Peptidomics of Circular Cysteine-Rich Plant Peptides: Analysis of the Diversity of Cyclotides from Viola tricolor by Transcriptome and Proteome Mining

Abstract

Cyclotides are plant-derived mini proteins. They are genetically encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Because of this structural topology cyclotides resist enzymatic degradation in biological fluids, and hence they are considered as promising lead molecules for pharmaceutical applications. Despite ongoing efforts to discover novel cyclotides and analyze their biodiversity, it is not clear how many individual peptides a single plant specimen can express. Therefore, we investigated the transcriptome and cyclotide peptidome of Viola tricolor. Transcriptome mining enabled the characterization of cyclotide precursor architecture and processing sites important for biosynthesis of mature peptides. The cyclotide peptidome was explored by mass spectrometry and bottom-up proteomics using the extracted peptide sequences as queries for database searching. In total 164 cyclotides were discovered by nucleic acid and peptide analysis in V. tricolor. Therefore, violaceous plants at a global scale may be the source to as many as 150 000 individual cyclotides. Encompassing the diversity of V. tricolor as a combinatorial library of bioactive peptides, this commercially available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.

Oral activity of a nature-derived cyclic peptide for the treatment of multiple sclerosis

“…Historically, cyclosporine A was instrumental to the development of modern immunopharmacology and it is still in clinical use today (29,36). Despite the limitations of such comparisons, we believe that the rich diversity of cyclotides (17,43) justifies their position as a treasure trove for drug discovery.…”

Cyclotides: Overview and Biotechnological Applications

“…[28] In a recent study, the use of in silico transcriptomic and proteomic screening identified 164 cyclotides in Viola tricolor , which led to the authors of this study to estimate the total number of cyclotides in the Violaceae family alone to be around 150,000. [29] In silico mining of genomes combined with molecular biology approaches has been used for the discovery of a large number of new ribosomal natural products, including cyclotides. [27] For example, database mining approaches were recently for the identification of cyclotide-like sequences in the plant Zea mays (maize), that were later confirmed to be expressed at the mRNA level in the root system of the plant.…”

Cyclotides, a versatile ultrastable micro-protein scaffold for biotechnological applications

“…29 In a recent study, the use of in silico transcriptomic and proteomic screening identified 164 cyclotides in Viola tricolor , which led to the authors of this study to estimate the total number of cyclotides in the Violaceae family alone to be around 150,000. 30 The CyBase database was recently created to allow easy access to the large number of cyclotide sequences that have been found thus far. 31 This database can be accessed through a publicly available website (http://CyBase.org.au) and contains above 300 known cyclotides as well as useful proteomic tools for their analysis.…”