Characterization of Four Medically Important Toxins from Centruroides huichol Scorpion Venom and Its Neutralization by a Single Recombinant Antibody Fragment

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD₅₀) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD₅₀ of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.     

Isolation and primary structure of a potent toxin from the venom of the scorpion Centruroides sculpturatus Ewing

A potent toxin has been purified from the venom of the scorpion Centruroides sculpturatus Ewing using the ion-exchange resin CM-Sepharose CL-6B at basic pH. The toxin, designated CsE M₁, comprised 65 amino acid residues and its primary structure was established as: Lys-Glu-Gly-Tyr-Leu-Val-Asn-Ser-Tyr-Thr¹⁰-Gly-Cys-Lys-Tyr-Glu-Cys-Leu-Lys-Leu-Gly²⁰-Asp-Asn-Asp-Tyr-Cys-Leu-Arg-Glu-Cys-Arg³⁰-Gln-Gln-Tyr-Gly-Lys-Ser-Gly-Gly-Tyr-Cys⁴⁰-Tyr-Ala-Phe-Ala-Cys-Trp-Cys-Thr-His-Leu⁵⁰-Tyr-Glu-Gln-Ala-Val-Val-Trp-Pro-Leu-Pro⁶⁰-Asn-Lys-Thr-Cys-Asn. CsE M₁ is the most lethal protein to be identified in C. sculpturatus venom and the LD₅₀ of the toxin, determined by subcutaneous injection into Swiss mice, is 87 μg/kg. CsE M₁ shows strong structural similarity (92% positional identity) to the most potent β-toxin, Css II, from the Mexican scorpion, Centruroides suffusus suffusus but is quite dissimilar to the previously characterized toxins with low potency isolated from C. sculpturarus Ewing.     

Venom components of the scorpion Centruroides limpidus modulate cytokine expression by T helper lymphocytes: Identification of ion channel-related toxins by mass spectrometry

The study of the effector mechanisms of T helper cells has revealed different phenotypic characteristics that can be manipulated for designing new therapeutic schemes in different pathological scenarios. Ion channels are significant targets in T lymphocyte modulation since they are closely related to their effector activity. Remarkably, some toxins produced by scorpions specifically affect the function of these membrane proteins. For that reason, these toxins are important candidates in the search for new immunomodulators. Here, the effect of two venom fractions of the scorpion Centruroides limpidus was assessed on T lymphocyte proliferation and cytokine production. The venom fractions ClF8 and ClF9 were separated by reversed-phase high-performance liquid chromatography (RP-HPLC) and cultured at 25 and 35 µg/ml with murine T lymphocytes. The results indicate that the fraction ClF8 increased both production and secretion levels of IFN-γ, IL-4, IL-17A and IL-10 by CD4⁺ T cells at 24 h. In contrast, fraction ClF9 only promoted the secretion of IL-17A and IL-10 at its highest concentration (35 µg/ml). Both fractions did not show any effect on T cell proliferation. Subsequent analyses by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed seventeen toxins in the fraction ClF8 and five toxins in the fraction ClF9, most of them with voltage-gated sodium (NaScTx) and potassium (KScTx) channels as molecular targets. These toxins might probably interact with ion channels involved in T lymphocyte activity. Our findings suggest that the difference in composition between the two fractions could be related to the observed effects, and the components identified could be isolated to search for possible immunomodulatory molecules.     

In Vitro Neurotoxicity of Chinese Krait (Bungarus multicinctus) Venom and Neutralization by Antivenoms

Bungarus multicinctus, the Chinese krait, is a highly venomous elapid snake which causes considerable morbidity and mortality in southern China. B. multicinctus venom contains pre-synaptic PLA₂ neurotoxins (i.e., β-bungarotoxins) and post-synaptic neurotoxins (i.e., α-bungarotoxins). We examined the in vitro neurotoxicity of B. multicinctus venom, and the efficacy of specific monovalent Chinese B. multicinctus antivenom, and Australian polyvalent elapid snake antivenom, against venom-induced neurotoxicity. B. multicinctus venom (1–10 μg/mL) abolished indirect twitches in the chick biventer cervicis nerve-muscle preparation as well as attenuating contractile responses to exogenous ACh and CCh, but not KCl. This indicates a post-synaptic neurotoxic action but myotoxicity was not evident. Given that post-synaptic α-neurotoxins have a more rapid onset than pre-synaptic neurotoxins, the activity of the latter in the whole venom will be masked. The prior addition of Chinese B. multicinctus antivenom (12 U/mL) or Australian polyvalent snake antivenom (15 U/mL), markedly attenuated the neurotoxic actions of B. multicinctus venom (3 μg/mL) and prevented the inhibition of contractile responses to ACh and CCh. The addition of B. multicinctus antivenom (60 U/mL), or Australian polyvalent snake antivenom (50 U/mL), at the t₉₀ time point after the addition of B. multicinctus venom (3 μg/mL), did not restore the twitch height over 180 min. The earlier addition of B. multicinctus antivenom (60 U/mL), at the t₂₀ or t₅₀ time points, also failed to prevent the neurotoxic effects of the venom but did delay the time to abolish twitches based on a comparison of t₉₀ values. Repeated washing of the preparation with physiological salt solution, commencing at the t₂₀ time point, failed to reverse the neurotoxic effects of venom or delay the time to abolish twitches. This study showed that B. multicinctus venom displays marked in vitro neurotoxicity in a skeletal muscle preparation which is not reversed by antivenom. This does not appear to be related to antivenom efficacy, but due to the irreversible/pseudo-irreversible nature of the neurotoxins.     

