Plant Cyclotides: an Unusual Class of Defense Compounds

peptides 28 (2007) 1475–1481

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Review Plant cyclotides: An unusual class of defense compounds

Patrı´cia B. Pelegrini a, Betania F. Quirino a, Octa´ vio L. Franco a,b,* a Centro de Ana´ lises Proteoˆmicas e Bioquı´micas, Programa de Po´s-Graduac¸a˜ o em Cieˆncias Genoˆmicas e Biotecnologia, Universidade Cato´lica de Brası´lia, Brası´lia, Brazil b Departamento de Biologia, Universidade Federal de Juiz de Fora, Minas Gerais, Brazil article info abstract

Article history: Plant cyclotides are unusual peptides with low molecular masses and a three-dimensional Received 3 March 2007 structure characterized by the presence of a cyclic fold. Synthetic peptides can adopt this Received in revised form circular conformation, but it is not a common feature for most members of other peptide 26 April 2007 groups. Cyclotides present a wide range of functions, such as the ability to induce stronger Accepted 30 April 2007 contractions during childbirth and anti-tumor activity. Additionally, some cyclotides pre- Published on line 16 May 2007 sent anti-viral, insecticidal or proteinase inhibitory activity. In this paper, we describe the structural and functional characteristics of plant cyclotides, their most conserved features Keywords: and the development of these peptides for human health and biotechnological applications. # Cyclotides 2007 Published by Elsevier Inc. Plant defense Anti-viral Insecticide

Contents

1. Introduction...... 1475 2. Biological activities ...... 1476 3. Sequence alignment and phylogenetic analysis ...... 1476 4. Structure characterization...... 1479 5. Concluding remarks and perspectives ...... 1480 Acknowledgements ...... 1480 References ...... 1480

1. Introduction appeared in the 1990s [29]. The plant cyclotide family consists of peptides of approximately 30 amino acids and, unlike Plant cyclotides are unusual proteins first reported in the early conventional proteins; do not have N- or C-termini, since 1970s, in studies of the medical properties of an African plant these extremities are linked, forming a cyclic structure named kalata-kalata [12]. Although their discovery occurred [10,27,36]. Cyclotides can be classified into two subfamilies: about 30 years ago, findings on their structural features and Mo¨ bius and bracelet. The first group is characterized by a twist studies reporting possible mechanisms of action only formation in the backbone of the peptide, and by the possible

* Corresponding author at: Centro de Analises Proteomicas e Bioquimicas, Universidade Catolica de Brası´lia, SGAN 916 Norte, Modulo B, Av. W5, 70.790-160, Asa Norte, DF- Brazil, Brası´lia. Fax: +55 61 3347-4797. E-mail address: [email protected] (O.L. Franco). 0196-9781/$ – see front matter # 2007 Published by Elsevier Inc. doi:10.1016/j.peptides.2007.04.025 1476 peptides 28 (2007) 1475–1481

presence of a cis-Pro motif [4,6,22]. The second group, the insecticidal activity. A protein from O. affinis, named kalata B2, bracelet subfamily, is characterized by the absence of this is able to inhibit growth and development of Helicoperva twist feature [5,10]. Cyclization seems to play an important armigera larvae [22]. Although its mode of action is not yet role in peptide stability and activity. This observation was understood, kalata B2 shows structure similarities to kalata reinforced by a study with kalata-B1, a Mo¨ bius member, where B1, a cyclotide with insecticidal and uterotonic activities as the cystine knot motif was shown to be important in the previously mentioned [22]. Earlier reports identified and thermal stability, while the cyclic backbone was responsible characterized a cyclotide from V. hederaceae, named vhl-1, for the complete enzymatic stability [5]. which has anti-HIV activity [2]. Recently, a group of cyclotides, Cyclotides have a wide variety of roles in plant defense, such called cycloviolins, was isolated from Leonia cymosa and also as proteinase inhibition, cytotoxicity to tumor cells, anti-viral demonstrated activity against HIV-1 [17]. Moreover, palicour- effects and insecticidal activities [2,8,21–23,34]. Moreover, ein, a 37 amino acid cyclotide from Palicourea condensata, has uterotonic activity can be induced by cyclotides, showing their been shown to inhibit HIV-1 infection of human T-lympho- potential as medical drugs [12,13]. Although the mechanisms of blastoid cells [20]. Earlier studies revealed that kalata B1 from action of cyclotides are still not very well understood, some O. affinis, cyclopsychotride from P. longipes and circulins A and information has been published. In order to elucidate the B from C. parvifolia also demonstrated antimicrobial activities mechanism of action of Mo¨bius cyclotides, kalata B1 with against bacteria. While circulin A and kalata B1 showed dodecylphosphocholine were studied using NMR spectroscopy activity against Gram-positive bacteria, such as Staphylococcus [31]. The data showed that the cyclotide binds to the micelle aureus, circulin B and cyclopsychotride were able to inhibit surface, with relatively high affinity, via two hydrophobic loops. growth of both Gram-negative and Gram-positive bacteria [35].

