SjAPI, the First Functionally Characterized Ascaris-Type Protease Inhibitor from Animal Venoms

Yingliang Wu,Bin Wang,Wenxin Li,Paulo Lee Ho,Zongyun Chen,Renxi Zhuo,Jun Hu,Zhijian Cao,Weishan Yang

doi:10.1371/journal.pone.0057529

Abstract

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.

Highlights

Serine protease inhibitors are of broad interest and can act as tools and modulators for serine proteases [1]
From the sequenced ESTs of scorpion cDNA libraries, we identified four genes encoding the Ascaris-type peptides 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) (Fig. 1A)
Multiple sequence alignments showed that the four Ascaris-type peptides from scorpion venom share homology with typical Ascaris-type venom peptides from other animals, such as Ascaris sp. trypsin inhibitor (ATI) from Ascaris lumbricoides var. suum [27], Apis mellifera cathepsin G/ chymotrypsin inhibitor-1 (AMCI-1) from Apis mellifera [18], bombina skin trypsin inhibitor (BSTI) from the skin secretions of Bombina bombina [17], chymotrypsin/elastase inhibitor-1 (C/E-1) from Ascaris suum [16], and NAP5 from Ancylostoma caninum [28]

Summary

Introduction

Serine protease inhibitors are of broad interest and can act as tools and modulators for serine proteases [1]. Diverse interactions between these inhibitors and proteases play key roles in a variety of physiological functions, such as blood coagulation, fibrinolysis, apoptosis, development, inflammation, and complement activation in humans [2,3,4]. Previous studies have indicated that protease inhibitors from venom glands might protect toxin peptides from degradation by various proteases and play important roles for venomous animal survival [8,9,10]. Ascaris-type peptides constitute an important class of serine protease inhibitors. Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. All known serine protease inhibitors discovered 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

Methods

Results

Conclusion