SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Crystal Structure of Isoform CBd of the Basic Phospholipase A2 Subunit of Crotoxin: Description of the Structural Framework of CB for Interaction with Protein Targets.

Crotoxin, from the venom of the South American rattlesnake Crotalus durissus terrificus, is a potent heterodimeric presynaptic ß-neurotoxin that exists in individual snake venom as a mixture of isoforms of a basic phospholipase A2 (PLA2) subunit (CBa2, CBb, CBc, and CBd) and acidic subunit (CA1-4). Specific natural mutations in CB isoforms are implicated in functional differences between crotoxin isoforms. The three-dimensional structure of two individual CB isoforms (CBa2, CBc), and one isoform in a crotoxin (CA2CBb) complex, have been previously reported. This study concerns CBd, which by interaction with various protein targets exhibits many physiological or pharmacological functions. It binds with high affinity to presynaptic receptors showing neurotoxicity, but also interacts with human coagulation factor Xa (hFXa), exhibiting anticoagulant effect, and acts as a positive allosteric modulator and corrector of mutated chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), implicated in cystic fibrosis. Thus, CBd represents a novel family of agents that have potential in identifying new drug leads related to anticoagulant and anti-cystic fibrosis function. We determined here the X-ray structure of CBd and compare it with the three other natural isoforms of CB. The structural role of specific amino acid variations between CB isoforms are analyzed and the structural framework of CB for interaction with protein targets is described.

Neuromuscular paralysis by the basic phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit acetylcholine release and produce muscle blockage.

BACKGROUND AND PURPOSE: Crotoxin (CTX), a heterodimeric phospholipase A2 (PLA2) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. EXPERIMENTAL APPROACH: Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [3H]-acetylcholine ([3H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. KEY RESULTS: Both CTX (5µg/mL) and its basic PLA2 subunit (CB, 20µg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [3H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. CONCLUSION AND IMPLICATIONS: Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer.

Insights on the structure of native CNF, an endogenous phospholipase A2 inhibitor from Crotalus durissus terrificus, the South American rattlesnake.

Several snake species possess endogenous phospholipase A2 inhibitors (sbPLIs) in their blood plasma, the primary role of which is protection against an eventual presence of toxic phospholipase A2 (PLA2) from their venom glands in the circulation. These inhibitors have an oligomeric structure of, at least, three subunits and have been categorized into three classes (α, ß and γ) based on their structural features. SbγPLIs have been further subdivided into two subclasses according to their hetero or homomeric nature, respectively. Despite the considerable number of sbγPLIs described, their structures and mechanisms of action are still not fully understood. In the present study, we focused on the native structure of CNF, a homomeric sbγPLI from Crotalus durissus terrificus, the South American rattlesnake. Based on the results of different biochemical and biophysical experiments, we concluded that, while the native inhibitor occurs as a mixture of oligomers, tetrameric arrangement appears to be the predominant quaternary structure. The inhibitory activity of CNF is most likely associated with this oligomeric conformation. In addition, we suggest that the CNF tetramer has a spherical shape and that tyrosinyl residues could play an important role in the oligomerization. The carbohydrate moiety, which is present in most sbγPLIs, is not essential for the inhibitory activity, oligomerization or complex formation of the CNF with the target PLA2. A minor component, comprising no more than 16% of the sample, was identified in the CNF preparations. The amino-terminal sequence of that component is similar to the B subunits of the heteromeric sbγPLIs; however, the role played by such molecule in the functionality of the CNF, if any, remains to be determined.

Structural studies with crotoxin B from Crotalus durissus collilineatus venom suggest a heterodimeric assembly formed by two new isoforms.

In accidents involving Crotalus snakes, the crotoxin complex (CTX) plays lethal action due to its neurotoxic activity. On the other hand, CTX have potential biotechnological application due to its anti-tumoral, anti-inflammatory, antimicrobial, analgesic and immunomodulatory properties. CTX is a heterodimer composed of Crotoxin A (CA or crotapotin), the acidic nontoxic and non-enzymatic component and; Crotoxin B (CB), a basic, toxic and catalytic PLA2. Currently, there are two classes of CTX isoforms, whose differences in their biological activities have been attributed to features presented in CB isoforms. Here, we present the crystal structure of CB isolated from the Crotalus durissus collilineatus venom. It amino acid sequence was assigned using the SEQUENCE SLIDER software, which revealed that the crystal structure is a heterodimer composed of two new CB isoforms (colCB-A and colCB-B). Bioinformatic and biophysical analyses showed that the toxin forms a tetrameric assembly in solution similar to CB from Crotalus durissus terrificus venom, despite some differences observed at the dimeric interface. By the previously proposed classification, the colCB-B presents features of the class I isoforms while colCB-A cannot be classified into classes I and II based on its amino acid sequence. Due to similar features observed for other CB isoforms found in the NCBI database and the results obtained for colCB-A, we suggest that there are more than two classes of CTX and CB isoforms in crotalic venoms.

