Keywords

Introduction

Snake venom is a complex mixture of components that exhibit numerous pathophysiological effects. The venom of the South American rattlesnake Crotalus durissus terrificus contains four proteic components: gyroxin, crotoxin, convulxin, and crotamine, of which crotoxin represents the principal toxic component. Crotoxin is a potent β-neurotoxin that acts primarily at the presynaptic level of the neuromuscular junction and inhibits acetylcholine release; blockage of neuromuscular transmission leads to muscle paralysis and death by asphyxia (Brazil and Excell 1971; Hawgood and Smith 1977). Crotoxin can also act at the postsynaptic level, stabilizing a desensitized form of the acetylcholine receptor (AChR) (Bon et al. 1979; Brazil et al. 2000).

In addition to neurotoxicity, crotoxin exhibits other significant pharmacological properties and biological effects such as myotoxic (Gutiérrez et al. 2008), cardiotoxic (potentiation of L-type Ca2+ channel) (Zhang et al. 2010), cytotoxic (chronic seizure effect by release of glutamate from cerebrocortical synaptosomes via N and P/Q Ca2+ channel in the central nervous system) (Lomeo Rda et al. 2014), bactericidal (Oliveira et al. 2002; Toyama et al. 2003; Perumal Samy et al. 2006), anti-inflammatory (Cardoso et al. 2001; Sampaio et al. 2005; Zambelli et al. 2008; Nunes et al. 2010), antitumoral (Rudd et al. 1994; Costa et al. 1998; Cura et al. 2002; Yan et al. 2007), and analgesic (Zhang et al. 2006; Nogueira-Neto Fde et al. 2008) effects and antiviral activity (Muller et al. 2012; Muller et al. 2014).

Crotoxin from Crotalus durissus terrificus venom was the first animal neurotoxin isolated and characterized (Slotta and Fraenkel-Conrat 1938). Other crotoxin-like proteins commonly occur in the venoms of Crotalus species, e.g., Crotalus durissus cascavella (Beghini et al. 2000; Beghini et al. 2004; Rangel-Santos et al. 2004; Fonseca et al. 2006), Crotalus durissus collilineatus (Ponce-Soto et al. 2002; Rangel-Santos et al. 2004; Toyama et al. 2005; Ponce-Soto et al. 2007; Salvador et al. 2009), Crotalus durissus cumanensis (Pereañez et al. 2009; Cavalcante et al. 2015), Crotalus durissus ruruima (Dos-Santos et al. 2005), Crotalus scutulatus (Mojave toxin) (Bieber et al. 1975; Gopalakrishnakone et al. 1980), and Crotalus simus (Calvete et al. 2010; Castro et al. 2013; Durban et al. 2013), and Erotalus vegronolis (Viala et al. 2015). Heterodimeric complexes of crotoxin-like proteins have also been identified in the venom of non-rattlesnake species belonging to Crotalinae subfamily: pit vipers Gloydius intermedius (Gintexin) (Yang et al. 2015a, b) and Bothriechis nigroviridis (Nigroviriditoxin) (Fernández et al. 2010; Lomonte et al. 2015).

A comparision of the structure, biological properties, and toxicity of crotoxin from Crotalus durissus terrificus and other crotoxin-like proteins is presented here. Other biological targets of crotoxin and its basic PLA2 CB subunit are also described.

Crotoxin

Crotoxin is a heterodimeric protein complex formed by the non-covalent association of an acidic, nontoxic, nonenzymatic CA subunit with a basic, weakly toxic CB subunit with phospholipase A2 activity (group IIA sPLA2) (Hendon and Fraenkel-Conrat 1971). Individual snake venom contains up to 16 natural crotoxin complexes having different biochemical and biological properties (Faure and Bon 1988). These complexes result from the association of different isoforms of the CA and CB subunits (Faure et al. 1991, 1993). Moreover, the profiles of crotoxin isoforms vary between individual snakes of the same species (Faure and Bon 1987).

