Cerrophidion Godmani Research Articles

Comparative Analysis of Alpha-1 Orthosteric-Site Binding by a Clade of Central American Pit Vipers (Genera Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium).

The distribution and relative potency of post-synaptic neurotoxic activity within Crotalinae venoms has been the subject of less investigation in comparison with Elapidae snake venoms. No previous studies have investigated post-synaptic neurotoxic activity within the Atropoides, Metlapilcoatlus, Cerrophidion, and Porthidium clade. Given the specificity of neurotoxins to relevant prey types, we aimed to uncover any activity present within this clade of snakes that may have been overlooked due to lower potency upon humans and thus not appearing as a clinical feature. Using biolayer interferometry, we assessed the relative binding of crude venoms to amphibian, lizard, bird, rodent and human α-1 nAChR orthosteric sites. We report potent alpha-1 orthosteric site binding in venoms from Atropoides picadoi, Metlapilcoatlus occiduus, M. olmec, M. mexicanus, M. nummifer. Lower levels of binding, but still notable, were evident for Cerrophidion godmani, C. tzotzilorum and C. wilsoni venoms. No activity was observed for Porthidium venoms, which is consistent with significant alpha-1 orthosteric site neurotoxicity being a trait that was amplified in the last common ancestor of Atropoides/Cerrophidion/Metlapilcoatlus subsequent to the split by Porthidium. We also observed potent taxon-selective activity, with strong selection for non-mammalian targets (amphibian, lizard, and bird). As these are poorly studied snakes, much of what is known about them is from clinical reports. The lack of affinity towards mammalian targets may explain the knowledge gap in neurotoxic activity within these species, since symptoms would not appear in bite reports. This study reports novel venom activity, which was previously unreported, indicating toxins that bind to post-synaptic receptors may be more widespread in pit vipers than previously considered. While these effects appear to not be clinically significant due to lineage-specific effects, they are of significant evolutionary novelty and of biodiscovery interest. This work sets the stage for future research directions, such as the use of in vitro and in vivo models to determine whether the alpha-1 orthosteric site binding observed within this study confers neurotoxic venom activity.

Sequence Divergence in Venom Genes Within and Between Montane Pitviper (Viperidae: Crotalinae: Cerrophidion) Species is Driven by Mutation-Drift Equilibrium.

Snake venom can vary both among and within species. While some groups of New World pitvipers-such as rattlesnakes-have been well studied, very little is known about the venom of montane pitvipers (Cerrophidion) found across the Mesoamerican highlands. Compared to most well-studied rattlesnakes, which are widely distributed, the isolated montane populations of Cerrophidion may facilitate unique evolutionary trajectories and venom differentiation. Here, we describe the venom gland transcriptomes for populations of C. petlalcalensis, C. tzotzilorum, and C. godmani from Mexico, and a single individual of C. sasai from Costa Rica. We explore gene expression variation in Cerrophidion and sequence evolution of toxins within C. godmani specifically. Cerrophidion venom gland transcriptomes are composed primarily of snake venom metalloproteinases, phospholipase A[Formula: see text]s (PLA[Formula: see text]s), and snake venom serine proteases. Cerrophidion petlalcalensis shows little intraspecific variation; however, C. godmani and C. tzotzilorum differ significantly between geographically isolated populations. Interestingly, intraspecific variation was mostly attributed to expression variation as we did not detect signals of selection within C. godmani toxins. Additionally, we found PLA[Formula: see text]-like myotoxins in all species except C. petlalcalensis, and crotoxin-like PLA[Formula: see text]s in the southern population of C. godmani. Our results demonstrate significant intraspecific venom variation within C. godmani and C. tzotzilorum. The toxins of C. godmani show little evidence of directional selection where variation in toxin sequence is consistent with evolution under a model of mutation-drift equilibrium. Cerrophidion godmani individuals from the southern population may exhibit neurotoxic venom activity given the presence of crotoxin-like PLA[Formula: see text]s; however, further research is required to confirm this hypothesis.

Differential Antivenom and Small-Molecule Inhibition of Novel Coagulotoxic Variations in Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium American Viperid Snake Venoms.

