Production of a structurally validated cyclotide in rice suspension cells is enabled by a supporting biosynthetic enzyme.

MAIN CONCLUSION: We demonstrate the production of a structurally correct cyclotide in rice suspension cells with co-expression of a ligase-type AEP, which unlocks monocotyledons as production platforms to produce cyclotides. Cyclotides are a class of backbone-cyclic plant peptides that harbor a cystine knot composed of three disulfide bonds. These structural features make cyclotides particularly stable, and thus they have attracted significant attention for their use in biotechnological applications such as drug design. Currently, chemical synthesis is the predominant strategy to produce cyclotides for research purposes. However, synthetic production becomes costly both economically and environmentally at large scale. Plants offer an attractive alternative to chemical synthesis because of their lower cost and environmental footprint. In this study, rice suspension cells were engineered to produce the prototypical cyclotide, kalata B1 (kB1), a cyclotide with insecticidal properties from the African plant Oldenlandia affinis. Engineered rice cells produced structurally validated kB1 at yields of 64.21 µg/g (DW), which was dependent on the co-expression of a peptide ligase-competent asparaginyl endopeptidase OaAEP1b from O. affinis. Without co-expression, kB1 was predominantly produced as linear peptide. Through HPLC-MS co-elution, reduction, alkylation, enzymatic digestion, and proton NMR analysis, kB1 produced in rice was shown to be structurally identical to native kB1. This study reports the first example of an engineered plant suspension cell culture with the required molecular machinery for efficient production and cyclisation of a heterologous cyclotide.

Transcriptomic profiling of the medicinal plant Clitoria ternatea: identification of potential genes in cyclotide biosynthesis.

Clitoria ternatea a perennial climber of the Fabaceae family, is well known for its agricultural and medical applications. It is also currently the only known member of the Fabaceae family that produces abundant amounts of the ultra-stable macrocyclic peptides, cyclotides, across all tissues. Cyclotides are a class of gene-encoded, disulphide-rich, macrocyclic peptides (26-37 residues) acting as defensive metabolites in several plant species. Previous transcriptomic studies have demonstrated the genetic origin of cyclotides from the Fabaceae plant family to be embedded in the albumin-1 genes, unlike its counterparts in other plant families. However, the complete mechanism of its biosynthesis and the repertoire of enzymes involved in cyclotide folding and processing remains to be understood. In this study, using RNA-Seq data and de novo transcriptome assembly of Clitoria ternatea, we have identified 71 precursor genes of cyclotides. Out of 71 unique cyclotide precursor genes obtained, 51 sequences display unique cyclotide domains, of which 26 are novel cyclotide sequences, arising from four individual tissues. MALDI-TOF mass spectrometry analysis of fractions from different tissue extracts, coupled with precursor protein sequences obtained from transcriptomic data, established the cyclotide diversity in this plant species. Special focus in this study has also been on identifying possible enzymes responsible for proper folding and processing of cyclotides in the cell. Transcriptomic mining for oxidative folding enzymes such as protein-disulphide isomerases (PDI), ER oxidoreductin-1 (ERO1) and peptidylprolyl cis-trans isomerases (PPIases)/cyclophilins, and their levels of expression are also reported. In particular, it was observed that the CtPDI genes formed plant-specific clusters among PDI genes as compared to those from other plant species. Collectively, this work provides insights into the biogenesis of the medicinally important cyclotides and establishes the expression of certain key enzymes participating in peptide biosynthesis. Also, several novel cyclotide sequences are reported and precursor sequences are analysed in detail. In the absence of a published reference genome, a comprehensive transcriptomics approach was adopted to provide an overview of diverse properties and constituents of C. ternatea.

The life cycle of cyclotides: biosynthesis and turnover in plant cells.

