*2.2. TmC4-47.2 Is a Galactose-Binding Nattectin-Like Lectin*

Subsequently, the pool of the 3 fractions containing the toxin obtained after the last C8 column chromatography was also used for mass determination by MALDI-ToF spectrometry. After digestion, we found 9 peptides with masses ranging from 867.439 to 2065.025 (Figure 2A), which were sequenced and compared to the sequence of Nattectin present in the venom of the fish *T. nattereri* (GenBank LECG\_THANI Galactose-specific lectin Q66S03). In Figure 2B, in red, we can observe the sequences of the internal peptides overlapping in the Nattectin sequence with 100% homology and the conserved galactosebinding domain QPD (Gln-Pro-Asp) [19].

**Figure 2.** The last pool of the fractions containing the toxin obtained in the C8 column chromatography was used for mass determination and identification of internal peptides by MALDI-ToF spectrometry (**A**). The found peptides were sequenced and overlapped to the *Thalassophryne nattereri* Nattectin (GenBank LECG\_THANI Galactose-specific lectin Q66S03) (**B**). Enzymatic deglycosylation was tested to check the effect on the electrophoretic mobility of the native protein in a 12% gel (**C**).

The glycan structure of nattectin-like lectin was further studied by enzymatic deglycosylation [20]. We confirmed that treatment with O-glycosidase or N-glycosidase did not alter the electrophoretic mobility of the proteins compared to the native protein, which shows a single band of 15 kDa (Figure 2C).

After identifying the sequences similarities of the internal peptides of the toxin to those of Nattectin from *T. nattereri*, we confirmed the ability of serum from mice immunized with *T. nattereri* venom or Nattectin to recognize *T. maculosa* toxin (Figure 3A) as the recognition of Nattectin itself from *T. nattereri* venom, which indicates that it is a nattectin-like protein. The high affinity and narrow specificity of the nattectin-like lectin of *T. maculosa* for defined oligosaccharide structures were evaluated using the digoxigenin (DIG) Glycan Differentiation Kit, a competition assay (Figure 3B). The toxin/carbohydrate binding revealed by incubation with different DIG-labeled lectins demonstrated a weak interaction of both Nattectin and nattectin-like protein of *T. maculosa* with PNA, indicative that both Nattectin proteins recognized Gal-β(1–3)-N-acetylgalactosamine, which forms the core 1 structure of many O-glycans [21].

**Figure 3.** The nattectin-like toxin from *Thalassophryne maculosa* is specifically recognized by Antivenom and Anti-Natectitn antibodies from *Thalassophryne nattereri*. Serum from mice immunized with *T. nattereri* venom-VTN or Nattectin were tested to check their ability in recognizing *T. maculosa* toxin-TmC (**A**). 10 μg of nattectin-like toxin and *T. nattereri* venom were subjected to 12% SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with *T. nattereri* anti-Natectin anti-venom serum. They were subsequently incubated with peroxidase-labeled mouse anti-IgG and revealed with 4-α-chloro-naphtol. A digoxigenin (DIG) glycan differentiation competition assay kit (Roche Applied Science, Germany) was used to identify the binding specificity of the toxin. Toxin/carbohydrate binding was revealed by incubation with different DIG-labeled lectins and alkaline phosphatase-conjugated anti-DIG antibody (**B**).