Mechanism of Lethal Toxin Neutralization by a Human Monoclonal Antibody Specific for the PA(20) Region of Bacillus anthracis Protective Antigen

The primary immunogenic component of the currently approved anthrax vaccine is the protective antigen (PA) unit of the binary toxin system. PA-specific antibodies neutralize anthrax toxins and protect against infection. Recent research has determined that in humans, only antibodies specific for particular determinants are capable of effecting toxin neutralization, and that the neutralizing epitopes recognized by these antibodies are distributed throughout the PA monomer. The mechanisms by which the majority of these epitopes effect neutralization remain unknown. In this report we investigate the process by which a human monoclonal antibody specific for the amino-terminal domain of PA neutralizes lethal toxin in an in vitro assay of cytotoxicity, and find that it neutralizes LT by blocking the requisite cleavage of the amino-terminal 20 kD portion of the molecule (PA(20)) from the remainder of the PA monomer. We also demonstrate that the epitope recognized by this human monoclonal does not encompass the (166)RKKR(169) furin recognition sequence in domain 1 of PA.     

In-Vitro Neutralization of the Neurotoxicity of Coastal Taipan Venom by Australian Polyvalent Antivenom: The Window of Opportunity

Coastal taipan (Oxyuranus scutellatus) envenoming causes life-threatening neuromuscular paralysis in humans. We studied the time period during which antivenom remains effective in preventing and arresting in vitro neuromuscular block caused by taipan venom and taipoxin. Venom showed predominant pre-synaptic neurotoxicity at 3 µg/mL and post-synaptic neurotoxicity at 10 µg/mL. Pre-synaptic neurotoxicity was prevented by addition of Australian polyvalent antivenom before the venom and taipoxin and, reversed when antivenom was added 5 min after venom and taipoxin. Antivenom only partially reversed the neurotoxicity when added 15 min after venom and had no significant effect when added 30 min after venom. In contrast, post-synaptic activity was fully reversed when antivenom was added 30 min after venom. The effect of antivenom on pre-synaptic neuromuscular block was reproduced by washing the bath at similar time intervals for 3 µg/mL, but not for 10 µg/mL. We found an approximate 10–15 min time window in which antivenom can prevent pre-synaptic neuromuscular block. This time window is likely to be longer in envenomed patients due to the delay in venom absorption. Similar effectiveness of antivenom and washing with 3 µg/mL venom suggests that antivenom most likely acts by neutralizing pre-synaptic toxins before they interfere with neurotransmission inside the motor nerve terminals.     

不同地域东亚钳蝎蝎毒的双向电泳鉴定

摘要 目的利用蛋白质组学中双向电泳技术,定性鉴定、分析不同地域东亚钳蝎蝎毒活性的差别,探索不同产地蝎毒的蛋白质组成及功能差异。方法将不同产地的冷冻干燥蝎毒粉经溶解、除盐、浓缩后测定蝎毒蛋白质含量,进行定量的蝎毒鉴定。采用pH梯度等电聚焦和SDS PAGE凝胶电泳技术分离蝎毒蛋白质。银染后通过凝胶成像系统获得双向电泳凝胶图谱,用PD Quest图像分析软件比较分析,确定差异的特征蛋白点,从而定性鉴定不同产地蝎毒。结果获得3个样品的蛋白质指纹图谱。分别检测出80,69和77个点,特征差异蛋白点依次为56,46和55个。结论不同产地的东亚钳蝎蝎毒通过双向电泳分离蛋白后,表现出明显不同的蛋白点分布。     

