Biomedical subjects

F Ducancel

Publications and source records attributed to F Ducancel.

At least 37 records · Page 2Linked to original sources

PubMed1996

Structural model of the anti-snake-toxin antibody, M alpha 2,3.

We present results of structural modeling of the variable fragment of M alpha 2,3, an antibody capable of neutralizing all short snake toxins. Three different methods were used to model the hypervariable loops: the conformational search algorithm CONGEN (Bruccoleri and Karplus, Biopolymers 26:137-168, 1987), high-temperature molecular dynamics (Bruccoleri and Karplus, Biopolymers 29:1847-1862, 1990), and a combined knowledge-based and energy-based algorithm (Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272, 1989). Ninety plausible conformations were generated and were clustered into 13 classes. The clustering results indicate that there was little overlap of the conformational space explored by the different methods. Canonical loop structures were found by all methods for two of the loops, in agreement with previously established empirical modeling criteria. Nine of the 13 classes of structure were rejected on the ground of their lacking common features of antibody combining-site structure. The remaining four models were refined using restrained molecular dynamics. It was found that interconversion between the four resulting structures is possible with no significant energy barriers, suggesting that they are in thermodynamic equilibrium at 300 K. Features of the combining-site structure likely to be particularly important for antigen binding are discussed.

Algorithms↗

PubMed1995

Recombinant antibody-alkaline phosphatase conjugates for diagnosis of human IgGs: application to anti-HBsAg detection.

We have designed an expression vector permitting the production in the periplasm of Escherichia coli of a fusion protein comprising a dimer of bacterial alkaline phosphatase (PhoA) and two Fab or scFv fragments of a monoclonal antibody directed against human IgG. Each hybrid protein expressed both high specificity for the antigen and full PhoA activity. We show that crude periplasmic extracts containing these conjugates can be used as such in enzyme immunoassays for the detection of human IgG, as exemplified in the case of anti-hepatitis B immunoglobulin.

Alkaline Phosphatase↗

PubMed1995

Genetic engineering of snake toxins. The functional site of Erabutoxin a, as delineated by site-directed mutagenesis, includes variant residues.

Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F. Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A. (1993) J. Biol. Chem. 268, 909-916). We have now studied the effect of mutating 26 new positions on the affinity of Ea for AcChoR. The mutations are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A, T13V, T14A, K15A, T16A, delta S18, E21A, Y25F, Q28A, S30A, T35A, I36R, P44V, T45A, V46A, K47A, P48Q, I50Q, and S53A. Binding affinity decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at His-6, Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36. Other mutations have no effect on Ea affinity. In addition, new mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38 better explain the functional role of these residues in Ea. The previous and present mutational analysis suggest that the "functional" site of Ea covers a homogeneous surface of at least 680 A2, encompassing the three toxin loops, and includes both conserved and variant residues. The variable residues might contribute to the selectivity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.

Amino Acid Sequence↗

PubMed1994

On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.

Amino Acid Sequence↗

PubMed1994

Precursors of vertebrate peptide antibiotics dermaseptin b and adenoregulin have extensive sequence identities with precursors of opioid peptides dermorphin, dermenkephalin, and deltorphins.

