Spatial Venomics─Cobra Venom System Reveals Spatial Differentiation of Snake Toxins by Mass Spectrometry Imaging.

Among venomous animals, toxic secretions have evolved as biochemical weapons associated with various highly specialized delivery systems on many occasions. Despite extensive research, there is still limited knowledge of the functional biology of most animal toxins, including their venom production and storage, as well as the morphological structures within sophisticated venom producing tissues that might underpin venom modulation. Here, we report on the spatial exploration of a snake venom gland system by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), in combination with standard proteotranscriptomic approaches, to enable in situ toxin mapping in spatial intensity maps across a venom gland sourced from the Egyptian cobra (Naja haje). MALDI-MSI toxin visualization on the elapid venom gland reveals a high spatial heterogeneity of different toxin classes at the proteoform level, which may be the result of physiological constraints on venom production and/or storage that reflects the potential for venom modulation under diverse stimuli.

Modern venomics-Current insights, novel methods, and future perspectives in biological and applied animal venom research.

Venoms have evolved >100 times in all major animal groups, and their components, known as toxins, have been fine-tuned over millions of years into highly effective biochemical weapons. There are many outstanding questions on the evolution of toxin arsenals, such as how venom genes originate, how venom contributes to the fitness of venomous species, and which modifications at the genomic, transcriptomic, and protein level drive their evolution. These questions have received particularly little attention outside of snakes, cone snails, spiders, and scorpions. Venom compounds have further become a source of inspiration for translational research using their diverse bioactivities for various applications. We highlight here recent advances and new strategies in modern venomics and discuss how recent technological innovations and multi-omic methods dramatically improve research on venomous animals. The study of genomes and their modifications through CRISPR and knockdown technologies will increase our understanding of how toxins evolve and which functions they have in the different ontogenetic stages during the development of venomous animals. Mass spectrometry imaging combined with spatial transcriptomics, in situ hybridization techniques, and modern computer tomography gives us further insights into the spatial distribution of toxins in the venom system and the function of the venom apparatus. All these evolutionary and biological insights contribute to more efficiently identify venom compounds, which can then be synthesized or produced in adapted expression systems to test their bioactivity. Finally, we critically discuss recent agrochemical, pharmaceutical, therapeutic, and diagnostic (so-called translational) aspects of venoms from which humans benefit.

Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani.

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-µHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-µHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Hexapod Assassins' Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus.

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

Old World Vipers-A Review about Snake Venom Proteomics of Viperinae and Their Variations.

Fine-tuned by millions of years of evolution, snake venoms have frightened but also fascinated humanity and nowadays they constitute potential resources for drug development, therapeutics and antivenoms. The continuous progress of mass spectrometry techniques and latest advances in proteomics workflows enabled toxinologists to decipher venoms by modern omics technologies, so-called 'venomics'. A tremendous upsurge reporting on snake venom proteomes could be observed. Within this review we focus on the highly venomous and widely distributed subfamily of Viperinae (Serpentes: Viperidae). A detailed public literature database search was performed (2003-2020) and we extensively reviewed all compositional venom studies of the so-called Old-World Vipers. In total, 54 studies resulted in 89 venom proteomes. The Viperinae venoms are dominated by four major, four secondary, six minor and several rare toxin families and peptides, respectively. The multitude of different venomics approaches complicates the comparison of venom composition datasets and therefore we differentiated between non-quantitative and three groups of quantitative workflows. The resulting direct comparisons within these groups show remarkable differences on the intra- and interspecies level across genera with a focus on regional differences. In summary, the present compilation is the first comprehensive up-to-date database on Viperinae venom proteomes and differentiating between analytical methods and workflows.

Extended Snake Venomics by Top-Down In-Source Decay: Investigating the Newly Discovered Anatolian Meadow Viper Subspecies, Vipera anatolica senliki.

Herein, we report on the venom proteome of Vipera anatolica senliki, a recently discovered and hitherto unexplored subspecies of the critically endangered Anatolian meadow viper endemic to the Antalya Province of Turkey. Integrative venomics, including venom gland transcriptomics as well as complementary bottom-up and top-down proteomics analyses, were applied to fully characterize the venom of V. a. senliki. Furthermore, the classical top-down venomics approach was extended to elucidate the venom proteome by an alternative in-source decay (ISD) proteomics workflow using the reducing matrix 1,5-diaminonaphthalene. Top-down ISD proteomics allows for disulfide bond counting and effective de novo sequencing-based identification of high-molecular-weight venom constituents, both of which are difficult to achieve by commonly established top-down approaches. Venom gland transcriptome analysis identified 96 toxin transcript annotations from 18 toxin families. Relative quantitative snake venomics revealed snake venom metalloproteinases (42.9%) as the most abundant protein family, followed by several less dominant toxin families. Online mass profiling and top-down venomics provide a detailed insight into the venom proteome of V. a. senliki and facilitate a comparative analysis of venom variability for the closely related subspecies, Vipera anatolica anatolica.

