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Frédéric Ducancel

Publications and source records attributed to Frédéric Ducancel.

12 recordsLinked to original sources
PubMed2005

Characterization of toxins within crude venoms by combined use of Fourier transform mass spectrometry and cloning.

The standard analytical procedure for screening the proteomic profile of a venom often relies on an appropriate combination of sample extraction, electrophoresis, reversed-phase high-performance liquid chromatography, mass spectrometry, and Edman degradation. We present in this study a new approach for venom screening based on Fourier transform mass spectrometry (FTMS) analysis directly on the crude venom. The venom chosen is a unique sample from Atractaspis irregularis, a species never studied at the molecular level previously. This snake belongs to the Atractaspidae family that is known to produce highly toxic venoms containing endothelin-like peptides called sarafotoxins (SRTXs). Nanoelectrospray-FTMS spectrum of the crude venom allowed the identification of 60 distinct compounds with molecular masses from 600 to 14,000 Da, which would have been impossible without the resolution of this kind of instrument. De novo sequencing within the entire venom confirmed the sequences of two new families of sarafotoxins, whose precursors had been cloned, and allowed the characterization of a third one. One particularly interesting point was that the propolypeptides appeared processed not in one unique compound, but rather in different length molecules ranging from 15 for the shorter to 30 amino acids for the longer. Moreover, our results clearly establish that in the case of A. irregularis only one copy of mature sarafotoxin emerges from each precursor, which is a totally different organization in comparison of other precursors of SRTXs.

Amino Acid Sequence↗
PubMed2005

Crystal structure of the complex between the monomeric form of Toxoplasma gondii surface antigen 1 (SAG1) and a monoclonal antibody that mimics the human immune response.

Toxoplasma gondii, the intracellular parasite responsible for toxoplasmosis infects more than one-third of the world population and can be life-threatening for fetuses and immunocompromised patients. The surface protein SAG1 is an important immune target, which provides a strong immune response against the invasive tachyzoite while the other forms of the parasite, devoid of SAG1 at their surface, are multiplying. In addition to this role as a "hot spot" decoy, SAG1 is predicted to act as an adhesin during host-cell attachment through its binding to proteoglycans. To begin to understand the relationships between SAG1 epitopes and the ligand-binding site, we have solved the crystal structure of the monomeric form of T.gondii SAG1 complexed to a Fab derived from a monoclonal antibody raised against tachyzoite particles. This antibody competes strongly with human Toxoplasma-specific sera, suggesting that its epitope is part of an immunodominant region present on the surface of SAG1. The structure reveals that this conformational epitope, located within the SAG1 N-terminal domain, does not overlap with the proposed ligand-binding pocket. This study provides the first structural description of the monomeric form of SAG1, and significant insights into its dual role of adhesin and immune target during parasite infection.

Amino Acid Sequence↗
PubMed2005

Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering.

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5beta- or 5alpha-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5beta- or 5alpha-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

5-alpha-Dihydroprogesterone↗
PubMed2005

A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant-negative activity.

Thyrotropin-releasing hormone is inactivated in the extracellular space by a membrane-bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14-intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C-terminal domain. PPII* was expressed in COS-7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co-transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose-dependent reduction in PPII activity and the formation of covalent PPII-PPII* heterodimers. PPII* is therefore a powerful dominant-negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.

Alternative Splicing↗
PubMed2004

Long-sarafotoxins: characterization of a new family of endothelin-like peptides.

Sarafotoxins (SRTXs) constitute a family of vasoactive peptides that were initially isolated from the venom of Atractaspis engaddensis, and that are structurally and functionally related to endothelins (ETs). Analysis of the venom of Atractaspis microlepidota microlepidota revealed several new SRTX molecules manifesting some new structural and functional characteristics. These novel SRTXs are longer by three amino acids than the previously described SRTXs, and are designated here "long-SRTXs". Six isoforms, derived from new poly-cistronic precursors, have been identified so far in the venom of this snake. One of these isoforms, designated SRTX-m, was chemically synthesized and its biological properties were studied. Our results show that SRTX-m induces toxicity in mice, mostly due to vasoconstriction, and also that it has a lower toxicity and potency than the more potent SRTX described up to now: sarafotoxin-b (SRTX-b) from A. engaddensis.

