Cyclotides from the Indian Medicinal Plant Viola odorata (Banafsha

Article pubs.acs.org/jnp

Cyclotides from the Indian Medicinal Plant Viola odorata (Banafsha): Identification and Characterization M. Narayani, Anju Chadha, and Smita Srivastava* Department of Biotechnology, Bhupat and Jyoti Mehta School of Biosciences, Indian Institute of Technology Madras, Chennai 600036, India S Supporting Information *

ABSTRACT: Cyclotides are cyclic cystine knotted macrocyclic plant peptides that have several promising applications. This study was undertaken to detect and identify known and new cyclotides in Viola odorata, a commercially important medicinal plant, from three geographical locations in India. The number of cyclotides in the plant varied with the tissue (leaves, petioles, flowers, runners, and roots) and with geographical locations in India. Using liquid chromatography coupled to Fourier transform mass spectrometry (FTMS), 166 cyclotide-like masses were observed to display cyclotide-diagnostic mass shifts following reduction, alkylation, and digestion, and 71 of these were positively identified based on automated spectrum matching. Of the remaining 95 putative cyclotides observed, de novo peptide sequencing of three new cyclotides, namely, vodo I1 (1), vodo I2 (2), and vodo I3 (3), was carried out with tandem mass spectrometry.

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several bioactive applications of cyclotides have been reported including antimicrobial, immunosuppressant, antifouling, cytotoxic, anti-HIV, nematocidal, anthelmintic, and hemolytic effects and use of molecular scaffolds.9−17 In India, traditional systems of medicine cater to the primary health care needs of the majority of its population. Viola odorata L. (Violaceae) [commonly known as “Banafsha” in India] is being used commercially owing to its large number of ethnobotanical uses that include the treatment of headaches, coughs, colds, bronchitis, and nervousness.18 Its medicinal applications and supportive literature indicate that one of the possible key principles involved could be cyclotides.9,10,13 However, there are no reports available to date on the discovery and characterization of cyclotides in any plant variety of Indian origin. Cyclotides are believed to be present in plants as natural defense agents, mainly against pests.19 It is interesting to note that the number of cyclotides discovered until now is around 300, a figure expected to increase to over 50 000.8 This is because each plant is likely to express hundreds of different cyclotides catering to its specific defense needs. Therefore, in

yclotides are mini plant proteins comprising a head-to-tail cyclic backbone with 27−38 amino acids.1 They have six conserved cysteine residues that contribute to the formation of three disulfide bonds in a distinct knotted topology. Circular proteins are seen in animals and other microorganisms, but cyclotides (which have a cyclic cystine knot) are unique to plants.2 To date, cyclotides have been reported from five plant families, namely, Violaceae, Rubiaceae, Cucurbitaceae, Fabaceae, and Solanaceae. However, the maximum number of cyclotides discovered to date has been reported from plants belonging to the family Violaceae.3 The first cyclotide, kalata B1, was isolated in the 1970s from an African plant called kalata-kalata (Oldenlandia af f inis), which was used by African women as an indigenous medicine to accelerate the process of childbirth.4 However, the threedimensional structure of kalata B1 was not determined until the mid-1990s.5 Since then, the discovery of many other similar plant proteins with varying amino acid sequences and numbers has led to a classification of plant proteins called “cyclotides”.6 Cyclotides are further divided into three subfamilies as möbius, bracelet, and cyclic trypsin inhibitors.7 Cyclotides have exceptional thermal, chemical, and enzymatic stability, which may lead to promising applications in drug design and agriculture.8 As a result, in the past decade © 2017 American Chemical Society and American Society of Pharmacognosy