Within Neotropical pit-vipers, the Mexican/Central-American clade consisting of Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium is a wide-ranging, morphologically and ecologically diverse group of snakes. Despite their prevalence, little is known of the functional aspects of their venoms. This study aimed to fill the knowledge gap regarding coagulotoxic effects and to examine the potential of different therapeutic approaches. As a general trait, the venoms were shown to be anticoagulant but were underpinned by diverse biochemical actions. Pseudo-procoagulant activity (i.e., thrombin-like), characterized by the direct cleavage of fibrinogen to form weak fibrin clots, was evident for Atropoides picadoi, Cerrophidion tzotzilorum, Metlapilcoatlus mexicanus, M. nummifer, M. occiduus, M. olmec, and Porthidium porrasi. In contrast, other venoms cleaved fibrinogen in a destructive (non-clotting) manner, with C. godmani and C. wilsoni being the most potent. In addition to actions on fibrinogen, clotting enzymes were also inhibited. FXa was only weakly inhibited by most species, but Cerrophidion godmani and C. wilsoni were extremely strong in their inhibitory action. Other clotting enzymes were more widely inhibited by diverse species spanning the full taxonomical range, but in each case, there were species that had these traits notably amplified relatively to the others. C. godmani and C. wilsoni were the most potent amongst those that inhibited the formation of the prothrombinase complex and were also amongst the most potent inhibitors of Factor XIa. While most species displayed only low levels of thrombin inhibition, Porthidium dunni potently inhibited this clotting factor. The regional polyvalent antivenom produced by Instituto Picado Clodomiro was tested and was shown to be effective against the diverse anticoagulant pathophysiological effects. In contrast to the anticoagulant activities of the other species, Porthidium volcanicum was uniquely procoagulant through the activation of Factor VII and Factor XII. This viperid species is the first snake outside of the Oxyuranus/Pseudonaja elapid snake clade to be shown to activate FVII and the first snake venom of any kind to activate FXII. Interestingly, while small-molecule metalloprotease inhibitors prinomastat and marimastat demonstrated the ability to prevent the procoagulant toxicity of P. volcanicum, neither ICP antivenom nor inhibitor DMPS showed this effect. The extreme variation among the snakes here studied underscores how venom is a dynamic trait and how this can shape clinical outcomes and influence evolving treatment strategies.

Preclinical assessment of a polyspecific antivenom against the venoms of Cerrophidion sasai, Porthidium nasutum and Porthidium ophryomegas: Insights from combined antivenomics and neutralization assays

A polyspecific antivenom is used in Central America for the treatment of envenomings by viperid snakes. This antivenom is generated in horses hyperimmunized with a mixture of venoms from Bothrops asper, Crotalus simus and Lachesis stenophrys. The present study analyzed the ability of this antivenom to neutralize the venoms of three Central American viperid species of the ‘Porthidium group’, i.e. Porthidium nasutum, Porthidium ophryomegas and Cerrophidion sasai, formerly classified as Cerrophidion godmani. In addition, the immunorecognition of the components of these venoms was assessed by immunoaffinity antivenomics. The antivenom proved effective in neutralizing the lethal, hemorrhagic, myotoxic, phospholipase A2 (PLA2) and proteinase activities of the three venoms, albeit exhibiting quantitative differences in the values of the Median Effective Doses (ED50). Excepting for certain low molecular mass bands corresponding to disintegrins, and some PLA2s and PI-metalloproteinases, Western blotting and immunoaffinity chromatography revealed immunorecognition of most Porthidium and Cerrophidion venom proteins. In agreement with in vivo neutralization assays, immobilized antivenom IgGs showed higher immunocapturing activity of toxins from both Porthidium taxa than from C. sasai. Overall our results demonstrate a significant paraspecific protection of the Costa Rican polyspecific antivenom against the three venoms sampled. They also stress the need to search for novel ways to enhance the immune response of horses against several weakly immunogenic venom components.

Cryptic diversity in disjunct populations of Middle American Montane Pitvipers: a systematic reassessment of Cerrophidion godmani

Jadin, R.C., Townsend, J.H., Castoe, T.A. & Campbell, J.A. (2012). Cryptic diversity in disjunct populations of Middle American Montane Pitvipers: a systematic reassessment of Cerrophidion godmani. —Zoologica Scripta, 41, 455–470.The discovery and taxonomic recognition of cryptic species has become increasingly frequent with the application of molecular phylogenetic analyses, particularly for species with broad geographic distributions. In this study we focus on the venomous pitviper species Cerrophidion godmani that is widely distributed throughout the highlands of Central America. We provide evidence based on both molecular phylogenetic analyses and morphological data that C. godmani represents three deeply divergent lineages and is possibly non‐monophyletic. These three lineages are relatively conserved in their morphology and tend to be highly variable among individuals, but we do find sufficient morphological characters to diagnose them as evolutionarily distinct. We apply these data, together with known geographic distributions of populations, to infer boundaries of these three divergent evolutionary lineages. Based on the body of evidence, we formally name and describe two new species of Cerrophidion and redescribe C. godmani sensu stricto.

