RESUMEN
This study investigated geographic variability in the venom of Centruroides sculpturatus scorpions from different biotopes. Venom from scorpions collected from two different regions in Arizona; Santa Rita Foothills (SR) and Yarnell (Yar) were analyzed. We found differences between venoms, mainly in the two most abundant peptides; SR (CsEv2e and CsEv1f) and Yar (CsEv2 and CsEv1c) identified as natural variants of CsEv1 and CsEv2. Sequence analyses of these peptides revealed conservative amino acid changes between variants, which may underlie biological activity against arthropods. A third peptide (CsEv6) was highly abundant in the Yar venom compared to the SR venom. CsEv6 is a 67 amino acid peptide with 8 cysteines. CsEv6 did not exhibit toxicity to the three animal models tested. However, both venoms shared similarities in peptides that are predicted to deter predators. For example, both venoms expressed CsEI (lethal to chick) in similar abundance, while CsEd and CsEM1a (toxic to mammals) displayed only moderate variation in their abundance. Electrophysiological evaluation of CsEd and CsEM1a showed that both toxins act on the human sodium-channel subtype 1.6 (hNav 1.6). Complete sequencing revealed that both toxins are structurally similar to beta-toxins isolated from different Centruroides species that also target hNav 1.6.
Asunto(s)
Proteínas de Artrópodos , Variación Genética , Venenos de Escorpión , Escorpiones , Animales , Arizona , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/toxicidad , Células CHO , Pollos , Cricetulus , Gryllidae , Células HEK293 , Humanos , Ratones , Canal de Sodio Activado por Voltaje NAV1.6/genética , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/genética , Venenos de Escorpión/toxicidad , Escorpiones/química , Escorpiones/genética , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: In T cells, the Kv1.3 and the KCa3.1 potassium channels regulate the membrane potential and calcium homeostasis. Notably, during TEM cell activation, the number of Kv1.3 channels on the cell membrane dramatically increases. Kv1.3 blockade results in inhibition of Ca2+ signaling in TEM cells, thus eliciting an immunomodulatory effect. Among the naturally occurring peptides, the Vm24 toxin from the Mexican scorpion Vaejovis mexicanus is the most potent and selective Kv1.3 channel blocker known, which makes it a promissory candidate for its use in the clinic. We have shown that addition of Vm24 to TCR-activated human T cells inhibits CD25 expression, cell proliferation and reduces delayed-type hypersensitivity reactions in a chronic inflammation model. Here, we used the Vm24 toxin as a tool to investigate the molecular events that follow Kv1.3 blockade specifically on human CD4+ TEM cells as they are actively involved in inflammation and are key mediators of autoimmune diseases. METHODS: We combined cell viability, activation, and multiplex cytokine assays with a proteomic analysis to identify the biological processes affected by Kv1.3 blockade on healthy donors CD4+ TEM cells, following TCR activation in the presence or absence of the Vm24 toxin. RESULTS: The peptide completely blocked Kv1.3 channels currents without impairing TEM cell viability, and in response to TCR stimulation, it inhibited the expression of the activation markers CD25 and CD40L (but not that of CD69), as well as the secretion of the pro-inflammatory cytokines IFN-γ and TNF and the anti-inflammatory cytokines IL-4, IL-5, IL-9, IL-10, and IL-13. These results, in combination with data from the proteomic analysis, indicate that the biological processes most affected by the blockade of Kv1.3 channels in a T cell activation context were cytokine-cytokine receptor interaction, mRNA processing via spliceosome, response to unfolded proteins and intracellular vesicle transport, targeting the cell protein synthesis machinery. CONCLUSIONS: The Vm24 toxin, a highly specific inhibitor of Kv1.3 channels allowed us to define downstream functions of the Kv1.3 channels in human CD4+ TEM lymphocytes. Blocking Kv1.3 channels profoundly affects the mRNA synthesis machinery, the unfolded protein response and the intracellular vesicle transport, impairing the synthesis and secretion of cytokines in response to TCR engagement, underscoring the role of Kv1.3 channels in regulating TEM lymphocyte function.