Received: November 3, 2016 Published: June 16, 2017 1972

DOI: 10.1021/acs.jnatprod.6b01004 J. Nat. Prod. 2017, 80, 1972−1980

Journal of Natural Products

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before and after S-alkylation with IAA. Each S-alkylated cysteine (half-cystine) residue caused a mass increase of 58.03 Da. Thus, upon reduction and alkylation, most of the peptides (which were presumed to be both previously known and new cyclotides) showed a mass shift of 348.18 Da. This confirmed that these peptides contained three disulfide bonds, suggesting that they could be cyclotides. The disulfide bond connectivity in cyclotides has been reported in earlier work as CysI−CysIV, CysII−CysV, and CysIII−CysVI.5 Compared to the native cyclotides, the reduced and alkylated cyclotides had an early retention time signifying a decrease in the hydrophobicity of cyclotides. Furthermore, the reduced and alkylated cyclotides exhibited poor fragmentation, suggesting the presence of a head-to-tail cyclic backbone.22 In addition to the knotted topology, cyclotides have a circular backbone. Identification of a circular backbone can be determined by the hydrolysis of the peptide bond and then further analyzing the mass shift using mass spectrometry. Most cyclotides contain only one conserved glutamic acid residue present in loop-1.6 Therefore, the reduced and alkylated peptides were further treated with endoproteinase Glu-C, which subsequently resulted in an increased mass shift of 18 Da. This observed mass shift was due to the hydrolysis of the peptide bond (leading to the addition of a water molecule), causing linearization of the cyclotides. The results were in agreement with the previous reports indicating the circular nature of the cyclotides.6,23 Most of the peptides that had cyclotide-like masses demonstrated a gain in mass of 18 Da after treatment with endoproteinase Glu-C, thereby confirming their cyclic structure. However, it should be noted that performing peptide bond hydrolysis only with endoproteinase Glu-C may result in incomplete profiling of cyclotides in V. odorata, as some cyclotides tend to have more than one glutamic acid, such as circulin D, circulin E,24 cter K,25 and cter L.25 Such cyclotides might not be detected in a routine investigation. Hence, to obtain the complete profile of cyclotides in each plant sample, three other endoproteinases, namely, trypsin, Lys-C, and chymotrypsin, were also used independently to linearize any cyclotides that had more than one glutamic acid residue but only one cleavage site. This approach facilitated the identification of the cyclotides with more than one glutamic acid residue. In this study, 95 putative new cyclotides have been detected in the Indian variety of V. odorata (Table S1, Supporting Information). Two or more cyclotides are known to have similar masses; for instance, cycloviolacin O2 and cycloviolacin O9 share a mass of 3138.37 Da, yet have different sequences. In addition, cyclotides can also undergo several post-translation modification stages, resulting in a different mass with the same underlying sequence.3 Such isobaric cyclotides or modified cyclotides can be differentiated only by comparing their sequences. However, the lack of a free N- or C-terminus in the cyclic peptides makes it impossible to employ conventional sequencing approaches such as Edman degradation or the more recent MS-based top-down proteomic approaches. Owing to the complex topology of cyclotides, the fact that they are poorly fragmented by collision-induced dissociation (CID) necessitates certain chemical modifications to make them amenable for MS/MS sequencing. It was also noted in this study that the native cyclotides remained intact when directly subjected to proteolysis, confirming the fact that cyclotides are not readily susceptible to enzymatic treatment. Hence, the cyclotides in the crude extract were reduced, alkylated, and then subjected to any

addition to identification and characterization of different cyclotides in plants, it is important to understand variations in cyclotide profiles produced in response to varying environmental conditions, for targeted extraction and improved yields from native plants and in vitro culture systems. Hence, the Indian variety of V. odorata was selected in this study to identify known and new cyclotides and their variation with plant tissue type and its geographical locations.

