**3. Discussion**

The *T. nattereri* envenomation in humans and the recapitulation of the injury in mice have been extensively studied by our group [24–26]. We identified in the Natterin group of venom toxins the aerolysin proteins being responsible for the main effects of envenomation [27,28], and their immunopharmacological activities were determined [29–32]. Besides the Natterin proteins, *T. nattereri* venom also contains Nattectin, a lectin with Ca2+ independent hemagglutinating and immunomodulatory activities [4]. Nattectin shows the particular potential to bind types I and V collagen and improve integrin-mediated HeLa cell adhesion and apoptosis protection by its binding to RGD-dependent integrins, mainly the β1 subunit [33]. We further demonstrated its ability to activate antigen-presenting cells [34] and trigger Th1-type immune response with IgG1 production [35].

On the other hand, just a few studies have been conducted with *T. maculosa*, one of the *T. nattereri*'s closest relatives. Sosa-Rosales et al. [17] partly identified a biologically active protein by chromatography. To obtain the toxin TmC4-47.2 from the venom of the *T. maculosa*, we used reverse-phase HPLC-based workflow with the application of the crude venom to the chromatography system coupled to a semi-preparative reverse-phase C18 column, followed by subsequent chromatography of the fractions containing the toxin with a molecular weight of 15 kDa on a reverse-phase C8 analytical column. Finally, the protein was purified into three lyophilized fractions for verification of purity and exact mass by LC/MS. Interestingly we confirmed the presence of two proteins with very close molecular weights, one with a calculated molecular weight of 15,135.9 Da and the other with 15,634.3 Da, unlike the migrated single band ~15 kDa via reducing SDS-PAGE.

To determine the number of cysteine residues and, consequently, of disulfide bridges, we proceeded with the reduction and alkylation of the toxins. After applying the sample to the same LC/MS system, we once more detected the presence of two proteins, now with 15,484.8 Da and 15,982.8 Da, respectively. From the difference in the masses of the pure proteins, we inferred that both proteins have 6 cysteines each and consequently form 3 disulfide bridges, as described in the sequence of Nattectin from *T. nattereri* [28]. Our findings that the anti-venom serum and the anti-Nattectin serum from *T. nattereri* recognize the TmC4-47.2 toxin from *T. maculosa* support the similarity of the sequences.

The sequencing of internal fragments of the toxins generated by enzymatic digestion with trypsin and analysis by MALDI-ToF revealed 100% similarity of the sequenced fragments with the Nattectin sequence, which correspond to 40% of its entire sequence. The high homology rate might reflect a relatively small evolutionary divergence between the two congeneric species and the fact that they harbor the same biological properties. Batrachoididae, the only family in the ray-finned fish order Batrachoidiformes, arose on Earth in the Miocene, which is the first geological epoch of the Neogene Period that extends from about 23 to 5 million years ago (mya). There are about 83 species of toadfishes grouped into 21 genera. The subfamily Thalassophryninae has six phenotypically alike species in the genus *Thalassophryne*, which suggests the time of divergence among species within this genus must be little, although there is no precise description of the time of divergence in the literature. Using peptide mass fingerprinting, we identified a unique carbohydrate recognition domain composed of the amino acids glutamine, proline, and aspartate (QPD), a specific galactose-binding site [36]. We have designated the TmC4-47.2 toxin as a galactose-binding protein.

Glycan structures are common post-translational modifications of proteins, assisting in protein folding and defining the timing for protein disposal as a part of the quality control function in the early secretory pathway [37]. Our data shows that the nattectin-like protein of *T. maculosa* is non-glycosylated since we determined the absence of GlcNAc-β-Ser/Thr or N-linked glycan residues.

Among the distinct post-translational modifications, glycosylation is the most recurrent, and virtually 50% of all known proteins are thought to be glycosylated. In contrast, the galectins are generally small, soluble, non-glycosylated proteins and, unlike the C-Type lectins, do not require structural stabilization via Ca2+ complexing for their activity. However, many C-type lectins are Ca2+-independent and may not necessarily bind to sugar ligands [38]. Our findings showing the nattectin-like lectin as a non-glycosylated protein with the ability to interact with the core and terminal galactose-type ligands point to a functional similarity with proto-type galectins that contains only one CRD. Galectins, a family of animal lectins with an affinity for β-galactosides, can form multivalent complexes with cell surface glycoconjugates and trigger several intracellular signals to modulate cell activation, differentiation, and survival [39].