Solenodon genome reveals convergent evolution of venom in eulipotyphlan mammals.

Venom systems are key adaptations that have evolved throughout the tree of life and typically facilitate predation or defense. Despite venoms being model systems for studying a variety of evolutionary and physiological processes, many taxonomic groups remain understudied, including venomous mammals. Within the order Eulipotyphla, multiple shrew species and solenodons have oral venom systems. Despite morphological variation of their delivery systems, it remains unclear whether venom represents the ancestral state in this group or is the result of multiple independent origins. We investigated the origin and evolution of venom in eulipotyphlans by characterizing the venom system of the endangered Hispaniolan solenodon (Solenodon paradoxus). We constructed a genome to underpin proteomic identifications of solenodon venom toxins, before undertaking evolutionary analyses of those constituents, and functional assessments of the secreted venom. Our findings show that solenodon venom consists of multiple paralogous kallikrein 1 (KLK1) serine proteases, which cause hypotensive effects in vivo, and seem likely to have evolved to facilitate vertebrate prey capture. Comparative analyses provide convincing evidence that the oral venom systems of solenodons and shrews have evolved convergently, with the 4 independent origins of venom in eulipotyphlans outnumbering all other venom origins in mammals. We find that KLK1s have been independently coopted into the venom of shrews and solenodons following their divergence during the late Cretaceous, suggesting that evolutionary constraints may be acting on these genes. Consequently, our findings represent a striking example of convergent molecular evolution and demonstrate that distinct structural backgrounds can yield equivalent functions.

Investigating the cytotoxic effects of the venom proteome of two species of the Viperidae family (Cerastes cerastes and Cryptelytrops purpureomaculatus) from various habitats.

Animal secretions are of great interest in terms of drug development due to their complex protein and peptide composition. Especially, in the field of therapeutic medications such as anti-cancer drugs snake venoms receive attention. In this study, we address two Viperidae species from various habitats with a particular focus on the cytotoxic potential along with the decomplexation of the venom proteome: the horned desert viper (Cerastes cerastes), native to desert regions of North Africa and the mangrove pit viper (Cryptelytrops purpureomaculatus), found in coastal forests of Southeast Asia. Initial cytotoxic screenings of the crude venoms revealed diverse activity, with the highest effect against SHSY5Y human glioblastoma carcinoma cells compared to other cancerous and non-cancerous cell lines. In-depth cytotoxicity studies of SHSY5Y cells with purified venom fractions revealed heterodimeric disintegrins from C. cerastes venom, which exerted a high cytotoxic activity with IC50 values from 0.11 to 0.58 µM and a disintegrin-like effect on SHSY5Y morphology was observed due to cell detachment. Furthermore, two polyproline BPP-related peptides, one PLA2 and a peptide-rich fraction were determined for C. purpureomaculatus with moderate IC50 values between 3 and 51 µM. Additionally, the decryption of the venom proteomes by snake venomic mass spectrometry and comparison of the same species from different habitats revealed slight differences in the composition.

Intact protein mass spectrometry reveals intraspecies variations in venom composition of a local population of Vipera kaznakovi in Northeastern Turkey.

We report on the variable venom composition of a population of the Caucasus viper (Vipera kaznakovi) in Northeastern Turkey. We applied a combination of venom gland transcriptomics, de-complexing bottom-up and top-down venomics. In contrast to sole bottom-up venomics approaches and gel or chromatography based venom comparison, our combined approach enables a faster and more detailed comparison of venom proteomes from multiple individuals. In total, we identified peptides and proteins from 15 toxin families, including snake venom metalloproteinases (svMP; 37.8%), phospholipases A2 (PLA2; 19.0%), snake venom serine proteinases (svSP; 11.5%), C-type lectins (CTL; 6.9%) and cysteine-rich secretory proteins (CRISP; 5.0%), in addition to several low abundant toxin families. Furthermore, we identified intraspecies variations of the venom composition of V. kaznakovi, and find these were mainly driven by the age of the animals, with lower svSP abundance detected in juveniles. On the proteoform level, several small molecular weight toxins between 5 and 8 kDa in size, as well as PLA2s, drove the differences observed between juvenile and adult individuals. This study provides novel insights into the venom variability of V. kaznakovi and highlights the utility of intact mass profiling for fast and detailed comparison of snake venom. BIOLOGICAL SIGNIFICANCE: Population level and ontogenetic venom variation (e.g. diet, habitat, sex or age) can result in a loss of antivenom efficacy against snakebites from wide ranging snake populations. The current state of the art for the analysis of snake venoms are de-complexing bottom-up proteomics approaches. While useful, these have the significant drawback of being time-consuming and following costly protocols, and consequently are often applied to pooled venom samples. To overcome these shortcomings and to enable rapid and detailed profiling of large numbers of individual venom samples, we integrated an intact protein analysis workflow into a transcriptomics-guided bottom-up approach. The application of this workflow to snake individuals of a local population of V. kaznakovi revealed intraspecies variations in venom composition, which are primarily explained by the age of the animals, and highlighted svSP abundance to be one of the molecular drivers for the compositional differences observed.

