A continent of plant defense peptide diversity: cyclotides in Australian Hybanthus (Violaceae)

Cyclotides are plant-derived miniproteins that have the unusual features of a head-to-tail cyclized peptide backbone and a knotted arrangement of disulfide bonds. It had been postulated that they might be an especially large family of host defense agents, but this had not yet been tested by field data on cyclotide variation in wild plant populations. In this study, we sampled Australian Hybanthus (Violaceae) to gain an insight into the level of variation within populations, within species, and between species. A wealth of cyclotide diversity was discovered: at least 246 new cyclotides are present in the 11 species sampled, and 26 novel sequences were characterized. A new approach to the discovery of cyclotide sequences was developed based on the identification of a conserved sequence within a signal sequence in cyclotide precursors. The number of cyclotides in the Violaceae is now estimated to be >9000. Cyclotide physicochemical profiles were shown to be a useful taxonomic feature that reflected species and their morphological relationships. The novel sequences provided substantial insight into the tolerance of the cystine knot framework in cyclotides to amino acid substitutions and will facilitate protein engineering applications of this framework.     

A novel suite of cyclotides from Viola odorata: sequence variation and the implications for structure, function and stability

Cyclotides are a fascinating family of plant-derived peptides characterized by their head-to-tail cyclized backbone and knotted arrangement of three disulfide bonds. This conserved structural architecture, termed the CCK (cyclic cystine knot), is responsible for their exceptional resistance to thermal, chemical and enzymatic degradation. Cyclotides have a variety of biological activities, but their insecticidal activities suggest that their primary function is in plant defence. In the present study, we determined the cyclotide content of the sweet violet Viola odorata, a member of the Violaceae family. We identified 30 cyclotides from the aerial parts and roots of this plant, 13 of which are novel sequences. The new sequences provide information about the natural diversity of cyclotides and the role of particular residues in defining structure and function. As many of the biological activities of cyclotides appear to be associated with membrane interactions, we used haemolytic activity as a marker of bioactivity for a selection of the new cyclotides. The new cyclotides were tested for their ability to resist proteolysis by a range of enzymes and, in common with other cyclotides, were completely resistant to trypsin, pepsin and thermolysin. The results show that while biological activity varies with the sequence, the proteolytic stability of the framework does not, and appears to be an inherent feature of the cyclotide framework. The structure of one of the new cyclotides, cycloviolacin O14, was determined and shown to contain the CCK motif. This study confirms that cyclotides may be regarded as a natural combinatorial template that displays a variety of peptide epitopes most likely targeted to a range of plant pests and pathogens.     

From nature to creation: Going around in circles,the art of peptide cyclization

Cyclic peptides and cyclotides are becoming common identities within the present efforts seen in peptide engineering – as we seek approaches to achieve potent biological activity, pharmacological selectivity, structurally stability and oral bioavailability. Yet this unique family of peptides has faced uncommon hurdles in their discovery, synthesis and bioengineering, retaining to characteristics that truly deviate these from their linear counterparts. In this mini-review we take a board spectrum approach to introduce this novel family of biomolecules and the troubles that they face in their sequence and disulfide connectivity assignment, together highlighting the present combined strategies involved in cyclic peptide/cyclotide synthesis and modification. These efforts have circumvented otherwise impossible hurdles in their manipulation and production that are only now advancing cyclic peptides/cyclotides as research probes and future pharmaceutical templates.     

Analysis of cyclotides in Viola ignobilis by Nano liquid chromatography fourier transform mass spectrometry

Cyclotides are macrocyclic knotted peptides originating from plants. They are extremely stable and have a range of bioactivities including anti-HIV and insecticidal activity. Given the stability of the cyclotide framework, there is interest in using these peptides as scaffolds in drug design. In the current study, we have shown that nano-LC Fourier transform mass spectrometry (FTMS) is an effective method of analyzing cyclotides in plants. In addition, we have used this technique to find cyclotides in a novel species, Viola ignobilis (Violaceae plant family), which was collected from the West Azerbaijan province of Iran. Varv peptide A, cycloviolacin B2, and cycloviolacin O8 were found in this species. This study provides a novel method for directly analyzing cyclotide sequences without enzymatic digestion and further information regarding the distribution of cyclotides in plant species.     

Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1

The plant cyclotides are a family of 28 to 37 amino acid miniproteins characterized by their head-to-tail cyclized peptide backbone and six absolutely conserved Cys residues arranged in a cystine knot motif: two disulfide bonds and the connecting backbone segments form a loop that is penetrated by the third disulfide bond. This knotted disulfide arrangement, together with the cyclic peptide backbone, renders the cyclotides extremely stable against enzymatic digest as well as thermal degradation, making them interesting targets for both pharmaceutical and agrochemical applications. We have examined the expression patterns of these fascinating peptides in various Viola species (Violaceae). All tissue types examined contained complex mixtures of cyclotides, with individual profiles differing significantly. We provide evidence for at least 57 novel cyclotides present in a single Viola species (Viola hederacea). Furthermore, we have isolated one cyclotide expressed only in underground parts of V. hederacea and characterized its primary and three-dimensional structure. We propose that cyclotides constitute a new family of plant defense peptides, which might constitute an even larger and, in their biological function, more diverse family than the well-known plant defensins.     

Cyclotide proteins and precursors from the genus Gloeospermum: Filling a blank spot in the cyclotide map of Violaceae

Cyclotides are disulfide-rich plant proteins that are exceptional in their cyclic structure; their N and C termini are joined by a peptide bond, forming a continuous circular backbone, which is reinforced by three interlocked disulfide bonds. Cyclotides have been found mainly in the coffee (Rubiaceae) and violet (Violaceae) plant families. Within the Violaceae, cyclotides seem to be widely distributed, but the cyclotide complements of the vast majority of Violaceae species have not yet been explored. This study provides insight into cyclotide occurrence, diversity and biosynthesis in the Violaceae, by identifying mature cyclotide proteins, their precursors and enzymes putatively involved in their biosynthesis in the tribe Rinoreeae and the genus Gloeospermum. Twelve cyclotides from two Panamanian species, Gloeospermum pauciflorum Hekking and Gloeospermum blakeanum (Standl.) Hekking (designated Glopa A-E and Globa A-G, respectively) were characterised through cDNA screening and protein isolation. Screening of cDNA for the oxidative folding enzymes protein-disulfide isomerase (PDI) and thioredoxin (TRX) resulted in positive hits in both species. These enzymes have demonstrated roles in oxidative folding of cyclotides in Rubiaceae, and results presented here indicate that Violaceae plants have evolved similar mechanisms of cyclotide biosynthesis. We also describe PDI and TRX sequences from a third cyclotide-expressing Violaceae species, Viola biflora L., which further support this hypothesis.     

Expression of Viola cyclotides by liquid chromatography-mass spectrometry and tandem mass spectrometry sequencing of intercysteine loops after introduction of charges and cleavage sites by aminoethylation

The expression of cyclotides-macrocyclic plant peptides-was profiled in six violets, Viola cotyledon, V. biflora, V. arvensis, V. tricolor, V. riviniana, and V. odorata, by LC-MS. All were found to express notably complex mixtures, with single species containing >50 cyclotides. To facilitate their sequencing by MS-MS, an analytical strategy is presented involving aminoethylation of cysteines. This overcomes a number of problems intimately associated with the cyclotide core structure-that is, their joined N and C termini, disulfide knot, and low or clustered content of positively charged amino acids and enzymatic cleavage sites. As a result, charges as well as cleavage sites are introduced at the most conserved part of their sequence, the cysteines. Combined with tryptic digestion, all intercysteine loops are then of suitable size and charge for MS-MS sequencing. The utility of this strategy is shown by the sequencing of two novel cyclotides isolated from V. cotyledon; vico A (cyclo-(AESCVYIPCFTGIAGCSCKNKVCYYNGSIPC)) and vico B (cyclo-(AESCVYIPCITGIAGCSCKNKVCYYNGSIPC)); their complete sequence could be determined by nanospray MS-MS. The strategy for converting conserved cysteines to enzymatic cleavage sites might also benefit the study of other peptides and proteins displaying similar structural problems for MS analysis.     

The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity

The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a cystine knot, the combination of which produces the cyclic cystine knot motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC(50)-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 microM while the M?bius cyclotide vibi D was not cytotoxic at 30 microM.     

