SdPI, The First Functionally Characterized Kunitz-Type Trypsin Inhibitor from Scorpion Venom

Abstract

BackgroundKunitz-type venom peptides have been isolated from a wide variety of venomous animals. They usually have protease inhibitory activity or potassium channel blocking activity, which by virtue of the effects on predator animals are essential for the survival of venomous animals. However, no Kunitz-type peptides from scorpion venom have been functionally characterized.Principal FindingsA new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus. It codes for a signal peptide of 21 residues and a mature peptide of 59 residues. The mature SdPI peptide possesses a unique cysteine framework reticulated by three disulfide bridges, different from all reported Kunitz-type proteins. The recombinant SdPI peptide was functionally expressed. It showed trypsin inhibitory activity with high potency (Ki = 1.6×10−7 M) and thermostability.ConclusionsThe results illustrated that SdPI is a potent and stable serine protease inhibitor. Further mutagenesis and molecular dynamics simulation revealed that SdPI possesses a serine protease inhibitory active site similar to other Kunitz-type venom peptides. To our knowledge, SdPI is the first functionally characterized Kunitz-type trypsin inhibitor derived from scorpion venom, and it represents a new class of Kunitz-type venom peptides.

Unique diversity of the venom peptides from the scorpion Androctonus bicolor revealed by transcriptomic and proteomic analysis

“…Until now, a total of seven Kunitz-type toxins have been identified from different species of scorpions, including BmKTT-1, BmKTT-2 and BmKTT-3 from M. martensii Karsch, Hg1 from Hadrurus gertschi, and LmKTT-1a, LmKTT-1b and LmKTT-1c from L. mucronatus [65][66][67]. These toxins can be classified into two groups: toxins with three disulfide bridges, and those with four disulfide bridges [66].…”

Discoveries of Serine Protease Inhibitors from Scorpions

“…Nevertheless, rLmKTT-1b along with the scorpion peptides rLmKTT-1a, rLmKTT-1c, and rBmKTT-1 [11,39] contain a unique cysteine framework, different from the classical Kunitz-type motif, where the normal C2-C4 disulfide bridge is absent, but two additional cysteine residues are present at the C-terminus of the mature peptide, which might create a new disulfide bond. Consequently, a distinct disulfide bridge may be generated [22,39]. Additionally, among other scorpion protease inhibitors, recombinant BmKTT-2 was found to possess eight cysteine residues connected by four disulfide bridges, which is different architectural property from all known Kunitz-type animal toxins [11,39] (Figure 1).…”

“…First, a novel Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from a venom gland cDNA library of the scorpion Lychas mucronatus [22]. It codes for a signal peptide of 21 amino acid residues and a mature peptide of 59 residues.…”

“…The recombinant SdPI peptide, being a thermo-stable peptide, showed potent inhibitory activity against trypsin with K i value of 1.6 × 10 −7 M. SdPI is the first functionally characterized Kunitz-type trypsin inhibitor from scorpion. Another Kunitz-type protease inhibitor, named SdPI-2, was identified from venom gland cDNA library of Lychas mucronatus [22], but its functional characterization has not yet been reported details. A Kunitz-type protease inhibitor named BmKPI was characterized from the venom gland of the scorpion Buthus martensi [10].…”

“…Many studies reported that animal venoms contain Kunitz-type serine protease inhibitors [12,[17][18][19]. Similarly, Kunitz-type as well as Ascaris-type serine protease inhibitors have been reported to be present in scorpions, and most of them have been identified by cDNA cloning and transcriptomic analysis [10,11,[20][21][22].…”