Literature has portrayed the protective role of galectins against infections when binding to the glycoconjugates on the surface of invasive microbes (virus, bacteria, fungi, and parasites) acting as pattern recognition receptors in innate immunity [40]. In fish, they have also been demonstrated to participate in pathogen recognition. Gal-1, a proto-type galectin, from rock bream (*Oplegnathus fasciatus*) bound and agglutinate many bacteria [41] and the Gal-1 of zebrafish bound infectious hematopoietic necrosis virus (IHNV) in a carbohydrate-dependent way [42]. AJL-1, a proto-type galectin of Japanese eel (*Anguilla japonica*), was reported to hinder the biofilm formation of *Aggregatibacter actinomycetemcomitans* [43]. The galectin-8 and -9 of mandarin fish (*Siniperca chuatsi*) were shown to inhibit the growth of some pathogens [44]. Our results corroborate these findings, and through functional analogies we confirmed the ability of nattectin-like protein to induce carbohydrate-independent agglutination of human erythrocytes and exert antimicrobial activity against gram-negative and gram-positive bacterial pathogens as well as against *C. albicans*.

A substantial number of lectins have been identified in other fish. Notably the galectins, which is unusual compared to venom from other animals, like snakes, that commonly present C-type lectins [45]. Probably due to the complex environment, fish have evolved natural immune modulators, such as galectins and C-type lectins. In the fish lectins, evolution likely led to the acquisition of galectin-like properties. Thus, the functional similarities between Nattectins and Galectins studied herein might be the result of convergent evolution and because Nattectin belongs, from an evolutionary and structural perspective, to the C-type lectin family. This is also corroborated by the similarity of *T. maculosa*'s and *T. nattereri*'s Nattectin that present highly conserved residues of carbohydrate-recognition domains found in the C-type lectins.

In addition to the role of galectins in microbial death, several reports show their importance in the transendothelial migration of neutrophils and eosinophils [46,47]. Intravital imaging has yielded relevant understandings into the regulation of inflammatory responses to pathogens and sterile insults in a variety of tissues [48]. Such imaging approaches were useful in characterizing individual steps of the leukocyte recruitment cascade. Moreover, our data show that nattectin-like lectin modulates leukocyte rolling and adherence to vascular endothelium dependent on α5β1 integrin. This crucial adhesion molecule mediates the adherence of many cell types to the extracellular matrix by recognizing its classic ligand fibronectin [49]. It has been reported that the increased expression of α5β1 integrin and fibronectin enrichment in the epidermis are seemingly features of chronic neutrophilic inflammation [50]. Furthermore, polymorphonuclear neutrophils (PMNs) express several surface integrins, including α5β1 [51].

Additionally, we noted that nattectin-like toxin increased eosinophil recruitment to the peritoneal cavity of mice and the production of the pro-inflammatory chemokine, eotaxin. The trafficking of primed mature eosinophils from the bloodstream into inflamed tissues is finely regulated by adhesion molecules, several cytokines, and chemokines with

overlapping functions. Studies with mouse and human eosinophils have shown that rolling along the vascular endothelium and the activation-dependent stable adhesion to the vascular endothelium are supported by β1 integrins. Trans-endothelial migration is under the control of eotaxin and its receptor, CCR3 [52].

Together, our findings show that *T. maculosa* possesses two non-glycosylated nattectinlike proteins with similar galactose-binding specificity. Nattectin-like lectin retained their ability to induce carbohydrate-independent agglutination of human erythrocytes. Furthermore, it exerted potent antimicrobial activity against gram-negative and gram-positive bacterial pathogens, as well *C. albicans*. The non-glycosylation status of nattectin-like proteins did not impact protein function since lectins showed a substantial effect on leukocytes rolling and adherence to the endothelial wall of the mice's cremaster muscle. This interaction was dependent on β1 integrin binding. The inflammation of the peritoneal cavity was characterized by eosinophils and neutrophils influx recruited in response to IL-6 and eotaxin mediators produced by nattectin-like protein. Our results point to an immune function of nattectin-like toxin participating in the antimicrobial response through the recognition of pathogens via carbohydrate-binding and modulation of eosinophil and neutrophil infiltration in inflammation.
