Venomous snakes of highest medical importance (category 1); The table was modified classified sub-area in each of four broad geographical regions in tables 3-6 of Fifty-ninth report/WHO Expert Committee.
Abstract
Venomous snakebite is a major human health issue in many countries and has been categorized as a neglected tropical disease by the World Health Organization. Venomous snakes have evolved to produce venom, which is a complex mixture of toxic proteins and peptides, both enzymatic and nonenzymatic in nature. In this current era of high-throughput technologies, venomics projects, which include genome, transcriptome, and proteome analyses of various venomous species, have been conducted to characterize divergent venom phenotypes and the evolution of venom-related genes. Additionally, venomics can also inform about mechanisms of toxin production, storage, and delivery. Venomics can guide antivenom and therapeutic strategies against envenomations and identify new toxin-derived drugs/tools. One potentially promising drug development direction is the use of endogenous inhibitors present in snake venom glands and serum that could be useful for snakebite therapeutics. These inhibitors suppress the activity of venom proteases, enzymatic proteins responsible for the irreversible damage from snakebite. This book chapter will focus on insights from venomous snake adaptations, such as the evolution of venom proteases to generate diverse activities and snake natural resistance to inhibit activity, and how this information can inform and have applications in the treatment of venomous snakebite.
Keywords
- venomous snake
- snake venom metalloprotease
- hemorrhagic
- nonhemorrhagic
- toxin resistance
- natural inhibitor
- endogenous inhibitor
1. Introduction
There are over 3700 extant snake species, but only approximately 200 in 600 venomous snake species, belonging to families Viperidae, Elapidae, Atractaspididae, and some of Colubridae, are considered medically important on public health aspects of snakebite [https://www.who.int/snakebites/disease/en/; http://www.reptile-database.org/, Accessed: 2019-11-22]. The World Health Organization (WHO) has recognized snakebite envenomation as a neglected tropical disease and has characterized a subset of venomous snake species as being of higher medical importance in the four geographical areas of the world snakebite is most frequent. The definition of highest medical importance to human public health (category 1) is “highly venomous snakes which are common or widespread and cause numerous snakebites, resulting in high levels of morbidity, disability, or mortality” [1]. These species are predominately from Elapidae and Viperidae families, but the majority of these species are from the family Viperidae (vipers and pit vipers). Viperidae species consist of approximately 50–100% of listed species in each geographical area and make up just over 60% of the entire list (Table 1).
Atractaspididae | 1 | ||
Elapidae | 17 | ||
Viperidae | 20 | ||
Elapidae | 25 | ||
Viperidae | 17 | ||
Viperidae | 3 | ||
Viperidae | 31 |
Venom variation results in pharmacological and clinical symptomology differences across venomous snake species, primarily varying in the extent of snakebite tissue damage and toxicity. In recent years, venom has been investigated using comprehensive venomics approaches, combining proteomics, transcriptomics, and genomics, in an attempt to better understand venom components responsible for variation. Next-generation sequencing (NGS) has greatly accelerated the pace of venomics, making high-throughput outputs possible; several venom gland transcriptomes can be sequenced together using multiplexed barcoded libraries, with little difference in cost, and third-generation longer read technologies, such as Oxford Nanopore (minION), are now available to correct transcriptome assembly errors [2]. Venom characterization that integrates both transcriptomics with proteomics has optimized proteomics by providing a species-specific database (venom gland transcriptome) for toxin identification, an ideal method to better distinguish unique toxins present in each species [3]. Current venom gland transcriptomes, completed using NGS technologies (Illumina and Roche) and
Elapidae | [4, 5] | Unpublished | ||
[6] | Unpublished | |||
[6] | ||||
[6] | ||||
[6] | ||||
[7] | ||||
[7] | ||||
Viperidae | [2, 8] | [33] | ||
[9] | [34] | |||
[10] | [35] | |||
[11, 12] | [36] | |||
[13, 14] | [37] | |||
[4, 13, 15, 16, 17] | [38] | |||
[18] | ||||
[19, 20, 21] | ||||
[19, 20, 22, 23] | ||||
[24, 25] | ||||
[12, 24] | ||||
[26] | ||||
[26] | ||||
[11, 27, 28] | ||||
[29] | ||||
[29, 30] | ||||
[66, 31] | ||||
[29] | ||||
[32] | ||||
[11] | ||||
Transcriptome and proteome analyses of Viperidae species have consistently found that snake venom metalloproteinases (SVMPs), phospholipases A2 (PLA2s), serine proteinases, and L-amino acid oxidases are the most abundant toxins in these venoms [39]. These toxins are all enzymatic, providing immobilization and digestive roles in prey capture. Snakebites from these species result in local tissue damage, hemorrhage, and impaired coagulation symptoms in humans, which can lead to disability and mortality [40].
