Venomics: The Intricate Dance of Venom Proteins

Venomics, a specialized field of study, delves into the complex world of proteins found within venom . This potent substance, secreted by a diverse array of animals, serves as a biological weapon, injected into prey or adversaries for offensive or defensive purposes. Understanding venomics is not merely an academic pursuit; it’s a critical endeavor with profound implications for medicine, agriculture, and our fundamental comprehension of the natural world.

Background: The Potent World of Venom

At its core, venom is a sophisticated biological cocktail, meticulously crafted within specialized glands and delivered through specialized structures such as hollow fangs or stingers, a process known as envenomation. The primary objective of venom is to incapacitate its target by disrupting vital physiological processes. This disruption can manifest through a variety of mechanisms, including neurotoxic effects that target the nervous system, cytotoxic actions that damage cells, myotoxic effects on muscles, or haemotoxic mechanisms that interfere with blood coagulation. These actions are instrumental in aiding the predator in capturing prey or serving as a formidable defense against potential threats.

The evolutionary trajectory of venom is a testament to nature’s ingenuity, having emerged independently multiple times across various phyla. Each lineage has independently developed its own unique venom compositions and sophisticated delivery systems. The sheer abundance and diversity of venomous creatures worldwide underscore their significant impact on global health. While non-venomous animals account for a smaller number of fatalities, venomous animals are responsible for a substantial burden, with estimates suggesting around 57,000 deaths annually in 2013 alone. The statistics surrounding snakebites are particularly alarming: globally, an individual is bitten by a snake every ten seconds. Snakes are implicated in over 5.4 million biting incidents, leading to an estimated 1.8 to 2.7 million cases of envenomation and, tragically, between 81,410 and 137,880 deaths each year. The consequences of venomous snakebites can be dire, ranging from acute medical emergencies such as severe paralysis that compromises respiration, to bleeding disorders culminating in fatal hemorrhages, irreversible kidney failure, and extensive local tissue damage that can necessitate permanent disability or limb amputation. Children, due to their smaller body mass, are particularly vulnerable and can experience the severe effects of envenomation more rapidly than adults.

However, the narrative of venom is not solely one of danger. The very components that make venom so potent can be harnessed for beneficial purposes. Through the lens of venomics, these toxic substances are being investigated and co-opted into the development of novel pharmaceuticals and highly effective insecticides. A prime example of this therapeutic potential lies in drugs derived from snake venoms. Medications such as Captopril® (Enalapril), Integrilin® (Eptifibatide), and Aggrastat® (Tirofiban) have received approval from the FDA and are actively used in medical practice. Beyond these established treatments, numerous other venom components are currently undergoing preclinical and clinical trials for a wide array of therapeutic applications, highlighting the immense medicinal promise held within these complex biological mixtures.

The Creation and History of Venomics Techniques

The intricate tapestry of venom is woven from a multitude of proteinaceous components, each possessing its own unique structural complexity. This intricate composition can range from simple peptides to proteins with secondary structures like α-helices and β-sheets , and further to proteins exhibiting tertiary structures, forming intricate crystalline arrangements. The strategies employed by different organisms in assembling their venom arsenals exhibit profound variations, with significant distinctions observed between invertebrates and vertebrates. For instance, the venom of many funnel-web spiders is predominantly composed of peptides weighing between 3 to 5 KDa, accounting for approximately 75% of the total venom mass, with the remaining peptides falling within the 6.5 to 8.5 KDa range. In stark contrast, snake venom is characterized by the presence of more complex proteins, including modified salivary proteins such as CRISPs and kallikreins, as well as protein families whose genes have been recruited from other tissue groups, such as acetylcholinesterase, crotasin, defensin, and cystatin.

The sheer diversity and vast number of bioactive molecules found within venom necessitated the development of a new field dedicated to their identification and categorization. Thus, by integrating methodologies from various disciplines, including genomics , transcriptomics , proteomics , and bioinformatics , the aptly named field of venomics emerged.