The charged residues (Glu3 and Arg24), along with the cation- Plant proteins belonging to the cyclotide family can have binding site (near Glu3) are segregated on the other side of the different biological functions. Recently, six bracelet and seven molecule and in contact with the detergent polar head groups. cyclotides from the Mo¨ bius subfamily were isolated from V. The spatial structure of kalata B1 is only slightly changed during odorata [20]. They were tested and showed stability against incorporation into micelles and represents a distorted triple- proteolytic degradation by pepsin, trypsin and thermolysin. stranded b-sheet cross-linked by a cystine knot [31]. Moreover, cycloviolacin O24, from the Mo¨ bius subfamily The biosynthesis of cyclotides has also been studied and, presented 75% of hemolytic activity, while O14, another although there are not any conclusive findings, there is an Mo¨ bius member, showed just 11% of hemolytic activity and hypothesis that better explains its folding mechanism. Studies no proteolytic activity. Further studies focusing on the with kalata B1 leaded to the hypothesis that disulfide bond elucidation of their three-dimensional structure revealed that formation would occur before cleavage and cyclization [21,29]. minor variations in primary sequence could be the cause for Moreover, this theory was also confirmed by Trabi and Craik changes in hemolytic activity [20]. [36], but the process of excision and cyclization, as well the enzymes involved are still unclear. This review focuses on novel insights on plant cyclotides, showing the recent 3. Sequence alignment and phylogenetic discoveries about their wide range of functions and the most analysis current hypothesis about their mechanisms of action. Furthermore, this report also intends to correlate amino acid Analysis of the primary sequences of plant cyclotides shows sequence and structure to conserved residues and describe that cysteine residues are well conserved in all plant species similarities between different species. (Fig. 1A). Three glycines at positions 2, 14 and 26 are also well

conserved in the cyclotides, while Gly7 and Gly8 showed conservation only in Mo¨ bius members and in some bracelet 2. Biological activities cyclotides from Viola species, such as Varv proteins. The same

cyclotides also presented a conserved Glu3, which has been The ﬁrst cyclotide described was isolated from the plant shown to be important for structure stabilization [22]. Loop 1 is kalata-kalata (Oldenlandia afﬁnis), and showed uterotonic conserved in both cyclotide subfamilies, while loop 5 is activity [12,13] stimulating stronger contractions during child- conserved within the cyclotide subfamilies, but not between birth, thereby shortening the delivery time [12]. Today, a range them. The primary sequence of the Mo¨ bius members have 80% of activities has been described for cyclotides. Table 1 identity, while for bracelet cyclotides from the Viola species, summarizes the plant cyclotides discovered in the last 20 there is approximately 54% identity. years. In particular, circular peptides from Momordica cochinci- A phylogenetic tree of plant cyclotides was constructed in nensis (MCoTI-I and II) have demonstrated ability to inhibit order to evaluate the existence of a common ancestor between trypsin-like enzymes [18,19]. Mo¨bius and bracelet members (Fig. 2). In general, bracelet Some species that are used in carcinoma treatments, such members were grouped at the top part of the tree, while Mo¨bius as Viola arvensis, contain cyclotides known as Varv peptides members were mostly concentrated at the bottom of the tree. [3,11]. Vitri A isolated from V. tricolor, demonstrated cytotoxi- One exception was Vhl-2, a protein from V. hederaceae which city to human lymphoma and myeloma cells [34]. Similarly, was grouped with Mo¨bius proteins. Varv proteins, which come cycloviolacin H4 isolated from V. hederaceae is able to cause from Violaceae, and Kalata proteins, present in Rubiaceae hemolysis in human erythrocytes. Despite the well-charac- plants were quite well separated within the Mo¨bius subfamily. terized cytotoxic activity toward tumor cells, their mechanism Vhl-2 function was not reported yet, however, the fact that it is of action has not yet been elucidated. Cyclotides can also have placed near VarvF protein suggests the possibility that this peptides 28 (2007) 1475–1481 1477