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RESULTS AND DISCUSSION Identification and Characterization of Cyclotides. The traditional proteomic approach for the identification of conventional proteins (with free N- and C-termini) relies on a first step of gel electrophoresis; however, this method is not amenable for circular proteins.20 Hence, in this study, highresolution Fourier transform mass spectrometry (FTMS) coupled to liquid chromatography (LC) was used for the identification and characterization of known and new cyclotides present in the Indian variety of V. odorata. Cyclotides were analyzed and confirmed based on five different approaches: (i) the late eluting property of cyclotides by reversed-phase LC; (ii) molecular mass scanning ranging from 2500 to 4000; (iii) the presence of three disulfide bonds; (iv) the presence of a cyclic structure; and (v) the primary sequence of cyclotides with defined number of amino acids in each loop. The profiles of cyclotides present in V. odorata plants cultivated in various geographical locations in India were investigated. The crude extract from the aerial parts (i.e., leaves and petioles) of V. odorata procured from Harwan, Emerald, and Chennai in India was investigated for the presence of cyclotides. Additionally, tissues including the leaves, petioles, flowers, runners, and roots of V. odorata were harvested from the Chennai-grown plants, and the crude extracts were subjected to LC-FTMS analysis. These extracts demonstrated several peaks (of possible peptides) in the reference mass range (2500−4000 Da) of cyclotides. Unlike most of the proteins, these peptides had a late retention time on a C18 reversed-phase LC, specifically eluting between 35% and 65% of acetonitrile in the mobile phase, which is a typical characteristic of cyclotides. This is because the cystine knot of the cyclotides occupies the core, forcing the hydrophobic residues to be exposed at the surface of the protein.8 The experimentally obtained masses of these peptides were compared with the masses of the previously reported cyclotides listed in CyBase,21 and it was observed that the masses of most of the putative cyclotides matched. CyBase is a database that provides a variety of information about the cyclic proteins, including cyclotides.21 Additionally, many unreported peptides falling well within the characteristic mass range of cyclotides were also present in the three varieties, which also exhibited late retention times by reversed-phase LC. Thus, preliminary identification of the putative cyclotides present based on their intact (native) masses resulted in the detection of 845 cyclotide-like masses, out of which 82 were consistent with the known cyclotides. Cyclotides contain six cysteine residues and three conserved disulfide bonds forming a cystine knot motif.8 Hence, the presence of three disulfide bonds in a structure can be used as one of the confirmatory analysis steps to identify cyclotides. Thus, reduction of disulfide bonds and subsequent capping of the free thiol groups (S-alkylation) was done using iodoacetamide (IAA) to prevent oxidation of the thiol groups. The disulfide bonds present in the putative cyclotides in V. odorata were examined by comparing the mass difference 1973



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Figure 1. De novo peptide sequencing of 1 using collision-induced dissociation (CID)-based MS/MS spectra of the precursor ion with m/z (A) 1038.433+ from Lys-C digest; (B) 692.772+ and (C) 874.382+ from endoproteinase Glu-C digests; and (D) 1122.942+ from chymotrypsin digests. The b and y ions are indicated in a blue and red font, respectively. The peptide sequence deduced from each MS/MS data set is indicated in green font in the respective panel, where “X” represents the presence of I/L and C* represents carbamidomethyl-cysteine.

C, whereas digestion with Glu-C resulted in two fragments of monoisotopic mass 1383.52 Da (m/z 692.772+) and 1746.74 Da (m/z 874.382+). This gave a preliminary indication of the presence of one lysine (Lys) residue and two glutamic acid (Glu) residues in the sequence of 1. Further, the full sequence of 1 was deciphered by the CID fragmentation pattern of the precursor ions obtained from both Lys-C (Figure 1A) and GluC (Figure 1B and C) digests. The sequence deciphered contained three residues of either isoleucine (Ile)/leucine (Leu). Since MS/MS sequencing cannot distinguish between

one of the proteolytic treatments (by Lys-C/trypsin/chymotrypsin/Glu-C). These treatments yielded one or more peptide fragments that were subjected to further fragmentation in MS/ MS. De novo sequencing of three new cyclotides was done manually on the resulting fragment ions (termed b-ions and yions) that had mass differences corresponding to the residual masses of the respective amino acid. One of the new cyclotides identified in this study, named vodo I1 (1), yielded one fragment of monoisotopic mass 3112.27 Da (m/z 1038.433+) upon digestion with trypsin/Lys1974



