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Venoms of the New World Vinesnakes (Oxybelis aeneus and O. fulgidus)
Abstract
Species of Oxybelis are extremely elongate arboreal snakes that are broadly distributed in the Americas, from extreme southeastern Arizona (USA) to central South America. Primarily feeding on lizards and birds, Oxybelis venoms are poorly known in general, but a prominent taxon-specific three-finger toxin (fulgimotoxin) was isolated from and is a prominent component of O. fulgidus venom; a homolog is also present in O. aeneus venom. As part of ongoing characterization of venoms from rear-fanged snakes, we describe here the composition of two broadly distributed species, O. aeneus and O. fulgidus. Venom proteomes were of very low complexity, and four protein families (LAAO, PIII SVMP, CRiSP and 3FTx) account for more than 90% of total protein composition. Venoms from both species are moderately toxic to mice and to Hemidactylus geckos, but they are nearly an order of magnitude more toxic to Anolis lizards (a native prey species). These results reflect a trend in colubrid venom composition that is becoming increasingly more common: the presence of taxon-specific toxins, specifically three-finger toxins, preferentially targeting lizards and/or birds.
... We sequenced the venom gland transcriptome of individuals of four colubrine species: Chironius multiventris, Oxybelis aeneus, Rhinobothryum bovallii, and Spilotes sulphureus. Of the four species studied in this paper, three have had previous venom proteomic or transcriptomic studies where 3FTxs were the most abundant protein family [13,27,28]. C. multiventris had no previous comparable studies. ...
... The highest-ranking 3FTx transcript had more than one order of magnitude greater relative abundance (FPKM) than the highest-ranking non-3FTx transcript ( Figure 2 The simple venom gene expression of 3FTx profiles of our snakes (Tables S6-S10) are consistent with transcriptomic and proteomic data in previously analyzed conspecifics [13,28] and other rear-fanged colubrines [7,8,29,30]. Studies of Oxybelis species show a simple venom proteome comprising 3FTXs (including fulgimotoxins from Oxybelis fulgidus), SVMP III, L-amino acid oxidase, and CRiSP [27]. Although we recovered members of SVMP III, L-amino acid oxidase, and CRiSP from our sample of O. aeneus, the majority of toxin transcripts produced by this individual were 3FTxs ( Figure 2). ...
Snake venom research has historically focused on front-fanged species (Viperidae and Elapidae), limiting our knowledge of venom evolution in rear-fanged snakes across their ecologically diverse phylogeny. Three-finger toxins (3FTxs) are a known neurotoxic component in the venoms of some rear-fanged snakes (Colubridae: Colubrinae), but it is unclear how prevalent 3FTxs are both in expression within venom glands and more broadly among colubrine species. Here, we used a transcriptomic approach to characterize the venom expression profiles of four species of colubrine snakes from the Neotropics that were dominated by 3FTx expression (in the genera Chironius, Oxybelis, Rhinobothryum, and Spilotes). By reconstructing the gene trees of 3FTxs, we found evidence of putative novel heterodimers in the sequences of Chironius multiventris and Oxybelis aeneus, revealing an instance of parallel evolution of this structural change in 3FTxs among rear-fanged colubrine snakes. We also found positive selection at sites within structural loops or “fingers” of 3FTxs, indicating these areas may be key binding sites that interact with prey target molecules. Overall, our results highlight the importance of exploring the venoms of understudied species in reconstructing the full evolutionary history of toxins across the tree of life.
... No neurologic complication was observed during his hospitalization. Both hemotoxic and neurotoxic proteins such as L-amino acid oxidase (LAAO), PIII-SVMP, cysteine-Rich Secretory Proteins (CRiSP), and three-finger toxins (3FTx, fulgimotoxin) were identified in the venom of O. fulgidus [60,61]. ...
... Like Viperiade snakes, SVMP is one of the main toxins in the venom, which was discovered in several colubrid species including Dispholidus typus [64], Thamnodynastes strigatus [65] and Philodryas species [66]. In addition to SVMP, other toxins such as serine proteases, LAAOs, PLA 2 , C-type lectin-like proteins and 3FTx were identified in colubrid snakes [60,61,67]. Although no clinical case of cerebral complications was reported by colubrid bite due to a tiny amount of envenoming, an animal model of venom (Philodryas patagoniensis) injection demonstrates the hemotoxic features of the venom, leading to multiple hemorrhages in the cerebellum and cerebrum [68]. ...
There are an estimated 5.4 million snakebite cases every year. People with snakebite envenoming suffer from severe complications, or even death. Although some review articles cover several topics of snakebite envenoming, a review of the cases regarding cerebral complications, especially rare syndromes, is lacking. Here, we overview 35 cases of snakebite by front-fanged snakes, including Bothrops, Daboia, Cerastes, Deinagkistrodon, Trimeresurus, and Crotalus in the Viperidae family; Bungarus and Naja in the Elapidae family, and Homoroselaps (rare cases) in the Lamprophiidae family. We also review three rare cases of snakebite by rear-fanged snakes, including Oxybelis and Leptodeira in the Colubridae family. In the cases of viper bites, most patients (17/24) were diagnosed with ischemic stroke and intracranial hemorrhage, leading to six deaths. We then discuss the potential underlying molecular mechanisms that cause these complications. In cases of elapid bites, neural, cardiac, and ophthalmic disorders are the main complications. Due to the small amount of venom injection and the inability to deep bite, all the rear-fanged snakebites did not develop any severe complications. To date, antivenom (AV) is the most effective therapy for snakebite envenoming. In the six cases of viper and elapid bites that did not receive AV, three cases (two by viper and one by elapid) resulted in death. This indicates that AV treatment is the key to survival after a venomous snakebite. Lastly, we also discuss several studies of therapeutic agents against snakebite-envenoming-induced complications, which could be potential adjuvants along with AV treatment. This article organizes the diagnosis of hemotoxic and neurotoxic envenoming, which may help ER doctors determine the treatment for unidentified snakebite.
... When considered a single species, Oxybelis aeneus was thought to be a specialised lizard predator, occasionally preying on birds (Keiser Jr., 1967, 1975Henderson, 1982;Wilson and Cruz-Díaz, 1993;Lee, 1996;Campbell, 1998;Marques et al., 2001Marques et al., , 2005Marques et al., , 2015Marques et al., , 2017Savage, 2002;Argôlo, 2004;Diener, 2007;Fraga et al., 2013;Castro and Silva-Soares, 2016;Heyborne and Mackessy, 2021;Oliveira-Souza et al., 2021). However, some records in the literature also included insects (Keiser Jr, 1967, 1975Campbell, 1998;Mesquita et al., 2012), fishes (Hetherington, 2006), anurans (Mole, 1924;Rohl, 1942Rohl, , 1956Beebe, 1946;Wehekind, 1955;Keiser Jr., 1967, 1975Hardy and McDiarmid, 1969;Amaral, 1977;Campbell, 1998;Argôlo, 2004;Mesquita et al., 2012;Fraga et al., 2013;Guedes et al., 2014;Sellmeijer and van den Burg, 2020) and even mammals (Villa, 1962;Keiser Jr, 1975;Campbell, 1998). ...
