Nematicidal Activity of Cyclotides: Toxicity Against Caenorhabditis elegans.

Cyclotides are a unique family of stable and cyclic mini-proteins found in plants that have nematicidal and anthelmintic activities. They are distributed across the Rubiaceae, Violaceae, Fabaceae, Cucurbitaceae, and Solanaceae plant families, where they are posited to act as protective agents against pests. In this study, we tested the nematicidal properties of extracts from four major cyclotide-producing plants, Oldenlandia affinis, Clitoria ternatea, Viola odorata, and Hybanthus enneaspermus, against the free-living model nematode Caenorhabditis elegans. We evaluated the nematicidal activity of the cyclotides kalata B1, cycloviolacin O2, and hyen D present in these extracts and found them to be active against the larvae of C. elegans. Both the plant extracts and isolated cyclotides exerted dose-dependent toxicity on the first-stage larvae of C. elegans. Isolated cyclotides caused death or damage upon interacting with the worms' mouth, pharynx, and midgut or membrane. Cycloviolacin O2 and hyen D produced bubble-like structures around the C. elegans membrane, termed blebs, implicating membrane disruption causing toxicity and death. All tested cyclotides lost their toxicity when the hydrophobic patches present on them were disrupted via a single-point mutation. The present results provide a facile assay design to measure and explore the nematicidal activities of plant extracts and purified cyclotides on C. elegans.

In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis.

Cyclotides are plant-derived peptides that have attracted interest as biocides and scaffolds for the development of stable peptide therapeutics. Cyclotides are characterized by their cyclic backbone and cystine knot framework, which engenders them with remarkably high stability. This study reports the cystine knot-related peptidome of Rinorea bengalensis, a small rainforest tree in the Violaceae family that is distributed from Australia westward to India. Surprisingly, many more acyclic knotted peptides (acyclotides) were discovered than cyclic counterparts (cyclotides), with 32 acyclotides and 1 cyclotide sequenced using combined transcriptome and proteomic analyses. Nine acyclotides were isolated and screened against a panel of mammalian cell lines, showing they had the cytotoxic properties normally associated with cyclotide-like peptides. NMR analysis of the acyclotide ribes 21 and 22 and the cyclotide ribe 33 confirmed that these peptides contained the cystine knot structural motif. The bracelet-subfamily cyclotide ribe 33 was amenable to chemical synthesis in reasonable yield, an achievement that has long eluded previous attempts to synthetically produce bracelet cyclotides. Accordingly, ribe 33 represents an exciting new bracelet cyclotide scaffold that can be subject to chemical modification for future molecular engineering applications.

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus.

Cyclotides are plant-derived peptides characterized by an ∼30-amino acid-long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide-membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

Butterfly Pea (Clitoria ternatea), a Cyclotide-Bearing Plant With Applications in Agriculture and Medicine.

The perennial leguminous herb Clitoria ternatea (butterfly pea) has attracted significant interest based on its agricultural and medical applications, which range from use as a fodder and nitrogen fixing crop, to applications in food coloring and cosmetics, traditional medicine and as a source of an eco-friendly insecticide. In this article we provide a broad multidisciplinary review that includes descriptions of the physical appearance, distribution, taxonomy, habitat, growth and propagation, phytochemical composition and applications of this plant. Notable amongst its repertoire of chemical components are anthocyanins which give C. ternatea flowers their characteristic blue color, and cyclotides, ultra-stable macrocyclic peptides that are present in all tissues of this plant. The latter are potent insecticidal molecules and are implicated as the bioactive agents in a plant extract used commercially as an insecticide. We include a description of the genetic origin of these peptides, which interestingly involve the co-option of an ancestral albumin gene to produce the cyclotide precursor protein. The biosynthesis step in which the cyclic peptide backbone is formed involves an asparaginyl endopeptidase, of which in C. ternatea is known as butelase-1. This enzyme is highly efficient in peptide ligation and has been the focus of many recent studies on peptide ligation and cyclization for biotechnological applications. The article concludes with some suggestions for future studies on this plant, including the need to explore possible synergies between the various peptidic and non-peptidic phytochemicals.

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions.

BACKGROUND: Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. OBJECTIVE: We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). METHODS: The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. RESULTS: Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. CONCLUSION: Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.