The fluorescence of the fluorescamine-treated proteins (Fig. 1) indicated the modification of 14 lysines in JBU-Lys, out of a total of 49 found in JBU, and of 22 acidic residues in JBU-Ac, from a total of 99 found in the native protein. Similar numbers of modified residues were detected after two independent modification assays for each derivatized protein. In order to analyze the effect of lysine and acidic residues modification on the ureolytic activity of JBU, the kinetic parameters (Km, Vmax and Kcat) of native and derivatized JBU were calculated ( Supplementary Table 1).
No significant alterations of these parameters were observed for both modified proteins, in comparison to the native JBU. As previously described (Follmer et al., 2004), JBU is highly toxic to the cotton stainer bug D. peruvianus, Palbociclib datasheet GSK2118436 cost with a LD50 value of 0.017% (w/w) of protein added to the cotton meal, when administrated in feeding trials. Here, we have used both native and the two derivatized JBU to verify the effect of the modifications upon the insecticidal activity. Both chemical modifications affected the entomotoxic activity of JBU,
drastically reducing this effect ( Fig. 2). After 17 days, the survival rate for JBU-fed groups was reduced to 18% of the control group, while JBU-Lys and JBU-Ac-fed groups survival rates were 46% and 58%, respectively ( Fig. 2, inset). There was no statistical difference between the lethalities observed for JBU-Ac and JBU-Lys when compared to each other. It was previously demonstrated that an essential step for the entomotoxic Calpain effects of plant ureases is their hydrolysis by insects’ digestive enzymes, releasing toxic peptides (Carlini et al., 1997; Defferrari et al., 2011; Ferreira-DaSilva et al., 2000; Piovesan et al., 2008). The in vitro digestion of JBU with D. peruvianus enzymes resulted in the release of several fragments from the protein, including peptide(s) in the 10 kDa range, as expected ( Fig. 3, lane 2). When the derivatized
proteins were subjected to the same digestion process, JBU-Lys showed no alteration in the pattern of the released fragments ( Fig. 3, lane 4) when compared to the native protein. In contrast, JBU-Ac was resistant to hydrolysis by the gut homogenate, thus preventing the release of the toxic peptide(s) ( Fig. 3, lane 6). Analysis of the location of the entomotoxic peptide (Jaburetox) within JBU sequence showed two aspartic acid residues flanking this region (Fig. 4). The three dimensional structure of the trimeric JBU revealed that Asp-229 (at the N-terminal of Jaburetox) is localized at the protein surface and therefore is potentially susceptible to chemical modification (Supplementary Fig. 1).