RESUMEN
The pursuit of novel treatment alternatives to address the accumulated resistance to antimicrobials over the years has prompted the scientific community to explore biodiversity, particularly animal venom, as a potential source of new antimicrobial drugs. Snake venoms, with their complex mixtures of components, are particularly promising targets for investigation in this regard. The search for novel molecules exhibiting antimicrobial activity against multidrug-resistant strains is of paramount importance for public health and numerous research groups worldwide. High expectations within the healthcare field are supported by the scientific literature, which highlights the potential development of innovative drugs through in vivo and in vitro application, depending on dose titration. Snake venoms and their molecules and peptides offer exponential possibilities for biotechnological applications as antimicrobial agents. However, many uncertainties and unexplored avenues remain, presenting opportunities for discoveries and research.
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In order to address the global antivenom crisis, novel antivenoms need to present high therapeutic efficacy, broad neutralization ability against systemic and local damage, sufficient safety, and cost-effectiveness. Due to biological characteristics of camelid single-domain antibodies (VHH) such as high affinity, their ability to penetrate dense tissues, and facility for genetic manipulation, their application in antivenoms has expanded considerably. VHHs that are active against the metalloprotease BjussuMP-II from the snake Bothrops jararacussu were selected. After isolation of BjussuMP-II, a camelid was immunized with the purified toxin in order to construct the recombinant phage library. Following a round of biopanning, 52% of the selected clones were able to recognize BjussuMP-II in an ELISA assay. After sequencing, seven sequence profiles were identified. One selected clone (VHH61) showed cross-reactivity to B. brazili venom, but did not recognize the Crotalus and Lachesis genera, indicating specificity for the Bothrops genus. Through in vitro tests, the capacity to neutralize the toxicity triggered by BjussuMP-II was observed. Circular dichroism spectroscopy indicated a robust secondary structure for VHH61, and the calculated melting temperature (T M) for the clone was 56.4°C. In silico analysis, through molecular docking of anti-BjussuMP-II VHHs with metalloprotease, revealed their potential interaction with amino acids present in regions critical for the toxin's conformation and stability. The findings suggest that anti-BjussuMP-II VHHs may be beneficial in the development of next-generation antivenoms.
Asunto(s)
Bothrops , Venenos de Crotálidos , Anticuerpos de Dominio Único , Mordeduras de Serpientes , Animales , Antivenenos/uso terapéutico , Bothrops/metabolismo , Metaloproteasas/metabolismo , Simulación del Acoplamiento Molecular , Pruebas de Neutralización , Anticuerpos de Dominio Único/farmacología , Mordeduras de Serpientes/tratamiento farmacológicoRESUMEN
Bothrops asper is one of the most important snake species in Central America, mainly because of its medical importance in countries like Ecuador, Panama and Costa Rica, where this species causes a high number of snakebite accidents. Several basic phospholipases A2 (PLA2s) have been previously characterized from B. asper venom, but few studies have been carried out with its acidic isoforms. In addition, since snake venom is a rich source of bioactive substances, it is necessary to investigate the biotechnological potential of its components. In this context, this study aimed to carry out the biochemical characterization of PLA2 isoforms isolated from B. asper venom and to evaluate the antiparasitic potential of these toxins. The venom and key fractions were subjected to different chromatographic steps, obtaining nine PLA2s, four acidic ones (BaspAc-I, BaspAc-II, BaspAc-III and BaspAc-IV) and five basic ones (BaspB-I, BaspB-II, BaspB-III, BaspB-IV and BaspB-V). The isoelectric points of the acidic PLA2s were also determined, which presented values ranging between 4.5 and 5. The findings indicated the isolation of five unpublished isoforms, four Asp49-PLA, corresponding to the group of acidic isoforms, and one Lys49-PLA2-like. Acidic PLA2s catalyzed the degradation of all substrates evaluated; however, for the basic PLA2s, there was a preference for phosphatidylglycerol and phosphatidic acid. The antiparasitic potential of the toxins was evaluated, and the acidic PLA2s demonstrated action against the epimastigote forms of T. cruzi and promastigote forms of L. infantum, while the basic PLA2s BaspB-II and BaspB-IV showed activity against P. falciparum. The results indicated an increase of up to 10 times in antiplasmodial activity, when the Asp49-PLA2 and Lys49-PLA2 were associated with one another, denoting synergistic action between these PLA2 isoforms. These findings correspond to the first report of synergistic antiplasmodial action for svPLA2s, demonstrating that these molecules may be important targets in the search for new antiparasitic agents.
