A consensus recombinant elapid long-chain α-neurotoxin and how protein folding matters for antibody recognition and neutralization of elapid venoms.

Antivenoms are essential in the treatment of the neurotoxicity caused by elapid snakebites. However, there are elapid neurotoxins, e.g., long-chain α-neurotoxins (also known as long-chain three-finger toxins) that are barely neutralized by commercial elapid antivenoms; so, recombinant elapid neurotoxins could be an alternative or complements for improving antibody production against the lethal long-chain α-neurotoxins from elapid venoms. This work communicates the expression of a recombinant long-chain α-neurotoxin, named HisrLcNTx or rLcNTx, which based on the most lethal long-chain α-neurotoxins reported, was constructed de novo. The gene of rLcNTx was synthesized and introduced into the expression vector pQE30, which contains a proteolytic cleavage region for exscinding the mature protein, and His residues in tandem for affinity purification. The cloned pQE30/rLcNTx was transfected into Escherichia coli Origami cells to express rLcNTx. After expression, it was found in inclusion bodies, and folded in multiple Cys-Cys structural isoforms. To observe the capability of those isoforms to generate antibodies against native long-chain α-neurotoxins, groups of rabbits were immunized with different cocktails of Cys-Cys rLcNTx isoforms. In vitro, and in vivo analyses revealed that rabbit antibodies raised against different rLcNTx Cys-Cys isoforms were able to recognize pure native long-chain α-neurotoxins and their elapid venoms, but they were unable to neutralize bungarotoxin, a classical long-chain α-neurotoxin, and other elapid venoms. The rLcNTx Cys-Cys isoform 2 was the immunogen that produced the best neutralizing antibodies in rabbits. Yet to neutralize the elapid venoms from the black mamba Dendroaspis polylepis, and the coral shield cobra Aspidelaps lubricus, it was required to use two types of antibodies, the ones produced using rLcNTx Cys-Cys isoform 2 and antibodies produced using short-chain α-neurotoxins. Expression of recombinant elapid neurotoxins as immunogens could be an alternative to improve elapid antivenoms; nevertheless, recombinant elapid neurotoxins must be well-folded to be used as immunogens for obtaining neutralizing antibodies.

Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family.

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Nav). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Nav receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Nav.

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A2 from Bothrops ammodytoides venom.

A mRNA transcript that codes for a phospholipase (PLA2) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA2 transcript was cloned onto a pCR®2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLA2s, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA2 from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLA2s in other venoms from regional species of Bothrops.

Antimicrobial, toxicological, and antigenic characteristics of three scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus and Tityus n. sp. aff. metuendus.

The venom fractions of three buthid scorpion species from Colombia, C. margaritatus, T. pachyurus and T. n. sp. aff. metuendus, were examined for antimicrobial and toxicity toward mice and insects. The three venoms were separated into individual fractions using RP-HPLC, resulting in 85 fractions from C. margaritatus, 106 from T. pachyurus, and 70 from T. n. sp. aff. metuendus. The major fractions from the three scorpion venoms, which were eluted between 35 and 50 min, were tested for antimicrobial activity and toxicity. It was confirmed that the venom of the three species contains fractions with antimicrobial peptides that were evaluated against two bacterial strains of public health importance, Pseudomonas aeruginosa and Staphylococcus aureus. The venom of C. margaritatus had two antimicrobial fractions that showed activity against the named tested strains. The venom of T. pachyurus had three fractions that showed activity against S. aureus and two against both bacterial strains. Finally, the venom of T. n. sp. aff. metuendus had one fraction that showed activity against S. aureus, and five fractions showed activity against both bacterial strains. Also, some peptide fractions from the three venoms were toxic to mice. Last, the venoms of C. margaritatus and T. pachyurus were used as immunogens to obtain neutralizing antibodies against its respective venoms and to observe antibody recognition to related and unrelated scorpion venoms. A total of 15 mg of lyophilized antibodies were able to neutralize 1.5⋅LD50 of the venoms from T. n. sp. aff. metuendus, T. pachyurus and C. margaritatus, respectively. This information provides valuable insights into the diversity of each species' venom and their potential role in antimicrobial and venom toxicity.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus.

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

Identification and Venom Characterization of Two Scorpions from the State of Chihuahua Mexico: Chihuahuanus coahuliae and Chihuahuanus crassimannus.

