**2. The Search for the Lethal Factor: Identification and/or Purification of Fish Cytolysins**

Fish venoms can be collected through different methods, which may vary depending on the presence of defined glands or more primitive secretory systems. For those with defined glands—e.g., stonefish—the venom can be collected through puncture of the gland after removal of the tegument [46]. For those with primitive secretory cells—e.g., scorpionfish—the venom is usually obtained through the batch method, in which the

spines are stripped and immersed in buffered solution, or the aspiration method, in which the spines are stripped and the venom aspirated from the grooves [44]. More recent methods, such as the sponge-in-a-tube method [47], in which a microtube containing a sponge is pressed against the spine to rupture the tegument and collect the venom, have the advantage of not requiring the removal of the spines and the sacrifice of specimens.

The search for a lethal factor in fish venoms started with the study of stonefish species, the most venomous of them all. The nature of the molecule responsible for the lethal activity associated with the venom of *Synanceia horrida* was first glimpsed in 1961, when it was found that only one of the seven protein fractions obtained through starch gel electrophoresis of the venom contained the heat-labile lethal material [46]. This fraction was almost twice as lethal as the crude venom when injected intravenously (i.v.) into mice tails (LD50: 18 versus 30 μg of nitrogen/kg). Ten years later, the lethal activity of the venom of the scorpionfish *Scorpaena guttata* was associated to a semi-purified instable fraction, which was almost three times as lethal as the crude venom (LD50: 0.9 versus 2.8 mg/kg, i.v.) [44]. The extreme lability of these fractions hindered the establishment of isolation processes, representing a major bottleneck for their research, which remained stagnant for many years.

By the end of the 20th century, when some of the difficulties imposed by the aforesaid lability of fish cytolysins were overcome through the establishment of proper purification and storage conditions [13,25,41,43], the biochemical and pharmacological characterization of these toxins was greatly accelerated. In addition, the combination of different methods, including liquid chromatography, cDNA cloning, automated Edman degradation, mass spectrometry (MS), and X-ray crystallography resulted in significant advances in the biochemical characterization of these molecules.

Cytolysins have been successfully purified from fish venoms through chromatographic steps using gel filtration, ion exchange, hydrophobic interaction, or adsorption, combined or not with fractionation by saline precipitation. Hemolytic and/or lethal activities were usually employed as a way of tracking the success of the purification process of these proteins [11–13,19,32,39,41].

From the venoms of the stonefish *S. horrida* and *Synanceia trachynis*, were purified stonustoxin (SNTX) [12] and trachynilysin (TLY) [32], respectively. The native molecular weights of SNTX and TLY were estimated in 148 kDa and 158 kDa, respectively. Both are heterodimeric proteins composed of two distinct subunits, named α and β, with masses of 71 kDa and 79 kDa for SNTX, and 76 kDa and 83 kDa for TLY. SNTX accounted for 9% of the protein content of the crude venom and was 22 times more lethal to mice (LD50 0.017 μg/g; i.v.) [12,25,32].

Two different cytolysins, verrucotoxin (VTX) and neoverrucotoxin (neoVTX), were isolated from the venom of the reef stonefish *Synanceia verrucosa* [19,39]. VTX is a 320-kDa glycoprotein that in native form is organized in a tetrameric scaffold comprising two 83-kDa α and two 78-kDa β subunits [19]. It was twice as potent as the crude venom, being immediately lethal to mice at less than 60 ng/g (i.v.). NeoVTX, on the other hand, is a dimeric 166-KDa protein that comprises two distinct subunits (75-kDa α and 80-kDa β) and lacks carbohydrate moieties, showing structural features comparable to those of SNTX and TLY, while considerably differing from VTX [39]. It was also lethal to mice, with an LD50 of 47 μg/kg (i.v.).

The venom of the spotted scorpionfish *Scorpaena plumieri*, although considerably less harmful to humans than that of stonefish, also contains a lethal factor, named *S. plumieri* cytolytic toxin (Sp-CTx), which was first purified with very low yields (1%) [41]. Sp-CTx was then shown to be 12.3-fold more hemolytic (EC50 56 ng/mL) than the crude venom. A molecular mass of 121 kDa was estimated for the native Sp-CTx, while a mass of 65,251 Da *m/z* was revealed by its mass spectrum, pointing to it having a dimeric nature with subunits of very similar molecular masses, much like SNTX and TLY [41]. However, unlike TLY and SNTX, Sp-CTx is a glycoprotein, displaying typical N- and O- linked glycoconjugate residues [41,43]. Three years later, the same group optimized the purification protocol of Sp-CTx and this new method increased the final yield by 13-fold when compared to the previous one [42]. The dimeric nature of Sp-CTx was then confirmed by crosslinking studies using bis-(sulfosuccinimidyl) suberate (BS3). Although lethality was not directly assessed in either of these previous reports, Sp-CTx was proved to be lethal when experiments conducted on anesthetized rats revealed that the animals eventually died upon receiving 70 μg/kg (i.v.) of the toxin [15].

