A2 found in the blood of snakes are associated with the resistance of venomous snakes to their own highly neurotoxic venom proteins. Three classes of phospholipases A2 inhibitors (PLI-α, PLI-β, PLI-γ) have been identified. These inhibitors display diverse structural motifs related to innate immune proteins including carbohydrate recognition domains (CRD), leucine rich repeat domains (found in Toll-like receptors) and three finger domains, which clearly differentiate them from components of the adaptive immune system. Thus, in structure, function and phylogeny, venomous function in both vertebrates and invertebrates are clearly interrelated with innate immune function.

9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L−agarose beads yielding 1–2 mg·L−1 of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.

Les venins de scorpions sont caracterises par le grand nombre et la diversite des neurotoxines actives sur les canaux ioniques membranaires. Ces neurotoxines constituent de veritables familles de molecules peptidiques polymorphes de haute specificite auxquelles s'apparentent structurellement les def ensines, famille de peptides antimicrobiens circulants presents dans l'hemolymphe des scorpions et dans celle d'autres ordres d'arthropodes. La diversification et la specificite fonctionnelles a partir d'un schema stucturel commun sont discutees d'un point de vue physiologique et evolutif.

A102 is a monoclonal antibody raised against the hemocyanin of the Tunisian scorpion Androctonus australis. It is directed against the subunit Aa6 and does not cross-react when tested against a variety of similar scorpion hemocyanins. Here, we report the construction of a plasmid encoding a recombinant enzyme-linked antigen-binding protein with the antigen-binding specificity of antibody A102. The DNA fragments encoding the variable domains of A102 were inserted into a prokaryotic expression vector so as to produce a single chain antibody variable fragment (scFv) fused to the bacterial alkaline phosphatase. The fusion protein preserved the IgG binding and alkaline phosphatase activities. Immunoelectron microscopic analysis showed that the recombinant protein bound antigen bivalently as is the case for natural antibodies. Crude preparations containing the conjugate were used in a rapid visual immunoassay for the specific detection of A. australis hemocyanin, using a droplet of hemolymph removed from live animals by puncture. The simplicity of the test made it suitable for the direct identification of animals belonging to this species. It could be useful in areas where A. australis, the most dangerous African scorpion, is found with other species from which it is not easy to distinguish using morphological criteria.

An electrophoretic study using polyacrylamide gradient gels and isoelectric focusing has confirmed that the ctenostome bryozoan Alcyonidium mytili Dalyell, 1847, to date considered a valid species, in fact comprises a number of cryptic species, of which three are present on the French, Danish, and Swedish coasts.

In order to determine the reactivity on hemocyanin from Androctonus australis, the reaction of superoxide anion has been investigated using pulse radiolysis. The kinetics of O2− decays have been studied in aqueous buffered media at various basic pH (8, 8.5 and 9), first in the absence and then in the presence of hemocyanin (in oxygenated solutions containing formate anion 0.16 mol•1−. We have shown that, in the presence of hemocyanin, O2− decay is a first-order process whose apparent rate constant is proportional to protein concentration (10−7 to 10−6 mol·1−1) and pH independent between 8 to 9. A second-order rate constant of 3.5±0.1·107 mol−1·1·s−1, has been deduced for the catalytic rate constant of hemocyanin with O2 • . Meanwhile, this activity is smaller than that described for free copper, eukaryotic Cu-Zn-SOD or some copper chelates. We have verified that apohemocyanin — the copper deprived protein — does not exhibit such an activity vs. SOD (superoxide dismutase).

In order to establish a venom fingerprint and a peptide profile of the Lasiodora parahybana tarantula venom gland, we used conventional methods such as reversed phase liquid chromatography coupled to an electrospray-ionisation hybrid quadrupole time of flight mass spectrometer (LC/ESI-QqTOFMS), matrix-assisted laser desorption/ionization time-of-flight-MS (MALDI-TOFMS) and direct study of L. parahybana venom by nanospray-ionization QqTOFMS (nanoESI-QqTOFMS) and a new technology for the direct analysis of fresh tissues using MALDI-TOFMS. The analysis of the crude venom allowed the characterization of specific juvenile and adult biomarkers. In situ MALDI analysis of L. parahybana venom gland sections revealed different peptide expression levels all along the gland and non-processed peptide precursors, demonstrating the power of the method for the dynamic investigation of peptide evolution in the venom gland of spiders.

