MSc Chemistry Track Analytical Sciences
Literature thesis
SNAKEBITE: A GLOBAL HEALTH CRISIS – PATHOLOGIES
By Ingrida Bagdonaite UvA # 12780367 October 2020 12 ECTS June 2020 – October 2020 Supervisor: Mátyás Bittenbinder Examiner: Dr. Jeroen Kool Examiner: Dr. Rob Haselberg
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Acknowledgements
I want to thank Mátyás Bittenbinder for giving me a chance to work on this project. I not only enjoyed working on this literature thesis, but also learned a lot. I improved my research and writing skills, so this was a truly useful experience.
Also, I would like to thank Jeroen Kool and Rob Haselberg for being my examiners and hope you all enjoyed reading this paper.
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Abstract
Snakebite is a global tropical disease that is an important public health hazard in many regions. Despite it being a major cause of morbidity and mortality, mainly in impoverished areas, where health care system is poor, not a lot has been published on pathologies resulting upon envenomation, toxins involved and snake species capable of causing these effects. According to World Health Organization there are at least 1.8 – 2.7 million snake envenomations occurring every year, with more than 80,000 of them being lethal. Around 200 species are classified as medically important, as their envenomations can be the cause of pharmacological effects on humans, such as neurotoxicity, hemotoxicity and cytotoxicity. This paper will be one of the first to provide a comprehensive overview of these pathologies and most common toxins that cause these pathologies. Snake venom is a complex toxic secretion and most widely studied analytical methods for analysis of snake venom composition will also be discussed in more detail.
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Content
Acknowledgements 2 Abstract 3 List of abbreviations 5 1. Introduction 62. Medically important snakes 8
2.1. Snake venom composition 12
3. Pathologies 15
3.1. Neurotoxicity 15
3.2. Cytotoxicity 17
3.3. Hemotoxicity 19
4. Snake venom toxins 21
4.1. Multitoxins 21 4.2. Toxins 21 4.2.1. Phospholipases A2 21 4.2.2.1. Phospholipase A2 complexes 22 4.2.2.2. Covalent complexes 23 4.2.2.3. Non-covalent complexes 24 4.2.2. Three-finger toxins 25 4.2.3. Metalloproteases 26
4.2.4. Serine proteases and hyaluronidases 28
4.2.5. L-amino acid oxidases 28
5. Analytical methods to asses pathology 30
5.1. Top-down and Bottom-up proteomics 32
5.2. Gel-based approaches 34
5.3. Mass Spectrometry 35
5.4. Liquid Chromatography with Mass Spectrometry 35
5.5. Isotope Labelling 37
6. Conclusions 40
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List of abbreviations
2-D PAGE – Two-Dimensional Polyacrylamide Electrophoresis 3FTX – Three-finger toxin 3D – Three-dimensional Ach - Acetylcholine ACHE - Acetylcholinesterase ACN – AcetonitrileADAM – A Disintegrin And Metalloproteinase
Asp49 – Aspartic Acid-49 ATP – Adenosine Triphosphate b-BuTX – Beta-Burgarotoxin
CID – Collision Induced Dissociation CRISP – Cysteine-Rich Secretory Protein DNA – Deoxyribonucleic Acid
dTC – d-Tubocurarine DNTx – Deboia Neurotoxin ECM – Extracellular Matrix ESI – Electrospray Ionisation FA – Formic acid
FT – Fourier Transform Gln48 – Glutamine-48 HA – Hemagglutinin H2O2 – Hydrogen Peroxide
HPLC – High Performance Liquid Chromatography
ICAT – Isotope-Coded Affinity Tag iTRAQ – Isobaric Tagging for Relative and Absolute Quantitation
LAAO – L-amino Acid Oxidase
LC – Liquid Chromatography Lys49 – Lysine-49
MALDI – Matrix Assisted Laser Desorption Ionization
MMP – Matrix Metallopeptidase MS – Mass Spectrometry
nAChR – nicotinic Acetylcholine Receptor NMJ – Neuromuscular Junction ‘
pI – Isoelectric Point PL - Phospholipid
PLA2 – Phospholipase A2
PTM – Post Translational Modification PVDF – Polyvinylidene Difluoride RBC – Red Blood Cells
RP – Reversed Phase
SDS-PAGE – Sodium Dodecyl Sulfate Polyacrylamide Electrophoresis
SI-LAC –To Stable Isotope Labelling using Amino Acids in Cell Culture SNARE – SNAP Receptor
SoPIL – Soluble Polymer-based Isotope Labelling
SVH – Snake Venom Hyaluronidase SVMP – Snake Venom Metalloprotease SVSP – Snake Venom Serine Protease TFA – Trifluoroacetic Acid
TOF – Time-of-Flight TOF – Train-of-four
VAP1 – Vascular Apoptosis Protein 1 WHO – World Health Organization
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1. Introduction
Snakebite envenoming is a significant health issue, in many regions of the world, especially in rural regions of tropical and subtropical countries [1]. Snakebite is a major cause of morbidity and mortality, mainly in the impoverished areas, where health care systems are rather poor [2]. There are at least 1.8 – 2.7 million snake envenomations occurring every year, with estimated deaths ranging from 81,000, up to 138,000.[3] According to World Health Organization, currently there are around 3,000 snake species in the world with over 600 being classified as venomous and around 200 are considered to be medically significant species [4]. Snake venoms can produce a wide range of local and systematic effects in humans, with some causing permanent disability and some being life-threatening.
This neglected tropical disease is a result of injection of a highly complex mixture of toxic compounds that may cause a wide range of clinical effects when delivered into the victim. Snake venom is made up of a rich variety of peptides, enzymatic and non-enzymatic proteins, carbohydrates and minerals [5]. Peptides and proteins make up 90 – 95 % of the dry weight of venom and they show toxic activities that may result in pharmacological effects. Those effects include neurotoxicity, which affects nervous signal transfer, hemotoxicity, which affects blood clotting, cytotoxicity, which affects cells, and cardiotoxicity, that results in damage to the heart muscle [5][6]. The most potent and widely studied snake venom toxins that are responsible for causing severe pathophysiological effects are phospholipase A2, Zn2+ dependent
metalloproteinases, serine proteases and three-finger toxins. Interestingly, venom compositions vary both interspecifically and intraspecifically depending on many factors, such as geographical location, age and gender of the snake [6]. Toxic effect can also be influenced by the age and general health of the victim. Certain pathologies may have the same results, but the course of envenomation can vary extensively.
Snakebite pathologies and related toxins have been extensively studied; a lot has been published on this topic. Nevertheless, not many papers give a comprehensive overview on various pathologies, toxins causing them and medically important species responsible for these pathologies. The purpose of this paper is to provide an overview of these toxicities for most affected regions, namely Sub-Saharan Africa, Southeast Asia and Latin America. This could be a reference guide for the development of effective snakebite treatments.
7 Goals of this paper:
• Overview of medically important snake species.
