A Cyclotide Isolated from Noisettia orchidiflora (Violaceae)^[*]

• Abstract
• Introduction
• Results and Discussion
• Materials and Methods
• General procedures
• Plant material
• Extraction and isolation of cyclotides
• Reduction, alkylation, and enzymatic digestion of cyclotides
• De novo sequencing of cyclotides
• LC-MS analysis
• Amino acid analysis
• Sequence alignment
• References

Abstract

Biologically active cyclotides have been found on some flowering plants species and are involved in the role of the plant protection. As part of studies focusing on peptides from Brazilian plant species, we are reporting the detection by LC-MS of several cyclotides from leaves and stems of Noisettia orchidiflora (Violaceae). From stems it was possible to isolate and characterize a cyclotide named Nor A. Its primary structure (amino acid sequence) was established by MALDI-TOF-MS, based on the y- and b-type ion series, after reduction and alkylation reactions, as well as enzymatic digestion using the enzymes endoproteinase glutamic acid (endoGlu-C), trypsin, and chymotrypsin. Furthermore, the amino acid analysis was also described.

Introduction

Cyclotides (cyclo-peptides) are cysteine-rich peptides from plants with molecular weights in the range of 2.8 – 3.7 kDa, with six conserved cysteine residues involved in the formation of a unique arrangement known as the cyclic cystine knot (CCK) motif [1], [2]. This arrangement is responsible for their resistance to thermal, chemical, and enzymatic stability [3]. Cyclotides can be classified into two subfamilies based on the presence (Möbius) or absence (bracelet) of a cis-proline (Pro) bond in Loop 5 [4].

Several biological activities have been reported for these compounds, including insecticidal [5], anthelmintic [6], molluscicidal [7], and antifouling [8] properties, suggesting that their biological function is related with plant defense [9], [10]. Furthermore, cyclotides exhibit uterotonic [11], [12], antimicrobial [13], [14], anti-HIV [15], [16], cytotoxic [17], [18], and protease-inhibiting activities [19]. Recently have attracted attention as immunosuppressive and oral active drug for therapy of multiple sclerosis [20], [21], [22] as well as modulators of G protein-coupled receptors [23], [24].

Until now, these molecules have been identified in five major families of angiosperms: Violaceae, Rubiaceae, Cucurbitaceae [25], Solanaceae [26], and Fabaceae [27], [28]. Between them, the Violaceae family stands out as containing these CCK proteins in all studied species.

In 2015, Burman et al. [29] mapped the occurrence and distribution of cyclotides in 200 samples covering 17 genera of Violaceae by combining material sampling from archived herbarium collections and analytical techniques. Among them, Noisettia longifolia Kunth (synonym Noisettia orchidiflora [Rudge] Gingins) [30], deposited at the herbarium of the Swedish Museum of Natural History, was observed to contain cyclotides, but no structure was characterized from this species.

N. orchidiflora, popularly known as “tetum rau” by the local population of the Acre state (Brazil), is used for treating of swelling [31]; their roots are used as a vomitive in French Guiana [32].

In the current study, we investigated the leaves and stems of the Brazilian species N. orchidiflora, searching for possible cyclotides biosynthesized by this plant. This work allowed the identification of several cyclotides and the characterization of a new cyclotide (Nor A), the first cyclotide that is described from the Noisettia genus.

Results and Discussion

Previously, Burman et al. [29] described N. orchidiflora as a cyclotide-containing species, but no sequence has been identified. Searching to identify and characterize these molecules, in this study, first, we used LC-MS to detect the possible cyclotide masses. This technique allowed the identification of 25 late-eluting compounds that were compared to the data available in the literature as shown in [Table 1]. The major reported cyclotides here were not detected in the study previously published by Burman et al. [29]. Several endo- and exo-factors may contribute for this variability as well as differences in collection place [33], but more experimental studies should be necessary to substantiate this difference.

Separately, dried stems (55.6 g) and leaves (15.1 g) were extracted, defatted, and lyophilized. The crude extracts were dissolved and applied on SPE-C18 cartridges and eluted with CH₃CN/H₂O 2 : 8 (v/v) and CH₃CN/H₂O 8 : 2 (v/v). These last fractions were considered to be rich in cyclotides. The peptide-rich fraction (CH₃CN 80%) from stems (213 mg) was subjected to repeated analytical and preparative RP-HPLC-DAD, yielding one pure substance, named Nor A (5 mg). Range-eluted cyclotides, as well as pure compound Nor A, are shown ([Fig. 1]).