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cyclotides, compounds 1−3 have a glycine residue preceding Cys IV. In all of the cyclotides discovered so far, loops 2, 3, 5, and 6 exhibit extensive variations in their composition, although asparagine (or occasionally aspartic acid) is a conserved residue that is found along with other residues in loop 6. Undoubtedly, 1−3 contain an asparagine residue in loop 6. An asparagine or aspartic residues in loop 6 are presumed to be required for the cyclization of cyclotides via an asparaginyl endopeptidase.8 Although 1−3 have similar compositions of amino acids in loop 4, loop 5, and loop 6, they differ from each other in terms of the composition of some amino acids in the other loops. “Ala” in loop 1 and loop 3 of 1 was replaced by “Leu” and “Pro” in 2 in the respective positions. Similarly, “Ala-Gln” in loop 2 of 1 was replaced by “Ile-Lys” in 2 at the respective position. Cyclotides 2 and 3 differed from each other by only one amino acid; that is, “Leu” in loop 1 of 2 was replaced by “Pro” in 3. Cyclotides 1−3 were classified within the bracelet subfamily based on the absence of a cis-Pro peptide bond in loop 5 in the circular peptide backbone. The bracelet subfamily is known to be more prevalent than the möbius subfamily and hence represents a large suite of cyclotides. In this study, de novo sequencing using a tandem mass spectrometry approach enabled the detection of known and three new cyclotides in the crude extract of V. odorata (a complex mixture of small molecules and other proteins). Such an approach helped in the identification of several new cyclotides, which can otherwise remain undetected during the multiple purification steps routinely involved in the isolation of individual cyclotides owing to their low natural yields. Moreover, in this study the identification of glutamic acid residue in loop 4, which is rare, explains the natural variation in the framework of cyclotides that can be exploited for several protein-engineering applications. The manually deciphered sequences of the cyclotides were verified independently using two different database search engines, PEAKS DB and SEQUEST. These software tools identify proteins using algorithms that compare the experimental MS/MS spectra with the theoretically derived spectra for the peptides contained in a protein sequence database. Although database matches were anticipated for the 82 masses observed via FTMS that were consistent with previously identified cyclotides, only 71 known cyclotides were collectively validated from the LC-MS/MS data using multiple search engines. Of the 71 cyclotides identified by the search engines, 62 (87.32%) cyclotides were identified using both the search engines, whereas two (2.82%) cyclotides were identified exclusively by SEQUEST (Table S2, Supporting Information) and seven (9.86%) cyclotides were identified exclusively by PEAKS DB (Table S3, Supporting Information). In addition to this, both search engines supported the identification of the three new cyclotides, vodo I1, vodo I2, and vodo I3 (1−3), that were determined in this study. The combined use of multiple search engines helped in the identification and confirmation of a larger number of cyclotides than by using a single search engine. The cyclic nature of compounds 1−3 proposed in this study was also confirmed using the CyPred tool. CyPred is a high-throughput sequence-based predictor of cyclic proteins.8 The scores obtained for 1−3 were 0.7, 1.5, and 1.5, respectively. A positive score indicates the cyclic nature of the peptide sequence examined. Variation in Cyclotides with Geographical Location. The aerial parts (i.e., leaves and petioles) of V. odorata procured from Harwan (Srinagar, Jammu, and Kashmir, India) and

