- Open Access
Alpha-type phospholipase A2 inhibitors from snake blood
Journal of Venomous Animals and Toxins including Tropical Diseases volume 23, Article number: 19 (2017)
It is of popular and scientific knowledge that toxins from snake venom (among them the PLA2 and myotoxins) are neutralized by various compounds, such as antibodies and proteins purified from animal blood. Venomous and nonvenomous snakes have PLA2 inhibitory proteins, called PLIs, in their blood serum. One hypothesis that could explain the presence of these PLIs in the serum of venomous snakes would be self-protection against the enzymes of their own venom, which eventually could reach the circulatory system. However, the presence of PLIs in non-venomous snakes suggests that their physiological role might not be restricted to protection against PLA2 toxins, but could be extended to other functions, as in the innate immune system and local regulation of PLA2s. The present study aimed to review the currently available literature on PLA2 and myotoxin alpha inhibitors present in snake plasma, thus helping to improve the research on these molecules. Furthermore, this review includes current information regarding the mechanism of action of these inhibitors in an attempt to better understand their application, and proposes the use of these molecules as new models in snakebite therapy. These molecules may help in the neutralization of different types of phospholipases A2 and myotoxins, complementing the conventional serum therapy.
Between 2009 and 2013, the World Health Organization (WHO) included envenomation by snakes among the neglected tropical diseases given the large number of accidents, the complexity of the clinical condition and the fact that the most affected population consists mainly of workers from poor rural communities in tropical countries [1–4]. However, nowadays experts in Toxinology call on WHO and governments to re-establish snakebite as a neglected tropical disease, since each year, approximately 421,000 cases of snakebite occur, of which approximately 20,000 result in death .
Generally, the lethality of bites is low, though the frequency of sequelae related to local complications is higher, especially when associated with risk factors such as the use of a tourniquet, bite in extremities (fingers and toes) and delayed treatment . It is important to note that some sequelae – especially those that lead to partial or total limb amputation – despite been a public health problem, also constitute social problems, since they may provoke various disorders, including the disability to work . Snake venoms are a complex mixture of components, and more than 90% of their dry weight consists of proteins with a large variety of enzymes, and a non-protein portion comprising carbohydrates, lipids, metals, free amino acids, nucleotides and others . The protein components of snake venoms include cytotoxins, cardiotoxins, nerve growth factors, lectins, enzyme inhibitors and various enzymes, such as phospholipase A2 (PLA2), metalloproteases, serine proteases, phosphodiesterases, cholinesterases, aminotransferases, L-amino acid oxidases, catalases, ATPases, hyaluronidases, etc. .
Thus, considering the search for natural inhibitors that neutralize snake venom toxins is of extreme importance for the production of more efficient antivenoms, the present study aims to review the currently available literature on alpha inhibitors present in snake plasma, thus helping to improve the current knowledge about these molecules.
Phospholipases A2 (PLA2)
Phospholipases are a superfamily of enzymes that act on phospholipids in the cell membrane leading to their cleavage in fatty acids and lysophospholipids. Phospholipases A2 (PLA2) (EC 22.214.171.124) were the first phospholipases to be known and their discovery was based on observation of the action of pancreatic fluid of mammals and snake venom in the hydrolysis of phosphatidylcholine .
These enzymes play an important role in several cellular functions including maintenance of cellular phospholipids, generation of prostaglandins (PGs) and leukotrienes, cell proliferation and muscle contraction. Furthermore, it is known that these enzymes are involved in human inflammatory processes and due to their central role in many cellular processes, they have been extensively studied [7, 10–12].
The PLA2s are a superfamily of enzymes belonging to 16 groups and subgroups that can also be divided into six distinct types: the secreted PLA2 (sPLA2), among them PLA2s found in snake venoms; the cytosolic PLA2 (cPLA2); the Ca2+ independent PLA2s (iPLA2); the acetyl-hydrolases activating factors of platelets (PAF-AH); lysosomal PLA2 and the lipoprotein-associated phospholipase A2 (Lp-PLA2) [13, 14].
According to Schaloske and Dennis  and Dennis et al. , the sPLA2s are enzymes with a molecular weight between 14,000 and 18,000 Da, usually containing from 5 to 8 disulfide bridges. These enzymes have a histidine in their active site and require the presence of Ca2+ ion for catalysis. The phospholipase A2 from groups IA, IB, IIA, IIB, IIC, IID, IIE, IIF, III, V, IX, X, XIA, XIB, XII, XIII and XIV are representatives of sPLA2s.
The PLA2s from snake venoms (svPLA2s) are classified into groups I and II, and those from the Viperidae family belong to group IIA [11, 13–15]. The svPLA2s belonging to group IIA are subdivided into subgroups based on the presence of a conserved residue on position 49, being the most studied: (i) PLA2s Asp49, enzymes that usually have high catalytic activity, and (ii) homologous PLA2s (or PLA2-like) Lys49, which have no enzymatic activity [16, 17]. It is important to point out that other variants in snake venom group II PLA2s have been reported, e.g., Ser49, Asn49 and Arg49 [18–23].
Interestingly, despite having no catalytic activity, the homologous PLA2s Lys49 have a wide variety of pharmacological and/or toxic effects, including myotoxicity, cytotoxicity, antibacterial, antifungal, muscle necrotic and anticoagulant activities [7, 24–27]. According to some authors, the main structural domain responsible for the toxic effect, particularly cytotoxic, in homologous Lys49-PLA2 is the C-terminal region (amino acids 115–129) .
