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

Roland Hellinger, Johannes Koehbach, Douglas E. Soltis, Eric J. Carpenter, Gane Ka-Shu Wong, Christian W. Gruber

Cyclotides are plant-derived mini proteins. They are genetically-encoded as precursor proteins that become post-translationally modified to yield circular cystine-knotted molecules. Due to 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 Viola tricolor. Therefore, violaceous plants at a global scale may be source to as many as 150,000 individual cyclotides. Encompassing the diversity of Viola tricolor as combinatorial library of bioactive peptides, this commercially-available medicinal herb may be a suitable starting point for future bioactivity-guided screening studies.