Table 1 – Relation of plant cyclic peptides function and molecular masses Peptide Source Function Molecular mass (Da) Reference

MCoTI M. cochinchinensis Trypsin inhibitor 3480.00 [16,17] MCoTII M. cochinchinensis Trypsin inhibitor 3453.00 [16,17] Varv peptide A V. tricolor Cytotoxic 2902.32 [11] Varv peptide E V. tricolor Cytotoxic 2916.34 [11] Varv peptide B V. arvensis Cytotoxic 3093.52 [19] Varv peptide C V. arvensis Cytotoxic 2902.32 [19] Varv peptide D V. arvensis Cytotoxic 2902.32 [19] Varv peptide E V. arvensis Cytotoxic 2983.43 [19] Varv peptide F V. arvensis Cytotoxic 3047.43 [19] Varv peptide G V. arvensis Cytotoxic 2916.34 [19] Varv peptide H V. arvensis Cytotoxic 3079.47 [19] Cycloviolacin O2 V. odorata Cytotoxic 3145.05 [33] Vitri A V. tricolor Cytotoxic 3178.78 [11] Kalata B2 O. affinis Insecticidal activity 2954.80 [7] Kalata B1 O. affinis Oxitocic agent, antimicrobial activity 2890.38 [1,23] Kalata B3 O. affinis Not reported 3106.50 [6] Kalata B4 O. affinis Not reported 2917.33 [6] Kalata S O. affinis Nor reported 2902.32 [6] Cyclopaychotride A P. longipes Antimicrobial activity 4165.93 [38] Circulins A C. parviflora Anti-HIV and antimicrobial activity 3175.78 [30,31] Circulins B C. parviflora Anti-HIV and antimicrobial activity 3307.98 [30,31] Circulins C C. parviflora Anti-HIV activity 3379.06 [31] Circulins D C. parviflora Anti-HIV activity 3125.72 [31] Circulins E C. parviflora Anti-HIV activity 3282.89 [31] Circulins F C. parviflora Anti-HIV activity 3075.70 [31] Palicourein P. condensata Anti-HIV activity 3928.43 [22] Cycloviolin A-D L. cymosa Anti-HIV activity 2946.57 [21] Cycloviolin A-D L. cymosa Anti-HIV activity 2910.33 [21] Cycloviolin A-D L. cymosa Anti-HIV activity 3166.77 [21] Cycloviolin A-D L. cymosa Anti-HIV activity 3071.63 [21] Vhl-1 V. hederaceae Anti-HIV activity 3330.61 [12] Vhl-2 V. hederaceae Not reported 3172.58 [12] Cycloviolacin O1 V. odorata Not reported 3140.69 [6] Cycloviolacin O3 V. odorata Not reported 3178.78 [6] Cycloviolacin O4 V. odorata Not reported 3191.78 [6] Cycloviolacin O5 V. odorata Not reported 3137.69 [6] Cycloviolacin O6 V. odorata Not reported 3207.82 [6] Cycloviolacin O7 V. odorata Not reported 3178.82 [6] Cycloviolacin O8 V. odorata Not reported 3194.82 [6] Cycloviolacin O9 V. odorata Not reported 3164.75 [6] Cycloviolacin O10 V. odorata Not reported 3141.72 [6] Cycloviolacin O11 V. odorata Not reported 3235.87 [6] Cycloviolacin H1 V. hederaceae Not reported 3155.74 [6] Cycloviolacin H4 V. hederaceae Hemolytic activity 3121.64 [20]

protein might have a cytotoxic activity. Experiments need to be These modifications probably occurred in a coordinated fashion performed with vhl-2 to address this possibility. Interestingly, in order to preserve an adaptive phenotype [30]. kalata B8 was not clearly grouped with either Mo¨bius or bracelet Proteins from the bracelet subfamily did not group proteins. This confirms the structural analysis that indicates according to the botanical genera of the plant from which that kalata B8 may be the missing link between both families [9]. they were reported, Rubiaceae and Violaceae. In general, Studies of kalata B8 may be interesting from an evolutionary cycloviolacins, circulins and cycloviolins present anti-HIV perspective and may give support to the idea that Mo¨bius activity and this may explain why they did not form separate proteins can evolve into a bracelet protein or vice-versa. There groups. However, there are already examples that they can are studies with plant biocide peptides called defensins have other functions such as antimicrobial activity presented showing that a single mutation can lead to function modifica- by circulin A [15]. Rubiacea and Violacea are distantly related tion such as an amylase inhibitor being converted into a plant families. Other possibility that cannot be ruled out to proteinase inhibitor and vice-versa [27]. Furthermore, not only explain why peptides from the bracelet or Mo¨bius subfamilies functions can be easily altered but a protein classified as a b- did not group according to their botanical family of origin is defensin can be converted into a a-defensin, groupings based on that there might have been a common ancestral protein before structure and organism source of the protein. Preliminary the separation of Rubiacea and Violacea families. studies suggest that the sequences of defensin genes are When the phylogenetic tree is analyzed regarding to suffering radical and rapid changes to allow this to occur [30]. function, only cytotoxic cyclotides appear grouped. The other 1478 peptides 28 (2007) 1475–1481