The dermaseptins are a family of broad spectrum antimicrobial peptides, 27-34 amino acids long, involved in the defense of the naked skin of frogs against microbial invasion. They are the first vertebrate peptides to show lethal effects against the filamentous fungi responsible for severe opportunistic infections accompanying immunodeficiency syndrome and the use of immunosuppressive agents. A cDNA library was constructed from skin poly(A+) RNA of the arboreal frog Phyllomedusa bicolor and screened with an oligonucleotide probe complementary to the COOH terminus of dermaseptin b. Several clones contained a full-length DNA copy of a 443-nucleotide mRNA that encoded a 78-residue dermaseptin b precursor protein. The deduced precursor contained a putative signal sequence at the NH2 terminus, a 20-residue spacer sequence extremely rich (60%) in glutamic and aspartic acids, and a single copy of a dermaseptin b progenitor sequence at the COOH terminus. One clone contained a complete copy of adenoregulin, a 33-residue peptide reported to enhance the binding of agonists to the A1 adenosine receptor. The mRNAs encoding adenoregulin and dermaseptin b were very similar: 70 and 75% nucleotide identities between the 5'- and 3'-untranslated regions, respectively; 91% amino acid identity between the signal peptides; 82% identity between the acidic spacer sequences; and 38% identity between adenoregulin and dermaseptin b. Because adenoregulin and dermaseptin b have similar precursor designs and antimicrobial spectra, adenoregulin should be considered as a new member of the dermaseptin family and alternatively named dermaseptin b II. Preprodermaseptin b and preproadenoregulin have considerable sequence identities to the precursors encoding the opioid heptapeptides dermorphin, dermenkephalin, and deltorphins. This similarity extended into the 5'-untranslated regions of the mRNAs. These findings suggest that the genes encoding the four preproproteins are all members of the same family despite the fact that they encode end products having very different biological activities. These genes might contain a homologous export exon comprising the 5'-untranslated region, the 22-residue signal peptide, the 20-24-residue acidic spacer, and the basic pair Lys-Arg.

Amino Acid Sequence↗

PubMed1994

Recombinant technology in the preparation of immunogen and enzymatic tracer. Application to the development of an enzyme immunoassay for rat prolactin.

A competitive enzyme immunoassay for rat prolactin using an immunogen and a tracer obtained by recombinant DNA technology is described. Polyclonal antibodies were raised in rabbits immunized with a purified chimeric protein consisting of rat prolactin fused with two synthetic immunoglobulin G binding domains derived from staphylococcal Protein A. The enzymatic tracer was obtained using an expression system which permits insertion of rPRL DNA sequence in the alkaline phosphatase gene. Antibodies and tracer were used to develop a solid-phase competitive immunoassay for the measurement of rat prolactin in plasma with a minimal detectable concentration of 0.5 ng/ml. The mean intra- and inter-assay coefficients of variation were 7.8 and 13.2%, respectively. Rat plasma concentrations measured with this assay correlated well with those obtained with a conventional enzyme immunoassay (r = 0.993, slope = 1.037, n = 24).

Alkaline Phosphatase↗

PubMed1994

Engineering of protein epitopes: a single deletion in a snake toxin generates full binding capacity to a previously unrecognized antibody.

Structural features associated with the ability of a monoclonal antibody (mAb) to discriminate between protein variants are identified and engineered. The variants are the curaremimetic toxin alpha from Naja nigricollis and erabutoxin a or b from Laticauda semifasciata, which differ from each other by 16 substitutions and one insertion. The neutralizing mAb M alpha 1 recognizes with high affinity a topographical epitope on the surface of toxin alpha, but fails to recognize the erabutoxins although they possess most of the residues forming the presumed epitope. Examinations of the toxin alpha and erabutoxin 3-D structures and molecular dynamics simulations reveal several differences between the variants. In particular, the region involving the beta-turn 17-24 is organized differently. Analysis of the differences found in this region suggest that the insertion (or deletion) at position 18 of the variant amino acid sequences is particularly important in determining the differential cross-reactivity. To test this proposal, residue 18 was deleted in one erabutoxin using site-directed mutagenesis, and the biological properties of the resulting mutant were examined. We found that full antigenicity was restored in the previously unrecognized variant. The implications of this finding are discussed.

Amino Acid Sequence↗

PubMed1993

Enzyme immunoassay using a rat prolactin-alkaline phosphatase recombinant tracer.

