Team:DTU-Denmark/ExperimentalDesign
Experimental Design
Venom composition
Snake venom is a complex mixture of proteins, enzymes and other substances with toxic and lethal properties. Its function it to defend against threats, immobilize the prey, and help the digestion. Some of the proteins in the snake venom have very specific effects on biological functions, such as blood coagulation, blood pressure regulation and transmission of nervous or muscular impulses.
Proteins constitute 90-95% of venom’s dry weight and are responsible for most of its biological effects. Enzymes make up 80-90% of the Viperidae family venom and 25-70% of the Elapidae family venom [1]. The most common enzymes are oxidases, phospholipases, metalloproteinases, hydrolases and serine proteases. Polypeptide toxins include cytotoxins (effects mainly on local tissue), cardiotoxins (effects on heart tissue) and neurotoxins (effects on nerve tissue).
Venomic studies have enabled the characterization of venom composition, from snakes species of medical importance [2]. From these, it can be shown that the venom between snake species, even within families, is more similar than expected [3]. Major protein families like metalloproteinases (SVMPs), Phospholipases A2 (PLA2), cysteine rich secretory proteins (CRISPs) are present in all snake families, with the relative abundance in the venom composition being the only difference [4].
However, these studies have also shown that there are certain enzymes that are probably innate to snake families, for example the prominent serine proteases in the Viperidae venom, or the much more abundant three finger toxins (3FTx) in Elapidae venom.
Choice of enzymatic assay
In Australia, immunoassays based on venom-specific antibodies have been developed to distinguish between the most lethal snakes in that region [5]. Immunoassays have been used or suggested to be used as a detection mechanism for snake venom for many years [5,6].
We apply an alternative approach to detect snake venom circumventing immunoassay based methods. We want to create a proteolytic enzyme assay based on a linker sequence which contains a cleavage site for proteases that are characteristic for the venom of different snakes.
Proteins that give off signals when a linker sequence is cleaved, are not a new invention. FRET (Föster resonance energy transfer) has been used for many years in research, to gain a better understanding of protein-protein interactions.
Our approach to snake venom detection, utilising the proteolytic activity of different venoms, has several potential advantages compared to current methods used for snake venom detection:
- - The recombinant proteins can be produced at a lower price compared to the current immunological methods, which involves inoculating snake venom in horses.
- - With this approach, we are able to find new potential target peptides for snake venom cleavage in a relatively fast and cheap way. Screening of these peptides is much less time consuming and easier than trying to identify suitable antibodies for the venom enzymes and toxins.
- - If reliable measurements can be made, we might be able to not only detect which snake people have been bitten by, but also to determine the level of venom injected into the blood.
Choice of snakes
The focus of our project was sub-Saharan Africa, where snakebites are more common than other regions of the continent. Approximately 1 million snakebites occur in sub-Saharan Africa annually, resulting in up to 500.000 envenomations, 25.000 deaths and another 20.000 permanent disabilities. Due to no reliable reporting system in place, victims often never report their injury to clinical facilities, which makes these numbers uncertain [7].
In sub-Saharan Africa, over 50% of snakebite incidents are not appropriately treated. Most victims, who receive treatment by healthcare professionals, have nevertheless delayed seeking medical attention sometimes up to 1 to 2 weeks. In many sub-Saharan countries, poor availability of expensive antivenom contributes to increased morbidity and mortality, whilst snakebites continue to remain a neglected health problem.
In that region, approximately 60% of all bites are caused by vipers, with the puff adder (Bitis Arietans) being responsible for the most fatalities overall. For that reason, we chose these species to be of primary focus in our experiments.
Sub-Saharan Africa also hosts cobra species of the Elapidae family, which in forested areas, cause 30% of all venomous bites. One of the most prominent of them is black-necked spitting cobra (Naja nigricollis), which was also chosen as a representative of the Elapidae family for our project.
