Technological advances in mass spectrometry have enabled the extensive identification, characteri... more Technological advances in mass spectrometry have enabled the extensive identification, characterization, and quantification of proteins in any biological system. In disease processes proteins are often altered in response to external stimuli; therefore, proteomics, the large-scale study of proteins and their functions, represents an invaluable tool for understanding the molecular basis of disease. This review highlights the use of mass spectrometry–based proteomics to study the pathogenesis, etiology, and pathology of several neglected tropical diseases (NTDs), a diverse group of disabling diseases primarily associated with poverty in tropical and subtropical regions of the world. While numerous NTDs have been the subject of proteomic studies, this review focuses on Buruli ulcer, dengue, leishmaniasis, and snakebite envenoming. The proteomic studies highlighted provide substantial information on the pathogenic mechanisms driving these diseases; they also identify molecular targets for drug discovery and development and uncover promising biomarkers that can assist in early diagnosis.
The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the ... more The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P–III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5′-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper (T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom–venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.
Human herpesvirus-6A (HHV-6A) and 6B (HHV-6B) are two closely related betaherpesviruses that are ... more Human herpesvirus-6A (HHV-6A) and 6B (HHV-6B) are two closely related betaherpesviruses that are associated with various diseases including seizures and encephalitis. The HHV-6A/B genomes have been shown to be present in an integrated state in the telomeres of latently infected cells. In addition, integration of HHV-6A/B in germ cells has resulted in individuals harboring this inherited chromosomally integrated HHV-6A/B (iciHHV-6) in every cell of their body. Until now, the viral transcriptome and the epigenetic modifications that contribute to the silencing of the integrated virus genome remain elusive. In the current study, we used a patient-derived iciHHV-6A cell line to assess the global viral gene expression profile by RNA-seq, and the chromatin profiles by MNase-seq and ChIP-seq analyses. In addition, we investigated an in vitro generated cell line (293-HHV-6A) that expresses GFP upon the addition of agents commonly used to induce herpesvirus reactivation such as TPA. No viral gene expression including miRNAs was detected from the HHV-6A genomes, indicating that the integrated virus is transcriptionally silent. Intriguingly, upon stimulation of the 293-HHV-6A cell line with TPA, only foreign promoters in the virus genome were activated, while all HHV-6A promoters remained completely silenced. The transcriptional silencing of latent HHV-6A was further supported by MNase-seq results, which demonstrate that the latent viral genome resides in a highly condensed nucleosome-associated state. We further explored the enrichment profiles of histone modifications via ChIP-seq analysis. Our results indicated that the HHV-6 genome is modestly enriched with the repressive histone marks H3K9me3/H3K27me3 and does not possess the active histone modifications H3K27ac/H3K4me3. Overall, these results indicate that HHV-6 genomes reside in a condensed chromatin state, providing insight into the epigenetic mechanisms associated with the silencing of the integrated HHV-6A genome.
As trophic adaptations, rattlesnake venoms can vary in composition depending on several intrinsic... more As trophic adaptations, rattlesnake venoms can vary in composition depending on several intrinsic and extrinsic factors. Ontogenetic changes in venom composition have been documented for numerous species, but little is known of the potential age-related changes in many rattlesnake species found in México. In the current study, venom samples collected from adult and neonate Crotalus polystictus from Estado de México were subjected to enzymatic and electrophoretic analyses, toxicity assays (LD 50), and MALDI-TOF mass spectrometry, and a pooled sample of adult venom was analyzed by shotgun proteomics. Electrophoretic profiles of adult males and females were quite similar, and only minor sex-based variation was noted. However, distinct differences were observed between venoms from adult females and their neonate offspring. Several prominent bands, including P-I and P-III snake venom metalloproteinases (SVMPs) and disintegrins (confirmed by MS/MS) were present in adult venoms and absent/greatly reduced in neonate venoms. Age-dependent differences in SVMP, kallikrein-like, phospholipase A 2 (PLA 2), and L-amino acid oxidase (LAAO) activity levels were confirmed by enzymatic activity assays, and like many other rattlesnake species, venoms from adult snakes have higher SVMP activity than neonate venoms. Conversely, PLA 2 activity was approximately 2.5 × greater in venoms from neonates, likely contributing to the increased toxicity (neonate venom LD 50 = 4.5 µg/g) towards non-Swiss albino mice when compared to adult venoms (LD 50 = 5.5 µg/g). Thrombin-like (TLE) and phosphodiesterase activities did not vary significantly with age. A significant effect of sex (between adult male and adult female venoms) was also observed for SVMP, TLE, and LAAO activities. Analysis of pooled adult venom by LC-MS/MS identified 14 toxin protein families, dominated by bradykinin-inhibitory peptides, SVMPs (P-I, P-II and P-III), disintegrins, PLA 2 s, C-type-lectins, CRiSPs, serine proteinases, and LAAOs (96% of total venom proteins). Neonate and adult C. polystictus in this population consume almost exclusively mammals, suggesting that age-based differences in composition are related to physical differences in prey (e.g., surface-to-volume ratio differences) rather than taxonomic differences between prey. Venoms from adult C. polystictus fit a Type I pattern (high SVMP activity, lower toxicity), which is characteristic of many larger-bodied rattlesnakes of North America. Key Contribution: We provide a detailed analysis of the venom of a rattlesnake that occupies mid-elevation habitat in México. Venoms from neonate and adult Crotalus polystictus show distinct differences in SVMP, kallikrein-like, LAAO, and PLA 2 activities and disintegrin content. Venomics of pooled adult C. polystictus venom identified many major venom components that are common in rattlesnake venoms, with bradykinin-inhibitory peptide constituting 36% of the venom proteome. Crotalus polystictus shows a pattern of venom ontogeny similar to other rattlesnakes that produce a type I venom.
