A structural and functional analogue of a Bowman–Birk-type protease inhibitor from Odorrana schmackeri

Yuxin Wu,Tianbao Chen,Qilin Long,Ying Xu,Brian Walker,Chris Shaw,Lei Wang,Shaodong Guo,Mei Zhou,Yingqi Zhang

doi:10.1042/bsr20160593

Abstract

Frog skin secretions contain complex peptidomes and peptidic protease inhibitors that are one of the biologically and structurally described groups of components. In the present study, by use of molecular ‘shotgun’ cloning and LC MS/MS fractionation sequencing, a novel Bowman–Birk-type heptadecapeptide (AALKGCWTKSIPPKPCF-amide), named Odorrana schmackeri Trypsin Inhibitor (OSTI), with a canonical Cys6–Cys16 disulfide bridge, was isolated and identified in piebald odorous frog (O. schmackeri) skin secretion. A synthetic replicate of OSTI-exhibited trypsin inhibitory activity with a Ki value of 0.3 ± 0.04 nM and also a tryptase inhibitory effect with a Ki of 2.5 ± 0.6 μM. This is the first time that this property has been reported for a peptide originating from amphibian sources. In addition, substituting lysine (K) with phenylalanine (F) at the presumed P1 position, completely abrogated the trypsin and tryptase inhibition, but produced a strong chymotrypsin inhibition with a Ki of 1.0 ± 0.1 μM. Thus, the specificity of this peptidic protease inhibitor could be optimized through modifying the amino acid residue at the presumed P1 position and this novel native OSTI, along with its analogue, [Phe9]-OSTI, have expanded the potential drug discovery and development pipeline directed towards alleviation of serine protease-mediated pathologies.

Highlights

Inhibitors of serine proteinases are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms, where they function by binding to their cognate enzymes in a substrate-like manner, forming stable complexes
Amphibian skin secretions have proven to be a rich source of biologically active peptides and proteins, including Birk-type inhibitor (BBI), such as HV-BBI from the Chinese Bamboo odorous frog, Huia versabilis [9] and HJTI from O. hejiangensis [11]
As the sequence of the reactive site determines the specificity of the inhibition, based on the elegant research of many scientists, the P1 position lysine residue is optimal for trypsin inhibition and the phenylalanine is optimal for chymotrypsin inhibition [7]

Summary

Introduction

Inhibitors of serine proteinases are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms, where they function by binding to their cognate enzymes in a substrate-like manner, forming stable complexes. The Bowman–Birk-type inhibitor (BBI) family is a typical canonical serine protease inhibitor family, which was originally found in the seeds of leguminous (dicots) and gramineous (monocot) plants [2,3]. The rigid structures of BBI proteins are maintained by a series of highly conserved disulfide bridges. The majority of BBI proteins have a symmetrical ‘double-headed’ structure, each ‘head’ having two tricyclic domains and each domain has an independent canonical proteinase-binding site. One BBI molecule can form a 1:1:1 stoichiometric complex with two different proteinases [4]

Methods

Results

Conclusion