CA Subunit

The CA subunit of crotoxin (crotapotin, component A, or crotoxin A) is an acidic, nontoxic protein comprising three disulfide-linked polypeptide chains α, β, and γ. The CA subunit does not possess catalytic activity and in snake venom is present only in complex with the basic CB subunit, enhancing its lethal potency. CA prevents the formation of oligomers between PLA2 subunits and targets CB to reach the specific crotoxin receptor. CA also inhibits the PLA2 and anticoagulant activities of CB (Faure et al. 1991, 1993, 2007).

Four CA isoforms (CA1-4) have been identified in Crotalus durissus terrificus venom . Briefly, crotoxin was isolated from crude venom by gel filtration chromatography, and the CA and CB subunits were separated by ion-exchange chromatography in the presence of 6 M urea. Four CA isoforms were purified from a mixture of isoforms on Mono Q column by an anion-exchange chromatography. The three chains of each purified CA isoforms from Crotalus durissus terrificus were separated by reverse-phase (RP)-HPLC (Vydac C18 column) and the sequences determined by Edman degradation (Faure et al. 1991). Several isoforms of an acidic CA-like protein were found in the non-rattlesnake species Gloydius intermedius (Yang et al. 2015a) and Bothriechis nigroviridis (Lomonte et al. 2015) and were isolated from the crude venoms using directly RP-HPLC in acetonitrile gradient (Yang et al. 2015a; Lomonte et al. 2015). Gel filtration chromatography was performed in the case of Gintexin-A (Yang et al. 2015a). The molecular masses of CA subunit isoforms isolated from Crotalus durissus terrificus, Gloydius intermedius, and Bothriechis nigroviridis are shown in Table 1.

Table 1 Molecular masses of CA subunit isoforms of crotoxin and crotoxin-like proteins
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All CA subunit isoforms of crotoxin derive from a pro-CA PLA2-like precursor protein homologous to nontoxic acidic group IIA sPLA2 and result from post-translational modifications by removal of three peptide segments (Faure et al. 2011) (the enzymes responsible for this process have not yet been identified). During protein maturation a conversion of N-terminal glutamine residues to pyrrolidone carboxylic acid occurs in the β and γ chains. This modification increases protection of the protein from proteolytic degradation. The mature CA protein is composed of three polypeptide chains (α, β, γ) linked by two intra- and five interchain disulfide bonds (Bouchier et al. 1991; Faure et al. 1991; Faure et al. 2011). Differences between CA isoforms of crotoxin and crotoxin-like proteins result from slightly different lengths and amino acid sequences of the individual polypeptide chains (Fig. 1) (Faure et al. 1991). The post-translational maturation of pro-CA is a prerequisite for an appropriate assembly of the three independent CA chains with the basic CB subunit to form the crotoxin complex (Faure et al. 1991).

Fig. 1
figure 1

Multiple sequence alignment of precursors and mature proteins of CA from Crotalus durissus terrificus (Faure et al. 1991) and Gintexin-A from Gloydius intermedius (Yang et al. 2015a). pro-CA precursor of the acidic subunit of crotoxin from Crotalus durissus terrificus, pro-Gintexin-A precursor of the acidic subunit of the crotoxin-like protein from Gloydius intermedius. Amino acid numbering is according to Renetseder et al. 1985. In blue, point mutations between sequences; black boxes, chain α, β, and γ in mature proteins; red boxes, pyrrolidone carboxylyl residues, converted from glutamine by posttranslational modification; underlined, residues determined only by amino acid composition (not sequenced). Sequence alignment was performed with CLC Sequence Viewer 6

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The crystal structure of only one CA isoform has been solved, the isoform CA2 in complex with isoform CBb in the 3D structure of crotoxin (PDB 3R0L) (Faure et al. 2011). This structure revealed that the two long α-helices C and D in the positions expected for group IIA sPLA2 are preserved in the CA subunit, but the long α-helix A and the short α-helix B are absent (as expected from posttranslational cleavage of pro-CA) (Faure et al. 2011). Despite the preservation of the amino acid sequence in the predicted Ca2+-binding loop, an extended conformation unlike the canonical Ca2+-binding loop of group IIA PLA2S structures was revealed, explaining in part why the CA subunit does not perform a catalytic function. The amino acid sequence of the precursor of the acidic subunit from Gloydius intermedius (Gintexin-A) displays 76% identity with pro-CA from Crotalus durissus terrificus (Fig. 1). Similar to the CA subunit of crotoxin, mature Gintexin-A consists of three polypeptide chains resulting from post-translational modifications of a precursor protein, and the structure is stabilized by seven disulfide bonds (Yang et al. 2015a). The α, β, and γ chains of this protein show high amino acid sequence identity (87% and 69% with CA1,2,4 for chain α and 85% and 66% with CA3 for chain β of crotoxin and 78% for chain γ of CA).

