Manuela Berto Pucca

Electrophysiological Characterization of Ts6 and Ts7, K+ Channel Toxins Isolated through an Improved Tityus serrulatus Venom Purification Procedure

In Brazil, Tityus serrulatus (Ts) is the species responsible for most of the scorpion related accidents. Among the Ts toxins, the neurotoxins with action on potassium channels (α-KTx) present high interest, due to their effect in the envenoming process and the ion channel specificity they display. The α-KTx toxins family is the most relevant because its toxins can be used as therapeutic tools for specific target cells. The improved isolation method provided toxins with high resolution, obtaining pure Ts6 and Ts7 in two chromatographic steps. The effects of Ts6 and Ts7 toxins were evaluated in 14 different types of potassium channels using the voltage-clamp technique with two-microelectrodes. Ts6 toxin shows high affinity for Kv1.2, Kv1.3 and Shaker IR, blocking these channels in low concentrations. Moreover, Ts6 blocks the Kv1.3 channel in picomolar concentrations with an IC50 of 0.55 nM and therefore could be of valuable assistance to further designing immunosuppressive therapeutics. Ts7 toxin blocks multiple subtypes channels, showing low selectivity among the channels analyzed. This work also stands out in its attempt to elucidate the residues important for interacting with each channel and, in the near future, to model a desired drug.

A gamut of undiscovered electrophysiological effects produced by Tityus serrulatus toxin 1 on NaV-type isoforms.

In the last decades, Ts1 has not only been the subject of many studies, it has also been considered as a very useful tool to investigate NaV channels and to explore the exact role of NaV channels in channelopathies. Ts1 is believed to modulate the activation process of NaV upon interaction at the neurotoxin binding site 4. Our aim was to carry out an in depth functional characterization of Ts1 on a wide array of Nav channels, in order to investigate its mechanism of action and to verify if Ts1 can indeed be considered as a prototype site 4 selective toxin, valid for all the Nav isoforms we know currently. Ts1 has been subjected to an in-depth functional investigation on 9 NaV isoforms expressed in Xenopus laevis oocytes. Ts1 does not only interfere with the activation process but also modulates the inactivation in a bell-shaped voltage-dependent matter. Furthermore, Ts1 altered the ion selectivity through insect NaV. without influencing the tetrodotoxin selectivity of the channels. Finally, Ts1 was also found to inhibit the sodium current through the cardiac Nav1.5 isoform. On the basis of the totally unexpected plethora of Nav modulations as induced by Ts1, we demonstrate that caution is required in interpretation the in vivo experiments when using Ts1. The electrophysiological characterization of Ts1 indeed shows that the general accepted contours of NaV binding sites are much more obscure than believed and that interpretation of NaV pharmacology upon toxin binding is more complex than believed thus far.

Production of Human Antibody Fragments Binding to Melittin and Phospholipase A2 in Africanised Bee Venom: Minimising Venom Toxicity

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

Tityus serrulatus venom - a lethal cocktail

Tityus serrulatus (Ts) is the main scorpion species of medical importance in Brazil. Ts venom is composed of several compounds such as mucus, inorganic salts, lipids, amines, nucleotides, enzymes, kallikrein inhibitor, natriuretic peptide, proteins with high molecular mass, peptides, free amino acids and neurotoxins. Neurotoxins are considered the most responsible for the envenoming syndrome due to their pharmacological action on ion channels such as voltage-gated sodium (Nav) and potassium (Kv) channels. The major goal of this review is to present important advances in Ts envenoming research, correlating both the crude Ts venom and isolated toxins with alterations observed in all human systems. The most remarkable event lies in the Ts induced massive releasing of neurotransmitters influencing, directly or indirectly, the entire body. Ts venom proved to extremely affect nervous and muscular systems, to modulate the immune system, to induce cardiac disorders, to cause pulmonary edema, to decrease urinary flow and to alter endocrine, exocrine, reproductive, integumentary, skeletal and digestive functions. Therefore, Ts venom possesses toxins affecting all anatomic systems, making it a lethal cocktail. However, its low lethality may be due to the low venom mass injected, to the different venom compositions, the body characteristics and health conditions of the victim and the local of Ts sting. Furthermore, we also described the different treatments employed during envenoming cases. In particular, throughout the review, an effort will be made to provide information from an extensive documented studies concerning Ts venom in vitro, in animals and in humans (a total of 151 references).

Serrumab: A human monoclonal antibody that counters the biochemical and immunological effects of Tityus serrulatus venom

In Brazil, the species Tityus serrulatus is responsible for the most severe cases of scorpion envenomation. There is currently a need for new scorpion anti-venoms that are more effective and less harmful. This study attempted to produce human monoclonal antibodies capable of inhibiting the activity of T. serrulatus venom (TsV), using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Four rounds of phage antibody selection were performed, and the round with the highest phage antibody titer was chosen for the production of monoclonal phage antibodies and for further analysis. The scFv 2A, designated serrumab, was selected for the production and purification of soluble antibody fragments. In a murine peritoneal macrophage cell line (J774.1), in vitro assays of the cytokines interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-10 were performed. In male BALB/c mice, in vivo assays of plasma urea, creatinine, aspartate transaminase, and glucose were performed, as well as of neutrophil recruitment and leukocyte counts. It was found that serrumab inhibited the TsV-induced increases in the production of IL-6, TNFα, and IL-10 in J774.1 cells. The in vivo inhibition assay showed that serrumab also prevented TsV-induced increases in the plasma levels of urea, creatinine, aspartate transaminase, and glucose, as well as preventing the TsV-induced increase in neutrophil recruitment. The results indicate that the human monoclonal antibody serrumab is a candidate for inclusion in a mixture of specific antibodies to the various toxins present in TsV. Therefore, serrumab shows promise for use in the production of new anti-venom.

