ABSTRACT
BACKGROUND: The 5'-methylthioadenosine phosphorylase (MTAP), an enzyme involved in purine and polyamine metabolism and in the methionine salvage pathway, is considered as a potential drug target against cancer and trypanosomiasis. In fact, Trypanosoma and Leishmania parasites lack de novo purine pathways and rely on purine salvage pathways to meet their requirements. Herein, we propose the first comprehensive bioinformatic and structural characterization of the putative Leishmania infantum MTAP (LiMTAP), using a comparative computational approach. RESULTS: Sequence analysis showed that LiMTAP shared higher identity rates with the Trypanosoma brucei (TbMTAP) and the human (huMTAP) homologs as compared to the human purine nucleoside phosphorylase (huPNP). Motifs search using MEME identified more common patterns and higher relatedness of the parasite proteins to the huMTAP than to the huPNP. The 3D structures of LiMTAP and TbMTAP were predicted by homology modeling and compared to the crystal structure of the huMTAP. These models presented conserved secondary structures compared to the huMTAP, with a similar topology corresponding to the Rossmann fold. This confirmed that both LiMTAP and TbMTAP are members of the NP-I family. In comparison to the huMTAP, the 3D model of LiMTAP showed an additional α-helix, at the C terminal extremity. One peptide located in this specific region was used to generate a specific antibody to LiMTAP. In comparison with the active site (AS) of huMTAP, the parasite ASs presented significant differences in the shape and the electrostatic potentials (EPs). Molecular docking of 5'-methylthioadenosine (MTA) and 5'-hydroxyethylthio-adenosine (HETA) on the ASs on the three proteins predicted differential binding modes and interactions when comparing the parasite proteins to the human orthologue. CONCLUSIONS: This study highlighted significant structural peculiarities, corresponding to functionally relevant sequence divergence in LiMTAP, making of it a potential drug target against Leishmania.
Subject(s)
Leishmania infantum/enzymology , Molecular Docking Simulation/methods , Purine-Nucleoside Phosphorylase/chemistry , Purine-Nucleoside Phosphorylase/metabolism , Sequence Analysis, DNA/methods , Trypanosoma brucei brucei/ultrastructure , Adenosine/analogs & derivatives , Adenosine/metabolism , Amino Acid Motifs , Amino Acid Sequence , Antibodies/metabolism , Binding Sites, Antibody , Catalytic Domain , Deoxyadenosines/metabolism , Humans , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Conformation , Sequence Homology , Static Electricity , Substrate Specificity , Thionucleosides/metabolismABSTRACT
KAaH1 and KAaH2 are non-toxic peptides, isolated from the venom of the Androctonus australis hector (Aah) scorpion. In a previous study, we showed these peptides to be the most abundant (approximately 10% each) in the toxic fraction (AahG50) of the Aah venom. KAaH1 and KAaH2 showed high sequence identities (approximately 60%) with birtoxin-like peptides, which likewise are the major peptidic components of Parabuthus transvaalicus scorpion venom. Here, we report the immunological characterization of KAaH1 and KAaH2. These peptides were found to be specifically recognized by polyclonal antibodies raised against AahII, the most toxic peptide of Aah venom, and represents the second antigenic group, including toxins from different scorpion species in the world. Moreover, KAaH1 partially inhibits AahII binding to its specific antibody, suggesting some common epitopes between these two peptides. The identification of possible key antigenic residues in KAaH1 was deduced from comparison of its 3-D model with the experimental structure of AahII. Two clusters of putative antigenically important residues were found at the exposed surface; one could be constituted of V3 and D53, the other of D10, T15 and Y16. Polyclonal antibodies raised against KAaH1 in mice were found to cross-react with both AahII and AahG50, and neutralizing 5LD(50)/ml of the toxic fraction. Mice vaccinated with KAaH1 were protected against a challenge of 2LD(50) of AahG50 fraction. All these data suggest that KAaH1 has clear advantages over the use of the whole or part of the venom. KAaH1 is not toxic and could produce sera-neutralizing scorpion toxins, not only from Aah venom, but also toxins of other venoms from Buthus, Leiurus, or Parabuthus scorpion species presenting antigenically related toxins.
Subject(s)
Peptides/immunology , Peptides/pharmacology , Scorpion Venoms/antagonists & inhibitors , Scorpion Venoms/toxicity , Scorpions , Amino Acid Sequence , Animals , Epitopes/chemistry , Epitopes/immunology , Male , Mice , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Scorpion Venoms/chemistry , Scorpion Venoms/immunologyABSTRACT
A new depressant insect toxin Buthus occitanus tunetanus insect-toxin 6 (BotIT6) was purified by high-performance liquid chromatography from Buthus occitanus tunetanus (Bot) venom. BotIT6 is very active against Blatella germanica (LD50=10ng/100mg body mass) thus being one of the most potent anti-insect toxin so far characterised. When compared to other insect toxin sequences, BotIT6 present high similarities with depressant insect toxins with an additional arginine residue at the C-terminus and a methionine at position 27. The calculated net charge of BotIT6 is positive (+3) whereas it is negative for classical depressant toxins: this might be associated with its high toxicity. Voltage current clump studies show that BotIT6 is not a very potent depressant insect toxin despite its high toxicity in vivo. BotIT6 is able to fully inhibit the specific binding of 125I AaHIT and 125I-BotIT2 on Periplaneta americana synaptosomal membrane vesicles with high affinities. Despite its higher toxicity BotIT6 is a weaker competitor with 125I AaHIT and 125I BotIT2 as compared to the other beta toxins.Altogether, these results may suggest that BotIT6 probably defines a novel sub-group of depressant anti-insect toxins for which the receptor site can be overlapping, but not identical to that for classical depressant insect toxins.
Subject(s)
Scorpion Venoms/chemistry , Action Potentials/drug effects , Amino Acid Sequence , Animals , Central Nervous System Depressants/pharmacology , Chromatography, High Pressure Liquid , Electrophysiology , Enzyme-Linked Immunosorbent Assay , Injections, Intraventricular , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Patch-Clamp Techniques , Periplaneta/drug effects , Scorpion Venoms/administration & dosage , Scorpion Venoms/pharmacology , Scorpions/physiology , Synaptosomes/drug effects , Synaptosomes/metabolismABSTRACT
Scorpion envenoming is a real health problem. The only specific treatment is immunotherapy with antibodies from immunized horses. The severity of scorpion envenoming and the rapid diffusion of the toxins into the blood compartment require an improvement of the present antivenom therapy. In this study, we report successful immunization of dromedaries (Camelus dromedarius) against the small weakly antigenic neurotoxins of Androctonus australis hector scorpion. Camel immune sera was tested for its specific antigenic reactivity and neutralizing capacity against Aah toxic fraction and AahII toxin. We demonstrate that a substantial proportion of polyclonal heavy chain antibodies bind to Aah toxins and in particular to AahII, the most toxic one scorpion venom component. Furthermore, we show that both dromedary sera and heavy chain antibody subclasses are capable of neutralizing the toxicity of Aah toxins in mice.