... Moreover, taxon-specific toxins specialized for different prey resources, ontogenetic shifts in venom composition, and geographic variation are now known to occur in NFFS venoms (e.g., Heyborne and Mackessy, 2021;Hofmann et al., 2021;Mackessy et al., 2006;Modahl et al., 2018b). Unfortunately, NFFSs remain underappreciated in toxinological studies despite representing the majority of extant snake diversity-with over 2400 species (Uetz, 2010;Uetz et al., 2020;Uetz & Stylianou, 2018;Zaher et al., 2019). ...
This thesis can be downloaded from: https://tigerprints.clemson.edu/all_theses/3913/
--------------------------------- The material of this thesis has been published in the following article: Schramer, T. D., Rautsaw, R. M., Bayona Serrano, J. D., Nystrom, G. S., West, T. R., Ortiz-Medina, J. A., Sabido-Alpuche, B., Meneses-Millán, M., Borja, M., Junqueira de Azevedo, I. L. M., Rokyta, D. R., & Parkinson, C. L. (2022). An integrative view of the toxic potential of Conophis lineatus (Dipsadidae: Xenodontinae), a medically relevant rear-fanged snake. Toxicon, 205(2022), 38–52. https://doi.org/10.1016/j.toxicon.2021.11.009
... Moreover, taxon-specific toxins specialized for different prey resources, ontogenetic shifts in venom composition, and geographic variation are now known to occur in NFFS venoms (e.g., Heyborne and Mackessy, 2021;Hofmann et al., 2021;Mackessy et al., 2006;Modahl et al., 2018b). Unfortunately, NFFSs remain underappreciated in toxinological studies despite representing the majority of extant snake diversity-with over 2400 species (Uetz, 2010;Uetz et al., 2020;Uetz and Stylianou, 2018;Zaher et al., 2019). ...
[https://doi.org/10.1016/j.toxicon.2021.11.009] Most traditional research on snake venoms has focused on front-fanged snake families (Viperidae, Elapidae, and Atractaspididae). However, venom is now generally accepted as being a much more broadly possessed trait within snakes, including species traditionally considered harmless. Unfortunately, due to historical inertia and methodological challenges, the toxin repertoires of non-front-fanged snake families (e.g., Colubridae, Dipsadidae, and Natricidae) have been heavily neglected despite the knowledge of numerous species capable of inflicting medically relevant envenomations. Integrating proteomic data for validation, we perform a de novo assembly and analysis of the Duvernoy's venom gland transcriptome of the Central American Road Guarder (Dipsadidae: Xenodontinae: Conophis lineatus), a species known for its potent bite. We identified 28 putative toxin transcripts from 13 toxin families in the Duvernoy's venom gland transcriptome, comprising 63.7% of total transcriptome expression. In addition to ubiquitous snake toxin families, we proteomically confirmed several atypical venom components. The most highly expressed toxins (55.6% of total toxin expression) were recently described snake venom matrix metalloproteases (svMMPs), with 48.0% of svMMP expression contributable to a novel svMMP isoform. We investigate the evolution of the new svMMP isoform in the context of rear-fanged snakes using phylogenetics. Finally, we examine the morphology of the venom apparatus using μCT and explore how the venom relates to autecology and the highly hemorrhagic effects seen in human envenomations. Importantly, we provide the most complete venom characterization of this medically relevant snake species to date, producing insights into the effects and evolution of its venom, and point to future research directions to better understand the venoms of ‘harmless’ non-front-fanged snakes.
The venom glands of reptiles, particularly those of front-fanged advanced snakes, must satisfy conflicting biological demands: rapid synthesis of potentially labile and highly toxic proteins, storage in the gland lumen for long periods, stabilization of the stored secretions, immediate activation of toxins upon deployment and protection of the animal from the toxic effects of its own venom. This dynamic system could serve as a model for the study of a variety of different phenomena involving exocrine gland activation, protein synthesis, stabilization of protein products and secretory mechanisms. However, these studies have been hampered by a lack of a long-term model that can be propagated in the lab (as opposed to whole-animal studies). Numerous attempts have been made to extend the lifetime of venom gland secretory cells, but only recently has an organoid model been shown to have the requisite qualities of recapitulation of the native system, self-propagation and long-term viability (>1 year). A tractable model is now available for myriad cell- and molecular-level studies of venom glands, protein synthesis and secretion. However, venom glands of reptiles are not identical, and many differ very extensively in overall architecture, microanatomy and protein products produced. This Review summarizes the similarities among and differences between venom glands of helodermatid lizards and of rear-fanged and front-fanged snakes, highlighting those areas that are well understood and identifying areas where future studies can fill in significant gaps in knowledge of these ancient, yet fascinating systems.
Snake venom research has focused on front-fanged venomous snakes because of the high incidence of human morbidity and mortality from envenomations and larger venom yields of these species, while venoms from rear-fanged snakes have been largely neglected. Rear-fanged snakes (RFS) are a phylogenetically diverse collection of species that feed on a variety of prey and show varying prey capture strategies, from constriction to envenomation. In general, RFS venoms share many toxin families with front-fanged snakes, and venoms generally are either a neurotoxic three-finger toxin (3FTx)-dominated venom or an enzymatic metalloproteinase-dominated venom. These venoms have also been discovered to contain several unique venom protein families. New venom protein superfamilies in RFS venoms include matrix metalloproteinases, distinct from but closely related to snake venom metalloproteinases, veficolins, and acid lipases. Specialized three-finger toxins that target select prey taxa have evolved in some RFS venoms, and this prey capture strategy has appeared in multiple RFS species, from Old World Boiga to New World Spilotes and Oxybelis. Though this same protein superfamily is commonly found in the venoms of elapid (front-fanged) snakes, no elapid 3FTxs appear to show prey-specific toxicity (with the exception of perhaps Micrurus). Neofunctionalization of Spilotes sulphureus 3FTx genes has even resulted in the evolution within a single venom of 3FTxs selectively neurotoxic to different prey taxa (mammals or lizards), allowing this non-constricting RFS to take larger mammalian prey. The large number of 3FTx protein sequences available, together with a growing database of RFS venom 3FTxs, make possible predictions concerning structure-function relationships among these toxins and the basis of selective toxicity of specific RFS venom 3FTxs. Rear-fanged snake venoms are therefore of considerable research interest due to the evolutionary novelties they contain, providing insights into the evolution of snake venom proteins and potential predator-prey coevolution in a broader phylogenetic context. Because of the limited complexity of these venoms, they represent a more tractable source to inform about the biological roles of specific venom proteins that are found in the venoms of this rich diversity of snakes.
Abstract A case of a bite inflicted by Oxybelis fulgidus in the wilds of Amazon is reported. The patient was a 67-year-old man who presented with dizziness, tachycardia and local pain, with erythema and bleeding in his left arm. The venom of Oxybelis fulgidus, a neotropical rear-fanged snake, contains one of the four three-finger toxins already isolated from colubrid snakes, called fulgimotoxin. He was treated with oral analgesics and returned to the ambulatory in 48 hours, with good evolution. We report tirst authenticated case of adult Oxybelis fulgidus with signs of mild local envenoming without evidence of systemic envenoming.
