Scorpion Venom Research Around the World: Tityus serrulatus
Tityus serrulatus is considered the most dangerous scorpion in Brazil. It is widely distributed, especially in the Southeast region, and is responsible for the highest number and most severe accidents. This chapter focuses on Tityus serrulatus scorpion venom (Tsv) and aspires to unravel its complex composition with emphases on its isolated proteins, their targets, structures, and functions. It takes a closer look at the peptides related to the Na+ and K+ channel toxin families, NaTx and KTx, respectively, including their toxin precursors. Additionally, a hyaluronidase, a serine proteinase, metalloproteinases, and many other proteins/peptides, such as a nontoxic protein (Ts4), PAPE peptides, bradykinin-potentiating peptides (BPP), antimicrobial peptides (AMP), anionic peptides, and venom peptides with undetermined functions, were reported.
KeywordsDisulfide Bridge Venom Gland Scorpion Venom Scorpion Toxin Scorpion Sting
Scorpion venoms are extremely versatile, being effective offensively against insect preys and defensively against vertebrate enemies. It has been suggested that their outstanding and dramatic effects on mammals are due to strong conservation of receptors and ion channels across diverse taxa.
The effects of Tityus serrulatus venom (Tsv) in various animal species were reported by Magalhães in 1946 (Almeida et al. 2002). Just after 20 years, the first separation of its components was reported by Gomez and Diniz in 1966. Ever since, the progression of knowledge about the Tsv composition, its effects, and its biotechnological application has risen. Scorpion toxins have been used as molecular probes to study the structure and function of Na+ and K+ channels, as well as to study the gating mechanisms of these channels, to isolate them from native tissues, and to understand the physiological role of specific channel ligands. In addition, scorpion toxins may be used as tools in physiopathological studies for the full comprehension of several channels related to diseases such as cancer, Alzheimer, and Parkinson. Since some of these toxins victimize insects in a specific way, they also contributed to the development of new pesticides. Lastly, the full characterization of T. serrulatus components and their effects will lead for more efficient envenoming treatments (for review, see Cologna et al. 2009).
Toxicity of Tsv
The sting of T. serrulatus causes local pain and, depending on the envenoming severity, paresthesia, sweating, vomiting, hypertension, neurological manifestations, alternation between agitation and exhaustion, cardiorespiratory alterations, pulmonary edema, and circulatory failure triggering death are also observed (for review, see Cologna et al. 2009). This plethora of symptoms and signs are attributed to the effects of toxins interacting with voltage-gated sodium channels of excitable cell membranes, causing them to release a large amount of neurotransmitters (Possani et al. 1999).
In healthy adults, most scorpion stings are little severe and may not need medical treatment. However, children and elderly people with pre-existing heart disease are at risk of death. All scorpion victims should always be kept under observation during 4–6 h after the sting and in case of moderate to severe accidents be monitored for at least 24 h.
The lethal dose of Tsv required to kill 50 % (LD50) of rats or mice has been experimentally attested by several researcher groups. The LD50 of Tsv is 25 μg/mouse (20 g) after intraperitoneal injection of soluble venom (Possani et al. 1977), 24.6 μg/mouse (20 g) after subcutaneous injection (Pucca et al. 2011), 7.5 μg/mouse (20 g) after intravenous (i.v.) injection, and 0.098 μg/mouse (20 g) after intracerebroventricular (i.c.v.) injection (Arantes et al. 1989). On the other hand, the i.v. and i.c.v. LD50 of toxins Ts2, Ts6, Ts3, and Ts1 were, respectively, 6.42 and 0.074, 16.52 and 0.22, 2.04 and 0.034, 1.52 and 0.022 μg/mouse (20 g) (Arantes et al. 1989).
The LD50 is 10.8 ± 0.6 mg/kg for adult male rats after subcutaneous injection of a saline extract of crude Tsv, while adult female rats and weanling (male and female) rats are, respectively, around 2 and 3.5 times more sensitive to the venom (Nunan et al. 2001). Thus, the effect of the scorpion envenoming in children is probably related to body maturation factors, such as number of receptor sites and pharmacokinetic parameters when compared with adults (Nunan et al. 2001). Besides that, children’s blood–brain barrier is more permeable to small peptides, such as toxins able to act on voltage-gated Na+ or K+ channels (Nunan et al. 2003).
The LD50 values for manually extracted venom are approximately twice or three times lower than those for venom extracted by electrical stimulation. The contraction of the venom gland can be controlled by the scorpion during manual extraction of the venom, being closer to reality than electrical stimulation. Manual extraction releases only toxins, while electrical stimulation releases also nontoxic components (Kalapothakis and Chávez-Olórtegui 1997 apud Pucca et al. 2011).
An adequate assessment of scorpion LD50 is an important step for accurate evaluation of antivenom sera potencies and the optimization of serotherapy (Krifi et al. 1988 apud Pucca et al. 2011). In retrospect to comparing LD50 values, several parameters have to be taken into account as there are: the administration via of venom injected, the animal model (strain, species, body weight, sex and age), the venom batch and the venom extraction procedure.
Isolation of Toxins
In order to extract the venom from Tityus serrulatus scorpions kept in captivity, an electric charge equivalent to 12 V is applied on the tail segment with the hazardous telson, stimulating the release of the venom. Hundreds of small droplets of venom from several specimens of Ts are accumulated in a glass tube or plate and desiccated on a vacuum pump or freeze-dried. The collected dried venom is a complex mixture as mentioned before and should be dispersed in a suitable buffer solution and centrifuged to separate the mucus from the protein solution.
Ts crude soluble venom has been fractionated by size exclusion chromatography, followed by ion exchange chromatography and rechromatography. Ts toxins have also been isolated through preparative reversed phase HPLC and rechromatography on a C18 column. Another reliable purification procedure consists in applying Ts venom on a carboxymethyl-cellulose-52 column (Arantes et al. 1989). This protocol assures a swift recuperation of pure Ts1, the major toxin from Ts venom, in just one purification step. After this first fractionation step, the desired fraction is rechromatographed on an ion exchange column or on a reversed phase C18 column (Pessini et al. 2001; Cologna et al. 2011).
Composition of the Venom
Tsv is composed of insoluble mucus, many neurotoxic proteins composed of 60–70 or 30–42 amino acid residues that affect, respectively, Na+ or K+ channels, bioactive amines, hypotensins, proteinases, a hyaluronidase, a bradykinin-potentiating peptide, a kallikrein inhibitor, allergenic proteins, and other peptides whose biological functions are still not clarified.