KEY MESSAGE: Turnover rates have implications for understanding cyclotide biology and improving plant cell culture-based production systems. Cyclotides are a family of polypeptides recognized for a broad spectrum of bioactivities. The cyclic, cystine knot structural motif imparts these peptides with resistance to temperature, chemicals and proteolysis. Cyclotides are found widely distributed across the Violaceae and in five other plant families, where their presumed biological role is host defense. Violets produce mixtures of different cyclotides that vary depending on the organ, tissue or influence of environmental factors. In the present study, we investigated the biosynthesis and turnover of cyclotides in plant cells. Viola uliginosa suspension cultures were grown in media where all nitrogen containing salts were replaced with their 15N counterparts. This approach combined with LC-MS analysis allowed to separately observe the production of 15N-labelled peptides and decomposition of 14N cyclotides present in the cells when switching the media. Additionally, we investigated changes in cyclotide content in V. odorata germinating seeds. In the suspension cultures, the degradation rates varied for individual cyclotides and the highest was noted for cyO13. Rapid increase in production of 15N peptides was observed until day 19 and subsequently, a plateau of production, indicating an equilibrium between biosynthesis and turnover. The developing seedling appeared to consume cyclotides present in the seed endosperm. We show that degradation processes shape the cyclotide pattern present in different tissues and environments. The results indicate that individual cyclotides play different roles-some in defense and others as storage proteins. The turnover of cyclotides should be accounted to improve cell culture production systems.

Recombinant Expression of Cyclotides Using Expressed Protein Ligation.

Cyclotides are naturally occurring microproteins (≈30 residues long) present in several families of plants. All cyclotides share a unique head-to-tail circular knotted topology containing three disulfide bridges forming a cystine knot topology. Cyclotides possess high stability to chemical, physical, and biological degradation and have been reported to cross cellular membranes. In addition, naturally occurring and engineered cyclotides have shown to possess various pharmacologically relevant activities. These unique features make the cyclotide scaffold an excellent tool for the design of novel peptide-based therapeutics by using molecular evolution and/or peptide epitope grafting techniques. In this chapter, we provide protocols to recombinantly produce a natively folded cyclotide making use of a standard bacterial expression system in combination with an intein-mediated backbone cyclization with concomitant oxidative folding.

Production of bioactive cyclotides in somatic embryos of Viola odorata.

Viola odorata L. (Violaceae), an Indian medicinal plant, contains a plethora of cyclotides, which are a class of cyclic peptides derived from plants, possessing several applications. Somatic embryo culture of V. odorata was developed, via indirect somatic embryogenesis, to serve as an alternative to natural plant biomass for sustainable and continuous production of its bioactive ingredients, such as cyclotides. Among the various combinations of phytohormones tested, Murashige and Skoog medium supplemented with 1 mg/l thidiazuron gave rise to the maximum frequency of induction (86.7%) and a high number of somatic embryos (3) from an embryogenic callus. Identification and characterization of cyclotides in the somatic embryos were carried out using a Fourier transform mass spectrometer coupled with liquid chromatography (LC-FTMS). Among the cyclotides identified in the study, few were found to be exclusively present in the somatic embryo culture. Furthermore, the relative abundance of the cyclotides was higher in somatic embryo extract than in the natural plant extract. The biological activities (cytotoxic, haemolytic and antimicrobial) of the somatic embryos and the parent plant were compared. Unlike the natural plants, the somatic embryo extracts demonstrated specificity i.e. they were found to be potent against cancerous cells but not against non-cancerous cell line or red blood cells. In contrast to the plant extract, the somatic embryos extracts were found to be potent against Escherichia coli and Staphylococcus aureus. These results suggest that somatic embryos of V. odorata (rich in cyclotides) can be used as an alternative to plant biomass for its therapeutic applications and germplasm conservation.

Violacin A, a new chromanone produced by Streptomyces violaceoruber and its anti-inflammatory activity.

A new chromanone derivative, named violacin A (1), was isolated from the fermentation broth of Streptomyces violaceoruber as a potential anti-inflammatory compound. The structure of violacin A was established using comprehensive NMR spectroscopic data analysis together with UV, IR, and MS data. The anti-inflammatory effects and action mechanisms of violacin A were investigated in vitro. The results demonstrated that violacin A attenuated the production of NO, IL-1ß, IL-6, and TNF-α as well as inhibited the expression of iNOS in LPS-induced RAW 264.7 cells. Additionally, Western blot and qRT-PCR results revealed that 1 down-regulated pro-inflammatory cytokines expression correlated with the suppression of NF-κB signaling pathway.

Recombinant Expression of Cyclotides Using Split Inteins.

Cyclotides are fascinating microproteins (≈30 residues long) present in several families of plants that share a unique head-to-tail circular knotted topology of three disulfide bridges, with one disulfide penetrating through a macrocycle formed by the two other disulfides and inter-connecting peptide backbones, forming what is called a cystine knot topology. Naturally occurring cyclotides have shown to posses various pharmacologically relevant activities and have been reported to cross cell membranes. Altogether, these features make the cyclotide scaffold an excellent molecular framework for the design of novel peptide-based therapeutics, making them ideal substrates for molecular grafting of biological peptide epitopes. In this chapter we describe how to express a native folded cyclotide using intein-mediated protein trans-splicing in live Escherichia coli cells.