The foundational principles of venomics began to take shape in the latter half of the 20th century, coinciding with the burgeoning popularity of various ‘-omic’ technologies. However, the progress of venomics has always been intrinsically linked to, and often constrained by, the advancements in technological capabilities. Juan Calvete keenly illustrates this dependency when recounting the history of venomics, asserting that the significant breakthroughs in venomic research over the past decade (referring to the period between 1989 and 1999) were a direct consequence of progress in proteomic-centered methods and an indirect benefit from the increasing accessibility and cost-effectiveness of transcriptomic and bioinformatic analyses. Early research in venomics largely focused on the pharmacological properties of polypeptide toxins found in snake venom, particularly those from the Elapidae and Hydrophidae families, owing to their potent neurotoxic effects and their capacity to induce respiratory failure. However, the limited technological sophistication of the era, which relied on less complex techniques such as dialysis for venom separation, followed by rudimentary chromatography and electrophoresis analysis, significantly restricted the scope of research.

(Left) The amino acid structure, (Middle) diagram and (Right) Stereodiagram of k-Bungarotoxin. [13]

Early indications of scientific interest in snake venom can be traced back to the early 20th century, with a notable breakthrough occurring in the mid-1960s. For instance, Halbert Raudonat pioneered early efforts by fractionating the venom of the Cobra (Naja nivea ) using a combination of dialysis and paper chromatography , employing techniques considered sophisticated for the time. Subsequently, Evert Karlsson and David Eaker achieved a significant milestone by successfully purifying specific neurotoxins from the venom of the Black-necked spitting cobra (Naja nigricollis ), determining that these isolated polypeptides possessed a consistent molecular weight of approximately 7000 Da.

Further investigations in this field eventually paved the way for the development of indirect predictive models and, subsequently, the direct determination of crystal structures for numerous important protein superfamilies. [16] [13] For example, Barbara Low was among the first to elucidate the three-dimensional structure of a three-finger protein (TFP), specifically Erabutoxin-b. TFPs represent a class of α-Neurotoxins ; they are relatively small in structure, typically comprising around 60-80 amino acids, and constitute a dominant component in the venoms of many snake species, often making up 70% to 95% of all toxins present. [18] [19]

The Current State and Methodology of Venomics

In retrospect, venomics has witnessed remarkable advancements in its capacity to sequence and construct accurate models of toxic molecules, largely driven by contemporary analytical methods. These sophisticated techniques have facilitated a global cataloging of venoms, with previously studied venoms now meticulously documented and widely accessible. A prime example of this initiative is the ‘Animal toxin annotation project,’ maintained by UniProtKB/Swiss-Prot. This database endeavors to provide a high-quality, freely accessible repository of protein sequences, three-dimensional structures, and functional information pertaining to thousands of animal venoms and poisons. To date, this project has cataloged over 6,500 toxins at the protein level, while the broader UniProt organization has reviewed more than 500,000 proteins and provided proteomic data for 100,000 organisms.

However, even with the sophisticated technology available today, the exhaustive deconstruction and cataloging of the individual components within a single venom sample remain a time-consuming and resource-intensive undertaking due to the sheer volume and complexity of molecules involved. This complexity is further amplified by the existence of certain animals, such as cone snails, which possess the remarkable ability to dynamically alter the complexity and composition of their venom depending on the specific context, whether it be for offensive predation or defensive purposes. [20] Moreover, inter-specific variations are commonly observed between male and female individuals of the same species, with their venoms exhibiting differences in both quantity and toxicity. [21]

A typical workflow for the isolation and screening of compounds found in venom. [22]

Professor Juan J. Calvete, a distinguished researcher in the field of venomics at the biomedical institute in Valencia, has meticulously detailed the intricate process involved in unraveling and analyzing venom. His insights, presented in publications from 2007 and more recently in 2017, outline a systematic approach comprising four key stages: (1) venom collection, (2) separation and quantification, (3) identification, and (4) representation of the identified components. [23] [22]

(1) Venom Collection Methods

Venom milking represents the most straightforward method for acquiring a venom sample. This typically involves inducing a venomous animal, most commonly a snake, to deliver a bite into a collection receptacle. For invertebrate subjects, such as insects and arachnids, electrical stimulation can be employed to elicit venom release. [24] This practice has been instrumental in revealing the fundamental properties of venoms and in elucidating the biological factors governing venom production, including the mechanisms of venom regeneration. Alternative methods involve the post-mortem dissection of venom glands to procure the necessary materials, either venom itself or gland tissue for further analysis.

