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Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 µg of recombinant nanobody could neutralize the toxic effects of 20 µg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.
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Objectives: In this study, the adjuvant activity of aqueous and alcoholic extracts of propolis was examined on the inactivated herpes simplex virus-1 (HSV-1). Materials and Methods: BALB/C mice were administered with inactivated (HSV-1; the KOS strain) plus alcoholic and aqueous extracts, followed by assessment of the cellular and humoral immune responses. Results: Alcoholic and aqueous extracts, as an adjuvant, revealed a significant increase in lymphocyte proliferation and cytotoxic T lymphocyte (CTL) responses versus the HSV-1 group. In addition, HSV-1 plus alcoholic extract showed a remarkable increase in IFN-γ cytokine and IFN-γ/IL-4 ratio. On the other hand, both alcoholic and aqueous extracts in the HSV-1 vaccine suppressed the IL-4 cytokine response as compared with the HSV-1 vaccine. In addition, HSV-1 plus alcoholic extract showed a significant increment in IgG1, IgG2a, and IgG2b isotypes as compared with the HSV-1 vaccine. Conclusion: Propolis extracts seem to modulate the immune response against inactivated HSV-1 model and can be used as a suitable vaccine adjuvant or a component of a complex adjuvant against infectious diseases.
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INTRODUCTION: Hemiscorpius lepturus envenomation is a serious health problem in the southern provinces of Iran. The antiserum produced in Iran to counteract this scorpion venom is not entirely effective due to the risk of anaphylactic shock and other adverse effects. METHODS: Therefore, more efficient alternatives to treat patients deserve attention, and plants are extensively good candidates to be studied. This study aimed to assess the potential of the aqueous fraction of Malva sylvestris in inhibiting the toxic effects of H. lepturus venom. Injection of sub-lethal dose of H. lepturus venom leads to severe tissue damage in vital organs including the kidney, liver, heart and intestine, after 24 hours. RESULTS: By injecting 80 mg of the aqueous extract of M. sylvestris into the peritoneum helped treat the damaged tissues caused by H. lepturus venom in mice. CONCLUSION: Thus, Malva sylvestris could serve as an alternative treatment for scorpion sting envenomation and may be used as a drug to neutralize relevant toxic effects in patients stung by H. lepturus.
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Malva , Camundongos Endogâmicos BALB C , Extratos Vegetais , Venenos de Escorpião , Escorpiões , Animais , Extratos Vegetais/farmacologia , Extratos Vegetais/isolamento & purificação , Venenos de Escorpião/toxicidade , Camundongos , Malva/química , Masculino , Picadas de Escorpião/tratamento farmacológico , Animais PeçonhentosRESUMO
BACKGROUND: Development of promising medicines from natural sources, specially venom, is of highly necessitated to combat against life-threatening cancers. Non-small cell lung cancer (NSCLC) has a significant percentage of mortalities. Melittin, from bee venom, is a potent anticancer peptide but its toxicity has limited its therapeutic applications. Accordingly, this study aims to synthesize niosomes with suitable stability and capacity for carrying melittin as a drug. Additionally, it seeks to evaluate the anti-cancer activity of melittin-loaded niosomes on non-small cell lung cancer. METHODS: The niosome was prepared by thin film hydration method. Cytotoxicity and apoptosis were assessed on A549, Calu-3, and MRC5 cells. Real-time PCR was used to determine expression of apoptotic and pro-apoptotic Bax, Bcl2, and Casp3 genes. Immunocytochemistry (ICC) was also used to confirm expression of the abovementioned genes. Furthermore, wound healing assay was performed to compare inhibition effects of melittin-loaded niosomes with free melittin on migration of cancer cells. RESULTS: IC50 values of melittin-loaded niosomes for A549, Calu-3, and MRC5 cells were respectively 0.69 µg/mL, 1.02 µg/mL, and 2.56 µg/mL after 72 h. Expression level of Bax and Casp3 increased '10 and 8' and '9 and 10.5' fold in A549 and Calu-3, whereas Bcl2 gene expression decreased 0.19 and 0.18 fold in the mentioned cell lines. The cell migration inhibited by melittin-loaded niosomes. CONCLUSIONS: Melittin-loaded niosomes had more anti-cancer effects and less toxicity on normal cells than free melittin. Furthermore, it induced apoptosis and inhibited cancer cells migration. Our results showed that melittin-loaded niosomes may be a drug lead and it has the potential to be future developed for lung cancer treatment.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Meliteno/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Lipossomos , Caspase 3 , Proteína X Associada a bcl-2/genética , Neoplasias Pulmonares/tratamento farmacológicoRESUMO
Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.