Estimates indicate that Tsv comprises over 300 toxins, most of which are small molecules (molecular mass <10 kDa) (Pimenta et al. 2001). This diversity of molecular masses from Tsv has been investigated using MS-based proteomic approaches, which allowed to detect posttranslational modifications, such as phosphorylation and N-glycosylation in toxins from Tsv. Proteomics led to a huge amount of new information considering the absence of information on the complete genome sequence of Tityus sp. (Verano-Braga et al. 2013).
Some bioactive amines, including histamine and 5-hydroxytryptamine, were found at low concentrations in Tsv. The most common function of histamine in venom is to produce pain at the site of injection, an advantage when the venom is used for defense. Tsv also contains compounds that induce release of histamine from mast cells, causing an increase in vascular permeability which facilitates the access of its toxic components into the blood circulation and contributes to profound hypotension.
Despite the description of phospholipases A2 in scorpion venoms, no activity had been detected in Tsv (Possani et al. 1977; Venancio et al. 2013) till its transcript is identified in the T. serrulatus cDNA library (Alvarenga et al. 2012).
The absence of l-amino acid oxidase and phosphodiesterase activities has been noted in scorpion venoms. In addition to the absence of phosphodiesterase activity (Possani et al. 1977), Tsv contains neither catecholamines nor acetylcholine. However, after intravenous injection of Tsv, a great increase of epinephrine and norepinephrine release was reported with concomitant increase of the mean arterial pressure in rats previously catheterized (Vasconcelos et al. 2005).
Because of their interactions with ionic channels in excitable membranes and their role in the envenoming, neurotoxins with low molecular mass became the most studied components of Tsv.
Tsv neurotoxins are represented by long-chain Na+ channel toxins (NaTx) , such as α-toxins which inhibit the inactivation and β-toxins responsible for decreasing the excitation threshold of this channel. This triggers depolarization and mediators’ release. The venom also contains short-chain K+ channel toxins (KTx) with low toxicity acting on K+ channels, which might be used as potential medicines (for review, see Cologna et al. 2009).
In 2009 a nomenclature for TsV components was proposed where the isolated proteins of which the amino acid sequences are completely determined are named consecutively, to avoid confusion and systematize the nomenclatures adopted previously by Dr. Giglio’s and Dr. Possani’s groups (for review, see Cologna et al. 2009). Following this systematized nomenclature, here it is proposed to appoint not completely mature toxins using the toxin name followed by the single-letter code of each amino acid residue that appear after the C-terminal of the mature protein, aiming to distinguish the immature toxin (e.g., Ts1-G) from its precursor (containing the signal peptide).
It is interesting to note that all the toxins belonging to the same toxin group are closely related to each other (Fig. 2), such as NaTxs (in red and orange) and KTxs (in violet and pink). Ts4 (black), a nontoxic protein, is closely related to Ts1 and shares 65.57 % of identity with it. The β-KTxs (in pink in Fig. 2) were joined together in the same branch and are near the α-KTxs (in violet). An outstanding observation is that Ts10 shares 92.3 % of identity with Ts5 and Ts17, α-NaTxs; however, it is a bradykinin-potentiating peptide and it is in a branch close to hypotensins (in blue in Fig. 2). The components of the latter belong to a new structural family of peptides able to potentiate the hypotensive effects of bradykinin (Verano-Braga et al. 2008).
Tityus serrulatus venom (Tsv) has been studied for many years, and several toxins have already been described. The classification of the different components of Tsv presented in this chapter is based upon their pharmacological action. However, some peptides identified in the venom have not been functionally characterized yet, and they have been classified according to the primary sequence similarity with other well-characterized venom components.
Na+ Channel Toxins (NaTx)
Voltage-gated sodium, calcium, and potassium channels play a key role in electrical signaling of excitable cells. They are critical elements required for action potential generation and conduction. Since ion channels play important roles in many physiological processes, they are molecular targets for a broad range of potent neurotoxins. Scorpion neurotoxins have been shown to affect the ion permeability of excitable cells and have been intensively studied because they represent excellent models for investigating structure/function relationships and they are also fine probes for studying ionic channel functions.
Voltage-gated sodium (NaV) channels are a large superfamily of transmembrane proteins that play a key role in the initiation and propagation of action potential in excitable cells and are responsible for the initial depolarization of the membrane. Nine mammalian NaV channels isoforms (NaV1.1–1.9) have been identified, and they are targets of neurotoxins which strongly alter their basic functions: conductance, voltage-dependent activation, and voltage-dependent inactivation. NaV channels consist of a pore-forming α-subunit and auxiliary β-subunits. The α-subunit contains four homologous domains (DI-DIV), each made out of six hydrophobic transmembrane segments (S1–S6), connected by both intracellular and extracellular loops. The membrane reentrant loop between S5 and S6 forms the narrow extracellular end of the pore, and the intracellular loop between DIII and DIV, in particular the IFM sequence, is believed to act as an inactivation gate. The voltage sensor domain consists of the S1–S4 segments. The S4 segments, which contain repeated motifs of a positively charged amino acid residues followed by two hydrophobic residues, move outward in response to depolarization to initiate a conformational change that opens the pore (for review, see Catterall et al. 2007).
Scorpion toxins targeting voltage-gated sodium (NaTx) channels are widely used as powerful tools to study the molecular structure and function of these channels. They are known as long-chain toxins (60–76 amino acid residues), cross-linked by four disulfide bridges. NaTx can be divided into two groups (α- and β-toxins), according to their mechanism of action and binding to different sites on the extracellular surface of the NaV channel.
α-Scorpion toxins bind to the extracellular receptor site-3 on the NaV channel and slow or block its fast inactivation by preventing outward movement of the S4 segment from domain IV of these channels. Basic amino acids of the α-toxins and amino acids located on the S3–S4 loop of domain IV (highlighting hydrophobic residues and Glu residues in S3b and Arg residues in S4), as well as on the loops S5–S6 (domains I and IV) of the NaV channel pore are involved in toxin binding (Rogers et al. 1996; Bosmans et al. 2008). (a) ‘α-classic’, highly active in mammal, their binding affinity to sodium channels is reduced by membrane depolarization; (b) ‘anti-insect’, that show high toxicity towards NaV channels of insects, their binding to neuronal membranes is independent of membrane potential, and (c) ‘α-like’, that are active in both mammals and insects NaV channels, with a preference for insects (Gordon et al. 2007).