CB Subunit

The CB subunit of crotoxin (component B or crotoxin B) is a basic, weakly neurotoxic protein that possesses PLA2 activity and catalyzes the hydrolysis of the sn-2 ester bond of phospholipids, producing free fatty acids and lysophospholipids (Hendon and Fraenkel-Conrat 1971; Faure and Bon 1988). In snake venom, isoforms of the CB subunit of crotoxin occur in complex with CA. The isolated CB subunit also acts at the presynaptic level of the neuromuscular junction and blocks neuromuscular transmission, but tenfold higher doses of this protein are needed compared to the CACB complex.

Four isoforms of the CB subunits (CBa2/CB2, CBb, CBc/CB1, and CBd) in Crotalus durissus terrificus venom have been identified. These isoforms were purified from crude venom using a four-step chromatographic procedure: (i) gel filtration (isolation of crotoxin), (ii) ion-exchange chromatography in the presence of 6 M urea (separation of the CA and CB subunits each containing a mixture of isoforms), (iii) cation-exchange column using double gradient (0–1 M NaCl and 0–3 M urea, purification of CB isoforms), and (iv) RP-HPLC, Vydac C4 column (additional purification step) (Faure and Bon 1988). The CB isoforms are products of different mRNAs (Bouchier et al. 1991; Faure et al. 2011). However, in Crotalus durissus terrificus venom, several additional CB isoforms (CB-like proteins: F15 (Toyama et al. 2003), F16 (Hernandez-Oliveira et al. 2005), F17 (Oliveira et al. 2002), and Intercro (Vieira et al. 2013)) have been identified. Proteins F15, F16, and F17 were isolated from the crotoxin fraction by RP-HPLC. Intercro was eluted from the column as a fraction located between crotamine and crotoxin during gel filtration chromatography. In the case of non-rattlesnake species, the presence of basic CB-like isoforms has also been observed. Two isoforms of Nigroviriditoxin B, a CB-like basic protein, were purified by RP-HPLC from the crude venom of Bothriechis nigroviridis (Lomonte et al. 2015). A single isoform of Gintexin-B was isolated from crude venom of Gloydius intermedius using gel filtration and RP-HPLC (Yang et al. 2015a). Both rattlesnake and non-rattlesnake CB and CB-like proteins contain 122 amino acid residues with a molecular weight of approximately 14 kDa and pI values around 9 (Table 2).

Table 2 Biochemical properties of CB and CB-like proteins from Crotalus durissus terrificus, Bothriechis nigroviridis, and Gloydius intermedius
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All CB isoforms from Crotalus durissus terrificus are secreted group IIA phospholipases structurally homologous to the inflammatory, non-pancreatic human group IIA sPLA2 (EC 3.1.1.4). The crystal structures of isoforms CBa2, CBb, and CBc show the presence of highly conserved canonical structural features of group IIA sPLA2 stabilized by seven disulfide bonds (Marchi-Salvador et al. 2008; Faure et al. 2011). The 3D structures of CB-like PLA2s have not yet been determined experimentally, but as shown in Fig. 2, they share a high degree of sequence homology with CB. CB isoforms and other CB-like proteins isolated from Crotalus durissus terrificus venom (F15, F16, F17, and Intercro) show sequence identities of more than 90% (except CBc/F17) (Table 3). The high sequence identity between CBa2 and Intercro (97.5%) is surprising since the latter is not part of the crotoxin fraction (Vieira et al. 2013). These proteins differ by only three residues (positions CBa2/Intercro: W70/F, Y117/L, and Y120/F) (Fig. 2), resulting in lower PLA2 activity and toxicity of Intercro and preventing complex formation with CA (Table 4). The amino acid sequences of CB-like subunits isolated from non-rattlesnake species Gintexin-B and Nigroviriditoxin B reveal the identities up to 90% and 80%, respectively, to all PLA2s isolated from Crotalus durissus terrificus (Table 3). Several sequence differences occur in the C-terminal region often considered to be responsible for the neurotoxicity of the venom PLA2s. The phylogenetic relationship of the CB and CB-like proteins analyzed here is shown in Fig. 3.