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Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 µg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value.
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Antineoplásicos , Neoplasias do Colo , Animais , Humanos , Elapidae , Naja naja , Venenos Elapídicos/farmacologia , Venenos Elapídicos/química , Células HT29 , Irã (Geográfico) , Proteômica , Células HEK293 , Venenos de Serpentes , Antineoplásicos/farmacologia , Peptídeos/farmacologia , Neoplasias do Colo/tratamento farmacológicoRESUMO
Objectives: One of the important interactions in controlling the human immune system is the reaction between checkpoint proteins such as programmed cell death-1 (PD-1) and its ligand, PD-L1. These are negative immunoregulatory molecules that promote immune evasion of tumor cells. PD-L1 expression is an immune-mediated mechanism used by various malignant cells in order to down-regulate the immune system. Checkpoint inhibitors (CPIs) are a new class of anti-cancer agents that stimulate immune cells to elicit an antitumor response by blocking the ligand and receptor interactions. Nanobody (Nb) as a new type of antibody fragment, has some potential as CPI. Materials and Methods: A female camel was immunized with recombinant PD-L1 protein, nanobody library was constructed and PD-L1 specific Nb was selected. The selected Nb was characterized in terms of affinity, specificity, and binding potency in ELISA, Western blotting, and flow cytometry. Results: Developed nanobody, A22 binds to its cognate target with high specificity and affinity. Western blot and flow cytometry techniques showed that nanobody A22 was able to specifically detect and attach to human PD-L1 protein on the cell surface and in the cell lysate. MTT assay showed the inhibitory effect of PD-L1 by specific Nb on A431 and HEK293 cells, with no cytotoxic effect on cell growth. Conclusion: The results highlighted the potential of anti-PD-L1 Nb as a novel therapeutic in cancer therapy without undesirable cytotoxicity.
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Cancer is one of the leading causes of mortality in the world. Unfortunately, the present anticancer chemotherapeutics display high cytotoxicity. Accordingly, the discovery of new anticancer agents with lower side effects is highly necessitated. This study aimed to discover an anticancer compound from Hemiscorpius lepturus scorpion venom. Bioactivity-guided chromatography was performed to isolate an active compound against colon and breast cancer cell lines. 2D electrophoresis and MALDI-TOF were performed to identify the molecule. A partial protein sequence was obtained by mass spectrometry, while the full-length was deciphered using a cDNA library of the venom gland by bioinformatics analyses and was designated as leptulipin. The gene was cloned in pET-26b, expressed, and purified. The anticancer effect and mechanism action of leptulipin were evaluated by MTT, apoptosis, and cell cycle assays, as well as by gene expression analysis of apoptosis-related genes. The treated cells displayed inhibition of cell proliferation, altered morphology, DNA fragmentation, and cell cycle arrest. Furthermore, the treated cells showed a decrease in BCL-2 expression and an increase in Bax and Caspase 9 genes. In this study, we discovered a new anticancer protein from H. lepturus scorpion venom. Leptulipin showed significant anticancer activity against breast and colon cancer cell lines.
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Venenos de Escorpião , Escorpiões , Sequência de Aminoácidos , Animais , Linhagem Celular , Biologia Computacional , Venenos de Escorpião/farmacologia , Escorpiões/metabolismoRESUMO
Background: Hemiscorpius lepturus is one of the most dangerous scorpions in Iran and the world. Numerous studies have been conducted on phospholipases, especially phospholipase D, in this scorpion's venom, and the results have shown this protein to be the main cause of death. Therefore, one of the most effective ways of preventing fatalities is to produce a toxoid vaccine from the deadly toxin of the venom. The present study was conducted to assess the non-toxicity of this toxoid and the safety of the vaccine candidate in BALB/c mice. Methods: The production of interferon-gamma and interleukin-4 cytokines in the spleen cells of the mice was measured using ELISpot assay 28 days following immunization with rPLD toxoid. Results: The unpaired t-test results showed a significant increase in the concentration of IFN-γ cytokine in the vaccinated mice (P= 0.001), indicating that the immune system is directed toward the Th1 pattern, while no significant difference was observed in the levels of IL-4 (P= 0.16) despite an increase in this cytokine. The in-vivo tests showed that the mice immunized with interval doses of 80µg of toxoid were completely protected against 10 × the LD100 of the venom. Moreover, the toxoid had no dermonecrotic effects and caused no necrotic and inflammatory complications in the rabbit skin. Conclusion: As a vaccine, the toxoid has the potential to increase the Th1 cytokine response and, subsequently, increase acquired cellular immunity. Thus, this toxoid appears to be able to provide an effective vaccine against the venom of Hemiscorpius lepturus.