β-Scorpion toxins bind to the extracellular loop of segments S3–S4 of domain II, the receptor site-4 of NaV channels, and induce both a shift in the voltage dependence of NaV channel activation in the hyperpolarizing direction and a reduction of the peak sodium current amplitude. These toxins trap segment S4, keeping it in the activated position, producing spontaneous and repetitive action potentials. β-toxins are classified according to their pharmacological preference for insect and mammalian NaV channels into four groups: (a) βm, active on mammalian NaV channels; (b) βi, which selectively act on insect NaV channels; (c) β-like, for those without preference between mammalian and insect NaV; and (d) β α , for toxins that present primary structure of β-toxins, but with a functional α-effect (Bosmans et al. 2007).
Ts1, the major toxin of Tsv, corresponds to 16 % of the crude soluble venom and contributes significantly to venom toxicity (Vasconcelos et al. 2005). It is also the best-studied toxin from Tsv and was initially named Toxin γ by Possani et al. (1977) and Ts VII by Bechis et al. (1984), who first established its amino acid sequence.
Ts1 is classified as a β-like toxin, binds at site 4 of the NaV channels, and is active on both mammalian and insect cells. This toxin has been reported to have high affinity for NaV channels using binding studies on membrane synaptosomes (Barhanin et al. 1982). Electrophysiological studies performed with Ts1 on peripheral nerve membrane of Xenopus laevis, under current- and voltage-clamp conditions, demonstrated that it depolarizes the membrane, induces spontaneous activity, reduces the amplitude of the action potential, and increases its duration. Ts1 (440 nmol/L) induces inward Na+ current flow at resting potential, shifts the voltage dependence of activation toward more negative potential values (~10 mV), and reduces the maximum Na+ permeability to about 20 % (Jonas et al. 1986).
Ts1 can interact with other domains in addition to the canonical interaction with domain II. Bosmans et al. (2008) showed that the transfer of the paddle motifs from domains II, III, or IV from rNaV1.2a renders the KV2.1 channel sensitive to Ts1 (TsVII), revealing that this toxin can interact with multiple paddle motifs from rNaV1.2a channel. Similar effects were observed with the transfer of the domain II, III, or IV paddle motifs from hNav1.9, with the domain II chimera exhibiting the largest inhibition in the presence of Ts1 (Bosmans et al. 2011). In contrast, Ts1 selectively binds to the voltage sensor in domain II from rNaV1.4 channel, showing that its interactions can differ between NaV channel subtypes (Bosmans et al. 2008). Additionally, Ts1 (100 nM) produces a dramatic facilitation of rNav1.9 currents with little or no effect on rNav1.8, showing that this toxin can discriminate between these two TTX-r NaV channels (Bosmans et al. 2011).
Study performed by Campos et al. (2007) showed that the binding of the Ts1 to skeletal muscle sodium channels, NaV 1.4, not only immobilizes domain II voltage sensor in an activated state, but also causes large hyperpolarizing shifts in the activation of other voltage sensors. These findings reveal the cooperative or coupled behavior of voltage sensors in NaV channels and explain how Ts1 has such profound effects on the voltage-dependent gating process.
Ts1 also exhibits strong toxicity to insects. It binds with high affinity to the same site as AaH IT on the insect sodium channel (De Lima et al. 1986). Therefore, in addition to its important application in studies of structure/function of NaV channels it can also be used to design new bioinsecticides.
In vivo studies performed with Ts1 showed that it is able to induce an increase in plasma levels of aminotransferase, amylase, creatine kinase, and lactate dehydrogenase, liver congestion, pulmonary and renal hemorrhage, hypertrophy, and degeneration of cardiac areas. Ts1 (30 μg/kg) induces pronounced hypertension, with the maximal pressor effect at 2.0–3.5 min after toxin administration, with concomitant increase in plasma catecholamines. The hypertensive effect is considered the major aetiological factor responsible for the development of cardiac failure and pulmonary edema (Vasconcelos et al. 2005).
In vitro assays show that macrophages exposed to Ts1 increase the production of proinflammatory cytokines IL-1α and IL-1β after 12 h; IL-6 and TNF after 24 h; as well as IFN-γ and NO after 72 h. In contrast, increase of IL-10, an anti-inflammatory cytokine, was observed after 120 h, indicating that Ts1 has an important immunomodulatory effect on macrophages (Petricevich et al. 2007, apud Zoccal et al. 2011). Similar results were obtained by Zoccal et al. (2011) using J774.1 murine macrophage cell line.
Ts1 is a basic protein composed of 61 amino acids cross-linked by four disulfide bridges (Table 1 and Fig. 3). Ten of its amino acid residues are positively charged (Arg, Lys, His); five are negatively charged (Asp, Glu) and there are nine aromatic residues (Trp,Tyr, Phe). Its amino acid sequence deduced from the cDNA nucleotide sequence shows that the mature toxin is the product of a precursor (Table 1) containing a signal peptide of 20 residues, the mature toxin and an extra Gly-Lys-Lys tail at the C-terminal region. Thus, the posttranslational modifications of Ts1 involve the removal of the signal peptide by a signal peptidase, cleavage of C-terminal Lys residues by a carboxypeptidase, and the conversion of the remaining Gly-extended toxin into a des-Gly protein by an α-amidating enzyme (Martin-Eauclaire et al. 1992).
Polikarpov et al. 1999 (apud Cologna et al. 2009) showed that positively charged Lys at positions 1 and 12 as well as a negatively charged Glu at position 2 are likely determinants of the specificity of β-toxins. They observed that all residues identified as functionally important are located at one side of the molecule (Face A), which is therefore considered as the NaV channel recognition site. Face A is also characterized by a number of aromatic interactions.
The α-toxin Ts2 was previously described as a β-neurotoxin based on receptor binding assays (Mansuelle et al. 1992 apud Cologna et al. 2012) and on the high sequence identity (72 %) shared with Ts1 (Table 1 and Fig. 3). However, despite sharing a very similar primary structure, Ts1 and Ts2 present opposite effects regarding to NO, TNF-a, IL-6, and IL-10 production (Zoccal et al. 2011). Ts2 stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Additionally, Sampaio et al. 1991 (apud Cologna et al. 2009) demonstrated that Ts2 induces prolongation of the action potential of myelinated fibers of rabbit vagus nerve, a classic effect of α-neurotoxins. More recently, Ts2 was assayed against eight subtypes of NaV channels (rNaV1.2, rNaV1.3, rNaV1.4, hNaV1.5, mNaV1.6, rNaV1.7, rNaV1.8, and the insect channel DmNaV1) and showed to be able to inhibit rapid inactivation of NaV1.2, NaV1.3, NaV1.5, NaV1.6, and NaV1.7, although not affecting NaV1.4, NaV1.8, or DmNaV1 (Cologna et al. 2012). These results confirm that it is able to discriminate between mammal and insect channels, with no activity for DmNaV1. De Lima et al. 1986 (apud Cologna et al. 2012) had already found that Ts2 is nontoxic for insects when injected in blowfly larvae (Sarcophaga argyrostoma).