PLA2 inhibitory proteins (PLIs) from snake blood
Venomous and non-venomous snakes have PLA2 inhibitory proteins, called PLIs, in their blood serum [28–30]. These PLA2 inhibitory proteins are produced by the liver, as indicated by Northern blot analysis and RT-PCR analysis of genetic material extracted from different tissues. This PLI production by the liver (and not by the venom glands or other organ) makes it possible for these proteins to enter the bloodstream, since the liver is the main organ producing plasma proteins, thus improving and accelerating the protection mechanism against poisoning [31–33]. Furthermore, it has been known that some secreted PLA2 receptors, which have structural similarity with PLIs, also exist in soluble forms, showing that PLIs, as well as PLA2 endogenous receptors, could have a regulatory role of proinflammatory activity of sPLA2s .
Several PLIs were purified from the plasma of different species of snakes, and their structures have been determined [28–30, 34, 35]. So far, for the isolation of PLA2 inhibitors described in the literature, two different methods were used. One of these purification methods is the bioaffinity chromatography, which is based on the immobilization of different proteins, PLA2 in this case (for example BthTX-I and BthTX-II, from Bothrops jararacussu), on a stationary phase [32, 36–40]. Another method used in purification of PLIs from snake plasma is a sequence of chromatographic steps such as gel filtration, ion exchange and hydrophobic chromatography [35, 41, 42].
The blood used for plasma separation is typically collected by cardiac puncture, by puncturing the tail vein or after decapitation of the snake. It is noteworthy that in recent years concern about the ethics in the use animals for experimentation is growing and therefore the least aggressive method that does not require animal death is the blood collection from the tail vein of the snake, being the most indicated. After collecting the blood, plasma and serum are separated, then plasma is lyophilized and stored. During purification, the inhibitory activity of these PLIs is monitored by biological assays based on inhibition activity of PLA2 and myotoxins, depending on the inhibitor of interest.
The PLA2 and myotoxin inhibitors from the blood of snakes are globular, acid and oligomeric proteins, which form soluble complexes with PLA2 and myotoxins, thus inhibiting the action of these molecules [34, 43–46]. Blood inhibitors found in snakes are classified into types alpha (α), beta (β) and gamma (γ) according to structural aspects [30, 47, 48].
One of the PLIs classes, the βPLIs, have repeated leucine-rich structures and show similarity to human α2-glycoprotein . βPLIs inhibit only basic group II PLA2s isolated from snake venoms and have been isolated from plasma of Agkistrodon blomhoffii siniticus, Elaphe quadrivirgata and E. climacophora snakes, which belong to the Viperidae and Colubridae family [33, 49, 50].
Another type of PLIs, known as γPLIs, is the most abundant to date. The γPLIs are acidic glycoproteins with a mass of 90–130 kDa consisting of 3 to 6 noncovalent subunits. Their amino acid sequences contain two sets of standards cysteine residues, responsible for the formation of the three-finger motif . This type of inhibitor has been reported in different snakes, as Crotalus durissus terrificus [52–54], Naja naja kaouthia [55, 56], Agkistrodon blomhoffii siniticus , Trimeresurus flavoviridis , Laticauda semifasciata , Elaphe quadrivirgata , E. climacophora , Cerrophidion godmani , Notechis ater, Notechis ater serventyi , Oxyuranus scutellatus and O. microlepidotus , Pseudonaja textilis , Python reticulates , Notechis scutatus , Lachesis muta muta , Protobothrops flavoviridis , Bothrops alternatus, B. erythromelas, B. jararaca, B. moojeni, B. neuwiedi , Bothrops jararacussu  and Crotalus durissus collilineatus  and these γPLIs appear to be less specific, since they inhibit PLA2 from groups I, II and III.
Alpha-type PLA2 inhibitor
The alpha-type PLA2 inhibitors (αPLIs) from the snake blood are found mainly as trimers in solution and have a region with high similarity with the carbohydrate recognition domain (CRD) of C-type lectins and pulmonary surfactant protein [30, 36, 37, 40, 67–70]. This region covers approximately 67% of the primary sequence of the monomers of αPLIs and is the most conserved portion of these molecules, with approximately 46% of sequence identity between species . The CRD of αPLIs lacks the amino acid residues involved in Ca2+ binding, making the interaction with their respective ligands Ca2+-independent [40, 42]. Moreover, several studies have shown that the carbohydrate motif present in αPLIs is not necessary for the connection with PLA2 [32, 38].
αPLIs studied to date
Various αPLIs were purified to date (Table 1), such as the plasma PLI from the snake Trimeresurus flavoviridis, which was purified by a combination of chromatographic steps through Sephadex gel filtration column G-200, DEAE-cellulose anion exchange and Blue Sepharose CL-6B . The purified inhibitor was found as a glycoprotein with an approximately molecular weight of 100,000 Da, with non-homologous subunits of approximately 20,000 to 24,000 Da. Subsequently, it was verified the ability of this inhibitor to interact with venom phospholipase A2 of T. flavoviridis, and Agkistrodon halys blomhoffii, besides the enzyme and the porcine pancreatic phospholipase C of Bacillus cereus. According to Kogaki et al. , this inhibitor showed specificity to T. flavoviridis PLA2, and an independent inhibitory activity of Ca2+.
Afterward, Inoue et al.  purified two distinct but homologous subunits (PLIα-A and PLIα-B) of the PLI from Trimeresurus flavoviridis. These subunits were separated by reversed-phase HPLC and showed molecular weights around 21,000–22,000 Da when glycosylated and 17,000 after deglycosylation. Furthermore, the sequences were significantly homologous to CRD portions of pulmonary surfactant apoprotein and animal lectins. Then, Shimada et al.  studied this αPLI, which was purified into different subspecies of two homologous subunits. Before this work, it was expected that this αPLI was a tetramer, composed of two molecules of αPLI-A and two molecules of αPLI-B . However, in this last study, it was showed that this αPLI is a trimeric protein. Curiously, all the αPLIs except that from P. flavoviridis are multimers composed of a single subunit.