Chihuahua is the largest state in Mexico. The ecosystem of this region is composed of large area of bushes, forests, and grasslands, which allows for a specific diversity of fauna; among them are interesting species of non-lethal scorpions. Most of the Chihuahuan scorpions have been previously morphologically and molecularly described; however, this manuscript could be the first to describe the composition of those venoms. This work aimed at the collection of two scorpion species from the region of Jiménez (Southwest of the State of Chihuahua), which belong to the species Chihuahuanus cohauilae and Chihuahuanus crassimanus; the two species were taxonomically and molecularly identified using a 16S DNA marker. Reverse-phase high-performance liquid chromatography (RP-HPLC) of C. coahuilae and C. crassimanus venoms allowed the identification of three fractions lethal to mice. Additionally, three fractions of each scorpion displayed an effect on house crickets. In the end, three new fractions from the venom of C. coahuilae were positive for antimicrobial activity, although none from C. crassimanus venom displayed growth inhibition. Despite being a preliminary study, the venom biochemical analysis of these two uncharacterized scorpion species opens the opportunity to find new molecules with potential applications in the biomedical and biotechnological fields.

Heterologous Expression of an Insecticidal Peptide Obtained from the Transcriptome of the Colombian Spider Phoneutria depilate.

Spider venoms are composed, among other substances, of peptide toxins whose selectivity for certain physiological targets has made them powerful tools for applications such as bioinsecticides, analgesics, antiarrhythmics, antibacterials, antifungals and antimalarials, among others. Bioinsecticides are an environmentally friendly alternative to conventional agrochemicals. In this paper, the primary structure of an insecticidal peptide was obtained from the venom gland transcriptome of the ctenid spider Phoneutria depilata (Transcript ID PhdNtxNav24). The peptide contains 53 amino acids, including 10 Cys residues that form 5 disulfide bonds. Using the amino acid sequence of such peptide, a synthetic gene was constructed de novo by overlapping PCRs and cloned into an expression vector. A recombinant peptide, named delta-ctenitoxin (rCtx-4), was obtained. It was expressed, folded, purified and validated using mass spectrometry (7994.61 Da). The insecticidal activity of rCtx-4 was demonstrated through intrathoracic injection in crickets (LD50 1.2 µg/g insect) and it was not toxic to mice. rCtx-4 is a potential bioinsecticide that could have a broad spectrum of applications in agriculture.

Antimicrobial and Immunomodulatory Effects of Selected Chemokine and Antimicrobial Peptide on Cytokine Profile during Salmonella Typhimurium Infection in Mouse.

The antimicrobial and immunomodulatory capacities of the peptide Css54 and the chemokine MCP-1 were tested. The first, a peptide isolated from the venom of the scorpion Centruroides suffusus suffusus was synthesized chemically. In contrast, the second is a monocyte chemoattractant expressed as a recombinant protein in our lab. It was observed in vitro that Css54 inhibited the growth of Salmonella enterica serovar Typhimurium (6.2 µg/mL). At high concentrations, it was toxic to macrophages (25 µg/mL), activated macrophage phagocytosis (1.5 µg/mL), and bound Salmonella LPS (3 µg/mL). On the other hand, the recombinant MCP-1 neither inhibited the growth of Salmonella Typhimurium nor was it toxic to macrophages (up to 25 µg/mL), nor activated macrophage phagocytosis or bound Salmonella LPS (up to 3 µg/mL). Although it was observed in vivo in mice Balb/C that both Css54 and MCP-1 did not resolve the intraperitoneal infection by S. Typhimurium, Css54 decreased the expression of IL-6 and increased IL-10, IL-12p70, and TNF-α levels; meanwhile, MCP-1 decreased the expression of IFN-γ and increased IL-12p70 and TNF-α. It was also observed that the combination of both molecules Css54 and MCP-1 increased the expression of IL-10 and TNF-α.

Transcriptomic Analysis of the Venom Gland and Enzymatic Characterization of the Venom of Phoneutria depilata (Ctenidae) from Colombia.

The transcriptome of the venom glands of the Phoneutria depilata spider was analyzed using RNA-seq with an Illumina protocol, which yielded 86,424 assembled transcripts. A total of 682 transcripts were identified as potentially coding for venom components. Most of the transcripts found were neurotoxins (156) that commonly act on sodium and calcium channels. Nevertheless, transcripts coding for some enzymes (239), growth factors (48), clotting factors (6), and a diuretic hormone (1) were found, which have not been described in this spider genus. Furthermore, an enzymatic characterization of the venom of P. depilata was performed, and the proteomic analysis showed a correlation between active protein bands and protein sequences found in the transcriptome. The transcriptomic analysis of P. depilata venom glands show a deeper description of its protein components, allowing the identification of novel molecules that could lead to the treatment of human diseases, or could be models for developing bioinsecticides.