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Antiprotozoarios/farmacología , Fosfolipasas A2/química , Plasmodium falciparum/efectos de los fármacos , Venenos de Serpiente/metabolismo , Secuencia de Aminoácidos , Animales , Antiprotozoarios/química , Antiprotozoarios/aislamiento & purificación , Bothrops/metabolismo , Sinergismo Farmacológico , Punto Isoeléctrico , Leishmania infantum/efectos de los fármacos , Panamá , Pruebas de Sensibilidad Parasitaria , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/farmacología , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/farmacología , Alineación de SecuenciaRESUMEN
We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34-40% of the total venom proteins) and PI (8-18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8-10%); bradykinin-potentiating-like peptides (BBPs, 10.7-15%); snake venom serine proteinases (SVSP, 5.5-14%); C-type lectin-like proteins (CTL, 3-10%); phospholipases A2 (PLA2, 2.8-7.6%); cysteine-rich secretory proteins (CRISP, 0.9-2.8%); l-amino acid oxidases (LAO, 0.9-5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops â³jararacaâ³ and â³taeniatusâ³ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.
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Bothrops , Venenos de Crotálidos , Crotalinae , Animales , Antivenenos , Proteoma/genética , Proteómica , Venenos de VíborasRESUMEN
We report a structural and functional proteomics characterization of venoms of the two subspecies (Bothrops bilineatusbilineatus and B. b. smaragdinus) of the South American palm pit viper from the Brazilian state of Rondônia and B. b. smaragdinus from Perú. These poorly known arboreal and mostly nocturnal generalist predators are widely distributed in lowland rainforests throughout the entire Amazon region, where they represent an important cause of snakebites. The three B. bilineatus spp. venom samples exhibit overall conserved proteomic profiles comprising components belonging to 11 venom protein classes, with PIII (34–40% of the total venom proteins) and PI (8–18%) SVMPs and their endogenous tripeptide inhibitors (SVMPi, 8–10%); bradykinin-potentiating-like peptides (BBPs, 10.7–15%); snake venom serine proteinases (SVSP, 5.5–14%); C-type lectin-like proteins (CTL, 3–10%); phospholipases A2 (PLA2, 2.8–7.6%); cysteine-rich secretory proteins (CRISP, 0.9–2.8%); l-amino acid oxidases (LAO, 0.9–5%) representing the major components of their common venom proteomes. Comparative analysis of the venom proteomes of the two geographic variants of B. b. smaragdinus with that of B. b. bilineatus revealed that the two Brazilian taxa share identical molecules between themselves but not with Peruvian B. b. smaragdinus, suggesting hybridization between the geographically close, possibly sympatric, Porto Velho (RO, BR) B. b. smaragdinus and B. b. bilineatus parental populations. However, limited sampling does not allow determining the frequency of this event. The toxin arsenal of the South American palm pit vipers may account for the in vitro recorded collagenolytic, caseinolytic, PLA2, l-amino acid oxidase, thrombin-like and factor X-activating activities, and the clinical features of South American palm pit viper envenomings, i.e., local and progressively ascending pain, shock and loss of consciousness, spontaneous bleeding, and profound coagulopathy. The remarkable cross-reactivity of the Brazilian pentabothropic SAB antivenom toward the heterologous B. b. bilineatus venom suggests that the paraspecific antigenic determinants should have been already present in the venom of the last common ancestor of the Bothrops ″jararaca″ and ″taeniatus″ clades, about 8.5 Mya in the mid-late Miocene epoch of the Cenozoic era. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifiers PXD020043, PXD020026, and PXD020013.
