Understanding the Diversity and Distribution of Cyclotides from Plants

Article pubs.acs.org/jnp

Understanding the Diversity and Distribution of Cyclotides from Plants of Varied Genetic Origin Anjaneya S. Ravipati,⊥,† Aaron G. Poth,⊥,† Sónia Troeira Henriques,† Murari Bhandari,† Yen-Hua Huang,† Jaime Nino,‡ Michelle L. Colgrave,§ and David J. Craik*,† †

Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Queensland Australia Universidad Tecnológica de Pereira, Cra 27 No 10-02-Los Á lamos, Pereira, Risaralda, Colombia § Commonwealth Scientific and Industrial Research Organization, Agriculture and Food, St Lucia 4067, Queensland, Australia ‡

S Supporting Information *

ABSTRACT: Cyclotides are a large family of naturally occurring plant-derived macrocyclic cystine-knot peptides, with more than 400 having been identified in species from the Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae families. Nevertheless, their specialized distribution within the plant kingdom remains poorly understood. In this study, the diversity of cyclotides was explored through the screening of 197 plants belonging to 43 different families. In total, 28 cyclotides were sequenced from 15 plant species, one of which belonged to the Rubiaceae and 14 to the Violaceae. Every Violaceae species screened contained cyclotides, but they were only sparsely represented in Rubiaceae and nonexistent in other families. The study thus supports the hypothesis that cyclotides are ubiquitous in the Violaceae, and it adds to the list of plants found to express kalata S and cycloviolacin O12. Finally, previous studies suggested the existence of cyclotide isoforms with either an Asn or an Asp at the C-terminal processing site of the cyclotide domain within the precursor proteins. Here we found that despite the discovery of a few cyclotides genuinely containing an Asp in loop 6 as evidenced by gene sequencing, deamidation of Asn during enzymatic digestion resulted in the artifactual presence of Asp isoforms. This result is consistent with studies suggesting that peptides can undergo deamidation after being subjected to external factors, including pH, temperature, and enzymatic digestion.

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reported.9 Chemical analysis of the decoction led to the discovery of a heat-stable uterotonic polypeptide, which was named kalata B1.10 The structure of kalata B1 remained unknown until 1995, when it was determined by NMR spectroscopy,2 revealing the CCK motif. Discoveries of other similar macrocyclic peptides in the 1990s led to the definition of the cyclotide family. More than 400 cyclotides11 have now been discovered from species of the Rubiaceae,12 Violaceae,13 Cucurbitaceae,14 Fabaceae,15 and Solanaceae plant families,16 and they are reported in CyBase, a database (www.cybase.org. au) dedicated to cyclic peptides.17,18 The biological activities of cyclotides have been explored. For example, early screening for anti-HIV agents led to the discovery of circulins A and B from Chassalia parvifolia19 and similar studies led to the discovery of several other biological

yclotides are plant-derived disulfide-rich cyclic peptides, typically 28−37 amino acids in size, which feature a cyclic cystine knot (CCK) formed by six cysteine residues and a headto-tail cyclic backbone.1 Their three-dimensional structure is represented in Figure 1 by the prototypic cyclotide, kalata B1.2 Recent cyclotide research has focused on the use of these peptides in drug discovery applications.3 Their potential for such applications stems from their extraordinary stability against chemical denaturants, high temperatures, or proteases,4 as well as their natural sequence diversity, biological activities, and amenability to peptide engineering via sequence substitutions.5 The first cyclotide was discovered in the early 1970s by Norwegian physician Lorents Gran who noticed pregnant Congolese women drinking a tea, called “kalata−kalata”, prepared from the leaves of Oldenlandia affinis (Rubiaceae family).7 A pharmacological study revealed the O. affinis extract to have uterotonic activity on human uterine muscle and rat uterus,8 and a molecular basis for this activity was recently © 2017 American Chemical Society and American Society of Pharmacognosy