• Overview of the most important snakebite pathologies: neurotoxicity, cytotoxicity, hemotoxicity.
• Overview of major toxins that cause toxicities.
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2. Medically important snakes
Many parts of the world are having problems with number of snakebites being underreported, in some parts many snake bites remain completely unreported. Patient treatment and manufacturing of antivenoms is completely dependent on the correct understanding and recognition of venomous snakes. Like any other scientific field, snake venom taxonomy is constantly changing, as new species are being discovered and many known species are comprising multiple new species, which makes the classification of venomous snakes a subject to change.
Venomous snakes are occurring in many regions of the world and are a big threat to public health, especially in less advanced tropic regions, where snakebites are most abundant. Out of 3000 registered species of snakes, over 600 species are venomous and around 200 are classified as medically important [7].
It is possible to establish the species of the greatest medical importance in different regions with the use of current literature, reported snakebites and registered snake species. There is a lack of information for the detailed statistics of snake species that are responsible for envenoming and fatalities in developing countries, as many snakebites there are not boing reported. So, classification and establishment of medically important snakes relies on extrapolation from the few known studies as well as the biology of the species, where all closely related snake species with similar history are likely to be medically important, if one of the related species is classified as medically important. World Health Organization listed the species of snakes that are considered as of greatest medical importance in four broad geographical regions, in this study we only consider three, Europe is left out, as the highest estimated burden of snake bites is from sub-Saharan Africa, southeast Asia and Latin America. This WHO list includes snakes that are widespread in largely populated areas and cause numerous snakebites as well as snake species that are poorly known but are suspected to fall into this category. It should be noted that some groups of these venomous snakes are highly under-studied and that new studies might bring some changes into nomenclature.[8]
In 1981, the World Health Organization developed a methodology for the identification of snakes of high medical importance. Snake species were classified into three classes [9]:
• Class I – Species, that commonly cause death or serious disability.
• Class II – Species, that uncommonly cause bites, but cause serious effects, such as death or local necrosis.
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• Class III – Species, that commonly cause bites, but cause mild effects, more serious effects are uncommon.
The WHO method for listing medically significant snake species is a useful tool for medical professionals, as it is not reliant on specific numbers. This method also allows the possibility that some new snake species may yet emerge.[9]
More recently, the classification of snake species was updated and adapted for all users, a database was created which was published online. In assessment of the relative risks of snakebite, the World Health Organization considered two major categories of medically important snakes [8]:
• Category 1: Highest medical importance. This category includes highly venomous snake species that are common, widespread and cause numerous snakebites with serious effects, such as morbidity, disability or mortality.
• Category 2: Secondary medical importance. This category includes highly venomous snakes, on which there is a lack of clinical data or their occurrence is less frequent, in comparison with first category. These snakes cause numerous snakebites with serious effects, such as morbidity, disability or mortality.
Snake species listed in the Category 1 are considered to cause the largest burden and should be considered as a priority for production of antivenom [8].
Based on their biological characteristics all venomous into four different families: Viperidae, Artractaspididae, Elapidae and Colubridae.[10] Tables 1-3 below indicate the venomous snakes of highest medical importance in different regions. These tables include either snake species that are most common or widespread in largely populated areas and cause numerous snakebites, are poorly known, but suspected to fall into this category, or snake species, that cause life-threatening envenoming, but are relatively uncommon.
Snakes within the Elapidae family are mostly dominant in Africa and the Middle East, together with snakes of Viperidae family they cause the majority of envenomations in Asia. Viperidae is a dominant family in Latin America, viperids are the only venomous snakes considered of high medical importance in this region. Less appearing snake families are Artractaspididae and Colubridae, therefore they are not significant part of overall envenomations. Artractaspididae snakes only appear in Middle East and North Africa and are classified as significant in these regions. Colubridae snakes appear in many regions though are classified as not medically important.[7][8]
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Table 1. Venomous snakes of highest medical importance: Africa and Middle East.[8]
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Table 3. Venomous snakes of highest medical importance: the Americas.[8]
Lethality is extreme outcome, not the only effect of snake bite, therefore this is one of the factors that contributes to the medical importance of certain snake species. Potential outcome of snakebite could be influenced by geographical variations, age and gender of the snake, snake venom composition of the same species in different regions can vary and therefore snakebite can have different outcome. The effect of snakebites on humans are not only dependent on the venom characteristics, but also is influenced by other factors, such as age and health of the victim, early treatment, whether the antivenom is available and general quality of medical care. Dangerousness of a venomous snake species, when talking about medically important snakes, varies in different geographical regions and human populations. [11]
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2.1. Snake venom composition
Snake venoms are complex mixtures of biologically active low and high molecular mass proteins and peptides, additionally venoms may also contain more than 100 components, including metal ions, carbohydrates, nucleosides, amines, lipids, free amino acids. Proteins and peptides comprise up to 90 – 95 percent of snake venom total content. Some studies have shown that actions of different toxins in the same snake venom can be very complex. [12]
Usually, snake venoms are classified according to an entire snake family, but this method can often be misleading, as the variability of venom compositions can be found even within the same families or species [12]. Snake venoms can vary in composition and variations can be found within the same species and depend on age, sex, diet, geographical location and seasonal variation, which in turn reflects in different clinical manifestations.[13] Table 4 contains information on most common venomous snake families and a comparison of the venom components present in their venom [14].
Snake venom consist of a diverse array of large proteins and peptides, these venoms can comprise up to 200 components that are assigned as dominant and secondary families. The dominant families in snake venom include phospholipases A2 (PLA2s), snake venom
metalloproteinases (SVMP), snake venom serine proteases (SVSPs) and three-finger peptides (3FTXs). Secondary families that influence the venom toxicity include L-amino acid oxidases, C-type lecitins, disintegrins and cysteine-rich secretory proteins (CRISP). [15]
Characterization of Colubrid, Elapid and Viperid snake venom and venom characteristics can be seen in table 4. This table shows information on dominant snake venom toxins and their characteristics, the majority of these toxins will be discussed later in this paper.
Figure 1 shows the relative complexity of snake venom [12]. It can be seen that venom toxins are only a small part of a complex snake venom “cocktail”.
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Table 4. Composition of venoms from Colubrid, Elapid and Viperid snakes and their characteristics.[16]
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Snake venoms consist amounts of phospholipases A2, three-finger toxins,
metalloproteinases, hyaluronidases, phosphodiesterases, acetylcholinesterases, L-amino acid dehydrogenases and L-amino oxidases in varying relative abundance. The proteins listed may have different functions, and can be cytotoxic, hemotoxic and neurotoxic. Considering the complex toxin variety in snake venom, scientific classification of venoms is done to better evaluate the effect of snakebite on patients and to determine the best course of treatment.[17]
Venom compositions may vary between species, their habitat, geographical location, age, though there are three main types of venom classified according to their effect.
