Identification of continuous interaction sites in PLA(2)-based protein complexes by peptide arrays

Crotoxin (CA.CB) is a beta-neurotoxin from Crotalus durissus terrificus snake venom that is responsible for main envenomation effects upon biting by this snake. It is a heterodimer of an acidic protein (CA) devoid of any biological activity per se and a basic, enzymatically active, PLA(2) counterpart (CB). Both lethal and enzymatic activities of crotoxin have been shown to be inhibited by CNF, a protein from the blood of C. d. terrificus snakes. CNF replaces CA in the CA-CB complex, forming a stable, non-toxic complex CNF.CB. The molecular sites involved in the tight interfacial protein-protein interactions in these PLA(2)-based complexes have not been clearly determined. To help address this question, we used the peptide arrays approach to map possible interfacial interaction sites in CA.CB and CNF.CB. Amino acid stretches putatively involved in these interactions were firstly identified in the primary structure of CB. Further analysis of the interfacial availability of these stretches in the presumed biologically active structure of CB, suggested two interaction main sites, located at the amino-terminus and beta-wing regions. Peptide segments at the carboxyl-terminus of CB were also suggested to play a secondary role in the binding of both CA and CNF. (C) 2009 Elsevier Masson SAS. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A Heterologous Multiepitope DNA Prime/Recombinant Protein Boost Immunisation Strategy for the Development of an Antiserum against Micrurus corallinus (Coral Snake) Venom.

BACKGROUND:Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. METHODS AND FINDINGS:In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. CONCLUSION:Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity

Phage-displayed peptides as capture antigens in an innovative assay for Taenia saginata-infected cattle

Bovine cysticercosis is detected during the routine post mortem examination of carcasses by visual inspection (knife and eye method). However, the sensitivity of this procedure is several times lower than immunoassays, even when it is performed by qualified professionals. In the present study, a new generation capture antigens were screened from a phage display peptide library using antibodies from Taenia saginata-infected animals. Eight phage clones were selected, and one, Tsag 3 (VHTSIRPRCQPRAITPR), produced similar results to the T. saginata metacestode crude antigen (TsCa) when used as a capture antigen in an ELISA. The phage-displayed peptides competed with TsCa for binding sites, reducing the reactivity by approximately 30 %. Alanine scanning indicated that proline, arginine, and serine are important residues for antibody binding. Tsag 1 (HFYQITWLPNTFPAR), the most frequent affinity-selected clone, and Tsag 6 (YRWPSTPSASRQATL) shared similarity with highly conserved proteins from the Taeniidae family with known immunogenicity. Due to their epitopic or mimotopic properties, these affinity-selected phages could contribute to the rational design of an ante mortem immunodiagnosis method for bovine cysticercosis, as well as an epitope-based vaccine to interrupt the taeniosis/cysticercosis complex.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Biological and structural comparison of recombinant phospholipase D toxins from Loxosceles intermedia (brown spider) venom

The clinical features of brown spider bites are the appearance of necrotic skin lesions, which can also be accompanied by systemic involvement, including weakness, vomiting, fever, convulsions, disseminated intravascular coagulation, intravascular hemolysis and renal disturbances. Severe systemic loxoscelism is much less common than the cutaneous form, but it may be the cause of clinical complications and even death following envenomation. Here, by using three recombinant dermonecrotic toxins, LiRecDT1, LiRecDT2 and LiRecDT3 (the major toxins found in the venom), we report the biological, immunological and structural differences for these members of this toxin family. Purified toxins evoked similar inflammatory reactions following injections into rabbit skin. Recombinant toxin treatments of MDCK cells with LiRecDT1 and LiRecDT2 changed cell viability, as evaluated by neutral red uptake and assessment of cell morphology through inverted microscopy, whereas LiRecDT3 caused only residual activity. Differences in cell cytotoxicity triggered by recombinant toxins were confirmed through a human red blood lysis assay, during which LiRecDT1 and LiRecDT2 caused a high degree of hemolysis compared to LiRecDT3, which induced only a small hemolytic effect. Additionally, biological differences for recombinant toxins were corroborated through mice lethality experiments, which showed animal mortality after LiRecDT1 and LiRecDT2 treatments, but an absence of lethality following LiRecDT3 exposure. Moreover, in experiments for edema, both the LiRecDT1 and the LiRecDT2 toxins evoked similar results, causing edema following toxin exposure, whereas LiRecDT3 caused only residual effects. Characterization of antigenic cross-reactivity using sera against crude venom toxins by immunoWestern blotting and immunodot blotting with recombinant LiRecDT1, LiRecDT2 and LiRecDT3 compared among themselves pointed to a higher cross-reactivity for LiRecDT1 compared to LiRecDT2 and LiRecDT3, corroborating structural and antigenic differences for these three toxins. Finally, evidence for structural differences among the recombinant toxins was strengthened by circular dichroism spectra, which suggested that the toxins were folded, and not aggregated or denatured proteins. (c) 2007 Elsevier B.V. All rights reserved.Univ Fed Parana, Dept Cell Biol, BR-81531990 Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniv Estadual Ponta Grossa, Dept Struct Mol Biol & Genet, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Med, São Paulo, BrazilUniv Fed Parana, Dept Basic Pathol, BR-81531990 Curitiba, Parana, BrazilUniv Fed Minas Gerais, Dept Biochem & Immunol, Belo Horizonte, MG, BrazilCatholic Univ Parana, Hlth & Biol Sci Inst, Curitiba, Parana, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, São Paulo, BrazilWeb of Scienc