Interestingly, Ts2 affects the inactivation and shifts the voltage dependence of activation of NaV1.3 channels (an effect that is characteristic of β-toxins), with no reduction in the sodium peak amplitude. Its dual behavior (α-type/β-type effect) for this channel shows that the pharmacological sensitivity of NaV channel subtypes to different modulators is very complex (Cologna et al. 2012).
The 3D structure to Ts2 was modeled using the Ts1 structure as a template, based on their high sequence identity (72 %). Its fold presents three antiparallel β-strands and one α-helix interlinked by four disulfide bridges (cysteine-stabilized α-helix/β-sheet motif), forming two opposite faces (A and B). Comparison between Ts1 and Ts2 structures shows that four substitutions are located in face A: Gly24Asp (comparable to Ts3 and Ts5 α-toxins), Asn50Asp (an acidic residue at position 50 is frequent in α-toxins, while β-like toxins contain a highly conserved Asn at this position), Trp51His (this Trp residue is important for Ts1 activity), and Arg57Tyr. Together, these substitutions induce important changes to the face A surface that may contribute to recognition of target sites in NaV channels. Ts2 represents the newest member of a small group of toxins with the structural features of β-toxins but displaying α-like activity (Cologna et al. 2012).
The effects of the α-neurotoxin Ts3 (previously named TsTX, Tityustoxin, or TsIV-5) on NaV channel inactivation have been studied by Kirsch et al. (1989). A model (stop model) proposed to explain the effect of Ts3 on NaV channel considers that the binding of site three toxins acts as a stop that prevents the complete movement of the segment S4 of domain IV (IVS4), slowing the inactivation, but not interfering with the channel activation (Campos et al. 2004).
Campos et al. (2008) analyzed the effects of Ts3 on the muscle NaV channels (NaV1.4) expressed in Xenopus oocytes using fluorescence–voltage relationship. Ts3 (200 nM) specifically impairs the conformational change that leads to fast inactivation of NaV1.4 channels; consequently, the decay of the currents became slower. Ts3 acts by partially blocking the movement of the S4 segment of domain IV, which would be enough to inhibit the inactivation but still allowing a normal activation to occur. The toxin significantly shifted the fluorescence–voltage relationship of domain I to more positive potentials, showing a strong coupling between domains I and IV. Additionally, Ts3 increased the amplitude of the sodium current compared to control conditions, but did not change significantly the voltage dependence of the activation.
Ts3 contains 62 amino acid residues, has a molecular mass of 7.2 kDa, and forms four disulfide bridges (Possani et al. 1999). Many studies have been conducted to elucidate Ts3 function and structure. This toxin induces the release of several mediators, such as catecholamines, acetylcholine, NO, GABA, aspartate, and glutamate, due to their primary action on the NaV channels (for review, see Cologna et al. 2009).
The precursors of Ts3 (Table 1), containing the C-terminal sequence Gly-Lys-Lys, are processed by caboxypeptidases. The Lys residues are removed and the remaining Gly-extended peptide is converted into a des-Gly peptide amine by an α-amidating enzyme to give a serine amide as C-terminal end (Martin-Eauclaire et al. 1994). Although the biological relevance of this posttranslational modification remains unclear, it can provide possible distinct molecular targets and effects.
Ts5 is an α-neurotoxin able to increase the duration of the composite action potential of rabbit vagus B fibber, an effect abolished by tetrodotoxin. The α-type effect of this toxin was confirmed by a study performed using the rate of 86Rb+ release from depolarized rat pancreatic β-cells as a measure of K+ permeability changes. Ts5 increases the rate of the marker outflow in the presence of 8.3 mM glucose. This effect was persistent and slowly reversible, showing similarity to that induced by veratridine, a toxin that delays NaV channels inactivation. By extending the depolarized period, Ts5 indirectly affects β-cell KV channels, thus increasing K+ permeability (Marangoni et al. 1995).
Ts5 represents 2 % of the soluble venom and shows high toxicity (LD50 = 94 ± 7 μg/kg, i.v.) in mice. In vivo assays shows that Ts5 is able to induce an increase in plasma levels of catecholamines, with concomitant increase in arterial pressure on conscious unrestrained rats (Vasconcelos et al. 2005). It is also able to induce the release of NO in isolated retractor penis muscle. Additionally, Ts5 inhibited both 3H-GABA (γ-aminobutyric acid) and 3H-DA (dopamine) uptake from rat synaptosomes, in a Ca2+-dependent manner, as consequence of depolarization, involving NaV channels, but it is not able to affect the 3H-Glu (glutamate) uptake (Cecchini et al. 2006).
Most of the effects of scorpion toxins are indirect and due to the release of adrenergic and cholinergic neurotransmitters. However, Ts5 is also able to interact directly with NaV channel in vascular smooth muscle cells, inducing an increase in Ca2+ cytosolic concentration (Neto et al. 2012).
Ts17 (U-BUTX-Ts1a) is a probable new NaTx since it presents 86 % identity with Ts5 (Fig. 2). It was found in the transcriptome profiling of the T. serrulatus venom gland (Alvarenga et al. 2012). Figure 3 shows the alignment of the Ts17 sequence with the NaTx from Tsv already described.
Ts18 (U-BUTX-Ts1b) was identified in the T. serrulatus cDNA library and was considered a NaTx, because its sequence matched significantly with the U1-buthitoxin-Hj1a (identities = 63 %), a predicted NaTx derived from Hottentotta judaicus (Alvarenga et al. 2012).
K+ Channel Toxins (KTx)
Potassium channels (K+ channels) play a crucial role in numerous biological processes such as blood pressure regulation, immunity, neurotransmitters release, heart rate, insulin secretion, smooth muscle contraction, cell volume regulation, and cell proliferation (to review see Mouhat et al. 2008). They are responsible for keeping the equilibrium of the membrane potential of excitable cells, contributing to membrane repolarization during the action potential. K+ channels represent the largest and most diverse family of ion channels in terms of subtypes, structure, and function (Mouhat et al. 2008) and have been implicated in a number of human pathologies such as asthma, cardiac arrhythmia, T-cell-mediated autoimmune disease, immune response to infection and inflammation, and hypertension (Bergeron and Bingham 2012). Due to their importance in many biological processes, K+ channels became a significant target for several compounds including animal venoms and toxins (Catterall et al. 2007).