Exploring venom diversity in Mixcoatlus browni and Mixcoatlus barbouri: A comparative analysis of two rare Mexican snake species with crotoxin-like presence.

The genus Mixcoatlus is composed of three species: Mixcoatlus barbouri, M. browni, and M. melanurus, of which the venom composition of M. melanurus, the most common species of the three, has only recently been described. However, very little is known about the natural history of M. barbouri and M. browni, and the venom composition of these two species has remained thus far unexplored. In this study we characterize the proteomic profiles and the main biochemical and toxic activities of these two venoms. Proteomic data obtained by shotgun analysis of whole venom identified 12 protein families for M. barbouri, and 13 for M. browni. The latter venom was further characterized by using a quantitative 'venomics' protocol, which revealed that it is mainly composed of 51.1 % phospholipases A2 (PLA2), 25.5 % snake venom serine proteases (SVSP), 4.6 % l-amino oxidases (LAO), and 3.6 % snake venom metalloproteases (SVMP), with lower percentages other six protein families. Both venoms contained homologs of the basic and acidic subunits of crotoxin. However, due to limitations in M. barbouri venom availability, we could only characterize the crotoxin-like protein of M. browni venom, which we have named Mixcoatlutoxin. It exhibited a lethal potency in mice like that described for classical rattlesnake crotoxins. These findings expand knowledge on the distribution of crotoxin-like heterodimeric proteins in viper snake species. Further investigation of the bioactivities of the venom of M. barbouri, on the other hand, remains necessary.

Literature Review on Crotalus durissus terrificus Toxins: From a Perspective of Structural Biology and Therapeutic Applications.

BACKGROUND: The venom of Crotalus durissus terrificus, as well as its fractions, has intrigued research groups worldwide who are working to isolate, characterize, and find possible biotechnological applications. A number of studies have elucidated that these fractions and their derivatives possess pharmacological properties, which can enable the development of new drug prototypes with anti-inflammatory, antinociceptive, antitumor, antiviral, and antiparasitic applications. OBJECTIVE: This review presents a systematic study on Crotalus durissus terrificus, the most notable crotalid subspecies in South America, focusing on the composition, toxicological mechanisms, structural aspects, and applications of the main venom toxins (convulxin, gyroxin, crotamine, crotoxin, and their subunits). CONCLUSION: The authors have found that research on this snake and its toxins is still an area of focus, despite that almost a century has passed since the isolation of crotoxin. Several applications of these proteins in the development of novel drugs and bioactive substances have also been demonstrated.

Design of Crotoxin-Based Peptides with Potentiator Activity Targeting the ΔF508NBD1 Cystic Fibrosis Transmembrane Conductance Regulator.

We have previously shown that the CBb subunit of crotoxin, a ß-neurotoxin with phospholipase A2 (PLA2) activity, targets the human ΔF508CFTR chloride channel implicated in cystic fibrosis (CF). By direct binding to the nucleotide binding domain 1 (NBD1) of ΔF508CFTR, this neurotoxic PLA2 acts as a potentiator increasing chloride channel current and corrects the trafficking defect of misfolded ΔF508CFTR inside the cell. Here, for a therapeutics development of new anti-cystic fibrosis agents, we use a structure-based in silico approach to design peptides mimicking the CBb-ΔF508NBD1 interface. Combining biophysical and electrophysiological methods, we identify several peptides that interact with the ΔF508NBD1 domain and reveal their effects as potentiators on phosphorylated ΔF508CFTR. Moreover, protein-peptide interactions and electrophysiological studies allowed us to identify key residues of ΔF508NBD1 governing the interactions with the novel potentiators. The designed peptides bind to the same region as CBb phospholipase A2 on ΔF508NBD1 and potentiate chloride channel activity. Certain peptides also show an additive effect towards the clinically approved VX-770 potentiator. The identified CF therapeutics peptides represent a novel class of CFTR potentiators and illustrate a strategy leading to reproducing the effect of specific protein-protein interactions.

Intraspecific venom variation of Mexican West Coast Rattlesnakes (Crotalus basiliscus) and its implications for antivenom production.