• Neurotoxic venoms – target the nervous system. • Cytotoxic venoms – target specific cellular sites. • Hemotoxic venoms – target the cardiovascular system.
PLA2 are the most widely studied snake venom toxins both as hemotoxins and
pre-synaptic neurotoxic snake venom [18]. These enzymes can be found in practically all venomous snake families, although the relative abundance and enzymatic activity of PLA2s is
overall higher for snakes within the Elapidae family [19]. 3FTx is another toxin family that is represented by low-molecular weight toxins and that is widely present in elapids. Higher molecular mass enzymatic toxins such as SVMPs and SVSPs are generally found in venoms of viperid snakes.
Not all existing snake venoms are characterized, new discovered types of venom often have both pre- and synaptic toxins. For example, venom of Russell’s viper contains post-synaptic toxin DNTx-I together with pre-post-synaptic PLA2 toxin, venom of kraits contains several
types of neurotoxins, such as beta-bungarotoxin, alpha-bungarotoxin and a kappa-bungarotoxin, which binds to receptors in post-synaptic level. [19]
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3. Pathologies
Snake venoms mainly consist of proteins and peptides, which are also referred to as toxins, that may result in a range of different pathologies, that can be either neurotoxic, cytotoxic or hemotoxic in nature [20]. These three types of toxicity will be discussed in more detail in the following section.
3.1. Neurotoxicity
Neurotoxicity is described as having a negative effect to central and peripheral nervous system, caused by exposure to certain toxic substances, namely neurotoxins. These toxins may alter the activity of the nervous system, killing or disrupting nerve cell endings that are essential for transmitting and processing signals to the brain and to other parts of the nervous system. Neurotoxins have direct or indirect effect on nervous system, direct neurotoxins act directly on neuronal cells and axons, while indirect toxins interfere with metabolic processes [21]. Depending on the characteristics of a particular neurotoxin, it can cause damage to specific parts or certain cellular elements of the nervous system. The effect of the toxin on the nervous systems may depend on various factors, such as the characteristics of the toxin, the dose a person has been exposed to, person’s ability to metabolize the toxin and recover, and how vulnerable the affected cells are [22]. Symptoms that may be observed include respiratory distress, lacrimation, hind limb paralysis, convulsions and profuse urination, cell death can also result in a loss of motor control [23]. Acute neuromuscular paralysis with respiratory involvement is the most clinically important neurotoxic effect, it is a major cause of morbidity and mortality related to snake envenomation. There are several other acute neurological features that are reported after snakebite and it is most likely because of direct neurotoxicity. The mechanisms that underlie the pathophysiological effects of direct neurotoxicity are not well studied, as the only available date comes from case reports [24].[25]
Neurotoxicity is a well-known feature in bite victims of elapid species such as kraits, cobras, taipans, coral snakes, death adders and tiger snakes. Vipers such as rattlesnakes, Russell’s viper, the asp viper, the adder and the nose-horned viper also characterize neurotoxicity well [24].
For a while it was considered that snake venom toxins can cause two types of neuromuscular blockade, these toxins are classified according to the site of action, namely pre-synaptic and post-pre-synaptic [24].This classification however is overly simplistic and technically
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there are three types of neuromuscular blockades, on the pre-synapse, the post-synapse and blockage within the synaptic cleft, although the latter is considered as the interstitial space into which the neurotransmitters diffuse [26]. Further we will discuss the simplistic classification of these toxins.
• Pre-synaptic neurotoxins bind to the motor nerve terminals, which leads to depletion of synaptic Acetylcholine (Ach) vesicles, disabled release of Ach and then deterioration of the motor nerve terminal. The binding of these toxins to the nerve terminal is irreversible, clinical recovery is rather slow and is fully dependent on regeneration of the nerve terminal, this effect does not respond to antivenom. [24] Beta-bungarotoxins is the best representation of pre-synaptic toxins. Effects produced by taipoxin, notexin and textilotoxin also result in a similar mechanism of action as for pre-synaptic neurotoxins. Pre-synaptic neurotoxins were identified in the venom of Crotalinae, Elapidae, Hydrophiidae and Viperidae snake families. [27]
• Post-synaptic neurotoxins, also known as alpha-neurotoxins, bind to the post-synaptic muscle nAChRs, producing a reversible, non-depolarizing post-synaptic by competitive inhibition, this effect can be reversed by antivenom [24]. These toxins belong to the group of so called three-finger toxins and are classified into three main groups- long chain, short chain and non-conventional alpha-neurotoxins.
bungarotoxin is the best representation of post-synaptic toxins [24]. Alpha-neurotoxins were identified in the venom of Elapidae and Hydrophiidae snake families [27].
With the rich and growing diversity in snake venom, many types of venom now appear to contain both the pre-synaptic and post-synaptic neurotoxins.
Some examples of neurotoxic effect of some snake venom can be seen in the table 5 below. What can be concluded from this table, is that a variety of toxins van have similar neurotoxic effects upon envenoming.
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Table 5. Examples of toxin diversity in snake venom [27]
3.2. Cytotoxicity
Cytotoxicity is defined as having a potential of being toxic to cells, cytotoxins are basic amphipathic proteins, containing both polar and nonpolar portions in their structure [28]. Cytotoxins have the capability to damage a vast variety of cells. Cells exposed to cytotoxic compounds may undergo a variety of effects, such as necrosis, apoptosis, disruption of cell membrane integrity, cells can also stop growing and dividing. Bite victims of snakes with cytotoxic venom components can be at risk of internal bleeding without having any previous injuries [28]. Cytotoxins can affect the activity of different cell membrane channels or receptors (including Na+/K+ -ATPase and integrin receptors).
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• Necrosis is the loss of membrane integrity which can later result in cell death. Cells that undergo necrosis usually experience rapid swelling, loss of membrane integrity, their metabolism shuts down and they start releasing their contents into the environment. Necrotic cell death is result of a range of cellular events. Intracellular organelles experience inflammation and destruction, that eventually can cause rapture of the interconnected cell membranes.[29]
• Apoptosis is defined as a programmed cell death. Cells undergoing apoptosis experience cytoplasmic shrinkage, condensation of nuclei and DNA cleavage into fragments of regular size. Distinguishing necrosis from apoptosis might be challenging sometimes, as these two processes often occur independently, simultaneously or sequentially.[30]
Cells that undergo apoptosis most likely will undergo necrosis as well, though cells that are undergoing a rapid necrosis will not show apoptotic effects [31]. It is accepted that nearly all pathological activities of cytotoxins are based on them binding to cell membranes and disrupting lipid bilayers upon binding [31].