Crotoxin B: Heterologous Expression, Protein Folding, Immunogenic Properties, and Irregular Presence in Crotalid Venoms.

Crotoxin complex CA/CB and crotamine are the main toxins associated with Crotalus envenomation besides the enzymatic activities of phospholipases (PLA2) and proteases. The neutralization at least of the crotoxin complex by neutralizing the subunit B could be a key in the production process of antivenoms against crotalids. Therefore, in this work, a Crotoxin B was recombinantly expressed to evaluate its capacity as an immunogen and its ability to produce neutralizing antibodies against crotalid venoms. A Crotoxin B transcript from Crotalus tzabcan was cloned into a pCR®2.1-TOPO vector (Invitrogen, Waltham, MA, USA) and subsequently expressed heterologously in bacteria. HisrCrotoxin B was extracted from inclusion bodies and refolded in vitro. The secondary structure of HisrCrotoxin B was comparable to the secondary structure of the native Crotoxin B, and it has PLA2 activity as the native Crotoxin B. HisrCrotoxin B was used to immunize rabbits, and the obtained antibodies partially inhibited the activity of PLA2 from C. tzabcan. The anti-HisrCrotoxin B antibodies neutralized the native Crotoxin B and the whole venoms from C. tzabcan, C. s. salvini, and C. mictlantecuhtli. Additionally, anti-HisrCrotoxin B antibodies recognized native Crotoxin B from different Crotalus species, and they could discriminate venom in species with high or low levels of or absence of Crotoxin B.

Heterologous expression of four recombinant toxins from Panamanian scorpions of the genus Tityus and Centruroides for production of antivenom.

BACKGROUND: The development of more effective antivenoms remains a necessity for countries where scorpionism is a public health problem. Also, the regionalization of antivenoms may be important for some countries with special scorpionism characteristics. OBJECTIVE: Production of antibodies capable of neutralizing the lethal effect of the venom of three scorpion species from Panama. METHODS: The primary structures of two neurotoxins from T. pachyurus, one from T. cerroazul and another from C. bicolor were elucidated using N-terminal amino acid degradation and Sanger gene cloned sequencing. The obtained mRNA transcripts were cloned and expressed using E. coli vectors. Different bacterial expression conditions were tested and the best culture conditions for each expressed protein is reported. The expressed scorpion toxins were purified by chromatographic methods and used as immunogens in rabbits. RESULTS: The antibodies produced under the reported immunization scheme show better neutralization (ED50) than other reported commercial antivenoms used to neutralize similar species scorpion venoms under similar LD50 conditions. CONCLUSION: The information reported here shows the proof of concept for selecting recombinant immunogens with the ability to produce antibodies for neutralizing the lethal effects of the most important medical species of scorpions in Panama.

Recombinant expression and antigenicity of two peptide families of neurotoxins from Androctonus sp.

Background: Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings. Methods: In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami. Results: Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms. Conclusions: Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.

Intraspecific Differences in the Venom of Crotalus durissus cumanensis from Colombia.

Biochemical and biological differences in the venom of Crotalus durissus cumanensis from three ecoregions of Colombia were evaluated. Rattlesnakes were collected from the geographic areas of Magdalena Medio (MM), Caribe (CA) and Orinoquía (OR). All three regionally distributed venoms contain proteases, PLA2s and the basic subunit of crotoxin. However, only crotamine was detected in the CA venom. The highest lethality, coagulant, phospholipase A2 and hyaluronidase activities were found in the MM venom. Also, some differences, observed by western blot and immunoaffinity, were found in all three venoms when using commercial antivenoms. Furthermore, all three eco-regional venoms showed intraspecific variability, considering the differences in the abundance and intensity of their components, in addition to the activity and response to commercial antivenoms.

Target promiscuity and heterogeneous effects of tarantula venom peptides affecting Na+ and K+ ion channels.

Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their "canonical" gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na(+) channels, the three human ether-à-go-go (hERG)-related K(+) channels, and two human Shaker-related K(+) channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na(+) channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na(+) channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K(+) channels at concentrations well above the IC(50) value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs.