Immunolocalization of cyclotides in plant cells, tissues and organ supports their role in host defense.

MAIN CONCLUSION: The distribution of cyclotides was visualized in plant cells, tissues and organs using immunohistochemistry. Finding of cyclotides in tissues potentially vulnerable to pathogen attacks supports their role as defense molecules. The cyclotide family of plant peptides is characterized by the cyclic cystine knot motif and its diverse biological activities. Given their insecticidal and antimicrobial properties, the role of cyclotides in planta is probably associated with host defense. Our current understanding of the cellular compartmentalization of cyclotides in the vacuole is based on indirect studies on transgenic model plants that do not express cyclotides naturally. Matrix-assisted laser desorption ionization (MALDI) imaging has also been used to study the distribution of cyclotides, but the technique's resolution was insufficient to determine their tissue or cell distribution. To avoid the limitations of these approaches, immunohistochemical visualization methods were used. Antibodies were raised in rabbits using cycloviolacin O2 (cyO2), and their specificity was determined by Western and dot blot experiments. Slides for immunohistochemical analysis were prepared from leaf, petiole and root fragments of Viola odorata and Viola uliginosa, and specimens were visualized using indirect epifluorescence microscopy. The antibodies against cyclotides were specific against selected bracelet cyclotides with high similarity (cyO2, cyO3, cyO8, cyO13) and suitable for immunohistochemistry. The tissue distribution of the cyclotides visualized in this way is consistent with their proposed role in host defense-relatively large quantities were observed in the leaf and petiole epidermis in both Viola species. Cyclotides were also found in vascular tissue in all the assessed plant organs. The vacuole storage of cyclotides was directly shown.

Cyclotide biosynthesis.

Cyclotides are bioactive macrocyclic peptides from plants that are characterized by their exceptional stability and potential applications as protein engineering or drug design frameworks. Their stability arises from their unique cyclic cystine knot structure, which combines a head-to-tail cyclic peptide backbone with three conserved disulfide bonds having a knotted topology. Cyclotides are ribosomally synthesized by plants and expressed in a wide range of tissues, including leaves, flowers, stems and roots. Here we describe recent studies that have examined the biosynthesis of cyclotides and in particular the mechanism associated with post-translational backbone cyclization.

An overview of proteomics approaches applied to biopharmaceuticals and cyclotides research.

The evolution in proteomics approaches is notable, including quantitative proteomics and strategies for elucidation of post-translational modifications. Faster and more accurate mass spectrometers as well as cleverer bioinformatics tolls are making the difference in such advancement. Among the wide range of research in plant proteomics, biopharmaceutical production using plants as "biofactories" and the screening of new activities of new molecules, in this case, peptides, are quite important regarding translational proteomics. The present review is focused on "recombinant proteins and bioactive peptides", with biopharmaceuticals and cyclotides chosen as examples. Their application and challenges are focused on a "translational proteomics" point of view, in order to exemplify some new areas of research based on proteomics strategies. This article is part of a Special Issue entitled: Translational Plant Proteomics.

Making ends meet: chemically mediated circularization of recombinant proteins.

A selective N→S acyl transfer reaction facilitates semi-synthesis of the plant cyclotide kalata B1 from a linear precursor peptide of bacterial origin, through simple appendage of N-terminal cysteine and a thiol-labile C-terminal Gly-Cys motif. This constitutes the first synthesis of a ribosomally derived circular miniprotein, without recourse to protein splicing elements.

Insights into processing and cyclization events associated with biosynthesis of the cyclic Peptide kalata B1.

