**1. Introduction**

Lectins are a comprehensive group of proteins with carbohydrate-binding properties. Due to their binding singularity, many natural lectins, purified from several sources such as plants, algae, and fungi, have prospective applications in biotechnology, medical research, and crop protection. Additionally, many groups have identified lectins in organs and tissues (gills, eggs, electric organ, stomach, intestine, liver, skin, mucus, and plasma)

**Citation:** Lopes-Ferreira, M.; Sosa-Rosales, I.; Silva Junior, P.I.; Conceicao, K.; Maleski, A.L.A.; Balan-Lima, L.; Disner, G.R.; Lima, C. Molecular Characterization and Functional Analysis of the Nattectin-like Toxin from the Venomous Fish *Thalassophryne maculosa*. *Toxins* **2022**, *14*, 2. https://doi.org/10.3390/ toxins14010002

Received: 14 October 2021 Accepted: 15 November 2021 Published: 21 December 2021

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of various fish species [1]. Lectins have been reported to have a wide array of functions, including immune-relevant ones such as pathogen recognition, agglutination, opsonization, complement activation, phagocytosis, and other functions such as splicing of RNA, protein folding, trafficking of molecules, control of cell proliferation, and roles in development [2,3].

Interestingly, our group has previously described a lectin, named Nattectin, in the venom gland of the fish *Thalassophryne nattereri* [4]. Nattectin is a basic monomeric protein, non-glycosylated, galactose-specific lectin from the C-type family, presenting remarkable pro-inflammatory activity. *T. nattereri* and *Thalassophryne maculosa* are venomous toadfish that belong to the family Batrachoididae. They are benthic ambush predators that favor from sandy or muddy substrates where their cryptic coloration, or the habit of burying under sand and mud, helps them avoid detection by their prey. Their omnivorous diet is composed of sea worms, crustaceans, mollusks, and other fish. They are found in temperate and tropical waters throughout the coast of America, Europe, Africa, and India. These species present one of the most adapted teleost venom apparatus composed of four canaliculated spines coupled to venom glands at their base capable of delivering a painful wound to predators. Thus, the venom apparatus has more of a defense function rather than predation.

Studies on *T. nattereri* envenomation and its venom composition have been carried out in Brazil by our group since 1998 at the Butantan Institute [5,6]. *T. nattereri* stands out among the venomous animals of medical importance in Brazil for the number of accidents it causes in the North and Northeast regions and the seriousness of the cases [7–9]. One of the main symptoms of *T. nattereri* envenomation is the immediate, intense pain that persists over 24 h. Erythema and edema are also shortly noticed with the efflorescence of bubbles with serous content. These lesions evolve to long-remaining necrosis with a delayed healing process devoid of specific drug treatment [10–13].

However, few studies have been conducted with *T. maculosa*, mainly found in Venezuela, Colombia, and the islands of Aruba, Curaçao, and Trinidad and Tobago. It is known that its venom presents a mixture of bioactive toxins differently expressed in females and males [14]. *T. maculosa* venom induces a significant necrotic lesion characterized by delayed neutrophil influx to the footpad of mice. An acute production of IL-1β, IL-6, and later secretion of TNF-α, MCP-1, KC, and mediators from arachidonic acid metabolism such as LTB4 and PGE2 were detected in the exudate of inflamed footpads [15]. In addition, we demonstrated that bone marrow-derived macrophages and dendritic cells were strongly stimulated by the venom, which demonstrates its ability to stimulate a specific and systemic immune response [16]. Furthermore, Sosa-Rosales et al. [17] performed a partial identification of two proteins with myotoxic activity in *T. maculosa* venom using reverse-phase HPLC and named them according to column retention time as TmC4-47.2. SDS-Page analysis of the crude venom showed a few weighty bands (one located above 97 Mw, one between 68 and 97 Mw, one major band between 29 and 43 Mw, and the last one located below 18.4 Mw). Then, it seems that the isolated nattectin-like protein is one of the main components of the venom, which includes a significant mixture of bioactive molecules involved in the local inflammatory lesion [17].

We understand that the advance in the knowledge of the pattern of ischemic and necrotic lesions induced by *T. maculosa* and the description of the main toxins can lead to the identification of targets for therapeutic intervention. Therefore, the study of the TmC4-47.2 toxin becomes an important tool for understanding the mechanisms of action involved in envenomation. In the present study, we performed the characterization of the TmC4-47.2 protein sequence using sequential C18 and C8 affinity column chromatography and mass spectrometry and determined its functions as a lectin.