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BACKGROUND: Functional and structural diversity of proteins of snake venoms is coupled with a wide repertoire of pharmacological effects. Snake venoms are targets of studies linked to searching molecules with biotechnological potential. METHODS: A homologue phospholipase A2 (BmatTX-IV) was obtained using two chromatographic techniques. Mass spectrometry and two-dimensional gel electrophoresis were used to determine the molecular mass and isoelectric point, respectively. By means of Edman degradation chemistry, it was possible to obtain the partial sequence of amino acids that comprise the isolated toxin. Trypanocidal, leishmanicidal and cytoxic activity against Trypanosoma cruzi, Leishmania infantum and murine fibrobasts was determinated. RESULTS: Combination of both chromatographic steps used in this study demonstrated efficacy to obtain the PLA2-Lys49. BmatTX-IV showed molecular mass and isoelectric point of 13.55 kDa and 9.3, respectively. Amino acid sequence of N-terminal region (51 residues) shows the presence of Lys49 residue at position 49, a distinctive trait of enzymatically inactive PLA2. Bothrops mattogrossensis snake venom showed IC50 values of 11.9 µg/mL against Leishmania infantum promastigotes and of 13.8 µg/mL against Trypanosoma cruzi epimastigotes, respectively. On the other hand, the venom showed a high cytotoxic activity (IC50 value of 16.7 µg/mL) against murine fibroblasts, whereas the BmatTX-IV showed IC50 value of 81.2 µg/mL. CONCLUSION: Physicochemical and biological characterization of snake venoms components is critically important, since these complex mixtures provide a source of molecules with antiparasitic potential, making further studies necessary to identify and characterize components with higher efficacy and selectivity.
Asunto(s)
Antiparasitarios/farmacología , Leishmania infantum/efectos de los fármacos , Fosfolipasas A2/farmacología , Venenos de Serpiente/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Antiparasitarios/química , Antiparasitarios/aislamiento & purificación , Bothrops , Relación Dosis-Respuesta a Droga , Fibroblastos/efectos de los fármacos , Ratones , Paraguay , Pruebas de Sensibilidad Parasitaria , Fosfolipasas A2/química , Fosfolipasas A2/aislamiento & purificación , Venenos de Serpiente/química , Venenos de Serpiente/aislamiento & purificación , Relación Estructura-ActividadRESUMEN
BACKGROUND: Research involving snake venom has gradually surpassed the simple discovery of new molecules using purification and structural characterization processes, and extended to the identification of their molecular targets and the evaluation of their therapeutic potential. Nevertheless, this only became possible due to constant progress in experimental biology and protein purification approaches. OBJECTIVE: This review aims to discuss the main components of snake venoms that have been investigated for biotechnological purposes, and to discover how these promising biomolecules were obtained with the satisfactory degree of purity that have enabled such studies. Advances in purification technologies of various snake venom molecules have allowed for important discoveries of proteins and peptides with different biomedical and biotechnological applications. RESULT AND CONCLUSION: It is believed that significant experimental and computational advances will arise in similar proportions in the coming years that will allow researchers to map the molecular regions responsible for their pharmacological actions, their respective mechanisms of action and their cell targets.
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Venenos de Serpiente/química , Venenos de Serpiente/farmacología , Serpientes/fisiología , Animales , Descubrimiento de Drogas , Humanos , Proteínas/química , Venenos de Serpiente/genética , Venenos de Serpiente/uso terapéuticoRESUMEN
Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.