The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake nat... more The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake native to northern and eastern regions of Australia, Papua New Guinea and the Solomon Islands. It was inadvertently introduced onto the island of Guam during the late 1940's to early 1950's, and it has caused massive declines and ex-tirpations of the native bird, lizard, and mammal populations. In the current study, we report the characterization of the venom proteome of an adult and a neonate B. irregularis specimens from Guam by a combination of venom gland transcriptomic and venomic analyses. Venom gland transcriptomic analysis of an adult individual identified toxins belonging to 18 protein families, with three-finger toxin isoforms being the most abundantly expressed transcripts, comprising 94% of all venom protein transcript reads. Transcripts for PIII-metallopro-teinases, C-type lectins, cysteine-rich secretory proteins, acetylcholinesterases, natriuretic peptides, ficolins, phospholipase A 2 (PLA 2) inhibitors, PLA 2 s, vascular endothelial growth factors, Kunitz-type protease inhibitors, cystatins, phospholipase Bs, cobra venom factors, waprins, SVMP inhibitors, matrix metalloproteinases, and hyaluronidases were also identified, albeit, at very low abundances ranging from 0.05% to 1.7% of the tran-scriptome. The venom proteomes of neonate and adult B. irregularis were also both overwhelmingly (78 and 84%, respectively) dominated by monomeric and dimeric 3FTxs, followed by moderately abundant (21% (N) and 13% (A)) CRISPs, low abundance (1% (N), 3% (A)) PIII-SVMPs, and very low abundance (< 0.01%) PLA 2 and SVMP inhibitors. The differences in relative toxin abundances identified between neonate and adult snakes likely correlates to shifts in prey preference between the two age classes, from nearly-exclusively lizards to lizards, birds and small mammals. Immunoaffinity antivenomics with experimentally designed rabbit anti-Brown Treesnake (anti-BTS) venom IgGs against homologous venom from adult snakes demonstrated that CRISPs, PIII-SVMPs, and 60–70% of 3FTxs were effectively immunocaptured. Western blot analysis showed that all venom proteins were recognized by anti-BTS IgGs, and cross-reactivity with other rear-fanged snake venoms was also observed. Incubation of anti-BTS venom IgGs with crude B. irregularis venom resulted in a significant decrease in proteolytic (SVMP) activity against azocasein. These results provide the first comparative venomic and anti-venomic analysis of neonate and adult B. irregularis from Guam, further highlighting evolutionary trends in venom composition among rear-fanged venomous snakes. Significance paragraph: The Brown Treesnake (Boiga irregularis) has caused extensive ecological and economic damage to the island of Guam where it has become a classic example of the negative impacts of invasive species. In the current study, we report the first combined transcriptomic and proteomic analysis of B. irregularis venom of Guam origin. The transcriptome of an adult snake contained toxin sequences belonging to 18 protein families, with three-finger toxin (3FTx) isoforms being the most abundant and representing 94% of all venom protein transcript reads. Our bottom-up and top-down venomic analyses confirmed that 3FTxs are the major components of B. irregularis venom, and a comparative analysis of neonate and adult venoms demonstrate a clear ontogenetic T shift in toxin abundance, likely driven by dietary variation between the two age classes. Second-generation antivenomics and Western blot analysis using purified anti-Brown Treesnake rabbit serum IgGs (anti-BTS IgGs) showed strong immunoreactivity toward B. irregularis venom. Interestingly, our anti-BTS IgGs did not cross-react with 3FTxs found in several other rear-fanged snake venoms, or against 3FTxs in the venom of the elapid Ophiophagus hannah, indicating that epitopes in these 3FTx molecules are quite distinct.