Ohkura et al.  purified an alpha inhibitor from the snake Agkistrodon blomhoffii siniticus, using a similar method described by Kogaki et al. . In this case, this αPLI purification was performed by sequential chromatography through Sephadex G-200 column, Mono Q and Sepharose Blue CL-6B. The purified PLI showed up as a glycoprotein with a molecular mass from 75,000 to 24,000 Da for the trimer and the monomer.
After, Inoue et al.  studied the specificity of the two previously purified (and cited above) PLA2 inhibitors from T. flavoviridis and A. b. siniticus plasma, purified by Kogaki et al. , and Ohkura et al. , respectively. Both αPLI showed a high specificity for group II acidic PLA2s from their own venom. In this work, the authors draw a parallel between PLI from snake plasma and PLA2 receptors of rabbit, bovine, and human, suggesting that the CRD-like domain would be involved in the binding to the PLA2 molecule.
Regarding the αPLI from Bothrops genus, other α inhibitors were purified, for example, BaMIP, a PLI isolated from the plasma of Bothrops asper by affinity chromatography in Sepharose 4B CNBr-activated with myotoxins immobilized . BaMIP presented monomers with a molecular weight of approximately 24,000 Da and a structure in solution composed of five subunits. The BaMIP showed inhibition on myotoxic, edema and cytolytic activity of the myotoxins I and III of B. asper snake. Structural studies have also shown that BaMIP, as well as all α phospholipase A2 inhibitors has a homologous domain to CRD of C-type lectins.
Another snake inhibitor studied is CgMIP-II, an αPLI, purified from plasma of Cerrophidion (Bothrops) godmani snake by affinity column containing myotoxins . The inhibitor is an acidic protein (pI 4.0), glycosylated, the monomeric subunits with a molecular weight between 20,000 Da and 25,000 Da, forming a polymer of about 180,000 Da.
Soares et al.  purified a protein that neutralizes the enzymatic, toxic and pharmacological activity of a variety of toxins (acidic or basic) of different venoms. This inhibitor, called BmjMIP, was isolated from the plasma of the snake Bothrops Moojeni, by affinity chromatography. BmjMIP presented similar biochemical and structural characteristics to those already described for αPLIs, besides being stable at a wide range of pH and temperature.
Okumura et al.  purified the αPLI-like protein (PLIα-LP) from a non-venomous snake E. quadrivirgata serum by sequential chromatography on Hi-trap Blue, Mono Q and Superdex 200 columns. The PLIα-LP showed the highly conserved C-type lectin-like domain (CTLD) and 51 kDa, being a trimer. Although this protein has about 70% similarity with other inhibitors previously studied, this protein did not demonstrate any inhibitory activity against different PLA2s. It is important to cite that Shirai et al.  also purified an αPLI-like protein (PLIα-LP) from E. climacophora snake. According to Okumura et al. , the high homology with αPLIs and the lack of inhibitory activity on αPLI-like proteins may provide important information concerning the structure/function of these αPLIs.
Quirós et al. , purified an αPLI (AnMIP) from the plasma of Atropoides nummifer by affinity matrix, prepared by coupling a mixture of myotoxins I and II from A. nummifer to an NHS-activated column. According to the work, this trimeric inhibitor neutralized the activity of basic PLA2 myotoxins and showed specificity towards group II PLA2, either belonging to the catalytically active (Asp49 PLA2) or inactive (Lys49 PLA2-like) subtypes.
Oliveira et al.  and Santos-Filho et al.  purified two different αPLIs (named αBjussuMIP and αBaltMIP), from B. jararacussu and B. alternatus, respectively. These molecules were purified through affinity chromatography using BthTX-I immobilized on Sepharose gel and neutralize enzymatic, toxic and pharmacological activities of several phospholipases A2. Santos-Filho et al. [74, 75] subsequently expressed an active recombinant alpha inhibitor, named rBaltMIP, in Pichia pastoris heterologous system. According to these works, heterologous expression would enable large-scale obtainment of these αPLI, thus allowing further investigations for the elucidation of possible mechanisms of inhibition of PLA2s, which have not yet been fully clarified.
Mechanism of action of αPLIs
In the last 30 years, several studies have been published aiming to biochemically, structurally and functionally characterize αPLIs. However, the mechanism of action of these αPLIs is still unknown. Some authors have suggested that the αPLI/PLA2 binding site is probably related to the CRD region of the molecule, which recognizes and binds to the enzyme, preventing its toxic activity. One factor that supports this idea is that these CRD domains are present in endogenous PLA2 receptors, such as the human receptor of group I pancreatic PLA2 and receptors of group II secretory PLA2 from rabbits, mice, cattle and humans [38, 73, 76–78]. Nevertheless, the molecular nature of the interaction between the CRD region and PLA2 is still unknown and efforts towards the elucidation of the structure of αPLIs and their complexes are being performed .
Studying the deletion of amino acid residues, Nobuhisa et al.  mapped the interaction between an αPLI and an acidic PLA2 from T. flavoviridis, noting that the binding capacity was more restricted to the C-terminal region between residues 136 and 147. In this region, two hydrophobic tripeptides and Tyr144 residue appear to be involved in the interaction PLI/PLA2 [37, 69, 79].
Thereafter, Okumura et al.  studied the relationship of the structure/function of the αPLI previously purified from the snake Agkistrodon blomhoffii siniticus, named GbPLIα, and the αPLI-like protein EqPLIα-LP, purified from the nonvenomous snake Elaphe quadrivirgata, and which does not show inhibitory activity against PLA2s [42, 68]. In that work, by constructing chimeric proteins, they mapped important residues to the inhibitory activity of the αPLIs; for example, the region 13-36 of the neck C-terminal portion of the trimer. Interestingly, the region found as the responsible for PLA2 inhibition was distinct from the carbohydrate-binding site. Furthermore, other residues were pointed as candidate, including Asn26, Lys28, Asp29, and Tyr144 .
