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:

1. - The recombinant proteins can be produced at a lower price compared to the current immunological methods, which involves inoculating snake venom in horses.
2. - 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.
3. - 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.

Substrate screning

In order to distinguish different snake species, we needed to be able to detect enzymatic activity that was unique to the given species. Our study of literature indicated that some proteases found in the snake venom, are unique to the venom of the Viperidae family [2]. For this reason, we decided to focus specifically on these proteases.

One of the most efficient ways to study protease activities and substrate specificity is the incubation of a collection of potential substrate peptides with the proteases, or in our case, the venom of interest.

As a collection of these substrates, JPT Peptide Technologies’ Protease Substrate Set was found to be the one more suited to our needs. This set is comprised of 360 peptides derived from cleavage sites described in the scientific literature. These peptide derivatives contain the cleavage site sequences, flanked by a quencher molecule (DABCYL) and a fluorophore Glu(EDANS)-amide at the N-terminus and the C-terminus, respectively. Subsequent to incubation with the snake venom, these two moieties are separated. The emitted fluorescence can be detected using standard microtiter plate readers.

The results of this experiment can be found here.

Biosensor design

Our team aims to develop a novel biosensor to detect and distinguish between snake venoms from different families of snakes. Our biosensor is based on the different proteolytic activity of snake venom from different species.

The biosensor consists of a fusion protein. Inspired by the design of different FRET reporters, our fusion protein contains two larger protein domains, separated by a flexible linker containing one or more substrate sequences. However, unlike FRET reporters, where fluorescent proteins make up the two larger domains, the two large protein domains in our biosensor are a chromoprotein domain and a binding domain. The linker between the two domains is designed to contain the specific cleavage sites found through the previously described AMC- and substrate screening experiments.

In this project, our team has used the RFC 25 standard (Freiburg standard) to assemble our protein domains in-frame. The standard RFC 10 standard of the iGEM competition does not give the option to assemble parts in-frame, as the ligation between XbaI and SpeI will leave an asymmetric scar.

Complying with the iGEM paradigm of protein domains, we fused the DNA sequences of our different parts in silico, and ordered most of our fusion-proteins as single DNA fragments.

Ideally, when using our BioBrick device to detect snake venom in a clinical setting, the output signal should be sufficiently strong to give a definite result within a short amount of time. In order to make the output signal of our test easy to analyse without unnecessary equipment, we chose to incorporate a reporter molecule that could be analysed without the use of expensive equipment. Using chromoproteins, it would in theory be possible to visually analyse whether or not a person has been bitten by a specific type of snake.

AmilCP is a chromoprotein isolated from Acropora millepora, a coral native to the indo-pacific ocean. The chromoprotein absorbs light at 588 nm, giving it a purple-blue colour [12].

The Uppsala iGEM team has been working with a collection of different chromoproteins since 2011. From their continuous collection of chromoprotein BioBricks, AmilCP was chosen from Uppsalas collection, as it has a high molar extinction coefficient and can absorb light at wavelengths that are far away from the red blood cells from the blood we are testing on. Furthermore, our team used AmilCP as a reporter during the BioBrick Tutorial, and had thereby gained experience with expressing this particular BioBrick from a very early starting point.

Avidin can form non-covalent bonds to biotin with an unusually low association constant ( K_a=10¹⁵ M^-1 ) [13], which is a much higher affinity compared to normal protein-ligand interactions that have association constant of ~ 10¹¹) M⁻¹. This high affinity has been utilised for years for different chromatography purposes.

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] Nikai T, Momose M, Okumura Y, Ohara A, Komori Y and Sugihara H. (1993). TKallidin-Releasing Enzyme from Bitis arietans (Puff Adder) Venom Archives of Biochemistry and Biophysics, 307(2), pp 304-310
[11] Vaiyapuri S, Harrison RA, Bicknell AB, Gibbins JM and Hutchinson G (2010). Purification and Functional Characterisation of Rhinocerase, a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros PLoS ONE, 5(3): e9687.