Snake venomics of the pit vipers Porthidium nasutum, Porthidium ophryomegas, and Cerrophidion godmani from Costa Rica: Toxicological and taxonomical insights

Within the Neotropical pit vipers, a lineage of primarily Middle American snake species referred to as the "Porthidium group" includes the genera Atropoides, Cerrophidion, and Porthidium. In this study, the venom proteomes of Porthidium nasutum, P. ophryomegas, and Cerrophidion godmani from Costa Rica were analyzed, and correlated to their toxic and enzymatic activities. Their HPLC profiles revealed a higher similarity between the two Porthidium species than between these and C. godmani. Proteins belonging to nine (P. nasutum), eight (P. ophryomegas), and nine (C. godmani) families were identified by mass spectrometry or N-terminal sequencing. Final cataloging of proteins and their relative abundances confirmed the close relationship between venoms of P. nasutum and P. ophryomegas, departing from that of C. godmani. Since the latter species had been taxonomically classified as Porthidium godmani previously, our venomic analyses agree with its current generic status. Venoms of P. nasutum and P. ophryomegas, despite containing abundant metalloproteinases and serine proteinases, lack procoagulant activity on human plasma, in contrast to venom of C. godmani. The latter induced strong myotoxicity in mice, which correlates with its high proportion of phospholipases A(2), whereas venoms from the two Porthidium species, containing lower amounts of these enzymes, induced only mild muscle damage.

Isolation, characterization and molecular cloning of AnMIP, a new α-type phospholipase A 2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae)

A new phospholipase A 2 (PLA 2)-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA 2 myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the α group of phospholipase A 2 inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247–22,301 and an isoelectric point of 4.1–4.7. This trimeric structure is the first observed in a PLIα from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIαs isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA 2s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper myotoxin I and A. nummifer myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.

Differential susceptibility of C2C12 myoblasts and myotubes to group II phospholipase A2 myotoxins from crotalid snake venoms

Group II phospholipase A(2) (PLA(2)) myotoxins isolated from Viperidae/Crotalidae snake venoms induce a rapid cytolytic effect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletal muscle myotubes than on other cell types, including undifferentiated myoblasts. This study utilized the murine skeletal muscle C2C12 cell line to investigate whether differentiated myotubes are more susceptible than myoblasts, and if this characteristic is specific for the group II myotoxic PLA(2)s. The release of lactic dehydrogenase was quantified as a measure of cytolysis, 3 h after cell exposure to different group II PLA(2)s purified from Bothrops asper, Atropoides nummifer, Cerrophidion godmani, and Bothriechis schlegelii venoms. In addition, susceptibility to lysis induced by synthetic melittin and group III PLA(2) from bee (Apis mellifera) venom, as well as by anionic, cationic, and neutral detergents, was comparatively evaluated on the two cultures. Myotubes were significantly more susceptible to group II PLA(2) myotoxins, but not to the other agents tested, under the same conditions. Moreover, the increased susceptibility of myotubes over myoblasts was also demonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA(2) myotoxins, that reproduce the action of their parent proteins. These results indicate that fusion and differentiation of myoblasts into myotubes induce changes that render these cells more susceptible to the toxic mechanism of group II PLA(2) myotoxins, but not to general perturbations of membrane homeostasis. Such changes are likely to involve myotoxin acceptor site(s), which remain(s) to be identified.

Molecular evolution and structure-function relationships of crotoxin-like and asparagine-6-containing phospholipases A2 in pit viper venoms.