(2) Separation and Quantification Methods

Separation techniques serve as the initial crucial step in deconstructing the complex venom sample. A widely adopted and versatile method is reverse-phase high-performance liquid chromatography (RP-HPLC ). This technique is broadly applicable to most venoms, serving as an effective crude fractionation method and enabling the detection of peptide bonds. Less commonly employed, but still valuable, are techniques like 1D/2D gel electrophoresis, which are particularly useful for venoms containing heavy, complex peptides, typically those exceeding 10 KDa in molecular weight. In conjunction with RP-HPLC, gel electrophoresis can aid in the identification of large molecules, such as enzymes, and further refine the venom sample prior to more advanced analytical procedures. [2] Following fractionation, N-terminal sequencing is utilized to determine the precise order of amino acids in the purified proteins or peptides, starting from the N-terminal end. [25] Additionally, SDS‐PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis) can be performed on proteins isolated via RP-HPLC to identify proteins of particular interest before proceeding to the identification stage. [23]

(3) Identification Methods

(Left) Representation of Bottom-up and Top-down proteomic analysis. (Right) Similarities and differences between the Proteomic and the Transcriptomics/Genomics analytical methods. [26]

Two primary proteomic methodologies are predominantly employed for identifying the structure of peptides and proteins within venom: Top-down proteomics (TDP ) and Bottom-up proteomics (BUP ). TDP involves the direct analysis of fractionated venom samples using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS ). This approach yields the identification and characterization of all peptides and proteins present in the original sample. In contrast, BUP entails the fractionation and subsequent breakdown of peptides and proteins into smaller fragments before analysis by LC-MS/MS. This fragmentation is typically achieved through chemical reduction, alkylation, and enzymatic digestion, most commonly employing trypsin. BUP is more frequently utilized than TDP because breaking down the samples allows the resulting components to fall within the optimal mass range for LC-MS/MS analysis. [26] [2]

However, both identification methods present their own set of disadvantages and limitations. BUP results can be susceptible to protein inference issues, where large toxins might be fragmented into smaller components that appear in the output but do not exist as distinct entities within the natural venom sample. TDP, while a more recent and capable method for filling the gaps left by BUP, requires instruments with exceptionally high resolving power, typically 50,000 or above. Consequently, most studies opt to employ both methods concurrently to achieve the most accurate and comprehensive results. Furthermore, transcriptomic and genomic methods offer an alternative approach. These techniques involve creating complementary DNA (cDNA ) libraries from messenger RNA (mRNA ) molecules extracted from the venom glands of venomous animals. By providing the DNA sequences of all proteins expressed within the venom glands, these methods can significantly optimize the protein identification process. A significant hurdle in applying transcriptomic/genomic analysis to venomic studies is the frequent lack of complete genome sequences for many venomous species. Nevertheless, this challenge is progressively being overcome due to the increasing number of full genome sequencing projects targeting venomous animals, such as the ‘venomous system genome project’ initiated in 2003. [27] Through these ambitious projects, diverse fields of study, including ecological, evolutionary, and venomic research, can derive invaluable supporting information and conduct systematic analyses of toxins.

