Novel inhibitor cystine knot peptides from Momordica charantia

PLoS One. 2013 Oct 8;8(10):e75334. doi: 10.1371/journal.pone.0075334. eCollection 2013.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Magnetic Resonance Spectroscopy
  • Momordica charantia*
  • Plant Proteins / isolation & purification*
  • Plant Proteins / metabolism
  • Protein Conformation
  • Protein Folding
  • Tandem Mass Spectrometry
  • Trypsin Inhibitors / isolation & purification*
  • Trypsin Inhibitors / metabolism


  • Plant Proteins
  • Trypsin Inhibitors

Grant support

This work was supported by the National Basic Research Program of China (2013CB127505, 2009CB522300), the National New Drug Innovation Great Project of China (2011ZX09307-002-02), the National Natural Science Foundation of China (21202174, U1032602, 91013002, 30725048), the foundation of Chinese Academy of Sciences (Hundred Talents Program), and the Natural Science Foundation of Yunnan Province (2012GA003). WJH is a recipient of a fellowship from the China Scholarship Council. NLD was supported by a Queensland Smart State Fellowship and is currently an ARC Future Fellow. DJC is a National Health and Medical Research Council Professorial Research Fellow. OLF is supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Fundação de Apoio a Pesquisa do Distrito Federal. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.