Antibody-based therapy is the only specific treatment for scorpion envenomation. However, there are still major drawbacks associated with its use; mainly because antivenoms are still prepared from immune equine serum raised against crude venoms, whereas only a limited number of neurotoxins are responsible for the lethality of the venom. Using a murine hybridoma that secretes a well-characterized neutralizing IgG directed to neurotoxins AahI and AahIII from the venom of the scorpion Androctonus australis, we constructed a recombinant Fab (rFab) fragment, which was produced and purified from transformed bacteria. It recognized toxin AahI with a high affinity ðKD ¼ 8:2 £ 10 211 MÞ equivalent to the homologous pFab prepared by papain digestion of whole IgG. Although the AahI-neutralizing capacity of protein L-purified rFab was low compared to other recombinant antibody formats (scFv and diabody) investigated in parallel, the antibody engineering approach presented here provides an innovative way to synthesize novel toxin-neutralizing molecules. It may serve as a strategy for designing a new generation of antivenoms. q 2003 Elsevier Ltd. All rights reserved.

Diabodies are recombinant, dimeric, antibody-based molecules composed of two non-covalently associated single-chain antibody fragments that bind to an antigen in a divalent manner. In an attempt to develop more effective therapeutic molecules against scorpion venoms, we designed a diabody derived from monoclonal antibody 9C2, which neutralizes the toxicity of scorpion neurotoxin AahI in mammals. The recombinant diabody produced in the periplasm of Escherichia coli was purified to homogeneity in a single step by protein L-agarose affinity chromatography. It was functional, and possessed a high binding affinity to AahI (8 x 10(-11) M). The bivalence of the diabody was confirmed by size-exclusion chromatography, isoelectrofocussing and electron microscopic observations. Finally, the diabody showed high thermal stability in serum and demonstrated protective activity when injected intraperitoneally in mice experimentally envenomed with toxin AahI. In conclusion, the diabody format gives the 9C2 molecule advantageous properties that are particularly important for potential clinical applications in the treatment of envenomations.

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.

We have isolated, from the hemolymph of unchallenged scorpions of the species Androctonus australis, three distinct antimicrobial peptides, which we have fully characterized by Edman degradation, electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry. Two are novel molecules: (i) androctonin, a 25-residue peptide with two disulfide bridges, active against both bacteria (Gram-positive and Gram-negative) and fungi and showing marked sequence homology to tachyplesins and polyphemusins from horseshoe crabs; and (ii) buthinin, a 34-residue antibacterial (Gram-positive and Gram-negative) peptide with three disulfide bridges. The third peptide contains 37 residues and three disulfide bridges and clearly belongs to the family of anti-Gram-positive insect defensins. We have synthesized androctonin and explored its activity spectrum and mode of action.

1. 1. Desert scorption are exceedingly resistant to ionizing irradation. It has previously been assumed that scorpion haemocyanin(blue blood pigment) plays an important role in this radioresistance because of its copper content, oxyphoric properties and catalase-like activity. 2. 2. In the present paper we describe eight monomers and a 55,000 mol. wt proteolytic fragment that have high catalase-like activity. 3. 3. Purified apohaemocyanin (copper-free protein) does not display oxyphoric and/or catalase-like properties. The concerted synthesis of nucleotide triphosphate in the presence of H 2 O 2 and catalase-like activity is reported for haemocyanin and its subnits. 4. 4. Measurements were carried out using luciferin-luciferase assays and HPLC in order to analyze the mechanism of 3 H-ADP transformation into 3 H-ATP. 5. 5. The catalase-like activity and H 2 O 2 disproportionation of haemocyanin and its subunits, which are thus strictly copper dependent, seem to be the two main functions of haemocyanin and its subunits (monomers and the proteolytic fragments) in the resistance of the scorpion to ionizing radiation. 6. 6. This is probably because ionizing radiation induces production of superoxide ions, hydroxyl-free radicals and hdyrogen peroxide, which oxidize susceptible target molecules, and hence induces biological changes. 7. 7. Haemocyanin, representing 80–90% of the haemolymph proteins in the scorpion, may play an important role in cellular detoxication of H 2 O 2 due to its catalase-like activity. 8. 8. Thus it is possible that this pigment and its subunits play a role in the high resistance of the scorpion to ionizing radiation.