According to Okumura et al. , the trimer is formed through the interactions of the helical neck regions, forming a central pore, responsible for PLA2 binding. Furthermore, as Tyr144 is expected to be located in this central pore, this residue may be one of the responsibles for the direct interaction to the PLA2 molecule. In a complementary study, Nishida et al.  created heterotrimers of αPLI composed of two different subunits derived from the recombinant GbPLIα, EqPLIα-LP, and chimeras of GbPLIα-EqPLIα-LP homotrimers, in order to estimate the contribution of each subunit to the total inhibitory activity as a trimeric PLA2 inhibitory protein. Summing up, in this work, it was observed, once more, the importance of the residues 13–36 for the trimer formation, and consequently for the αPLI inhibitory activity. Furthermore, the interactions between residues Glu23 and Lys28 of GbPLIα were also suggested to be important to stabilize the trimeric structure.
Lastly, in a recent study, Estevão-Costa et al.  studied the importance of αPLI trimerization for the binding and inhibition to acidic PLA2s. Furthermore, they suggested that the central pore, which is composed by positive charged residues, especially Arg57, Lys71, Arg108 and His109, could be a significant part of the binding site of αPLIs to acidic PLA2s. In addition, these authors pointed the importance of the hydrophobic core (Leu158 to Val161), which may be the responsible for the central pore structural integrity. However, the positive surface of the basic PLA2 could prevent the PLA2/PLI interaction at the central pore and according to these authors, the mechanism of inhibition of basic PLA2 by αPLIs remains to be understood. It is interesting to point out that, considering the sequence of the native protein, obtained through Edman degradation sequencing , the numbering of central pore important residues should be Arg38, Lys52, Arg89 and His90 (Fig. 1).
So far, it is possible to observe that the mechanism of action of these inhibitors and the region responsible for their inhibitory properties are not yet fully elucidated in the literature, requiring further study concerning these macromolecules and their interactions with PLA2s.
Potential complement of antiophidic serum therapy
Currently, antiserum composed of specific immunoglobulins is the only treatment for snake envenomation, but there are ongoing issues with availability, effectiveness and dosing [81–83]. These antivenoms neutralize the toxicity and lethality of specific venoms, but their administration is often related with significant clinical side effects [84, 85]. Additionally, the production of antivenoms is associated with high costs related to animal maintenance and also comes across animal welfare concerns, which instigates the search for innovative products for snakebite therapy [82, 86].
Interestingly, the production of specific antivenom was started by Vital Brazil in the 1900’s and it was Vital Brazil who also discovered the effectiveness of the polyvalent antivenom [87, 88]. At that time, antivenom was prepared with crude plasma of hyperimmunized animals. However, it was thereafter discovered that antibodies (immunoglobulins) were the active therapeutic molecules responsible for the action of the antivenom. Therefore, only the antibodies started to be purified and used in antivenom therapy.
Nowadays, despite advances in the production of antivenoms, this production is still similar to the methods originally described by Vital Brazil [87, 88]. Currently, immunoglobulins or immunoglobulin fragments [F(ab’)2 or Fab] purified from serum are used in antivenom . Other innovations have been proposed on traditional antiserum, as the use of the single chain variable fragment (scFv) or the use of recombinant antigen binding domains derived from camelid heavy chain antibodies (VHH) [82, 89–91]. However, there are numerous challenges on antivenom improvement, for example, the high cost of monoclonal antibodies production or the lower affinity and the short serum half-life profiles of some immunoglobulin fragments [82, 92].
Although serum therapy effectively reverses the systemic effects of venom into the victim’s body, avoiding death many times, it has some disadvantages including a number of side effects (anaphylactic shock, renal failure and serum sickness, for example). The inefficiency to combat the local effects of the envenomation (increasing the chances of sequelae in the stricken member), the need for careful storage and the short shelf life of the serum are also other limiting factors.
PLA2 enzymes and PLA2-like myotoxins are the main responsible for myonecrosis, an important medical complication of snake envenomation, and which, in severe cases can lead to drastic consequences such as permanent loss of tissue or limb amputation. These outcomes provoke severe problems for both the affected individual and public health, since the victim may become incapable of working and lose life quality. In addition, these sequelae burden the public health once they increase the length of hospitalization and surgeries and, in some cases, can lead to early retirement of the individual affected by the envenomation.
The search for natural inhibitors that neutralize snake venom toxins is of extreme importance for the production of more efficient antivenoms, especially considering that several toxins induce weak immunogenic responses, making traditional serum therapy unable to inhibit local effects such as the myotoxicity induced by phospholipases A2 and PLA2-like enzymes [46, 93].
In conclusion, the traditional antivenom is not completely able to inhibit local effects of envenomation, mainly caused by myotoxins. Thus, the search for proteins, such as αPLIs, that neutralize myotoxins present in snake venom is extremely important for the production of a more efficient treatment.
Carbohydrate recognition domain
C-type lectin-like domain
Ca2+ independent PLA2s
Lipoprotein-associated phospholipase A2
Acetyl-hydrolases activating factors of platelets
PLA2 inhibitory proteins
Alpha-type PLA2 inhibitor
Harrison RA, Hargreaves A, Wagstaff SC, Faragher B, Lalloo DG. Snake envenoming: a disease of poverty. PLoS Negl Trop Dis. 2009;3(12):e569.
WHO. WHO Expert Committee on Biological Standardization. 59th report. WHO Technical Report Series, no. 964. 2012. Available: http://www.who.int/biologicals/WHO_TRS_964_web.pdf.