Ile and Leu, which are isobaric residues, assignment of Leu was done based on the preferential cleavage of Leu by chymotrypsin. As evident from the sequence drawn for 1, there are five potential cleavage sites for chymotrypsin (as it cleaves amide bonds C-terminal to Leu, Trp, Tyr, and Phe). Treatment with chymotrypsin resulted in a larger detectable fragment (Figure 1D) of monoisotopic mass 2243.86 Da (m/z 1122.942+), indicating the presence of only one Leu residue in the cyclotide sequence, while other fragments were undetectable by MS. Another new cyclotide identified in this study, vodo I2 (2), upon digestion with Glu-C, yielded two fragments of monoisotopic mass 1746.74 Da (m/z 874.382+) and 1493.66 Da (m/z 747.842+). Similarly, trypsin/Lys-C treatment resulted in fragments of monoisotopic mass 1295.60 Da (m/z 648.812+) and 1944.82 Da (m/z 973.422+), indicating the presence of two Glu and two Lys residues in the sequence of 2. The full sequence of 2 was deciphered from the CID fragmentation pattern of the precursor ions obtained from both trypsin and Glu-C (Figure S1, Supporting Information) digests. The chymotrypsin digestion resulted in one detectable fragment of monoisotopic mass 1894.84 Da (m/z 948.432+), indicating the presence of one Leu residue and three Ile residues in this fragment (Figure S1, Supporting Information), while other fragments were undetectable by MS. Further, detection of the fragment (with mass 1894.84 Da) also suggests that “X” (Ile/ Leu) preceding CII should be Leu for the cleavage to occur before CII, explaining the presence of an additional Leu in the sequence of 2. A third new cyclotide, vodo I3 (3), upon treatment with Glu C yielded one fragment of monoisotopic mass 3206.38 Da (m/ z 1069.803+). Similarly, digestion with trypsin/Lys-C yielded two fragments of monoisotopic mass 1279.56 Da (m/z 640.792+) and 1944.82 Da (m/z 973.422+). This gave a preliminary indication of the presence of one Glu and two Lys residues in the cyclotide sequence. However, de novo sequencing revealed the presence of two Glu residues that was evident from the CID fragmentation pattern (Figure S2, Supporting Information) obtained for the fragment with a mass 3206.38 Da. This is because one of the Glu residues preceded a Pro in the sequence. Thus, only one fragment resulted after Glu-C treatment, as no cleavage occurs if a Pro residue is on the carboxylic acid side.26 Similar sequence results were obtained when de novo sequencing of the tryptic digests was done (Figure S2, Supporting Information). The chymotrypsin digestion yielded one detectable fragment of monoisotopic mass 2337.98 Da (m/z 1170.002+), indicating the presence of one Leu residue and three Ile residues in the sequence, while the other fragments were undetectable in MS. The six-cysteine residues that form the framework of the cyclotides were present in the three new cyclotides sequenced in this study. The six backbone segments between successive cysteine residues termed as “loops” (1−6) have the greatest variations in loop size and/or composition.6 All cyclotides reported so far are known to contain only one amino acid in loop 4, which is normally any one of the residues Tyr, Lys, Thr, Ser, Ile, or Ala. However, the newly discovered cyclotides 1−3 were found to contain a Glu in loop 4, as reported earlier for the cyclotide vitri peptide 3.27 Among the cyclotides discovered so far, loop 1 is found to contain three amino acids, including a conserved Glu. The new cyclotides 1−3 were also found to contain three amino acids in loop 1 with a Glu occupying the second position, as mentioned in the literature.6 Like most 1975



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Figure 2. LC-MS chromatograms depicting differences in relative abundance of cyclotides in Viola odorata from three geographical locations in India. Cy: cycloviolacin.