Received: January 20, 2017 Published: May 4, 2017 1522

DOI: 10.1021/acs.jnatprod.7b00061 J. Nat. Prod. 2017, 80, 1522−1530

Journal of Natural Products

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or bracelet cyclotides because of the presence of a greater number of positively charged residues and a lower proportion of hydrophobic residues.38 Until recently, few naturally occurring acyclic derivatives of cyclotides had been found in plants. Acyclic permutants of cyclotides have been chemically synthesized,39 and it is generally observed that linearization of the peptide backbone reduces or eliminates the biological activity of the native cyclotide.40,41 However, recently a number of naturally occurring “acyclotides” have been reported in plants.16,42−44 Mostly these are the result of deleterious mutations at Asn or Asp residues pivotal in their biosynthetic processing mechanism, raising questions about what the evolutionary advantages of cyclization might be. Thus, there is ongoing interest in both discovering new cyclotide sequences as well as understanding the distribution of known cyclotides sequences to better understand their biosynthesis, evolution and range among plant species, and potential for agronomic and therapeutic applications.11,45−55 Recently, combined proteomic and transcriptomic analytical approaches have been applied in largescale chemical screens for cyclotides to expand knowledge of their occurrence, sequence diversity, and phylogenetics. Koehbach et al.51 investigated more than 296 species from 43 plant families with a special emphasis on Rubiaceae (100 species), finding evidence of cyclotides in only nine species, supporting their sporadic expression in this family. A recent investigation of over 140 species selected from the majority of Violaceous genera highlighted the ubiquity of cyclotides therein and also the widespread representation of selected cyclotides among them, kalata S (Varv A) being present in 70% of the analyzed species.53 Analyses of Viola species collected from regions in all continents sampled including Asia, South-East Asia, Europe, South America, Africa, and Australia have also supported the ubiquity of cyclotides among Violaceae.56 Thus, to accelerate discoveries on the origins and evolution of cyclotides, it is of particular interest to focus on the distribution of cyclotides from the “sporadic” cyclotide-containing families, while continuing to capture data on Violaceous species endemic to isolated geographies. In the current study, 197 plants, including some utilized in Traditional Chinese Medicine (TCM) were subjected to highthroughput screening using a combination of liquid chromatography−mass spectrometry (LC-MS) and matrix-assisted laser desorption ionization (MALDI) techniques (Table S1, Supporting Information). The screen included both wild-type species and hybrid species, including 15 Viola cultivars originating from the Violaceae family, and we report the discovery of 24 novel cyclotides from among one of 16 Rubiaceous species investigated, and 14 Violaceous species.

Figure 1. Sequence and three-dimensional structure of the prototypic cyclotide kalata B1. (A) The amino acid sequence of kalata B1 showing the six Cys residues (labeled I−VI), the disulfide connectivity, and the cyclic backbone. (B) Three-dimensional structure of kalata B1 (Protein Data Bank ID code: 1NB1).6 The segments between two adjacent Cys residues are termed loops and are numbered 1−6. The circular backbone is formed by a peptide bond in loop 6 between Gly1 and Asn-29. Potential protease cleavage sites of reduced cyclotides, which are used in sequencing studies, are indicated by arrows.

activities, including antimicrobial activities,20 cancer cell cytotoxicity,21 immunosuppressive22−24 and hemolytic activities.25 Aside from having a broad spectrum of biological activities of pharmaceutical relevance, the natural function of cyclotides is thought to be as plant defense agents.26−28 The first study to suggest such a role was published in 2001, whereby Helicoverpa punctigera larvae fed with an artificial diet containing kalata B1 showed impaired development and increased mortality rate.27 Since that original study, insecticidal activity against Helicoverpa spp. has been observed for a range of other cyclotides and against other larval insects,15,26,27,29 as well as against mollusks30 and nematodes,31 with their mode of action involving membrane disruption.32 Cyclotides were originally classified as being either Möbius or bracelet,1 based on the presence or absence, respectively, of a conserved cis-Pro residue in loop 5.33 In addition to this topological difference, the subfamilies are further distinguished by variations in loop size and sequence, as well as global net charge. Bracelet subfamily members typically have a higher net positive charge due to the presence of Lys and/or Arg residues in loops 5 and 6,18,34 whereas many Möbius cyclotides are neutral and contain few charged residues.34 For example, cycloviolacin O2, the prototypic bracelet, has a global charge of +2, and its hydrophobic residues are located mainly in loops 2 and 3. On the other hand, kalata B1, the prototypic Möbius, has a neutral global charge and its hydrophobic residues are located mainly in loops 2, 5, and 6.35,36 Trypsin inhibitors are a third subfamily of cyclotides and were first identified in squash plants.14 MCoTI-I and MCoTI-II are examples of cyclic trypsin inhibitors isolated from the seeds of Momordica cochinchinensis, a tropical vine belonging to the Cucurbitaceae family.14 Although members of this subfamily share little sequence homology with Möbius and bracelet subfamilies,37 their CCK motif and similar biosynthetic mechanism justify their inclusion in the cyclotide family. Trypsin inhibitors are typically more hydrophilic than Möbius