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Camélidos del Nuevo Mundo/inmunología , Crotoxina/inmunología , Anticuerpos de Dominio Único/inmunología , Animales , Crotoxina/toxicidad , Escherichia coli/genética , Masculino , Ratones , Simulación del Acoplamiento Molecular , Enfermedades Musculares/inducido químicamente , Enfermedades Musculares/tratamiento farmacológico , Anticuerpos de Dominio Único/genética , Anticuerpos de Dominio Único/uso terapéutico , Mordeduras de Serpientes/diagnóstico , Mordeduras de Serpientes/terapiaRESUMEN
BACKGROUND: Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. METHODS: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. RESULTS: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896.47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. CONCLUSION: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.
RESUMEN
Phospholipases A2 (PLA2s) are important enzymes present in snake venoms and are related to a wide spectrum of pharmacological effects, however the toxic potential and therapeutic effects of acidic isoforms have not been fully explored and understood. Due to this, the present study describes the isolation and biochemical characterization of two new acidic Asp49-PLA2s from Bothrops brazili snake venom, named Braziliase-I and Braziliase-II. The venom was fractionated in three chromatographic steps: ion exchange, hydrophobic interaction and reversed phase. The isoelectric point (pI) of the isolated PLA2s was determined by two-dimensional electrophoresis, and 5.2 and 5.3 pIs for Braziliase-I and II were observed, respectively. The molecular mass was determined with values ââof 13,894 and 13,869Da for Braziliase-I and II, respectively. Amino acid sequence by Edman degradation and mass spectrometry completed 87% and 74% of the sequences, respectively for Braziliase-I and II. Molecular modeling of isolated PLA2s using acid PLA2BthA-I-PLA2 from B. jararacussu template showed high quality. Both acidic PLA2s showed no significant myotoxic activity, however they induced significant oedematogenic activity. Braziliase-I and II (100µg/mL) showed 31.5% and 33.2% of cytotoxicity on Trypanosoma cruzi and 26.2% and 19.2% on Leishmania infantum, respectively. Braziliase-I and II (10µg) inhibited 96.98% and 87.98% of platelet aggregation induced by ADP and 66.94% and 49% induced by collagen, respectively. The acidic PLA2s biochemical and structural characterization can lead to a better understanding of its pharmacological effects and functional roles in snakebites pathophysiology, as well as its possible biotechnological applications as research probes and drug leads.
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Fosfolipasas A2/química , Inhibidores de Agregación Plaquetaria/química , Agregación Plaquetaria/efectos de los fármacos , Venenos de Serpiente/química , Secuencia de Aminoácidos/genética , Animales , Bothrops/genética , Leishmania infantum/efectos de los fármacos , Leishmania infantum/patogenicidad , Modelos Moleculares , Fosfolipasas A2/genética , Fosfolipasas A2/aislamiento & purificación , Fosfolipasas A2/farmacología , Inhibidores de Agregación Plaquetaria/aislamiento & purificación , Inhibidores de Agregación Plaquetaria/farmacología , Homología de Secuencia de Aminoácido , Trypanosoma cruzi/efectos de los fármacos , Trypanosoma cruzi/patogenicidadRESUMEN
Snake venom phospholipases A2 (PLA2 s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA2 homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA2 s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 µg/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA2 from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.