Emerald (Nilgiris district, Tamilnadu, India) were investigated for their cyclotide constituents. Additionally, the Emerald variety that was transplanted and cultivated in Chennai (Tamilnadu, India), referred to as the Chennai variety below, was investigated for its cyclotide constituents. In this study, known cyclotides were identified using cyclotide-diagnostic mass shifts and automated spectrum matching, while putative new cyclotides were observed based on cyclotide-diagnostic mass shifts alone. Interestingly, the cyclotide profiles from different geographical locations in India were found to vary, as evident from the LC data of the crude extracts from three varieties of V. odorata (Figure 2). The data showed that some of the cyclotides found in the aerial parts of the Harwan variety were not present in the aerial parts of the Chennai and the Emerald varieties and vice versa (Table S4, Supporting Information). For example, cycloviolacin O16 and 1−3 were identified in the Harwan variety but not in the other varieties. Similarly, compounds including cycloviolacin O18, cycloviolacin O14, and cycloviolacin O23 were identified in both the Chennai and the Emerald varieties but not in the Harwan variety. It was also observed that among the aerial parts of the three varieties studied the Chennai variety demonstrated the largest suite of cyclotides followed by the Harwan variety and the Emerald variety. Among the cyclotide-like masses detected in the Chennai variety, 13 known and 24 putative new cyclotides were present exclusively in this variety (Figure 3A and B). This substantiates that when the same plant variety is transplanted from one geographical location to another, the production pattern of cyclotides varies. This change in cyclotide production pattern with geographical location can be attributed to the large variations in environmental factors such as the type of soil, altitude, water, temperature, humidity, and sunlight and the related stress conditions.28 It can be hypothesized that the genes for differential cyclotide production remain cryptic, which can get triggered depending upon external stimuli regulated by several abiotic and biotic factors, thereby leading to variation in the cyclotide production pattern with the change in the geographical location.

Figure 3. Venn diagram representing the number of shared and unique cyclotides in Viola odorata at different geographical locations. (A) Known and (B) putative new cyclotides found in this study.

Apart from cyclotides, both the Chennai variety and the Emerald variety of V. odorata had several other uncharacterized secondary metabolites that also had their retention times close to those of the cyclotides. The most prominent (intense) secondary metabolite peaks had m/z values corresponding to 593.19 and 634.20. Interestingly, these compounds were not detected in the V. odorata plant material obtained from Harwan, India. This further substantiated the possible role of environmental conditions on the expression of secondary metabolites (including cyclotides) in plant cells. Tissue-Specific Variation in the Cyclotide Profile. Secondary metabolite biosynthesis and storage in plants is known to be tissue specific; hence the expression of cyclotides in plants can also vary with the type of tissue. To test this hypothesis, crude extracts of different tissues including that of leaves, petioles, flowers, runners, and roots of V. odorata plants maintained in Chennai were subjected to LC-MS analysis (Figure 4) for the detection of cyclotides (known and new) as described in the earlier section. The suite of cyclotides detected varied with the type of plant tissue investigated, and a few of the molecular masses observed in one type of tissue were not observed in another type (Table S5, Supporting Information). Among the different types of plant tissues investigated, the roots accumulated the highest number of known cyclotides (33) and putative new cyclotides (40). Among those detected in the roots, six known cyclotides 1976



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geographical locations. These results were in agreement with previous reports on cyclotides.29−31 Moreover, as evident from Figures 2 and 4, the relative concentration of cyclotides also varied with geographical locations and tissue type. However, it should be noted that a comparative analysis based on complex crude extracts is challenging and nonconclusive due to overlapping peaks in LC and large variations in their relative abundance.32 Although over 300 cyclotides have been identified in plants, the literature suggests that only 39 cyclotides (cycloviolacins O1−O36,6,23 violacin A,33 vodo N,34 and vodo M34) have been identified in V. odorata. The peptidomics workflow (Scheme 1) used in this study resulted in the determination of 71 known cyclotides and 95 putative new cyclotides from V. odorata plants of Indian origin. Among the 71 known cyclotides identified in this study, 23 cyclotides correspond to those reported in V. odorata from other geographical locations, whereas the remaining 48 cyclotides occur in other Viola species or in different plant families. It is noteworthy that this is the first report representing such a large suite of cyclotides from a single plant species (V. odorata). The amino acid sequences of three new cyclotides 1−3 were established in this study via de novo sequencing (using tandem mass spectrometry). This study has expanded knowledge about the occurrence and sequence diversity of cyclotides found in plants. The leads obtained can facilitate the development of a targeted chemical extraction process and in the development of a plant cell based bioprocess, for enhanced production of known high-value cyclotides identified in V. odorata plants of Indian origin.