Some myotoxic or neurotoxic PLA2s (phospholipases A2) from pit viper venoms contain characteristic N6 substitutions. Our survey of the venoms of more than ten pit viper genera revealed that N6-PLA2s exist only in limited Asian pit vipers of two genera, Protobothrops and Gloydius, and exist as either monomers or the basic subunits of heterodimers in some New World pit vipers. For the newly identified N6-PLA2s, the neuromuscular blocking activities were assayed with the chick biventer cervicis neuromuscular tissue, whereas the increased serum creatine kinase level assessed their myotoxicities. The purified N6-PLA2s from Protobothrops mangshanensis and Gloydius intermedius saxatilis were found to be presynaptic neurotoxins. In contrast, all N6-PLA2s from the venoms of Sistrurus miliarius strackeri, S. m. barbouri, Crotalus viridis viridis, C. lepidus lepidus, Cerrophidion godmani and Bothreichis schlegelii were myotoxins without neurotoxicity even in the presence of crotoxin A. Crotoxin-like complexes were for the first time purified from the venoms of Sitrurus catenatus tergeminus, C. mitchelli mitchelli, C. horridus atricaudatus, C. basiliscus and C. durissus cumanensis. The cDNAs encoding six novel N6-PLA2s and subunits of the crotoxin-like complex from S. c. tergeminus were cloned and fully sequenced. Phylogeny analysis showed that two structural subtypes of N6-PLA2s with either F24 or S24 substitution have been evolved in parallel, possibly descended respectively from species related to present-day Protobothrops and Gloydius. Calmodulin binds all the N6-PLA2s but crotoxin A may inhibit its binding to crotoxin B and to other neurotoxic N6-PLA2s. Structure-activity relationships at various regions of the PLA2 molecules were extensively discussed.

Inhibitory effect of fucoidan on the activities of crotaline snake venom myotoxic phospholipases A 2

Myotoxic phospholipases A 2 account for most of the muscle necrosis that results from envenenomation by crotaline snakes. In this study, we investigated the protective effect of fucoidan, a natural sulfated polysaccharide obtained from the brown seaweed Fucus vesiculosus, against the cytotoxic and myotoxic activities of a group of phospholipase A 2 myotoxins from crotaline snake venoms: Bothrops asper myotoxins I, II, III, and IV, Cerrophidion godmani myotoxins I and II, Atropoides nummifer myotoxins I and II, and Bothriechis schlegelii myotoxin I. All of the toxins tested were efficiently inhibited by fucoidan, in both their cytotoxic and myotoxic effects. The basis for this inhibition appears to be the rapid formation of complexes between fucoidan and myotoxins, as evidenced by turbidimetric analysis. The possible binding site of fucoidan on the myotoxins was investigated using short synthetic peptides that represent the membrane-damaging region (residues 115–129) for three of these toxins. Fucoidan clearly inhibited the cytolytic activity of the peptides, indicating its ability to interact with the C-terminal myotoxic region of these phospholipases A 2. Fucoidan significantly inhibited muscle damage in mice, when administered locally, immediately after experimental envenomation with crude venom from B. asper. These results encourage further studies of sulfated fucans as compounds of potential use to improve the treatment of envenomations by crotaline snakes.

Structural characterization and phylogenetic relationships of myotoxin II from Atropoides ( Bothrops) nummifer snake venom, a Lys49 phospholipase A 2 homologue

In order to analyze its structure–function relationships, the complete amino acid sequence of myotoxin II from Atropoides ( Bothrops) nummifer from Costa Rica was determined. This toxin is a Lys49-type phospholipase A 2 (PLA 2) homologue, devoid of catalytic activity, structurally belonging to class IIA. In addition to the Asp49→Lys change in the (inactive) catalytic center, substitutions in the calcium-binding loop suggest that its lack of enzymatic activity is due to the loss of ability to bind Ca 2+. The toxin occurs as a homodimer of basic subunits of 121 residues. Its sequence has highest similarity to Lys49 PLA 2s from Cerrophidion, Trimeresurus, Bothrops and Agkistrodon species, which form a subfamily of proteins that diverged early from Asp49 PLA 2s present in the same species, as shown by phylogenetic analysis. The tertiary structure of the toxin was modeled, based on the coordinates of Cerrophidion godmani myotoxin II. Its exposed C-terminal region 115–129 shows several differences in comparison to the homologous sequences of other Lys49 PLA 2s, i.e. from Agkistrodon p. piscivorus and Bothrops asper. Region 115–129 of the latter two proteins has been implicated in myotoxic activity, on the basis of the direct membrane-damaging of their corresponding synthetic peptides. However, peptide 115–129 of A. nummifer myotoxin II did not exert toxicity upon cultured skeletal muscle cells or mature muscle in vivo. Differences in several amino acid residues, either critical for toxicity, or influencing the conformation of free peptide 115–129 from A. nummifer myotoxin II, may account for its lack of direct membrane-damaging properties.