卵黄抗体用于眼镜王蛇毒抗原检测的初步研究

目的探讨抗蛇毒卵黄抗体应用于蛇毒检测的可行性。方法以甲醛减毒的眼镜王蛇毒抗原免疫莱航母鸡,制备抗眼镜王蛇毒抗体;采用过碘酸钠法对特异卵黄抗体进行酶标;ELISA的实验条件及相关参数通过棋盘滴定法确定,并进行特异性、灵敏度、精确度及稳定性等测定。结果本检测方法的灵敏度可达32μg·L-1,并且眼镜王蛇毒抗原浓度在32～750μg·L-1的范围内,线性关系较好(r=0.963);卵黄抗体对眼镜王蛇毒具有较高的特异性,与蝰蛇科的五步蛇毒、园斑蝰蛇毒无交叉反应,与眼镜蛇科的金环及银环蛇毒仅在高剂量(剂量大于500μg·L-1)时存在轻度交叉反应,但与眼镜蛇毒之间交叉反应明显。应用本方法对不同浓度(100、300、1000μg·L-1)的眼镜王蛇毒样品检测,结果平均板内相对标准差在1%～3%之内,板间相对标准差在8%以内;稳定性实验显示:卵黄抗体置于37℃条件6天,其检测结果与4℃条件下无显著差异(P>0.05)。结论特异的卵黄抗体应用于微量蛇毒检测研究是可行的,但仍需要进一步研究以降低交叉反应。     

Ophiophagus hannah Venom: Proteome,Components Bound by Naja kaouthia Antivenin and Neutralization by N. kaouthia Neurotoxin-Specific Human ScFv

Venomous snakebites are an important health problem in tropical and subtropical countries. King cobra (Ophiophagushannah) is the largest venomous snake found in South and Southeast Asia. In this study, the O. hannah venom proteome and the venom components cross-reactive to N. kaouthia monospecific antivenin were studied. O. hannah venom consisted of 14 different protein families, including three finger toxins, phospholipases, cysteine-rich secretory proteins, cobra venom factor, muscarinic toxin, L-amino acid oxidase, hypothetical proteins, low cysteine protein, phosphodiesterase, proteases, vespryn toxin, Kunitz, growth factor activators and others (coagulation factor, endonuclease, 5’-nucleotidase). N. kaouthia antivenin recognized several functionally different O. hannah venom proteins and mediated paratherapeutic efficacy by rescuing the O. hannah envenomed mice from lethality. An engineered human ScFv specific to N. kaouthia long neurotoxin (NkLN-HuScFv) cross-neutralized the O. hannah venom and extricated the O. hannah envenomed mice from death in a dose escalation manner. Homology modeling and molecular docking revealed that NkLN-HuScFv interacted with residues in loops 2 and 3 of the neurotoxins of both snake species, which are important for neuronal acetylcholine receptor binding. The data of this study are useful for snakebite treatment when and where the polyspecific antivenin is not available. Because the supply of horse-derived antivenin is limited and the preparation may cause some adverse effects in recipients, a cocktail of recombinant human ScFvs for various toxic venom components shared by different venomous snakes, exemplified by the in vitro produced NkLN-HuScFv in this study, should contribute to a possible future route for an improved alternative to the antivenins.     

Development of a Monoclonal scFv against Cytotoxin to Neutralize Cytolytic Activity Induced by Naja atra Venom on Myoblast C2C12 Cells

The Taiwanese cobra, Naja atra, is a clinically significant species of snake observed in the wild in Taiwan. Victims bitten by N. atra usually experience severe pain and local tissue necrosis. Although antivenom is available for treatment of cobra envenomation, its neutralization potency against cobra-induced necrosis is weak, with more than 60% of cobra envenoming patients developing tissue necrosis after antivenom administration. The present study found that cytotoxin (CTX) is a key component of N. atra venom responsible for cytotoxicity against myoblast cells. Anti-CTX IgY was generated in hens, and the spleens of these hens were used to construct libraries for the development of single chain variable fragments (scFv). Two anti-CTX scFv, S1 and 2S7, were selected using phage display technology and biopanning. Both polyclonal IgY and monoclonal scFv S1 reacted specifically with CTX in cobra venom. In a cell model assay, the CTX-induced cytolytic effect was inhibited only by monoclonal scFv S1, not by polyclonal IgY. Moreover, the neutralization potency of scFv S1 was about 3.8 mg/mg, approximately three times higher than that of conventional freeze-dried neurotoxic antivenom (FNAV). Collectively, these results suggest that scFv S1 can effectively neutralize CTX-induced cytotoxicity and, when combined with currently available antivenom, can improve the potency of the latter, thereby preventing tissue damage induced by cobra envenoming.     

