Biomedical subjects
Publications and source records attributed to Christian Legros.
In this work, we used a panel prokaryote/eukaryote K+ channel chimeras to generate K+ channel arrays. Their behaviour in solution was compared with that when spotted on a nitrocellulose-supported film and their responses to selective high affinity ligands, including polypeptide toxins and TEA, were studied.
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.
The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.
The AaH II toxin from the scorpion Androctonus australis Hector is considered to be the standard alpha-toxin because it selectively binds with the highest known affinity to site 3 of mammalian voltage-activated Na+ channels (Na(v)) on rat brain synaptosomes but does not bind to insect synaptosomes. We generated two different constructs in pMALp allowing us to produce AaH II fused with the maltose-binding protein (MBP) in E. coli. We obtained reasonable amounts of recombinant AaH II after cleavage by enterokinase at the site DDDDK. We show that the introduction of a net negative charge at the C-terminus by the suppression of H64 amidation and the addition of an extra residue to the C-terminus (G65) led to fully active AaH II mutants, exhibiting exactly the same affinity as the native toxin for its target on rat brain synaptosomes. In contrast, the mutation of residue K58 into V, I or E residues drastically reduced toxin activity.
In order to find new peptide inhibitors for voltage-dependent potassium (Kv) channels, we examined the effects of venom from Theraphosa leblondi on Kv channel-mediated currents with the whole-cell patch-clamp technique. Both A-type currents in cultured hippocampal neurons and A-type currents recorded from HEK 293 cells transiently expressing recombinant Kv4.2 channels were selectively inhibited by T. leblondi venom. No venom activity was observed on recombinant Kv1.3, Kv1.4, Kv2.1 or Kv3.4 channels. We purified and sequenced three novel homologous peptides from this venom, which are related to previously identified Kv4 channel-specific peptide inhibitors and were named T. leblondi toxin (TLTx) 1, 2 and 3. The mode of action of TLTx1 on recombinant Kv4.2 channels was studied in more detail. TLTx1 inhibited Kv4.2-mediated currents with an IC50 of approximately 200 nM, and macroscopic current inactivation was slowed in the presence of TLTx1. Notably, TLTx1 also caused a shallower voltage dependence of Kv4.2 peak conductance and a shift of the activation midpoint to more positive potentials (DeltaV1/2 = +35 mV). TLTx1 caused a noticable slowing of Kv4.2 activation kinetics, and Kv4.2 deactivation kinetics were accelerated by TLTx1 as infered from Rb+ tail current measurements. Chimeric Kv2.1(4.2L3-4) channels, in which the linker region between S3 and S4 of the TLTx1-insensitive Kv2.1 channel was replaced by the corresponding Kv4.2 domain, were sensitive to TLTx1. Apparently, TLTx1 can act as a gating modifier of Kv4.2 channels.
Mass spectrometric methods, including matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS), on-line liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS), and nanospray ionisation/hybrid quadrupole time-of-flight mass spectrometry (nanoESI-QqTOFMS), were applied to characterize by mass fingerprinting the venom of the French Guyanese tarantula Theraphosa leblondi. Of these techniques direct nanoESI-QqTOFMS, which allowed the detection of 65 protonated molecules with high mass accuracy, appeared to give the best results. Three major peptides, TlTx1, TlTx2 and TlTx3, were sequenced using a combination of nanoESI-MS/MS and enzyme digestion/MS and MS/MS experiments. Each sequence was confirmed by automated Edman sequencing. In patch-clamp experiments these peptides were found to have a specific inhibitory effect on the voltage-dependent potassium channel, Kv4.2.
A recombinant peptidic spider toxin, HpTx2, was investigated directly by nanoelectrospray tandem mass spectrometry (MS/MS). This 30-residue toxin possesses a highly knotted structure with cystines arranged in close proximity. The low-energy collision-induced dissociation MS/MS spectrum of the [M+4H](4+) ion permitted characterization of the C-terminal sequence of HpTx2 up to Cys(26) that is involved in a disulfide bridge. Chemical pre-treatment with DTT or TCEP was then investigated, and it was found that an unexpected cleavage reaction of HpTx2 gave two smaller peptides which were completely sequenced by MS/MS experiments using a Qq-TOF mass spectrometer. This unusual hydrolysis reaction facilitated the determination of the complete sequence of the HpTx2 toxin.
A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.
Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K(+) currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron-exon organisation of the gene corresponding to the AaTX1 cDNAs was also depicted.
The bacterial potassium channel, KcsA, can be modified to express a high-affinity receptor site for the scorpion toxin kaliotoxin (KTX) by substituting subregion I in the P region of KcsA with the one present in the human voltage-gated potassium channel Kv1.3 [Legros, C., Pollmann, V., Knaus, H. G., Farrell, A. M., Darbon, H., Bougis, P. E., Martin-Eauclaire, M. F., and Pongs, O. (2000) J. Biol. Chem. 275, 16918-16924]. This approach opened the way to investigate whether sequence differences in subregion I of Kv1 channels correlate with the distinct pharmacological profiles of peptide inhibitors. A panel of six chimeras between KcsA and human Kv1.1-6 were constructed, expressed in Escherichia coli, purified to homogeneity, and assessed in filter binding assays using either monoiodo-tyrosine-KTX ([(125)I]KTX) or monoiodo-tyrosine-hongotoxin(1)(A19Y/Y37F) ([(125)I]HgTX(1)(A19Y/Y37F)). The KcsA-Kv1.X chimeras were found to have lower affinities for these ligands than the corresponding mammalian Kv1.X channels, indicating that other parts of the channels may contribute to binding or that subtle structural differences exist between these channels. The properties of the KcsA-Kv1.X chimeras were also characterized in surface plasmon resonance experiments. KcsA-Kv1.3 chimeras were immobilized on the surface of a sensor chip for determining, in real time, binding of the peptides. KTX binding properties to immobilized KcsA-Kv1.3 chimera were similar to those determined by filtration techniques. Taken together, our results demonstrate that the pharmacological profile of peptide toxins can be incorporated into KcsA-Kv1.X chimeras containing the subregion I of the corresponding mammalian Kv1.X channels. This innovative approach may facilitate the high-throughput screening of ligand libraries aimed at the discovery of novel potassium channel modulators.