Asunto(s)
Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Canal de Potasio Kv1.3/antagonistas & inhibidores , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Receptores de Antígenos de Linfocitos T/metabolismo , Venenos de Escorpión/farmacología , Animales , Citocinas/biosíntesisRESUMEN
While most scorpion venom components identified in the past are peptidic or proteinic in nature, we report here a new alkaloid isolated from the venom of the Mexican scorpion Megacormus gertschi. Nuclear magnetic resonance and mass spectrometric investigations elucidate the structure of the alkaloid as ( Z)- N-(2-(1 H-imidazol-4-yl)ethyl)-3-(4-hydroxy-3-methoxyphenyl)-2-methoxyacrylamide (1). A chemical method of synthesizing this alkaloid is also described. Although abundant in venom, the above alkaloid was not found to have insecticidal activity. Structural analysis suggests that this venom alkaloid might be of potential interest for evaluating its medicinal effect.
Asunto(s)
Alcaloides/química , Venenos de Escorpión/química , Alcaloides/toxicidad , Animales , Cromatografía Líquida de Alta Presión , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Estructura Molecular , Escorpiones , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
BACKGROUND: The venoms of several scorpion species have long been associated with pancreatitis in animal models and humans. Antarease, a Zn-metalloprotease from Tityus serrulatus, is able to penetrate intact pancreatic tissue and disrupts the normal vesicular traffic necessary for secretion, so it could play a relevant role in the onset of acute pancreatitis. METHODS: The cDNA libraries from five different scorpion species were screened for antarease homologs with specific primers. The amplified PCR products were cloned and sequenced. A structural model was constructed to assess the functionality of the putative metalloproteases. A phylogenetic analysis was performed to identify clustering patterns of these venom components. RESULTS: Antarease-like sequences were amplified from all the screened cDNA libraries. The complete sequence of the antarease from T. serrulatus was obtained. The structural model of the putative antarease from Tityus trivittatus shows that it may adopt a catalytically active conformation, sharing relevant structural elements with previously reported metalloproteases of the ADAM family. The phylogenetic analysis reveals that the reported sequences cluster in groups that correlate with the geographical localization of the respective species. CONCLUSIONS: Antareases are ubiquitous to a broad range of scorpion species, where they could be catalytically active enzymes. These molecules can be used to describe the evolution of scorpion venoms under different ecogeographic constrains. GENERAL SIGNIFICANCE: For the first time the complete sequence of the antareases is reported. It is demonstrated that antareases are common in the venom of different scorpion species. They are now proposed as targets for antivenom therapies.
Asunto(s)
Metaloproteasas/química , Venenos de Escorpión/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Metaloproteasas/genética , Metaloproteasas/fisiología , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , FilogeografíaRESUMEN
Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20-171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.
Asunto(s)
Perciformes , Escorpiones , Animales , Filogenia , Péptidos/farmacología , AminoácidosRESUMEN
A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K+ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K+ channel hKV1.2 with high affinity (Kd = 65 nM). The conductance-voltage relationship of KV1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca2+-activated KCa2.2 and KCa3.1 channels, with Kd = 502 nM, and Kd = 58 nM, respectively. However, the peptide does not inhibit hKV1.1, hKV1.3, hKV1.4, hKV1.5, hKV1.6, hKV11.1, mKCa1.1 K+ channels or the hNaV1.5 and hNaV1.4 Na+ channels at 1 µM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.
Asunto(s)
Venenos de Escorpión , Humanos , Animales , Venenos de Escorpión/química , Filogenia , Bloqueadores de los Canales de Potasio/química , Secuencia de Aminoácidos , Péptidos/química , Escorpiones/químicaRESUMEN
Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.