Figure 4. LC-MS chromatograms depicting differences in relative abundance of cyclotides in various plant parts of V. odorata. Cy: cycloviolacin, k: kalata.

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(Figure 5A) and 10 putative new cyclotides (Figure 5B) were found to be present exclusively. It is presumed that the accumulation of a greater number of cyclotides in the roots is to protect the plant from the invasion of different soil-borne microbes, arthropods, and nematodes. Moreover, extracts from the aerial parts of the plant also demonstrated accumulation of certain small molecules, as evident from high abundant peaks in the chromatograms corresponding to m/z 593.19 and m/z 634.20. These peaks were found to be absent in the extracts obtained from the underground tissues. Therefore, this study has revealed that the number of cyclotides in the plant varied with the type of tissue and its

EXPERIMENTAL SECTION

Plant Material. Whole plants of V. odorata were obtained from the Center of Medicinal Plants Research in Homeopathy (previously the Survey of Medicinal Plants and Collection Unit), Emerald (11°20′41″ N and 76°37′0″ E), Nilgiris district, Tamilnadu, India [courtesy: Dr. Suresh Baburaj, Central Council for Research in Homeopathy (CCRH)] (referred to as the Emerald variety). Whole plants procured were then transplanted in the horticulture unit at Indian Institute of Technology Madras, Chennai (12°59′27″ N and 80°14′2″ E), Tamilnadu, India, and maintained for more than three years for the study (Chennai variety). Another variety of V. odorata plant material for the study was obtained from Harwan (34°10′40″ N and 74°54′0″

Figure 5. Venn diagram representing the distribution of differentially expressed collective and unique cyclotides between each plant parts that have been (A) previously reported and (B) unreported in the leaves (L), petioles (P), flowers (F), runners (Rn), and roots (Rt). 1977



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Scheme 1. Peptidomics Workflow Undertaken in This Study for the Identification of Known and Three New Cyclotides (1−3) Present in Indian Varieties of V. odorata

E), Srinagar, Jammu, and Kashmir, India [courtesy: Dr. Sumit G. Gandhi, Indian Institute of Integrative Medicine (IIIM), Jammu, India] (Harwan variety). Voucher specimens (Chennai variety: 122002 and Emerald variety: 120177) were deposited at the Foundation for Revitalisation of Local Health Traditions (FRLH) herbarium, Bangalore, India. For the Harwan variety, a voucher specimen (23065) was deposited at Janaki Ammal Herbarium (RRLH), IIIM, Jammu, India. Extraction. V. odorata plant material was lyophilized and homogenized using a mortar and pestle. The homogenized plant material (100 mg) was then macerated in 4 mL of 60% ethanol and incubated at 25 °C for 6 h. It was then centrifuged at 9000 rpm for 10 min, and the supernatant was lyophilized. Reduction, Alkylation, and Enzymatic Digestion of Extracts. The crude extract prepared above was resuspended in 500 μL of 100

mM ammonium bicarbonate buffer (pH 8−8.3). Reduction of the disulfide bonds present in the peptides was then carried out by the addition of 75 μL of 100 mM dithiothreitol (Sigma-Aldrich, St. Louis, MO, USA) and further incubation at 60 °C for 1 h. Subsequent alkylation of the thiol groups present in the reduced peptides was carried out by the addition of 75 μL of 200 mM iodoacetamide (Sigma-Aldrich) at 25 °C, and the mixture was incubated in the dark for 45 min. Aliquots (10 μL) of the reduced and alkylated cyclotides in solution were treated separately with the following enzymes: either Endo Glu-C (Promega, Milan, Italy) for 2 h at 37 °C, TPCK-treated trypsin (Sigma-Aldrich) or Lys-C (Promega) for 3 h at 37 °C, or chymotrypsin for 3 h at 25 °C. All samples were desalted using C18 ZipTips (Millipore, Carrigtwohill, Ireland) before MS analysis, as per the manufacturer’s instructions. 1978