This paper describes a competitive enzyme immunoassay of rat prolactin (rPrl) using a recombinant conjugate as a colorimetric tracer. rPrl was inserted into the N-terminal end of Escherichia coli alkaline phosphatase (AP), using an expression vector which allows insertion of foreign DNA sequences between codons +6 and +7 of the phoA gene. The assay was performed in 96-well microtiter plates coated with a mouse monoclonal antibody raised against rabbit immunoglobulin G. Each component (recombinant tracer, rabbit antiserum against rPrl, and rPrl standard) was added in a volume of 50 microL. The sensitivity of the assay was sufficiently high to allow titration of rPrl in plasma. The detection threshold was 15 pg (0.3 ng/mL) and the B/B0 50% value was 150 pg (3 ng/mL). The intraassay coefficient of variation was less than 10% over a wide range of rPrl concentrations (2.9-50 ng/mL). The interassay coefficient of variation was less than 15% for rat plasma samples in the concentration range of 4-40 ng/mL. The good parallelism observed between the standard curve and sample dilution curves showed that the immunoreactivity in rat plasma behaves like standard rPrl. Together with recovery experiments, these results indicated that assay without extraction is possible. A single immunoreactive peak that comigrates with standard rPrl is observed after molecular sieve fractionation of plasma samples. The reliability of the assay was confirmed by good correlation with conventional radioimmunoassay (r = 0.996, slope 0.978).

Alkaline Phosphatase↗

PubMed1993

Molecular cloning of a cDNA encoding the precursor of adenoregulin from frog skin. Relationships with the vertebrate defensive peptides, dermaseptins.

Adenoregulin has recently been isolated from Phyllomedusa skin as a 33 amino acid residues peptide which enhanced binding of agonists to the A1 adenosine receptor. In order to study the structure of the precursor of adenoregulin we constructed a cDNA library from mRNAs extracted from the skin of Phyllomedusa bicolor. We detected the complete nucleotide sequence of a cDNA encoding the adenoregulin biosynthetic precursor. The deduced sequence of the precursor is 81 amino acids long, exhibits a putative signal sequence at the NH2 terminus and contains a single copy of the biologically active peptide at the COOH terminus. Structural and conformational homologies that are observed between adenoregulin and the dermaseptins, antimicrobial peptides exhibiting strong membranolytic activities against various pathogenic agents, suggest that adenoregulin is an additional member of the growing family of cytotropic antimicrobial peptides that allow vertebrate animals to defend themselves against microorganisms. As such, the adenosine receptor regulating activity of adenoregulin could be due to its ability to interact with and disrupt membranes lipid bilayers.

Amino Acid Sequence↗

PubMed1993

Production of recombinant notechis 11'2L, an enzymatically active mutant of a phospholipase A2 from Notechis scutatus scutatus venom, as directly generated by cleavage of a fusion protein produced in Escherichia coli.

We have constructed an expression vector to produce, in Escherichia coli, a fusion protein containing successively two IgG binding domains from staphyloccocal protein A, a nine-amino-acid linker peptide terminating in a methionine residue and the phospholipase A2 notechis 11'2L, an isoform of notexin of Notechis scutatus scutatus venom. Notechis 11'2L is a mutant of the naturally occurring notechis 11'2 [Bouchier, C., Boyot, P., Tesson, F., Trémeau, O., Bouet, F., Hodgson, D., Boulain, J. C. & Ménez, A. (1991) Eur. J. Biochem. 202, 493-500] in which Met8 has been replaced by Leu. The fusion protein was recovered in the periplasmic extract with a yield of 0.25 mg/l culture. It was hydrolyzed with cyanogen bromide, yielding a protein having the molecular mass, amino acid composition and N-terminal sequence of notechis 11'2L. Notechis 11'2L and the wild notechis 11'2 displayed identical circular dichroic spectra and shared similar enzymatic, myotoxic and antigenic properties, suggesting that the recombinant notechis 11'2L was directly generated in a correctly folded form.

Base Sequence↗

PubMed1993

Cloning and sequence analysis of cDNAs encoding precursors of sarafotoxins. Evidence for an unusual "rosary-type" organization.