Asunto(s)
Escorpiones , Ponzoñas , Humanos , Animales , Ratones , Escorpiones/genética , Cromatografía Liquida , Espectrometría de Masas en Tándem , Inhibidores de Proteasas , MamíferosRESUMEN
Ca2+ channel blockers (CCBs) are commonly used to treat different cardiovascular conditions. These drugs disrupt the intracellular Ca2+ signaling network, inhibiting numerous cellular functions in different cells, including T lymphocytes. We explored the effect of the CCB verapamil on normal human peripheral blood T cell activation, proliferation, and cytokine production. Cells were activated by ligating CD3 or CD3/CD28 in the presence or absence of verapamil, and the expression of activation-induced cell surface molecules (CD25, CD40L, CD69, PD-1, and OX40), cell proliferation, and cytokine release were assessed by flow cytometry. Verapamil exerted a dose-dependent inhibitory effect on the expression of all the activation-induced cell surface molecules tested. In addition, verapamil diminished T cell proliferation induced in response to CD3/CD28 stimulation. Likewise, the production of Th1/Th17 and Th2 cytokines was also reduced by verapamil. Our data substantiate a potent in vitro suppressive effect of verapamil on T lymphocytes, a fact that might be relevant in patients receiving CCBs.
RESUMEN
Epilepsy is one of the most common neurological diseases in the world. The objective of this research was to investigate a new peptide from the venom of the social wasp Chartergellus communis useful to the study or pharmacotherapy of epilepsy. The wasps were collected, and their venom was extracted. Afterward, the steps of fractionation, sequencing, and identification were carried out to obtain four peptides. These molecules were synthesized for behavioral evaluation tests and electroencephalographic assays to determine their antiseizure potential (induction of acute seizures using the chemical compounds, pentylenetetrazole - PTZ, and pilocarpine - PILO) and analysis of neuropharmacological profile (general spontaneous activity and alteration in motor coordination). Chartergellus-CP1 (i.c.v. - 3.0 µg/animal) caused beneficial alterations in some of the parameters evaluated in both models: PTZ (latency and duration of maximum seizures) and PILO (latency and duration of, and protection against, maximum seizures, and reduction of the median of the seizure scores. When evaluated in 3 doses in the seizure model induced by PILO, the dose of 3.0 µg/animal protected the animals against seizures, with an estimated ED50 of 1.49 µg/animal. Electroencephalographic evaluation of Chartergellus-CP1 showed an improvement in latency, quantity, and percentage of protection against generalized electroencephalographic seizures in the PILO model. Further, Chartergellus-CP1 did not cause adverse effects on general spontaneous activity and motor coordination of animals. This study demonstrated how compounds isolated from wasps' venom may be important resources in the search for new drugs. Such compounds can be considered valuable therapeutic and biotechnological tools for the study and future treatment of epileptic disorders. In this context, a peptide that is potentially useful for epilepsy pharmacotherapy was identified in the venom of C. communis.
Asunto(s)
Anticonvulsivantes/farmacología , Venenos de Avispas/farmacología , Avispas , Animales , Anticonvulsivantes/uso terapéutico , Modelos Animales de Enfermedad , Pentilenotetrazol/uso terapéutico , Pentilenotetrazol/toxicidad , Péptidos , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológicoRESUMEN
The Colombian scorpion Centruroides margaritatus produces a venom considered of low toxicity. Nevertheless, there are known cases of envenomation resulting in cardiovascular disorders, probably due to venom components that target ion channels. Among them, the humanether-à-go-go-Related gene (hERG1) potassium channels are critical for cardiac action potential repolarization and alteration in its functionality are associated with cardiac disorders. This work describes the purification and electrophysiological characterization of a Centruroides margaritatus venom component acting on hERG1 channels, the CmERG1 toxin. This novel peptide is composed of 42 amino acids with a MW of 4792.88 Da, folded by four disulfide bonds and it is classified as member number 10 of the γ-KTx1 toxin family. CmERG1 inhibits hERG1 currents with an IC50 of 3.4 ± 0.2 nM. Despite its 90.5% identity with toxin É£-KTx1.1, isolated from Centruroides noxius, CmERG1 completely blocks hERG1 current, suggesting a more stable plug of the hERG channel, compared to that formed by other É£-KTx.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Venenos de Escorpión/farmacología , Animales , Colombia , Canales de Potasio Éter-A-Go-Go/fisiología , Péptidos/aislamiento & purificación , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Venenos de Escorpión/aislamiento & purificación , EscorpionesRESUMEN
The scorpion venom is a cocktail of many components. Its composition can exhibit a level of plasticity in response to different behavioral and environmental factors, leading to intraspecific variation. The toxicity and specificity of scorpion venoms appear to be taxon-dependent, due to a co-evolutionary interaction with prey and predators, which shaped the composition at the molecular level. The venom regeneration by the venom glands is an asynchronous process, in which particular components are expressed at different stages and at different rates. According to this, it can be reasonably assumed that the regeneration of toxicity in the venom is also asynchronous. In this work, we studied the toxicity regeneration dynamics by the scorpion Centruroides limpidus after full venom depletion by electrical stimulation. For this, we evaluated the toxicity of venom samples extracted at different days post depletion, against insects (crickets) and mammals (humans, by assessing the venom activity on the human voltage-dependent Na+ channel Nav1.6). The regeneration of toxicity against humans lagged behind that against crickets (13 vs 10 days, respectively). Thirteen days after depletion the venom seems to be replenished. Our results show asynchrony in the regeneration of species-specific toxic activity in the venom of Centruroides limpidus. The understanding of the venom regeneration kinetics for the different scorpion species will help to design venom extraction protocols that could maximize the yield and quality of the collected venoms.