Immunochemical properties of the N-terminal helix of myotoxin II, a lysine-49 phospholipase A 2 from Bothrops asper snake venom

Myotoxic class II phospholipases A 2 from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/cationic region, near the C-terminus of myotoxin II (115–129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1–15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A 2, showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins ( Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus flavoviridis basic protein I) were not recognized by the antibodies, revealing a significant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with affinity-purified antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These findings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A 2, regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clarified.

Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties

Myotoxic phospholipases A(2) (PLA(2)s; group II) account for most of the muscle-tissue damage that results from envenomation by viperid snakes. In the venom of the Godman's viper (Cerrophidion godmani, formerly Bothrops godmani), an enzymically active PLA(2) (myotoxin I) and an inactive, Lys-49 variant (myotoxin II) induce extensive muscle damage and oedema. In this study, two distinct myotoxin inhibitor proteins of C. godmani, CgMIP-I and CgMIP-II, were purified directly from blood plasma by selective binding to affinity columns containing either myotoxin I or myotoxin II, respectively. Both proteins are glycosylated, acidic (pI=4) and composed of 20-25-kDa subunits that form oligomers of 110 kDa (CgMIP-I) or 180 kDa (CgMIP-II). In inhibition studies, CgMIP-I specifically neutralized the PLA(2) and the myotoxic, oedema-forming and cytolytic activities of myotoxins I, whereas CgMIP-II selectively inhibited the toxic properties of myotoxin II. N-terminal amino acid sequence analysis and sequencing of cDNAs encoding the two inhibitors revealed that CgMIP-I is similar to gamma-type inhibitors, which share a pattern of cysteine residues present in the Ly-6 superfamily of proteins, whereas CgMIP-II shares sequence identity with alpha-type inhibitors that contain carbohydrate-recognition-like domains, also found in C-type lectins and mammalian PLA(2) receptors. N-terminal sequencing of myotoxin I revealed a different primary structure from myotoxin II [De Sousa, Morhy, Arni, Ward, Díaz and Gutiérrez (1998) Biochim. Biophys. Acta 1384, 204-208], which provides insight into the nature of such pharmacological specificity.

Cerrophidion godmani in Costa Rica: A Case of Extremely Low Allozyme Variation?

Cerrophidion godmani in Costa Rica: A Case of Extremely Low Allozyme Variation?

Reassessment of Phylogenetic Relationships among Pitviper Genera Based on Mitochondrial Cytochrome b Gene Sequences

Crotalines are biomedically important, venomous snakes that are widely distributed across Asia and the Western Hemisphere. Phylogenetic relationships and contingent taxonomy of these pitvipers, however, remain problematic. This has been particularly true for snakes of the moccasin complex of the tribe Agkistrodontini (Gloyd and Conant, 1990). Species of this group were assigned to the genus Agkistrodon (senso lato), first described by de Beauvois (1799) but more recently have been divided into three Asian genera (Calloselasma, Deinagkistrodon, and Hypnale) and a more narrowly redefined Agkistrodon (Gloyd, 1979). This general division is supported by immunological evidence (Cadle, 1992; Minton, 1990). The redefined genus Agkistrodon remains controversial. Morphological evidence led Hoge and Romano-Hoge (1981) to group Old World Agkistrodon species in a separate genus, which they named Gloydius. Cranial musculoskeletal data were interpreted by Goombridge (1986) as favoring recognition of Gloydius, whereas Kardong (1990) viewed the same data as compatible with retention of Agkistrodon for Old World species. Using osteological characters, Brattstrom (1964) supported a monophyletic Agkistrodon and suggested that the bushmaster, Lachesis muta, and the rattlesnakes (genera Crotalus and Sistrurus) formed a monophyletic group. Finally, molecular data (Knight et al., 1992) indicate that the genus Agkistrodon, as defined by Gloyd (1979), is paraphyletic; restriction fragment length polymorphism (RFLP) of mitochondrial DNA and 16S ribosomal RNA sequences group New World species of Agkistrodon with the New World rattlesnake, Crotalus molossus, and the tropical New World pit viper, Cerrophidion godmani, rather than with Old World species of Agkistrodon. In this study, we used mitochondrial cytochrome b gene sequences to test two phylogenetic hypotheses: (1) the genus Agkistrodon as presently defined is monophyletic; and (2) the bushmaster, Lachesis muta, and the rattlesnakes, represented by Crotalus viridis, constitute a monophyletic clade.