In addition to the aforementioned toxins—which are the best chemically and functionally characterized fish cytolysins—a few such molecules have been purified from the venoms of other fish species.

From the lesser (*Echiichthys vipera*—also known as *Trachinus vipera*) and greater (*Trachinus draco*) weeverfish venoms were purified the cytolysins trachinine [11] and dracotoxin [13], respectively. Trachinine was purified by preparative electrophoresis and shown to be a 324-kDa molecule of tetrameric nature (81-kDa subunits), similar to VTX. It showed an LD100 of < 100 μg/kg in mice (i.v.) [11]. Dracotoxin, unlike all the other known fish cytolysins, is a monomeric protein with MW estimated in 105 kDa both by SDS-PAGE and gel filtration. It was purified with high yields (36%) and it was lethal to mice (minimum lethal dose of 11 μg/g; i.v.) and ∼20-fold more hemolytic than the crude venom of *T. draco* [13].

Semi-purified hemolytic fractions were obtained from lionfish (*Pterois lunulata*), devil stinger (*Inimicus japonicus*), and waspfish (*Hypodytes rubripinnis*) species. Based on the data obtained through gel filtration, immunoblotting, and cDNA cloning, these toxins were determined to be 160-kDa heterodimers composed of 80-kDa α and β subunits [40].

In spite of some variation as to the oligomeric functional state assumed by fish cytolysins purified so far, they are all high-molecular-mass proteins that appear to be active when assembled into oligomers, which could explain their extreme lability. Regarding the quaternary scaffold of these toxins, it is worthy of note that those described as dimeric had their native masses estimated by gel filtration chromatography, while, incidentally, those deemed tetrameric had their masses estimated by gel electrophoresis using the method described by [48].

Lethal and hemolytic activities have also been described in the venoms of the lionfish species *Pterois antennata* and *Pterois volitans* [14], although the actual molecules responsible for these activities have not yet been purified. Nevertheless, both venoms contain toxins identified through cDNA cloning and immunoblotting that share high sequence identity with stonefish cytolysins [14]. The venoms of the scorpaenoid fish species *Sebastapistes strongia*, *Scorpaenopsis oxycephala*, *Sebasticus marmoratus*, and *Dendrochirus zebra* also contain toxins, whose sequences were deduced from cDNA and genomic DNA data, that showed similarity with known fish cytolysins [45].

Finally, several studies have reported antigenic cross-reactivity between fish venoms. The antivenom raised against the venom of *S. trachynis* (SFAV)—the only commercially available fish antivenom—is effective in neutralizing not only the in vivo and in vitro effects of *S. trachynis* venom, but also those of other fish venoms [24,49–51]. In addition, it has been shown that the cytolysins isolated so far cross-react with SFAV and their effects can be neutralized by this antivenom [41,51], suggesting a close similarity between these molecules.

#### **3. Analyzing the Structural Features of Fish Cytolysins**

Before comparing and discussing the biological activities of different fish cytolysins, we will describe what is known so far regarding their structures. This overview should provide a useful background for a better understanding of such activities, particularly regarding the hemolytic activity associated with these toxins.

The complete amino acid sequences of several fish cytolysins were determined by cDNA cloning and/or genomic sequencing [13,14,39,40,45,52–54] (Figure 2).