Asunto(s)
Glándulas Exocrinas/fisiología , Venenos de Escorpión/biosíntesis , Venenos de Escorpión/toxicidad , Escorpiones/fisiología , Animales , Estimulación Eléctrica/métodos , Gryllidae/efectos de los fármacos , Células HEK293 , Humanos , Canal de Sodio Activado por Voltaje NAV1.6/efectos de los fármacosRESUMEN
The soluble venom of the scorpion Tityus macrochirus was separated by chromatographic procedures and three homogeneous peptides were obtained and their primary structures were determined. They were called: Tma1-Tma3, from the abbreviated name of the scorpion. Tma1 is a peptide containing 65 amino acids with four disulfide linkages and a molecular weight of 7386.2â¯Da. It is a mammalian toxin, shown to affect human sodium-channels sub-types hNav1.6 and hNav1.4. Tma2 and Tma3 are peptides containing 69 amino acids linked by four disulfide bonds, molecular weights 7819.7 and 7830.0â¯Da, respectively. They do not affect human sodium-channels but are lethal to insects (crickets). A phylogenic analysis of the three peptides and those of other toxic peptides isolated from the genus Tityus and Centruroides were grouped together and analyzed, permitting to obtain a topology with two main clades, one includes most sodium-channel anti-insect scorpion toxins and others includes mostly sodium-channel scorpion toxins anti-mammalian. Tma1 segregates among a group of well-studied ß-class toxins of other Tityus species such as T. discrepans, T. obscurus and T. pachyurus. Tma2 and Tma3 are associated with anti-insect toxins, particularly with one of T. obscurus. This phylogenetic analysis confirms and enforces our experimental results obtained with these three new sodium-channel scorpion toxins.
Asunto(s)
Venenos de Escorpión/química , Escorpiones/química , Animales , Femenino , Gryllidae , Péptidos/química , Péptidos/aislamiento & purificación , Péptidos/toxicidad , Filogenia , Venenos de Escorpión/aislamiento & purificación , Análisis de Secuencia de Proteína , Pruebas de ToxicidadRESUMEN
Voltage-gated potassium channels of the ether-a-go-go related gene (ERG) family are implicated in many important cellular processes. Three such genes have been cloned (erg1, erg2 and erg3) and shown to be expressed in the central nervous system (CNS) of mammalians. This communication describes the isolation and characterization of two isoforms of scorpion toxin (CeErg4 and CeErg5, systematic nomenclature gamma-KTx1.7 and gamma-KTx1.8, respectively) that can discriminate the various subtypes of ERG channels of human and rat. These peptides were purified from the venom of the Mexican scorpion Centruroides elegans elegans. They contain 42 amino acid residues, tightly folded by four disulfide bridges. Both peptides block in a reversible manner human and rat ERG1 channels, but have no effect on human ERG2. They also block completely and irreversibly the rat ERG2 and the human ERG3 channels hence are excellent tools for the discrimination of the various sub-types of ion-channels studied.