Chemical modification of histidine and lysine residues of myotoxic phospholipases A 2 isolated from Bothrops asper and Bothrops godmani snake venoms: Effects on enzymatic and pharmacological properties

Lysine and histidine residues of two myotoxic phospholipases A 2, Bothrops asper myotoxin III and Bothrops godmani myotoxin I, were chemically modified in order to study the effects of these treatments on enzymatic and pharmacological properties. After lysine acetylation the overall basicity of these toxins was lost and their enzymatic activity was significantly reduced, although a residual effect remained, which corresponded to 25% of the activity of native toxins. This treatment abolished both myotoxic and anticoagulant effects, and partially reduced liposome-disrupting activity. Histidine alkylation with P-bromophenacyl bromide affected phospholipase A 2, myotoxic and anticoagulant effects in a parallel way. After 24 hr of incubation with the alkylating reagent, these three activities were totally inhibited, in contrast to the liposome-disrupting effect which was only partially affected by this treatment. It is suggested that: (1) catalytic activity plays a role in the pharmacological effects of these myotoxins; (2) lysine residues are relevant for the toxic effects induced by these phospholipases A 2; and (3) despite the apparent relevance of enzymatic activity to the pharmacological properties of these toxins, the dissociation observed in lysine acetylation experiments suggests that these myotoxins have a molecular region, different from the catalytic site, which might be also involved in the toxic effects observed.

Isolation and characterization of basic myotoxic phospholipases A 2 from Bothrops godmani (Godman's pit viper) snake venom

Two basic myotoxic phospholipases A 2 were purified to homogeneity from the venom of Bothrops godmani from Costa Rica by ion-exchange chromatography on CM-Sephadex. They have molecular weights of 14,300 (myotoxin I) and 13,400 (myotoxin II) and isoelectric points of 8.2 (myotoxin I) and 8.9 (myotoxin II). They behave as amphiphilic proteins in charge-shift electrophoresis and have similar amino acid compositions. Both toxins induce drastic myotoxic effects when injected in the gastrocnemius muscle of mice and induce release of peroxidase trapped in negatively charged liposomes. In addition, myotoxin I has high phospholipase A 2 activity and is anticoagulant at doses higher than 0.3 μg/ml, whereas myotoxin II has a very low phospholipase A 2 activity and exerts anticoagulant effect only at concentrations higher than 50 μg/ml. Immunochemical data indicate that both toxins are immunologically related to Bothrops asper myotoxins, although B. godmani myotoxin II gives a stronger cross-reactivity when tested with antisera raised against B. asper myotoxins I and II.

Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper)

B. Lomonte, G. Rojas, J. M. Gutíerrez and G. Ramírez. Isolation of a galactose-binding lectin from the venom of the snake Bothrops godmani (Godmann's pit viper). Toxicon 28, 75–81, 1990.—A galactose-binding lectin, isolated from the venom of B. godmani by affinity chromatography. is an acidic protein (pI 4.9) with a subunit mol. wt of about 14,000, occurring mostly as a disulfide-linked dimer of 28,000. A small proportion of lectin appears as a monomer and as a tetramer. The lectin agglutinates erythrocytes from mice, rabbit, cow and human (all ABO types, either Rh positive or negative), but does not agglutinate horse, sheep, goat and snake ( Oxybelis aeneus, Colubridae) erythrocytes. The agglutinating activity is inhibited by 1 mM EDTA. The lectin is devoid of lethal, hemorrhagic, myotoxic, proteolytic and phospholipase A 2 activities. It is not mitogenic for human peripheral blood mononuclear cells. The only effect observed was a moderate induction of edema in the footpad of mice, with a minimal edema-forming dose of 22 μg. This effect developed rapidly, and was significantly inhibited by i.p. administration of cyproheptadine, a histamine and serotonin antagonist, before injection of the lectin. Despite the edema-forming activity observed, the low concentration of lectin in crude venom, together with its relatively low potency, suggest that this lectin is not a key component in the development of edema following envenomations by B. godmani.