The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-2... more The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-205) cells against apoptosis induction by Rusvinoxidase, an L-amino acid oxidase purified from Russell's Viper venom (RVV). The significantly lower cytotoxicity as well as apoptotic activity of Rusvinoxidase towards Colo-205 cells (compared to MCF-7 breast cancer cells) is correlated with lower depletion of cellular glutathione content and increased down-regulation of catalase activity of Colo-205 cells following Rusvinoxidase treatment. Exposure to Rusvinoxidase subsequently diminished reactive oxygen species (ROS) production and failed to impair mitochondrial membrane potential, resulting in apoptosis induction resistance in Colo-205 cells. Further, higher expression levels of caspase 8, compared to caspase 9, indicate that Rusvinoxidase preferentially triggers the extrinsic pathway of apoptosis in Colo-205 cells. A time-dependent lower ratio of the relative expression of Bax and Bcl-xL (pro-and anti-apoptotic proteins) in Colo-205 cells, compared to our previous study on MCF-7 cells, unambiguously supports a higher cellular resistance mechanism in Colo-205 cells against Rusvinoxidase-induced apoptosis.
Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the worl... more Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the world, studies on the species found throughout this region have been relatively scarce. This has led to challenges with examining venom phenotypic characteristics, as well as species misidentifications and mis-classifications. In the current study we investigated venom variation among four subspecies of Crotalus lepidus (C. l. klaluberi, C. l. lepidus, C. l. maculosus, C. l. morulus) and four subspecies of C. willardi (C. w. amabilis, C. w. obscurus, C. w. silus, and C. w. willardi) that inhabit regions of southwestern United States and central M exico. SDS-PAGE patterns show the presence of many of the major compounds found in other rattlesnake venoms, although minor variations in protein banding patterns and intensity are recognizable. Most notably, PI-metalloproteinase (SVMP) bands appear to be very faint to absent in northern C. l. lepidus and C. l. klauberi subspecies, but are fairly prominent in all other C. lepidus and C. willardi subspecies. Enzyme activity assays revealed that C. lepidus subspecies exhibit higher SVMP and thrombin-like activities when compared to C. willardi subspecies. Significant differences between sub-species were also observed for kallikrein-like serine protease, L-amino acid oxidase, and phosphodies-terase activities, although these differences appear to be random and fail to follow a geographical or phylogenetic trend. The same relationship was also observed for fibrinogenolytic and coagulation assays. Toxicity assays conducted on lab mice (Mus musculus), house geckos (Hemidactylus frenatus), and house crickets (Acheta domestica) revealed varying toxicities between subspecies, with C. l klauberi being the most toxic towards mice (LD 50 ¼ 1.36 mg/g) and house geckos (LD 50 ¼ 0.17 mg/g), and C. w. silus being most toxic to house crickets (LD 50 ¼ 1.94 mg/g). These results provide additional evidence that geographical isolation, natural selection, and adaptive evolution in response to diets may be driving forces contributing to population-level variation in venom composition.
Rattlesnakes use prey chemical cues for ambush site selection and for relocating envenomated (E) ... more Rattlesnakes use prey chemical cues for ambush site selection and for relocating envenomated (E) prey following a predatory strike. The ability to discriminate between E and non-envenomated (NE) prey cues has been widely studied in rattlesnake species that produce type I venoms, which show high levels of snake venom metal-loproteinase (SVMP) activity and low lethal toxicity [lethal dose which kills 50% of test animals (LD 50) [1.0 lg/g]. However, E vs. NE prey discrimination studies have not been conducted on rattlesnake species that produce a type II venom that consists of low SVMP activity and high lethal toxicity (LD 50 \1.0 lg/g). In the current study, long-term captive Crotalus oreganus concolor, which produce a type II venom, were tested for their ability to discriminate between chemical cues of natural (Sceloporus undulatus and Peromyscus maniculatus) and non-natural (Hemi-dactylus frenatus and Mus musculus) prey cues, as well as for their ability to discriminate between E and NE mouse carcasses, when prey envenomation occurred by a con-specific. Snakes showed significant levels of tongue flicking towards the chemical extracts of P. maniculatus and M. musculus, suggesting that C. oreganus concolor exhibit both innate and experience-based plasticity in response to prey chemical cues. In addition, C. oreganus concolor were able to discriminate between E and NE prey sources, when envenomation occurred by a conspecific, indicating that a type II venomous species can also discriminate between E and NE chemical cues.
Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical act... more Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeu-tics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and ''Colubri-dae'') in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells.
Integrins play an essential role in cancer survival and invasion, and they have been major target... more Integrins play an essential role in cancer survival and invasion, and they have been major targets in drug development and design. Disintegrins are small (4–16 kDa) viperid snake venom proteins that exhibit a canonical integrin-binding site (often RGD). These non-enzymatic proteins inhibit integrin-mediated cell–cell and cell-extracellular matrix interactions, making them potential candidates as therapeutics in cancer and numerous other human disorders. The present study examined the cytotoxic, anti-adhesion, and anti-migration effects of a recently characterized disintegrin, tzabcanin, towards melanoma (A-375) and lung (A-549) cancer cell lines. Tzabcanin inhibits adhesion of both cells lines to vitronectin and exhibited very weak cytotoxicity towards A-375 cells; however, it had no effect on cell viability of A-549 cells. Further, tzabcanin significantly inhibited migration of both cell lines in cell scratch/wound healing assays. Flow cytometric analysis indicates that both A-375 and A-549 cell lines express integrin v 3 , a critical integrin in tumor motility and invasion, and a major receptor of the extracellular matrix protein vitronectin. Flow cytometric analysis also identified v 3 as a binding site of tzabcanin. These results suggest that tzabcanin may have utility in the development of anticancer therapies, or may be used as a biomarker to detect neoplasms that over-express integrin v 3 .