The bird Catharus occidentalis (Russet Nightingale-Thrush) is reported for the first time in the diet of the snake Oxybelis fulgidus, in Oaxaca, Mexico.
Snake venoms represent an adaptive trophic response to the challenges confronting a limbless predator for overcoming combative prey, and this chemical means of subduing prey shows several dominant phenotypes. Many front-fanged snakes, particularly vipers, feed on various vertebrate and invertebrate prey species, and some of their venom components (e.g., metalloproteinases, cobratoxin) appear to have been selected for "broad-brush" incapacitation of different prey taxa. Using proteomic and genomic techniques, the compositional diversity of front-fanged snakes is becoming well characterized; however, this is not the case for most rear-fanged colubroid snakes. Because these species consume a high diversity of prey, and because venoms are primarily a trophic adaptation, important clues for understanding specific selective pressures favoring venom component composition will be found among rear-fanged snake venoms. Rear-fanged snakes typically (but not always) produce venoms with lower complexity than front-fanged snakes, and there are even fewer dominant (and, arguably, biologically most relevant) venom protein families. We have demonstrated taxon-specific toxic effects, where lizards and birds show high susceptibility while mammals are largely unaffected, for both Old World and New World rear-fanged snakes, strongly indicating a causal link between toxin evolution and prey preference. New data are presented on myotoxin a, showing that the extremely rapid paralysis induced by this rattlesnake toxin is specific for rodents, and that myotoxin a is ineffectual against lizards. Relatively few rear-fanged snake venoms have been characterized, and basic natural history data are largely lacking, but directed sampling of specialized species indicates that novel compounds are likely among these specialists, particularly among those species feeding on invertebrate prey such as scorpions and centipedes. Because many of the more than 2200 species of colubroid snakes are rear-fanged, and many possess a Duvernoy's venom gland, understanding the nature of their venoms is foundational to understanding venom evolution in advanced snakes.
Snake venoms have been subjected to increasingly sensitive analyses for well over 100 years, but most research has been restricted to front-fanged snakes, which actually represent a relatively small proportion of extant species of advanced snakes. Because rear-fanged snakes are a diverse and distinct radiation of the advanced snakes, understanding venom composition among “colubrids” is critical to understanding the evolution of venom among snakes. Here we review the state of knowledge concerning rear-fanged snake venom composition, emphasizing those toxins for which protein or transcript sequences are available. We have also added new transcriptome-based data on venoms of three species of rear-fanged snakes. Based on this compilation, it is apparent that several components, including cysteine-rich secretory proteins (CRiSPs), C-type lectins (CTLs), CTLs-like proteins and snake venom metalloproteinases (SVMPs), are broadly distributed among “colubrid” venoms, while others, notably three-finger toxins (3FTxs), appear nearly restricted to the Colubridae (sensu stricto). Some putative new toxins, such as snake venom matrix metalloproteinases, are in fact present in several colubrid venoms, while others are only transcribed, at lower levels. This work provides insights into the evolution of these toxin classes, but because only a small number of species have been explored, generalizations are still rather limited. It is likely that new venom protein families await discovery, particularly among those species with highly specialized diets.
Cysteine-rich secretory proteins (CRISPs) are found in a wide variety of animal tissues, particularly the epididymis of mammals, and most reptile venoms appear to contain at least one isoform. Although several venom CRISPs have been assigned specific functions, many have not, and the biological significance of this family of proteins in venoms is not clear. In many colubrid venoms, they are major protein constituents, suggesting that they have an important role in envenomation. Like many other families of reptile toxins, CRISPs show a highly conserved molecular scaffold, and the sixteen cysteines and eight disulfides they form are 100% conserved. Because they are widely distributed among reptile venoms, show structural conservation, and many have been sequenced, they may have utility as phylogenetic markers. In general, venom CRISP relationships reflect established phylogenetic relationships among the species from which they are derived. By analogy with the three-finger toxins of reptile venoms, which also have a highly conserved protein scaffold stabilized by disulfides, one can expect that venom CRISPs will also show myriad pharmacological activities. Future efforts should be directed toward the elucidation of these activities, as they are an excellent protein family for structure-activity studies.
Largely because of their direct, negative impacts on human health, the venoms of front-fanged snakesof the families Viperidae and Elapidae have been extensively characterized proteomically, transcriptomically,and pharmacologically. However, relatively little is known about the molecular complexityand evolution of the venoms of rear-fanged colubrid snakes, which are, with a few notable exceptions,regarded as harmless to humans. Many of these snakes have venoms with major effects on their preferredprey, and their venoms are probably as critical to their survival as those of front-fanged elapidsand viperids.
We sequenced the venom-gland transcriptomes from a specimen of Hypsiglena (Desert Night Snake;family Colubridae, subfamily Dipsadinae) and of Boiga irregularis (Brown Treesnake; family Colubridae,subfamily Colubrinae) and verified the transcriptomic results proteomically by means ofhigh-definition mass spectrometry. We identified nearly 3,000 nontoxin genes for each species. ForB. irregularis, we found 108 putative toxin transcripts in 46 clusters with <1% nucleotide divergence,and for Hypsiglena we identified 79 toxin sequences that were grouped into 33 clusters. Comparisonsof the venoms revealed divergent venom types, with Hypsiglena possessing a viper-like venom dominatedby metalloproteinases, and B. irregularis having a more elapid-like venom, consisting primarilyof three-finger toxins.
Despite the difficulty of procuring venom from rear-fanged species, we were able to complete all analysesfrom a single specimen of each species without pooling venom samples or glands, demonstratingthe power of high-definition transcriptomic and proteomic approaches. We found a high level of divergencein the venom types of two colubrids. These two venoms reflected the hemorrhagic/neurotoxicvenom dichotomy that broadly characterizes the difference in venom strategies between elapids andviperids.
Animal venoms represent a diverse source of potentially valuable therapeutic compounds due to the high specificity and the potent biological activity of many toxins. Snake venom toxins, particularly disintegrins and proteases from viper venoms, have yielded therapeutics with anti-cancer and hemostatic dysfunction activities. However, venoms from rear-fanged ''colubrid'' snakes have rarely been analyzed from the perspective of potential lead compound development. Here, we discuss recent progress in the analysis of these venoms, focusing on several studies of specific venom components as well as transcriptomic and proteomic surveys. Currently available –omic technologies largely circumvent the problematic low venom yields of most rear-fanged snakes, and because their basic biology is often very different from the well-studied front-fanged snakes, there is great potential for novel compound discovery in their venoms.