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RESULTS AND DISCUSSION Identification, Isolation, Purification, and de Novo Sequencing of Cyclotides. Dried or fresh plant material from selected plant species was subjected to small-scale extraction and analyzed using a combination of LC-MS and MALDI-TOF MS techniques. The extracts contained molecules with masses ranging from 1000−3500 Da. The characteristic features of cyclotides, including masses in the range 2500− 3500 Da, and late elution under standard HPLC conditions enabled the distinction of these peptides from other chemical constituents. Plants with cyclotide-like components (Figure 2) were subjected to large-scale extraction. Extracted peptides were then purified for de novo sequence characterization. 1523



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Figure 2. Plant species screened for the presence of cyclotides. In total, 197 plants (Table S1, Supporting Information), belonging to 43 plant families, were screened for the presence of cyclotides using a high-throughput method comprising LC-MS, LC, and MALDI-TOF MS. Fifteen plant species, belonging to the Violaceae (14 species) and Rubiaceae (one species) were found to contain cyclotides. For some Violaceae species, multiple varieties were examined, leading to a total of 27 cyclotide-positive plants.

triply charged ion (Figure 3B; m/z 1102.23+), which was selected as the precursor ion for MS/MS fragmentation (Figure 3C). On the basis of the b- and y-ion series (b2−b16 and y2− y16) annotated in the MS/MS spectrum, the complete sequence of mden A was determined to be TCTLGTCNTPGCTCSWPICTKNGIPTCGE (Figure 3C). One of the challenges involved in MS/MS is differentiating isobaric residues, including Gln/Lys and Ile/Leu, but this challenge can be overcome by analyzing data obtained following cleavage using different proteases (trypsin and chymotrypsin) and by acetylation of specific residues. For example, the full-length sequence data for mden A indicated the presence of one Gln/Lys. Acetylation of the native mden A resulted in a mass increase of 42 Da, suggesting the presence of

The absence of a free N-terminus and the presence of the CCK motif complicate the de novo sequencing of cyclotides, but full sequences can be delineated using an appropriate enzyme cleavage protocol. Here we illustrate the de novo sequencing of cyclotides, using data from the newly discovered cyclotide, mden A, reported here as shown in Figure 3. The native mden A (m/z 2937.3) was reduced with dithiothreitol to remove the disulfide bonds, followed by carboxyamidomethylation of free thiol groups with iodoacetamide. A mass increase of 348 Da (m/z 3285.2) was observed, which is equivalent to the alkylation of six cysteines (Figure 3A). The reduced and alkylated mden A was treated with endoproteinase GluC (endoGlu-C) to linearize the cyclic backbone, which resulted in a further mass increase of 18 Da (m/z 3303.3) (Figure 3A). The linear product was subjected to ESI-MS and observed as a 1524