**Figure 2.** *Cont.*

**Figure 2.** *Cont.*

**Figure 2.** Primary sequence alignment of α and β chains of the cytolysins SNTX [A (Q98989); B (Q91453)] from *S. horrida*, VTX [A (CAA69254.1-partial); B (Q98993)] and neoVTX [A (A0ZSK3), B (A0ZSK4)] from *S. verrucosa*, HrTx [A (BAM74459.1); B (BAM74460.1)] from *H. rubriprinnis*, IjTx [A (BAM74457.1); B (BAM74458.1)] from *I. japonicus*, SmTx [A (AIC84040.1); B (AIC84041.1)] from *S. marmoratus*, SsTx [A (AIC84036.1); B (AIC84037.1)] from *S. strongia*, SoTx [A (AIC84038.1); B (AIC84039.1)] from *S. oxycephala*, DzTx [A (AIC84042.1–partial); B (AIC84043.1–partial)] from *D. zebra*, trachinine [A (AHY22717.1)] from *E. vipera*, Sp-CTx [A (A0A2P1BRQ0), B (A0A2P1BRP3)] from *S. plumieri*, PlTx [A (BAM74455.1); B (BAM74456.1)] from *P. lunulata*, PvTx [A (BAK18814.1); B (BAK18815.1)] from *P. volitans*, and PaTx [A (BAK18812.1); B (BAK18813.1)] from *P. antennata*. Alignments were performed on Muscle server. The sequence of dracotoxin from *T. draco* was not found. The species *S. trachynis* has been reclassified as a synonym of *S. horrida*, so that the sequence of TLY is reported as an alternative name for SNTX, under the same access code. Trachinine is referred to as echiitoxin on NCBI and only the α chain was found. Amino acid residues mentioned at the text are colored as yellow (nonpolar side chain); pink (polar side chain); blue (negatively charged side chain); red (positively charged side chain); and cyan (aromatic side chain) and marked with \*. Domain marking was performed based on SNTX structure [55].

These sequences, along with others totaling one hundred sequences (cover > 83%), were acquired by submitting the primary sequences of the α and β subunits of SNTX to the BLASTP algorithm [56] on the NCBI webserver (https://blast.ncbi.nlm.nih.gov/Blast.cgi; last accessed on 6 October 2021), employing the non-redundant protein sequences (nr) database from fish (taxid 7898).

Fish cytolysins share high primary sequence similarity, with identity ranging from 45 to 94% and ∼20% of residues present at conserved positions (Figure 2), suggesting a strong structural correlation between these molecules. However, only SNTX had its three-dimensional structure determined [55]. The crystallographic model (3.1 Å resolution) of SNTX (Figure 3A) showed that the α and β chains form a stable dimer with an extensive parallel interface along its longitudinal axis, maintained through polar interactions, such as hydrogen bonds and electrostatic interactions.

**Figure 3.** Crystallographic model of stonustoxin (SNTX). (**A**)—SNTX heterodimer showing the α (SNTXA-orange) and β (SNTXB-green) chains. (**B**)—SNTX domains: N-terminal MACPF/CDC domain (residues 1–265-orange); FAT domain (residues 266-385-blue); THX domain (residues 386- 517-red), and C-terminal PRISPY domain (residues 518-703-green). Figure produced on Pymol using the model 4wvm deposited in the Protein Data Bank [55].

Fold recognition searches [55] showed that SNTX belongs to a branch of the "perforinlike" superfamily and revealed the presence of four domains (Figures 2 and 3B) in each chain: (i) N-terminal MACPF/CDC domain (residues 1–265), homologous to the Membrane Attack Complex -Perforin/Cholesterol-Dependent Cytolysin domain. This domain is found in several species and represents a superfamily of pore-forming toxins; (ii) FAT domain (residues 266–385), with high structural similarity to the focal adhesion-targeting domain of human kinase-1. This domain is also found in several proteins and plays a role in the assembly of signaling complexes; (iii) THX domain (residues 386–517), with high structural similarity with thioredoxin-3 (THX-3) from *Saccharomyces cerevisiae*; and (iv) C-terminal domain (residues 518–703), which is similar to the PRYSPRY (PRY SPla and the RYanodine Receptor) domain, member of the tripartite motif family (TRIM) that participates in immune recognition in intracellular bacteria and viruses.

The sequences of the other fish cytolysins were analyzed using the Conserved Domains tool of the NCBI server (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; last accessed on 6 October 2021) (Figure 2). Much like SNTX, they all showed the presence of the C-terminal SPRY (residues from 500 to 700, approximately) and the THX (residues from 380 to 500, approximately) domains on each subunit.

The similarity between these cytolysins and SNTX allows us to infer that some of the features described for the latter are shared among them all. For instance, the THX domain—which is usually associated with redox reactions—most likely plays a structural role, as the key cysteines of the THX catalytic motif are not conserved in these molecules. In addition, there appears to be electrostatic complementarity between the α and β chains through highly conserved charged residues (D314 and Q310 in SNTX-α; R269, N273, R272, and E276 in SNTX-β), which most likely participate in the polar interactions that take place in the interface between the chains. These interactions can be disrupted under the conditions used in purification and mass estimation processes, which may also account for the differences observed in the quaternary arrangements of fish cytolysins. Finally, four cysteine residues are conserved in all cytolysins analyzed, pointing to a possible role in heterodimer stability through disulfide bridges. However, the formation of these bonds was not observed in the three-dimensional structure of SNTX [55].