functional classes such as antimicrobial, anti-viral, oxytocic performed and these may also improve our knowledge about and hemolytic are totally dispersed throughout the tree (Fig. 2). the functional evolution. The history of the evolution of plant It is possible that proteins that are phylogenetically related do cyclotides was recently described in a study with genes from indeed share a common not yet tested function. Therefore, different sources, and similarities were observed among further studies of structure function relation need to be sequences from wheat, rice and maize [24].

Fig. 1 – (A) Alignment of 40 plant cyclotides from the more than 80 described. Disulfide bonds are shown as linked lines. Underlined proteins belong to Mo¨ bius subfamily; non-subtitled proteins belong to bracelet subfamily. Dark blue lines above indicate the loops. (B) Tertiary structure comparisons of bracelet and Mo¨ bius cyclotide subfamilies (labeled). Side chains indicate conservation in both subfamilies at residues level. 3D structures were drawn using Spdb-Viewer 3.7 (PDB code 1ZA8, bracelet; 1PT4, Mo¨bius) [30]. (C) Schematic illustration showing the differences between trans and cis geometries that determines the division of Mo¨ bius and bracelet groups as described by Jennings et al. [22]. peptides 28 (2007) 1475–1481 1479

Fig. 1. (Continued).

4. Structure characterization Saether et al. [29] reported the ﬁrst structure for kalata B1 that contained a compact cystine knot motif. The structure In recent years, several plant macrocyclic peptides have been also showed a distorted triple-stranded b-sheet and numerous reported and classiﬁed as plant cyclotides, and most of these turns. The plant cyclotide structure is mainly characterized by have been from the Violaceae family [26]. Nevertheless, the presence of a head-to-tail backbone composed by six

Fig. 2 – Phylogenetic tree of plant cyclotides, using the Tree View Program [35]. Protein sequences from Fig. 1A were used as a model to construct the tree. Underlined proteins correspond to Violaceae family members. Not underlined proteins correspond to Rubiaceae family members. (m) indicates Mo¨ bius members; (b) indicates bracelet members; asterisks indicate proteins from C. parviflora. Gray circles indicate proteins from O. affinis. White squares indicate protein from P. longipes. (+) indicates proteins from L. cymosa. Underlined proteins without any other symbol correspond to molecules from Viola species. White circles correspond to cytotoxic cyclotides; Gray squares correspond to proteins with anti-HIV activity; Four point stars correspond to antimicrobial cyclotides; Double dashed line corresponds to insecticidal proteins; Five points stars correspond to oxytocic cyclotides. White triangles correspond to proteins with hemolytic activity. 1480 peptides 28 (2007) 1475–1481

