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Two native peptides with disulfide-directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full-length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the "simple" DDH peptide originates from the "complex" ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH-fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.

Although numerous Kunitz-type toxins were isolated from snake venom, no bifunctional Kunitz-type snake toxins with protease and potassium channel inhibiting properties have been reported till now. With the help of bioinformatics analyses and biological experiments, we characterized Kunitz-type snake toxin BF9 as a bifunctional peptide. Enzyme and inhibitor reaction kinetics experiments showed that BF9 inhibited α-chymotrypsin with Ki value of 1.8 × 10−8 M. Electrophysiological experiments showed that BF9 inhibited the Kv1.3 potassium channel with an IC50 of 120.0 nM, which demonstrated that serine protease inhibitor BF9 could also inhibit potassium channels. In addition, the key amino acids of BF9 responsible for the unique bifunctional mechanism are further investigated. To the best of our knowledge, BF9 is the first Kunitz-type snake peptide with the unique bifunctionality of potassium channel and serine protease inhibiting properties, providing novel insights into divergent evolution and functional applications of snake Kunitz-type peptides.

Two native peptides with disulfide-directed hairpin (DDH) fold, LaIT1 and LITX, were recently isolated from scorpion venom, a development that offered insights into exploring the evolutionary linkage between DDH and inhibitor cystine knot (ICK) peptides. In this work, we isolated and identified the full-length cDNAs of LaTI1, a representative member with DDH fold, and further determined its complete gene structure. The precursor organization of LaIT1 is similar to that of ICK peptides. The LaIT1 gene contains four exons interrupted by three unique introns and differed from ICK peptides, suggesting divergent genomic organizations of DDH peptides and ICK peptides. Phylogenetic analysis further showed that the "simple" DDH peptide originates from the "complex" ICK peptide, rather than the reverse. To the best of our knowledge, this is the first report on the genomic organization of DDH-fold peptides, and it presents new evidence of an evolutionary linkage between ICK and DDH peptides.

Although numerous Kunitz-type toxins were isolated from snake venom, no bifunctional Kunitz-type snake toxins with protease and potassium channel inhibiting properties have been reported till now. With the help of bioinformatics analyses and biological experiments, we characterized Kunitz-type snake toxin BF9 as a bifunctional peptide. Enzyme and inhibitor reaction kinetics experiments showed that BF9 inhibited α-chymotrypsin with Ki value of 1.8 × 10⁻⁸ M. Electrophysiological experiments showed that BF9 inhibited the Kv1.3 potassium channel with an IC₅₀ of 120.0 nM, which demonstrated that serine protease inhibitor BF9 could also inhibit potassium channels. In addition, the key amino acids of BF9 responsible for the unique bifunctional mechanism are further investigated. To the best of our knowledge, BF9 is the first Kunitz-type snake peptide with the unique bifunctionality of potassium channel and serine protease inhibiting properties, providing novel insights into divergent evolution and functional applications of snake Kunitz-type peptides.

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure–function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC50 of 1.70 n± 1.35 µM, whereas 10 µM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 µM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca2+ channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K+ channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel. © 2011 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:244–251, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20382