The concept of "sensory drive' suggests that animal sensory systems, which have evolved to allow animals to function effectively in their environments, act as a directional selective force in the evolution of animal signals. This ideas is explored by examining the visual system and the visual displays of anoline lizards. The design of the visual system and its response to movements in the environment are explored. Display form (temporal pattern, amplitude, acceleration, velocity) depends on the response patterns of the visual system, the specific context in which a display is given, and the habituating effects of background vegetation motion. Motion patterns in other vertebrates designed to draw attention (eg alarm signals), exhibit some of the same properties found in Anolis displays. Cryptic motion patterns, which have a low probability of eliciting the attention of anoles, are found in the stalking behavior of a specilaized Anolis predator, the vine snake Oxybelis aeneus. -from Author
The Brown Vine snake,Oxybelis aeneus(Wagler, 1824), is widely distributed from the United States to Argentina. 113 specimens encountered in the field and 39 individuals from scientific collections were analyzed and we determined patterns of diet, habitat use, and daily and seasonal activity.Oxybelis aeneuscan be found to be active year round, especially during the dry and warmer months. Daily temperature poorly describes its activity as it seems to be more active around 31 to 35°C. During the hours of inactivity the species tends to chose higher branches than when active. The snakes choose spiny trees as retreat sites and spineless trees as foraging sites. We suggest that the high abundance of O. aeneus makes it an appropriate model organism for studies on activity patterns of snakes in semi-arid environments.
Trophic relationships and foraging strategies are examined in two New World arboreal, diurnal, snake communities: Leptophis mexicanus, Oxybelis aeneus, and O.fulgidus from Mexico's Yucatán Peninsula, and Uromacer catesbyi and U. oxyrhynchus from Isla Saona, República Dominicana. Active foragers (L. mexicanus and U. catesbyi) have similar body proportions and feed primarily on diurnally quiescent prey (hylid frogs, bird eggs). Sit-and-wait strategists (O. aeneus, O.fulgidus and U. oxyrhynchus) have similar body proportions and feed on diurnally active prey (primarily lizards). Active foragers take prey that is, on the average, larger than the prey of sit-and-wait foragers, but presumably, active foragers expend more energy in locating prey. Trophic niche breadth is widest among the active foragers and narrowest for the slender vine snakes (O. aeneus and U. oxyrhynchus). Trophic niche overlap values are low for species pairs that occur sympatrically (or syntopically) with the exception of O. aeneus-O. fulgidus, but O. fufgidus preys on a wider variety of lizard taxa, preys on birds (which are absent from the diet of O. aeneus), and takes prey items that are significantly larger in size. It seems likely that, among arboreal colubrids, the sit-and-wait foraging strategy is derived from one of active foraging.
Aspects of natural history of the vine snake Oxybelis fulgidus from the northern region of Brazil were inferred based on the analysis of 106 preserved specimens (55 males and 51 females), and from a review of records in the published literature. Males mature at smaller size than females. Differences in body length and shape were also found among adult females and males: adult females are larger in mean snout-vent length and have relatively larger heads and shorter tails. Females have an extended seasonal vitellogenic cycle from April to December (mainly throughout the dry season). Oviductal eggs and egg-laying were recorded from September to December, while births occurred from January to April. Fecundity varied from four to twelve eggs or vitellogenic follicles (n = 13), and was positively correlated with female body length. Testicle volume is significantly larger from February to July (mainly in April and May; end of the rainy season), however the deferent ducts diameter do not differ significantly throughout the year. We hypothesized that both males and females may have an associated reproductive pattern, both synchronized in the end of the rainy season. Oxybelis fulgidus feeds on lizards (mainly iguanian) and passerine birds. Apparently, females feed more frequently on birds than males.
Snake venoms are recognized here as a grossly under-explored resource in pharmacological prospecting. Discoveries in snake systematics demonstrate that former taxonomic bias in research has led to the neglect of thousands of species of potential medical use. Recent discoveries reveal an unexpectedly vast degree of variation in venom composition among snakes, from different species down to litter mates. The molecular mechanisms underlying this diversity are only beginning to be understood. However, the enormous potential that this resource represents for pharmacological prospecting is clear. New high-throughput screening systems offer greatly increased speed and efficiency in identifying and extracting therapeutically useful molecules. At the same time a global biodiversity crisis is threatening the very snake populations on which hopes for new venom-derived medications depend. Biomedical researchers, pharmacologists, clinicians, herpetologists, and conservation biologists must combine their efforts if the full potential of snake venom-derived medications is to be realized.
A new species of Oxybelis is described from Isla de Roatán, Honduras. It differs from all species of Oxybelis in its tan to golden yellow coloration, in having a higher number of subcaudals as well as in other details of color and scutellation. The new species is closely related to O. fulgidus and is thought to be derived from a fulgidus-like ancestor (i.e., is the sister species to fulgidus) that has been isolated in the area of its present range probably since the very late Pliocene. The presence of a yellow population of Oxybelis on Isla de Roatán, Honduras, has long been known (Keiser 1969). The status of this insular population has remained uncertain pending the acquisition of additional material (Keiser 1969, Wilson and Meyer 1985). We recently visited Isla de Roatán a number of times and obtained a large series of additional specimens. After examining this new material, and the previously known specimens from this island, we believe that this population represents an undescribed taxon, which should be known as.
Novel phenotypes are commonly associated with gene duplications and neofunctionalization, less documented are the cases of phenotypic maintenance through the recruitment of novel genes. Proteolysis is the primary toxic character of many snake venoms, and ADAM metalloproteinases, named Snake Venom Metalloproteinases (SVMPs), are largely recognized as the major effectors of this phenotype. However, by investigating original transcriptomes from 58 species of advanced snakes (Caenophidia) across their phylogeny, we discovered that a different enzyme, matrix metalloproteinase (MMP), is actually the dominant venom component in three tribes (Tachymenini, Xenodontini, and Conophiini) of rear-fanged snakes (Dipsadidae). Proteomic and functional analyses of these venoms further indicate that MMPs are likely playing an 'SVMP-like' function in the proteolytic phenotype. A detailed look into the venom-specific sequences revealed a new highly expressed MMP subtype, named snake venom MMP (svMMP), which originated independently on at least three occasions from an endogenous MMP-9. We further show that by losing ancillary non-catalytic domains present in its ancestors, svMMPs followed an evolutionary path toward a simplified structure during their expansion in the genomes, thus paralleling what has been proposed for the evolution of their Viperidae counterparts, the SVMPs. Moreover, we inferred an inverse relationship between the expression of svMMPs and SVMPs along the evolutionary history of Xenodontinae, pointing out that one type of enzyme may be substituting for the other, while the general (metallo)proteolytic phenotype is maintained. These results provide rare evidence on how relevant phenotypic traits can be optimized via natural selection on non-homologous genes, yielding alternate biochemical components.
Rear-fanged colubrid snakes include hundreds of species globally that possess a Duvernoy's venom gland and often one-several enlarged rear maxillary teeth. We investigated the venom proteome of the Central American Lyre Snake (Trimorphodon quadruplex), a moderate-sized rear-fanged colubrid snake and the southernmost Trimorphodon, using a bottom-up proteomic approach coupled with enzyme and inhibitor assays, cytotoxicity assays and lethal toxicity assays. Several enzymes uncommonly observed in colubrid venoms were purified and characterized further. Trimorphodon quadruplex has a rather low complexity venome, typical of many rear-fanged snakes, but its venom contains L-amino acid oxidase, phospholipase A2, and a dimeric 3FTx, and 3FTxs dominate the proteome. Its PLA2 is catalytically quite active, but it lacks myotoxicity or acute toxicity; LAAO exhibits conserved structure and appears to be highly labile. Several P-III metalloproteinases are present and hydrolyze azocasein and the α-subunit of fibrinogen but lack hemorrhagic activity. Trimorphodon quadruplex produces venom and retains constriction, utilizing both chemically-mediated and mechanical feeding modes.