Influence of post-starvation extraction time and prey-specific diet in Tityus serrulatus scorpion venom composition and hyaluronidase activity.

The role of diet in venom composition has been a topic of intense research interest. This work presents evidence that the variation in the venom composition from the scorpion Tityus serrulatus (Ts) is closely associated with post-starvation extraction time and prey-specific diet. The scorpions were fed with cockroach, cricket, peanut beetle or giant Tenebrio. The venoms demonstrated a pronounced difference in the total protein and toxins composition, which was evaluated by electrophoresis, reversed-phase chromatography, densitometry, hyaluronidase activity and N-terminal sequencing. Indeed, many toxins and peptides, such as Ts1, Ts2, Ts4, Ts5, Ts6, Ts15, Ts19 frag. II, hypotensins 1 and 3, PAPE peptide and peptide 9797 (first described in Ts venom), were all identified in different proportions in the analyzed Ts venoms. This study is pioneer on assessing the influence of the starvation time and the prey diet on hyaluronidase activity as well as to describe a modification of Tricine-gel-electrophoresis to evaluate this enzyme activity. Altogether, this study reveal a large contribution of the extraction time and diet on Ts venom variability as well as present a background to recommend the cockroach diet to obtain higher protein content and the cricket diet to obtain higher hyaluronidase specific activity.

Isolation and characterization of Ts19 Fragment II, a new long-chain potassium channel toxin from Tityus serrulatus venom

Ts19 Fragment II (Ts19 Frag-II) was first isolated from the venom of the scorpion Tityus serrulatus (Ts). It is a protein presenting 49 amino acid residues, three disulfide bridges, Mr 5534Da and was classified as a new member of class (subfamily) 2 of the β-KTxs, the second one described for Ts scorpion. The β-KTx family is composed by two-domain peptides: N-terminal helical domain (NHD), with cytolytic activity, and a C-terminal CSαβ domain (CCD), with Kv blocking activity. The extensive electrophysiological screening (16 Kv channels and 5 Nav channels) showed that Ts19 Frag-II presents a specific and significant blocking effect on Kv1.2 (IC50 value of 544±32nM). However, no cytolytic activity was observed with this toxin. We conclude that the absence of 9 amino acid residues from the N-terminal sequence (compared to Ts19 Frag-I) is responsible for the absence of cytolytic activity. In order to prove this hypothesis, we synthesized the peptide with these 9 amino acid residues, called Ts19 Frag-III. As expected, Ts19 Frag-III showed to be cytolytic and did not block the Kv1.2 channel. The post-translational modifications of Ts19 and its fragments (I-III) are also discussed here. A mechanism of post-translational processing (post-splitting) is suggested to explain Ts19 fragments production. In addition to the discovery of this new toxin, this report provides further evidence for the existence of several compounds in the scorpion venom contributing to the diversity of the venom arsenal.

Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

Abstract In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds’ maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera.

Revealing the Function and the Structural Model of Ts4: Insights into the “Non-Toxic” Toxin from Tityus serrulatus Venom

The toxin, previously described as a “non-toxic” toxin, was isolated from the scorpion venom of Tityus serrulatus (Ts), responsible for the most severe and the highest number of accidents in Brazil. In this study, the subtype specificity and selectivity of Ts4 was investigated using six mammalian Nav channels (Nav1.2→Nav1.6 and Nav1.8) and two insect Nav channels (DmNav1 and BgNav). The electrophysiological assays showed that Ts4 specifically inhibited the fast inactivation of Nav1.6 channels, the most abundant sodium channel expressed in the adult central nervous system, and can no longer be classified as a “non-toxic peptide”. Based on the results, we could classify the Ts4 as a classical α-toxin. The Ts4 3D-structural model was built based on the solved X-ray Ts1 3D-structure, the major toxin from Ts venom with which it shares high sequence identity (65.57%). The Ts4 model revealed a flattened triangular shape constituted by three-stranded antiparallel β-sheet and one α-helix stabilized by four disulfide bonds. The absence of a Lys in the first amino acid residue of the N-terminal of Ts4 is probably the main responsible for its low toxicity. Other key amino acid residues important to the toxicity of α- and β-toxins are discussed here.

Electrophysiological characterization of the first Tityus serrulatus alpha-like toxin, Ts5: Evidence of a pro-inflammatory toxin on macrophages

Tityus serrulatus (Ts) venom is composed of mainly neurotoxins specific for voltage-gated K(+) and Na(+) channels, which are expressed in many cells such as macrophages. Macrophages are the first line of defense invasion and they participate in the inflammatory response of Ts envenoming. However, little is known about the effect of Ts toxins on macrophage activation. This study investigated the effect of Ts5 toxin on different sodium channels as well as its role on the macrophage immunomodulation. The electrophysiological assays showed that Ts5 inhibits the rapid inactivation of the mammalian sodium channels Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6 and Nav1.7. Interestingly, Ts5 also inhibits the inactivation of the insect Drosophila melanogaster sodium channel (DmNav1), and it is therefore classified as the first Ts α-like toxin. The immunological experiments on macrophages reveal that Ts5 is a pro-inflammatory toxin inducing the cytokine production of tumor necrosis factor (TNF)-α and interleukin (IL)-6. On the basis of recent literature, our study also stresses a possible mechanism responsible for venom-associated molecular patterns (VAMPs) internalization and macrophage activation and moreover we suggest two main pathways of VAMPs signaling: direct and indirect. This work provides useful insights for a better understanding of the involvement of VAMPs in macrophage modulation.