Cytotoxin structure is characterized by the presence of the three-fingered fold, all cytotoxins are known to have a similar 3D structure, it is stabilized by four disulphide bridges. As a rule, most cytotoxins are molecules with a highly positive charge and they interact with negatively charged phospholipids on the outer leaflets on lipid bilayer.[31]
The principle of cytotoxins of snake venom is characterized based on the studies of cytotoxins from cobra venom and these cytotoxins non-selectively disrupt cell membranes killing the cells. Studies show that snake venoms exhibit a relatively broad cytotoxic activity spectrum, as all toxic effects, cytotoxicity is selective and varies depending on the snake species. Cytotoxins of cobra snake venoms are stable polypeptide molecules, that can be resistant to various denaturing agents.[31]
Cardiotoxins are also known to exhibit cytotoxicity, so these names are often used as synonyms. For example, venom of Indian snake N. kaouthia has a multifunctional toxin that shows both cardiotoxic and cytotoxic effects. [32] Structurally cardiotoxins are highly similar to short-chain neurotoxins, they adopt similar three-finger loop structure, although biological functions of these toxins differ significantly [33]. Cardiotoxicity is defined as damage to the heart muscle, cardiotoxins depolarize cardiac myocytes. Cytotoxicity caused by cardiotoxins
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in the heart muscle cells mostly results in opening of voltage-dependent Ca2+ channels, which
leads to a block of K+ channels and formation of new ion conductive pathways [34].
3.3. Hemotoxicity
Hemotoxic snake venoms can have cardiovascular and hemostatic effects, they affect cardiovascular system, cause destruction of red blood cells (RBCs) [35]. Cardiovascular effects caused by snake venoms can be best described as a dramatic fall in blood pressure, hemostatic effects can be characterized by local and systematic hemorrhage. Low blood pressure, hypotension, can be caused indirectly by SVMPs or directly by snakes injecting bradykinin potentiating peptides that enhanced by SVMPs cause ultimately the same result [36]. Hemotoxicity occurs when hemotoxins destroy red blood cells, slow down blood clotting or cause tissue damage and often organ degeneration. Clinical effects of hemotoxic snake venoms include inflammation, hemorrhage at systematic and local level, edema and, in some cases, necrosis, however not all venom show all these effects [36]. Injuries from these toxins can be very painful, can cause irreversible damage and in some rare cases even death, hemotoxicity is one of the most common clinical signs in victims of snakebites, especially when envenomings are caused by viperid snakes [37] Death cause by hemotoxicity is much slower than the process by which neurotoxins cause death.
Viperidae snake family such as rattle snakes, copper heads and cotton heads, including the subfamilies of Viperidae and Crotalinae is the biggest family known to contain hemotoxic venom components [38]. In addition, several cobra species, such as non-spitting Asian cobra and non-spitting African cobra, have been shown to produce anticoagulant effects upon envenomation [39].
Although this classification is not always very precise, as some hemotoxic venom can contain neurotoxic components and the other way around, for example venom of Elapidae snake family very often contains important neurotoxins as well as hemotoxins [37].
Hemotoxic effect can be produced by proteins with different structures, such as serine proteases, C-type lectin-like proteins, multitoxic phospholipases A2 and some other proteins.
There are four main hemotoxic enzyme families, namely SVSPs, SVHs, SVMPs and PLA2.
SVMP and SVSP enzymes are reported as the main proteases for some venoms of Viperidae snakes.[40]
Hemorrhagic toxins, also referred to hemorrhagins, comprise a major group of active principles in viperid snake venom and are mainly comprised of metalloproteases, enzymes that
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are capable of degrading proteins of the extracellular matrix [40]. The effect of these toxins depends on the severity of envenomation, in almost all the cases pure hemorrhagins are lethal at sufficiently low dose, in cases that hemorrhagins are not severe enough to cause patients mortality, it might lead to some serious pathophysiological conditions. Snake species have hemorrhagins that are not all equally hemorrhagic, lethal doses vary among toxins from 0.01 μg to 200 μg [41]. An interesting fact to mention, is that several hemorrhagic toxins can be absent in juvenile snakes and then reappear in adult snakes [41].
Snake venom components causing fibrinogenolysis make blood uncoagulable almost completely, clotting phenomenon is directly blocked by anticoagulant factors. The main step in the formation coagulation cascade in extrinsic and extrinsic pathways is the formation of the prothrombinase complex, that converts prothrombin into thrombin. Thrombin has many functions, such as cleaving fibrinogen to fibrin, which results in the formation of a clot and inducing platelet, thrombocyte, aggregation. [41]
There are some venoms with majority of neurotoxic properties containing metalloproteinases that affect the coagulation of blood in addition to neurotoxic effects they have [42]. PLA2 from several snake families can also have hemotoxic effects, it can affect the
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4. Snake venom toxins
Snake venom is not a composition of a single substance, but it is a mixture of dozens to over a hundred of different peptides and proteins. This toxic “cocktail” is present in snake venom in a wide variety of combinations and concentrations, though there are certain toxins that are more commonly found in certain snake families [44]. In this chapter most common multitoxins as well as toxins that cause neurotoxicity, cytotoxicity and hemotoxicity will be discussed in more detail.
4.1. Multitoxins
Phospholipase A2 is a major element of snake venom of many species, it is able to cause
various toxic effects and therefore it could be characterized as multitoxin. Over time of evolution this toxin acquired a wide variety of toxic effects, such as neurotoxicity, myotoxicity, cytotoxicity, anticoagulant and inflammatory effects [45]. Despite its diverse mechanism of action and its ability to cause numerous toxic effects, phospholipase A2 is classified as a
characteristic neurotoxin.
Another toxin that could be characterized as multitoxin is SVMP, as these are known to have both cytotoxic and hemotoxic properties.
4.2. Toxins
4.2.1. Phospholipases A2
Phospholipases A2 are particularly abundant proteins in snake venoms and one of the
most extensively studied protein families. PLA2s are low molecular weight proteins (around
14 kDa). [45] Mostly PLA2 enzymes exist as monomers, though some of them are known to
interact and form complexes with other PLA2s or other protein [45]. Complexes are formed
through covalent or non-covalent bonds and exhibit presynaptic neurotoxicity. [45]
Most PLA2s share common structural features and are further classified into two groups,
where group I includes toxins from elapids, such as beta-bungarotoxin, taipoxin and textilotoxin and group II includes myotoxins from viperids.[45]
This classification is made on the basis of their primary structure and disulphide bridge pattern. PLA2s can also we found in the venoms of Crotalidae and Hydrophiidae snakes.
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PLA2s in snake venom are the active component of presynaptic neurotoxic venoms,
causing a variety of pharmacological effects [46].
There are four different widely known pre-synaptic snake venom neurotoxic PLA2
toxins which are beta-bungarotoxin, taipoxin, notexin and textilotoxin. One study showed that neurotoxins taipoxin and notexin have similar effects to beta-bungarotoxin, it was shown that all pre-synaptically active PLA2s show similar effects, suggesting a similar mechanism of
action for pre-synaptic neurotoxins [20].
The second group of PLA2 can be further divided into two groups: Asp49 myotoxins
and Lys49 myotoxins. Venom of some snake species contain several isoforms of Lys49 myotoxins, that showed variation in both neuromuscular and myotoxic effects [20].