To date, the only effective snakebite treatment is intravenous administration of antibodies (often called antivenom), which come from animals immunized with toxins. However, using heterologous antibodies generated from numerous venom components has inherent weaknesses, such as an increased likelihood of an allergic reaction or life-threatening anaphylactic shock. Further, antivenom does not abolish local tissue damage, as it is intravenous and is usually not administered quickly enough. Thus, there is the issue of incomplete neutralization because of geographic venom variation, and high manufacturing costs and regulations have resulted in a struggle to properly match antivenom to venomous snakes of each locality and maintain antivenom stock [40]. To alleviate these issues, specific toxin inhibitors are actively being characterized and evaluated that neutralize snake venom toxicity and would work as an alternative antivenom snakebite therapy [41, 42, 43]. The challenge still remains to investigate the safety and efficiency of these toxin inhibitors to treat snakebite envenoming in humans.
Venomous snakes have endogenous inhibitors circulating in their plasma that provide resistance to their own venoms. These inhibitors can suppress the activity of enzymatic venom components, such as SVMPs, with high specificity. SVMPs are key venom components in viper venoms that contribute to hemorrhage and tissue damage; therefore, targeting these enzymes would greatly reduce human morbidity and mortality from snakebite. In this review, an overview of SVMPs is provided, with a focus on structure-function relationships within the various classes of SVMPS across snake families, and is followed by insights into how snake endogenous inhibitors function to abolish SVMP activity. The goal of our group is to design peptide inhibitors that bind to hemorrhagic SVMPs with high affinity and effectively neutralize these toxins. This chapter deeply understands the target SVMP behaviors on the snakebite issue and is a summary of our current work with historical studies in endogenous inhibitor of venomous snake.
2. Metalloproteinases
Metalloproteinases (MPs) are one of the most functionally diverse proteases in more than 50 families characterized in the MEROP database [44]. MPs play a significant role in organism homeostasis, and are involved in, cell invasion, cell fertilization, self-defense, and reproduction. Metalloproteinases are classified into two subgroups known by their enzyme commission numbers (ECNs), exopeptidases (ECN 3.4.17), and endopeptidases (ECN 3.4.24). The second group are enzymes from the metzincin family and include serralysins, astacins, adamalysins (a disintegrin and metalloproteinase domain; ADAMs), and matrix metalloproteinases (MMPs). There have been many studies documenting endogenous metalloproteinase dysregulation in cancer cells [45], especially mammalian ADAMs. ADAM proteins and SVMPs belonged to the same M12 family [46], and similar domain features are present for both. For SVMPs, three-dimensional structures and well-characterized effects on animal models in vivo have been published. Thus, the functional and structural insights provided by SVMPs have been useful for human ADAM inferences.
2.1 Structural classification of snake venom metalloproteinases (SVMPs)
All SVMP genes exhibit a conserved signal peptide region and a pro-(pre) domain. The number of domains following these conserved N-terminal regions varies, and the arrangements of the domains have resulted in the categorization of SVMPs into three main classes. SVMPs of the P-III class consist of the metalloproteinase domain (MD), disintegrin-like domain (DID), and cysteine-rich domain (CRD). P-IIs have a MD and DID, and P-I has only a MD. Further, each class has subclasses classified for different representation forms (P-Ia, P-IIa-e, P-IIIa-d). These subclasses include dimeric or truncate isoforms that have only been observed within the venom and are generated mainly by post-translational modifications.