Fig. 2
figure 2

Multiple sequence alignment of CB isoforms and CB-like proteins F17, F16, F15, and Intercro from Crotalus durissus terrificus and non-rattlesnake basic subunits Gintexin-B from Bothriechis nigroviridis and Nigroviriditoxin B from Gloydius intermedius. Amino acid numbering is according to Renetseder et al. 1985. In blue, point mutations occurring in sequences of analyzed proteins; black boxes, highly conserved regions of SPLA2-IIA: α-helix A, helix B, Ca2+-binding loop, α-helix C, the β-wing, and α-helix D.

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Table 3 Sequence identities (%) of sPLA2 isoforms from Crotalus durissus terrificus , Bothriechis nigroviridis, and Gloydius intermedius venom
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Table 4 Enzymatic activity of CB isoforms and CB-like PLA2s from Crotalus durissus terrificus , Gloydius intermedius, and Bothriechis nigroviridis
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Fig. 3
figure 3

Phylogenetic tree of CB and CB-like PLA2 isoforms F17, F16, F15, and Intercro from Crotalus durissus terrificus and non-rattlesnake basic subunits Gintexin-B from Gloydius intermedius and Nigroviriditoxin B from Bothriechis nigroviridis. The phylogenetic tree was calculated with the UPGMA algorithm.

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CB isoforms and CB-like proteins in the presence of acidic subunit CA form different classes of crotoxin complexes (see below), confirming that natural mutations in CB isoforms determine different properties of crotoxin complexes (Faure and Bon 1988). The phospholipase A2 activities of all CB isoforms are quite similar (Table 4) (Faure et al. 1993). However, significant differences appear in the presence of the CA subunit (except for isoform CBa2 where binding of CA does not influence PLA2 activity). The crystal structure of the crotoxin CA2CB6 complex (Faure et al. 2011) revealed that the natural point mutation at position 1 (Ser1 in CBa2, His1 in CBb,c) is crucial for the functional differences between crotoxin isoforms. The presence of Ser at position 1 in CBa2 associated with a displacement of Trp70 and Trp31 prevents their interaction with Asp89 and Asp99 of the CA subunit (chain β), leading to greater substrate access to the catalytic site of CB (His48 and Asp99) (Faure et al. 2011). These structural differences explain the higher PLA2 activity of the CA2CBa2 complex.

For the CBb, CBc, and CBd isoforms, a two- to fourfold decrease in enzymatic activity is observed after CA binding, depending on the CB isoform (Table 4) (Faure et al. 1993). Once again, these differences result from point mutations in the CB subunit (Fig. 2). For isoforms F15, F16, and F17, very weak inhibition of enzymatic activity is observed in complexes with the CA subunit (Table 4). Intercro does not form complexes with CA (Vieira et al. 2013). In agreement with previous results (Faure et al. 2011), the mutation at position 70 (Phe70 in Intercro, Trp70 in CBa2) explains the absence of binding of Intercro with CA. Nigroviriditoxin B alone shows very similar PLA2 activity to CB (Lomonte et al. 2015).

The enzymatic activity of all PLA2s is strictly dependent on the presence of Ca2+ ions (Mayer and Marshall 1993; Murakami and Kudo 2002). The calcium binding sites (Cys29, Gly30, Gly32), as well as Trp31 which affects stability in this region, are conserved in all CB isoforms and other crotoxin-like proteins analyzed here (Fig. 2). Moreover it has been shown that Mg2+ stimulates PLA2 activity of Intercro (Vieira et al. 2013).

