A multifaceted analysis of viperid snake venoms by two-dimensional gel electrophoresis: an approach to understanding venom proteomics

The complexity of Viperid venoms has long been appreciated by investigators in the fields of toxinology and medicine. However, it is only recently that the depth of that complexity has become somewhat quantitatively and qualitatively appreciated. With the resurgence of two-dimensional gel electrophoresis (2-DE) and the advances in mass spectrometry virtually all venom components can be visualized and identified given sufficient effort and resources. Here we present the use of 2-DE for examining venom complexity as well as demonstrating interesting approaches to selectively delineate subpopulations of venom proteins based on particular characteristics of the proteins such as antibody cross-reactivity or enzymatic activities. 2-DE comparisons between venoms from different species of the same genus (Bothrops) of snake clearly demonstrated both the similarity as well as the apparent diversity among these venoms. Using liquid chromatography/tandem mass spectrometry we were able to identify regions of the two-dimensional gels from each venom in which certain classes of proteins were found. 2-DE was also used to compare venoms from Crotalus atrox and Bothrops jararaca. For these venoms a variety of staining/detection protocols was utilized to compare and contrast the venoms. Specifically, we used various stains to visualize subpopulations of the venom proteomes of these snakes, including Coomassie, Silver, Sypro Ruby and Pro-Q-Emerald. Using specific antibodies in Western blot analyses of 2-DE of the venoms we have examined subpopulations of proteins in these venoms including the serine proteinase proteome, the metalloproteinase proteome, and the phospholipases A2 proteome. A functional assessment of the gelatinolytic activity of these venoms was also performed by zymography. These approaches have given rise to a more thorough understanding of venom complexity and the toxins comprising these venoms and provide insights to investigators who wish to focus on these venom subpopulations of proteins in future studies.     

Assembling an arsenal: origin and evolution of the snake venom proteome inferred from phylogenetic analysis of toxin sequences

We analyzed the origin and evolution of snake venom toxin families represented in both viperid and elapid snakes by means of phylogenetic analysis of the amino acid sequences of the toxins and related nonvenom proteins. Out of eight toxin families analyzed, five provided clear evidence of recruitment into the snake venom proteome before the diversification of the advanced snakes (Kunitz-type protease inhibitors, CRISP toxins, galactose-binding lectins, M12B peptidases, nerve growth factor toxins), and one was equivocal (cystatin toxins). In two others (phospholipase A(2) and natriuretic toxins), the nonmonophyly of venom toxins demonstrates that presence of these proteins in elapids and viperids results from independent recruitment events. The ANP/BNP natriuretic toxins are likely to be basal, whereas the CNP/BPP toxins are Viperidae only. Similarly, the lectins were recruited twice. In contrast to the basal recruitment of the galactose-binding lectins, the C-type lectins were shown to be Viperidae only, with the alpha-chains and beta-chains resulting from an early duplication event. These results provide strong additional evidence that venom evolved once, at the base of the advanced snake radiation, rather than multiple times in different lineages, with these toxins also present in the venoms of the "colubrid" snake families. Moreover, they provide a first insight into the composition of the earliest ophidian venoms and point the way toward a research program that could elucidate the functional context of the evolution of the snake venom proteome.     

Proteome and phosphoproteome of Africanized and European honeybee venoms

Honey bee venom toxins trigger immunological, physiological, and neurological responses within victims. The high occurrence of bee attacks involving potentially fatal toxic and allergic reactions in humans and the prospect of developing novel pharmaceuticals make honey bee venom an attractive target for proteomic studies. Using label‐free quantification, we compared the proteome and phosphoproteome of the venom of Africanized honeybees with that of two European subspecies, namely Apis mellifera ligustica and A. m. carnica. From the total of 51 proteins, 42 were common to all three subspecies. Remarkably, the toxins melittin and icarapin were phosphorylated. In all venoms, icarapin was phosphorylated at the ²⁰⁵Ser residue, which is located in close proximity to its known antigenic site. Melittin, the major toxin of honeybee venoms, was phosphorylated in all venoms at the ¹⁰Thr and ¹⁸Ser residues. ¹⁸Ser phosphorylated melittin—the major of its two phosphorylated forms—was less toxic compared to the native peptide.     