We report the elucidation of a mechanism of
apoptosis induction in breast cancer (MCF-7) cells by... more We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of *57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidaseinduced intrinsic pathway of apoptosis was accompanied by a
Here we describe and compare the venomic and antivenomic characteristics of both neonate and adul... more 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), Zn2+-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.
Background: Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fra... more Background: Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation. Results: During behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with sizefractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A2, showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions.
Conclusions: These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.
Technological advances in mass spectrometry have enabled the extensive identification, characteri... more Technological advances in mass spectrometry have enabled the extensive identification, characterization, and quantification of proteins in any biological system. In disease processes proteins are often altered in response to external stimuli; therefore, proteomics, the large-scale study of proteins and their functions, represents an invaluable tool for understanding the molecular basis of disease. This review highlights the use of mass spectrometry–based proteomics to study the pathogenesis, etiology, and pathology of several neglected tropical diseases (NTDs), a diverse group of disabling diseases primarily associated with poverty in tropical and subtropical regions of the world. While numerous NTDs have been the subject of proteomic studies, this review focuses on Buruli ulcer, dengue, leishmaniasis, and snakebite envenoming. The proteomic studies highlighted provide substantial information on the pathogenic mechanisms driving these diseases; they also identify molecular targets for drug discovery and development and uncover promising biomarkers that can assist in early diagnosis.
The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the ... more The genus Trimeresurus comprises a group of venomous pitvipers endemic to Southeast Asia and the Pacific Islands. Of these, Trimeresurus insularis, the White-lipped Island Pitviper, is a nocturnal, arboreal species that occurs on nearly every major island of the Lesser Sunda archipelago. In the current study, venom phenotypic characteristics of T. insularis sampled from eight Lesser Sunda Islands (Flores, Lembata, Lombok, Pantar, Sumba, Sumbawa, Timor, and Wetar) were evaluated via SDS-PAGE, enzymatic activity assays, fibrinogenolytic assays, gelatin zymography, and RP-HPLC, and the Sumbawa sample was characterized by venomic analysis. For additional comparative analyses, venoms were also examined from several species in the Trimeresurus complex, including T. borneensis, T. gramineus, T. puniceus, T. purpureomaculatus, T. stejnegeri, and Protobothrops flavoviridis. Despite the geographical isolation, T. insularis venoms from all eight islands demonstrated remarkable similarities in gel electrophoretic profiles and RP-HPLC patterns, and all populations had protein bands in the mass ranges of phosphodiesterases (PDE), l-amino acid oxidases (LAAO), P–III snake venom metalloproteinases (SVMP), serine proteases, cysteine-rich secretory proteins (CRISP), phospholipases A2 (PLA2), and C-type lectins. An exception was observed in the Lombok sample, which lacked protein bands in the mass range of serine protease and CRISP. Venomic analysis of the Sumbawa venom also identified these protein families, in addition to several proteins of lesser abundance (<1%), including glutaminyl cyclase, aminopeptidase, PLA2 inhibitor, phospholipase B, cobra venom factor, 5′-nucleotidase, vascular endothelial growth factor, and hyaluronidase. All T. insularis venoms exhibited similarities in thrombin-like and PDE activities, while significant differences were observed for LAAO, SVMP, and kallikrein-like activities, though these differences were only observed for a few islands. Slight but noticeable differences were also observed with fibrinogen and gelatin digestion activities. Trimeresurus insularis venoms exhibited overall similarity to the other Trimeresurus complex species examined, with the exception of P. flavoviridis venom, which showed the greatest overall differentiation. Western blot analysis revealed that all major T. insularis venom proteins were recognized by Green Pitviper (T. albolabris) antivenom, and reactivity was also seen with most venom proteins of the other Trimeresurus species, but incomplete antivenom–venom recognition was observed against P. flavoviridis venom proteins. These results demonstrate significant conservation in the venom composition of T. insularis across the Lesser Sunda archipelago relative to the other Trimeresurus species examined.