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Figure 3. De novo sequencing of deamidated mden A. (A) MALDI-TOF MS revealed a peak at m/z 2937.3. After reduction and alkylation, a mass increase of 348 Da was noted (m/z 3285.2). EndoGlu-C digestion resulted in a further mass increase of 18 Da (m/z 3303.3); however, the linearized peptide was observed to undergo extensive deamidation (of Asn in loop 6, Figure 4) and this was the major product after enzymatic digestion (m/z 3304.3). (B) Nanoelectrospray ionization (nESI) TOF MS showing the [M + 3H]3+ precursor m/z 1102.23+ (3304.6 Da) representing the deamidated peptide. (C) NanoESI product ion MS, revealing the annotation of the b- and y-ion series, which allows for the deduction of the complete sequence, as described previously.57

a single Lys, a finding that was further confirmed by tryptic digestion, which resulted in cleavage at the Lys in loop 6. In another example, the de novo sequencing of ltri A suggested the presence of two Gln or Lys residues, or a combination of both. Acetylation of native ltri A (m/z 2883.2) resulted in an increase in mass of 42 Da (m/z 2925.0), revealing the presence of a single Lys, which was confirmed by tryptic digestion. Furthermore, amino acid analysis (AAA) provided independent evidence for the presence of one Lys and one Gln. Using this strategy, 28 cyclotides were characterized, and their full-length sequences are shown in Table 1. On the basis of the availability of pure peptides, amino acid analysis of 14 cyclotides was conducted and the data are presented in Tables S2−S15, Supporting Information. A detailed list of

additional experiments done to deduce the de novo sequence of the cyclotides is given in Table S16, Supporting Information. Sequence Diversity of Novel Cyclotides. Among known cyclotides, loop 1 is the least diverse in terms of size and sequence,53 and one of the highly characteristic features of cyclotides is the presence of a conserved Glu in the second position of this loop. This feature is often exploited in cyclotide sequencing efforts by opening the peptide backbone at a single site, yielding a full-length linear peptide via enzymatic digestion with endoGlu-C. Positions 1 and 3 in loop 1 of the newly discovered cyclotides shown in Table 1 were found to contain Gly/Ala and Ser/Thr, respectively, as is frequently encountered in known cyclotides. Loops 2 and 3 of the new cyclotides differ in size and sequence in bracelet cyclotides compared to Möbius cyclotides. 1525



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Table 1. Sequences of Cyclotides Identified in this Study

a

Mass (Da) = theoretical mass. bM = Möbius subfamily, B = bracelet subfamily. cA list of 15 Viola cultivars (175−180, 184, 187−194) is provided in Table S1, Supporting Information. dKnown peptides. eThis sequence was confirmed on the basis of data obtained from digestion with endoproteinase Glu-C, trypsin, and chymotrypsin. Furthermore, the isobaric residues I/L were unambiguously identified based on AAA results. fThis sequence was confirmed on the basis of data obtained from digestion with endoproteinase Glu-C, trypsin, and chymotrypsin. Furthermore, the isobaric residues I/L were determined by homology with known sequences. f*Has been considered as Lys-rich on the basis of sequence similarities.

For instance, loop 2 of the bracelet cyclotides possess the sequence motif VXI [/L] P (X = F/Y/W), whereas the Möbius cyclotides showed a higher incidence of lysine incorporation, with the sequences AKGK, FKGK, FFGK, TLGT, VGGT, and FTGK (Table 1). Loop 3 of Möbius subfamily members is typically highly conserved in size, comprising four residues. Bracelet subfamily members, on the other hand, show greater diversity in sequence and size (four to six residues) in this loop (Table 1). Both subfamilies possess a highly conserved Gly residue in the last position of loop 3, except mden F, mden H, and mobo B, which all contain a Lys at this position. Loop 4, the smallest of the loops and containing only a single residue, is occupied by Ser, Thr, or Ala in the new cyclotides, residues which are all commonly observed in known cyclotides (www. cybase.org.au).

Loop 5 is pivotal in the classification of cyclotides into the bracelet and Möbius subfamilies, as it is this loop that can contain the twist-inducing cis-Pro, typically as the penultimate residue. Of the 28 cyclotides reported in the current study, 16 can be identified as bracelet cyclotides and 12 as Möbius cyclotides. The bracelet and Möbius subfamily members were classified without ambiguity, with the exception of ltri A. In contrast to the other cyclotides identified in this study, ltri A has TSSQ in loop 5, which is not typical of either bracelet or Möbius cyclotides. Nevertheless, loop 2 contains the VYLP motif, which is highly conserved in bracelet family members, suggesting its inclusion in bracelet subfamily. Loop 6 of the new cyclotides invariably contains a highly conserved Asn or Asp residue, which has been demonstrated to be the key recognition site for asparaginyl endoproteinase enzymes that mediate the cyclization step in cyclotide 1526