MALDI-TOF-MS analysis was employed for the sequencing of Nor A. After purification, the peptide with an m/z of 3282.472 [M + H]⁺ ([Fig. 1]) was reduced and alkylated, a procedure that increased the mass of each cyclotide by 348 Da, confirming the presence of six cysteine moieties and resulting in a modified structure m/z 3630.368 (Fig. 1S, Supporting Information).

After reduction and alkylation, Nor A was separately subjected to enzymatic digestion with endoproteinase Glu-C, trypsin, and chymotrypsin.

Endoproteinase Glu-C digestion, which typically cleaves after a conserved glutamic acid residue (Glu, E) in Loop 1 that leads to an increase of 18 Da, results in m/z 3648.561; this peak was analyzed by MALDI-MS/MS. Thus, de novo sequencing of this fragment allowed the identification of the y-ion series: SCVFXPCXSTXXGCSCKNKVCYRN ([Fig. 2]). Here, the letter X represents the isobaric residues leucine (Leu, L) or isoleucine (Ile, I). For full sequence coverage, enzymatic digestion was done with trypsin, which cleaves lysine and arginine, resulting in two main fragments, m/z 2828.260 and 3407.530 (Fig. 2SA, Supporting Information). MS/MS of these peaks allowed the identification of the sequences NGVXPCGESCVFXPCXSTXXGCS (Fig. 2SB, Supporting Information) and CVFXPCXSTXXGCSCK (Fig. 2SC, Supporting Information).

The isobaric residues Leu and Ile cannot be distinguished by the MS/MS experiment. Thus, amino acid analysis was performed and revealed the presence of one Leu and four Ile residues (Table 1S, Supporting Information). To determine the position of the Leu/Ile residues, the chymotrypsin enzyme, which cleaves amide bonds C-terminal to Leu (when they are not preceded by Pro), was used. As evident from the sequence of Nor A, there are three potential cleavage sites, all of them in Loop 3. The chymotrypsin digestion produced complementary peptide fragments m/z 2261.057 and 1388.610. Both peaks were submitted to tandem mass analysis. The MS/MS experiment of the m/z 2261.057 ion (NGVXPCGESCVFXPCISTL) indicates the presence of one Leu¹⁸ residue (Loop 3) and one Ile residue (Ile¹⁵, Loop 3) (Fig. 3SB, Supporting Information); the other two residues in this fragment (Ile¹², Loop 2, and Ile³, Loop 6) were assigned based on the amino acid analysis results (Table 1S, Supporting Information). The other fragment m/z 1388.602 (IGCSCKNKVCY) allowed assignment of Ile¹⁹ (Loop 3) (Fig. 3SA, Supporting Information).

Assignment of cysteine connectivity (CCK arrangement) C_I – IV, C_II – V and C_{III – VI} was established by homology with previously reported cyclotides [34]. Thus, a novel cyclotide was proposed, named Nor A ([Fig. 3]), following the naming system proposed by Brossalius et al. [35]. As a result of the lack of the cis-Pro amide bond in Loop 5, Nor A was classified as belonging to the bracelet subfamily.

To identify the homology of Nor A with other cyclotides, an alignment using the BlastP suite (NCBI) was done. This study revealed that the sequence of circulin B, a potent anti-HIV cyclotide isolated from Chassalia parvifolia K. Schum. (Gentianales order) [36], is more similar to Nor A isolated from N. orchidiflora (Malpighiales order). The amino acid alignment of Nor A has 97%, of identity with circulin B, differing only in the 19th residue, where in circulin B, it is Leu, and in Nor A, it is Ile ([Fig. 3]). It is known that the amino acid homology does not represent a large implication for biodiversity of cyclotides, and thus, further studies are necessary to try to explain the similarity take into account evolutionary features. However, the presence of a highly homologous cyclotides in Brazilian species opens a door to future investigations to understand this remarkable kind of natural product in Brazilian biodiversity.

In summary, this work reported the identification of a variety of cyclotides occurring in the stems and leaves from N. orchidiflora by LC-MS. It was possible to identify a group of 25 late-eluting compounds with masses in the range of 3.0 – 3.6 kDa. One new bracelet cyclotide, named Nor A, was purified and characterized by MALDI-MS/MS analysis, employing de novo sequencing and amino acid analysis. MALDI-TOF-MS spectra of Nor A before and after reduction/alkylation reactions and after tryptic and chymotryptic digestion, as well as the amino acid analysis data, are available as Supporting Information.