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Antibacterianos/farmacología , Venenos de Crotálidos/enzimología , Fosfolipasas A2/química , Viperidae , Animales , Antibacterianos/síntesis química , Cromatografía en Gel/métodos , Cromatografía de Fase Inversa/métodos , Venenos de Crotálidos/química , Diseño de Fármacos , Pruebas de Enzimas , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Péptidos/farmacología , Fosfolipasas A2/aislamiento & purificación , Pseudomonas aeruginosa/efectos de los fármacosRESUMEN
This study aimed to evaluate the in vivo anti-Leishmania amazonensis activity of a Phospholipase A2 (Asp49-PLA2), isolated from Bothrops jararacussu venom, encapsulated in liposomes as a modified toxin release system. The activity of the liposomes was evaluated in BALB/c mice, previously infected with 1×105 of the parasite's promastigotes. The size of the paw lesion in Asp49-PLA2-liposomal-treated animals, after 21days, was observed as decreasing by 16% relative to the untreated control group and 12% by the Glucantime®-treated animals, which was used as a reference drug. At the end of the treatment, the animals were sacrificed and the paw and lymph node tissues were collected. Part of the collection was used to recover amastigotes and another to quantify cytokines and nitrites. In the group treated with Asp49-PLA2-liposomes the parasitic load was observed to be reduced by 73.5% in the macerated lymph node, compared to the control group. Comparatively, in the paw tissue was observed a reduction of 57.1%. The infected groups treated with Asp49-PLA2-liposomes showed significant production in TNF-α measured in lymph nodes and paw (43.73pg/mL±2.25 and 81.03pg/mL±5.52, respectively) and nitrite levels (31.28µM±0.58 and 35.64µM±5.08) also measured in lymph nodes and paw tissues, respectively, compared to untreated groups. These results indicate that the Asp49-PLA2-loaded liposomes were able to activate the production of some cellular components of the protective TH1 response during the infection, constituting a promising tool for inducing the microbicidal activity of the Leishmania-infected macrophages.
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Venenos de Crotálidos/metabolismo , Leishmania/fisiología , Leishmaniasis Cutánea/terapia , Liposomas/metabolismo , Ganglios Linfáticos/inmunología , Macrófagos/inmunología , Fosfolipasas A2/metabolismo , Proteínas de Reptiles/metabolismo , Animales , Antiinfecciosos/metabolismo , Bothrops , Modelos Animales de Enfermedad , Humanos , Liposomas/uso terapéutico , Ganglios Linfáticos/parasitología , Macrófagos/parasitología , Masculino , Ratones , Ratones Endogámicos BALB C , Nitritos/metabolismo , Carga de Parásitos , Fosfolipasas A2/uso terapéutico , Proteínas de Reptiles/uso terapéutico , Células TH1/inmunología , Terapias en Investigación , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Background: Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. Methods: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. Results: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896. 47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. Conclusion: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.(AU)
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Animales , Venenos de Avispas , Fosfolipasas A2/química , Fosfolipasas A2/aislamiento & purificación , Avispas/enzimologíaRESUMEN
Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. Methods: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. Results: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896. 47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. Conclusion: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.(AU)
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Animales , Avispas , Receptores de Fosfolipasa A2/aislamiento & purificación , Receptores de Fosfolipasa A2/química , Intoxicación , Espectrometría de Masas/métodos , Receptores de Fosfolipasa A2/química , Cromatografía de Fase Inversa/métodosRESUMEN
Snake venom phospholipases A(2) (PLA(2)s) are responsible for numerous pathophysiological effects in snakebites; however, their biochemical properties favour antimicrobial actions against different pathogens, thus constituting a true source of potential microbicidal agents. This study describes the isolation of a Lys49 PLA(2) homologue from Lachesis muta muta venom using two chromatographic steps: size exclusion and reverse phase. The protein showed a molecular mass of 13,889 Da and was devoid of phospholipase activity on an artificial substrate. The primary structure made it possible to identify an unpublished protein from L. m. muta venom, named LmutTX, that presented high identity with other Lys49 PLA(2)s from bothropic venoms. Synthetic peptides designed from LmutTX were evaluated for their cytotoxic and antimicrobial activities. LmutTX was cytotoxic against C2C12 myotubes at concentrations of at least 200 g/mL, whereas the peptides showed a low cytolytic effect. LmutTX showed antibacterial activity against Gram-positive and Gram-negative bacteria; however, S. aureusATCC 29213 and MRSA strains were more sensitive to the toxin's action. Synthetic peptides were tested on S. aureus, MRSA and P. aeruginosaATCC 27853 strains, showing promising results. This study describes for the first time the isolation of a Lys49 PLA(2) from Lachesis snake venom and shows that peptides from specific regions of the sequence may constitute new sources of molecules with biotechnological potential.