Gutiérrez JM. Current challenges for confronting the public health problem of snakebite envenoming in Central America. J Venom Anim Toxins Incl Trop Dis. 2014;20(1):7.
Chippaux JP. Epidemiology of envenomations by terrestrial venomous animals in Brazil based on case reporting: from obvious facts to contingencies. J Venom Anim Toxins Incl Trop Dis. 2015;21:13.
Bagcchi S. Experts call for snakebite to be re-established as a neglected tropical disease. BMJ. 2015;351:h5313.
Warrell DA. Venomous bites, stings, and poisoning. Infect Dis Clin North Am. 2012;26(2):207–23.
Santos-Filho NA, Silveira LB, Oliveira CZ, Bernardes CP, Menaldo DL, Fuly AL, et al. A new acidic myotoxic, anti-platelet and prostaglandin I2 inductor phospholipase A2 isolated from Bothrops moojeni snake venom. Toxicon. 2008;52(8):908–17.
Tu AT. Overview of snake venom chemistry. Adv Exp Med Biol. 1996;391:37–62.
Barros GA, Pereira AV, Barros LC Jr AL, Calvi SA, Santos LD, et al. In vitro activity of phospholipase A2 and of peptides from Crotalus durissus terrificus venom against amastigote and promastigote forms of Leishmania (L.) infantum chagasi. J Venom Anim Toxins Incl Trop Dis. 2015;21:48.
Dennis EA, Rhee SG, Billah MM, Hannun YA. Role of phospholipase in generating lipid second messengers in signal transduction. FASEB J. 1991;5(7):2068–77.
Six DA, Dennis EA. The expanding superfamily of phospholipase A2 enzymes: classification and characterization. Biochim Biophys Acta. 2000;1488(1–2):1–19.
Murakami M, Kudo I. Diversity and regulatory functions of mammalian secretory phospholipase A2s. Adv Immunol. 2001;77:163–94.
Schaloske RH, Dennis EA. The phospholipase A2 superfamily and its group numbering system. Biochim Biophys Acta. 2006;1761(11):1246–59.
Dennis EA, Cao J, Hsu YH, Magrioti V, Kokotos G. Phospholipase A2 enzymes: physical structure, biological function, disease implication, chemical inhibition, and therapeutic intervention. Chem Rev. 2011;111(10):6130–85.
Rodrigues VM, Marcussi S, Cambraia RS, de Araújo AL, Malta-Neto NR, Hamaguchi A, et al. Bactericidal and neurotoxic activities of two myotoxic phospholipases A2 from Bothrops neuwiedi pauloensis snake venom. Toxicon. 2004;44(3):305–14.
Ownby CL, Selistre de Araujo HS, White SP, Fletcher JE. Lysine 49 phospholipase A2 proteins. Toxicon. 1999;37(3):411–45.
Soares AM, Sestito WP, Marcussi S, Stábeli RG, Andrião-Escarso SH, Cunha OA, et al. Alkylation of myotoxic phospholipases A2 in Bothrops moojeni venom: a promising approach to an enhanced antivenom production. Int J Biochem Cell Biol. 2004;36(2):258–70.
Krizaj I, Bieber AL, Ritonja A, Gubensek F. The primary structure of ammodytin L, a myotoxic phospholipase A2 homologue from Vipera ammodytes venom. Eur J Biochem. 1991;202(3):1165–8.
Polgár J, Magnenat EM, Peitsch MC, Wells TN, Clemetson KJ. Asp-49 is not an absolute prerequisite for the enzymic activity of low-M(r) phospholipases A2: purification, characterization and computer modelling of an enzymically active Ser-49 phospholipase A2, ecarpholin S, from the venom of Echis carinatus sochureki (saw-scaled viper). Biochem J. 1996;319(Pt 3):961–8.
Pan H, Liu X, Yang G, Zhou Y, Wu X. Research on the diversity of PLA2 gene from Agkistrodon halys pallas. Acta Biochim Biophys Sin Shanghai. 1998;30(1):91–5.
Tsai IH, Wang YM, Chen YH, Tsai TS, Tu MC. Venom phospholipases A2 of bamboo viper (Trimeresurus stejnegeri): molecular characterization, geographic variations and evidence of multiple ancestries. Biochem J. 2004;377(Pt 1):215–23.
Mebs D, Kuch U, Coronas FI, Batista CV, Gumprecht A, Possani LD. Biochemical and biological activities of the venom of the Chinese pitviper Zhaoermia mangshanensis, with the complete amino acid sequence and phylogenetic analysis of a novel Arg49 phospholipase A2 myotoxin. Toxicon. 2006;47(7):797–811.
Murakami MT, Kuch U, Betzel C, Mebs D, Arni RK. Crystal structure of a novel myotoxic Arg49 phospholipase A2 homolog (zhaoermiatoxin) from Zhaoermia mangshanensis snake venom: insights into Arg49 coordination and the role of Lys122 in the polarization of the C-terminus. Toxicon. 2008;51(5):723–35.
Koduri RS, Grönroos JO, Laine VJ, Le Calvez C, Lambeau G, Nevalainen TJ, et al. Bactericidal properties of human and murine groups I, II, V, X, and XII secreted phospholipases A2. J Biol Chem. 2002;277(8):5849–57.
Murillo LA, Lan CY, Agabian NM, Larios S, Lomonte B. Fungicidal activity of a phospholipase-A2-derived synthetic peptide variant against Candida albicans. Rev Esp Quimioter. 2007;20(3):330–3.
Costa TR, Menaldo DL, Oliveira CZ, Santos-Filho NA, Teixeira SS, Nomizo A, et al. Myotoxic phospholipases A2 isolated from Bothrops brazili snake venom and synthetic peptides derived from their C-terminal region: cytotoxic effect on microorganism and tumor cells. Peptides. 2008;29(10):1645–56.