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biosynthesis. Additionally, this loop in the new cyclotides generally contains one or more positively charged residues. Given the recent identification of a Lys-rich group of cyclotides in other Melicytus species,58 it is not surprising to observe cyclotides with such charged residues in M. angustifolius, M. dentatus, and M. obovatus. These positively charged residues are typically adjacent to the conserved Asn/Asp in loop 6, as illustrated by the sequences FKNGVA (ltri A), KKNGLPI, KKNGKPI (mden B and mden C), TKNGIPT, TRNGFPT (mden A and mobo A), YKNGL(/I)PI (mden F), YKNGKPI (mobo B), TRNGIPV (vinc A), and AKNGSIPA (vinc B). Overall, the main message to emerge from the sequencing studies is that every species of the Violaceae family screened to date has been shown to contain cyclotides, and new sequences continue to emerge in the various loops. Accordingly, the Violaceae family continues to be a rich source of diverse cyclotides.24,59 Deamidation of Cyclotides. Deamidation occurs frequently in proteins via the conversion of certain amino acids (i.e., Asn, Gln) to acidic amino acids (i.e., Asp, Glu) through liberation of ammonia.60 In particular, it has been found that plant proteins can undergo deamidation in response to heat, acidic/basic conditions, or enzymatic hydrolysis.60 Because cyclotides have a conserved Asn/Asp in loop 6 and are subjected to pH (∼8), moderate temperatures (37 °C), and enzymatic treatments in the process of de novo sequencing, they are potentially susceptible to deamidation. Indeed, here we observed that Asn residues undergo rapid deamidation in the buffers used for proteolysis (Figure 4). This phenomenon is observed in other proteins, particularly when Asn precedes a Gly residue,61,62 as is indeed the case for cyclotides. However, when both Asp and Asn isoforms of a cyclotide are detected in an extracted mixture, how can artifactual deamidation versus the natural presence of both forms be discriminated? Although the pH and heating steps above are a prerequisite in sample preparation for primary sequence determination, elucidating the native isoform of an Asx residue can be achieved through analysis of the peptide masses present in fresh extracts prepared at low pH and low temperature.63 Using this method, Poth et al.15 recently reported evidence for the natural (i.e., genetically encoded) occurrence of both the Asn and Asp isoforms of certain cyclotides. In the present study, MS/MS fragmentation of several cyclotide ions produced atypical isotopic distributions for both the precursor and selected fragment ions, suggesting the presence of Asp and Asn isoform mixtures. For example, the theoretical mass of mden A corresponds to 3302.3 Da (detected as m/z 3303.3, [M + H]+) after reduction, alkylation and linearization. As illustrated in Figure 4, the isotopic distribution for the linear product’s signal is skewed with detection of a peptide 1 Da higher than the expected theoretical mass, suggesting the presence of a mixture of Asn and Asp isoforms. Furthermore, the isotopic distribution of the b21 and y7 fragment ions are as expected, whereas the subsequent ions display altered isotopic distributions corresponding to a 1 Da mass shift, indicating deamidation of the Asn in loop 6 (TCTLGTCNTPGCTCSWPICTKD). Similarly, the y8 ion at m/z 847.3 corresponds to NGIPTCGE, whereas m/z 848.3 corresponds to the same fragment with an Asp substitution at the N-terminus. Taken together, these data clearly demonstrate that rapid artifactual deamidation occurs in cyclotides, highlighting the importance of careful sample collection procedures. Whether this modification can occur in planta, (e.g., in hot climates) and

Figure 4. Deamidation of Asn during enzyme digestion. (A) MALDITOF MS of native mden A (m/z 2937.3; left panel), and after reduction, alkylation and endoGlu-C digestion (right panel). A small peak is detected at the expected m/z 3303.3 for the monoisotopic [M + H]+ ion, but the deamidated form dominates the spectrum at m/z 3304.3. (B) Regions of product ion mass spectra showing the b-ion series: b21 at m/z 1229.02+, b22 at m/z 1286.02+, and b23 at m/z 1314.52+. (C) Regions of the product ion mass spectrum showing the y-ion series: y7 at m/z 733.3, y8 at m/z 847.3, and y9 at m/z 975.4. The isotopic distributions of the b21 and y7 ions are as expected. The successive ions, including b22, b23, y8, and y9, reveal the presence of a peptide with a mass increase of 1 Da, corresponding to deamidation of Asn in loop 6.