RESUMEN
Background: Wasp venoms constitute a molecular reservoir of new pharmacological substances such as peptides and proteins, biological property holders, many of which are yet to be identified. Exploring these sources may lead to the discovery of molecules hitherto unknown. This study describes, for the first time in hymenopteran venoms, the identification of an enzymatically inactive phospholipase A2 (PLA2) from the venom of the social wasp Polybia occidentalis. Methods: P. occidentalis venom was fractioned by molecular exclusion and reverse phase chromatography. For the biochemical characterization of the protein, 1D and 2D SDS-PAGE were performed, along with phospholipase activity assays on synthetic substrates, MALDI-TOF mass spectrometry and sequencing by Edman degradation. Results: The protein, called PocTX, was isolated using two chromatographic steps. Based on the phospholipase activity assay, electrophoresis and mass spectrometry, the protein presented a high degree of purity, with a mass of 13,896. 47 Da and a basic pI. After sequencing by the Edman degradation method, it was found that the protein showed a high identity with snake venom PLA2 homologues. Conclusion: This is the first report of an enzymatically inactive PLA2 isolated from wasp venom, similar to snake PLA2 homologues.
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Animales , /aislamiento & purificación , /química , Venenos de Avispas , Avispas/enzimologíaRESUMEN
Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIß and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment.
Asunto(s)
Proteínas Sanguíneas/química , Proteínas Sanguíneas/genética , Bothrops/genética , Serpientes de Coral/genética , Modelos Moleculares , Secuencia de Aminoácidos , Animales , Clonación Molecular , Conformación ProteicaRESUMEN
Snake venoms contain various proteins, especially phospholipases A2 (PLA2s), which present potential applications in diverse areas of health and medicine. In this study, a new basic PLA2 from Bothrops marajoensis with parasiticidal activity was purified and characterized biochemically and biologically. B. marajoensis venom was fractionated through cation exchange followed by reverse phase chromatographies. The isolated toxin, BmajPLA2-II, was structurally characterized with MALDI-TOF (Matrix-assisted laser desorption/ionization-time of flight) mass spectrometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by two-dimensional electrophoresis, partial amino acid sequencing, an enzymatic activity assay, circular dichroism, and dynamic light scattering assays. These structural characterization tests presented BmajPLA2-II as a basic Lys49 PLA2 homologue, compatible with other basic snake venom PLA2s (svPLA2), with a tendency to form aggregations. The in vitro anti-parasitic potential of B. marajoensis venom and of BmajPLA2-II was evaluated against Leishmania infantum promastigotes and Trypanosoma cruzi epimastigotes, showing significant activity at a concentration of 100µg/mL. The venom and BmajPLA2-II presented IC50 of 0.14±0.08 and 6.41±0.64µg/mL, respectively, against intraerythrocytic forms of Plasmodium falciparum with CC50 cytotoxicity values against HepG2 cells of 43.64±7.94 and >150µg/mL, respectively. The biotechnological potential of these substances in relation to leishmaniasis, Chagas disease and malaria should be more deeply investigated.
Asunto(s)
Antiprotozoarios/química , Antiprotozoarios/farmacología , Bothrops , Venenos de Crotálidos/enzimología , Fosfolipasas A2/química , Fosfolipasas A2/farmacología , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Antiprotozoarios/metabolismo , Fosfolipasas A2/metabolismo , Tripsina/metabolismoAsunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/metabolismo , Leishmania/fisiología , Leishmaniasis Cutánea/tratamiento farmacológico , Liposomas/farmacología , Macrófagos/efectos de los fármacos , Fosfolipasas A2/farmacología , Animales , Línea Celular , Supervivencia Celular , Sistemas de Liberación de Medicamentos , Liposomas/metabolismo , Macrófagos/parasitología , Macrófagos/fisiología , Ratones , Nitritos/metabolismo , Fosfolipasas A2/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.