4.2.2.1. Phospholipase A2 complexes
Phospholipase A2 complexes play an important role in envenomation, so another
method for PLA2 classification is complex based. There are two classes of complexes that could
be formed, namely covalent and non-covalent complexes [47].
Figure 2. Schematic representation of covalent and non-covalent snake venom PLA2 complexes. [48]
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Figure 2 shows a schematic structural representation of most important covalent and non-covalent complexes of PLA2s. Beta-bungarotoxin is the only covalent complex that is well
studied and will be discussed in more detail [48].
4.2.2.2. Covalent complexes
Beta-neurotoxins are covalently linked heterodimers, that are pre-synaptically active. They bind to the motor nerve terminals and this binding leads to depletion of synaptic neurotransmitter acetylcholine (Ach) vesicles, impaired release of Ach and further to degeneration of the whole motor nerve terminal. These toxins produce neuromuscular block, it occurs in phases: an immediate depression of Ach release and a period of enhanced ACh release, later followed by complete inhibition of neuromuscular junction transmission (NMJ). The effect of this neuromuscular transmission results in latency period of 20 to 60 minutes [24]. Neurotoxic phospholipase A2 toxins, PLA2s are mostly known to have this working
principle, the binding of these toxins to the nerve terminals is irreversible and treatment with antivenom is more likely to be ineffective [24].
Beta-bungarotoxin (b-BuTX) has an effect of PLA2 enzymatic activity, it produces
pre-synaptic toxicity, which is characterized by pre-synaptic vesicle depletion, motor nerve terminal destruction, axonal degeneration and reinnervation. Beta-bungarotoxins produce calcium influx through calcium channels and at the same time increase the release of Ach via mechanisms, dependent on SNARE protein complexes, that lead to depletion of synaptic vesicles. There is a correlation between the neuromuscular transmission failure induced by pre-synaptic toxin beta-bungarotoxin and pathological changes in cells. These changes were studied using rats, rat muscles were injected with beta-bungarotoxin, it resulted in paralysis within 3 hours, after 3-6 hours nerve terminals showed evidence of degeneration and by 12 hours all muscle fibers had lost all physiological functions. Regeneration in nerve terminals started appearing after 3 days, some muscles started getting back the neuromuscular function and after 7 days full recovery occurred. Authors suggested that all pre-synaptically active PLA2s have a similar, reversible effect. [24]
Alpha-bungarotoxin produces purely post-synaptic and non-depolarizing neuromuscular blockade that is almost irreversible. This toxin is not able to block the pre-synaptic neuronal receptors. Candoxin is a toxin that is structurally similar to alpha-bungarotoxin, though in contrast this toxin produces a readily and completely reversible post-synaptic blockade. Furthermore, candoxin is able to block pre-post-synaptic receptors and produce
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characteristic tetanic fade and train-of-four (TOF) fade during the recovery from neuromuscular blockade. This fade phenomenon is a pre-synaptic event involving a blockade of putative pre-synaptic nAChRs at a neuromuscular junction, to maintain an adequate transmitter release during a rapid and continuous nerve activity [49].
Other pre-synaptic neurotoxins may interfere with neuromuscular junction transmission using different mechanisms of action. Dendrotoxins enhance Ach release by inhibition of potassium channels and then produce a neuromuscular block that is similar to a depolarizing blockage. If muscle preparation is performed with dendrotoxins, the neuromuscular blockade by beta-bungarotoxin will be much lower. Fasciculin toxin acts as the inhibitor of AChE to increase the availability of Ach at the neuromuscular junction. [24][49]
4.2.2.3. Non-covalent complexes
• Homodimeric complexes
Trimucrotoxin is the only known homodimeric complex of PLA2 enzyme. This
complex displays presynaptic neurotoxicity. Belong to the group II of PLA2 enzymes.
Structurally, pharmacologically and immunologically similar to crotoxins and agkistrodoxins. No more information on this complex is known. [3]
• Heterodimeric complexes
Crotoxin is the main toxic component of this group. This toxin is composed from two parts, the non-toxic acidic subunit crotapotin (CA) and the weakly toxic basic subunit (CB). CA lacks Phospholipase A2 activity and CB exhibits this activity. This complex belongs to
group II of PLA2 enzymes. [3]
Functionally this toxin is a strong neurotoxin, that acts at neuromuscular junction blocking nerve signal transmission. To be more precise, CB causes a blockade of nerve signal transmission and CA is pharmacologically inactive and works as chaperone for CB, allowing binding of this unit to specific target sites only. [3]
Crotoxin complexes not only cause neurotoxicity, but may also induce local and systemic myotoxicity, myoglobinuria, initiation of platelet aggregation, analgesic effects and cytotoxicity on tumor cell lines or even necrosis. All in all, crotoxin is a multifunctional complex. [3]
Vipoxin and viperotoxin F are toxins from a group of heterodimeric complexes. All toxins in this group are 60 % similar to crotoxin and even though vipoxin and viperoxin F are
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structurally very similar, their modes of action are different: vipoxin is a pre-synaptic neurotoxin and viperotoxin has post-synaptic properties. [3]
• Heterotrimeric complexes
Taipoxin and paradoxin belong to this group, paradoxin is a homolog of taipoxin. Taipoxin is one of the strongest animal toxins, it exhibits toxicity through presynaptic blockade. This toxin is a combination of three subunits, alpha, beta and gamma, where alpha is the most toxic subunit, gamma is moderately toxin and beta has no toxicity. The gamma subunit is the most important part of this complexes structure, as it stabilizes it and protects alpha subunit for dissociation. [3]
• Heteropentameric complexes Textilotoxin belongs to this group.
Structurally textilotoxin is comprised of four subunits, A,B,C and D, where subunit A has highest toxicity, subunit B is non-toxic and structurally is similar to beta unit from taipoxin complex, subunit C is similar to A, though its enzymatic activity is lower and subunit D is similar to C, but with much lower enzymatic activity. [3]
• Other complexes
Taicatoxin is oligometic complex that is the only non-neurotoxic complex of PLA2, that
consists of three non-covalently bound subunits, namely alpha-neurotoxin-like peptide, serine protease inhibitor and neurotoxic PLA2. [3]
Taicatoxin causes toxicity simply by blocking the Ca2+ channels and it doesn’t lose its
channel blocking activity even when neurotoxic PLA2 unit is removed. [3]
Non-covalent complexes are not well studied and only the basic working principles of these toxin complexes are known.
4.2.2. Three-finger-toxins
Three-finger toxins are non-enzymatic proteins, that can mostly be found in venom of Elapidae, although 3FTxs have been also found in venom of Colubridae and Cerotalinae.[50] The name of this toxin class comes from the particular toxin structure, consisting of three beta-stranded loops, also called “fingers”, stretching from their hydrophobic core, cross-linked by four disulphide bridges [50].