(4) Accurate Representation of Components

The findings derived from (Left) proteomic practices and (Right) transcriptomic practices when analyzing the venom of Bothropoides pauloensis. [28]

Renata Rodrigues conducted a comprehensive study detailing both the proteome and the transcriptome of the Neuwied’s Lancehead (Bothropoides pauloensis), employing all the aforementioned analytical methods. [28] The proteomic analysis revealed the presence of nine distinct protein families, with the majority of venom components belonging to snake venom metalloproteinases (38%), phospholipase A2 (31%), and Bradykinin-potentiating peptides/C-type natriuretic peptides (12%). The transcriptomic analysis yielded over 1100 expressed sequence tags (ESTs ), of which 688 sequences were identified as originating from the venom gland. Similarly, the transcriptome data corroborated the proteomic findings, with SVMPs constituting the largest proportion of ESTs (36%), followed by PLA2 (26%) and BPP/C-NP sequences (17%). Crucially, this study demonstrates that by integrating both proteomic and transcriptomic approaches, a complete understanding of venom composition can be achieved. This comprehensive knowledge facilitates the elucidation of the molecular structure and functions of numerous bioactive components, which in turn opens avenues for bioprospecting venom components into new therapeutic agents and aids in the development of more effective antivenoms.

The Future Possibilities of Venomics

The field of venomics has undergone a profound transformation since its inception in the 20th century and continues to evolve with the integration of contemporary techniques such as next-generation sequencing and nuclear magnetic resonance spectroscopy . This ongoing trend suggests that venomics will witness progressively enhanced capabilities driven by the relentless technological advancements of the 21st century. As previously highlighted, a particularly promising avenue for further venomic exploration lies in the co-option of venom-specific molecules into specialized medicines. The pioneering example of this therapeutic paradigm emerged in the early 1970s with the discovery of Captopril , an inhibitor of angiotensin-converting enzymes (ACE ), which proved effective in treating hypertension. [29] Glenn King’s research illuminates the current landscape of venom-derived drugs, noting that six such drugs have received FDA approval, with an additional ten currently undergoing clinical trials. [30] Michael Pennington provides a detailed update on the evolving landscape of venom-derived pharmaceuticals and the future potential of this field, as summarized in Table 1. [6]

The development of improved anti-venoms represents another critical area where venomics can make significant contributions, particularly addressing the challenges faced by developing countries grappling with high incidences of venomous animal encounters. Regions such as South/Southeast Asia and sub-Saharan Africa bear a disproportionate burden of morbidity, often manifesting as limb amputation, and mortality due to venomous bites. [31] Snakes, especially those from the Elapidae and Viperidae families, are the primary culprits behind envenomings. Antivenoms, essential for treatment, remain in critically short supply in high-risk areas due to the strenuous and costly production methods (involving the immunization of animals) and the stringent storage requirements (requiring constant refrigeration below 0°C). Compounding these issues, the antivenoms themselves often exhibit limited efficacy against localized tissue damage and can provoke adverse reactions in patients, ranging from acute responses like anaphylaxis or pyrogenic reactions to delayed serum sickness-type reactions. [32] However, the application of various ‘omic’ technologies, particularly through the development of ‘Antivenomics,’ holds the potential to revolutionize antivenom production, leading to safer, more cost-effective, and less time-consuming methods for creating antivenoms effective against a broader spectrum of toxic organisms. Novel antivenom strategies are currently under investigation, including the use of monoclonal antibodies (mAbs ) and the expansion of comprehensive venomous databases, which facilitate more efficient screening for antivenom cross-reactivity. [33] [34]

Finally, venomic techniques offer promising avenues for enhancing agricultural practices through the development of insect-specific biopesticides derived from venom. Insects pose a significant threat to agriculture and horticulture, acting as pests and vectors for numerous parasites and diseases. [35] Consequently, effective insecticides are perpetually in demand to mitigate the detrimental impact of various insect species. However, many insecticides employed in the past have fallen out of favor and have been banned due to their adverse effects, such as harming non-target species (e.g., DDT ) or exhibiting high toxicity towards mammals (e.g., Neonicotinoids ). [36] Monique Windley proposes that venom from arachnids presents a compelling solution to this challenge. Arachnid venoms are rich in neurotoxic compounds, with an estimated ten million bioactive peptides identified, and are often highly specific in their action against insects. [7]

Table 1. Venom-derived medicines discussed by Pennington, Czerwinski et al., (2017). [6]