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SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom.

ABSTRACT: BACKGROUND: Kunitz-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 FINDINGS: A 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 (K(i)?=?1.6×10(-7) M) and thermostability. CONCLUSIONS: The 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.

SUBMITTER: Zhao R

PROVIDER: S-EPMC3210814 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom.

Zhao Ruiming R Dai Hui H Qiu Su S Li Tian T He Yawen Y Ma Yibao Y Chen Zongyun Z Wu Yingliang Y Li Wenxin W Cao Zhijian Z

PloS one 20111108 11

<h4>Background</h4>Kunitz-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.<h4>Principal findings</h4>A new Kunitz-type venom peptide gene precursor, SdPI, was cloned and characterized from ...[more]

PMID: 22087336

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Project description:BackgroundSerine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear.Principal findingsHere, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI), Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2), Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI), and Buthus martensii Ascaris-type protease inhibitor (BmAPI). The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors.Conclusions/significanceTo our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the development of diagnostic and therapeutic agents for human diseases that target diverse proteases.

Project description:BackgroundProteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L.MethodsThe low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program.ResultsThe fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate.ConclusionThis work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant.

Project description:A Kunitz-type trypsin inhibitor protein has been purified and characterized from seeds of Acacia nilotica L. LC-MS/MS analysis of Acacia nilotica trypsin inhibitor (AnTI) provided the N-terminal fragment of 11 amino acids which yielded 100% identity with already reported Kunitz-type trypsin inhibitor protein of Acacia confusa (AcTI) in UniProtKB database search. SDS-PAGE showed a single band of ~21 kDa under nonreduced condition and appearance of a daughter band (17 kDa) in the presence of β-mercaptoethanol indicating the presence of interchain disulfide linkage typical for Kunitz-type trypsin inhibitors. AnTI was purified from seed extract by using a combination of anion exchange and gel filtration chromatography. Since AnTI showed maximum homology with AcTI, a molecular structure of AcTI was predicted which showed highly β-sheeted molecular conformation similar to crystallographic structure of Enterolobium contortisiliquum trypsin inhibitor (EcTI). AnTI (20 µg) produces significant population inhibition against different human pathogenic bacteria along strong antifungal activity (50 µg). Entomotoxin potential of AnTI was evaluated against two stored grain insect pests Tribolium castaneum (Herbst) (Tenebrionidae: Coleoptera) and Sitophilus oryzae (Linnaeus) (Curculionidae: Coleoptera). Statistically significant mortality of T. castaneum adults was observed at 1.5 mg after 15 days in comparison to control. Additionally, number of total eggs, larvae, pupae, adults, and their male/female ratio were also severely reduced in comparison to control. Similarly, two generation progeny of S. oryzae was studied after mixing AnTI with rice kernels. Mean percent mortality of adult population was significantly higher after 9 days of exposure in comparison to control group. AnTI significantly reduced the F1 generation while little mortality was observed for F2 generation. Exploration of such potent molecules is the prerequisite of our time regarding the anticipation of postantibiotic era and the development of insect resistance against chemical pesticides.

Dataset Information

SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom.

Publications

SdPI, the first functionally characterized Kunitz-type trypsin inhibitor from scorpion venom.

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