Scorpion toxins acting on K+ channels (KTx) generally are compact peptides composed of 23–42 amino acid residues, reticulated by 3–4 disulfide bonds (Rodríguez De La Vega and Possani 2004). These toxins act in synergism with those active on Na+ channels resulting in abnormal nerve functioning. The combination of both types of toxins causes changes in the neurotransmitters’ release resulting in paralysis and death of the prey (Mouhat et al. 2008). Based on their molecular target, sequence similarities, and cysteine distribution, the scorpions’ toxins active on K+ channels (KTx) were classified in four families named α, β, γ, and κ-KTx. In addition, a systematic nomenclature was also proposed (Rodríguez De La Vega and Possani 2004). The α-KTx, the largest subfamily, comprises peptides of 23–43 amino acids stabilized by 3–4 disulfide bonds and folded within the CSα/β scaffold, the most common for scorpion toxins (Mouhat et al. 2008). The unusual β-KTx group is characterized by long peptides (~60 amino acid residues) linked by three disulfide bridges with the same CSα/β architecture of α-KTx. γ-KTx’s are hERG (ether-à-go-go) channel blockers with 36–47 amino acid residues connected by three or four disulfide bridges. This subfamily has the same CSα/β framework of the previous subfamilies (Rodríguez De La Vega and Possani 2004). The representatives of the last family, the κ-KTx, are relatively poor K+ blockers and display a different tertiary arrangement with two α-helices stabilized by two disulfide bonds also known as CSα/α scaffold (Mouhat et al. 2008).
This structural diversity of scorpion toxins represents a valuable research tool in different areas of knowledge. Scorpion toxins active on K+ channels have been essential to the investigation and understanding of the physiological role of these channels. The persevering research on this field resulted in the identification, localization, and classification of novel K+ channel subtypes as well as its structural and pharmacological elucidation (Bergeron and Bingham 2012). In addition, considering the role of K+ channels in numerous diseases, scorpion toxins active on this channel family may assist in the bioengineering of therapeutic scaffolds and development of novel drugs.
Among the wide variety of compounds predicted to compose Tsv, up to this date, only seven were described to act on Kv channels (Fig. 4).
Ts6, previously named as TsTx-IV, was first isolated and described in 1989 by Arantes and coworkers (Arantes et al. 1989) as a 41-amino-acid-residue peptide. Later on, the absence of the last residue Asn41 was verified, confirming that Ts6 and the 40-amino-acid-residue-long peptide butantoxin were actually the same toxin (Oyama et al. 2005). Ts6 was shown to block the high-conductance Ca2+-activated K+ channels in Leydig cells (Novello et al. 1999) and to inhibit the binding of 125 I-kaliotoxin into its receptors in rat brain synaptosomes, suggesting an additional affinity for Kv1.3 channels with an IC50 of 46 nM. This effect was reduced about 100-fold when the first six N-terminal residues, including the unusual disulfide bridge which forms an N-terminus ring, were cleaved (Pimenta et al. 2003a). In addition, the blocking effect of Ts6 on Shaker B K+ channels with Kd of 660 nM was described, highlighting the ability of this toxin to interact with diverse types of K+ channels with different affinities (Coronas et al. 2003).
Structural studies reveal the presence of the global CSαβ scaffold on the tertiary arrangement of Ts6. Interestingly, Ts6 structure is linked by four disulfide bridges, a characteristic first elucidated for short peptides from T. serrulatus for this striking toxin (Novello et al. 1999; Oyama et al. 2005). This novel feature, the presence of an extra disulfide bridge in the N-terminal of the toxin (Fig. 4), seems not to be related with the structural stability, as observed for other disulfide bonds, but it may play an important role in receptor specificity. Moreover, Ts6 has the ability to preserve its active conformation even when submitted to drastic pH variation, thus expanding the range of cellular conditions in which the toxin may exhibit its function (Oyama et al. 2005).
The immunomodulatory effects of Ts6 were also investigated and the results show that Ts6 can stimulate the production of NO, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in J774.1 cell, which are enhanced under LPS co-stimulation. Those effects observed were suggested to be independent of toxin/ion channel interaction, since Ts6 has shown similar inflammatory effects to Ts1, a specific Na+ channel modulator (Zoccal et al. 2011).
Formerly known as TsTx-Kα, Tityustoxin K-alpha, TsII-9, and TSK4, Ts7 was first described as a selective blocker of the 86Rb efflux through non-inactivating delayed-rectifier-type K+ channels present in synaptosome preparations, with an IC50 of 8 nM. Its action on voltage-gated non-inactivating K+ current was also confirmed in hippocampal and cerebellar neurons. In contrast, Ts7 was reported to block low-voltage-activated, partially inactivating K+ currents in neurons of dorsal root ganglion (DRG) (Matteson and Blaustein 1997). In addition, Ts7 was assayed on fibroblast cells transformed to express Kv1.2 subtype channel resulting in a block effect with very high affinity. This toxin has already been used as a pharmacological tool to evaluate the involvement of Kv1.2 subunits in the generation of total K+ currents in native cells (Dodson et al. 2002). The action of Ts7 on Kv1.3 channels expressed both in mammalian cell lines (L929) and in Xenopus laevis oocytes were also investigated and revealed high affinity for both expression systems (Kd = 19.8 at pH 7.4; Kd = 3.9 at pH 7.5, respectively). The authors also reported a pH dependence of the blocking activity of Ts7. These results indicated that Ts7, or α-KTx4.1, may also represent a useful tool for probing the physiological role of Kv1.3 channels (for review, see Cologna et al. 2009).
The interaction of Ts7 with a cloned K+ channel from Squid (sqKv1A) was also evaluated. To further study the interaction between this toxin and the mentioned channel, the 3D structure of Ts7 was determined by NMR spectroscopy and a model Ts7/sqKv1A complex was generated. The importance of lysine at position 27 (K27) (Fig. 4) was confirmed, and this residue seems to be inserted into the ion conducting pathway, causing the block of the channel pore. The model also corroborates to the hypothesis that the pH-dependent block observed is related to a histidine residue (H351) present in the outer vestibule of the channel, which seems to repels the positively charged residues of the toxin at low pH (Ellis et al. 2001 apud Cologna et al. 2009).
Ts8 or TsTx-K beta is a 60-amino-acid-residue peptide, linked by three disulfide bridges with an experimental molecular mass of 6,716 Da (Legros et al. 1998). Despite the unusual long chain, Ts8 was characterized as a selective blocker of voltage-gated non-inactivation K+ channels in synaptosome preparations (Rogowski et al. 1994). The complete sequence was very different from those of the 60–70-residue toxins active on Na+ channels or those of the 23–42-residue toxins active on K+ channels and therefore was the first member of the β-KTx subfamily. Its amino acid sequence was also confirmed by cDNA which encodes a precursor consisted of a signal peptide (19 amino acid residues), a propeptide (8 amino acid residues), and the mature chain with 60 amino acid residues (Legros et al. 1998).