Comprehensive Snake Venomics of the Okinawa Habu Pit Viper, Protobothrops flavoviridis, by Complementary Mass Spectrometry-Guided Approaches.

The Asian world is home to a multitude of venomous and dangerous snakes, which are used to induce various medical effects in the preparation of traditional snake tinctures and alcoholics, like the Japanese snake wine, named Habushu. The aim of this work was to perform the first quantitative proteomic analysis of the Protobothrops flavoviridis pit viper venom. Accordingly, the venom was analyzed by complimentary bottom-up and top-down mass spectrometry techniques. The mass spectrometry-based snake venomics approach revealed that more than half of the venom is composed of different phospholipases A2 (PLA2). The combination of this approach and an intact mass profiling led to the identification of the three main Habu PLA2s. Furthermore, nearly one-third of the total venom consists of snake venom metalloproteinases and disintegrins, and several minor represented toxin families were detected: C-type lectin-like proteins (CTL), cysteine-rich secretory proteins (CRISP), snake venom serine proteases (svSP), l-amino acid oxidases (LAAO), phosphodiesterase (PDE) and 5'-nucleotidase. Finally, the venom of P. flavoviridis contains certain bradykinin-potentiating peptides and related peptides, like the svMP inhibitors, pEKW, pEQW, pEEW and pENW. In preliminary MTT cytotoxicity assays, the highest cancerous-cytotoxicity of crude venom was measured against human neuroblastoma SH-SY5Y cells and shows disintegrin-like effects in some fractions.

Comparative Venomics of the Vipera ammodytes transcaucasiana and Vipera ammodytes montandoni from Turkey Provides Insights into Kinship.

The Nose-horned Viper (Vipera ammodytes) is one of the most widespread and venomous snakes in Europe, which causes high frequent snakebite accidents. The first comprehensive venom characterization of the regional endemic Transcaucasian Nose-horned Viper (Vipera ammodytes transcaucasiana) and the Transdanubian Sand Viper (Vipera ammodytes montandoni) is reported employing a combination of intact mass profiling and bottom-up proteomics. The bottom-up analysis of both subspecies identified the major snake protein families of viper venoms. Furthermore, intact mass profiling revealed the presence of two tripeptidic metalloprotease inhibitors and their precursors. While previous reports applied multivariate analysis techniques to clarify the taxonomic status of the subspecies, an accurate classification of Vipera ammodytestranscaucasiana is still part of the ongoing research. The comparative analysis of the viper venoms on the proteome level reveals a close relationship between the Vipera ammodytes subspecies, which could be considered to clarify the classification of the Transcaucasian Nose-horned Viper. However, the slightly different ratio of some venom components could be indicating interspecific variations of the two studied subspecies or intraspecies alternations based on small sample size. Additionally, we performed a bioactivity screening with the crude venoms against several human cancerous and non-cancerous cell lines, which showed interesting results against a human breast adenocarcinoma epithelial cell line. Several fractions of Vipera a. transcaucasiana demonstrated a strong cytotoxic effect on triple negative MDA MB 231 breast cancer cells.

Combined venom profiling and cytotoxicity screening of the Radde's mountain viper (Montivipera raddei) and Mount Bulgar Viper (Montivipera bulgardaghica) with potent cytotoxicity against human A549 lung carcinoma cells.

Here we report the first characterization of the endemic Mount Bulgar Viper (Montivipera bulgardaghica) and Radde's mountain viper (Montivipera raddei) venom by a combined approach using intact mass profiling and bottom-up proteomics. The cytotoxicity screening of crude venom as well as isolated serine proteases revealed a high activity against A549 human lung carcinoma cells. By means of intact mass profiling of native and reduced venom we observed basic and acidic phospholipases type A2. Moreover, the analysis revealed snake venom metalloproteases, cysteine-rich secretory proteins, disintegrins, snake venom serine proteases, C-type lectins, a vascular endothelial growth factor and an L-amino acid oxidase.