Scorpion BmK venom induces nociceptive response of rats by plantar injection

In this study, the nociceptive responses induced by subcutaneous injection of scorpion BmK venom into the plantar surface of rat hind paw were quantified. The suitable dose of the venom is between 0.01 and 0.05 mg. The venom injection could induce local edema and tonic-pain responses, which had lasted more than 1 h. The pain scores reached peak intensity within 20 min, and then decreased slowly. Morphine, as a classic analgesic drug, could inhibit the spontaneous nociceptive response induced by the venom. With Morphine pretreatment, the mean pain scores for the entire 60 min were significantly decreased (P<.05). BmK I, a main lethal component, was also found to induce nociceptive responses with dose-dependent means. The nociceptive responses induced by BmK I could be partially inhibited by morphine.     

Scorpion Peptide Smp24 Exhibits a Potent Antitumor Effect on Human Lung Cancer Cells by Damaging the Membrane and Cytoskeleton In Vivo and In Vitro

Smp24, a cationic antimicrobial peptide identified from the venom gland of the Egyptian scorpion Scorpio maurus palmatus, shows variable cytotoxicity on various tumor (KG1a, CCRF-CEM and HepG2) and non-tumor (CD34⁺, HRECs, HACAT) cell lines. However, the effects of Smp24 and its mode of action on lung cancer cell lines remain unknown. Herein, the effect of Smp24 on the viability, membrane disruption, cytoskeleton, migration and invasion, and MMP-2/-9 and TIMP-1/-2 expression of human lung cancer cells have been evaluated. In addition, its in vivo antitumor role and acute toxicity were also assessed. In our study, Smp24 was found to suppress the growth of A549, H3122, PC-9, and H460 with IC₅₀ values from about 4.06 to 7.07 µM and show low toxicity to normal cells (MRC-5) with 14.68 µM of IC₅₀. Furthermore, Smp24 could induce necrosis of A549 cells via destroying the integrity of the cell membrane and mitochondrial and nuclear membranes. Additionally, Smp24 suppressed cell motility by damaging the cytoskeleton and altering MMP-2/-9 and TIMP-1/-2 expression. Finally, Smp24 showed effective anticancer protection in a A549 xenograft mice model and low acute toxicity. Overall, these findings indicate that Smp24 significantly exerts an antitumor effect due to its induction of membrane defects and cytoskeleton disruption. Accordingly, our findings will open an avenue for developing scorpion venom peptides into chemotherapeutic agents targeting lung cancer cells.     

Appraisal of Antiophidic Potential of Marine Sponges against Bothrops jararaca and Lachesis muta Venom

Snakebites are a health problem in many countries due to the high incidence of such accidents. Antivenom treatment has regularly been used for more than a century, however, this does not neutralize tissue damage and may even increase the severity and morbidity of accidents. Thus, it has been relevant to search for new strategies to improve antiserum therapy, and a variety of molecules from natural sources with antiophidian properties have been reported. In this paper, we analyzed the ability of ten extracts from marine sponges (Amphimedon viridis, Aplysina fulva, Chondrosia collectrix, Desmapsamma anchorata, Dysidea etheria, Hymeniacidon heliophila, Mycale angulosa, Petromica citrina, Polymastia janeirensis, and Tedania ignis) to inhibit the effects caused by Bothrops jararaca and Lachesis muta venom. All sponge extracts inhibited proteolysis and hemolysis induced by both snake venoms, except H. heliophila, which failed to inhibit any biological activity. P. citrina inhibited lethality, hemorrhage, plasma clotting, and hemolysis induced by B. jararaca or L. muta. Moreover, other sponges inhibited hemorrhage induced only by B. jararaca. We conclude that Brazilian sponges may be a useful aid in the treatment of snakebites caused by L. muta and B. jararaca and therefore have potential for the discovery of molecules with antiophidian properties.     

Systemic Effects of Hemorrhagic Snake Venom Metalloproteinases: Untargeted Peptidomics to Explore the Pathodegradome of Plasma Proteins

Hemorrhage induced by snake venom metalloproteinases (SVMPs) is a complex phenomenon that involves capillary disruption and blood extravasation. HF3 (hemorrhagic factor 3) is an extremely hemorrhagic SVMP of Bothrops jararaca venom. Studies using proteomic approaches revealed targets of HF3 among intracellular and extracellular proteins. However, the role of the cleavage of plasma proteins in the context of the hemorrhage remains not fully understood. The main goal of this study was to analyze the degradome of HF3 in human plasma. For this purpose, approaches for the depletion of the most abundant proteins, and for the enrichment of low abundant proteins of human plasma, were used to minimize the dynamic range of protein concentration, in order to assess the proteolytic activity of HF3 on a wide spectrum of proteins, and to detect the degradation products using mass spectrometry-based untargeted peptidomics. The results revealed the hydrolysis products generated by HF3 and allowed the identification of cleavage sites. A total of 61 plasma proteins were identified as cleaved by HF3. Some of these proteins corroborate previous studies, and others are new HF3 targets, including proteins of the coagulation cascade, of the complement system, proteins acting on the modulation of inflammation, and plasma proteinase inhibitors. Overall, the data indicate that HF3 escapes inhibition and sculpts the plasma proteome by degrading key proteins and generating peptides that may act synergistically in the hemorrhagic process.     