Sarafotoxins (SRTXs) are 21-amino acid peptides structurally and functionally similar to endothelins (ETs). To understand how SRTXs are overproduced in venom glands of the snakes Atractaspis engaddensis and hence used as toxins, we cloned cDNAs encoding SRTXs and elucidated their nucleotide sequences. We predict that SRTX precursors are large prepropolypeptide chains with an unusual "rosary-type" structure made of 12 successive similar stretches of 40 residues (39 in the first stretch). Each stretch begins with a "spacer" of 19 invariant residues (18 in the first stretch) immediately followed by the sequence of one SRTX isoform. Six different isoforms are identified within a single precursor molecule. Maturation of the precursor may require endopeptidases that cleave the Leu-Cys bond and the Trp-Arg/Lys bond invariably found at the SRTX N and C termini, respectively.

Amino Acid Sequence↗

PubMed1993

Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis.

To study the site by which erabutoxin a (Ea) from Laticauda semifasciata binds to the nicotinic acetylcholine receptor, we mutated most residues that are shared with other curaremimetic toxins and studied the structural and biological consequences of introduced mutations. By site-directed mutagenesis, we changed Ser-8 into Gly (EaS8G), Lys-27 into Glu (EaK27E), Trp-29 into Phe (EaW29F) and His (EaW29H), Asp-31 into His (EaD31H), Phe-32 into Leu (EaF32L), Arg-33 into Lys (EaR33K) and Glu (EaR33E), Gly-34 into Ser (EaG34S), Glu-38 into Gln (EaE38Q) and Lys (EaE38K), Gly-49 into Val (EaG49V), and Leu-52 into Ala (EaL52A). All mutants were homogeneous as judged by various analytical procedures. EaE38Q, EaG49V, and EaL52A bound the nicotinic acetylcholine receptor with apparent Kd values close to 10(-10) M, virtually identical to wild Ea. Therefore, Glu-38, Gly-49, and Leu-52 are not important elements in the expression of curaremimetic function in Ea. Mutations of Phe-32 and Gly-34 provoked a 7-fold affinity decrease, suggesting that these residues moderately contribute to function. The 176-fold affinity decrease due to mutation of Ser-8 may reflect some structural change that operates in the polypeptide chain of the mutant, as detected by circular dichroism. Decreases in affinity by a factor of 175, 67, 46, and 318 were seen upon mutations of Lys-27 into Glu, Trp-29 into Phe, Asp-31 into His, and Arg-33 into Glu, with no concomitant change in secondary structure. These residues appear to be important elements of the curaremimetic function of Ea. Thus, a picture of the contribution of conserved residues to the function of a curaremimetic toxin is proposed on the basis of experimental evidence.

Amino Acid Sequence↗

PubMed1993

Recombinant colorimetric antibodies: construction and characterization of a bifunctional F(ab)2/alkaline phosphatase conjugate produced in Escherichia coli.

We have designed a vector which allows the synthesis in Escherichia coli of bifunctional F(ab)2-alkaline phosphatase conjugates. The vector contains a di-cistronic operon encoding truncated heavy chain (Fd or VH-CH1) of an IgG inserted between residues +6 and +7 of bacterial alkaline phosphatase (PhoA), and the light chain of the same IgG. We demonstrate the utility of this approach with the heavy and light chain domains of a snake toxin-specific monoclonal antibody, M alpha 2-3. We show that the VH-CH1-PhoA hybrid and VL-CL are concomitantly expressed and exported to the periplasm of E. coli where they form a disulfide-linked chimeric protein. The hybrid has the same affinity as M alpha 2-3 for the snake toxin antigen and possesses PhoA enzymatic activity.

Alkaline Phosphatase↗

PubMed1992

Role and environment of the conserved Lys27 of snake curaremimetic toxins as probed by chemical modifications, site-directed mutagenesis and photolabelling experiments.