Asunto(s)
Canales de Potasio Éter-A-Go-Go/metabolismo , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Bloqueadores de los Canales de Potasio , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/metabolismo , Escorpiones/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Canales de Potasio Éter-A-Go-Go/genética , Humanos , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Péptidos/genética , Bloqueadores de los Canales de Potasio/aislamiento & purificación , Bloqueadores de los Canales de Potasio/metabolismo , Ratas , Venenos de Escorpión/genética , Alineación de SecuenciaRESUMEN
To understand the diversity of scorpion venom, RNA from venomous glands from a sawfinger scorpion, Serradigitus gertschi, of the family Vaejovidae, was extracted and used for transcriptomic analysis. A total of 84,835 transcripts were assembled after Illumina sequencing. From those, 119 transcripts were annotated and found to putatively code for peptides or proteins that share sequence similarities with the previously reported venom components of other species. In accordance with sequence similarity, the transcripts were classified as potentially coding for 37 ion channel toxins; 17 host defense peptides; 28 enzymes, including phospholipases, hyaluronidases, metalloproteases, and serine proteases; nine protease inhibitor-like peptides; 10 peptides of the cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 protein superfamily; seven La1-like peptides; and 11 sequences classified as "other venom components". A mass fingerprint performed by mass spectrometry identified 204 components with molecular masses varying from 444.26 Da to 12,432.80 Da, plus several higher molecular weight proteins whose precise masses were not determined. The LC-MS/MS analysis of a tryptic digestion of the soluble venom resulted in the de novo determination of 16,840 peptide sequences, 24 of which matched sequences predicted from the translated transcriptome. The database presented here increases our general knowledge of the biodiversity of venom components from neglected non-buthid scorpions.
Asunto(s)
Proteínas de Artrópodos/análisis , Venenos de Escorpión/química , Animales , Bloqueadores de los Canales de Calcio/análisis , Bloqueadores de los Canales de Calcio/química , Femenino , Perfilación de la Expresión Génica , Hialuronoglucosaminidasa/análisis , Hialuronoglucosaminidasa/química , Masculino , Péptido Hidrolasas/análisis , Péptido Hidrolasas/química , Péptidos/análisis , Péptidos/química , Fosfolipasas A2/análisis , Fosfolipasas A2/química , Bloqueadores de los Canales de Potasio/análisis , Bloqueadores de los Canales de Potasio/química , Proteoma , Proteómica , Escorpiones , Bloqueadores de los Canales de Sodio/análisis , Bloqueadores de los Canales de Sodio/químicaRESUMEN
Venom from male and female scorpions of the species Centruroides limpidus were separated by HPLC and their molecular masses determined by mass spectrometry. The relative concentration of components eluting in equivalent retention times from the HPLC column shows some differences. A new peptide with 29 amino acids, cross-linked by three disulfide bonds was found in male scorpions and its structure determined. Another unknown peptide present in female venom, with sequence identity similar to K+-channel blocking peptide was isolated. This peptide contains 39 amino acid residues linked by three disulfide bonds. Due to sequence similarities, a systematic number (αKTx2.18) was assigned. Venom from male and female scorpions was separated by Sephadex G-50 gel filtration. Components of fraction I of this chromatogram were analyzed by two-dimensional gel electrophoresis and 41 spots were selected (20 from female and 21 from male). The spots were excised from the gel, enzymatically digested and sequenced by LC-MS/MS. This procedure allowed the identification of several proteins containing similar amino acid sequence of other known proteins registered on UniProt database. Among these proteins the presence of metalloproteinases (proteolytic enzymes), hyaluronidases and phosphatases were experimentally determined and shown to be present in both venom samples. The results shown here should help further work aimed at fully identification of the structure and function of venom components form C. limpidus male and female scorpions.