Ts9, Ts kappa, 1TSK, Neurotoxin Ts kappa, or α-KTx4.2 is a short peptide (3784.4 Da) purified from Tsv, considered as a very high potent ligand for small-conductance apamin-sensitive calcium-activated K+ channels (SK). Ts9 is able to efficiently compete with apamin for binding on these channels. The solution structure of Ts9 has been determined by NMR techniques, which led to the full description of its 3D conformation: a short alpha helix and a three-stranded antiparallel beta sheet (Blanc et al. 1997).
Ts15 or α-KTx21.1 is described as a short peptide with 36 amino acid residues, cross-linked by three disulfide bridges, with a molecular mass of 3,961 Da. This toxin was assayed on a wide range of K+ channel subtypes and has shown a blocking effect on Kv1.2, Kv1.3, Kv1.6, and Shaker IR in a nanomolar range, while it does not block the other KV isoforms tested (Kv1.1, Kv1.4, Kv1.5, Kv2.1, Kv3.1, Kv4.2, Kv4.3, and hERG). It was demonstrated that Ts15 preferentially blocks Kv1.2 and Kv1.3 channels with an IC50 value of 196 ± 25 and 508 ± 67 nM, respectively (Cologna et al. 2011). The preference of Ts15 for Kv1.2 was related with the quantity of basic amino acid residues in the C-terminal region of the toxin (Fig. 4), which was proposed to be less in Kv1.2 high-affinity toxins when compared with the ones with high affinity for Kv1.3. Ts15 did not share high similarity with any of the 14 Ts toxins previously described, neither with KTxs deposited in the data bank, and therefore was considered a bona fide novel type of toxin (Cologna et al. 2011). Recently, a posttranslational modification in the structure of Ts15 was reported, which became the first N-glycosylated toxin ever described in Tsv (Verano-Braga et al. 2013).
Ts16, as well as Ts15, was screened on a wide variety of K+ channels such as Kv1.1–Kv1.6, Kv2.1, Kv3.1, Kv4.2, Kv4.3, and Kv7.1 and Shaker IR and hERG using the two-electrode voltage-clamp technique. Interestingly this toxin selectively blocks Kv1.2 channels, without any effect on the other K+ subtypes (Bordon et al. 2011). Since this toxin shares 62 % of identity with Tt28 or α-KTx20.1 from T. trivittatus venom, it would be reasonable to classify it at the same α-KTx20 subfamily and therefore name it as α-KTx20.2 (Saucedo et al. 2012). However, Saucedo and collaborators (2012) have stated an unexpected new cysteine pattern accompanied by a different arrangement of the secondary structure topology into a CSα/α scaffold, the characteristic scaffold of the κ-KTx. The unconventional structure, which consists of an antiparallel helix–loop–helix topology stabilized by three disulfide bonds, was determined for the recombinant version of Ts16 (rTs16) and provides new insights on the structural versatility of scorpion peptides. These findings could suggest the classification of Ts16 as a member of the κ-KTx subfamily, the only group of scorpion toxins which adopt the same structural pattern. However, by structural analyses, the authors hesitated to classify it since the helix–loop–helix topology of rTs16 seems to be an elaboration of CSα/β scaffold, rather than a variation of the κ-KTx α-hairpin (Saucedo et al. 2012).
The partial sequence of Ts19 was first detected by peptidomic analysis as two overlapping peptide fragments of 12 and 9 amino acid residues and was suggested to be fragments from the propeptide region of a β-KTx-like toxin (Rates et al. 2008). In a newly transcriptomic study, Ts19 precursor was fully determined and consists of a signal peptide of 25 amino acid residues followed by the mature toxin composed of 66 amino acid residues (Alvarenga et al. 2012). Just recently, two mature fragments of Ts19 were sequenced by Edman degradation and deposited in the UniProt data bank as Ts19 fragment I (Carmanhan et al. 2013) and fragment II (Cerni et al. 2013). The determined sequences suggest a presence of a propeptide region in the precursor described by Alvarenga and coauthors (2012) composed of eight amino acid residues. The toxin Ts19 fragment I has nine additional residues in the N-terminal region, starting with residues KIK, when compared with Ts19 fragment II (Table 1). The predicted Ts19, Ts19 fragment I, and Ts19 fragment II all share high identity with TsTKMK and TtrKIK, both β-KTx-like toxins (98 % and 94 % of identity, respectively) from T. stigmurus and T. trivittatus (Alvarenga et al. 2012). Up to this date, no experimental data were published to confirm the activity of this toxin and to evaluate the functional differences among the two described fragments.
Proteinases, such as exopeptidases, are linked with the posttranslational processing of peptides and proteins from the venom and their precursors. Besides that, they are involved in the inhibition of platelet aggregation, activation of the complement system, modulation of cytokine production, and diffusion of toxic components, since they degrade proteins of interstitial matrix (Cologna et al. 2009).
Proteolytic activity has been detected in some scorpion genera. In 1946, some effects observed in various animal species after a Ts sting, such as necrosis, hemolysis, and gangrene, were attributed to proteolytic enzymes in the venom. Probably these enzymes process and activate the toxins, facilitate the diffusion of toxic components, and activate trypsinogen, contributing to pancreatitis often observed in the victims. However, their functions in the venom are still unknown (Almeida et al. 2002). Concerning the pancreatitis, study performed with alpha (Ts2 and Ts3) and beta (Ts1) toxins from T. serrulatus venom shows that the acinar cells are stimulated by these peptides to discharge their zymogen granules. Additionally, the appearance of large vacuoles and some loss of morphological integrity was observed. These results indicate that probably a synergistic action between the venom components is responsible by the pancreatitis (Possani et al. 1991).
There are few studies about serine proteinases from Tsv. Gelatinolytic activity was detected in the first fraction of Tsv filtered on Sephadex G-50, whose activity was inhibited by PMSF, suggesting this fraction contains a serine proteinase; however, its isolation was unsuccessful (Almeida et al. 2002). In another study, the gelatinase activity was not found in Tsv, leading to speculation that these discrepancies could be due to the sensitivity of the methods of measurement or intraspecific/interspecific variations in venom composition (Venancio et al. 2013).