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Members of this toxin family are similar in structure but can exhibit a variety of pharmacological functions including neurotoxicity, cardiotoxicity, cytotoxicity, anticoagulant effects and platelet aggregation. Three-finger-toxins exist mostly as monomers, though covalent and non-covalent dimers can also be formed. [50]
Alpha-neurotoxins belong to the group of 3FTXs. Alpha-neurotoxins are also known as post-synaptically active neurotoxins and they bind to the post-synaptic muscle nicotinic acetylcholine receptors (nAChRs). These toxins are classified into groups – long-chain, short-chain and non-conventional alpha- neurotoxins [50].
Post-synaptically active neurotoxins follow the action of d-tubocurarine (dTC), which classically produces a reversible and non-depolarizing post-synaptic block by competitive inhibition of Ach binding to the muscle nicotinic acetylcholine receptors (nAChRs). There can be some variations in the effect of these neurotoxins on the post-synaptic receptors. In this toxicity the antivenom may accelerate recovery, however most of the alpha-neurotoxins, especially long-chain 3FTXs, bind to the post-synaptic nAChRs almost irreversibly. These neurotoxins also may inhibit the pre-synaptic neuronal nAChRs and produce TOF or tetanic fade as a characteristic of this action. [51]
Kapa-bungarotoxin is a non-covalent complex that belongs three-finger-toxins and exhibits neurotoxicity.
4.2.3. Metalloproteases
Snake venom from Viperidae and Crotalinae contain protease complexes that affect the coagulation of blood and help with the digestion of the prey [18]. Metalloproteases and serine proteases are the two characteristic groups of proteases and will be discussed in more detail in the following section.
Snake venom metalloproteases are a large multi-domain protein and one of the major causes of venom-induced hemotoxicity, these enzymes belong to the superfamily known as zinc-dependent snake venom metallo-proteinases (SVMP/ metzincins/ hemorrhagins), that consist of grouped families of zinc endopeptidases. [18]
SVMPs have been found to that have a rather specific mechanism of action, in terms of both substrate preference and peptide bond cleavage pattern. In terms of similarity, metalloproteases share a sequence motif of three histidines binding to zinc at a catalytic site and a methionine that sits under the active site. [18]
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This family is further divided into four subclasses: P-I to P-IV and can also be referred to as seralysins, astacins, ADAMs/adamlysins and MMPs. This classification is solely dependent on the different composition of domain, more specifically, on the presence or absence of non-catalytic domains [19]. The calassification scheme and more detailed characteristics of each class can be found in table 5. It was concluded that first subclass of SVMP in general is less hemotoxic than P-II and P-III and the P-III class of hemorrhagins is the most potent group in terms of biological activity. However, there are still gaps of knowledge when talking about these classes of hemorrhagins, as there is a limited availability of sequence data. [41]
Table 5. Classification of Metalloproteases [41]
Protein P-III of snake venom metalloproteinase is a vascular apoptosis inducing protein 1 (VAP1), it also has high hemorrhagic activities. This enzyme promotes apoptosis by interaction with integrins and eventhough VAP1 contains a hemorrhagic domain, it is thought to be atleast partly involved in stimulating apoptosis. Not much is yet known about the working principle of VAP1 induced apoptosis and further research needs to be conducted [52].
Metalloproteinases can evoke a particular type of apoptosis, namely “anoikis”, simply by degrading the extracellular matrix. The extracellular matrix controls cell behaviour and regulates growth, differentiation, shape, cell adhesion and even death functions. Snake venom
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metalloproteinases can also degrade extracellular matrix proteins and disturb some of the interactive processes between cells and ECM by inhibiting integrin receptors. [53]
Overall Snake Venom Metalloproteases are known to exhibit fibrinogenolytic, apoptotic, inflammatory, factor X and prothrombin activating effects [54]. There is no doubt that local effects of SVMPs are enhanced in the presence of other major toxins, such as PLA2s
and LAAOs.
4.2.4. Serine proteases and hyaluronidases
Snake venom serine proteases (SVSP) are an important coagulant enzymes, also referred to as thrombin-like enzymes, as they exhibit thrombin like fibrinogenolytic functional activities. SVSPs are able to induce defibrinogenation that leads to consumptive coagulopathy, that potentially leads to renal hypoperfusion and ischemia. [19] It is present in practically all snake venom and is known as a spreading or dispersion factor, though majority of these enzymes have been isolated from viperid snakes.
Serine proteases are known to degrade fibrinogen into fibrinopeptides by selectively cleaving either alpha or beta chain of fibrinogen. This leads to polymerization of fibrin monomers and unstable clots that are quickly dissolved by plasmin, this continuous generation and destruction of fibrin blood clot results in a bleeding disorder that periodically uses fibrinogen. [19]
Snake venom hyaluronidase (SVH) is an endogycosidase, it degrades the beta-N-acetyl-glucosaminidic linkages in HA polymer. Hyaluronidase induces rapid spreading of the venom toxins by degrading hyaluronic acid and in that way destroying the integrity of extracellular matrix of the tissue and indirectly inducing hemorrhage. [55][44]
4.2.5. L-amino acid oxidases
L-amino acid oxidase (LAAO) is a dimeric flavoprotein and is capable of producing cytotoxic effects and antipathogenic activities by inducing the programmed cell death, apoptosis. [56] LAAOs catalyze the oxidative deamination of L-amino acids and produce alpha-keto acids together releasing the contaminants, such as hydrogen peroxide and ammonia. L-amino acid oxidases can occur in many different organisms, though they are widely distributed in venoms of many snake species, but mainly viperids and elapids. LAAOs are known to be responsible for the yellow color of the venom. [57]
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LAAOs from the venomous snakes have been proven to induce various toxic effects and even cell death on a mammalian organism, such as platelet aggregation, cytotoxicity, edema and hemorrhage. [57]. These flavoproteins contribute to the toxicity of snake venoms, possibly when snake venom L-amino acid oxidases oxidize amino acids and produce a localizes high concentrations of hydrogen peroxide [58].
Most of biological effects of L-amino acid oxidases are due to the secondary effect of enzymatic reaction biproduct- hydrogen peroxide. At low levels H2O2 is believed to have
control in the transcription of some genes, which initially means it could prevent cell death. Although, with an increase in H2O2 concentration, the control is lost, growth arrest and
apoptotic cell death is induced. [59] This suggests that it is important the way in which H2O2
is introduced to the cells.
It is important to note, that apoptosis induced by LAAOs is different than apoptosis cause by the exogenous hydrogen peroxide [59].
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5. Analytical methods to asses pathology
As discussed previously, snake venoms have a complex nature and because of this, the analysis, identification and biological characterization of individual snake venom toxins is challenging and therefore requires advanced analytical techniques. [54] In the following section the most widely used and promising techniques will be discussed in more detail.
