Isolation of Cysteine-Rich Peptides from Citrullus colocynthis.

Behzad Shahin-Kaleybar,Ghorbanali Nematzadeh,Reza Yousefi,Christian W Gruber,Edin Muratspahić,Ali Niazi,Roland Hellinger,Bernhard Retzl,Alireza Afsharifar

doi:10.3390/biom10091326

Abstract

The plant Citrullus colocynthis, a member of the squash (Cucurbitaceae) family, has a long history in traditional medicine. Based on the ancient knowledge about the healing properties of herbal preparations, plant-derived small molecules, e.g., salicylic acid, or quinine, have been integral to modern drug discovery. Additionally, many plant families, such as Cucurbitaceae, are known as a rich source for cysteine-rich peptides, which are gaining importance as valuable pharmaceuticals. In this study, we characterized the C. colocynthis peptidome using chemical modification of cysteine residues, and mass shift analysis via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. We identified the presence of at least 23 cysteine-rich peptides in this plant, and eight novel peptides, named citcol-1 to -8, with a molecular weight between ~3650 and 4160 Da, were purified using reversed-phase high performance liquid chromatography (HPLC), and their amino acid sequences were determined by de novo assignment of b- and y-ion series of proteolytic peptide fragments. In silico analysis of citcol peptides revealed a high sequence similarity to trypsin inhibitor peptides from Cucumis sativus, Momordica cochinchinensis, Momordica macrophylla and Momordica sphaeroidea. Using genome/transcriptome mining it was possible to identify precursor sequences of this peptide family in related Cucurbitaceae species that cluster into trypsin inhibitor and antimicrobial peptides. Based on our analysis, the presence or absence of a crucial Arg/Lys residue at the putative P1 position may be used to classify these common cysteine-rich peptides by functional properties. Despite sequence homology and the common classification into the inhibitor cysteine knot family, these peptides appear to have diverse and additional bioactivities yet to be revealed.

Highlights

Small molecules have been the dominant class of chemicals in drug discovery and development, in particular due to their favorable pharmacokinetic properties and low production costs [1]
We confirmed the presence of at least 23 CRPs in this plant based on peptide mass fingerprinting, which were de novo sequenced by enzymatic digests, mass spectrometry and amino acid analysis
Genome/transcriptome mining and a subsequent bioinformatics analysis enabled us to elucidate the phylogenetic distribution and to propose a classification of Cucurbitaceae CRPs into trypsin inhibitors and antimicrobial peptides providing a better understanding of the distribution of these two subgroups of the manifold ‘knottins’ peptide family

Summary

Introduction

Small molecules have been the dominant class of chemicals in drug discovery and development, in particular due to their favorable pharmacokinetic properties and low production costs [1]. They often lack target selectivity, which is generally associated with unwanted side-effects in clinical applications. One drawback associated with using proteins as pharmaceutical drugs is their low metabolic and structural stability [2,3] To overcome these limitations, small peptides (~5–50 amino acids in length) appear to be ideal molecules: peptide and medicinal chemists are able to design peptides with drug-like properties, such as metabolic stability, great efficacy and low off-target effects [4]. To highlight two examples: the cone snail toxin-derived ziconotide [10] has been on the market since 2000 for management of neuropathic pain, and the plant-derived peptide T20K is in clinical development for multiple sclerosis [11]

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Results

Conclusion