Proteome and peptidome profiling of spider venoms

Spider venoms are an important source of novel molecules with different pharmacological properties. Recent technological developments of proteomics, especially mass spectrometry, have greatly promoted the systematic analysis of spider venom. The enormous diversity of venom components between spider species and the lack of complete genome sequence, and the limited database of protein and peptide sequences make spider venom profiling a challenging task and special considerations for technical strategies are required. This review highlights recently used methods for spider venom profiling. In general, spider venom profiling can be achieved in two parts: proteome profiling of the components with molecular weights above 10 kDa, and peptidome profiling of the components with a molecular weight of 10 kDa or under through the use of different methods. Venom proteomes are rich in various enzymes, hemocyanins, toxin-like proteins and many unknown proteins. Peptidomes are dominated by peptides with a mass of 3-6 kDa with three to five disulfide bonds. Although there are some similarities in peptide superfamily types of venoms from different spider species, the venom profile of each species is unique. The linkage of the peptidomic data with that of the cDNA approach is discussed briefly. Future challenges and perspectives are also highlighted in this review.     

Snake venomics – from low-resolution toxin-pattern recognition to toxin-resolved venom proteomes with absolute quantification

Introduction: Venoms are integrated phenotypes used by a wide range of organisms for predatory and defensive purposes. The study of venoms is of great interest in diverse fields, such as evolutionary ecology and biotechnology. Omics technologies have contributed to understanding the evolutionary mechanisms that molded snake venoms to their present-day structural and functional variability landscape.

Areas covered: This review article reflects on two recent implementations in venomics: absolute quantification of intact proteins by elemental mass spectrometry, and top-down molecular mass spectrometry.

Expert commentary: Leveraging on a new way of polyatomic interference removal, a triple quadrupole inductively coupled plasma mass spectrometry configuration has proven feasible for the absolute quantification of venom toxins via sulfur detection. A major advantage of this approach over quantitative molecular mass spectrometry techniques is that only a generic S-standard is required to quantify all the chromatographically separated sulfur-containing fractions. Top-down venomics is in its infancy but, due to recent hardware and software developments, is gaining momentum. Proteoform-resolved venom proteomes are needed to understand the spatio-temporal variability landscape underlying the adaptations that drive intraspecific venom evolution. Integrating top-down venomics and absolute proteoform quantification into a novel elemental and molecular mass spectrometry configuration will represent a quantitative leap in the study of individual venoms.     

Snake venomics: comparative analysis of the venom proteomes of the Tunisian snakes Cerastes cerastes, Cerastes vipera and Macrovipera lebetina

The protein composition of the crude venoms of the three most important vipers of Tunisia was analyzed by RP-HPLC, N-terminal sequence analysis, MALDI-TOF mass determination, and in-gel tryptic digestion followed by PMF and CID-MS/MS of selected peptide ions in a quadrupole-linear IT instrument. Our results show that the venom proteomes of Cerastes cerastes, Cerastes vipera, and Macrovipera lebetina are composed of proteins belonging to a few protein families. However, each venom showed distinct degree of protein composition complexity. The three venoms shared a number of protein classes though the relative occurrence of these toxins was different in each snake species. On the other hand, the venoms of the Cerastes species and Macrovipera lebetina each contained unique components. The comparative proteomic analysis of Tunisian snake venoms provides a comprehensible catalogue of secreted proteins, which may contribute to a deeper understanding of the biological effects of the venoms, and may also serve as a starting point for studying structure-function correlations of individual toxins.     