Over the past few years, a variety of quantitative and analytical techniques for snakebite analysis have been implemented and thoroughly studied, a number of them can be seen in table 6. Proteomics approaches combined with mass spectrometry greatly increased the identification of the proteins in the proteome of snake venom. [60]
Up until 1990s, the main approach for snake venom analysis was typical biochemical characterization, where individual proteins were isolated and analyzed. [54] With implementation of 2D-PAGE more global approaches started appearing that enabled to visualize venom complexity fort the first time. After this, 2-D PAGE coupling to MS and to LC-MS/MS started and a variety of methods appeared. Some of the methods showed here proved to be more promising than the others, they were improved and applied in more analysis. [60]
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5.1. Top-down and Bottom-up proteomics
Proteomics is a rapidly growing technology; it has been widely used for studying protein profiles and was successfully implemented for studying snake venom composition [62]. Even though labelling and label-free quantitative approached in proteomics show great advances, large-scale quantitation of all different molecular forms of protein products, proteoforms, requires a lot of time and research.
Figure 3 shows a schematic representation of the basic principles of two-dimensional top-down and bottom-up down proteomics. As can be seen in the figure, the amount of data acquired from top-down proteomics is higher than with bottom-up proteomics. In top-down proteomics proteins are identified and characterized by the intact protein fragmentation and in bottom-up proteomics the sample is proteolytically digested upon analysis, so the relation of peptides to their intact proteins is lost. Further these approaches will be discussed in more detail. [62][63]
To note, in figure proteomics approaches are introduced as top-down and bottom-up venomics. Venomics is the global study of the venom and venom gland, it is a modern approach that combines transcriptomics and proteomics to explore and analyze the toxins of venom. [63]
Figure 3. Schematic representation of Top-down and Bottom-up proteomics. [64]
Bottom-up proteomics is proteomic profiling using liquid chromatography coupled
with mass spectrometry, that relies on the separation of peptides, when the sample (e.g. snake venom) is first proteolytically digested. This also known as ‘shotgun’ proteomics. High resolution data is obtained using reversed phase HPLC columns at the nano-flow scale, it can
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also be coupled with additional LC media like ion-exchange, to make it a two-dimensional separation. [2]
Attractive features of this method [65]: • Simple sample preparation. • High speed and high throughput. • LC-MS/MS runs are automated.
• Deep detection of trace protein components.
Bottom-up proteomics most often provides a qualitative information on composition of snake venom.
This method is well developed to identify the components of the venom, but there is a downside to it. As protein digestion is done for the whole venom sample, the relationship of identified peptides to their parent molecules is lost. Because of this reason, conversion of obtained data into qualitative information is very challenging. Snake venom samples generally contain a few protein families and different peptides have different variations in their ionization efficiencies, only a relative comparison of identical components can be done. [66]
Top-down proteomics is another protein identification approach that is based on
analysis of intact proteins. In top-down proteomics strategy, proteins are identified and characterized by the intact protein fragmentation. This type of analysis provides a rich data that can cover almost complete protein sequence, it can help identify a rich diversity of snake venom protein families.[67]
Separation of intact proteins is the biggest challenge in proteomics, extracting proteins from samples requires buffers that can affect the ionization process. Sample preparation here is very important and so far, the most trusted separation is reversed-phase liquid chromatography. Other techniques, such as hydrophobic or hydrophilic interaction chromatography, ion exchange chromatography, mixed mode chromatography and size-exclusion chromatography have been used for better separation and resolution prior to MS analysis. Two-dimensional MS is used to get even better resolution, higher quality data and higher sequence coverage. [67]
The information obtained from this analysis generally is very complex, due to the nature of the sample there are often overlaps of charged states, so data analysis is also an important part of this method. [67]
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As already mentioned before, information acquired from this analysis is rather rich. When testing the method scientists were able to determine isotope-averaged masses of the venom proteomes, number of disulphide linkages and sequence tags. High number of specific toxins can be identified in the snake venom, as well as proteins and proteoforms.
The main shortcoming of this approach is its low performance when analysing proteins with high molecular weight. Top-down proteomics performance is reduced when protein masses are over 30 kDa. [67]
To conclude, bottom-up venomics has some limitations that top-down approach is able to overcome with the ability to give more qualitative and quantitative information on venom toxins. A great number of proteoforms have been identified and characterized with top-down MS, which gives a great promise for the future of proteome profiling and maybe even detailed localization of post-translational modifications.
5.2. Gel-based approaches
Gel-based approaches have been used in the first reported studies of snake venoms and since then have been used for numerous proteomic studies. All gel-based methods follow the same working principle, where individual spots are taken out, are in-gel digested and for analysis submitted to tandem mass spectrometer (MS/MS). [68]
2D PAGE
This is the traditional and most frequently used gel-based method for investigation of differential protein abundances on a large scale and samples of different sources. Two-dimensional Polyacrylamide gel electrophoresis (2D PAGE) is a separation method for proteins, where separation is based on isoelectric point in the first dimension and molecular mass in the second dimension. [69] Separated spots are analysed by tandem mass spectrometry (MS/MS) and resulting chromatograms are used for quantitative analysis. [70]
The main advantage of this approach is that with a single 2D-PAGE analysis a full pattern of venom sample decomplexation is obtained. The information we get on the isoelectric point of first dimension and molecular weight on second dimension can be instantly determined for each spot. Major advantage of these methods is the possibility to stain the gel not only for proteins, but also for post-translational modifications (PTMs).
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• Only large peptides and proteins are retained, peptides smaller than 2-3 kDA are lost. • There is a limited dynamic range of concentrations that can be resolved, which means
there are maximal limits to sample loads.
• Some proteins exhibit pI’s close to the limit of pH gradient.
• Unstable proteins, that tend to aggregate can be lost or affect the overall resolution. • Low reproducibility. [71]
In comparison to 2D-PAGE, simple native 1-D PAGE is not able to perfectly summarize the complexity of crude venom. [61]
SDS-PAGE
A combined LC and gel-based proteomics strategy called ‘snake venomics’ was introduced as an alternative approach for analysis of snake venom composition. In this method, the first dimension LC separation is combined with one-dimensional electrophoresis (SDS-PAGE) in a second dimension. This method has a slightly different working principle than 2D-PAGE, first venom decomposition by reversed-phase LC is done, then resolved fractions are collected manually and further separated by one-dimensional SDS-PAGE. In SDS-PAGE protein samples are in-gel digested and are finally submitted for analysis with MS/MS. [72]
In comparison to other gel-based approaches, this method rather slow and it requires a significant amount of manual work when collecting fractions. Also, venom components that are present in trace amount are generally more likely to be lost or overlooked due to the proteins that are more noticeable in chromatograms and gels. [73]
However, there are several advantages which might compensate the shortcomings: • Small peptides are recovered.
• Loading of LC-resolved fractions onto gels for SDS-PAGE can be adjusted or normalized to obtain adequate intensity of protein bands. This is not passible in 2D or ‘shotgun’ proteomics.
• Considerable sample loads, fractions can be recovered for complimentary analysis. • Relative abundance of proteins can be estimated from integrated peak areas.