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LC-MS Analysis. Crude extracts of native, reduced, alkylated, and digested samples (each comprising two biological replicates) were analyzed in triplicate on an Orbitrap Elite mass spectrometer (Thermo, Bremen, Germany) coupled to an EASY-nLC liquid chromatography unit (Thermo Fisher Scientific, Odense, Denmark) via a Nano electrospray ion source equipped with a stainless steel nanoemitter. Reversed-phase liquid chromatography was performed using a Thermo EASY-nLC with a binary buffer system consisting of water/acetonitrile (95:5) with 0.1% formic acid (solvent A) and water/acetonitrile (5:95) with 0.1% formic acid (solvent B) over a 100 min gradient at a flow rate of 300 nL/min. Samples were automatically loaded from a 96-well microplate autosampler by using an EASY-nLC system at 3 μL/min. The peptides were concentrated on a trapping column (Easy-column, L 2 cm, i.d. 100 μm, 120 Å, C18-A1; Thermo Scientific) and were eluted and directed onto a reversed-phase column (Easy-column, L 10 cm, i.d. 75 μm, 120 Å, C18-A2; Thermo Scientific). The Orbitrap Elite instrument was operated in data-dependent mode, automatically switching between MS and MS/MS. Peptides were detected in the Orbitrap with the FT survey scan set to scan a range from m/z 500 to 2000 for intact mass analysis and m/z 100 to 2000 for endoproteinase digest at a resolution of 120 000. The lock-mass option was used to obtain accurate mass measurements. The six most intense peaks were subject to collision-induced dissociation (CID). Normalized collision energy was set to 30%. Monoisotopic masses of native and chemically modified cyclotides were determined using Protein Deconvolution software (version 1.0; Thermo Fisher Scientific, San Jose, CA, USA) and were verified manually by direct calculation from the peak positions and charge states. For de novo sequencing, the MS/MS spectra were manually examined and sequenced based on the presence of both b and y series of ions (N- and C-terminal fragments). For further validation of cyclotide sequences, MS/MS data files were searched against (i) the “cyclotides” entry in the CyBase database (http://cybase.org.au/) and (ii) manually interpreted sequences of new cyclotides, using two different search engines, namely, “PEAKS DB” in Peaks (version 7; Bioinformatics Solutions Inc., Waterloo, Ontario, Canada) and “SEQUEST” in Proteome Discoverer software (version 1.4.0.228; Thermo Fisher Scientific). The following parameters were used for searching on the databases: 15 ppm precursor tolerance; 0.5 Da fragment tolerance; static modification: cysteine carbamidomethylation; dynamic modifications: methionine oxidation. A standalone application of CyPred (http://biomine.cs.vcu.edu/servers/CyPred/) was used for sequence-based prediction of the cyclic proteins.8

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HPLC facility at IIT Madras, India. The authors thank Prof. P. Balaram (Indian Institute of Science, Bangalore, India) for his valuable suggestions in de novo sequencing of cyclotides.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b01004. Additional Information (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Tel: 91-44-2257 4127. Fax: 91-44-2257 4102. E-mail: smita@ iitm.ac.in. ORCID

Smita Srivastava: 0000-0001-9582-3904 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Financial support from the Department of Biotechnology, Ministry of Science and Technology, Government of India, is gratefully acknowledged by the authors (project sanction order no. BT/PR6829/GBD/27/489/2012). The authors thank Dr. N. Madhavan for providing access to the semipreparative 1979



Article

(34) Svangard, E.; Goransson, U.; Smith, D.; Verma, C.; Backlund, A.; Bohlin, L.; Claeson, P. Phytochemistry 2003, 64, 135−142.

1980


Cyclotides from the Indian Medicinal Plant Viola odorata (Banafsha

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