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The discovery of novel anti-cancer agents from natural resources is highly necessary. Concerning the above problem, the purpose of this study was to discover an anti-cancer compound from Caspian cobra venom. Fractionation of Caspian cobra venom was performed by gel filtration and IEX chromatography. The results showed an anti-cancer protein nominated as Oxilipin. Activity and toxicity of Oxilipin were studied on the colon SW480 cancer cell line using MTT, LDH release, PI staining, morphological cell analysis, hemolysis, and anti-proliferation assays. Oxilipin, an 11kDa protein purified from the venom of the Caspian cobra. LC/MS/MS analysis of obtained protein showed homology with Phospholipase A2 from Naja naja oxiana. 40 µg/ml of Oxilipin can induce an apoptotic effect in SW480 cell line up to 90%; meanwhile, this amount can induce only one-third of cytotoxicity on a normal cell. In this study, Iranian cobra venom was found to have cytotoxic effects on SW480 colon cancer tumor cells, with the least cytotoxicity on normal cells on HEK-293. Given that Oxilipin has slight toxicity on normal cells, it can be hypothesized that the obtained peptide can be considered as a drug lead in an animal model study of colon cancer.
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OdTx12, a beta excitatory toxin from Odontobothus doriae had previously been identified and characterized. It had been shown that OdTx12 causes significant lethal effects on insects by injection but does not show any toxicity on mice. Due to the natural ineffectiveness of scorpion toxins to act as oral toxins, OdTx12 was fused to Galanthus nivalis agglutinin (GNA), a protein with the potential to cross the insect gut. The sequence of OdTx12/GNA gene was chemically synthesized, cloned in Escherichia coli, and expressed. The effect of the purified fusion protein (OdTx12/GNA) was assessed on the insect and mammalian cell lines, insect larvae and mice. Toxicity assay on insect cell culture (SF9 cell line) showed comparable toxicity between OdTx12 and OdTx12/GNA (LD50 of 0.0030 and 0.0048 µM, respectively). Also very similar mortality rates were observed by injecting OdTx12 and OdTx12/GNA to Locusta migratoria and Tenebrio molitor. Oral administration of OdTx12/GNA, after five days of feeding, resulted in 96.6% and 98.3% mortality of L. migratoria and T. molitor larvae with an LC50 of 0.69 and 0.43 nmol/g of insect food, respectively, while OdTx12 alone did not cause any toxic effects on the larvae orally, suggesting the role of GNA in delivering the toxin to the insect's haemolymph. No toxicity or mortality was observed after toxicity testing of OdTx12/GNA on a mammalian cell line (HEP-2) or any mortality in vivo, by testing the protein in the laboratory mouse. Herein, we demonstrated that the fusion protein OdTx12/GNA could be considered an effective toxin for the biological control of insects.
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Inseticidas , Locusta migratoria , Lectinas de Ligação a Manose , Tenebrio , Animais , Lectinas de Ligação a Manose/toxicidade , Camundongos , Lectinas de PlantasRESUMO
BACKGROUND: The use of venom fractions from the Iranian cobra could be useful adjunct treatments of malaria with chloroquine. A metabolomic investigation with 1HNMR spectroscopy was conducted on an effective fraction tested earlier using Plasmodium berghei as an experimental murine model. PURPOSE: We sought to ascertain both safety and anti-parasitic effects of experimental therapies. METHODS: After purification of the venom fractions, 25 mice were infected, then treated for 4 days with 0.2 ml of 5 mg/kg, 2.5 mg/kg and 1 mg/kg of the effective fraction, chloroquine, and a drug vehicle. An ED50 was obtained using Giemsa staining and real-time PCR analysis. The toxicity tests inspecting both liver and kidney tissues were performed. RESULTS: A clear inhibitory effect on parasitaemia was observed (with 75% inhibition with 5 mg/kg and 50% reduction when 2.5 mg/kg dosage used). ED50 obtained 2.5 mg/kg. The metabolomics were identified as differentiation of aminoacyl-t-RNA biosynthesis, valine, leucine, isoleucine biosynthesis and degradation pathways were observed. CONCLUSION: Upon therapeutic effects of cobra venom fraction, further optimization of dose-dependent response of pharmacokinetics would be worthwhile for further exploration in adjunct experimental venom therapies.
