Christiane Devaux

Characterization of Amm VIII from Androctonus mauretanicus mauretanicus: a new scorpion toxin that discriminates between neuronal and skeletal sodium channels

The venom of the scorpion Androctonus mauretanicus mauretanicus was screened by use of a specific serum directed against AaH II, the scorpion alpha-toxin of reference, with the aim of identifying new analogues. This led to the isolation of Amm VIII (7382.57 Da), which gave a highly positive response in ELISA, but was totally devoid of toxicity when injected subcutaneously into mice. In voltage-clamp experiments with rat brain type II Na+ channel rNa(v)1.2 or rat skeletal muscle Na+ channel rNa(v)1.4, expressed in Xenopus oocytes, the EC50 values of the toxin-induced slowing of inactivation were: 29+/-5 and 416+/-14 nM respectively for AmmVIII and 2.6+/-0.3 nM and 2.2+/-0.2 nM, respectively, for AaH II interactions. Accordingly, Amm VIII clearly discriminates neuronal versus muscular Na+ channel. The Amm VIII cDNA was amplified from a venom gland cDNA library and its oligonucleotide sequence determined. It shows 87% sequence homology with AaH II, but carries an unusual extension at its C-terminal end, consisting of an additional Asp due to a point mutation in the cDNA penultimate codon. We hypothesized that this extra amino acid residue could induce steric hindrance and dramatically reduce recognition of the target by Amm VIII. We constructed a model of Amm VIII based on the X-ray structure of AaH II to clarify this point. Molecular modelling showed that this C-terminal extension does not lead to an overall conformational change in Amm VIII, but drastically modifies the charge repartition and, consequently, the electrostatic dipole moment of the molecule. At last, liquid-phase radioimmunassays with poly- and monoclonal anti-(AaH II) antibodies showed the loss of conformational epitopes between AaH II and Amm VIII.

CD26 modulates nociception in mice via its dipeptidyl-peptidase IV activity.

BACKGROUNDnCD26 is a multifunctional cell surface glycoprotein expressed by T and B cells. It exhibits a dipeptidyl-peptidase activity (DPP-IV) that cleaves the penultimate proline from the N-terminus of polypeptides, thereby regulating their activity and concentration.nnnMETHODSnUsing CD26-/- mice resulting from targeted inactivation of the gene, we examined the consequences of a DPP-IV defect on behavioural response to nociceptive stimuli and concentration of the pain modulator peptides substance P (SP) and endomorphin 2, two DPP-IV substrates.nnnRESULTSnCD26 inactivation induced a three-fold decrease in circulating endopeptidase activity while that found in brain extracts was normal, albeit very weak. CD26-/- mice had high SP concentrations in plasma (3.4+/-1 pg/ml versus 1.5+/-0.3 pg/ml, P<10(-3)) but not in brain extracts (35+/-12 pg/ml versus 32+/-9 pg/ml, P>0.05). Endomorphin-2 levels in the two groups were in the same range for plasma and brain extracts. CD26-/- mice displayed short latencies to nociceptive stimuli (hot plate test: 6.6+/-1.2 s versus 8.6+/-1.5 s, P<10(-4); tail pinch test: 3.1+/-0.6 s versus 4.2+/-0.8 s, P<10(-3)). Administration of an SP (NK1) receptor antagonist or DPP-IV to CD26-/- mice normalised latencies. DPP-IV inhibitors decreased latencies only in CD26+/+ mice.nnnCONCLUSIONSnOur observations represent the first fundamental evidence showing that DPP-IV influences pain perception via modulation of the peripheral SP concentration. Our work also highlights the role of peripheral NK1 receptors in nociception.

Monoclonal antibodies against the Androctonus australis hector scorpion neurotoxin I: characterisation and use for venom neutralisation

A series of monoclonal antibodies (mAbs) specific for the α‐neurotoxin I (Aah I) from the venom of the dangerous Androctonus australis hector scorpion were obtained using carrier protein‐coupled toxin. Competitive RIA, receptor assays and mouse toxicity tests were performed to characterise mAbs in terms of affinity and neutralisation. Cross‐reactivity studies and two‐site ELISA results allowed some classification of mAbs into three groups. One mAb, 9C2, was particularly interesting since it recognised the parent toxin I with a K D of 0.15 nM and was also reactive with toxins of the same immunological group. Its ability to neutralise the toxic effect of the parent toxin and the venom fraction has been investigated. This anti‐Aah I mAb 9C2, associated with anti‐Aah II mAb 4C1, provides a valuable tool to neutralise the toxicity of the venom.

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 (Cys13) and C4 (Cys19), 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, [A12,A20]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.

In vivo neurotoxicity of Androctonus australis hector scorpion venom: evidence that the supra-thoracic nervous system is not implicated in the clinical manifestations.

The severity of scorpion stings is related to the highly active neurotoxins in the venom. In this study, rats whose supra-spinal central nervous system was deprived of its peripheral connections were experimentally poisoned by the venom of Androctonus australis hector scorpion. Clinical signs of severity were not modified when the rats had previously undergone high medullar section. These results suggest that the supra-thoracic nervous system is not implicated in the neurotoxicity manifestations of scorpion envenomation.