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

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 gating modifier, 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.