Human herpesvirus-6A (HHV-6A) and 6B (HHV-6B) are two closely related betaherpesviruses that are ... more Human herpesvirus-6A (HHV-6A) and 6B (HHV-6B) are two closely related betaherpesviruses that are associated with various diseases including seizures and encephalitis. The HHV-6A/B genomes have been shown to be present in an integrated state in the telomeres of latently infected cells. In addition, integration of HHV-6A/B in germ cells has resulted in individuals harboring this inherited chromosomally integrated HHV-6A/B (iciHHV-6) in every cell of their body. Until now, the viral transcriptome and the epigenetic modifications that contribute to the silencing of the integrated virus genome remain elusive. In the current study, we used a patient-derived iciHHV-6A cell line to assess the global viral gene expression profile by RNA-seq, and the chromatin profiles by MNase-seq and ChIP-seq analyses. In addition, we investigated an in vitro generated cell line (293-HHV-6A) that expresses GFP upon the addition of agents commonly used to induce herpesvirus reactivation such as TPA. No viral gene expression including miRNAs was detected from the HHV-6A genomes, indicating that the integrated virus is transcriptionally silent. Intriguingly, upon stimulation of the 293-HHV-6A cell line with TPA, only foreign promoters in the virus genome were activated, while all HHV-6A promoters remained completely silenced. The transcriptional silencing of latent HHV-6A was further supported by MNase-seq results, which demonstrate that the latent viral genome resides in a highly condensed nucleosome-associated state. We further explored the enrichment profiles of histone modifications via ChIP-seq analysis. Our results indicated that the HHV-6 genome is modestly enriched with the repressive histone marks H3K9me3/H3K27me3 and does not possess the active histone modifications H3K27ac/H3K4me3. Overall, these results indicate that HHV-6 genomes reside in a condensed chromatin state, providing insight into the epigenetic mechanisms associated with the silencing of the integrated HHV-6A genome.
As trophic adaptations, rattlesnake venoms can vary in composition depending on several intrinsic... more As trophic adaptations, rattlesnake venoms can vary in composition depending on several intrinsic and extrinsic factors. Ontogenetic changes in venom composition have been documented for numerous species, but little is known of the potential age-related changes in many rattlesnake species found in México. In the current study, venom samples collected from adult and neonate Crotalus polystictus from Estado de México were subjected to enzymatic and electrophoretic analyses, toxicity assays (LD 50), and MALDI-TOF mass spectrometry, and a pooled sample of adult venom was analyzed by shotgun proteomics. Electrophoretic profiles of adult males and females were quite similar, and only minor sex-based variation was noted. However, distinct differences were observed between venoms from adult females and their neonate offspring. Several prominent bands, including P-I and P-III snake venom metalloproteinases (SVMPs) and disintegrins (confirmed by MS/MS) were present in adult venoms and absent/greatly reduced in neonate venoms. Age-dependent differences in SVMP, kallikrein-like, phospholipase A 2 (PLA 2), and L-amino acid oxidase (LAAO) activity levels were confirmed by enzymatic activity assays, and like many other rattlesnake species, venoms from adult snakes have higher SVMP activity than neonate venoms. Conversely, PLA 2 activity was approximately 2.5 × greater in venoms from neonates, likely contributing to the increased toxicity (neonate venom LD 50 = 4.5 µg/g) towards non-Swiss albino mice when compared to adult venoms (LD 50 = 5.5 µg/g). Thrombin-like (TLE) and phosphodiesterase activities did not vary significantly with age. A significant effect of sex (between adult male and adult female venoms) was also observed for SVMP, TLE, and LAAO activities. Analysis of pooled adult venom by LC-MS/MS identified 14 toxin protein families, dominated by bradykinin-inhibitory peptides, SVMPs (P-I, P-II and P-III), disintegrins, PLA 2 s, C-type-lectins, CRiSPs, serine proteinases, and LAAOs (96% of total venom proteins). Neonate and adult C. polystictus in this population consume almost exclusively mammals, suggesting that age-based differences in composition are related to physical differences in prey (e.g., surface-to-volume ratio differences) rather than taxonomic differences between prey. Venoms from adult C. polystictus fit a Type I pattern (high SVMP activity, lower toxicity), which is characteristic of many larger-bodied rattlesnakes of North America. Key Contribution: We provide a detailed analysis of the venom of a rattlesnake that occupies mid-elevation habitat in México. Venoms from neonate and adult Crotalus polystictus show distinct differences in SVMP, kallikrein-like, LAAO, and PLA 2 activities and disintegrin content. Venomics of pooled adult C. polystictus venom identified many major venom components that are common in rattlesnake venoms, with bradykinin-inhibitory peptide constituting 36% of the venom proteome. Crotalus polystictus shows a pattern of venom ontogeny similar to other rattlesnakes that produce a type I venom.