conserved cysteine residues, forming a knot motif [14,22,32]. Until now approximately 80 cyclotides have been char- Thus, two disulfide bonds form a ring with their connecting acterized and the 3D structures of only a few have been backbone segments, while the third disulfide bond appears to determined using NMR technology. All structures elucidated penetrate the ring formed by the first two bonds [37]. indicate the peptides are stable and compact and are Furthermore, a cis-Pro motif was found in the structure of associated with several flexible residues, which could be some cyclotides, which formed a twist in the backbone targeted for drug design. Therefore, the cyclotide family of (Fig. 1C). As previously mentioned, this is the distinguishing plant proteins is of interest not only due to their unique feature of Mo¨ bius cyclotides (Fig. 1B) [6,22]. The main example topology and amazing biological properties, but also for their is the three-dimensional structure of kalata B2, which was potential in drug development. An example of this approach solved by NMR [22]. In this protein, several structural elements for exploitation of the cyclotide framework is the work with are stabilized by hydrophobic interactions and hydrogen kalata B1, where polar and/or charged residues were bonds. On the other hand, the absence of this small peculiarity modified and the effects on biological function and structure leads to the classification of the cyclotide in a second studied [4]. subfamily named the bracelet cyclotides (Fig. 1B). Examples of bracelet subfamily members are circulin A [8], the cycloviolacins [28] and cycloviolins [17]. Bracelet cyclotides 5. Concluding remarks and perspectives displaying anti-HIV activity have been found in V. hederacea, L. cymosa and Palicourea condensate [1,17]. One of these peptides, Cyclotides appear to have high sequence similarities, as well named vhl-1, adopts a compact and well defined structure that as a structural identity. This high degree of homology suggests includes a distorted triple-strand b-sheet, a short helical conservation during evolution, possibly due to an important segment and several turns (Fig. 1B). role in plant defense against pests and pathogens. There are Besides bracelets and Mo¨ bius, there is a third group within studies where in vivo assays have been performed that support the cyclotide family, which is composed by proteinase this idea [22]. The mechanisms of action of cyclotides have not inhibitors. These peptides were isolated from M. cochincinensis, yet been elucidated and are still under discussion. The which could not be classified into either Mo¨ bius or bracelet opportunities for studying cyclotides abound, as there are subfamilies. Moreover, another type of cyclotide structure, still many questions that need to be answered. The informa- recently discovered and isolated from O. affinis, was named tion about this family of peptides gathered so far may prove to kalata B8, and appears to be a hybrid between Mo¨ bius and be invaluable in the development of novel antibiotics and bio- bracelet subfamilies, suggesting that these families are insecticides. Biochemical screening will most likely continue evolutionarily related [37]. to play an important role in the search for cyclotides with But what are the differences and similarities of Mo¨bius and desirable characteristics. However, a thorough understanding bracelet subfamilies? Both contain similar disulphide bridge of the structural basis of these peptides and their interaction patterns that are strongly conserved, and this is the main with possible receptor molecules will enable site directed feature used to classify peptides as cyclotides [2]. Some studies mutagenesis of existing cyclotides or the design of synthetic have focused their attention on extremely conserved residues peptides to yield cyclotides with specific activities [4]. Natural in both Mo¨ bius and bracelets subfamilies, such as Glu3 in loop circular proteins found in bacteria, plants, and mammals

1 and Gly17 in loop 3. The Glu3 side chain appears to be show antimicrobial activity and exceptional stability, making involved in extensive hydrogen bonding, while Gly17 is them ideal templates for engineering better drugs [22,25]. thought to be important for flexibility, both contributing to Furthermore, the discovery of different cyclotides will con- the folding scaffold [37]. Furthermore, the glycine residue is tribute to the development of novel antibiotics and anti-viral involved in connecting sites in both families: helix and strands drugs with low side effects, which could be utilized to control in bracelets, and turn and b-sheets in the Mo¨ bius subfamily [2]. infectious diseases. Asn in loop 6 is extremely conserved and seems to be essential for post-translational modification. However, this information must be proved and correlated to post-translational processes Acknowledgements such as cleavage and cyclization in further studies. One important difference between bracelet and Mo¨bius members The authors are thankful for the financial support of is that, while the first subfamily contains 5–8 amino acid Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior residues in loop 6, the second subfamily always has 7. (CAPES), Conselho Nacional de Desenvolvimento Cientı´fico e Therefore, small sequence differences could be observed as Tecnolo´ gico (CNPq), Fundaçaõ de Amparo a Pesquisa do the presence of a short 310 helical segment in bracelet Estado de Minas Gerais (FAPEMIG) and Universidade Catolica subfamily components and a cis-Pro motif in Mo¨ bius sub- de Brasilia (UCB). family cyclotides. While in bracelets, six or seven residues form this loop; in the Mo¨bius subfamily only four residues are present in the same loop [22]. Another feature that may help references distinguish bracelet and Mo¨ bius cyclotides is the number of hydrophobic residues. Hydrophobic residues constitute approximately 57% of a bracelet cyclopeptide molecular [1] Bokesch HR, Pannel LK, Cochran PK, Sowder 2nd RC, McKee surface but only approximately 40% of a Mo¨ bius cyclotide TC, Boyd MR. A novel anti-HIV macrocyclic peptide from surface [2]. Palicourea condensate. J Nat Prod 2001;64:249–50. peptides 28 (2007) 1475–1481 1481

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