All CB isoforms display neurotoxic properties, CBa2 being the least toxic (Table 5). Binding of the acidic CA subunit increases the lethal potency of the CB subunit, and this toxic effect is correlated with the stability of CACB complexes (Faure et al. 1993, 2011). CBa2 in complex with CA forms less stable and less neurotoxic complexes, with affinity to CA sixfold lower compared to the other CB isoforms (Table 6). CBb, CBc, and CBd have comparable affinity to CA (KD = 3.5–5 nM), and these complexes are at least four times more toxic. However, the stability of these complexes is different. The half-life of CACBd is two times greater than that observed for CACBb (Table 6) (Faure et al. 1993). Slight variations in the neurotoxic activities of these isoforms are shown in Table 5. Differences in the stability and neurotoxicity of crotoxin complexes result from the point mutations in CB isoforms (see below). F15, F16, and F17 PLA2s in complex with CA reveal much lower toxicity (Oliveira et al. 2002; Toyama et al. 2003; Hernandez-Oliveira et al. 2005), whereas Intercro does not display neurotoxic properties (Vieira et al. 2013). Surprisingly, the toxicity of the Gintexin complex is similar to that observed for crotoxin (Yang et al. 2015a). Uncomplexed Nigroviriditoxin B revealed almost sixfold lower potency in comparison with CB isoforms. The toxicity of the complex (Nigroviriditoxin B with Nigroviriditoxin A) increases only slightly and is 20-fold lower than that determined for crotoxin (Table 5) (Lomonte et al. 2015).

Table 5 Neurotoxic properties of CB isoforms and CB-like PLA2s from Crotalus durissus terrificus, Gloydius intermedius, and Bothriechis nigroviridis
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Table 6 Stability of CACB complexes
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Based on their enzymatic and neurotoxic properties, crotoxin isoforms can be divided into two classes (Faure and Bon 1988). Class I includes complexes formed by CBb, CBc, CBd, and CA isoforms, forming stable complexes with high toxicity and low phospholipase A2 activity. CBa2, which belongs to crotoxin complexes of class II, forms less toxic and less stable complexes with higher enzymatic activity.

The F15, F16, F17, and Intercro proteins show very weak or no toxicity and low phospholipase A2 activity. However, binding of the CA subunit has little effect on their enzymatic activity. Based on these data, complexes of F15, F16, and F17 with CA can be classified as class II crotoxin-like complexes similar to the CA2CBa2 crotoxin complex.

Complexes of CB with Different Protein Targets

The CA and CB subunits of crotoxin are present in snake venom as non-covalent, high (nanomolar) affinity complexes. However, CB isoforms obtained in vitro after separation of the two subunits from a mixture of crotoxin isoforms can also interact with other protein targets such as human factor Xa (hFXa), CICS (a natural crotoxin inhibitor from snake blood), GLIC pentameric receptor, and CFTR chloride channel, affecting their pharmacological properties and biological functions. The crystal structure of crotoxin (pdb 3R0L) provides a detailed model (Fig. 4) to explore possible modes of binding of CB to different protein targets.

Fig. 4
figure 4

Molecular binding interface of the CA and CB subunits of crotoxin. The crystal structure of crotoxin (pdb 3R0L) provides a detailed model to explore possible modes of binding of CB to other protein targets. Left: surface representation of the CB subunit (in blue, amino acid residues in contact with the CA subunit). Center: the assembled CACB complex. Right: surface representation of the CA subunit (in yellow, α-chain residues in contact with CB; in green, β-chain residues in contact with CB; in pink, the γ-chain of the CA subunit makes no contact with CB)

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CB Interaction with Human Coagulation Factor Xa

It has been shown that CB can form complexes with human coagulation factor Xa (hFXa) (Faure et al. 2007). This interaction influences the coagulation pathway by inhibiting the prothrombinase complex formation and retarding the formation of thrombin. Surface Plasmon Resonance (SPR) analysis showed that binding affinity depends on the CB isoform. CBc interacts with hFXa with very high affinity (KD = 0.6±0.3 nM) compared to CBa2 (KD = 52±4 nM), which is correlated with anticoagulant activity (stronger inhibition of prothrombinase complex formation) (Faure et al. 2007).