5.3. Mass Spectrometry
MALDI-TOF MS was introduced to analyze patterns of major peptides in crude venom
and showed great results in samples where peptides masses were smaller than 8 kDa. Using this method, complex snake venom samples can be analyzed for peptides of low molecular
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mass. This is a rather simple one-step approach for investigation of low molecular mass compounds in raw high complexity venom samples. MALFI-TOF MS is fast and efficient method for preliminary studies of crude venom mixtures. [74]
Tandem mass spectrometry (MS/MS) so far is the fastest and most reliable technique for determination of peptide structures and their PTMs. [75] The use of MS/MS to analyze crude extracts should make the classification and understanding of biological activity of new, unknown compounds possible.
5.4. Liquid Chromatography with Mass Spectrometry
RP-HPLC separation is used as an initial separation of crude snake venom samples for
a considerable amount of studies. Every laboratory usually develops their own method, a common method for chromatographic separation could standardize the results among different research teams and make them more comparable. There are some general chromatographic conditions used by many, these conditions are presented in figure 4. [68]
Separation of venom proteins is done using a very common analytical reverse-phase C18
column, using a linear gradient with acetonitrile. Dashed line on the figure indicated the gradient.
The figure also indicates approximate elution times of some of the most commonly found and analysed protein components of snake venom. [68]
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LC-nanofractionation with parallel MS is a straightforward method that shows
effective screening of snake venoms for selected bioactivities. In this method snake venom is separated by liquid chromatography, then the column effluent is split. One part of the effluent is collected by nanofractionation into high-resolution 96- or 384- well plates, the other part is simultaneously analyzed by mass spectrometry. Fractions collected by nanofractionation can be exposed to various bioassays and resulting bioactivity chromatograms can then be successfully correlated with the MS chromatogram obtained in parallel.[76] This correlation is based on elution times and peak shapes of bioactive peaks [77].
Sensitivity of MS is dependent on the venom toxin characteristics, toxins will differ in ionization efficiency and MS sensitivity. Also, with increasing size of the protein sensitivity decreases, this approach is successful for venom proteins with masses up to 15 kDa. [76]
Some technical details about the method:
• Bioassay analysis and optimization is performed using high-performance liquid chromatography system (HPLC). To optimize the separation, gradient elution is used. The most efficient separation so far was done with trifluoroacetic acid (TFA) as mobile phase, though some methods use acetonitrile (ACN) or formic acid (FA). [76]
• After LC, 90/10 split is used, in which 90 % of the column effluent is directed to nanofractionation system and 10 % is analyzed by quadrupole time-of-flight mass spectrometer (Q-TOF MS) with electrospray ionization (ESI). Protein identification is performed using the Mascot search engine. [76]
The authors concluded that this is a reliable method for uncovering individual proteins present in snake venoms and a high-throughput screening tool for discovery of new drug leads.[76]
5.5. Isotope Labelling
Stable isotopic labelling was and still is the most popular and most widely used method for quantitative proteomics. Chemical approaches for introduction of stable isotopes [78]:
1. Chemical derivatization of proteins (e.g. via the ICAT), isobaric tagging for relative and absolute quantitation (iTRAQ), isotope-coded labelling.
2. Enzyme catalyzed labelling.
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Out of these chemical approaches, ICAT was known to be the best. Several improvements were attempted to be made to make this method more practical, but the adaptations have notable disadvantages. To overcome issues, new quantitative strategy and reagents were introduced, soluble polymer-based isotope labelling (SoPIL). [78]
SoPIL is a quantitative strategy for the identification and quantification of protein
complexes in snake venom. The SoPOL reagents capture and isolate peptides that contain cysteine, the peptides are tagged with isotopic labels and released for the analysis with nanoflow LC-MS/MS [79]. Reagents in this method are based on soluble nanopolymers- dendrimers.
This new method overcome issues that previous methods were facing: • No extra purification steps for removal of excessive reagents. • No solid phase extraction that is limited by heterogeneous reactions.
These issues are overcome by building the function groups for reaction, isotopic labelling and isolation on a soluble nanopolymer. [79]
Soluble polymer-based isotope labelling has a potential to be an efficient tool for analysis of snake venom, it could be widely applicable for quantitative proteomics. Implementing stable isotope labelling would give a detailed and comprehensive analysis of variations is snake venom. [79]
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To conclude this part, there has been many exciting developments in the field of snake venom proteomics. We were able to look into compositions of venoms, characterize toxins and learn more about complex structures of snake venom. Although current technologies initiated many possibilities, the ultimate goal of characterization and quantification of snake venom proteins has not been reached, many advancements in this field are expected to be made in the future.
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6. Conclusions
This literature thesis provides detailed overview of the most important pathologies and toxins causing those pathologies. In addition to this, the complexity and challenges of studying snake venom composition was discussed as well as analytical methods that are being used to study snake venom composition and biological activities.
Snakebite is an important public health hazard and a major cause of morbidity and mortality in subtropical and tropical regions of the developing world. The highest estimated burden of snake envenomation is in sub-Saharan Africa, Southeast Asia and central and Latin America.
Based on the biological characteristics all venomous snakes can be classified into four families, namely Viperidae, Artractaspididae, Elapidae and Colubridae. Elapids are most common in Africa, the countries of the Middle East and Southeast Asia regions, whereas viperids are widely spread throughout Africa and Southeast Asia and are the main family of venomous snakes in Latin America, Artractaspididae snakes are only significant in Middle East and snakes within the Colubridae family are classified as not medically significant.
Snake venoms are complex mixtures, that can be composed of various amounts of phospholipases A2, three-finger toxins, metalloproteinases, serine proteases, hyaluronidases,
and L-amino oxidases. These toxins may produce a range of different pathologies, that can be neurotoxic, cytotoxic or hemotoxic of nature. Neurotoxic venom components target nervous system, cytotoxic components target specific cellular sites and hemotoxic toxins target the cardiovascular system. Some toxins are able to cause multiple toxic effects and they are therefore characterized as multitoxins. Phospholipases A2 and Snake Venom Metalloproteases
can acquire a variety of toxic effects and can be classified as multitoxins.
Because of the complex nature of snake venoms, identification and characterization of individual toxins is rather difficult. There is a handful of techniques that are studied for analyzing snake venom composition and that have been widely studied. Top-down proteomics coupled with MS is able to overcome all limitations of bottom-down proteomics and this approach is able to identify characterize a great number of proteoforms. Gel-based approaches are the traditional and most frequently used for snake venom analysis. Although having its limitations, these methods are able to give information on relative abundance of proteins and also some information on PTMs. Some studies are also very optimistic about using different LC methods for uncovering individual proteins present in snake venoms. These methods are
41
straightforward, reliable and seem to be effective. Nevertheless, Stable Isotopic labelling was and still is the most widely used quantitative proteomics method.
Finally, this overview is a minor, but extremely important step in development of effective snakebite treatment, many advancements are expected to be made in the future.
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