There have been many observations of gene neofunctionalization generating large families of venom proteins with multiple functionalities, and the SVMP gene family is an example of this. SVMPs originated from the gene duplication of an ancestral ADAM gene. The ADAM 28 precursor gene is the closed SVMP homolog present in nonvenomous snakes and is also present in mammalian species [47, 48]. Sequence comparisons between the lizard (
SVMPs constitute more than 30% of the total venom proteins present in many Viperidae species. While these proteins are less abundant in the venoms of Elapidae, Atractaspididae, and Colubridae, they appear to be ubiquitously occurring [50, 51]. These observations suggest that P-III SVMP in Elapidae, Atractaspididae, and Colubridae venoms may have a conserve ancestral function and serve a common biological role in snake envenomation. Therefore, the functional roles of the diversified SVMPs are important for the clinical symptomology associated with Viperidae envenoming in humans. Domain structure, at least the topological shape, and sequence, especially the catalytic motif on the MD, are very similar among SVMPs, but SVMP activities vary, including their target substrates. Elucidating the structure-function relationships within this protein superfamily has applications for both protein evolution and snakebite treatment.
2.2 SVMP structure-function relationships and mechanism of action
Viperid snakebites are characterized by severe hemorrhagic, microvessel damage and inflammation, both local and systemic [52]. There is strong evidence from in vivo and in vitro studies of isolated SVMPs that these proteins are responsible for snakebite hemorrhage. Mitigating hemorrhage is critical in snakebite treatment; therefore studying the SVMP molecular mechanism of inducing hemorrhage is of critical importance. Hemorrhage results from SVMP proteolysis, targeted cleavage of extracellular matrix components, transmembrane receptors, and fibrinogen, mostly around microvessels. Interestingly, despite sharing similar catalytic activity, not all SVMPs induce hemorrhage in vivo. SVMP effects also include blood coagulation irregularities, platelet aggregation, cell infiltration, apoptosis-induced activity, and alternations in vascular permeability, even if these SVMPs do not show hemorrhagic activity [52]. These additional functionalities and targets likely result from C-terminal P-III SVMP binding, not only the catalytic activity of the N-terminally located MP domain [53]. To design inhibitors to neutralized hemorrhagic effects of SVMPs, we must first understand SVMP targets, as well as the tissue distribution and localization of SVMPs upon envenomation.
Both of P-III and P-I SVMPs have hemorrhagic activities, but P-III SVMPs tend to show greater hemorrhagic activity than P-I SVMPs [54, 55]. It has been proposed that the occurrence of hemorrhage results from the degradation of the vascular basement membrane of capillaries. Immunofluorescence confocal microscopy, immunochemical and proteomic analyses of tissue, and exudate in vivo have revealed a distinct pattern of P-I and P-III SVMP distribution in tissue. Observations from SVMPs labeled with Alexa Fluor 647 have found that P-III SVMP co-localized with capillary collagen IV, especially in those of microvesicles. P-I SVMPs applied to whole tissues appear to function primarily in the degradation of basement membrane components [56]. Hemorrhagic P-I and P-III SVMPs show a preference for type IV collagen in targeted degradation. J.M. Gutiérrez et al. provided a two-step hypothesis for SVMP mechanism of hemorrhage [56]. First, SVMPs hydrolyze type IV collagen and perlecan at the basement membrane components of capillaries and surrounding endothelial cells, resulting in a weakening of the mechanical stability of the basement membrane and microvessel wall. The second step occurs when the biophysical hemodynamic forces operating in microcirculation induce a distention in the wall, causing capillaries to disrupt, followed by consequent extravasation.
SVMP hemorrhage disrupts capillary networks, facilitating toxin dispersion. In 2016, reviews by Sanhajariya and colleagues investigated snake venom pharmacokinetics using an ELISA time course, varying venom concentrations mixed with the plasma of laboratory animals (rat, rabbit, and sheep) and humans [57]. In laboratory animals, two phases were observed in the metabolism of snake venom by intravenous injection of the venoms or toxins: the first phase consisting of rapid distribution with half-lives of 5–48 min and a second a slow elimination phase with half-lives of 0.8–28 h. Half-lives of the second phase did not show a significant difference among the species (
3. Toxin resistance in venomous snakes
Given the toxic and proteolytic nature of venom, it is of the utmost importance that venomous snakes are protected against the activity of their own venom. This is an interesting area of research because venoms, especially viper venoms, have high concentrations of proteases that must be stored in an inactive state in the venom gland to prevent degradation of both the snake’s own tissue and other proteins present in the venom. These proteases must then be readily activated when delivered into prey, requiring a finely tuned on/off switch. Here, we briefly summarize how these toxins are regulated in snake venom glands, with a focus on endogenous inhibitors, especially SVMP inhibitors, as SVMPs are one of the major venom compounds in Viperid snake venoms.
