Identification and Target-Modification of SL-BBI: A Novel Bowman-Birk Type Trypsin Inhibitor from Sylvirana latouchii.

Xi Chen,Tianbao Chen,Chengbang Ma,Lei Wang,Xiaoling Chen,Xinping Xi,Mei Zhou,Dong Chen,Linyuan Huang

doi:10.3390/biom10091254

Xi Chen, Tianbao Chen + Show 7 more

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https://doi.org/10.3390/biom10091254

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Abstract

The peptides from the ranacyclin family share similar active disulphide loop with plant-derived Bowman–Birk type inhibitors, some of which have the dual activities of trypsin inhibition and antimicrobial. Herein, a novel Bowman–Birk type trypsin inhibitor of the ranacyclin family was identified from the skin secretion of broad-folded frog (Sylvirana latouchii) by molecular cloning method and named as SL-BBI. After chemical synthesis, it was proved to be a potent inhibitor of trypsin with a Ki value of 230.5 nM and showed weak antimicrobial activity against tested microorganisms. Modified analogue K-SL maintains the original inhibitory activity with a Ki value of 77.27 nM while enhancing the antimicrobial activity. After the substitution of active P1 site to phenylalanine and P2′ site to isoleucine, F-SL regenerated its inhibitory activity on chymotrypsin with a Ki value of 309.3 nM and exhibited antiproliferative effects on PC-3, MCF-7 and a series of non-small cell lung cancer cell lines without cell membrane damage. The affinity of F-SL for the β subunits in the yeast 20S proteasome showed by molecular docking simulations enriched the understanding of the possible action mode of Bowman–Birk type inhibitors. Further mechanistic studies have shown that F-SL can activate caspase 3/7 in H157 cells and induce apoptosis, which means it has the potential to become an anticancer agent.

Highlights

Serine protease inhibitors can regulate a variety of physiological reactions in the body by preventing unnecessary proteolytic functions
The cDNA encoding the biosynthetic precursor of a trypsin inhibitory peptide was successfully cloned from the skin secretion-derived cDNA library of Sylvirana latouchii and was named SL-Birk inhibitor (BBI)
Simulated structure of SL-BBI in stick model showed a similar configuration to peptide HV-BBI (PDB ID: 4U2W) which belongs to the Ranacyclin family (Figure 1d)

Summary

Introduction

Serine protease inhibitors can regulate a variety of physiological reactions in the body by preventing unnecessary proteolytic functions. Bowman–Birk inhibitor (BBI) is a peptide family rich in cysteine and supported by seven intramolecular disulphide bonds, with dual inhibitory activity of trypsin and chymotrypsin due to its two independent reaction ring exposure at the molecular terminal [1]. The more concise BBI sequences are usually within 20 amino acids and contain an 11-residue active reaction loop, almost all of which are present in amphibian skin secretions and were generalised as the ranacyclin family [2]. Ranacyclin is the unique family of serine protease inhibitors known from amphibian skin secretions and usually has the dual activities of trypsin inhibition and antibacterial [2,11] to combat the invasion of foreign invasive pathogens. Inhibitors of BBI families have been confirmed to bind to classical bradykinin and its analogues as a protective pathway to against the cleavage of plasmatic serine proteases, leading to an increase in the half-life of bradykinin [24]

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