Significance
We demonstrate that T. quadruplex venom proteins are similar to those found in front-fanged snake species are present but show different biological activities. Our results underscore the importance of considering the biological roles of venoms from more than a mammal-centric perspective.
Most colubrid snake venoms have been poorly studied, despite the fact that they represent a great resource for biological, ecological, toxinological and pharmacological research. Herein, we explore the venom delivery system of the Aesculapian False Coral Snake Erythrolamprus aesculapii as well as some biochemical and toxicological properties of its venom. Its Duvernoy's venom gland is composed of serous secretory cells arranged in densely packed secretory tubules, and the most striking feature of its fang is their double-curved shape, exhibiting a beveled bladelike appearance near the tips. Although E. aesculapii resembles elapid snakes of the genus Micrurus in color pattern, this species produces a venom reminiscent of viperid venoms, containing mainly tissue-damaging toxins such as proteinases. Prominent hemorrhage developed both locally and systemically in mice injected with the venom, and the minimum hemorrhagic dose was found to be 18.8 μg/mouse; the lethal dose, determined in mice, was 9.5 ± 3.7 μg/g body weight. This work has toxicological implications that bites to humans by E. aesculapii could result in moderately severe local (and perhaps systemic) hemorrhage and gives insight into future directions for research on the venom of this species.
High-throughput technologies were used to identify venom gland toxin expression and to characterize the venom proteomes of two rear-fanged snakes, Ahaetulla prasina (Asian Green Vine Snake) and Borikenophis portoricensis (Puerto Rican Racer). Sixty-nine complete toxin-coding transcripts from 12 venom protein superfamilies (A. prasina) and 50 complete coding transcripts from 11 venom protein superfamilies (B. portoricensis) were identified in the venom glands. However, only 18% (A. prasina) and 32% (B. portoricensis) of the translated protein isoforms were detected in the proteome of these venoms. Both venom gland transcriptomes and venom proteomes were dominated by P-III metalloproteinases. Three-finger toxins, cysteine-rich secretory proteins, and C-type lectins were present in moderate amounts, but other protein superfamilies showed very low abundances. Venoms contained metalloproteinase activity comparable to viperid snake venom levels, but other common venom enzymes were absent or present at negligible levels. Western blot analysis showed metalloproteinase and cysteine-rich secretory protein epitopes shared with the highly venomous Boomslang (Dispholidus typus). The abundance of metalloproteinases emphasizes the important trophic role of these toxins. Comprehensive, transcriptome-informed definition of proteomes and functional characterization of venom proteins in rear-fanged snake families help to elucidate toxin evolution and provide models for protein structure-function analyses.
Hybridization between divergent species can be analyzed to elucidate expression patterns of distinct parental characteristics, as well as to provide information about the extent of reproductive isolation between species. A known hybrid cross between two rattlesnakes with highly divergent venom phenotypes provided the opportunity to examine occurrence of parental venom characteristics in the F1 hybrids as well as ontogenetic shifts in the expression of these characters as the hybrids aged. Although venom phenotypes of adult rattlesnake venoms are known for many species, the effect of hybridization on phenotype inheritance is not well understood, and effects of hybridization on venom ontogeny have not yet been investigated. The current study investigates both phenomena resulting from the hybridization of a male snake with type I degradative venom, Crotalus oreganus helleri (Southern Pacific Rattlesnake), and a female snake with type II highly toxic venom, C. scutulatus scutulatus (Mojave Rattlesnake). SDS-PAGE, enzymology, Western blot and reversed phase HPLC (RP-HPLC) were used to characterize the venom of the C. o. helleri male, the C. s. scutulatus female and their two hybrid offspring as they aged. In general, Crotalus o. helleri × C. s. scutulatus hybrid venoms appeared to exhibit overlapping parental venom profiles, and several different enzyme activity patterns. Both hybrids expressed C. o. helleri father-specific myotoxins as well as C. s. scutulatus mother-specific Mojave toxin. Snake venom metalloprotease activity displayed apparent sex-influenced expression patterns, while hybrid serine protease activities were intermediate to parental activities. The C. s. scutulatus × C. o. helleri hybrid male's venom profile provided the strongest evidence that type I and type II venom characteristics are expressed simultaneously in hybrid venoms, as this snake contained distinctive characteristics of both parental species. However, the possibility of sex-influenced development of metalloprotease activity, as seen in the ontogenetic shifts of the hybrid female, may influence the levels of expression of both type I and type II characteristics in hybrid venoms. Ultimately, the chronological analysis of this known hybrid system reveals the most distinct characteristics that can be used in determining successful hybridization between snakes that follow the type I-type II trend in rattlesnake venom composition, namely the presence of metalloprotease activity and Mojave toxin.
This book is the first significant contribution to thoroughly examine the potential hazards associated with snakes of the former family, Colubridae. This family contained >65% of living snake species (approximately 3,000 taxa) and has recently been split into multiple families. Many of these snakes produce oral secretions that contain toxins and other biologically-active substances. A large variety of these snakes figure in the pet industry, yet little documented information or formal study of their potential medical importance has been published. Therefore, although the possible medical importance of many of these species has been subjected to speculation since the mid-nineteenth century, there is a limited amount of useful descriptive information regarding the real hazard (or lack thereof) of snakes belonging to this diverse, artificial family. There is a need for "one-stop shopping" offering information regarding their possible toxicity and clinical relevance as well as recommendations for medical management of their bites. This book is the first synthesis of this information and includes evidence-based risk assessment, hazard rankings and specific recommendations regarding important species, many common in captivity. Fills a gap in the toxinological, medical and herpetological literature by providing a comprehensive review of this entire assemblage of snakes, with particular attention given to their capacity, real or rumored, to cause harm to humans A patient-centered, evidence-based approach is applied to analyzing documented case reports of bites inflicted by approximately 100 species. Clinical management of medically significant bites from non-front-fanged colubroids is methodically reviewed, and specific recommendations are provided.