1. The effects of some neurotransmitter or precursor of neurotransmitters aminoacids on the spontaneous electrical activity of the central nervous system of the scorpions were studied. 2. L -glutamic acid and L -tryptophan acted as stimulating substances, glycine and taurine as inhibitors. GABA was slightly stimulating for low doses, inhibitor for high doses. 3. All the stimulating drugs tested were antagonized by glycine. 4. Owing to the difference in the activity between GABA and GHB, a structural analogue of GABA, the physiological role of the epineural connective structures of the cerebroid ganglia is discussed.

Summary Structural modifications of the scorpion haemocyanin induced by pH variations and salt addition are studied by U.V. absorption, fluorescence, circular dichroism and light scattering. Haemocyanin fluorescence is due to both aromatic aminoacids tyrosine and tryptophan. Deoxygenation or denaturation lead to a fourfold enhancement of its intensity. At acidic pH the active site is modified and the protein is dissociated, but at alkaline pH the haemocyanin aggregates. The addition of different salts (sodium citrate, potassium bromide and iodide…) involves protein dissociation, the amplitude of which depends on the anion. But pH variations and salt addition don't change the haemocyanin secondary structure as shown by circular dichroism. The C.D. spectrum of scorpion haemocyanin exhibits the characteristic bands of Arthropod haemocyanin.

1. 1. A monoclonal antibody has been produced against the external subunit Aa 6 of the haemocyanin of the scorpion Androctonus australis (Buthidae). 2. 2. This monoclonal antibody cross-reacts with some, but not all, the haemocyanins of the family Buthidae using ELISA and PAGE immunoblotting. 3. 3. The intramolecular localization of the epitope is well-preserved within all the haemocyanins on which it is present. 4. 4. These results are correlated with morphological characteristics of the animals and may be used for a revision of the scorpion systematics.

1. 1. Scorpions are exceedingly resistant to ionizing irradiations. 2. 2. Hemocyanin, the blue pigment of hemolymph, is undoubtedly an important factor in this resistance because of its copper content and oxyphoric properties, and of its catalatic activity demonstrated here. 3. 3. These characteristics, found in crude or purified hemocyanin and in its heavy dissociated products (i.e. dodecamers and hexamers) made it possible to show that hemocyanin neutralizes the effects of irradiation by disproportionation of the toxic H2O2 product. 4. 4. The finding that catalase is a component of this complex oxyphoric metalloprotein is an exciting discovery that warrants further study, since catalase is considered essential to cell protection against all types of irradiation.

1. The effects of carbaryl, a anticholinesterasic drug, on the spontaneous electrical activity of the prosomian nervous system and the electroretinograms of the median and lateral scorpion eyes were investigated. 2. Carbaryl increased the amplitude and the frequency of the spontaneous electrical activity, modified the median eyes response, but did not the lateral one. 3. There is no cholinergic synapses in the lateral optical path.

Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis. The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning. A single-chain Fv gene was engineered and expressed in Escherichia coli. The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo. We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations.

Hemocyanin, the copper-containing glycoprotein that serves as an oxygen carrier in the hemolymph of some arthropods and molluscs, was obtained from the blood of the scorpion Androctonus australis. Sugar analysis of the glycoprotein revealed that its carbohydrate moiety is of the N-glycosylic type. The carbohydrate chains were released from the protein by hydrazinolysis. Determination of the molecular weight and carbohydrate composition, in conjunction with methylation analysis and 500-MHz 1H-n.m.r. spectroscopy of the oligosaccharides, indicated that this hemocyanin contains glycans of the oligomannosidic-type. Their structures were found to be homogeneous; all isolated chains were identified as Man9GlcNAc2. The number of chains per molecule is 8. The finding of (non-processed) oligomannoside-type structures in scorpion hemocyanin fits the proposal [R. C. Hughes and T. D. Butters, Trends Biochem. Sci., (1981) 228–230] that glycosylation of a protein is an evolutionary marker.