Lomonte B, Angulo Y, Moreno E. Synthetic peptides derived from the C-terminal region of Lys49 phospholipase A2 homologues from viperidae snake venoms: biomimetic activities and potential applications. Curr Pharm Des. 2010;16(28):3224–30.
Fortes-Dias CL. Endogenous inhibitors of snake venom phospholipases A2 in the blood plasma of snakes. Toxicon. 2002;40(5):481–4.
Marcussi S, Sant’Ana CD, Oliveira CZ, Rueda AQ, Menaldo DL, Beleboni RO, et al. Snake venom phospholipase A2 inhibitors: medicinal chemistry and therapeutic potential. Curr Top Med Chem. 2007;7(8):743–56.
Lizano S, Domont G, Perales J. Natural phospholipase A2 myotoxin inhibitor proteins from snakes, mammals and plants. Toxicon. 2003;42(8):963–77.
Nobuhisa I, Deshimaru M, Chijiwa T, Nakashima K, Ogawa T, Shimohigashi Y, et al. Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake. Gene. 1997;191(1):31–7.
Lizano S, Angulo Y, Lomonte B, Fox JW, Lambeau G, Lazdunski M, et al. Two phospholipase A2 inhibitors from the plasma of Cerrophidion (Bothrops) godmani which selectively inhibit two different group-II phospholipase A2 myotoxins from its own venom: isolation, molecular cloning and biological properties. Biochem J. 2000;346(Pt 3):631–9.
Okumura K, Inoue S, Ikeda K, Hayashi K. Identification of beta-type phospholipase A2 inhibitor in a nonvenomous snake, Elaphe quadrivirgata. Arch Biochem Biophys. 2002;408(1):124–30.
Dunn RD, Broady KW. Snake inhibitors of phospholipase A2 enzymes. Biochim Biophys Acta. 2001;1533(1):29–37.
Faure G. Natural inhibitors of toxic phospholipases A2. Biochimie. 2000;82(9–10):833–40.
Soares AM, Marcussi S, Stábeli RG, França SC, Giglio JR, Ward RJ, et al. Structural and functional analysis of BmjMIP, a phospholipase A2 myotoxin inhibitor protein from Bothrops moojeni snake plasma. Biochem Biophys Res Commun. 2003;302(2):193–200.
Quirós S, Alape-Girón A, Angulo Y, Lomonte B. Isolation, characterization and molecular cloning of AnMIP, a new alpha-type phospholipase A2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae). Comp Biochem Physiol B Biochem Mol Biol. 2007;146(1):60–8.
Oliveira CZ, Menaldo DL, Marcussi S, Santos-Filho NA, Silveira LB, Boldrini-Franca J, et al. An alpha-type phospholipase A2 inhibitor from Bothrops jararacussu snake plasma: structural and functional characterization. Biochimie. 2008;90(10):1506–14.
Oliveira CZ, Santos-Filho NA, Menaldo DL, Boldrini-Franca J, Giglio JR, Calderon LA, et al. Structural and functional characterization of gamma-type phospholipase A2 inhibitor from Bothrops jararacussu snake plasma. Curr Top Med Chem. 2011;11(20):2509–19.
Santos-Filho NA, Fernandes CA, Menaldo DL, Magro AJ, Fortes-Dias CL, Estevao-Costa MI, et al. Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma. Biochimie. 2011;93(3):583–92.
Kogaki H, Inoue S, Ikeda K, Samejima Y, Omori-Satoh T, Hamaguchi K. Isolation and fundamental properties of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. J Biochem. 1989;106(6):966–71.
Ohkura N, Inoue S, Ikeda K, Hayashi K. Isolation and amino acid sequence of a phospholipase A2 inhibitor from the blood plasma of Agkistrodon blomhoffii siniticus. J Biochem. 1993;113(4):413–9.
Ovadia M, Kochva E. Neutralization of Viperidae and Elapidae snake venoms by sera of different animals. Toxicon. 1977;15(6):541–7.
Domont GB, Perales J, Moussatché H. Natural anti-snake venom proteins. Toxicon. 1991;29(10):1183–94.
Thwin MM, Gopalakrishnakone P. Snake envenomation and protective natural endogenous proteins: a mini review of the recent developments (1991–1997). Toxicon. 1998;36(11):1471–82.
Santos-Filho N, Silveira LB, Boldrini-França J. Myotoxin inhibitors. In: Toxins and drug discovery. 1st ed. Heidelberg: Chapter: Myotoxin Inhibitors. Springer Netherlands; 2015. p. 1–24.
Thwin MM, Satish RL, Chan ST, Gopalakrishnakone P. Functional site of endogenous phospholipase A2 inhibitor from python serum. Eur J Biochem. 2002;269(2):719–27.
Thwin MM, Samy RP, Satyanarayanajois SD, Gopalakrishnakone P. Venom neutralization by purified bioactive molecules: synthetic peptide derivatives of the endogenous PLA2 inhibitory protein PIP (a mini-review). Toxicon. 2010;56(7):1275–83.
Okumura K, Ohkura N, Inoue S, Ikeda K, Hayashi K. A novel phospholipase A2 inhibitor with leucine-rich repeats from the blood plasma of Agkistrodon blomhoffii siniticus. Sequence homologies with human leucine-rich alpha2-glycoprotein. J Biol Chem. 1998;273(31):19469–75.
Shirai R, Toriba M, Hayashi K, Ikeda K, Inoue S. Identification and characterization of phospholipase A2 inhibitors from the serum of the Japanese rat snake, Elaphe climacophora. Toxicon. 2009;53(6):685–92.