The polyphyletic family Colubridae contains approximately two-thirds of the described species of advanced snakes, and nearly half of these (∼700 species) produce a venom in a specialized cephalic gland, the Duvernoy's gland. Biochemical and pharmacological information is lacking for venoms of most species, and modest detailed information on venom composition is available for only a few species which represent a potential health threat to humans. However, colubrid venoms represent a vast source of novel compounds, and some toxins, such as the 20-26 kD CRISP-related venom proteins (helveprins), have only recently been identified in both colubrid and elapid/viperid venoms. Difficulties associated with extraction have been addressed, and it is now possible to obtain venom sufficient for many analyses from even small species. There appears to be a greater number of venom components shared among the colubrids and the front-fanged snakes than has been previously noted, and it is probable that as analytical methods improve, more similarities will emerge. It is clear that colubrid venoms are homologous with front-fanged snake venoms, but overall composition as well as biological role(s) of colubrid venoms may be quite different. Metallo- and serine proteases have been identified in several colubrid venoms, and phospholipase A2 is a more frequent component than has been previously recognized. Venom phosphodiesterase, acetylcholinesterase and prothrombin activator activities occur in some venoms, and postsynaptic neurotoxins and myotoxins have been partially characterized for venoms from several species. Some venoms show high toxicity toward inbred mice, and others are toxic to birds and/or frogs only. Because many colubrids feed on non-mammalian prey, lethal toxicity toward mice is likely only relevant as a measure of potential risk posed to humans. Development of a non-mammalian vertebrate animal model would greatly facilitate systematic comparisons of the pharmacology of colubrid venoms and their components, and such a model would be more appropriate for evaluation of colubrid venom toxicity. Proteomics has the potential to increase our understanding of these venoms rapidly, but classical approaches to toxinology can also contribute tremendously to this understudied field. As more colubrid venoms are analyzed, new compounds unique to colubrid venoms will be identified, and this work in turn will lead to a better understanding of the evolution and biological significance of snake venoms and venom components.
Unlabelled:
Here we describe and compare the venomic and antivenomic characteristics of both neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) venoms. Although both neonate and adult venoms contain unique components, similarities among protein family content were seen. Both neonate and adult venoms consisted of myotoxin, bradykinin-potentiating peptide (BPP), phospholipase A2 (PLA2), Zn(2+)-dependent metalloproteinase (SVMP), serine proteinase, L-amino acid oxidase (LAAO), cysteine-rich secretory protein (CRISP) and disintegrin families. Quantitative differences, however, were observed, with venoms of adults containing significantly higher concentrations of the non-enzymatic toxic compounds and venoms of neonates containing higher concentrations of pre-digestive enzymatic proteins such as SVMPs. To assess the relevance of this venom variation in the context of snakebite and snakebite treatment, we tested the efficacy of the common antivenom CroFab® for recognition of both adult and neonate venoms in vitro. This comparison revealed that many of the major protein families (SVMPs, CRISP, PLA2, serine proteases, and LAAO) in both neonate and adult venoms were immunodepleted by the antivenom, whereas myotoxins, one of the major toxic components of C. v. viridis venom, in addition to many of the small peptides, were not efficiently depleted by CroFab®. These results therefore provide a comprehensive catalog of the venom compounds present in C. v. viridis venom and new molecular insight into the potential efficacy of CroFab® against human envenomations by one of the most widely distributed rattlesnake species in North America.
Biological significance:
Comparative proteomic analysis of venoms of neonate and adult Prairie Rattlesnake (Crotalus viridis viridis) from a discrete population in Colorado revealed a novel pattern of ontogenetic shifts in toxin composition for viperid snakes. The observed stage-dependent decrease of the relative content of disintegrins, catalytically active D49-PLA2s, L-amino acid oxidase, and SVMPs, and the concomitant increase of the relative abundance of paralytic small basic myotoxins and ohanin-like toxin, and hemostasis-disrupting serine proteinases, may represent an age-dependent strategy for securing prey and avoiding injury as the snake switches from small ectothermic prey and newborn rodents to larger endothermic prey. Such age-dependent shifts in venom composition may be relevant for antivenom efficacy and treatment of snakebite. However, applying a second-generation antivenomics approach, we show that CroFab®, developed against venom of three Crotalus and one Agkistrodon species, efficiently immunodepleted many, but not all, of the major compounds present in neonate and adult C. v. viridis venoms.
Recent studies regarding the multiple origin of front-fanged venom delivery systems prompt a reconsideration of the role of venom delivery in ophidian history. Snakes are derived from lizards, which swallow large prey items by an inertial feeding sequence. Early snakes were poorly adapted for inertial feeding, but they may have taken advantage of their relatively rigid cranial structure to stun and immobilize their prey. Most henophidian snakes kill their prey by constriction, thereby involving the axial skeleton and musculature in both locomotion and feeding. Powerful constriction seems to preclude rapid locomotion. Henophidians dominated snake faunas until the mid-Tertiary, when caenophidian snakes suddenly underwent extensive radiation. The Duvernoy's gland, a serous gland occurring in most living colubrid snakes, may have provided the mechanism for uncoupling locomotor and feeding activities by providing a venomous secretion for the immobilization of prey. Thus, caenophidians were able to develop more rapid locomotor systems, which were especially advantageous during the mid-Tertiary expansion of open habitats. Duvernoy's gland is regarded as a characteristic of colubroid snakes. Although the gland is lost in some taxa (including certain constricting colubrids), in others it is hypertrophied and associated with additional morphological features in an integrated front-fanged venom delivery system. The multiple origin of the latter condition is not surprising, given the ancestral nature of the venom delivery system among colubroid snakes.
Snake venoms contain a variety of protein and peptide toxins, and the three-finger toxins (3FTxs) are among the best characterized family of venom proteins. The compact nature and highly conserved molecular fold of 3FTxs, together with their abundance in many venoms, has contributed to their utility in structure-function studies. Although many target the nicotinic acetylcholine receptor of vertebrate skeletal muscle, often binding with nanomolar Kds, several non-conventional 3FTxs show pronounced taxon-specific neurotoxic effects. Here we describe the purification and characterization of fulgimotoxin, a monomeric 3FTx from the venom of Oxybelis fulgidus, a neotropical rear-fanged snake. Fulgimotoxin retains the canonical 5 disulfides of the non-conventional 3FTxs and is highly neurotoxic to lizards; however, mice are unaffected, demonstrating that this toxin is taxon-specific in its effects. Analysis of structural features of fulgimotoxin and other colubrid venom 3FTxs indicate the presence of a "colubrid toxin motif" (CYTLY) and a second conserved segment (WAVK) found in Boiga and Oxybelis taxon-specific 3FTxs, both in loop II. Because specific residues in loop II conventional α-neurotoxic 3FTxs are intimately associated with receptor binding, we hypothesize that this loop, with its highly conserved substitutions, confers taxon-specific neurotoxicity. These findings underscore the importance of rear-fanged snake venoms for understanding the evolution of toxin molecules and demonstrate that even among well-characterized toxin families, novel structural and functional motifs may be found.
symptoms consistent with biochemical analyses, with numbness associated with the bite, coagulation abnormalities and essentially no tissue damage. Results suggest that the occurrence of potent neurotoxic component(s) in a venom minimizes prediges- tive components (metalloproteases). Further, concurrence of these functional com- ponents in the venom of an individual may be selected against, and highly toxic venom in both juvenile and adult C. o. concolor may represent a form of venom paedomorphosis.
From lizards to snakes, the trophic system of squamates exhibits at least six major modifications correlated with different feeding strategies. Beginning in lizards, these include 1) shift from tongue to jaws as the primary means of prey capture, accompanied by specialization of the tongue for chemoreception, and 2) increasing skull kineticism. These features continue into snakes along with 3) unilateral jaw displacement during swallowing accompanied by 4) increasing skull kineticism, 5) development of the cervical vertebrae into a lever system for launching the strike, 6) addition of sensory modalities (thermoreception) in some snakes, and in advanced snakes, 7) shift from mechanical to chemical means of predation. Many fundamental features elaborated into the highly kinematic and jaw-based feeding system of snakes actually appear first within lizards. However, the highly kinetic skull of snakes represents not so much an extrapolation of lizard kinesis, as it does a rebuilding, even redesign, of the skull to achieve its high level of kinesis.