Isolation and characterization of cytotoxic cyclotides from Viola philippica

Cyclotides are a large family of plant peptides characterized by a macrocyclic backbone and knotted arrangement of three disulfide bonds. This unique structure renders cyclotides exceptionally stable to thermal, chemical and enzymatic treatments. They exhibit a variety of bioactivities, including uterotonic, anti-HIV, cytotoxic and hemolytic activity and it is these properties that make cyclotides an interesting peptide scaffold for drug design. In this study, eight new cyclotides (Viphi A-H), along with eight known cyclotides, were isolated from Viola philippica, a plant from the Violaceae family. In addition, Viba 17 and Mram 8 were isolated for the first time as peptides. The sequences of these cyclotides were elucidated primarily by using a strategy involving reduction, enzymatic digestion and tandem mass spectroscopy sequencing. Several of the cyclotides showed cytotoxic activities against the cancer cell lines MM96L, HeLa and BGC-823. The novel cyclotides reported here: (1) enhance the known sequence variation observed for cyclotides; (2) extend the number of species known to contain cyclotides; (3) provide interesting structure-activity relationships that delineate residues important for cytotoxic activity. In addition, this study provides insights into the potential active ingredients of traditional Chinese medicines.     

Discovery of cyclotide-like protein sequences in graminaceous crop plants: ancestral precursors of circular proteins?

Cyclotides are peptides from plants of the Rubiaceae and Violaceae families that have the unusual characteristic of a macrocylic backbone. They are further characterized by their incorporation of a cystine knot in which two disulfides, along with the intervening backbone residues, form a ring through which a third disulfide is threaded. The cyclotides have been found in every Violaceae species screened to date but are apparently present in only a few Rubiaceae species. The selective distribution reported so far raises questions about the evolution of the cyclotides within the plant kingdom. In this study, we use a combined bioinformatics and expression analysis approach to elucidate the evolution and distribution of the cyclotides in the plant kingdom and report the discovery of related sequences widespread in the Poaceae family, including crop plants such as rice (Oryza sativa), maize (Zea mays), and wheat (Triticum aestivum), which carry considerable economic and social importance. The presence of cyclotide-like sequences within these plants suggests that the cyclotides may be derived from an ancestral gene of great antiquity. Quantitative RT-PCR was used to show that two of the discovered cyclotide-like genes from rice and barley (Hordeum vulgare) have tissue-specific expression patterns.     

Discovery and characterization of novel cyclotides originated from chimeric precursors consisting of albumin-1 chain a and cyclotide domains in the Fabaceae family

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1-T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants.     

Phosphatidylethanolamine Binding Is a Conserved Feature of Cyclotide-Membrane Interactions

Cyclotides are bioactive cyclic peptides isolated from plants that are characterized by a topologically complex structure and exceptional resistance to enzymatic or thermal degradation. With their sequence diversity, ultra-stable core structural motif, and range of bioactivities, cyclotides are regarded as a combinatorial peptide template with potential applications in drug design. The mode of action of cyclotides remains elusive, but all reported biological activities are consistent with a mechanism involving membrane interactions. In this study, a diverse set of cyclotides from the two major subfamilies, Möbius and bracelet, and an all-d mirror image form, were examined to determine their mode of action. Their lipid selectivity and membrane affinity were determined, as were their toxicities against a range of targets (red blood cells, bacteria, and HIV particles). Although they had different membrane-binding affinities, all of the tested cyclotides targeted membranes through binding to phospholipids containing phosphatidylethanolamine headgroups. Furthermore, the biological potency of the tested cyclotides broadly correlated with their ability to target and disrupt cell membranes. The finding that a broad range of cyclotides target a specific lipid suggests their categorization as a new lipid-binding protein family. Knowledge of their membrane specificity has the potential to assist in the design of novel drugs based on the cyclotide framework, perhaps allowing the targeting of peptide drugs to specific cell types.     

Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins

Cyclotides are mini-proteins of 28-37 amino acid residues that have the unusual feature of a head-to-tail cyclic backbone surrounding a cystine knot. This molecular architecture gives the cyclotides heightened resistance to thermal, chemical and enzymatic degradation and has prompted investigations into their use as scaffolds in peptide therapeutics. There are now more than 80 reported cyclotide sequences from plants in the families Rubiaceae, Violaceae and Cucurbitaceae, with a wide variety of biological activities observed. However, potentially limiting the development of cyclotide-based therapeutics is a lack of understanding of the mechanism by which these peptides are cyclized in vivo. Until now, no linear versions of cyclotides have been reported, limiting our understanding of the cyclization mechanism. This study reports the discovery of a naturally occurring linear cyclotide, violacin A, from the plant Viola odorata and discusses the implications for in vivo cyclization of peptides. The elucidation of the cDNA clone of violacin A revealed a point mutation that introduces a stop codon, which inhibits the translation of a key Asn residue that is thought to be required for cyclization. The three-dimensional solution structure of violacin A was determined and found to adopt the cystine knot fold of native cyclotides. Enzymatic stability assays on violacin A indicate that despite an increase in the flexibility of the structure relative to cyclic counterparts, the cystine knot preserves the overall stability of the molecule.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

