Venomics


Venomics is the study of proteins associated with venom, a toxic substance secreted by animals, which is typically injected either offensively or defensively into prey or aggressors, respectively.

Background

Venom is produced in a specialised gland and is delivered through hollow fangs or a stinger in a process called envenomation. The main function of venom is to disrupt the physiological processes of the wounded animal through neurotoxic, cytotoxic, myotoxic, or haemotoxic mechanisms. This can then help in certain processes such as procuring prey or in defense from predators. Venom has evolved many times in multiple phyla, each having developed their own unique types of venom and methods of delivery independently. However, due to the excessive amounts of venomous animals in the world, they are the major cause of animal-related deaths than non-venomous animals. For example, globally, someone is bitten by a snake every 10 seconds, according to estimates. Snakes are responsible for more than 5.4 million biting-injuries, resulting to 1.8 - 2.7 million envenomings and around 81,410 to 137,880 deaths annually. Bites by venomous snakes can cause acute medical emergencies involving severe paralysis that may prevent breathing, cause bleeding disorders that can lead to fatal haemorrhage, cause irreversible kidney failure and severe local tissue destruction that can cause permanent disability and limb amputation. Children may suffer more severe effects and can experience the effects more quickly than adults due to their smaller body mass. With venomic methods, venom can be co-opted into beneficial substances such as new medicines and effective insecticides. For instance, Captopril®, Integrilin® and Aggrastat® are drugs based on snake venoms, which have been approved by the FDA. In addition to these approved drugs, many other snake venom components are now involved in preclinical or clinical trials for a variety of therapeutic applications.

The Creation and History of Venomics Techniques

Venom is made up of multiple proteinous components, with each component differing in its structural complexity. Venom can be a mixture of simplistic peptides, secondary structured proteins and tertiary structured proteins. Furthermore, depending on the organism, there can be fundamental differences in the strategies they incorporate in their venom contents, the biggest difference being between invertebrates and vertebrates. For example, the majority of funnel-web spider's venom was made up of peptides between 3-5 KDa, with the remaining peptides being between 6.5 and 8.5 KDa in mass. Conversely, snake venom is made up of more complex protein such as modified saliva proteins and protein families that have had their genes recruited from other tissue groups. Due to this extraordinary amount of variation in the components that make up venom, a new field was needed to identify and categorise the millions of bioactive molecules that are found within the venom. Therefore, by combining the methods of multiple fields such as genomics, transcriptomics, proteomics and bioinformatics, an aptly named new field emerged named venomics.
Venomics was first established in the latter half of the 20th century as different ‘-omic’ technologies began to rise in popularity. However, the progression of venomics since its inception has always been reliant on and limited by the advancement of technology. Juan Calvete draws attention to this with explicitly when detailing the history of venomics. He declares that the last revolutions made in venomics research in the last decade are the direct result of advancements made in proteomic-centered methods and the indirect result of more widely available and cost-effective forms of transcriptomics and bio-informatics analysis. One of the first popular research topics of venomics was the pharmacological properties of the polypeptide toxins found in snake venom due to the neurotoxic properties and their ability to cause respiratory failure in animals. However, due to the lack of competent technology, less complex techniques, followed by simplistic chromatography and electrophoresis analysis, research was limited.
Evidence of early interest in snake venom was prevalent throughout the early 20th century with one of the first big breakthroughs being in the mid-1960s. For example, Halbert Raudonat was one of the first researchers to fractionate Cobra venom using a sophisticated dialysis and paper chromatography techniques. Furthermore, Evert Karlsson and David Eaker were able to successfully purify the specific neurotoxins found in Cobra venom and found that those isolated polypeptides had a consistent molecular weight of around 7000.
Future research in this field would eventually lead to indirect predictive models and then direct crystal structures of important many protein superfamilies. For example, Barbara Low was one of the first to release a 3D structure of the three-finger protein, Erabutoxin-b. TFPs are an example of α-Neurotoxins, they are small in structure and are a predominant component found in many snake venoms.

