Cycloviolacin O3

Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.

The cyclotides are a family of backbone-cyclised cystine-knot-containing peptides from plants that possess anthelmintic activity against Haemonchus contortus and Trichostrongylus colubriformis, two important gastrointestinal nematode parasites of sheep. In the current study, we investigated the in vitro effects of newly discovered natural cyclotides on the viability of larval and adult life stages of these pests. The natural variants cycloviolacin O2, cycloviolacin O3, cycloviolacin O8, cycloviolacin O13, cycloviolacin O14, cycloviolacin O15, and cycloviolacin O16 extracted from Viola odorata showed up to 18-fold greater potency than the prototypic cyclotide kalata B1 in nematode larval development assays. Cycloviolacin O2 and cycloviolacin O14 were significantly more potent than kalata B1 in adult H. contortus motility assays. The lysine and glutamic acid residues of cycloviolacin O2, the most potent anthelmintic cyclotide, were chemically modified to investigate the role of these charged residues in modulating the biological activity. The single glutamic acid residue, which is conserved across all known cyclotides, was shown to be essential for activity, with a sixfold decrease in potency of cycloviolacin O2 following methylation. The three lysine residues present in cycloviolacin O2 were acetylated to effectively mask the positive charge, resulting in a 18-fold decrease in anthelmintic activity. The relative anthelmintic activities of the natural variants assayed against nematode larvae correlated with the number of charged residues present in their sequence.