Finally, another important snake species of that area is a close relative of the puff adder, the gaboon viper (Bitis gabonica). This snake is not responsible for a major percentage of deaths, but is also very dangerous due to the enormous amount of venom that it injects with its bite, which is the highest among all snakes. This snake was chosen in order to explore the possibility of distinguishing snake venom at the species level.
AMC experiment
7-Amino-4-MethylCoumarin is a fluorescent compound that can be used to create fluorogenic oligopeptides for detection of proteolytic activity. AMC has been used for protease activity assays since 1976 [8], and other amine species, such as nitroanilides, have been used for even longer [9].
AMC emits light at 460 nm, with a maximum excitation at 366 nm.
Due to the primary amine in the aromatic ring, AMC is able to bind to the C-terminal end of peptides. Thus, the AMC molecule will emit light only if the bond between the C-terminal peptide and AMC is cleaved.
Among the major classes of proteases, serine proteases are one that stands out when analysing snake venoms. As explained in the background page, serine proteases are found predominantly in the venoms of snakes belonging in the viperidae family, with only a few exceptions in other families, such as the elapids. By targeting serine proteases with synthetically designed oligopeptides, our team hypothesised that it would be possible to distinguish snake venom from viperidae from other snake families.
To test our hypothesis, we identified some oligopeptides that have been used in the past to characterise snake venom from different Bitis species. Although studies have been able to document the ability of Bitis venom to cleave blood factors such as fibrin and bradykinin/kallidin [10, 11], no comparative study has ever been carried out, researching the ability to cleave these substrates by snake venoms from across taxonomical families.
We found a suitable peptide substrate in literature [11], and we conducted our initial experiment to test our hypothesis with that specific peptide. The results can be found here.
References
[1] Bauchot, R (1994). Snakes: A Natural History. Sterling Publishing Co., pp. 194–209.
[2] Fasoli E, Sanz L, Wagstaff S, Harrison RA, Righetti PG, Calvete JJ (2010). Exploring the venom proteome of the African puff adder, Bitis arietans, using a combinatorial peptide ligand library approach at different pHs. J Proteomics.,73(5):932-42.
[3]Calderón-Celis F, Cid-Barrio L, Encinar JR, Sanz-Medel A, Calvete JJ (2017). Absolute venomics: Absolute quantification of intact venom proteins through elemental mass spectrometry. J Proteomics., 164:33-42.
[4] Calvete JJ, Marcinkiewicz C, Sanz L (2007). Snake venomics of Bitis gabonica gabonica. Protein family composition, subunit organization of venom toxins, and characterization of dimeric disintegrins bitisgabonin-1 and bitisgabonin-2. J Proteome Res., 6(1):326-36.
[5] David R, Theakston G and Laing GD (2014). Diagnosis of Snakebite and the Importance of Immunological Tests in Venom Research Toxins, 6(5), 1667-1695.
[6] K Silamut, M Ho, S Looareesuwan, C Viravan, V Wuthiekanun, and D A Warrell (1987). Detection of venom by enzyme linked immunosorbent assay (ELISA) in patients bitten by snakes in Thailand. Br Med J (Clin Res Ed)., 294(6569): 402–404.
[7] Mallow D, Ludwig D and Nilson G (2004). True Vipers: Natural History and Toxinology of Old World Vipers. Krieger Publishing Company.
[8] Zimmerman M, Yurewicz E and Patel G (1976). A new fluorogenic substrate for chymotrypsin Analytical Biochemistry 70(1), pp258-262
[9] Erlanger BF, Kokowsky N and Cohen W (1961). The preparation and properties of two new chromogenic substrates of trypsin Archives of Biochemistry and Biophysics, 95(2),pp 271-278
[10] Mallow D, Ludwig D and Nilson G (2004). True Vipers: Natural History and Toxinology of Old World Vipers. Krieger Publishing Company.
[11] Mallow D, Ludwig D and Nilson G (2004). True Vipers: Natural History and Toxinology of Old World Vipers. Krieger Publishing Company.