Information on bites by snakes of the family Colubridae in Mexico and Central America is reviewed. Little is known of the biochemistry and pharmacology of the Duvernoy gland secretion (venom) of colubrids from this region, although some reports describe proteolytic, phosphodiesterase, phospholipase A2 and hemorrhagic activities. A search of published reports and an effort to obtain reliable unpublished information on colubrid snake bites in the region documented cases inflicted by species of the genera Conophis, Coniophanes, Crisantophis, Erythrolamprus, Pliocercus, Oxybelis and Dryadophis (=Mastigodryas). The following general pattern emerges from the analysis of these cases: 1) Bites occurred mainly in hands and fingers on people that frequently manipulate colubrids, i.e. herpetologists, herpetoculturists and people that take care of these snakes at museums, exhibits or zoos; and 2) In most cases, only mild local effects were described, i.e. pain, swelling and, in few cases, ecchymosis. In only one case by Erythrolamprus bizonus there was ecchymosis beyond the bitten region, whereas persistent bleeding at the bite site was reported in a Conophis lineatus case. No systemic alterations were described in any of the cases. Management of colubrid bites in Mexico and Central America includes cleaning and disinfection of the bitten area, together with administration of tetanus toxoid. In the case of local infection, antibiotics are administered. There is no experimental or clinical evidence supporting the use of Crotalinae antivenoms in these bites. Despite the lack of systemic alterations in the cases described, caution should be exercised when manipulating these snakes, and bitten people should be closely observed for the potential development of bleeding and coagulopathies, since these effects have been described in bites by colubrid snakes from other regions of the world.
The polyphyletic family Colubridae contains approximately two-thirds of the described species of advanced snakes, and nearly half of these (700 species) produce a venom in a specialized cephalic gland, the Duvernoy's gland. Biochemical and pharmacological information is lacking for venoms of most species, and modest detailed information on venom composition is available for only a few species which represent a potential health threat to humans. However, colubrid venoms represent a vast source of novel compounds, and some toxins, such as the 20–26 kD CRISP-related venom proteins (helveprins), have only recently been identified in both colubrid and elapid/viperid venoms. Difficulties associated with extraction have been addressed, and it is now possible to obtain venom sufficient for many analyses from even small species. There appears to be a greater number of venom components shared among the colubrids and the front-fanged snakes than has been previously noted, and it is probable that as analytical methods improve, more similarities will emerge. It is clear that colubrid venoms are homologous with front-fanged snake venoms, but overall composition as well as biological role(s) of colubrid venoms may be quite different. Metallo- and serine proteases have been identified in several colubrid venoms, and phospholipase A2 is a more frequent component than has been previously recognized. Venom phosphodiesterase, acetylcholinesterase and prothrombin activator activities occur in some venoms, and postsynaptic neurotoxins and myotoxins have been partially characterized for venoms from several species. Some venoms show high toxicity toward inbred mice, and others are toxic to birds and/or frogs only. Because many colubrids feed on non-mammalian prey, lethal toxicity toward mice is likely only relevant as a measure of potential risk posed to humans. Development of a non-mammalian vertebrate animal model would greatly facilitate systematic comparisons of the pharmacology of colubrid venoms and their components, and such a model would be more appropriate for evaluation of colubrid venom toxicity. Proteomics has the potential to increase our understanding of these venoms rapidly, but classical approaches to toxinology can also contribute tremendously to this understudied field. As more colubrid venoms are analyzed, new compounds unique to colubrid venoms will be identified, and this work in turn will lead to a better understanding of the evolution and biological significance of snake venoms and venom components.
A photometric method for determining acetylcholinesterase activity of tissue extracts, homogenates, cell suspensions, etc., has been described. The enzyme activity is measured by following the increase of yellow color produced from thiocholine when it reacts with dithiobisnitrobenzoate ion. It is based on coupling of these reactions: The latter reaction is rapid and the assay is sensitive (i.e. a 10 μ1 sample of blood is adequate). The use of a recorder has been most helpful, but is not essential. The method has been used to study the enzyme in human erythrocytes and homogenates of rat brain, kidney, lungs, liver and muscle tissue. Kinetic constants determined by this system for erythrocyte eholinesterase are presented. The data obtained with acetylthiocholine as substrate are similar to those with acetylcholine.
Rattlesnake venoms are complex biological products containing potentially autolytic components, and they provide a useful tool for the study of long-term maintenance of enzymes in a competent state, both in vivo and in vitro. To evaluate the stability of venom components, 15 aliquots of freshly extracted venom (from Crotalus molossus molossus) were subjected to 15 different temperature and storage conditions for 1 week and then lyophilized; conditions varied from storage at -80 degrees C (optimal preservation of activities) to dilution (1:24) and storage at 37 degrees C (maximal degradation potential). Effects of different storage conditions were evaluated using SDS-PAGE, metalloprotease zymogram gels, a cricket LD50 assay and enzyme assays (metalloprotease, serine proteases, phosphodiesterase, L-amino acid oxidase and phospholipase A2). Venom samples were remarkably refractive to widely varying conditions; enzyme activities of some samples were variable, particularly L-amino acid oxidase, and one sample treatment showed higher toxicity, but electrophoretic results indicated very little effect on venom proteins. This study suggests that most venom activities should remain stable even if stored or collected under potentially adverse conditions, and freezing samples is not necessarily advantageous. Proteins in the crude venom are not as labile as has been previously thought, and endogenous mechanisms present in the venoms likely inhibit autolysis during long-term storage that occurs in vivo in the gland.
"Doctoral dissertation, Louisiana State University, 1967: A monographic study of the neotropical vine snake, Oxybelis aenus (Wagler)." Literature cited: p. 45-51.
A novel heterodimeric three-finger neurotoxin, irditoxin, was isolated from venom of the brown treesnake Boiga irregularis (Colubridae). Irditoxin subunit amino acid sequences were determined by Edman degradation and cDNA sequencing. The crystal structure revealed two subunits with a three-finger protein fold, typical for "nonconventional" toxins such as denmotoxin, bucandin, and candoxin. This is the first colubrid three-finger toxin dimer, covalently connected via an interchain disulfide bond. Irditoxin showed taxon-specific lethality toward birds and lizards and was nontoxic toward mice. It produced a potent neuromuscular blockade at the avian neuromuscular junction (IC(50)=10 nM), comparable to alpha-bungarotoxin, but was three orders of magnitude less effective at the mammalian neuromuscular junction. Covalently linked heterodimeric three-finger toxins found in colubrid venoms constitute a new class of venom peptides, which may be a useful source of new neurobiology probes and therapeutic leads.