Intraspecific variation in snake venoms has been widely documented worldwide. However, there are few studies on this subject in Mexico. Venom characterization studies provide important data used to predict clinical syndromes, to evaluate the efficacy of antivenoms and, in some cases, to improve immunogenic mixtures in the production of antivenoms. In the present work, we evaluated the intraspecific venom variation of Crotalus basiliscus, a rattlesnake of medical importance and whose venom is used in the immunization of horses to produce one of the Mexican antivenoms. Our results demonstrate that there is variation in biological and biochemical activities among adult venoms and that there is an ontogenetic change from juvenile to adult venoms. Juvenile venoms were more lethal and had higher percentages of crotamine and crotoxin, while adult venoms had higher percentages of snake venom metalloproteases (SVMPs). Additionally, we documented crotoxin-like PLA2 variation in which specimens from Zacatecas, Sinaloa and Michoacán (except 1) lacked the neurotoxin, while the rest of the venoms had it. Finally, we evaluated the efficacy of three lots of Birmex antivenom and all three were able to neutralize the lethality of four representative venoms but were not able to neutralize crotamine. We also observed significant differences in the LD50 values neutralized per vial among the different lots. Based on these results, we recommend including venoms containing crotamine in the production of antivenom for a better immunogenic mixture and to improve the homogeneity of lots.

Biological and Biochemical Characterization of Coronado Island Rattlesnake (Crotalus helleri caliginis) Venom and Antivenom Neutralization.

The Baja California Peninsula has over 250 islands and islets with many endemic species. Among them, rattlesnakes are the most numerous but also one of the least studied groups. The study of island rattlesnake venom could guide us to a better understanding of evolutionary processes and the description of novel toxins. Crotalus helleri caliginis venom samples were analyzed to determine possible ontogenetic variation with SDS-PAGE in one and two dimensions and with RP-HPLC. Western Blot, ELISA, and amino-terminal sequencing were used to determine the main components of the venom. The biological and biochemical activities demonstrate the similarity of C. helleri caliginis venom to the continental species C. helleri helleri, with both having low proteolytic and phospholipase A2 (PLA2) activity but differing due to the absence of neurotoxin (crotoxin-like) in the insular species. The main components of the snake venom were metalloproteases, serine proteases, and crotamine, which was the most abundant toxin group (30-35% of full venom). The crotamine was isolated using size-exclusion chromatography where its functional effects were tested on mouse phrenic nerve-hemidiaphragm preparations in which a significant reduction in muscle twitch contractions were observed. The two Mexican antivenoms could neutralize the lethality of C. helleri caliginis venom but not the crotamine effects.

Correlating biological activity to thermo-structural analysis of the interaction of CTX with synthetic models of macrophage membranes.

The important pharmacological actions of Crotoxin (CTX) on macrophages, the main toxin in the venom of Crotalus durissus terrificus, and its important participation in the control of different pathophysiological processes, have been demonstrated. The biological activities performed by macrophages are related to signaling mediated by receptors expressed on the membrane surface of these cells or opening and closing of ion channels, generation of membrane curvature and pore formation. In the present work, the interaction of the CTX complex with the cell membrane of macrophages is studied, both using biological cells and synthetic lipid membranes to monitor structural alterations induced by the protein. Here we show that CTX can penetrate THP-1 cells and induce pores only in anionic lipid model membranes, suggesting that a possible access pathway for CTX to the cell is via lipids with anionic polar heads. Considering that the selectivity of the lipid composition varies in different tissues and organs of the human body, the thermostructural studies presented here are extremely important to open new investigations on the biological activities of CTX in different biological systems.

Protein complexes in snake venom.

Snake venom contains mixture of bioactive proteins and polypeptides. Most of these proteins and polypeptides exist as monomers, but some of them form complexes in the venom. These complexes exhibit much higher levels of pharmacological activity compared to individual components and play an important role in pathophysiological effects during envenomation. They are formed through covalent and/or non-covalent interactions. The subunits of the complexes are either identical (homodimers) or dissimilar (heterodimers; in some cases subunits belong to different families of proteins). The formation of complexes, at times, eliminates the non-specific binding and enhances the binding to the target molecule. On several occasions, it also leads to recognition of new targets as protein-protein interaction in complexes exposes the critical amino acid residues buried in the monomers. Here, we describe the structure and function of various protein complexes of snake venoms and their role in snake venom toxicity.

Use of a phosphopeptide as a ligand to purify phospholipase A2 from the venom of Crotalus durisuss terrificus by affinity chromatography.

The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.

Immunoprotection against lethal effects of Crotalus durissus snake venom elicited by synthetic epitopes trapped in liposomes.

Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.

Engineered protein containing crotoxin epitopes induces neutralizing antibodies in immunized rabbits.