its functional significance on any bioactivity has yet to be determined. Cyclotides Are a Naturally Occurring Diverse Class of Peptides. To date, ∼400 cyclotides have been discovered, with many being species-specific.11 Plants produce a multitude of cyclotides, only a few of which are so far known to be expressed in multiple plant species or phylogenetically distant families. In the current study, all new peptides were unique to a species, except that mden A was found in M. dentatus and M. obovatus, which belong to the same genus (Table 1), and cycloviolacin O12 and kalata S were expressed in all Viola cultivars examined. Combined with data in previous studies, these latter two peptides are the most extreme examples of “multiply hosted” cyclotides seen so far. Kalata B1 was initially discovered in O. af f inis (Rubiaceae), but later studies showed it to be present in half of the Violaceae species examined, and a recent analysis of Violaceae plants revealed that kalata S (Varv A) is produced in 70% of surveyed species.64 Thus, the current work confirms both the pervasiveness of a few cyclotides in many species of 1527



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removed by filtering the suspension first through cotton wool/filter paper and then transferred to a separating funnel. Milli-Q water was added until a clear separation between CH2Cl2 and aqueous methanol layers was visible. The methanol layer was collected, and any remaining methanol removed using a rotary evaporator followed by lyophilization. All lyophilized extracts were stored at −20 °C until required for further analysis. LC-MS Analysis of Cyclotides. The identification of cyclotides using LC-MS was carried out as described previously.15 The dry extracts were resuspended in 10% acetonitrile containing 1% (v/v) trifluoroacetic acid, before being separated on a Phenomenex C18 RPHPLC column (150 × 2.00 mm, 3 μm, 300 Å) with a linear 1% min−1 acetonitrile gradient (0 to 80% solvent B which consists of 90% acetonitrile, 0.5% formic acid) at a flow rate of 0.3 mL min−1 on Shimadzu LC-MS 2020. The eluents were monitored on a dual wavelength UV detector set to 214 and 280 nm. The mass spectra of all LC samples were obtained in positive ion mode with a mass range of m/z 400−2000 Da.15 MALDI-TOF Analysis. Molecular weights of all cyclotides characterized in this study were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on an Applied Biosystems 4700 TOF-TOF Proteomics Analyzer. The peptide samples were reconstituted in 50% acetonitrile and diluted at a 1:1 ratio with CHCA (α-cyano-4-hydroxycinnamic acid) matrix prepared at 5−6 mg mL−1 in 50% (v/v) acetonitrile and 2% (v/ v) formic acid. Peptide matrix mixture (∼1 μL) was spotted onto a stainless steel target and dried under a N2 stream or left to air-dry at room temperature. The MALDI spectra of samples were acquired in reflector positive mode with source voltage of 20 kV, mass range of 1000−4000 Da, focus mass of 3000 Da, laser shots of 1000−2000, and laser intensity of 2500−3500. The acquired spectra were analyzed using Data Explorer 4.1 software. Purification of Cyclotides. Dried solvent extracts were resuspended in approximately 20% (v/v) acetonitrile containing 2% (v/v) formic acid and separated on a Phenomenex C18 RP-HPLC column (250 × 21 mm, 10 μm, 300 Å) with a flow rate of 8 mL min−1 and a linear 1% min−1 acetonitrile gradient. Eluents were collected into individual 10 mL test tubes at 1 min intervals for between 20 and 83 min. All fractions were analyzed for cyclotide-like masses on the 4700 MALDI-TOF MS. The fractions containing cyclotide-like masses were pooled and lyophilized. The lyophilized peptide samples were resuspended in 20% (v/v) acetonitrile containing 2% (v/v) formic acid, and they were separated on a Phenomenex C18 RP-HPLC column (250 × 10 mm, 5 μm, 300 Å) with a flow rate of 3 mL min−1. The pure peptides were freeze-dried and stored at −20 °C until needed for future studies. Reduction, Alkylation and Enzymatic Digestion. The reduction and alkylation of pure cyclotides was carried out as described previously.15 Approximately ∼50 μg of lyophilized pure dry peptide was reconstituted with 40 μL of 100 mM NH4HCO3 before being vortexed. The disulfide bonds of native cyclotides were reduced by the addition of 5 μL of 0.1 M DTT prepared in deionized water and incubated at 37 °C for 1 h. The free thiol groups were alkylated by the addition of 5 μL of 0.1 M iodoacetamide prepared in deionized water and incubated at room temperature for 30 min. Desalting of reduced and alkylated cyclotides was carried out using C18 Ziptips (Millipore, Sydney, Australia). The reduced and alkylated cyclotides were analyzed by MALDI-TOF MS. De Novo Sequencing of Cyclotides. De novo sequencing of cyclotides was undertaken as described in previous studies.15 The reduced and alkylated cyclotides were digested using the proteolytic enzymes endoGlu-C, trypsin, and chymotrypsin, or combinations of these enzymes. Each individual digest was performed by the addition of 1 μL of 500 ng μL−1 of enzyme to 10 μL aliquots of reduced and alkylated cyclotide, followed by incubation at 37 °C for 3−4 h. The digested peptide samples were desalted using Ziptips (Millipore) followed by MALDI-TOF MS analysis. The reduced and alkylated samples were subjected to MS/MS analysis on a QStar Pulsar mass spectrometer (AB SCIEX). Approximately 5−7 μL of the desalted peptide, which was