A metalloproteinase from Tsv, known as antarease, whose name is derived from the principal star Antares in the constellation Scorpio, was isolated. Its primary structure was partially determined by the sequencing of its N-terminal and internal peptides cleaved with trypsin, Asp-N, Arg-C, CNBr, Glu-C, and Lys-C. Antarease cleaves SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which are involved in the essential step that leads to fusion of vesicles with cellular membranes and are responsible for selective transport between cellular compartments. Therefore, antarease could be used for treating muscle spasms and other disorders, e.g., diabetes (Fletcher et al. 2010).
Another metalloproteinase had its N-terminal partially determined (Richardson et al. 2008c) and a dynorphin-cleaving metalloproteinase similar to antarease VAMP2 was detected in Tsv (Venancio et al. 2013).
The proteinases MQ-5 and MQ-7, isolated from Tsv, were able to activate the complement system, playing an important role during the inflammatory process after scorpion sting (Bertazzi 2007).
Hyaluronidases have been detected in the venoms of snakes, bees, wasps, spiders, lizards, fishes, and scorpions. These enzymes possess high antiedematogenic activity and cleave hyaluronan, the major glycosaminoglycan of the extracellular matrix, being responsible for spreading toxic components through the tissues of the victim/prey. Since hyaluronidase inhibition would retard toxins’ diffusion, improving antivenom therapy and reducing its side effects, several classes of chemical compounds have been studied as potential hyaluronidase inhibitors, such as flavonoids, alkaloids, antioxidants, terpenoids, lanostanoids, antibiotics, glycosaminoglycans, polysaccharides, proteins, polyphenols, fatty acids, anti-inflammatory drugs, or synthetic organic compounds.
A 51 kDa hyaluronidase from Tsv was isolated (Pessini et al. 2001) and its 34 first amino acid residues from its N-terminal were determined (Richardson et al. 2008b). Ts hyaluronidase potentiated the action of Ts1, increasing the levels of serum enzymes, and its enzymatic activity was inhibited by some flavonoid compounds with determined structural characteristics (Pessini et al. 2001). It was also reported that Ts hyaluronidase activity is similar to that determined for some bothropic venoms (Venancio et al. 2013).
Hyaluronidase isolated from Tsv induced mononuclear increase in the bronchoalveolar space after intranasal inoculation of 16 U in C57Bl/6 mice (Bitencourt et al. 2011). It decreases the concentration of hyaluronan and thus prevents the bleomycin-induced pulmonary fibrosis development. Besides that, it ameliorated pneumofibrosis by mesenchymal-like cell recruitment and did not cause edema formation and neither induced the increase in lung vascular permeability. Considering those findings, hyaluronidase became a promising tool in the treatment of pulmonary fibrosis.
Other components include non-neurotoxic venom components previously described for T. serrulatus venom.
Ts4 (TsTX-VI, TsNTxP) presents a primary structure similar to Ts1 (Fig. 2, Table 1), but its hydropathic index indicates that it is more hydrophobic than Ts1. These characteristics can be useful in clarifying the relationship between primary structure and biological function of scorpion toxins (Marangoni et al. 1990; Chávez-Olórtegui et al. 1997).
Ts4 was considered nontoxic to mice since it was unable to induce the characteristic symptoms of toxicity produced by Tsv and the NaV scorpion toxins. However, it induced an allergic reaction, lacrimation, and spasm of the hind legs of mice and produced dose dependent GABA and Glu liberation of synaptosomes that were blocked by tetrodotoxin (Marangoni et al. 1990). Polyclonal antibodies elicited by Ts4 are cross-reactive with several toxins from Tsv. Antibodies raised against peptides corresponding to residues 1–15 and 47–61 were able to neutralize the venom toxic fraction, indicating that these residues might be involved in the toxic action of the scorpion neurotoxins . These results indicate that this protein may be of interest in the production of antivenoms for clinical use (Chávez-Olórtegui et al. 2002).
Ts11, Ts12, and Ts13
Ts11 (TsPep1, 2,936 Da), Ts12 (TsPep2, 2,991 Da), and Ts13 (TsPep3, 3,017 Da) are 29 amino acid residues long, highly reticulated by a new pattern of four disulfide bridges, which make them the most constrained structures of scorpion venom-derived peptides known up to date. They present high sequence similarities with some KTx, as can be observed in the phylogenetic tree of peptides and toxins from Tsv (Fig. 2), but their biological function has not been determined yet. They are devoid of toxicity in mice. Ts13 was found to be a close isoform of Ts12 and the difference between them is just a change in position 13 (Ala/Pro). Ts11 showed 58.6 % of sequence homology with Ts12. Interestingly, the sequence of Ts12 corresponds only partially to that of the precursor (Table 1), indicating a specific posttranslational maturation process with the cleavage of a non-negligible C-terminal portion of 13 amino acid residues (Pimenta et al. 2003b).
Ts10 and Ts14 (Hypotensins)
Bradykinin-potentiating peptides (BPPs) , which are able to inhibit the angiotensin-converting enzyme (ACE) activity, are random-coiled linear peptides characterized by the BPP amino acid signature (an N-terminal pyroglutamic acid and the sequence Ile-Pro-Pro at the C-terminal). The BPP first isolated from Bothrops jararaca venom was essential to develop the commercial antihypertensive drug, captopril. Since then, many other BPPs were found in venom of other snakes, spiders, and scorpions.
Surprisingly, Ts10 (peptide T) has not a typical BPP amino acid signature, but it was able to potentiate bradykinin, by inhibiting ACE activity (Ferreira et al. 1993). It presents 13 amino acid residues, 1603.7 Da (Table 1), and is very different from the other components of the venom with hypotensive action (hypotensins), as shown in the phylogenetic tree presented in Fig. 2.
Ts14, also known as hypotensins (TsHpt-I, II, III, and IV), are random-coiled linear peptides. Their primary structure (Table 1) presents a similar BPP amino acid signature, with a noncanonical Lys residue prior to the conservative Pro-Pro doublet. TsHpt-I (2,732 Da) induces hypotension and potentiates the bradykinin hypotensive effects when administrated to normotensive rats. It is able to induce endothelium-dependent vasorelaxation dependent on NO release, but could not inhibit ACE activity (Verano-Braga et al. 2008). Differently from other BPPs, TsHpt-I acts as an agonist of the B2 receptor, a subtype of kinin receptor, inducing NO synthesis and consequent vasodilatation. Synthetic peptides containing the Lys-Pro-Pro at C-terminal kept the ability to activate this receptor and could induce a transient hypotension (Verano-Braga et al. 2010).