Crotoxin (Ctx) is the main lethal component of Crotalus durissus terrificus venom. It is a neurotoxin, composed of two subunits associated by noncovalent interactions, the non-toxic acid subunit (CA), named Crotapotin, and the basic subunit (CB), with phospholipase A2 (PLA2) activity. Employing the SPOT synthesis technique, we determined two epitopes located in the C-terminal of each Ctx subunit. In addition, 3 other epitopes were mapped in different regions of Ctx using subcutaneous spot implants surgically inserted in mice. All epitopes mapped here were expressed together as recombinant multi-epitopic protein (rMEPCtx), which was used to immunize New Zealand rabbits. Anti-rMEPCtx rabbit serum cross-reacted with Ctx and crude venoms from C. d. terrificus, Crotalus durissus ruruima, Peruvian C. durissus and Bothrops jararaca (with lower intensity). Furthermore, anti-rMEPCtx serum was able to neutralize Ctx lethal activity. As the recombinant multiepitopic protein is not toxic, it can be administered in larger doses without causing adverse effects on the immunized animals health. Therefore, our work evidences the identification of neutralizing epitopes of Ctx and support the use of recombinant multiepitopic proteins as an innovation to immunotherapeutics production.

The Crotoxin:SBA-15 Complex Down-Regulates the Incidence and Intensity of Experimental Autoimmune Encephalomyelitis Through Peripheral and Central Actions.

Crotoxin (CTX), the main neurotoxin from Crotalus durissus terrificus snake venom, has anti-inflammatory, immunomodulatory and antinociceptive activities. However, the CTX-induced toxicity may compromise its use. Under this scenario, the use of nanoparticle such as nanostructured mesoporous silica (SBA-15) as a carrier might become a feasible approach to improve CTX safety. Here, we determined the benefits of SBA-15 on CTX-related neuroinflammatory and immunomodulatory properties during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis that replicates several histopathological and immunological features observed in humans. We showed that a single administration of CTX:SBA-15 (54 µg/kg) was more effective in reducing pain and ameliorated the clinical score (motor impairment) in EAE animals compared to the CTX-treated EAE group; therefore, improving the disease outcome. Of interest, CTX:SBA-15, but not unconjugated CTX, prevented EAE-induced atrophy and loss of muscle function. Further supporting an immune mechanism, CTX:SBA-15 treatment reduced both recruitment and proliferation of peripheral Th17 cells as well as diminished IL-17 expression and glial cells activation in the spinal cord in EAE animals when compared with CTX-treated EAE group. Finally, CTX:SBA-15, but not unconjugated CTX, prevented the EAE-induced cell infiltration in the CNS. These results provide evidence that SBA-15 maximizes the immunomodulatory and anti-inflammatory effects of CTX in an EAE model; therefore, suggesting that SBA-15 has the potential to improve CTX effectiveness in the treatment of MS.

Evaluation of the Effectiveness of Crotoxin as an Antiseptic against Candida spp. Biofilms.

The growing number of oral infections caused by the Candida species are becoming harder to treat as the commonly used antibiotics become less effective. This drawback has led to the search for alternative strategies of treatment, which include the use of antifungal molecules derived from natural products. Herein, crotoxin (CTX), the main toxin of Crotalus durissus terrificus venom, was challenged against Candida tropicalis (CBS94) and Candida dubliniensis (CBS7987) strains by in vitro antimicrobial susceptibility tests. Minimum inhibitory concentration (MIC), minimum fungicidal concentration (MFC), and inhibition of biofilm formation were evaluated after CTX treatment. In addition, CTX-induced cytotoxicity in HaCaT cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric assay. Native CTX showed a higher antimicrobial activity (MIC = 47 µg/mL) when compared to CTX-containing mouthwash (MIC = 750 µg/mL) and nystatin (MIC = 375 µg/mL). Candida spp biofilm formation was more sensitive to both CTX and CTX-containing mouthwash (IC100 = 12 µg/mL) when compared to nystatin (IC100 > 47 µg/mL). Moreover, significant membrane permeabilization at concentrations of 1.5 and 47 µg/mL was observed. Native CTX was less cytotoxic to HaCaT cells than CTX-containing mouthwash or nystatin between 24 and 48 h. These preliminary findings highlight the potential use of CTX in the treatment of oral candidiasis caused by resistant strains.

Biochemical and biological characterization of a PLA2 from crotoxin complex of Crotalus durissus cumanensis.