the Violaceae, and the single species distribution of the majority of individual cyclotides. A complete understanding of origin and distribution of cyclotides remains elusive but some understanding of their evolution is emerging. Plants that have been found to express cyclotides have no discernible all-encompassing phylogenetic link, suggesting that cyclotides may have evolved independently in distant families. One plausible explanation for the distribution of cyclotides among plants is convergent evolution.12 The structural similarity of knotted proteins among animals and plants is also supportive of this suggestion.65 Many animals, plants, bacteria, fungi and viruses express linear precursors of cyclic peptides containing the cystine knot motif, where they play crucial roles in defense mechanisms.65 The occurrence of functional, linear cystineknotted peptides among living organisms suggests backbone cyclization occurred after the evolutionary formation of the cystine knot motif. Conclusions. The current study supports the hypothesis that cyclotides are ubiquitous in the Violaceae family and expands our knowledge of their distribution and sequence diversity. The combination of analytical techniques used in this study enabled the rapid characterization of 24 novel cyclotide sequences from 197 screened plants across 43 plant families. Tellingly, cyclotides were found only in the Violaceae and Rubiaceae, suggesting that their distribution is highly localized in the plant kingdom, so far found only in a total of five families, with the Violaceae having particular prominence. Previous studies reported the existence of Asn and Asp isoforms of cyclotides as a natural phenomenon in plants. In this study, many of the cyclotides were indeed detected as mixtures of both Asn and Asp forms after enzymatic digestion. However, careful scrutiny of the MS data revealed that these mixtures were produced by deamidation of Asn during sample preparation. Thus, detection of cyclotide isoforms at the peptide level in future studies should where possible be supplemented with genetic studies or with careful consideration of sample workup procedures. Finally, the widespread the production of metabolically “expensive” molecules such as kalata S, kB1, and cycloviolacin O12 suggests they are important to the plants producing them, most likely for defense, serving to link the species producing them evolutionarily, and may support future refinements to Violaceae taxonomy.