Materials and Methods

General procedures

Analytical HPLC analysis was carried out on a Shimadzu Prominence LC-20A instrument, LC-6AD pump, DAD SPD-M20A detector, SIL-10AF autosampler, CBM-20A controller, and DGU 20-A_5R degassing unit with a Phenomenex C₁₈ column (250 × 4.6 mm, 100 Å pore size, 5 µm particle size, 1 mL/min). Preparative purifications were carried out on a Shimadzu Prominence LC-20A instrument, LC-20AT pump, DAD SPD-M20A detector, SIL-20A autosampler, CBM-20A controller, DGU 20-A₃ degassing unit, and CTO-20A oven with a Phenomenex Luna C₁₈ column (250 × 21.2 mm, 100 Å pore size, 5 µm particle size, 10 mL/min). In all analyses, two detection wavelengths were selected: 220 and 280 nm. The solvents consisted of H₂O/0.1% CF₃CO₂H (solvent A) or H₂O/CH₃CN/CF₃CO₂H, 10/90/0.08% (v/v/v) (solvent B). Amino acid analyses were carried out on a Shimadzu LC-10A/C-47A automated analyzer.

Plant material

The leaves and stems of N. orchidiflora were collected in Tijuca Park (Tijuca Forest), Rio de Janeiro, during April 2014. The specimen was identified by Dr. Marcelo Trovó, and a voucher is deposited at Rio de Janeiro Botanical Garden with collection number MT591 (JBRJ).

Extraction and isolation of cyclotides

Already dry and pulverized, the stems (55.6 g) and leaves (15.1 g) were extracted with 100 mL of MeOH/H₂O (6 : 4, v/v) at room temperature for 24 h (four times). The hydromethanolic extract was partitioned with 200 mL of CH₂Cl₂ (four times), and the aqueous phases were separated and concentrated on a rotary evaporator prior to freeze-drying, yielding what is further referred to as the aqueous extract. The stems aqueous extract was dissolved in CH₃CN/H₂O (1 : 9, v/v) and immediately used for solid-phase extraction (SPE). C₁₈ SPE cartridges (Strata-Phenomenex C₁₈ 55 µm, 70 Å, 500 mg) were activated with MeOH and subsequently equilibrated with aqueous 1% CF₃CO₂H. After application of the extract, the cartridge was washed with solvent A (H₂O/0.1% CF₃CO₂H) in B (H₂O/CH₃CN/CF₃CO₂H, 10/90/0.08%) 8/2 (v/v) and then eluted with solvent A in B, 2/8 (v/v). The fraction eluted in solvent B 80% was considered rich in peptides and was analyzed by analytical HPLC using a nonlinear gradient: 5 – 34% B in 10 min; 34 – 41% B in 28 min; 41 – 100% B in 2 min ([Fig. 1]).

For this peptide-rich fraction from stems, a preparative HPLC was done using a nonlinear gradient from 34% to 65% solvent B in A during 60 min, collecting peaks manually. With this separation, 12 fractions were obtained; the purified cyclotide, named Nor A, was obtained. This cyclotide was characterized by de novo peptide sequencing using enzymatic digestion, MALDI-MS/MS (time-of-flight analyzers), amino acid analysis, and homology modeling using CyBase tools [37].

Reduction, alkylation, and enzymatic digestion of cyclotides

For sequence analysis, the peptide Nor A (1 mg) was suspended in 200 µL of ammonium bicarbonate buffer (pH 8.2 – 8.4). An aliquot (20 µL) was reduced with 2 µL of dithiothreitol (3.2 mg in 0.1 M NH₄HCO₃) (Sigma-Aldrich) and incubated at 37 °C in the dark for 30 min. Subsequent alkylation of free thiols was done by addition of 4 µL of iodoacetamide (9.25 mg in 0.1 M NH₄HCO₃) at 25 °C for 10 min in the dark. The S-alkylation was quenched after 1 h by adding 1 µL of 1% CF₃CO₂H. The reduced and alkylated cyclotide was digested using individual endoGlu-C (Promega), trypsin, and chymotrypsin enzymes for 6 h at 37 °C, as described previously [40]. All samples were desalted using C₁₈ ZipTips (Millipore) before MS analysis, per the manufacturerʼs instructions.

De novo sequencing of cyclotides

The pure cyclotide Nor A was elucidated by MS using a Bruker Daltonics Ultraflex MALDI TOF/TOF mass spectrometer. The reflector mode was adjusted in positive ion mode and 1000 – 2000 laser shots were acquired per spectrum. The calibration was undertaken using Brukerʼs Peptide Calibration Standard II (Pepcal II). For acquisition of natural masses, the dried compound was dissolved in 0.1% CF₃CO₂H and mixed at a ratio of 1 : 3 (v/v) with a matrix solution of saturated α-cyano-4-hydroxycinnamic acid (Sigma-Aldrich) in H₂O/CH₃CN/CF₃CO₂H, 50/50/0.1% (v/v/v). A 2-µL aliquot of these mixtures were directly spotted onto the MALDI target plate and dried. Mass spectra were obtained in the spectral range 2500 – 4500 m/z. After spotted in the plate, the MS/MS spectra were recorded from fragments coming from enzymatic digestion on a Daltonics Ultraflex mass spectrometer (Bruker). The spectra were obtained with 1000 – 2000 laser shots, and the data were acquired over the mass range m/z 1000 – 4000 Da in positive ion reflectron mode. The cyclotide amino acid sequence was obtained by manual assignment of N-terminal b-ion and C-terminal y-ion series using Bruker flexAnalysis 3.3 software.