A new PLA2 (Cdcum6) from crotoxin complex of Colombian Crotalus durissus cumanensis rattlesnake was purified using molecular exclusion chromatography and RP-HPLC. The molecular mass of Cdcum6 was determined by SDS-PAGE approximately 14 KDa and confirmed by MALDI-TOF (14321.98 Da). The enzyme showed Km 6.0 mM, Vmax 3.44 nmol/min, optimum pH was 8.0 and temperature was between 30 and 45 degrees C, and it had a strict requirement of Ca2+ for its activity. The N-terminal sequence of PLA2 was SLVQF EKMIK EVAGK NGVPWY. Comparison of amino acid sequence data with other PLA2 from South American Crotalus durissus rattlesnakes showed that Cdcum6 shares the highest sequence identity with Cdr13 an isoform PLA2 from Crotalus durissus ruruima, nevertheless, Cdcum6 showed high content of basic and hydrophobic amino acids. In mice, Cdcum6 presented higher LD50 than crotoxin complex from C d. cumanensis. Additionally, Cdcum6 induced a conspicuous local myotoxic effect and moderate footpad edema; in vitro, it was antigoagulant in doses as low as 0.5 microg/l ml, and it was not cytotoxic on myoblast but Cdcum6 was able to lyse myotubes.

Crystallization and preliminary X-ray diffraction analysis of crotoxin B from Crotalus durissus collilineatus venom.

Crotoxin B is a basic phospholipase A(2) found in the venom of several Crotalus durissus ssp. rattlesnakes and is one of the subunits that constitute crotoxin, the main component of the venom of these snakes. This heterodimeric toxin is related to important envenomation effects such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, the first structural data only became available in 2007 (for crotoxin B from C. durissus terrificus) and showed an ambiguous result for the biological assembly, which could be either dimeric or tetrameric. In this work, the crystallization, X-ray diffraction data collection at 2.2 A resolution and molecular-replacement solution of a dimeric complex formed by two crotoxin B isoforms from C. durissus collilineatus venom is presented.

Venom characterization of the three species of Ophryacus and proteomic profiling of O. sphenophrys unveils Sphenotoxin, a novel Crotoxin-like heterodimeric ß-neurotoxin.

Venoms of the three species of Ophryacus (O. sphenophrys, O. smaragdinus, and O. undulatus), a viperid genus endemic to Mexico, were analyzed for the first time in the present work. The three venoms lacked procoagulant activity on human plasma, but induced hemorrhage and were highly lethal to mice. These venoms also displayed proteolytic and phospholipase A2 activities in vitro. The venom of O. sphenophrys was the most lethal and caused hind-limb paralysis in mice. Proteomic profiling of O. sphenophrys venom showed a predominance of metalloproteinase (34.9%), phospholipase A2 (24.8%) and serine protease (17.1%) in its composition. Strikingly, within its PLA2 components, 12.9% corresponded to a Crotoxin-like heterodimer, here named Sphenotoxin, which was not found in the other two species of Ophryacus. Sphenotoxin, like Crotoxin, is composed of non-covalently bound A and B subunits. Partial amino acid sequence was obtained for Sphenotoxin B and was similar (78-89%) to other subunits described. The mouse i.v. LD50 of Sphenotoxin at 1:1 M radio was 0.16 µg/g. Also, like Crotoxin, Sphenotoxin induced a potent neuromuscular blockade in the phrenic nerve-diaphragm preparation. Ophryacus is the fifth genus and O. sphenophrys the third non-rattlesnake species shown to contain a novel Crotoxin-like heterodimeric ß-neurotoxin. BIOLOGICAL SIGNIFICANCE: Ophryacus is an endemic genus of semi-arboreal pitvipers from Mexico that includes three species with restricted distributions. Little is known about the natural history of these species and nothing is known about the properties of their venoms. Research on these species' venoms could generate relevant information regarding venom composition of Mexican pitvipers. Additionally, research into the presence of neurotoxic Crotoxin-like molecules outside of rattlesnakes (genera Crotalus and Sistrurus) has identified this molecule in several new genera. Knowing which genera and species possess neurotoxic components is important to fully understand the repercussions of snakebites, the interaction with prey and predators, and the origin, evolution, and phylogenetic distribution of Crotoxin-like molecules during the evolutionary history of pitvipers. Our study expands current knowledge regarding venom's compositions and function from Mexican pitvipers, providing a comparative venom characterization of major activities in the three Ophryacus species. Additionally, the discovery and characterization of a novel Crotoxin-like molecule, here named Sphenotoxin, in O. sphenophrys, and the detailed protein composition of O. sphenophrys venom supports the hypotheses that Crotoxin-like -ß-neurotoxins are more widespread than initially thought.