RT-PCR from COS-7 cells transfected with all pSECTAG2A constructions.

<p>(M) 1kb Plus DNA ladder. (1) RT-PCR from pSECTAG2A-<i>ag1</i> transfected cells. (2) RT-PCR from pSECTAG2A-<i>ag2</i> transfected cells. (3) RT-PCR from pSECTAG2A-<i>ag3</i> transfected cells. (4) RT-PCR from pSECTAG2A-<i>ag4</i> transfected cells. (5) RT-PCR from pSECTAG2A-<i>ag5</i> transfected cells. (6) RT-PCR from pSECTAG2A (empty plasmid) transfected cells.</p

Three dimensional modelling with solvent-accessible surface area (SASA) of all five antigens described in this work.

<p>Reactive epitopes are highlighted in orange. (A) 3D model of Ag1 (Nxh8) based on the crystal structure (PDB ID: 3nds) of <i>Naja nigricollis</i> toxin alpha (GMQE: 0.80 / Seq. identity: 54.10 / Seq. similarity: 0.48). (B) 3D model of Ag2 (Nxh7/3/1) based on the NMR structure (PDB ID: 1nor) of neurotoxin II from <i>Naja naja oxiana</i> (GMQE: 0.78 / Seq. identity: 40.35 / Seq. similarity: 0.48). (C) 3D model of Ag3 (3FTx) based on the crystal structure (PDB ID: 2h8u) of Bucain, a cardiotoxin from the Malayan Krait <i>Bungarus candidus</i> (GMQE: 0.89 / Seq. identity: 57.63 / Seq. similarity: 0.51). (D) 3D model of Ag4 (3FTx) based on the crystal structure (PDB ID: 4iye) of the green mamba, <i>Dendroaspis angusticeps</i>, ρ-Da1a toxin (GMQE: 0.75 / Seq. identity: 40.00 / Seq. similarity: 0.40). (E) 3D model of Ag5 (PLA₂) based on the crystal structure (PDB ID: 1yxh) of a phospholipase A₂ from <i>Naja naja sagittifera</i> (GMQE: 0.82 / Seq. identity: 58.97 / Seq. similarity: 0.50). All images were generated using DeepView (Swiss PDB Viewer) [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004484#pntd.0004484.ref034" target="_blank">34</a>].</p

SPOT peptide synthesis scheme.

<p>(Black circles) Blank spots. (Cyan circles) Spots from Ag1 (Nxh8— 3FTx). (Red Circles) Spots from Ag2 (Nxh 7/3/1— 3FTx). (Yellow circles) Spots from Ag3 (3Ftx). (Blue circles) Spots from Ag4 (3Ftx). (Green circles) Spots from Ag5 (PLA₂). (Highlighted spot sequences) Signal peptides, which were not considered for multiepitope gene design.</p

Multiepitope DNA string sequence coding for epitopes detected from the selected 3FTx from <i>M</i>. <i>corallinus</i>.

<p>Cysteine codons were exchanged by serine codons to avoid the formation disulphide bond-mediated protein multimerisation. Epitopes were separated by a six residues linker. Epitopes 1 and 2 are from Ag1, while Epitopes 3, 4 and 5 are from Ags 2, 3 and 4, respectively. Restriction sites (red sequences) were inserted between epitopes to allow further DNA manipulation when required.</p