Plant cyclotides are the largest family of gene-encoded cyclic proteins. They act as host defense molecules to protect plants and are promising candidates as insecticidal and nematocidal agents in agriculture. For this promise to be realized a greater understanding of the post-translational processing of these proteins is needed. Cyclotides are cleaved from precursor proteins with subsequent ligation of the N and C termini to form a continuous peptide backbone. This cyclization step is inefficient in transgenic plants and our work aims to shed light on the specificity requirements at the excision sites for cyclic peptide production. Using the prototypic cyclotide kalata B1 (kB1) expressed from the Oak1 gene, MALDI-TOF mass spectrometry was used to examine the cyclization efficiency when mutants of the Oak1 gene were expressed in transgenic Nicotiana benthamiana. Cleavage at the N terminus of the cyclotide domain occurs rapidly with no strict specificity requirements for amino acids at the cleavage site. In contrast, the C-terminal region of the cyclotide domain in the P2, P1, P1', and P2' positions is highly conserved and only specific amino acids can occupy these positions. The cyclization reaction requires an Asn at position P1 followed by a small amino acid (Ala, Gly, Ser) at the P1' position. The P2' position must be filled by Leu or Ile; in their absence an unusual post-translational modification occurs. Substitution of the P2' Leu with Ala leads to hydroxylation of the neighboring proline. Through mutational analysis this novel proline hydroxylation motif was determined to be Gly-Ala-Pro-Ser.

Do plant cyclotides have potential as immunosuppressant peptides?

Cyclotides are an abundant and diverse group of ribosomally synthesized plant peptides containing a cyclic cystine-knotted structure that confers them with remarkable stability. They are explored for their distribution in plants, although little is known about the individual peptide content of a single species. Therefore, we chemically analyzed the crude extract of the coffee-family plant Oldenlandia affinis using a rapid peptidomics workflow utilizing nano-LC-MS, peptide reconstruct with database identification, and MS/MS automated sequence analysis to determine its cyclotide content. Biologically, cyclotides are mainly explored for applications in agriculture and drug design; here we report their growth-inhibiting effects on primary cells of the human immune system using biological and immunological end points in cell-based test systems. LC-MS quantification of the active O. affinis plant extract triggered the characterization of the antiproliferative activity of kalata B1, one of the most abundant cyclotides in this extract, on primary activated human lymphocytes. The effect has a defined concentration range and was not due to cytotoxicity, thus opening a new avenue to utilize native and synthetically optimized plant cyclotides for applications in immune-related disorders and as immunosuppressant peptides.

Circular micro-proteins and mechanisms of cyclization.

Transpeptidation reactions result in the formation of new peptide bonds and this can occur between two separate peptides or within the one peptide. These reactions are catalyzed by enzymes and when the N- and C-terminus of the one peptide are joined it results in the formation of cyclic proteins. Cyclization via head-to-tail linkage of the termini of a peptide chain occurs in only a small percentage of proteins but gives the resultant cyclic proteins exceptional stability. The mechanisms are not well understood and this review documents what is known of the events that lead to cyclization. Gene encoded cyclic proteins are found in both prokaryotic and eukaryotic species. The prokaryotic circular proteins include the bacteriocins and pilins. The eukaryotic circular proteins in mammals include the θ-defensins and retrocyclins. Small cyclic proteins are also found in fungi and a large range of cyclic proteins are expressed in cyanobacteria. Three types of cyclic proteins have been found in plants, the small cyclic proteins of 5-12 amino acids, the cyclic proteins from sunflower which are made up of 12-14 amino acids, and the larger group known as cyclotides which contain 28-37 amino acids. Three classes of enzymes are able to catalyse transpeptidation reactions, these include the cysteine, serine and threonine proteases. However only cysteine and serine proteases have been documented to cyclize proteins. The cyclotides from Oldenlandia affinis, the plant in which cyclotides were first discovered, are processed by an asparaginyl endopeptidase which is a cysteine protease. These proteases cleave an amide bond and form an acyl enzyme intermediate before nucleophilic attack of the amine group of the N-terminal residue to form a peptide bond, resulting in a cyclic peptide.

Subcellular targeting and biosynthesis of cyclotides in plant cells.

PREMISE OF THE STUDY: The cyclotide kalata B1 is found in the leaves of Oldenlandia affinis and is a potent insecticidal and nematocidal molecule. This peptide is cleaved from a precursor protein, Oak1, and ligation of the N- and C-termini occurs to form a continuous peptide backbone. The subcellular location of the excision and cyclization reactions is unknown, and there is debate as to which enzyme catalyzes the event. To determine where in the plant cell Oak1 is processed, we prepared constructs encoding GFP (green fluorescent protein) linked to the cyclotide precursor Oak1. METHODS: The GFP constructs were transiently expressed in the leaves of Nicotiana benthamiana, and GFP fluorescence was observed in living cells using confocal microscopy. A Fei Mao (FM) styryl dye was infiltrated into whole leaves that were still growing and expressing GFP constructs, enabling the plasma membrane and the tonoplast to be highlighted for visualization of the vacuole in living cells. KEY RESULTS: The full length Oak1 precursor directed GFP to the vacuole, suggesting that excision and cyclization of the cyclotide domain occurs in the vacuole where the cyclotides are then stored. The N-terminal propeptide and N-terminal repeat of Oak1 were both sufficient to target GFP to the vacuole, although the C-terminal propeptide, which is essential for cyclization, was not a targeting signal. CONCLUSIONS: The vacuolar location of cyclotides supports our hypothesis that the vacuolar processing enzyme, asparaginyl endoproteinase, has a pivotal role in excision and cyclization from cyclotide precursors.