Asunto(s)
Proteínas de Artrópodos/química , Proteoma , Venenos de Escorpión/química , Animales , Proteínas de Artrópodos/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Femenino , Masculino , Espectrometría de Masas , Análisis de Secuencia de Proteína , Caracteres SexualesRESUMEN
It is widely known that targeting a variety of antigens to the DEC-205 receptor on dendritic cells (DCs) significantly potentiate immunity. This communication reports the development of a new murine monoclonal antibody (mAb) against the chicken DEC-205, using as immunogen the carbohydrate recognition domain-2 (CRD-2) heterologously expressed. This mAb recognizes a protein band of 250kDa by immunoprecipitation analysis and shows strong cross-reactivity with human and pig DEC-205. Furthermore, the hemagglutinin (HA) of avian influenza H5N2 virus was cloned and expressed using insect cell-baculovirus expression system. We chemically conjugated the anti-chicken DEC-205 antibody with the highly purified HA to direct the antigen to the dendritic cells and evaluate the immune response elicited in vivo by this conjugate. A single dose of chemical conjugate was sufficient to elicit a strong immune response in chickens as early as fourteen days after priming. In addition, the conjugate induced an earlier and higher response compared to unconjugated HA. These results suggest that the strategy described here has potential to be used in the future design and development of successful vaccines against different chicken infectious diseases with direct impact in biotechnology and veterinary fields.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Células Dendríticas/metabolismo , Vacunas contra la Influenza/inmunología , Gripe Aviar/prevención & control , Lectinas Tipo C/inmunología , Antígenos de Histocompatibilidad Menor/inmunología , Receptores de Superficie Celular/inmunología , Animales , Pollos/inmunología , Hemaglutininas , Humanos , Subtipo H5N1 del Virus de la Influenza A/inmunología , Subtipo H5N2 del Virus de la Influenza A/inmunología , Gripe Aviar/metabolismo , PorcinosRESUMEN
Chatergellus communis is a wasp species endemic to the neotropical region and its venom constituents have never been described. In this study, two peptides from C. communis venom, denominated Communis and Communis-AAAA, were chemically and biologically characterized. In respect to the chemical characterization, the following amino acid sequences and molecular masses were identified: Communis: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-COOH (1340.9Da) Communis-AAAA: Ile-Asn-Trp-Lys-Ala-Ile-Leu-Gly-Lys-Ile-Gly-Lys-Ala-Ala-Ala-Ala-Val-Xle-NH2 (1836.3Da). Furthermore, their biological effects were compared, accounting for the differences in structural characteristics between the two peptides. To this end, three biological assays were performed in order to evaluate the hyperalgesic, edematogenic and hemolytic effects of these molecules. Communis-AAAA, unlike Communis, showed a potent hemolytic activity with EC50=142.6µM. Moreover, the highest dose of Communis-AAAA (2nmol/animal) induced hyperalgesia in mice. On the other hand, Communis (10nmol/animal) was able to induce edema but did not present hemolytic or hyperalgesic activity. Although both peptides have similarities in linear structures, we demonstrated the distinct biological effects of Communis and Communis-AAAA. This is the first study with Chartegellus communis venom, and both Communis and Communis-AAAA are unpublished peptides.
Asunto(s)
Alanina/química , Hemólisis/efectos de los fármacos , Péptidos/farmacología , Venenos de Avispas/farmacología , Secuencia de Aminoácidos/genética , Animales , Humanos , Oligopéptidos/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Péptidos/química , Péptidos/genética , Tripsina/química , Venenos de Avispas/química , Venenos de Avispas/genética , Avispas/química , Avispas/genéticaRESUMEN
This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.
Asunto(s)
Anticuerpos/genética , Anticuerpos/inmunología , Evolución Molecular Dirigida , Fragmentos de Inmunoglobulinas/inmunología , Biblioteca de Péptidos , Venenos de Escorpión/inmunología , Secuencia de Aminoácidos , Animales , Afinidad de Anticuerpos , Humanos , Fragmentos de Inmunoglobulinas/genética , Datos de Secuencia Molecular , Neurotoxinas/inmunología , Pruebas de Neutralización , Venenos de Escorpión/aislamiento & purificación , Escorpiones , Alineación de Secuencia , Resonancia por Plasmón de SuperficieRESUMEN
The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50=2.4ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph node tissue sections, mAb-1C4 marked cells in situ, some with apparent dendritic morphology. Thus the mAb generated to a recombinant epitope from horse CD11c identified the molecule in intact cells within horse lymphoid tissue. By the labelling intensity, the histological location (paracortical and interfollicular areas) and the apparent morphology of the marked cells, we can say that these are putative horse dendritic cells (DCs). The development of a mAb to horse CD11c provides a new tool to better study the horse DC biology and opens other biotechnological avenues, such as DC targeting-based vaccines.