3.1 Mechanisms of toxin resistance
Three toxin resistance mechanisms have been proposed for venomous snakes: (1) target receptor mutations, (2) venom gland physiological conditions, and (3) inhibitors present in the venom gland or blood circulation. For the first mechanism, limited mutations on target receptors in snakes prevent the binding of their own toxins [58, 59, 60]. An example of this has been described by Takacs et al., where resistance against conspecific α-neurotoxins, major lethal components of Elapidae venoms, has been shown to be mediated by a unique N-glycosylation of the nicotinic acetylcholine receptor ligand binding domain of Elapidae snakes [58]. This observation has only been made for a few Elapidae species, but there is currently a limited amount of research in this area. The second mechanism involves the physiological conditions within the venom gland and those required for enzymatic toxins to be active. A high concentration of citrate is present in venom, and this results in a low pH environment. It is estimated that 25% of dried crude venom (30–150 mM) from
In addition to inhibitors present in the venom and venom gland, serum proteins in some venomous snakes have also been found to bind toxins with high affinity and neutralize toxin pathophysiological effects [69]. These serum proteins and those found in the venom/venom gland are referred to as “endogenous inhibitors,” but kinds of their inhibitor are different. Serum inhibitors circulate in the blood to effectively bind and neutralize host toxins, but they are different than immune antibodies. The roles of these endogenous inhibitors and their classifications will be discussed in the next sections.
3.2 Endogenous inhibitor protein families
There are three main endogenous inhibitor classes; these are phospholipase A2 inhibitors (PLI) [70, 71], anti-hemorrhagic factors [72], and small serum proteins [73, 74, 75, 76], which have predominately been found in the blood of vipers. All endogenous inhibitors are stable at high temperatures and in acidic conditions and have been purified by reverse-phase high-performance liquid chromatography (RP-HPLC).
3.2.1 Phospholipase A2 inhibitors (PLIs)
Snake venom PLA2s are ubiquitous to Viperidae and Elapidae venoms and one of the primary components in viper venoms. These toxins are versatile and can induced a variety of effects, including neurotoxicity, myotoxicity, cardiotoxicity, hemolysis, and anticoagulation [77]. Inhibitors of PLA2s, PLIs, have been identified and characterized from the blood sera of both venomous and nonvenomous snakes. PLI genes have been found uniquely expressed in snake liver tissues [78]. This suggests that the secretion of PLIs into blood circulation could be to provide protection against accidental self-envenomation in venomous snakes.
PLIs are divided into three groups (PLIα, PLIβ, and PLIγ) based on structural characteristics. PLIαs are glycoproteins with molecular masses ranging from 75 to 120 kDa and more than three non-covalently associated subunits. Their structural features demonstrate sequence homology to the carbohydrate-recognition domain of Ca2+-dependent lectins (C-type lectin-like domain), but they lack the carbohydrate-binding ability. A highly conserved region (residues 49–143) shares 80–90% sequence identity between PLIαs and appears to be responsible for PLA2 binding [79]. PLIβs are 150–160 kDa glycoproteins composed of three non-covalently bonded subunits and have nine tandem leucine-rich repeats. A homology analysis by BLAST shows similarity to human leucine-rich α2-glycoproteins, which structurally forms horseshoe-shaped molecules, as observed in Toll-like receptors [80]. PLIβs have been purified and characterized from only one venomous snake,
Numerous studies have described highly effective inhibition of PLA2 toxicity in vitro and in vivo by PLIs purified as a highly soluble protein from snake serum. However, binding sites, as well as inhibitory mechanism, have not been fully elucidated for these proteins because each PLIs’ group targets different PLA2s. Currently, there are two review articles that have attempted to determine the PLA2 targets of PLI classes based on structural predictions [70, 71]. In 2015, Zhen and colleagues successfully established recombinant expression of PLIγ in
3.2.2 Anti-hemorrhagic factors
As previously detailed, hemorrhage, one of the main symptoms of viper envenomation, is induced by snake venom metalloproteinases (SVMPs). The first anti-hemorrhagic factor, habu serum factor (HSF), was identified from the serum of