The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake nat... more The Brown Treesnake (Boiga irregularis) is an arboreal, nocturnal, rear-fanged venomous snake native to northern and eastern regions of Australia, Papua New Guinea and the Solomon Islands. It was inadvertently introduced onto the island of Guam during the late 1940's to early 1950's, and it has caused massive declines and ex-tirpations of the native bird, lizard, and mammal populations. In the current study, we report the characterization of the venom proteome of an adult and a neonate B. irregularis specimens from Guam by a combination of venom gland transcriptomic and venomic analyses. Venom gland transcriptomic analysis of an adult individual identified toxins belonging to 18 protein families, with three-finger toxin isoforms being the most abundantly expressed transcripts, comprising 94% of all venom protein transcript reads. Transcripts for PIII-metallopro-teinases, C-type lectins, cysteine-rich secretory proteins, acetylcholinesterases, natriuretic peptides, ficolins, phospholipase A 2 (PLA 2) inhibitors, PLA 2 s, vascular endothelial growth factors, Kunitz-type protease inhibitors, cystatins, phospholipase Bs, cobra venom factors, waprins, SVMP inhibitors, matrix metalloproteinases, and hyaluronidases were also identified, albeit, at very low abundances ranging from 0.05% to 1.7% of the tran-scriptome. The venom proteomes of neonate and adult B. irregularis were also both overwhelmingly (78 and 84%, respectively) dominated by monomeric and dimeric 3FTxs, followed by moderately abundant (21% (N) and 13% (A)) CRISPs, low abundance (1% (N), 3% (A)) PIII-SVMPs, and very low abundance (< 0.01%) PLA 2 and SVMP inhibitors. The differences in relative toxin abundances identified between neonate and adult snakes likely correlates to shifts in prey preference between the two age classes, from nearly-exclusively lizards to lizards, birds and small mammals. Immunoaffinity antivenomics with experimentally designed rabbit anti-Brown Treesnake (anti-BTS) venom IgGs against homologous venom from adult snakes demonstrated that CRISPs, PIII-SVMPs, and 60–70% of 3FTxs were effectively immunocaptured. Western blot analysis showed that all venom proteins were recognized by anti-BTS IgGs, and cross-reactivity with other rear-fanged snake venoms was also observed. Incubation of anti-BTS venom IgGs with crude B. irregularis venom resulted in a significant decrease in proteolytic (SVMP) activity against azocasein. These results provide the first comparative venomic and anti-venomic analysis of neonate and adult B. irregularis from Guam, further highlighting evolutionary trends in venom composition among rear-fanged venomous snakes. Significance paragraph: The Brown Treesnake (Boiga irregularis) has caused extensive ecological and economic damage to the island of Guam where it has become a classic example of the negative impacts of invasive species. In the current study, we report the first combined transcriptomic and proteomic analysis of B. irregularis venom of Guam origin. The transcriptome of an adult snake contained toxin sequences belonging to 18 protein families, with three-finger toxin (3FTx) isoforms being the most abundant and representing 94% of all venom protein transcript reads. Our bottom-up and top-down venomic analyses confirmed that 3FTxs are the major components of B. irregularis venom, and a comparative analysis of neonate and adult venoms demonstrate a clear ontogenetic T shift in toxin abundance, likely driven by dietary variation between the two age classes. Second-generation antivenomics and Western blot analysis using purified anti-Brown Treesnake rabbit serum IgGs (anti-BTS IgGs) showed strong immunoreactivity toward B. irregularis venom. Interestingly, our anti-BTS IgGs did not cross-react with 3FTxs found in several other rear-fanged snake venoms, or against 3FTxs in the venom of the elapid Ophiophagus hannah, indicating that epitopes in these 3FTx molecules are quite distinct.
The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-2... more The present study highlights the cellular mechanism of resistance in human adenocarcinoma (Colo-205) cells against apoptosis induction by Rusvinoxidase, an L-amino acid oxidase purified from Russell's Viper venom (RVV). The significantly lower cytotoxicity as well as apoptotic activity of Rusvinoxidase towards Colo-205 cells (compared to MCF-7 breast cancer cells) is correlated with lower depletion of cellular glutathione content and increased down-regulation of catalase activity of Colo-205 cells following Rusvinoxidase treatment. Exposure to Rusvinoxidase subsequently diminished reactive oxygen species (ROS) production and failed to impair mitochondrial membrane potential, resulting in apoptosis induction resistance in Colo-205 cells. Further, higher expression levels of caspase 8, compared to caspase 9, indicate that Rusvinoxidase preferentially triggers the extrinsic pathway of apoptosis in Colo-205 cells. A time-dependent lower ratio of the relative expression of Bax and Bcl-xL (pro-and anti-apoptotic proteins) in Colo-205 cells, compared to our previous study on MCF-7 cells, unambiguously supports a higher cellular resistance mechanism in Colo-205 cells against Rusvinoxidase-induced apoptosis.
Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the worl... more Although the Mexican Highlands has the highest diversity of small-bodied rattlesnakes in the world, studies on the species found throughout this region have been relatively scarce. This has led to challenges with examining venom phenotypic characteristics, as well as species misidentifications and mis-classifications. In the current study we investigated venom variation among four subspecies of Crotalus lepidus (C. l. klaluberi, C. l. lepidus, C. l. maculosus, C. l. morulus) and four subspecies of C. willardi (C. w. amabilis, C. w. obscurus, C. w. silus, and C. w. willardi) that inhabit regions of southwestern United States and central M exico. SDS-PAGE patterns show the presence of many of the major compounds found in other rattlesnake venoms, although minor variations in protein banding patterns and intensity are recognizable. Most notably, PI-metalloproteinase (SVMP) bands appear to be very faint to absent in northern C. l. lepidus and C. l. klauberi subspecies, but are fairly prominent in all other C. lepidus and C. willardi subspecies. Enzyme activity assays revealed that C. lepidus subspecies exhibit higher SVMP and thrombin-like activities when compared to C. willardi subspecies. Significant differences between sub-species were also observed for kallikrein-like serine protease, L-amino acid oxidase, and phosphodies-terase activities, although these differences appear to be random and fail to follow a geographical or phylogenetic trend. The same relationship was also observed for fibrinogenolytic and coagulation assays. Toxicity assays conducted on lab mice (Mus musculus), house geckos (Hemidactylus frenatus), and house crickets (Acheta domestica) revealed varying toxicities between subspecies, with C. l klauberi being the most toxic towards mice (LD 50 ¼ 1.36 mg/g) and house geckos (LD 50 ¼ 0.17 mg/g), and C. w. silus being most toxic to house crickets (LD 50 ¼ 1.94 mg/g). These results provide additional evidence that geographical isolation, natural selection, and adaptive evolution in response to diets may be driving forces contributing to population-level variation in venom composition.
Rattlesnakes use prey chemical cues for ambush site selection and for relocating envenomated (E) ... more Rattlesnakes use prey chemical cues for ambush site selection and for relocating envenomated (E) prey following a predatory strike. The ability to discriminate between E and non-envenomated (NE) prey cues has been widely studied in rattlesnake species that produce type I venoms, which show high levels of snake venom metal-loproteinase (SVMP) activity and low lethal toxicity [lethal dose which kills 50% of test animals (LD 50) [1.0 lg/g]. However, E vs. NE prey discrimination studies have not been conducted on rattlesnake species that produce a type II venom that consists of low SVMP activity and high lethal toxicity (LD 50 \1.0 lg/g). In the current study, long-term captive Crotalus oreganus concolor, which produce a type II venom, were tested for their ability to discriminate between chemical cues of natural (Sceloporus undulatus and Peromyscus maniculatus) and non-natural (Hemi-dactylus frenatus and Mus musculus) prey cues, as well as for their ability to discriminate between E and NE mouse carcasses, when prey envenomation occurred by a con-specific. Snakes showed significant levels of tongue flicking towards the chemical extracts of P. maniculatus and M. musculus, suggesting that C. oreganus concolor exhibit both innate and experience-based plasticity in response to prey chemical cues. In addition, C. oreganus concolor were able to discriminate between E and NE prey sources, when envenomation occurred by a conspecific, indicating that a type II venomous species can also discriminate between E and NE chemical cues.
Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical act... more Snake venoms are mixtures of bioactive proteins and peptides that exhibit diverse biochemical activities. This wide array of pharmacologies associated with snake venoms has made them attractive sources for research into potentially novel therapeu-tics, and several venom-derived drugs are now in use. In the current study we performed a broad screen of a variety of venoms (61 taxa) from the major venomous snake families (Viperidae, Elapidae and ''Colubri-dae'') in order to examine cytotoxic effects toward MCF-7 breast cancer cells and A-375 melanoma cells.
Integrins play an essential role in cancer survival and invasion, and they have been major target... more Integrins play an essential role in cancer survival and invasion, and they have been major targets in drug development and design. Disintegrins are small (4–16 kDa) viperid snake venom proteins that exhibit a canonical integrin-binding site (often RGD). These non-enzymatic proteins inhibit integrin-mediated cell–cell and cell-extracellular matrix interactions, making them potential candidates as therapeutics in cancer and numerous other human disorders. The present study examined the cytotoxic, anti-adhesion, and anti-migration effects of a recently characterized disintegrin, tzabcanin, towards melanoma (A-375) and lung (A-549) cancer cell lines. Tzabcanin inhibits adhesion of both cells lines to vitronectin and exhibited very weak cytotoxicity towards A-375 cells; however, it had no effect on cell viability of A-549 cells. Further, tzabcanin significantly inhibited migration of both cell lines in cell scratch/wound healing assays. Flow cytometric analysis indicates that both A-375 and A-549 cell lines express integrin v 3 , a critical integrin in tumor motility and invasion, and a major receptor of the extracellular matrix protein vitronectin. Flow cytometric analysis also identified v 3 as a binding site of tzabcanin. These results suggest that tzabcanin may have utility in the development of anticancer therapies, or may be used as a biomarker to detect neoplasms that over-express integrin v 3 .