The complete sequence of hypotensin-like and hypotensin-1 precursors was identified in T. serrulatus venom gland transcriptome (Alvarenga et al. 2012). TsHpt-I and TsHpt-II are phosphorylated (Verano-Braga et al. 2013).
Second only to the neurotoxins , the PAPE peptide (8.5 % of the total transcripts) was the most highly expressed transcript in the T. serrulatus venom gland transcriptome (Alvarenga et al. 2012). In this protein rich in proline, the PAPE tetrapeptide (Pro-Ala-Pro-Glu) iteratively appears in the sequence. Additionally, it presents three proline-containing motifs (PEPAP, AAPE, and PEPAAAAPE). Rates et al. (2008) report the identification/sequencing of 28 peptides that could be identified as fragments from PAPE protein. While the signal peptide is highly conserved among these peptides, the N-terminal region is moderately conserved and the C-terminal region shows very little conservation. Despite its higher expression level, the function of the PAPE peptides in T. serrulatus venom remains unknown.
AMPs and Ponericin-Like and Anionic Peptides
Sequences of antimicrobial peptides (AMPs, 17 %) and anionic peptides (3 %) were abundant in the venom gland transcriptome of T. serrulatus. Although the precise function of these components has not been elucidated yet, their abundance suggests an important role in the biological function in the venom gland (Alvarenga et al. 2012). Three different sequences coded an AMP highly similar to the putative AMP of T. costatus. These AMP may act as protectors against bacterial infection or potentiators of neurotoxin action. One anionic peptide sequence was identified in the transcriptome of T. serulatus. This class of peptides has been reported as highly expressed and conserved among the Buthidae scorpion species. They might play antimicrobial activity or an important role in pH balance, since neurotoxins are basic peptides. Additionally, two ponericin-like sequences were found and they, probably, present antimicrobial function (Alvarenga et al. 2012).
Conclusion and Future Directions
Tityus serrulatus venom presents several underexplored compounds with promising pharmacological effect. Further characterization of these peptides will help to clarify their role in the envenoming and will propitiate its use as potential drugs or tools for biological systems’ studies.
Scorpion neurotoxins, due to their high affinity and specificity, have provided important information about the structure and the function of NaV and KV channels, affecting both permeation and gating properties. Moreover, neurotoxins turn out to be invaluable tools to distinguish and to reveal unique properties of different NaV channel isoforms. The demonstrated antimicrobial activities of AMPs have indicated their potential for use as anti-infective drugs.
Some bioactive proteins/peptides (such as KTx, AMPs, and hypotensins) present in Tityus serrulatus venom are an important resource for the investigation and characterization of molecules applicable in pharmaceutical research and biotechnology. Such richness can be useful to biotechnology in many ways, with the prospection of new drug candidates being the most promising.
- Barhanin J, Giglio JR, Leopold P, Schmid A, Sampaio SV, Lazdunski M. Tityus serrulatus venom contains two classes of toxins: Tityus ‘gamma’ toxin is a new tool with a very high affinity for studying the Na+ channel. J Biol Chem. 1982;257(21):2553–8.Google Scholar
- Bertazzi DT. Isolation and biochemical characterization of components from Tityus serrulatus venom with action on the complement system. Ribeirão Preto: University of São Paulo; 2007.Google Scholar
- Bordon KCF, Cologna CT, Tytgat J, Arantes EC. Purification and characterization of Ts16, new specific Kv1.2 blocker, from Tityus serrulatus scorpion venom. In: Calvete JJ, chairman. Proceedings of the 17th Congress of the European Section of the International Society of Toxinology; 2011 Sep 11–15; Valencia: Program & Abstracts Book, p. 240, Poster 128; 2011.Google Scholar
- Bosmans F, Martin-Eauclaire MR, Tytgat J. Differential effects of five ‘classical’ scorpion β-toxins on rNav1.2a and DmNav1 provide clues on species-selectivity. Toxicol Appl Pharmacol. 2007;218:45–51.Google Scholar
- Carmanhan PAS, Bordon KCF, Arantes EC. Isolation and characterization of a new probable potassium channel toxin from Tityus serrulatus venom. 2013;UniProtKB: P86822.Google Scholar
- Cerni FA, Amorim FG, Bordon KCF, Arantes EC. Isolation and electrophysiological characterization of a new potassium channel blocker from Tityus serrulatus venom. 2013;UniProtKB: P86822.Google Scholar
- Coelho VA, Cremonez CM, Anjolette FAP, Aguiar JF, Varanda WA, Arantes EC. Functional and structural study comparing the C-terminal amidated β-neurotoxin Ts1 with its isoform Ts1-G isolated from Tityus serrulatus venom. Toxicon. 2014. doi:10.1016/j.toxicon.2014.02.010.Google Scholar
- Lutz A, Mello-Campos O. Descripção de 5 espécies brasileiras dos gêneros Tityus e Rhopalurus. Folha Médica. 1922;4:25–26.Google Scholar
- Marangoni S, Toyama MH, Arantes EC, Giglio J, da Silva CA, Carneiro EM, et al. Amino acid sequence of TsTX-V, an alpha-toxin from Tityus serrulatus scorpion venom, and its effect on K+ permeability of beta-cells from isolated rat islets of Langerhans. Biochim Biophys Acta (BBA)-Gen Subj. 1995;1243(3):309–14.CrossRefGoogle Scholar
- Nunan EA, Cardoso VN, Moraes-Santos T. Lethal effect of the scorpion Tityus serrulatus venom: comparative study on adult and weanling rats. Braz J Pharm Sci. 2001;37(1):39–44.Google Scholar
- Richardson M, Borges MH, Cordeiro MN, Pimenta AMC, de Lima ME, Rates B. Allergenic protein from venom of Brazilian scorpion Tityus serrulatus. 2008a;UniProtKB: P85840.Google Scholar
- Richardson M, Borges MH, Cordeiro MN, Pimenta AMC, de Lima ME, Rates B. Hyaluronidase from venom of Brazilian scorpion Tityus serrulatus. 2008b;UniProtKB: P85841.Google Scholar
- Richardson M, Borges MH, Cordeiro MN, Pimenta AMC, de Lima ME, Rates B. Metaloproteinase from venom of Brazilian scorpion Tityus serrulatus. 2008c;UniProtKB: P85842.Google Scholar
- Sampaio SV, CoutinhoNetto J, Arantes EC, Toyama MH, Novello JC, Giglio JR. TsTX-VII, a new toxin from Tityus serrulatus scorpion venom able to induce the release of neurotransmitters from rat brain synaptosomes not blocked by tetrodotoxin. Biochem Mol Biol Int. 1997;41(6):1255–63.PubMedGoogle Scholar