The positive charge of Lys27 was suppressed by chemical means in two short-chain curaremimetic toxins, namely erabutoxin a (Ea) from Laticauda semifasciata and toxin alpha from Naja nigricollis. This modification leads to a decrease in the binding affinity of the toxins for the nicotinic acetylcholine receptor, which range 6-15-fold, as judged from both the data reported here and those previously described in the literature. A negatively charged glutamate residue has been introduced at position 27 of erabutoxin a by site-directed mutagenesis. This change provokes a 120-fold decrease in the affinity, which reflects a major alteration of toxin-receptor cognate events. Using toxin-alpha derivative harbouring a photoactive group at Lys27, we probed the toxin local environment in a receptor-bound state by photocoupling experiments. The delta chain was the predominant coupling target, in contrast to previous observations indicating that a photoactive probe on Lys47 predominantly labelled the alpha chain. The toxin derivative weakly labelled the alpha and gamma chains but not the beta chain. The toxin may therefore interact with subunits other than the alpha chain, at least in the vicinity of Lys27.

Amino Acid Sequence↗

PubMed1992

Insertion of a disulfide-containing neurotoxin into E. coli alkaline phosphatase: the hybrid retains both biological activities.

We have inserted a disulfide-containing snake neurotoxin into the N-terminal end of Escherichia coli alkaline phosphatase, between residues +6 and +7 of the mature enzyme. For this purpose, we have designed a cloning and expression vector which allows insertion of foreign DNA between the corresponding codons, and visual selection of the desired recombinant clones upon recovery of phosphatase activity. The hybrid protein is exported to the bacterial periplasm, the alkaline phosphatase signal peptide is correctly processed, and both domains are functionally conformed. The phosphatase domain displays catalytic activity, and the inserted toxin is able to bind to its biological target, the nicotinic acetylcholine receptor. The hybrid molecule is remarkably stable and resistant to proteolysis. Crude periplasmic extract containing the hybrid can be used as a tracer-containing reagent in competitive enzymo-immuno and enzymo-receptor assays. We propose to use the system described in this paper for fast preparation of properly folded disulfide-containing enzymatic probes.

Alkaline Phosphatase↗

PubMed1992

[Recombinant colorimetric antibodies: genetic construction and production in E. coli].

We constructed an expression vector comprising a transcription unit composed of (1) the gene of alkaline phosphatase from E. coli in which we inserted a DNA fragment encoding the VH and CH domains of an IgG2A; (2) a DNA fragment encoding the light chain of the same immunoglobulin. Bacteria, E. coli were transformed with the plasmid, and hence it produced a periplasmic and chimaeric protein having both the alkaline phosphatase enzymatic activity and the immunoglobulin binding function. The hybrid protein mimics a bivalent antibody whose Fc part is replaced by a dimer of alkaline phosphatase. Recombinant colorimetric antibodies offer interesting potentialities for future developments in the field of immuno-enzymatic diagnosis.

Alkaline Phosphatase↗

PubMed1991

Amino acid sequence of a muscarinic toxin deduced from the cDNA nucleotide sequence.

We prepared a cDNA library from venom glands of the green mamba Dendroaspis angusticeps. A cDNA clone was isolated using an appropriate nucleotide probe. The nucleotide sequence codes for a 21 residue signal peptide followed by a 65 residue protein having the amino acid sequence of muscarinic toxin 2, as confirmed in the accompanying paper (Karlsson, E., Risinger, C., Jolkkonen, M., Wernstedt, C. and Adem, A.). The cDNA encoding the muscarinic toxin has been compared with those encoding other snake toxins. There are close similarities with short-chain curaremimetic neurotoxins.

Amino Acid Sequence↗

PubMed1990

A recombinant snake neurotoxin generated by chemical cleavage of a hybrid protein recovers full biological properties.

We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E. coli. The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin. By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC. This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin. This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants.

Amino Acid Sequence↗