We report the elucidation of a mechanism of
apoptosis induction in breast cancer (MCF-7) cells by... more We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of *57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidaseinduced intrinsic pathway of apoptosis was accompanied by a
Here we describe and compare the venomic and antivenomic characteristics of both neonate and adul... more 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), Zn2+-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.
Background: Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fra... more Background: Vertebrate predators use a broad arsenal of behaviors and weaponry for overcoming fractious and potentially dangerous prey. A unique array of predatory strategies occur among snakes, ranging from mechanical modes of constriction and jaw-holding in non-venomous snakes, to a chemical means, venom, for quickly dispatching prey. However, even among venomous snakes, different prey handling strategies are utilized, varying from the strike-and-hold behaviors exhibited by highly toxic elapid snakes to the rapid strike-and-release envenomation seen in viperid snakes. For vipers, this mode of envenomation represents a minimal risk predatory strategy by permitting little contact with or retaliation from prey, but it adds the additional task of relocating envenomated prey which has wandered from the attack site. This task is further confounded by trails of other unstruck conspecific or heterospecific prey. Despite decades of behavioral study, researchers still do not know the molecular mechanism which allows for prey relocation. Results: During behavioral discrimination trials (vomeronasal responsiveness) to euthanized mice injected with sizefractionated venom, Crotalus atrox responded significantly to only one protein peak. Assays for enzymes common in rattlesnake venoms, such as exonuclease, L-amino acid oxidase, metalloproteinase, thrombin-like and kallikrein-like serine proteases and phospholipase A2, showed that vomeronasal responsiveness was not dependent on enzymatic activity. Using mass spectrometry and N-terminal sequencing, we identified the proteins responsible for envenomated prey discrimination as the non-enzymatic disintegrins crotatroxin 1 and 2. Our results demonstrate a novel and critical biological role for venom disintegrins far beyond their well-established role in disruption of cell-cell and cell-extracellular matrix interactions.
Conclusions: These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.
Synopsis Snake venoms represent an adaptive trophic response to the challenges confronting a limb... more Synopsis 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.
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Papers by Anthony Saviola
apoptosis induction in breast cancer (MCF-7) cells by an
L-amino acid oxidase (LAAO), Rusvinoxidase, purified
from the venom of Daboia russelii russelii. Peptide mass
fingerprinting analysis of Rusvinoxidase, an acidic monomeric
glycoprotein with a mass of *57 kDa, confirmed its
identity as snake venom LAAO. The enzymatic activity of
Rusvinoxidase was completely abolished after two cycles
of freezing and thawing; however, its cytotoxicity toward
MCF-7 cells remained unaffected. Dose- and time-dependent
induction of apoptosis by Rusvinoxidase on MCF-7
cells was evident from changes in cell morphology, cell
membrane integrity, shrinkage of cells and apoptotic body
formation accompanied by DNA fragmentation. Rusvinoxidase
induced apoptosis in MCF-7 cells by both the
extrinsic (death-receptor) and intrinsic (mitochondrial)
signaling pathways. The former pathway of apoptosis
operated through activation of caspase-8 that subsequently
activated caspase-7 but not caspase-3. Rusvinoxidaseinduced
intrinsic pathway of apoptosis was accompanied by a
Conclusions: These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.
apoptosis induction in breast cancer (MCF-7) cells by an
L-amino acid oxidase (LAAO), Rusvinoxidase, purified
from the venom of Daboia russelii russelii. Peptide mass
fingerprinting analysis of Rusvinoxidase, an acidic monomeric
glycoprotein with a mass of *57 kDa, confirmed its
identity as snake venom LAAO. The enzymatic activity of
Rusvinoxidase was completely abolished after two cycles
of freezing and thawing; however, its cytotoxicity toward
MCF-7 cells remained unaffected. Dose- and time-dependent
induction of apoptosis by Rusvinoxidase on MCF-7
cells was evident from changes in cell morphology, cell
membrane integrity, shrinkage of cells and apoptotic body
formation accompanied by DNA fragmentation. Rusvinoxidase
induced apoptosis in MCF-7 cells by both the
extrinsic (death-receptor) and intrinsic (mitochondrial)
signaling pathways. The former pathway of apoptosis
operated through activation of caspase-8 that subsequently
activated caspase-7 but not caspase-3. Rusvinoxidaseinduced
intrinsic pathway of apoptosis was accompanied by a
Conclusions: These findings reveal the evolutionary significance of free disintegrins in venoms as the molecular mechanism in vipers allowing for effective relocation of envenomated prey. The presence of free disintegrins in turn has led to evolution of a major behavioral adaptation (strike-and-release), characteristic of only rattlesnakes and other vipers, which exploits and refines the efficiency of a pre-existing chemical means of predation and a highly sensitive olfaction system. This system of a predator chemically tagging prey represents a novel trend in the coevolution of predator-prey relationships.