Conserved structural and sequence elements implicated in the processing of gene-encoded circular proteins

The cyclotides are the largest family of naturally occurring circular proteins. The mechanism by which the termini of these gene-encoded proteins are linked seamlessly with a peptide bond to form a circular backbone is unknown. Here we report cyclotide-encoding cDNA sequences from the plant Viola odorata and compare them with those from an evolutionarily distinct species, Oldenlandia affinis. Individual members of this multigene family encode one to three mature cyclotide domains. These domains are preceded by N-terminal repeat regions (NTRs) that are conserved within a plant species but not between species. We have structurally characterized peptides corresponding to these NTRs and show that, despite them having no sequence homology, they form a structurally conserved alpha-helical motif. This structural conservation suggests a vital role for the NTR in the in vivo folding, processing, or detoxification of cyclotide domains from the precursor protein.     

Optimization of cyclotide extraction parameters

Cyclotides are gene-encoded plant mini-proteins that contain a unique circular and cystine knotted amide backbone. Because of that ultra stable scaffold and the ability to harness a wide variety of sequences and biological activities within the scaffold, cyclotides find interesting potential applications for drug discovery and in agriculture. However, some fundamental knowledge is still missing to exploit these plant compounds, including finding the optimal process of their extraction from plant material. In the current work, the extraction parameters solvent type, time of extraction, number of re-macerations and the plant material to solvent ratio have been compared using the sweet violet (Viola odorata L.) as a model plant. That species is a well-characterized and rich source of cyclotides that contains prototypic cyclotides with different chemical and physical properties. We found that hydroalcoholic solutions of medium polarity give good yield of the cyclotide cocktail. In conclusion, single maceration with 50% MeOH for 6 h at a plant material to solvent ratio of 0.5:10 (g/mL) represents an optimum extraction method.     

Novel cyclotides and uncyclotides with highly shortened precursors from Chassalia chartacea and effects of methionine oxidation on bioactivities

Cyclotides are a new class of plant biologics that display a diverse range of bioactivities with therapeutic potentials. They possess an unusual end-to-end cyclic backbone combined with a cystine knot arrangement, making them exceptionally stable to heat, chemical and enzymatic degradation. Currently, >200 cyclotides have been discovered but only three naturally occurring linear variants (also known as uncyclotides) have been isolated. In this study, we report the discovery of 18 novel peptides, chassatides C1 to C18, composed of 14 new cyclotides and four uncyclotides from Chassalia chartacea (Rubiaceae family). Thus far, this is the largest number of uncyclotides being reported in a single species. Activity testing showed that the uncyclotides not only retain the effectiveness but also are the most potent chassatides in the assays for antimicrobial, cytotoxic, and hemolytic activities. Genetic characterization of novel chassatides revealed that they have the shortest precursors of all known cyclotides hitherto isolated, which represents a new class of cyclotide precursors. This is the first report of cyclotide genes in a second genus, the Chassalia, other than the Hedyotis (Oldenlandia) of the Rubiaceae family. In addition, we also report the characterization of two Met-oxidized derivatives of chassatides C2 and C11. The oxidation of Met residue causes loss of bioactivities, strengthening the importance of the hydrophobic patch for membrane interaction.     

Alanine scanning mutagenesis of the prototypic cyclotide reveals a cluster of residues essential for bioactivity

The cyclotides are stable plant-derived mini-proteins with a topologically circular peptide backbone and a knotted arrangement of three disulfide bonds that form a cyclic cystine knot structural framework. They display a wide range of pharmaceutically important bioactivities, but their natural function is in plant defense as insecticidal agents. To determine the influence of individual residues on structure and activity in the prototypic cyclotide kalata B1, all 23 non-cysteine residues were successively replaced with alanine. The structure was generally tolerant of modification, indicating that the framework is a viable candidate for the stabilization of bioactive peptide epitopes. Remarkably, insecticidal and hemolytic activities were both dependent on a common, well defined cluster of hydrophilic residues on one face of the cyclotide. Interestingly, this cluster is separate from the membrane binding face of the cyclotides. Overall, the mutagenesis data provide an important insight into cyclotide biological activity and suggest that specific self-association, in combination with membrane binding mediates cyclotide bioactivities.