Estevão-Costa MI, Rocha BC, de Alvarenga MM, Redondo R, Franco GR, Fortes-Dias CL. Prospection, structural analysis and phylogenetic relationships of endogenous gamma-phospholipase A2 inhibitors in Brazilian Bothrops snakes (Viperidae, Crotalinae). Toxicon. 2008;52(1):122–9.
Fortes-Dias CL, Jannotti ML, Franco FJ, Magalhães A, Diniz CR. Studies on the specificity of CNF, a phospholipase A2 inhibitor isolated from the blood plasma of the South American rattlesnake (Crotalus durissus terrificus). I. Interaction with PLA2 from Lachesis muta muta snake venom. Toxicon. 1999;37(12):1747–59.
Fortes-Dias CL, Lin Y, Ewell J, Diniz CR, Liu TY. A phospholipase A2 inhibitor from the plasma of the South American rattlesnake (Crotalus durissus terrificus). Protein structure, genomic structure, and mechanism of action. J Biol Chem. 1994;269(22):15646–51.
Perales J, Villela C, Domont GB, Choumet V, Saliou B, Moussatché H, et al. Molecular structure and mechanism of action of the crotoxin inhibitor from Crotalus durissus terrificus serum. Eur J Biochem. 1995;227(1–2):19–26.
Ohkura N, Inoue S, Ikeda K, Hayashi K. The two subunits of a phospholipase A2 inhibitor from the plasma of Thailand cobra having structural similarity to urokinase-type plasminogen activator receptor and LY-6 related proteins. Biochem Biophys Res Commun. 1994;204(3):1212–8.
Ohkura N, Inoue S, Ikeda K, Hayashi K. Isolation and characterization of a phospholipase A2 inhibitor from the blood plasma of the Thailand cobra Naja naja kaouthia. Biochem Biophys Res Commun. 1994;200(2):784–8.
Ohkura N, Okuhara H, Inoue S, Ikeda K, Hayashi K. Purification and characterization of three distinct types of phospholipase A2 inhibitors from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus. Biochem J. 1997;325(Pt 2):527–31.
Nobuhisa I, Inamasu S, Nakai M, Tatsui A, Mimori T, Ogawa T, et al. Characterization and evolution of a gene encoding a Trimeresurus flavoviridis serum protein that inhibits basic phospholipase A2 isozymes in the snake’s venom. Eur J Biochem. 1997;249(3):838–45.
Ohkura N, Kitahara Y, Inoue S, Ikeda K, Hayashi K. Isolation and amino acid sequence of a phospholipase A2 inhibitor from the blood plasma of the sea krait, Laticauda semifasciata. J Biochem. 1999;125(2):375–82.
Okumura K, Masui K, Inoue S, Ikeda K, Hayashi K. Purification, characterization and cDNA cloning of a phospholipase A2 inhibitor from the serum of the non-venomous snake Elaphe quadrivirgata. Biochem J. 1999;341(Pt 1):165–71.
Hains PG, Broady KW. Purification and inhibitory profile of phospholipase A2 inhibitors from Australian elapid sera. Biochem J. 2000;346(Pt 1):139–46.
Thwin MM, Gopalakrishnakone P, Kini RM, Armugam A, Jeyaseelan K. Recombinant antitoxic and antiinflammatory factor from the nonvenomous snake Python reticulatus: phospholipase A2 inhibition and venom neutralizing potential. Biochemistry. 2000;39(31):9604–11.
Hains PG, Nield B, Sekuloski S, Dunn R, Broady K. Sequencing and two-dimensional structure prediction of a phospholipase A2 inhibitor from the serum of the common tiger snake (Notechis scutatus). J Mol Biol. 2001;312(4):875–84.
Fortes-Dias CL, Barcellos CJ, Estevão-Costa MI. Molecular cloning of a gamma-phospholipase A2 inhibitor from Lachesis muta muta (the bushmaster snake). Toxicon. 2003;41(7):909–17.
So S, Chijiwa T, Ikeda N, Nobuhisa I, Oda-Ueda N, Hattori S, et al. Identification of the B subtype of gamma-phospholipase A2 inhibitor from Protobothrops flavoviridis serum and molecular evolution of snake serum phospholipase A2 inhibitors. J Mol Evol. 2008;66(3):298–307.
Gimenes SN, Ferreira FB, Silveira AC, Rodrigues RS, Yoneyama KA, Izabel dos Santos J, et al. Isolation and biochemical characterization of a γ-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum. Toxicon. 2014;81:58–66.
Inoue S, Kogaki H, Ikeda K, Samejima Y, Omori-Satoh T. Amino acid sequences of the two subunits of a phospholipase A2 inhibitor from the blood plasma of Trimeresurus flavoviridis. Sequence homologies with pulmonary surfactant apoprotein and animal lectins. J Biol Chem. 1991;266(2):1001–7.
Okumura K, Inoue S, Ikeda K, Hayashi K. Identification and characterization of a serum protein homologous to alpha-type phospholipase A2 inhibitor (PLIalpha) from a nonvenomous snake, Elaphe quadrivirgata. IUBMB Life. 2003;55(9):539–45.
Okumura K, Ohno A, Nishida M, Hayashi K, Ikeda K, Inoue S. Mapping the region of the alpha-type phospholipase A2 inhibitor responsible for its inhibitory activity. J Biol Chem. 2005;280(45):37651–9.
Nishida M, Okamoto M, Ohno A, Okumura K, Hayashi K, Ikeda K, et al. Inhibitory activities of the heterotrimers formed from two α-type phospholipase A2 inhibitory proteins with different enzyme affinities and importance of the intersubunit electrostatic interaction in trimer formation. Biochim Biophys Acta. 2010;1804(11):2121–7.