The anticoagulant binding site of CB was identified by mutagenesis, affinity-binding studies, functional assays, and molecular docking calculations (Faure et al. 2007; Faure and Saul 2011). Two adjacent regions were identified. The first region is composed of residues located in helices A and B of the CB subunit and a loop between helix C and the β-wing. The second region includes part of the Ca2+ binding loop and the C-terminal region of the protein. Seven regions in hFXa (five in the heavy chain and two in the light chain) are involved in binding with CB. The catalytic sites of both proteins are not involved in these interactions (Faure and Saul 2011). Moreover, analysis of crystallographic structures showed that His1, Arg34, and Gly128 may be essential in the binding of hFXa by isoform CBc. Mutations of these residues in isoform CBa2 (Ser1, Gln34, and Glu128) lead to conformational changes in adjacent residues and result in lower affinity for hFXa (Faure et al. 2007; Faure and Saul 2011).

CB Interaction with CICS

Several natural PLA2 inhibitors (PLI) have been identified in the blood of venomous snakes (Faure 2000). They can be divided into three types (PLI-α, PLI-β, and PLI-γ) according to their structure and properties. The crotoxin inhibitor from Crotalus durissus terrificus serum (CICS) belongs to PLI-γ type inhibitors, homologous to proteins of the Ly-6 superfamily. CICS is a 130 kDa acidic glycoprotein formed by the non-covalent association of six 23–25 kDa subunits (Perales et al. 1995; Faure et al. 2000). SPR analysis revealed a very high affinity of crotoxin and its CB subunit to CICS (nM affinity) (Faure et al. 2000). CICS neutralizes the toxic effect and inhibits the PLA2 activity of crotoxin by interacting with the CB subunit. Binding of CICS with crotoxin leads to dissociation of the crotoxin complex and the release of CA (Perales et al. 1995).

Using peptide arrays Fortes-Dias and co-workers (Fortes-Dias et al. 2009) identified three possible interaction regions involved in CICS/CNF binding. According to their studies, the CA-CB and CICS-CB interfaces partially overlap.

CB Interaction with Prokaryotic Receptor GLIC

Crotoxin is primarily a presynaptic neurotoxin. However, some studies have shown that it also displays postsynaptic action. Recently, the proton-gated ion channel GLIC from Gloeobacter violaceus, a bacterial homolog of pentameric ligand-gated ion channel receptors, was identified as a new protein target of the CB subunit of crotoxin, and a novel function of CB as an inhibitor of proton-gated ion channel activity was reported (Ostrowski et al. 2016). Using SPR studies, it was shown that the interaction PLA2-GLIC involves the extracellular domain of GLIC and that the enzymatic activity of PLA2 is enhanced during interaction with GLIC (Ostrowski et al. 2016). Formation of the CB-GLIC complex leads to conformational changes in the secondary structure of the proteins, and it was proposed that PLA2 would be a new negative allosteric modulator of GLIC (Ostrowski et al. 2016).

CB Interaction with CFTR

Numerous studies have shown that neurotoxic PLA2 can enter cells and interact with various intracellular protein targets, resulting in a wide range of pharmacological effects. PLA2s as multifunctional proteins could potentially interfere as binders with ion channels. It has recently been shown that crotoxin and its CB subunit interact with the nucleotide-binding domain (NBD1) of CFTR (cystic fibrosis transmembrane regulator, a cyclic AMP-regulated chloride channel) and increase its chloride channel currents (Faure et al. 2016). Interestingly, the CB subunit possesses high affinity for both wild type and ΔF508-CFTR (the most frequent mutation associated with cystic fibrosis). CB behaves as a dual modulator of CFTR activity, as a potentiator and as a corrector (Faure et al. 2016).

Conclusion

Knowledge at the molecular level of the crotoxin CACB-binding interface and the 3D structures of natural isoforms of CB and CB-like proteins may help to identify other biologically relevant PPI sites. Two examples cited here (the CB-FXa and CB-CFTR/ΔF508CFTR complexes) open interesting perspectives for the future development of noncompetitive inhibitors of human FXa as anticoagulant agents and new correctors and potentiators for the treatment of cystic fibrosis.