Discovery and Functional Characterization of Kunitz-Type Toxins from the Tetrodotoxin-Bearing Flatworm Planocera multitencaculata

Kunitz-type protease inhibitors are well-known protease inhibitors found in various organisms, consisting of approximately 60 amino acid residues and featuring three disulfide bridges with a unique cysteine (Cys) framework. Among venomous animals, KTTs inhibit voltage-gated ion channels. In this study, we identified eight novel KTTs in the TTX-bearing flatworm Planocera multitentaculata , naming them Pm -KTT-1 through − 8. These Pm -KTTs share 37.9–46.5% amino acid sequence identities with BPTI and DTX-K. Additionally, since Pm -KTTs contained conserved amino acid residues involved in the inhibition of trypsin and potassium channels, we speculated that Pm -KTTs act as inhibitors of trypsin and potassium channels. Recombinant expression of Pm -KTT-1 through − 5 inhibited trypsin activity. Moreover, Pm -KTT-4 regulated Drosophila K ⁺ channels in Shaker and Shal. Furthermore, Pm -KTT-4 functioned as a channel opener, unlike typical KTTs in other venomous animals. These findings provide the first molecular and functional characterization of toxic polypeptides in P. multitentaculata , expanding our understanding of the evolutionary diversity and biological roles of Kunitz-type toxins.