Shimada A, Ohkura N, Hayashi K, Samejima Y, Omori-Satoh T, Inoue S, et al. Subunit structure and inhibition specificity of alpha-type phospholipase A2 inhibitor from Protobothrops flavoviridis. Toxicon. 2008;51(5):787–96.
Inoue S, Shimada A, Ohkura N, Ikeda K, Samejima Y, Omori-Satoh T, et al. Specificity of two types of phospholipase A2 inhibitors from the plasma of venomous snakes. Biochem Mol Biol Int. 1997;41(3):529–37.
Lizano S, Lomonte B, Fox JW, Gutiérrez JM. Biochemical characterization and pharmacological properties of a phospholipase A2 myotoxin inhibitor from the plasma of the snake Bothrops asper. Biochem J. 1997;326(Pt 3):853–9.
Santos-Filho NA, Boldrini-França J, Santos-Silva LK, Menaldo DL, Henrique-Silva F, Sousa TS, et al. Heterologous expression and biochemical and functional characterization of a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus snake. Biochimie. 2014;105:119–28.
Santos-Filho NA, Sousa TS, Boldrini-França J, Santos-Silva LK, Menaldo DL, Henrique-Silva F, et al. rBaltMIP, a recombinant alpha-type myotoxin inhibitor from Bothrops alternatus (Rhinocerophis alternatus) snake, as a potential candidate to complement the antivenom therapy. Toxicon. 2016;124:53-62.
Nicolas JP, Lambeau G, Lazdunski M. Identification of the binding domain for secretory phospholipases A2 on their M-type 180-kDa membrane receptor. J Biol Chem. 1995;270(48):28869–73.
Lambeau G, Ancian P, Nicolas JP, Beiboer SH, Moinier D, Verheij H, et al. Structural elements of secretory phospholipases A2 involved in the binding to M-type receptors. J Biol Chem. 1995;270(10):5534–40.
Valentin E, Lambeau G. Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins. Biochim Biophys Acta. 2000;1488(1-2):59–70.
Nobuhisa I, Chiwata T, Fukumaki Y, Hattori S, Shimohigashi Y, Ohno M. Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes. FEBS Lett. 1998;429(3):385–9.
Estevão-Costa MI, Fernandes CA, Mudadu M de A, Franco GR, Fontes MR, Fortes-Dias CL. Structural and evolutionary insights into endogenous alpha-phospholipase A2 inhibitors of Latin American pit vipers. Toxicon. 2016;112:35–44.
Scheske L, Ruitenberg J, Bissumbhar B. Needs and availability of snake antivenoms: relevance and application of international guidelines. Int J Health Policy Manag. 2015;4(7):447–57.
Prado ND, Pereira SS, da Silva MP, Morais MS, Kayano AM, Moreira-Dill LS, et al. Inhibition of the myotoxicity induced by Bothrops jararacussu venom and isolated Phospholipases A2 by specific camelid single-domain antibody fragments. PLoS One. 2016;11(3):e0151363.
Bochner R. The international view of envenoming in Brazil: myths and realities. J Venom Anim Toxins Incl Trop Dis. 2013;19(1):29.
Morais VM, Massaldi H. Snake antivenoms: adverse reactions and production technology. J Venom Anim Toxins Incl Trop Dis. 2009;15(1):2–18.
Zolfagharian H, Dounighi NM. Study on development of Vipera lebetina snake anti-venom in chicken egg yolk for passive immunization. Hum Vaccin Immunother. 2015;11(11):2734–9.
Krifi MN, El Ayeb M, Dellagi K. The improvement and standardization of antivenom production in developing countries: comparing antivenom quality, therapeutical efficiency, and cost. J Venom Anim Toxins. 1999;5(2):128–41.
Brazil V. Dosagem do valor antitóxico dos serums antipeçonhentos. Brasil: Trib Med. 1908;14(3):39–44.
Bochner R. Paths to the discovery of antivenom serotherapy in France. J Venom Anim Toxins Incl Trop Dis. 2016;22:20.
Cardoso DF, Nato F, England P, Ferreira ML, Vaughan TJ, Mota I, et al. Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library. Scand J Immunol. 2000;51(4):337–44.
Fernandes I, Assumpção GG, Silveira CR, Faquim-Mauro EL, Tanjoni I, Carmona AK, et al. Immunochemical and biological characterization of monoclonal antibodies against BaP1, a metalloproteinase from Bothrops asper snake venom. Toxicon. 2010;56(6):1059–65.
Richard G, Meyers AJ, McLean MD, Arbabi-Ghahroudi M, MacKenzie R, Hall JC. In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody. PLoS One. 2013;8(7):e69495.
Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: successes, limitations and hopes for the future. Br J Pharmacol. 2009;157(2):220–33.
Kulkeaw K, Chaicumpa W, Sakolvaree Y, Tongtawe P, Tapchaisri P. Proteome and immunome of the venom of the Thai cobra, Naja kaouthia. Toxicon. 2007;49(7):1026–41.
The authors are grateful to Dr. Lucas Blundi Silveira for the careful and meticulous correction of this paper, scientific support and essential discussion upon the completion of the manuscript. Thanks are also due to the Center for the Study of Venoms and Venomous Animals (CEVAP) of UNESP for enabling the publication of this paper (Edital Toxinologia CAPES no. 063/2010, Process no. 230.38.006285/2011-21, AUXPE Toxinologia 1219/2011).
This work was supported by the São Paulo Research Foundation (FAPESP).
Both authors contributed with data compilation, writing and critical discussion of the manuscript. Both authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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Santos-Filho, N.A., Santos, C.T. Alpha-type phospholipase A2 inhibitors from snake blood. J Venom Anim Toxins Incl Trop Dis 23, 19 (2017). https://doi.org/10.1186/s40409-017-0110-2
- Phospholipases A2
- Myotoxin inhibitor
- Snake blood