N-glycome profiling of Bothrops jararaca newborn and adult venoms

Glycosylation is an important post-translational modification of snake venom proteins and contributes to venom proteome complexity. Many snake venom components are known to be glycosylated, however, very little is known about the carbohydrate structures present in venom glycoproteins. Previous studies showed that the ontogenetic shift in diet, from ectothermic prey in early life to endothermic prey in adulthood, and shift in animal size are associated with changes in the venom proteome of the snake Bothrops jararaca. In this study we explored the composition of the N-glycome released from newborn and adult B. jararaca venom proteins. We used an ion trap mass spectrometer (IT-MS) to disassemble glycan structures based on the use of several pathways of MS (MSn) and demonstrate the presence of some structural isomers in both newborn and adult venom B. jararaca N-glycans. The main N-glycans identified in both venoms are of the hybrid/complex type however some mannose-rich type structures were also detected. The N-glycan composition of newborn and adult venoms did not vary indicating that differences in the utilization of the N-glycosylation motif could be the explanation for the differences in the glycosylation levels indicated by the differential electrophoretic profiles previously reported for B. jararaca newborn and adult venoms.     

Proteomics analysis to compare the venom composition between Naja naja and Naja kaouthia from the same geographical location of eastern India: Correlation with pathophysiology of envenomation and immunological cross-reactivity towards commercial polyantivenom

ABSTRACT

Background : Cobra bite is frequently reported across the Indian subcontinent and is associated with a high rate of death and morbidity. In eastern India (EI) Naja naja and Naja kaouthia are reported to be the two most abundant species of cobra.

Research design and methods : The venom proteome composition of N. naja (NnV) and N. kaouthia (NkV) from Burdwan districts of EI were compared by separation of venom proteins by 1D-SDS-PAGE followed by LC-MS/MS analysis of protein bands. The potency of commercial polyantivenom (PAV) was assessed by neutralization, ELISA, immuno-blot and venom-PAV immunoaffinity chromatography studies.

Results : Proteomic analysis identified 52 and 55 proteins for NnV and NkV, respectively, when searched against the Elapidae database. A small quantitative difference in venom composition between these two species of cobra was observed. PAVs exhibited poor cross-reactivity against low molecular mass toxins (<20 kDa) of both cobra venoms, which was substantiated by a meager neutralization of their phospholipase A₂ activity. Phospholipase A₂ and 3FTx, the two major classes of nonenzymatic and enzymatic proteins, respectively, were partially recognized by PAVs.

Conclusions : Efforts must be made to improve immunization protocols and supplement existing antivenoms with antibodies raised against the major toxins of these venoms.     

Venoms, venomics, antivenomics

Venoms comprise mixtures of peptides and proteins tailored by Natural Selection to act on vital systems of the prey or victim. Here we review our proteomic protocols for uncoiling the composition, immunological profile, and evolution of snake venoms. Our long-term goal is to gain a deep insight of all viperid venom proteomes. Knowledge of the inter- and intraspecies ontogenetic, individual, and geographic venom variability has applied importance for the design of immunization protocols aimed at producing more effective polyspecific antivenoms. A practical consequence of assessing the cross-reactivity of heterologous antivenoms is the possibility of circumventing the restricted availability of species-specific antivenoms in some regions. Further, the high degree of target specificity makes toxins valuable scaffolds for drug development.     

Proteomic analysis of the venom of the predatory ant Pachycondyla striata (Hymenoptera: Formicidae)

The ants use their venom for predation, defense, and communication. The venom of these insects is rich in peptides and proteins, and compared with other animal venoms, ant venoms remain poorly explored. The objective of this study was to evaluate the protein content of the venom in the Ponerinae ant Pachycondyla striata. Venom samples were collected by manual gland reservoir dissection, and samples were submitted to two‐dimensional gel electrophoresis and separation by ion‐exchange and reverse‐phase high‐performance liquid chromatography followed by mass spectrometry using tanden matrix‐assisted laser desorption/ionization with time‐of‐flight (MALDI‐TOF/TOF) mass spectrometry and electrospray ionization‐quadrupole with time‐of‐flight (ESI‐Q/TOF) mass spectrometry for obtaining amino acid sequence. Spectra obtained were searched against the NCBInr and SwissProt database. Additional analysis was performed using PEAKS Studio 7.0 (Sequencing de novo). The venom of P. striata has a complex mixture of proteins from which 43 were identified. Within the identified proteins are classical venom proteins (phospholipase A, hyaluronidase, and aminopeptidase N), allergenic proteins (different venom allergens), and bioactive peptides (U10‐ctenitoxin Pn1a). Venom allergens are among the most expressed proteins, suggesting that P. striata venom has high allergenic potential. This study discusses the possible functions of the proteins identified in the venom of P. striata.     