The Effects of a Chactoid Scorpion Venom and Its Purified Toxins on Rat Blood Pressure and Mast Cells Histamine Release

The effect of the venom of the Chactoid family of scorpions on blood pressure was scantly investigated and was addressed in the present study using the venom of the Israeli scorpion, Scorpio maurus palmatus. Blood pressure in rats was monitored via cannulated femoral artery, while venom and toxins were introduced into femoral vein. Venom injection elicited a biphasic effect, expressed first by a fast and transient hypotensive response, which lasted up to 10 min, followed by a hypertensive response, which lasted up to one hour. It was found that these effects resulted from different venom components. Phospholipase A₂ produced the hypotensive effect, while a non-enzymatic neurotoxic polypeptide fraction produced the hypertensive effect. Surprisingly, the main neurotoxic polypeptide to mice had no effect on blood pressure. In vitro experiments indicated that the hypertensive factors caused histamine release from the peritoneal mast cells, but this effect is assumed to be not relevant to their in vivo effect. In spite of the cytotoxic activity of phospholipase A₂, it did not release histamine. These findings suggest that the effects of venom and isolated fractions on blood pressure parameters are mediated by different mechanisms, which deserve further pharmacological investigation.     

酚妥拉明减轻蟾酥注射液致血管刺激的临床观察

目的:观察酚妥拉明减轻蟾酥注射液致血管刺激的作用。方法:将45例静脉滴注蟾酥注射液的晚期肿瘤患者随机分成观察组23例和对照组22例。观察组采用酚妥拉明5mg+蟾酥注射液20mL,同时加入到5%葡萄糖注射液500mL中静脉滴注,对照组仅采用蟾酥注射液20mL,加入到5%葡萄糖注射液500mL中静脉滴注。2组均每日1次,21d为1个疗程,均观察1个疗程,比较2组的滴速减慢、血管疼痛和红肿发生率。结果:由每分钟50滴减慢至每分钟40～49滴,观察组发生率为39.13%,对照组为81.82%,2组比较有显著性差异(P<0.01);观察组的血管疼痛和红肿发生率为26.09%,对照组为68.18%,2组比较有显著性差异(P<0.01)。结论:蟾酥注射液联合酚妥拉明静脉滴注,可有效减轻蟾酥注射液所致血管刺激。     

双抗体夹心-酶联免疫吸附法测定家兔血清中短尾蝮蛇毒磷脂结合抗凝蛋白的浓度及其药动学研究

目的:建立测定家兔血清中短尾蝮蛇毒磷脂结合抗凝蛋白(PBAP)浓度的方法,并考察其药动学特征。方法:取家兔随机分为低、高剂量组(短尾蝮蛇毒PBAP,0.4、0.8mg·kg-1)和阴性对照组(生理盐水),每组5只,耳缘静脉单次给予相应药物,给药前及给药后1、5、15、30、60、120、240、480min分别从颈总动脉取血,加入到短尾蝮蛇毒PBAP抗体包被酶标板(浓度为100μg·mL-1)中,以给药前的正常血清作为空白对照,用双抗体夹心-酶联免疫吸附(ELISA)法测定不同时间点血清中短尾蝮蛇毒PBAP的浓度,并用DAS2.0软件计算其药动学参数。结果:短尾蝮蛇毒PBAP检测浓度的线性范围为0.0024～10μg·mL-(1r=0.9703),平均回收率为98.78%,平均板内变异系数为7.96%,平均板间变异系数为10.94%;低、高剂量短尾蝮蛇毒PBAP的主要药动学参数分别为t1/2α(9.553±4.668)、(9.873±1.433)min,t1/2β(201.295±66.060)、(205.798±76.834)min,cmax(0.685±0.160)、(2.333±0.478)mg·L-1,AUC0～∞(27.114±2.781)、(61.625±11.631)mg·min·L-1。结论:ELISA法准确、灵敏、特异性强,可用于短尾蝮蛇毒PBAP在家兔血清中的药动学研究。