Insecticidal peptides from the theraposid spider Brachypelma albiceps: an NMR-based model of Ba2.

Soluble venom and purified fractions of the theraposid spider Brachypelma albiceps were screened for insecticidal peptides based on toxicity to crickets. Two insecticidal peptides, named Ba1 and Ba2, were obtained after the soluble venom was separated by high performance liquid chromatography and cation exchange chromatography. The two insecticidal peptides contain 39 amino acid residues and three disulfide bonds, and based on their amino acid sequence, they are highly identical to the insecticidal peptides from the theraposid spiders Aphonopelma sp. from the USA and Haplopelma huwenum from China indicating a relationship among these genera. Although Ba1 and Ba2 were not able to modify currents in insect and vertebrate cloned voltage-gated sodium ion channels, they have noteworthy insecticidal activities compared to classical arachnid insecticidal toxins indicating that they might target unknown receptors in insect species. The most abundant insecticidal peptide Ba2 was submitted to NMR spectroscopy to determine its 3-D structure; a remarkable characteristic of Ba2 is a cluster of basic residues, which might be important for receptor recognition.

A Novel Insecticidal Spider Peptide that Affects the Mammalian Voltage-Gated Ion Channel hKv1.5.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hKv1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider Oculicosa supermirabilis, which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hKv1.5. Therefore, a peptide from the spider O. supermirabilis venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed de novo from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the E. coli SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hKv1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.

Comparison of the peptidome and insecticidal activity of venom from a taxonomically diverse group of theraphosid spiders.

We screened a panel of theraphosid venoms in two orders of insect in order to determine whether these bioassays would help in the selection of candidate venoms for future discovery of insecticidal toxins. Venoms from six different theraphosid genera were compared with venom from the Australian funnel-web spider Hadronyche infensa (Hexathelidae). The tarantulas included were Coremiocnemis tropix, Selenocosmia crossipes, and Selenotholus foelschei from Australia and Brachypelma albiceps and Brachypelma hamorii from Mexico. The insects assayed, Tenebrio molitor (Coleoptera: Tenebrionidae) and Acheta domesticus (Orthoptera: Gryllidae), were selected because of their relevance as model holometabolous and hemimetabolous insects, respectively, as well as their taxonomic relationship to economically important pest insects. Despite significant differences in their peptide/protein profiles as determined using SDS-PAGE, HPLC, and mass spectrometry, all of the theraphosid venoms exhibited remarkably similar LD50 values of 46-126 microg/g for crickets and 0.5-4.0 microg/g for mealworms. Notably, mealworms were on average 50-fold more susceptible than crickets to each of the crude theraphosid venoms and consequently they provide an excellent bioassay system when venom supply is limited. This study indicates that even closely related spiders have evolved quite different toxin repertoires that nevertheless have comparable efficiency with respect to killing their primary prey, namely insects.

Immunogenic Properties of Recombinant Enzymes from Bothrops Ammodytoides Towards the Generation of Neutralizing Antibodies against Its Own Venom.

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

An insecticidal peptide from the theraposid Brachypelma smithi spider venom reveals common molecular features among spider species from different genera.

The soluble venom of the Mexican theraposid spider Brachypelma smithi was screened for insecticidal peptides based on toxicity to house crickets. An insecticidal peptide, named Bs1 (which stands for Brachypelma smithi toxin 1) was obtained in homogeneous form after the soluble venom was fractionated using reverse-phase and cation-exchange chromatography. It contains 41 amino acids cross-linked by three disulfide bridges. Its sequence is similar to an insecticidal peptide isolated from the theraposid spider Ornithoctonus huwena from China, and another from the hexathelid spider Macrothelegigas from Japan, indicating that they are phylogenetically related. A cDNA library was prepared from the venomous glands of B. smithi and the gene that code for Bs1 was cloned. Sequence analysis of the nucleotides of Bs1 showed similarities to that of the hexathelid spider from Japan proving additional evidence for close genetic relationship between these spider peptides. The mRNAs of these toxins code for signal peptides that are processed at the segment rich in acidic and basic residues. Their C-terminal amino acids are amidated. However, they contain only a glycine residue at the most C-terminal position, without the presence of additional basic amino acid residues, normally required for post-translation processing of other toxins reported in the literature. The possible mechanism of action of Bs1 was investigated using several ion channels as putative receptors. Bs1 had minor, but significant effects on the Para/tipE insect ion channel, which could indirectly correlate with the observed lethal activity to crickets.