Cyclotide host-defense tailored for species and environments in violets from the Canary Islands.

Cyclotides are cyclic peptides produced by plants. Due to their insecticidal properties, they are thought to be involved in host defense. Violets produce complex mixtures of cyclotides, that are characteristic for each species and variable in different environments. Herein, we utilized mass spectrometry (LC-MS, MALDI-MS), transcriptomics and biological assays to investigate the diversity, differences in cyclotide expression based on species and different environment, and antimicrobial activity of cyclotides found in violets from the Canary Islands. A wide range of different habitats can be found on these islands, from subtropical forests to dry volcano peaks at high altitudes. The islands are inhabited by the endemic Viola palmensis, V. cheiranthifolia, V. anagae and the common V. odorata. The number of cyclotides produced by a given species varied in plants from different environments. The highest diversity was noted in V. anagae which resides in subtropical forest and the lowest in V. cheiranthifolia from the Teide volcano. Transcriptome sequencing and LC-MS were used to identify 23 cyclotide sequences from V. anagae. Cyclotide extracts exhibited antifungal activities with the lowest minimal inhibitory concentrations noted for V. anagae (15.62 µg/ml against Fusarium culmorum). The analysis of the relative abundance of 30 selected cyclotides revealed patterns characteristic to both species and populations, which can be the result of genetic variability or environmental conditions in different habitats. The current study exemplifies how plants tailor their host defense peptides for various habitats, and the usefulness of cyclotides as markers for chemosystematics.

Targeted Delivery of Cyclotides via Conjugation to a Nanobody.

Many naturally occurring peptides have poor proteolytic stability, which limits their therapeutic applications. Cyclotides are plant-derived cyclic peptides that resist proteolysis due to their highly constrained structure, comprising a head-to-tail cyclic backbone and three disulfide bonds that form a cystine-knotted core. This structure makes them useful as scaffolds onto which peptide sequences (epitopes) can be grafted. In this study, VHH7, an alpaca-derived nanobody that targets murine class II MHC molecules, was used for the targeted delivery of cyclotides to antigen-presenting cells (APCs). The cyclotides MCoTI-I, and MCoTI-I with a HA-tag (YPYDVPDYA) grafted into loop 6 (MCoTI-HA), were tested for immunogenic properties. To produce the requisite VHH7-peptide conjugates, a site-specific sortase A-catalyzed reaction in combination with a copper-free strain-promoted cycloaddition reaction was used. MCoTI-I alone did not display any obvious antibody response, thus showing the capacity of cyclotides as immunologically silent scaffolds. By contrast, MCoTI-I conjugated to VHH7 elicited antibodies against cyclic or linear MCoTI-I, thus suggesting a simple and robust approach for targeting cyclotides to APCs, and potentially to other cell types. A similar antibody response was observed when MCoTI-HA was conjugated to VHH7, but there was no reactivity toward a linear HA-tag itself, suggesting differences in conformational constraint between cyclotide-presented and linear epitopes. Studies of commercially available HA antibodies applied to MCoTI-HA confirmed that the conformation of peptide immunogens affects their reactivity. Thus, the production of antibodies that recognize constrained epitopes may benefit from engraftment onto scaffolds such as cyclotides. More broadly, this study validates that a prototypic cyclotide, a member of a peptide family that has proven to be useful as drug design scaffolds in many other studies, can efficiently reach a specific target in vivo.

Single-step purification of cyclotides using affinity chromatography.

Cyclotides are considered promising scaffolds for drug development owing to their inherent host defence activities and highly stable structure, defined by the cyclic cystine knot. These proteins are expressed as complex mixtures in plants. Although several methods have been developed for their isolation and analysis, purification of cyclotides is still a lengthy process. Here, we describe the use of affinity chromatography for the purification of cyclotides using polyclonal IgG antibodies raised in rabbits against cycloviolacin O2 and immobilized on NHS-activated Sepharose columns. Cycloviolacin O2 was used as a model substance to evaluate the chromatographic principle, first as a pure compound and then in combination with other cyclotides, that is, bracelet cyclotide cycloviolacin O19 and Möbius cyclotide kalata B1, and in a plant extract. We demonstrate that single-step purification of cyclotides by affinity chromatography is possible but cross reactivity may occur between homologue cyclotides of the bracelet subfamily.

Immunosuppressive peptides and their therapeutic applications.

The immune system is vital for detecting and evading endogenous and exogenous threats to the body. Failure to regulate this homeostasis leads to autoimmunity, which is often associated with malfunctioning T cell signaling. Several medications are available to suppress over-reactive T lymphocytes, but many of the currently marketed drugs produce severe and life-threatening side-effects. Ribosomally synthesized peptides are gaining recognition from the pharmaceutical industry for their enhanced selectivity and decreased toxicity compared with small molecules; in particular, circular peptides exhibit remarkable stability and increased oral administration properties. For example, plant cyclotides effectively inhibit T lymphocyte proliferation. They are composed of a head-to-tail cyclized backbone and a cystine-knot motif, which confers them with remarkable stability, thus making them attractive pharmaceutical tools.

Importance of the cell membrane on the mechanism of action of cyclotides.

Their distinctive structures, diverse range of bioactivities, and potential for pharmaceutical or agricultural applications make cyclotides an intriguing family of cyclic peptides. Together with the physiological role in plant host defense, cyclotides possess antimicrobial, anticancer, and anti-HIV activities. In all of the reported activities, cell membranes seem to be the primary target for cyclotide binding. This article examines recent literature on cyclotide-membrane studies and highlights the hypothesis that the activity of cyclotides is dependent on their affinity for lipid bilayers and enhanced by the presence of specific lipids, i.e., phospholipids containing phosphatidylethanolamine headgroups. There is growing evidence that the lipid composition of target cell membranes dictates the amount of cyclotides bound to the cell and the extent of their activity. After membrane targeting and insertion in the bilayer core, cyclotides induce disruption of membranes by a pore formation mechanism. This proposed mechanism of action is supported by biophysical studies with model membranes and by studies on natural biological membranes of known lipid compositions.

Cyclotides are a component of the innate defense of Oldenlandia affinis.

Cyclotides are small cysteine-rich plant peptides similar in size and processing to the defensins. Long-term growth of the Rubiaceae family plant Oldenlandia affinis under different conditions reveals a diverse cyclotide gene and peptide expression profile, including tissue specificity, suggesting that different cyclotides are regulated differently both spatially and in response to the environment. To determine whether cyclotide precursor gene regulation was dynamic we exposed O. affinis to a range of abiotic, biotic, and hormonal stimuli and monitored Oak1-4 expression over a 48-h period. Unlike some defensins, the genes for cyclotide precursor proteins Oak1-4 did not display dynamic change, indicating that they contribute to the basal defense of O. affinis. Despite this lack of dynamism, the cyclotide profile of plants grown on plates differed markedly from field-grown plants and so prompted attempts to discover novel cyclotides and precursor genes. The two most abundant cyclotides from plate-grown O. affinis were sequenced and one was found to be an unusual linear cyclotide derivative, kalata B20-lin. Degenerate PCR of plate-grown O. affinis obtained five novel cyclotide genes including Oak9 which encodes for kalata B20-lin and appears to have arisen by the presence of a premature stop codon.