Amino Acid Sequence↗
PubMed2004

Expression of recombinant alkaline phosphatase conjugates in Escherichia coli.

The methods described in this article are relative to the use of a positive cloning/screening recombinant system for the generation in Escherichia coli of foreign proteins fused to a highly active bacterial alkaline phosphatase (PhoA) variant as reporter enzyme. Appropriate insertion of the DNA encoding the foreign peptides, proteic domains, or proteins between codons +6 and +7 of the phoa gene restores the initial frame of the phoa gene in the vector. Consequently, only recombinant clones appear as blue colonies when plating onto an agar medium containing a chromogenic substrate for PhoA. The presence of an intact PhoA signal peptide yields to a systematic secretion of the fusion proteins into the periplasm where the PhoA dimerises to its active form, and disulfides can be formed if necessary. The resultant PhoA-tagged proteins are particularly convenient novel tools that can be used in a wide range of applications, including expression, epitope mapping, histochemistry, immunoblotting, mutant analysis, and competition or sandwich ELISAs. Expression of an scFv antibody fragment derived from an IgG2a/kappa immunoglobulin specific for curaremimetic toxins from snake (named M-alpha2-3), will be used to illustrate the methods utilized for its cloning, expression in E.coli, extraction, and functional characterization.

Alkaline Phosphatase↗
PubMed2003

Crystal structure of a hydrophobic immunodominant antigenic site on hepatitis C virus core protein complexed to monoclonal antibody 19D9D6.

The first crystal structure of a complex between a hepatitis C virus (HCV) core protein-derived peptide (residues 13-40) and the Ab fragment of a murine mAb (19D9D6) has been solved, allowing determination of the recognized epitope and elucidation of its conformation. This Ab, raised against the first 120 residues of the core protein, recognizes core particles and strongly competes with anticore human Abs, suggesting that it is highly representative of the human anti-HCV core response. Its epitope lies within the first 45 aa of the protein, the major antigenic segment of core recognized both by murine and human Abs. Surprisingly, the recognized epitope (29-37: QIVGGVYLL) has an unusual preponderance of hydrophobic residues, some of which are buried in a small hydrophobic core in the nuclear magnetic resonance structure of the peptide (2-45) in solution, suggesting that the Ab may induce a structural rearrangement upon recognition. The flexibility may reside entirely within the Ag, since the Fab'-peptide complex structure at 2.34 A shows that the Ab binding site is hardly perturbed by complexation. Given that the recognized residues are unlikely to be solvent exposed, we are left with the interesting possibility that Ab-core interactions may take place in a nonaqueous environment.

Amino Acid Sequence↗
PubMed2003

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.

Alkaline Phosphatase↗
PubMed2002

Characterization and tissue-specific expression of the Drosophila transaldolase gene.

Transaldolase (TAL) is an enzyme of the non-oxidative part of the pentose phosphate pathway which produces reductive potential in the form of NADPH as well as ribose 5-phosphate for incorporation into nucleotides. Developmental analysis via reverse transcriptase-polymerase chain reaction, immunoblots, enzymatic activity, in situ hybridization and immunocytochemistry demonstrate that TAL is expressed during all developmental stages in Drosophila. The tal locus is unique and contains two small introns. The first two introns in the human gene are situated at the same locations in the coding sequence as in Drosophila. Analysis of TAL protein levels and expression patterns reveals that it is also expressed in all larval tissues examined with particularly high levels in the fat body. Interestingly, we describe specific TAL expression only in non-neuronal cells in the larval brain.

Amino Acid Sequence↗
PubMed2002

The sarafotoxins.

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Amino Acid Sequence↗
PubMed2002

A chimeric scorpion alpha-toxin displays de novo electrophysiological properties similar to those of alpha-like toxins.

BotXIV and LqhalphaIT are two structurally related long chain scorpion alpha-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhalphaIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect alpha-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8-10, 19-22, and 38-43. To evaluate their functional role, three BotXIV/LqhalphaIT chimeras were designed by transferring the corresponding LqhalphaIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular alpha/beta fold. Interestingly, substitution of residues 8-10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of alpha-like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure-function relationships between insect alpha-toxins and alpha-like scorpion toxins are probably more complex than expected.

Amino Acid Sequence↗