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Naja naja , Plasmodium berghei , Animais , Irã (Geográfico) , Espectroscopia de Ressonância Magnética , Metabolômica , CamundongosRESUMO
Hemiscorpius lepturus scorpion stings do not induce considerable pain based on epidemiological surveys conducted in the southwest part of Iran. Accordingly, this study was aimed to identify the analgesic molecule in H. lepturus venom by analyzing a cDNA library of the scorpion venom gland looking for sequences having homology with known animal venom analgesic peptides. The analgesic molecule is a cysteine rich peptide of 55 amino acids. the synthetic peptide was deprotected and refolded. RP-HPLC, Ellman's, and DLS assays confirmed the refolding accuracy. Circular dichroism (CD) showed helix and beta sheet contents. This peptide, called leptucin, demonstrated 95% analgesic activity at the dose of 0.48 mg/kg in hot plate assay. Leptucin at the doses of 0.32, 0.48, and 0.64 mg/kg showed 100% activity in thermal tail flick test. No hemolysis or cytotoxicity was observed at 8 and 16 µg. Histopathology evaluations indicated no hepatotoxicity, nephrotoxicity, and cardiotoxicity. We thus report that leptucin is the analgesic agent of H. lepturus venom. Regarding the high in vivo efficacy of leptucin and the fact it shows no observable toxicity, it could be suggested as a drug lead in a preclinical study of acute pain as well as the study of its mechanism of action.
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Analgésicos/farmacologia , Peptídeos/farmacologia , Escorpiões/química , Sequência de Aminoácidos , Analgésicos/química , Animais , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Biologia Computacional/métodos , Hemólise/efeitos dos fármacos , Irã (Geográfico) , Dose Máxima Tolerável , Fases de Leitura Aberta , Peptídeos/química , Peptídeos/genética , Conformação Proteica , Picadas de Escorpião , Análise EspectralRESUMO
Snakebite is an important public health problem in tropical and subtropical regions. Macrovipera lebetina is one of the most dangerous snakes in Iran. Envenoming by this snake can lead to respiratory distress, heart attack, bleeding, and death. The specific treatment available is immunized equine serum, which has several side effects like serum sickness. Nowadays, single-chain fragment variable antibodies (scFvs) are one of the fast growing classes of monoclonal antibodies, which are suggested for treatment of envenoming. This study aimed to achieve a fully human scFv antibody against M. lebetina venom from human non-immune library. In this study, scFvs against M. lebetina venom were isolated by phage display technique. Using three rounds of biopanning, two specific scFvs (C37 and C69) with the highest affinity were selected. The selected scFvs purified by nickel affinity chromatography. The specific binding of purified antibodies were confirmed by enzyme-linked immunosorbent assay. The LD50 as well as HD50 concentration of the crude venom were obtained to be 45 µg and 120 µg/ml, respectively. C69 neutralized 48% of the hemolysis activity of M. lebetina venom and C37 survived 66% of mice after 115 min of envenoming. Taken together, the results indicate the potential of human non-immune libraries for selection of functional antibodies against M. lebetina venom.