By means of DEAE-Sephadex A-50 Column chromatography, Trimeresurus gramineus venom was separated into twelve fractions. The fibrinogenolytic activities were distributed in Fractions 1 and 10. These enzymes were further purified by gel filtration and were homogeneous as judged by cellulose acetate membrane, sodium dodecyl sulfate polyacrylamide gel electrophoresis and ultracentrifugal analysis. Both of them were single peptide chains. The sedimentation constants of alpha- (Fraction 1) and beta-fibrinogenases (Fraction 10) were 2.20 and 3.60, respectively. The molecular weights of alpha- and beta-fibrinogenases were 23 500 and 25 000 respectively. The contents of proline and glycine were higher in beta-fibrinogenase than in alpha-fibrinogenase. The isoelectric points of alpha-fibrinogenase and beta-fibrinogenase were pH greater than 10 and 4.5, respectively. The optimal pH of alpha-fibrinogenase was approx. 7.4 and that of beta-fibrinogenase was approx. 9.0. The activity of alpha-fibrinogenase was completely destroyed after 30 min at 60 degrees C, pH 5.4, 7.4 and 9.0, while that of beta-fibrinogenase was much less affected by the same treatment. The specific fibrinogenolytic activity alpha-fibrinogenase was 31 mg fibrinogen/min per mg protein, while that of beta-fibrinogenase was 9 mg fibrinogen/min per mg protein. alpha-Fibrinogenase cleaved specifically the alpha(A) chain of monomeric fibrinogen without cleaving the beta(B) chain and gamma-chain. beta-fibrinogenase preferentially cleaved the beta(B) chain, and the alpha(A) chain was also partially cleaved by beta-fibrinogenase, if the incubation time was prolonged. Both enzymes showed proteolytic activities toward fibrinogen, fibrin and casein, but were devoid of phospholipase A, alkaline phosphomonoesterase and phosphodiesterase activities found in the crude venom. The tosyl-L-arginine methylester esterase activity of beta-fibrinogenase was about 14 times that of crude venom, while alpha-fibrinogenase was completely devoid of this activity. The fibrinogenolytic activity of alpha-fibrinogenase was markedly inhibited by EDTA and cysteine, while that of beta-fibrinogenase was inhibited markedly by phenylmethanesulfonylfluoride. alpha- and beta-fibrinogenases exert their fibrinogenolytic activity by a direct action on fibrinogen or fibrin without activation of plasminogen.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
1. Venoms of 11 coral snake taxa, including Micrurus albicinctus, M. corallinus, M. frontalis altirostris, M. f. brasiliensis, M. f. frontalis, M. fulvius fulvius, M. ibiboboca, M. lemniscatus ssp., M. randonianus, M. spixii spixii, and M. surinamensis surinamensis, were examined for 13 enzymatic activities. 2. These were compared with venoms of three outgroup taxa: Naja naja kaouthia, Bungarus multicinctus, and Bothrops moojeni. 3. Enzyme activity levels in Micrurus venoms were highly variable from species to species. 4. All venoms possessed phospholipase activity. 5. Protease activity against synthetic or dyed natural substrates was generally negligible in all elapid venoms examined. By contrast, most Micrurus venoms displayed ample L-leucine aminopeptidase activity. 6. Venom of M.s. surinamensis was significantly different from those of its congeners in most assays.
This chapter deals with discussion of purification and properties of venom phosphodiesterase. With PM2 DNA as substrate and under favorable conditions phosphodiesterase produces not only the full-length linear double-stranded DNA (form III) but also a series of fragments of form III that result from two almost simultaneous cleavages, possibly occurring at the base of the supercoiled branch. A modest advantage of bis-p-nitrophenyl phosphate is its low cost and general use by commercial suppliers of phosphodiesterase. The use of p-nitrophenyl-pT is advantageous because of specificity toward 5'- monoester forming phosphodiesterases and exclusion phosphodiesterases and exclusion of 3'-monoester formers. The chapter also describes a non recording procedure.
The composition of rear-fanged colubrid snake venoms is largely unknown due primarily to the difficulty involved in venom collection. Several different methods have been used to maximize the yield of Duvernoy's secretions. The method proposed by Rosenberg in 1992, which includes the use of ketamine hydrochloride anesthetic and pilocarpine to induce Duvernoy's glands secretion, was used in the present study to collect venom from eight species of colubrids. Protein concentrations, using a dye-binding microassay technique, were determined for the venoms collected. Average protein concentrations ranged from 49.8 to 96.4%. Most yields (dry weight/snake) obtained from specimens in this study were significantly greater than yields previously reported. There was a wide range of effects that occurred due to the ketamine injections; however, all snakes recovered from the effects of the ketamine hydrochloride/pilocarpine with no apparent ill effects. Recommended doses of ketamine hydrochloride have thus been adjusted, depending on previous reactions to the drug. The use of ketamine/pilocarpine in the collection of Duvernoy's secretion has proven to be highly effective in increasing yields. Some caution should be observed when administering ketamine to various species of colubrids, as effects do not necessarily scale to body mass.
In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+-induced depolarization in the 0.1-1 microm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.
Spectrophotometric assays for snake venom l-amino acid oxidase have been developed based on the conversion of l-kynurenine (I) to kynurenic acid (III) and of 3,4-dehydro-l-proline (IV) to pyrrole-2-carboxylic acid (VI).
The Brown Treesnake (Boiga irregularis), a rear-fanged member of the polyphyletic family Colubridae, is an introduced predator on Guam which has been responsible for numerous human envenomations. Because little is known about this species' venom, we characterized venom proteins from B. irregularis using enzyme assays, one and 2D electrophoresis, Western blot analysis, mass spectrometry, HPLC and toxicity assays. Venom yields and protein content varied significantly with snake size, and large adult specimens averaged over 500 microl venom (19.2 mg, protein content approximately 90%). Only two enzymes, azocaseinolytic metalloprotease and acetylcholinesterase, were detected in venoms, and both activities increased with snake size/age. Western blot analysis demonstrated a 25 kDa CRiSP homolog in venoms from both neonate and adult snakes. 2D electrophoresis showed variation between venoms from neonate and adult snakes, especially with respect to metalloprotease and acetylcholinesterase. Analysis by MALDI-TOF mass spectrometry revealed the presence of numerous proteins with molecular masses of approximately 8.5-11 kDa. Adult B. irregularis venom was quite toxic to domestic chickens (Gallus domesticus; 1.75 microg/g) and lizards (Hemidactylus geckos: 2.5 microg/g and Carlia skinks: 4.5 microg/g), and intoxication was characterized by rapid paralysis of all species and neck droop in chickens. Toxicity of venom from neonates toward geckos was 1.1 microg/g, consistent with the presence of a greater diversity of 8-11 kDa proteins (suspected neurotoxins) in these venoms. All of these values were notably lower than murine LD50 values (neonate: 18 microg/g; adult: 31 microg/g). Like venoms of several front-fanged species, B. irregularis venom showed an ontogenetic shift in enzyme activities and toxicity, and neonate snakes produced more toxic venoms with lower protease and acetylcholinesterase activities. High toxicity toward non-mammalian prey demonstrated the presence of taxa-specific effects (and thus toxins) in B. irregularis venom, likely a characteristic of many colubrid snake venoms. We hypothesize that the lack of significant envenomation effects in humans following most colubrid bites results from this taxa-specific action of colubrid venom components, not from a lack of toxins.