Habu serum factor; HSF | 5.9 | 4 | 55 | (Crude venom) Protobothrops flavoviridis (SVMPs from Protobothrops flavoviridis) HR1A (P-III), HR1B (P-III), HR2a (P-III), HR2b (P-III), H2 proteinase (P-I) (SVMP from | Cystatin super family (Fetuin family) | [87, 109] | AB058635 | |
BJ46a | 5.7 | 5.2 | 55.4 | (Crude venom) | [88] | AF294836.1 | ||
Mamushi serum factor; jMSF (Japanese MSF) | 5.6 | N.D | 48 | (Crude venom) | [89] | AB200169 | ||
cMSF (Chainese MSF) | 5.6 | N.D | 48 | (Crude venom) | [89] | Q5KQS4.1 | ||
BaSAH | N.D | 5.2 | 66 | (Crude venom) | N.D | [98] | N.D | |
XX | N.D | 4.6 | 64–68 | (Crude venom) | N.D | [99] | N.D | |
XX | N.D | N.D | 65–80 | (Crude venom) | N.D | [100] | N.D | |
XX | N.D | 4.7 | 80 | (Crude venom) | N.D | [101] | N.D | |
TMI | N.D | N.D | 47 | (Crude venom) | N.D | [102] | N.D | |
BaltMPI | N.D | 5.27 | 60.5 and 42.4 | (SVMP) Batroxase (P-I MP from | N.D | [93] | N.D | |
NtAH (serum) | N.D | N.D | 880, 70, 100, 150 | (Crude venom) | N.D | [104] | N.D | |
XX | N.D | N.D | 59 and 52 | (Crude venom) | N.D | [105] | N.D | |
XXX | N.D | N.D | Whole serum | (Crude venom) | N.D | [106] | N.D |
Recently, BaltMPI [93] was found as a hemorrhagic inhibitor in
3.2.3 Small serum proteins (SSPs)
Since 2007, a new class of endogenous inhibitors, named small serum proteins (SSP-1 to SSP-5), has been characterized from Japanese vipers,
4. Undetermined proteinous inhibitors
Isolated proteins from the serum of
Venom resistant has been discovered in some species in mammals, birds, and reptiles, which are either predators or prey of venomous snakes. Natural inhibitors isolated from resistant animals are detailed in reviews by Domont et al. [69, 103] and Bastos et al. [72], which summarized toxin resistance corresponding to several snake species from the plasma, serum, and muscle of mammals. There are a few reports of natural inhibitors isolated from nonvenomous snakes, such as NtAH from
Endogenous inhibitor genes are expressed in the liver of venomous snakes, and these genes appear to be evolving by gene duplication and rapid diversification. This facilitates the neutralization of various toxins within venoms, which also are evolving under similar mechanism [97, 107]. Thus, a detailed characterization of inhibitors against species-specific toxins may help to decipher the evolution of endogenous natural resistance in venomous snakes. Unfortunately, the structural features that govern the inhibitor interaction are still unknown. Recently, there are reports that making a computational analysis predicted three-dimensional structure available [81], and one paper demonstrated that recombinantly expressed BJ46 was able to produce using the expression system of the methylotrophic yeast
5. Conclusions and remaining challenges
There are still many current challenges in the field of toxin inhibitors. Systemic effects of envenomation in humans by snakebite are often mitigated by antivenom therapy, the medically accepted treatment to date. Inhibitors have yet to gain acceptance in clinical use. However, local tissue damage is not neutralized by antivenom and results in permanent morbidity and disability in patients [109]. Local tissue damage is incurred by enzymatic toxins and thus is one of the reasons that Viperidae species occupy just over 60% of venomous snakes listed as Category 1 of highest importance to human health (Tables 1 and 2). Endogenous inhibitors isolated from almost all Viperidae show potent inhibition against their own venom and have also demonstrated to be selective toward highly lethal enzymes within these venoms. The serum inhibitor genes of venomous snakes might have evolved by gene duplication and rapid diversification to facilitate the neutralization of various venom toxins. These serum inhibitors are very stable (resistant to acidic, alkaline, and high temperature environments), selective in inhibitory activity against snake toxins, and are nontoxic, given they exist in blood serum and consist of amino acids (Table 3). However, the molecule mechanism of toxin neutralization involving endogenous inhibitors remains unclear due to a lack of three-dimensional structures detailing toxin and inhibitor complexes. By exploring molecular mechanisms responsible for natural toxin resistance in snakes, we may begin to understand the specificity and selectivity of endogenous inhibitors and use these insights in the design of better therapeutic agents for the treatment of snakebite victims.
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