LC-MS analysis

The detection of cyclotides using LC-MS was carried out in a Shimadzu chromatograph coupled to an amaZon-SL ion trap Bruker Daltonics. The HPLC comprised an LC-20AD solvent pump unit, a CTO-20A column oven, a DGU-20A_3R online degasser, a CBM-20A system controller, and a SPD-M20A (190 – 800 nm) DAD. The injection was performed automatically (5 µL) in a SIL-20A HT autosampler. The dry extract was resuspended in H₂O/CH₃CN/HCO₂H, 90/10/0.1% (v/v/v) and analyzed by RP-HPLC at 55 °C on a Kromasil C18 column (250 × 4.6 mm i. d., 5 µm particle size, 300 Å pore size) at a flow rate of 1 mL/min with a nonlinear gradient: 5 – 34% B in 10 min; 34 – 41% B in 28 min; 41 – 100% B in 2 min; 100% B for 3 min (A: H₂O/0.1% HCO₂H [v/v]; B: H₂O/CH₃CN/HCO₂H, 10/90/0.1% [v/v/v]). The eluents were monitored on a dual wavelength UV detector set to 220 and 280 nm. The mass spectrum was obtained in positive ion mode considering a mass range of 400 – 2000 m/z. The mass spectrometer source parameters were set as follows: capillary voltage at 3.0 kV. Nitrogen was used as the nebulizing and drying gas (7 psi, 4 L/min, 230 °C). The data was processed through Bruker Compass Data Analysis 4.1 software.

Amino acid analysis

This analysis was carried out as described previously [41]. The purified Nor A (1 mg) was hydrolyzed in 6 M HCl (1 mL) in a sealed tube at 110 °C for 72 h. After hydrolysis, HCl was removed under reduced pressure and the residual material was dissolved in a 0.2 M Na⁺-citrate buffer (pH 2.2). The amino acid content was determined by cation-exchange chromatography using an automatic amino acid analyzer (Shimadzu LC-10A/C-47A) with ortho-phthalaldehyde as the detection reagent.

Sequence alignment

The alignment was produced in QuickBLASTP, an accelerated version of BLASTP, using the one-letter code for amino acids. The sites of the conserved cysteines that are involved in disulfide bonds are shaded [42].

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by INCT Program (INCT BioNat, grant# 465637/2014-0 and 2014/50926-0) and FAPESP (CEPID CIBFar, grant# 2013/07600-3). The authors are also grateful for fellowships to FAPESP (grant# 2017/17098-4) and CNPq (grant# 142286/2016-8 and 162855/2015-0), as well as to Prof. M. Trovó (UFRJ) for the identification of N. orchidiflora plant material and to Dr. Jacqueline Nakau Mendonça (USP-Ribeirão Preto) for MALDI-TOF-MS measurements.

^* Dedicated to Professor Dr. Robert Verpoorte in recognition of his outstanding contribution to natural products research.

^** These authors contributed equally to this work.

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Correspondence

• References

• 1 Craik DJ, Daly NL, Bond T, Waine C. Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol 1999; 294: 1327-1336
  CrossrefPubMedSearch in Google Scholar
• 2 Craik DJ, Daly NL, Waine C. The cystine knot motif in toxins and implications for drug design. Toxicon 2001; 39: 43-60
  CrossrefPubMedSearch in Google Scholar
• 3 Colgrave ML, Craik DJ. Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: the importance of the cyclic cystine knot. Biochemistry 2004; 43: 5965-5975
  CrossrefPubMedSearch in Google Scholar
• 4 Göransson U, Malik S, Slazak B. Cyclotides in the Violaceae. Adv Bot Res 2015; 76: 15-49
  PubMedSearch in Google Scholar
• 5 Barbeta BL, Marshall AT, Gillon AD, Craik DJ, Anderson MA. Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proc Natl Acad Sci U S A 2008; 105: 1221-1225
  CrossrefPubMedSearch in Google Scholar
• 6 Colgrave ML, Kotze AC, Huang YH, OʼGrady J, Simonsen SM, Craik DJ. Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep. Biochemistry 2008; 47: 5581-5589
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