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TmC4-47.2 is a toxin with myotoxic activity found in the venom of Thalassophryne maculosa, a venomous fish commonly found in Latin America whose envenomation produces an injury characterized by delayed neutrophil migration, production of major pro-inflammatory cytokines, and necrosis at the wound site, as well as a specific systemic immune response. However, there are few studies on the protein structure and functions associated with it. Here, the toxin was identified from the crude venom by chromatography and protein purification systems. TmC4-47.2 shows high homology with the Nattectin from Thalassophryne nattereri venom, with 6 cysteines and QPD domain for binding to galactose. We confirm its hemagglutinating and microbicide abilities independent of carbohydrate binding, supporting its classification as a nattectin-like lectin. After performing the characterization of TmC4-47.2, we verified its ability to induce an increase in the rolling and adherence of leukocytes in cremaster post-capillary venules dependent on the α5β1 integrin. Finally, we could observe the inflammatory activity of TmC4-47.2 through the production of IL-6 and eotaxin in the peritoneal cavity with sustained recruitment of eosinophils and neutrophils up to 24 h. Together, our study characterized a nattectin-like protein from T. maculosa, pointing to its role as a molecule involved in the carbohydrate-independent agglutination response and modulation of eosinophilic and neutrophilic inflammation.

Recently our group described that Nattectin, a C-type lectin of the venom of Thalassophryne nattereri shows a potent pro-inflammatory capacity. Here, we demonstrated that Nattectin is able to induce M1 macrophage marker iNOS, and up-regulate the expression of MHC class II, CD80, CD86 and CD40 molecules. The increase in MHC class II and CD49a integrin expression with MMP-9 production and endocytic capacity depend on lectin function of Nattectin. Moreover, the polarization of peritoneal and bone marrow-derived macrophages induced by Nattectin to M1 profile is dependent on Th1 cytokines (IL-12 and IFN-γ), and negatively regulated by Th2 cytokines (IL-4, IL-10 and IL-13). Also we reveal that IL-4 play a dual role in this polarization: a regular action of IL-4 was seen in the negative regulation of the CD40 expression, but an unexpected positive regulation was seen in the expression of CCR7 and MHC class II. Finally, our in vivo studies showed that the influx of neutrophils and small peritoneal macrophage - F4/80lowMHCIIhi induced by Nattectin is totally dependent on IL-4 and IFN-γ cytokines. Furthermore, the induction of IL-6 release is negatively regulated by IL-4 and positively regulated by IL-12 and IFN-γ. Together, the results allowed us to expand the knowledge about the regulation of macrophage activation, as well as confirmed the ability of Nattectin, a fish C-type lectin, as an important immunomodulatory agent.

Considerable efforts are currently focused on the biology of DC in view of their possible clinical use as adjuvant for the generation of antigen-specific immunity and lifelong immunologic memory or for the treatment of tumors. We assessed the role of Nattectin a C-type lectin identified in the Thalassophryne nattereri fish venom in DC maturation. Nattectin induced a significant neutrophilic recruitment into peritoneal cavity of mice, followed by macrophages, with lipidic mediators and IL-12 p70 synthesis. Macrophages derived from 7day-Nattectin mice were CD11c+CD11b(low)Ly6(high)F4/80R(high) and express high levels of MHC class II and CD80 molecules. Culture of peritoneal exudates derived macrophages from 7day Nattectin-mice and immature BMDCs with Nattectin markedly increased the surface expression of CD40, CD80, CD86, and MHC class II in a dose-dependent manner, and the production of MMP-2 and MMP-9 distributed in nucleus and cytoplasm of cells, that was associated with strong activity in the culture supernatant. Nattectin treated DCs secreted IL-12 p70 and IL-10. The Nattectin-treated BMDC or macrophage-derived DCs were highly efficient at Ag capture. The specific immune response elicited by Nattectin was characterized by the production of specific antibodies IgG1 and mainly IgG2a with IL-10 and IFN-γ synthesis by splenic cells. These results enable us to address that Nattectin induces the recruitment of Ly6C(high) monocytes into the peritoneum, which exhibit a pro-inflammatory profile, where they differentiate into proliferating F4/80R(high) macrophages. Macrophage-derived DCs mature in the presence of the cytokine milieu generated against Nattectin, exhibiting T cell co-stimulatory molecule expression and induced a Th1 polarized response.

Combined proteomic and transcriptomic approaches to study the composition of the venom of Thalassophryne nattereri venomous fish revealed the primary structures of the major toxins as a family of proteases natterins, never described on venoms and a C-type lectin nattectin. To gain new insights into the mechanisms of venom pathogenesis and to further elucidate the role of its major toxins, the natterins and nattectin, we undertook in vitro investigations using these isolated toxins. Here we demonstrated the specific ability of the nattectin to bind types I and V collagen and natterins to bind and cleave type I collagen as well as type IV collagen, disrupting cell attachment and HeLa cells survival. Natterins have cytotoxic effect on both adherent cells or at in suspension, showing direct induction of necrosis that is followed by cell detachment. Nattectin improves integrin-mediated HeLa cell adhesion and resistance to apoptosis by its binding to RGD-dependent integrins, especially the β1 subunit. Based on our studies we now report that extracellular matrix (ECM) components as well as the integrin β1 subunit are targets for the natterins and nattectin. The ECM degradation or remodeling activities exerted by these toxins affect cell–cell and cell–ECM adhesion and survival and impair inflammatory cell migration into inflamed tissues.