The Current State and Methodology of Venomics

Retrospectively, venomics has made a lot of progress in sequencing and creating accurate models of toxic molecules through current advanced methods. Through these methods, global categorisation of venoms has also taken place, with previously studied venoms being documented and widely available. An example of this would be the ‘Animal toxin annotation project’, which is a database that aims to provide a high quality and freely available source of protein sequences, 3D structures and functional information on thousands of animal venom/poisons. So far, they have categorised over 6,500 toxins at the protein-level, with the overall UniProt organisation having reviewed over 500,000 proteins and provided the proteomes of 100,000 organisms. However, even with today's technology the deconstruction and cataloguing of the individual components of what makes up an animal's venom takes a large amount of time and resources due to the overwhelming amount of molecules that are found in a single venom sample. This is complicated further when there are some animals that can change the complexity and make-up of their venom depending on the circumstances of the envenoming. Furthermore, inter-specific differences exist between male and female of a species with their venoms varying in quantities and toxicity.
Professor Juan J. Calvete is a prolific researcher in venomics at the biomedical institute in Valencia and has extensively explained the process involved in untangling and analysing venom Venom collection, Separation and quantification, Identification and Representation of components found.

(1) Venom collection methods

Venom milking is the most simplistic way of collecting a venom sample. It usually involves a vertebrate animal to deliver a venomous bite into a container. Similarly, electrical stimulation can be used for invertebrate animal subjects. This practice has allowed for the discovery of the basic properties of venom and to understand the biological factors involved in venom production such as venom regeneration periods. Other methods involve post-mortem dissection of the venom glands to collect the required materials.

(2) Separation and quantification methods

Separation methods are the first step to decomplexify the venom sample, with a common method being reverse‐phase high performance liquid chromatography. This method can be applied broadly to nearly all venoms as a crude fractionation method and to detect the peptide bonds found. A less common techniques like 1D/2D gel electrophoresis can also be used in cases of venoms containing heavy, complex peptides. This means in additions to RP-HPLC, Gel electrophoresis can help identify large molecules and to help refine venom prior to further analytical methods. Next, N-terminal sequencing is used to find the amino acid order of the fractionated proteins/peptides starting with the N-terminal end. Furthermore, SDS‐PAGE can be performed on the isolated proteins from the RP-HPLC to identify proteins of interest before moving on to the identification stage.

(3) Identification methods

There are two predominantly used proteomic methods when identifying the structure of a peptide/protein, Top-down proteomics and Bottom-up proteomics. TDP involves taking fractionated venom samples and analysing those peptides/proteins with Liquid chromatography tandem-mass spectrometry. This results in the identification and characterisation of all peptides/proteins present in the initial sample. While, BUP consists of fractionating and breaking down the peptides/proteins before analysis using chemical reduction, alkylating and enzymatic digestion. BUP is more commonly used than TDP as breaking down the samples allows the components to meet the ideal mass range for LC-MS/MS analysis. However, there are disadvantages and limitations with both identification methods. BUP results are prone to protein inference problems as large toxins can be broken down into smaller toxins which are shown in the output, but do not exist naturally within the venom sample. While, TDP is the newer method and is able to fill-in the gaps BUP leaves, TDP needs instruments with high amounts of resolving power. Most studies will actually use both methods in parallel to obtain the most accurate results. Furthermore, transcriptomic/genomic methods can be used to create cDNA libraries from the extracted mRNA molecules expressed in the venom glands of a venomous animal. These methods optimise the protein identification process by producing the DNA sequences of all proteins expressed in the venom glands. A large problem in using transcriptomic/genomic analysis in venomic studies is the lack of full genome sequences of many venomous animals. However, this is a fleeting problem due to the amount of full genome projects involved in sequencing venomous animals such as the ‘venomous system genome project’. Through these projects, various fields of study such as ecological/evolutionary studies and venomic studies can provide supporting information and systematic analysis of toxins.