Snake venomics of the Lesser Antillean pit vipers Bothrops caribbaeus and Bothrops lanceolatus: correlation with toxicological activities and immunoreactivity of a heterologous antivenom

The venom proteomes of the snakes Bothrops caribbaeus and Bothrops lanceolatus, endemic to the Lesser Antillean islands of Saint Lucia and Martinique, respectively, were characterized by reverse-phase HPLC fractionation, followed by analysis of each chromatographic fraction by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. The venoms contain proteins belonging to seven ( B. caribbaeus) and five ( B. lanceolatus) types of toxins. B. caribbaeus and B. lanceolatus venoms contain phospholipases A 2, serine proteinases, l-amino acid oxidases and zinc-dependent metalloproteinases, whereas a long disintegrin, DC-fragments and a CRISP molecule were present only in the venom of B. caribbaeus, and a C-type lectin-like molecule was characterized in the venom of B. lanceolatus. Compositional differences between venoms among closely related species from different geographic regions may be due to evolutionary environmental pressure acting on isolated populations. The venoms of these two species differed in the composition and the relative abundance of their component toxins, but they exhibited similar toxicological and enzymatic profiles in mice, characterized by lethal, hemorrhagic, edema-forming, phospholipase A 2 and proteolytic activities. The venoms of B. caribbaeus and B. lanceolatus are devoid of coagulant and defibrinogenating effects and induce only mild local myotoxicity in mice. The characteristic thrombotic effect described in human envenomings by these species was not reproduced in the mouse model. The toxicological profile observed is consistent with the abundance of metalloproteinases, PLA 2s and serine proteinases in the venoms. A polyvalent (Crotalinae) antivenom produced in Costa Rica was able to immunodeplete approximately 80% of the proteins from both B. caribbaeus and B. lanceolatus venoms, and was effective in neutralizing the lethal, hemorrhagic, phospholipase A 2 and proteolytic activities of these venoms.     

Analysis of the subproteomes of proteinases and heparin‐binding toxins of eight Bothrops venoms

Viperid snakes show the most complex snake‐venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2‐DE, and their subproteomes of proteinases were explored by 2‐D immunostaining and 2‐D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti‐coagulant and anti‐inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high‐affinity for heparin as composed of mainly serine proteinases and C‐type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin‐binding toxins. Only the non‐bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin‐bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.     

2014蛋白质组学专刊序言

蛋白质组学研究是后基因组学时代最重要的功能基因组学研究之一,与医学生物学、化学、物理学、信息学以及现代技术等关系十分密切。为了检阅近年来国内外蛋白质组学某些重要研究进展,探索其可能的应用范围,讨论其存在的问题,展望其发展前景,特组织出版"蛋白质组学专刊"。本期专刊包括综述和研究论文两部分,内容主要涉及不同物种(包括人类、哺乳类动物、原核生物、放线菌等)蛋白质组学研究、蛋白质组学重要方法学与技术研究(包括串联质谱分析、尿蛋白膜保存法、定量蛋白质组学分折、meta分析等)和蛋白质组功能与应用研究(包括蜘蛛毒素蛋白质组、磷酸化蛋白质组、卵母细胞和早期胚胎蛋白质组、肝脏纤维化蛋白质组、分枝杆菌耐药的蛋白质组等)。     

Proteome analysis of snake venom toxins: pharmacological insights

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques. Detailed knowledge of the venom toxin composition and biological properties of the protein constituents should provide the scaffold for the design of new more effective drugs for the treatment of the hemostatic system and heart disorders, inflammation, cancer and consequences of snake bites, as well as new tools for clinical diagnostic and assays of hemostatic parameters.     

Snake venomics and antivenomics of Bothrops atrox venoms from Colombia and the Amazon regions of Brazil, Perú and Ecuador suggest the occurrence of geographic variation of venom phenotype by a trend towards paedomorphism

The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA₂ molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA₂ molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.