Novel Inhibitor Cystine Knot Peptides from Momordica charantia

Wen-Jun He,Richard J Clark,Cinzia Cantacessi,Jun Tang,Guang-Zhi Zeng,David J Craik,Lai Yue Chan,Norelle L Daly,Octavio L Franco,Ning-Hua Tan

doi:10.1371/journal.pone.0075334

Abstract

Two new peptides, MCh-1 and MCh-2, along with three known trypsin inhibitors (MCTI-I, MCTI-II and MCTI-III), were isolated from the seeds of the tropical vine Momordica charantia. The sequences of the peptides were determined using mass spectrometry and NMR spectroscopy. Using a strategy involving partial reduction and stepwise alkylation of the peptides, followed by enzymatic digestion and tandem mass spectrometry sequencing, the disulfide connectivity of MCh-1 was elucidated to be CysI-CysIV, CysII-CysV and CysIII-CysVI. The three-dimensional structures of MCh-1 and MCh-2 were determined using NMR spectroscopy and found to contain the inhibitor cystine knot (ICK) motif. The sequences of the novel peptides differ significantly from peptides previously isolated from this plant. Therefore, this study expands the known peptide diversity in M. charantia and the range of sequences that can be accommodated by the ICK motif. Furthermore, we show that a stable two-disulfide intermediate is involved in the oxidative folding of MCh-1. This disulfide intermediate is structurally homologous to the proposed ancestral fold of ICK peptides, and provides a possible pathway for the evolution of this structural motif, which is highly prevalent in nature.

Highlights

Small disulfide-rich peptides from plants and animals have diverse structures and bioactivities, and many have potential therapeutic applications [1]
Isolation and Characterization of Peptides To gain a better understanding of the structural diversity of peptides from M. charantia we screened the seed coats, decoated seeds, stems, fruits and vines for the presence of peptides
The seeds of Cucurbitaceae species are emerging as a rich source of novel disulfide-rich peptides

Summary

Introduction

Small disulfide-rich peptides from plants and animals have diverse structures and bioactivities, and many have potential therapeutic applications [1]. Several serine protease inhibitors have been isolated and characterized from the seeds [2,4,5,6,7] These inhibitors are classified as squash trypsin inhibitors and are small (,30 residue) disulfide-rich peptides containing three-disulfide bonds [2]. The traditional approach to assign the disulfide connectivity of peptides and proteins involves enzymatic digestion and disulfide mapping of the digestion fragments by mass spectrometry (MS) or N-terminal sequencing This is generally not feasible for cystine-rich peptides because of the compact packing of the cysteine residues and resistance to enzymatic digestion. In the current study a chemical and biochemical investigation of the seeds of M. charantia was undertaken This analysis led to the isolation and characterization of novel peptides that share no sequence homology with known peptides but adopt an ICK motif. The new peptides were screened in several biological assays, including trypsin inhibition, antimalarial and cytotoxicity assays

Experimental Procedures

Results

Discussion