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EXPERIMENTAL SECTION

General Experimental Procedures. The plant species screened in this study were obtained from field trips, through collaboration with academic institutions, from private botanical gardens, and from private companies. The complete list of plant species and their place of collection are provided in Table S1, Supporting Information. Small-Scale Plant Crude Extraction. Approximately 1−2 g of dry or fresh plant material was ground to powder in the presence of liquid N2 and extracted overnight with 5−10 mL of 50% acetonitrile (v/v) containing 2% formic acid (v/v). The extracts were lyophilized to evaporate solvent, before being stored at −20 °C until required for further analysis. The samples were resuspended in ∼100 μL of 10% acetonitrile (v/v) and analyzed for the presence of cyclotides using a combination of LC-MS and MALDI-TOF MS techniques. Large-Scale Plant Crude Extraction. Plants identified to contain cyclotides in the small-scale screen were later extracted on a large scale, using methods described previously.15 Dry or fresh plant material was ground to powder using a mortar and pestle in the presence of liquid N2 and extracted using dichloromethane-methanol (1:1, (v/v)) overnight at room temperature. Debris from the extraction was 1528



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reconstituted in 80% acetonitrile and 2% formic acid, was loaded into Proxeon ES380 nanospray tips. TOF MS and MS/MS spectra were acquired by applying a capillary voltage of 800−1000 V, with collision energy varied from 10−50 V. TOF mass spectra were acquired over the mass range m/z 500−2000 (MS) and m/z 100−2000 (MS/MS). The data were processed using Analyst QS 1.1 software, and product ion spectra were annotated for the b- and y-ion series. Quantification of Lysines Using Acetylation. The number of Lys residues in cyclotides were determined after modification by acetylation. The acetylation reagent was freshly prepared using 20 μL of acetic anhydride and 60 μL of methanol. Approximately 15−20 μg of dry peptide was reconstituted in 20 μL of 50 mM ammonium bicarbonate. The acetylation reagent (50 μL) was added to 20 μL of peptide solution and incubated at room temperature for 1 h, followed by lyophilization to remove organic content. The samples were desalted using Ziptips, and the acetylation products were analyzed using MALDI-TOF MS on a 4700 Proteomics Analyzer. Amino Acid Analysis. Duplicate samples of purified, lyophilized cyclotide samples were subject to gas-phase hydrolysis with 6 M HCl at 110 °C. Following this, the amino acid content present in each digest was determined via Waters AccQTag Ultra chemistry on a Waters Acquity HPLC.

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(DE120103152). The authors thank J. Allen, G. Bourke, S. Goodwin, and P. Symes for the provision of plant material from the Royal Botanical Gardens, Sydney and Melbourne, Australia; Y.-S. Liang and J-C. Wang for plant material from Taiwan. We also thank A. Jones for his assistance and access to the Molecular and Cellular Proteomics Mass Spectrometry facility at The University of Queensland. We thank A. Cooper for editorial assistance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00061. (PDF) (XLSX) Accession Codes

Cyclotide sequences reported herein can be accessed on the UniProt Knowledgebase under the following accessions: Ltri A, C0HKI2., Mang A, C0HKI3., Mden A, C0HKI4., Mden B, C0HKI5., Mden C, C0HKI6., Mden E, C0HKI7., Mden F, C0HKI8., Mden G, C0HKI9., Mden H, C0HKJ0., Mden I, C0HKJ1., Mden J, C0HKJ2., Mden K, C0HKJ3., Mden L, C0HKJ4., Mden M, C0HKJ5., Mden N, C0HKJ6., Mobo A, C0HKJ7., Mobo B, C0HKJ8., Pali A, C0HKJ9., Vdif A, C0HKK0., Vinc A, C0HKK1., Vinc B, C0HKK2., Vpub A, C0HKK3., Vpub B, C0HKK4., Vpub C, C0HKK5. Species found not to contain cyclotides appear on the “List of plant species that may not express cyclotides” on Cybase (http:// www.cybase.org.au/?page=no_cyclotide_plants).

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +61-7-3346-2019. Fax: +61-7-334-2101. ORCID

Sónia Troeira Henriques: 0000-0001-9564-9651 Michelle L. Colgrave: 0000-0001-8463-805X David J. Craik: 0000-0003-0007-6796 Author Contributions ⊥

(A.S.R. and A.G.P.) These authors contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported by the Australian Research Council (ARC; DP150100443). D.C. is funded by an ARC Australian Laureate Fellowship (FL150100146). S.T.H. was supported by an ARC Discovery Early Career Research Award 1529



Article

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