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Anticorpos de Cadeia Única , Mordeduras de Serpentes , Viperidae , Animais , Cavalos , Humanos , Irã (Geográfico) , Dose Letal Mediana , Camundongos , Mordeduras de Serpentes/tratamento farmacológico , Venenos de SerpentesRESUMO
BACKGROUND AND AIMS: Due to the lack of an effective vaccine and complexity of the control measures against vectors and reservoir hosts, the control of leishmaniasis depends primarily on chemotherapy. This study was aimed to assess the snake venom, Naja naja oxiana fraction 11(NNOVF11) on Leishmania infantum and its broad mode of action. METHODS: A wide range of in vitro advanced assays including high-performance liquid chromatography (HPLC), MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5diphenyltetrazolium bromide; Thiazolyl blue), macrophage assays, quantitative real-time polymerase chain reaction (qPCR), flow cytometry and enzyme- linked immunosorbent assay (ELISA) on L. infantum promastigote and amastigote stages were used. IC50 values of L. infantum stages, CC50 value, and apoptosis were also analyzed. RESULTS: The NNOV-F11 demonstrated strong antileishmanial activity against L. infantum stages in a dose-dependent manner compared to the untreated control group. Interleukin (IL)-12, TNF-α, and iNOS genes expression as the indicators of T helper(h)1 response significantly increased; in contrast, the expression level of IL-10, as the representative of Th2 response significantly decreased (p < 0.001). Reactive oxygen species (ROS) detection showed a significant increase (p < 0.001) after treatment with different concentrations of NNOV-F11, unlike arginase (ARG) activity, which displayed a significant reduction (p < 0.001). CONCLUSION: NNOV-F11 possessed a potent inhibitory effect on L. infantum stages with the multifunctional and broad mode of actions, which promoted the immunomodulatory role, induced ROS production, stimulated apoptotic-like mechanisms, and inhibited L-ARG activity, which collectively led to the parasite death. Further studies are crucial to assess the effect of the NNOV-F11 on animal models or clinical settings.
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Antiprotozoários/farmacologia , Venenos Elapídicos/farmacologia , Leishmania infantum/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/parasitologia , Animais , Antiprotozoários/isolamento & purificação , Células Cultivadas , Relação Dose-Resposta a Droga , Venenos Elapídicos/isolamento & purificação , Mediadores da Inflamação/antagonistas & inibidores , Mediadores da Inflamação/metabolismo , Leishmania infantum/metabolismo , Macrófagos/metabolismo , Camundongos , Naja najaRESUMO
PURPOSE: The aim of this study was to explore the activity of Naja naja oxiana venom on Leishmania tropica and its modes of action. METHODS: Different fractions of Naja naja oxiana venom (NNOV) were prepared and characterized by high-performance liquid chromatography. The superior component, fraction k (FK) was selected. The activity of the fraction was assessed using advanced assays. RESULTS: Interleukin (IL)-12, TNF-α and iNOS gene expression as the indicators of Th1 significantly increased. In contrast, the level of IL-10, as the marker of T helper 2 substantially decreased (p < 0.001). Reactive oxygen species (ROS) detection showed a significant increase (p < 0.001) after treatment with different concentrations of NNOV-FK, unlike arginase (L-ARG) activity which showed a significant reduction (p < 0.001). The NNOV-FK showed significant lethal activity on the L. tropica stages. CONCLUSION: The findings demonstrated that NNOV-FK represented a strong leishmanicidal activity on L. tropica stages. The major modes of NNOV-FK action are multidimensional, which perceives the induction of a synergistic response and upregulation of the immune-modulatory role towards Th1 response against L. tropica stages as well as apoptotic and anti-metabolic action as a model drug to generate ROS, block the polyamine synthesis and lead to parasite death.
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Leishmania tropica , Naja naja , Animais , Bioensaio , Venenos Elapídicos , Venenos de SerpentesRESUMO
Gloydius caucasicus (NIKOLSKY, 1916) is a member of the Viperidae family in Iran. Comprehensive understanding of the toxigenic characteristics of snake venom is important for clinical monitoring of snakebite patients and effective therapy. We compared the toxic activities of venoms and the neutralization capacity of antivenoms produced with venoms from wild adult (WA) with long-term captive adult (LCA) of G. caucasicus in order to obtain more effective antivenom from LCA in therapy, and subsequently protect G. caucasicus from overharvesting for its venom, which poses a real threat of extinction for the species. Our results showed that LD50 of WA and LCA were 16.8 µg/dose and 17.7 µg/dose, respectively. Lower hemorrhagic and necrotic (p ≥ 0.05), and higher coagulative and edematogenic activities (p ≤ 0.05) were observed in WA compared with LCA venom. Also, captive-born neonates exhibited weaker toxic activities compared with captive adult snakes, which could be an age-related difference. Study data illustrated that effective capacity of LCA antivenom to neutralize the toxic activities of WA viper venom. According to the results, about 0.4-4 µl of LCA antivenom is required to neutralize the toxic activities of 1 µg of WA venom, indicating its efficacy in treatment of snakebites in humans. On this basis, it is recommended that capture of wild snakes for their venom be discontinued to reduce their future extinction risk.
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The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.