Ribosomal synthesis of backbone macrocyclic peptides.

A wealth of knowledge has been accumulated on ribosomal synthesis of macrocyclic peptides in the past decade. In nature, backbone cyclization of the translated linear peptides is generally catalyzed by specific enzymes, giving them peptidase resistance, thermodynamic stability and various other physiological activities. Due to these biochemical traits, backbone cyclic peptides have become an attractive resource for the discovery of drug leads. Recently, various new methodologies have also been established to generate man-made cyclic peptides. Here, we describe the biosynthetic mechanisms of naturally occurring backbone macrocyclic peptides focusing on cyclotides, sunflower trypsin inhibitors (SFTIs) and cyanobactins as well as several new emerging methodologies, such as sortase mediated ligation, protein splicing method and genetic code reprogramming.

Naturally occurring circular proteins: distribution, biosynthesis and evolution.

Circular proteins, i.e., proteins with a backbone comprised of a continuous and seamless circle of amino acids, have been discovered over the last 15 years in bacteria, plants, fungi and animals. They function as defence tools in the organisms in which they are expressed and are exceptionally stable. The cyclotides are the largest known family of circular proteins and are expressed by plants of the Violaceae (violet), Rubiaceae (coffee) and Cucurbitaceae (cucurbit) families, where they have a role in plant defence against insect predation. So far there are fewer examples of cyclic peptides in bacteria or animals but we suggest that cyclic peptides are an underdiscovered class of molecules and that many more will be discovered in the near future. There is much interest in understanding the mechanism of cyclization of circular proteins and the role of the cyclic backbone in defining structure and activity. In this review, the families of ribosomally synthesized cyclic proteins reported to date are described and their common features are examined, providing information on their distribution, biosynthesis and evolution. The unusual structure of circular proteins confers them with high stability, and makes them very interesting as scaffolds for drug design, and this has led to the re-engineering of linear proteins to stabilise them and use them for such applications.

Cyclotides, a promising molecular scaffold for peptide-based therapeutics.

Cyclotides are a new emerging family of large plant-derived backbone-cyclized polypeptides (approximately 30 amino acids long) that share a disulfide-stabilized core (three disulfide bonds) characterized by an unusual knotted structure. Their unique circular backbone topology and knotted arrangement of three disulfide bonds make them exceptionally stable to thermal, chemical, and enzymatic degradation compared to other peptides of similar size. Currently, more than 100 sequences of different cyclotides have been characterized, and the number is expected to increase dramatically in the coming years. Considering their stability and biological activities like anti-HIV, uterotonic, and insecticidal, and also their abilities to cross the cell membrane, cyclotides can be exploited to develop new stable peptide-based drugs. We have recently demonstrated the intriguing possibility of producing libraries of cyclotides inside living bacterial cells. This opens the possibility to generate large genetically encoded libraries of cyclotides that can then be screened inside the cell for selecting particular biological activities in a high-throughput fashion. The present minireview reports the efforts carried out toward the selection of cyclotide-based compounds with specific biological activities for drug design.

Cyclotide synthesis and supply: from plant to bioprocess.

Cyclotides are disulfide-rich miniproteins with a circular backbone and a knotted arrangement ofdisulfide bonds. Because these plant-derived peptides are resistant to degradation and exhibit a diverse range of bioactivity they have become important agronomic and industrial objectives. They belong to a group of compounds with low market volume and high price that are poorly processed by microorganisms, are too complex for economic chemical synthesis, and thus are valuable candidates for the synthesis in plant cell bioprocesses. This review highlights current research aimed at production routes of cyclotides in Oldenlandia affinis plantlets and cell cultures, and summarizes recent advances in bioprocessing aspects, with particular emphasis on the development of suitable bioreactor configurations for plant cell culture-based processes, the optimization of culture environments as a powerful means to improve yields, bioreactor operational modes, and trends in protein recovery.