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• W2076008842 abstract "Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 (vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional 1H NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β-sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pKa of 3.0 for Glu3 and ∼5.0 for Glu14 to be determined. Met7 was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μm. Based on a newly established sequencing strategy featured by its efficiency, simplicity, and easy manipulation, the sequences of four novel cyclotides (macrocyclic knotted proteins) isolated from an Australian plant Viola hederaceae were determined. The three-dimensional solution structure of V. hederaceae leaf cyclotide-1 (vhl-1), a leaf-specific expressed 31-residue cyclotide, has been determined using two-dimensional 1H NMR spectroscopy. vhl-1 adopts a compact and well defined structure including a distorted triple-stranded β-sheet, a short 310 helical segment and several turns. It is stabilized by three disulfide bonds, which, together with backbone segments, form a cyclic cystine knot motif. The three-disulfide bonds are almost completely buried into the protein core, and the six cysteines contribute only 3.8% to the molecular surface. A pH titration experiment revealed that the folding of vhl-1 shows little pH dependence and allowed the pKa of 3.0 for Glu3 and ∼5.0 for Glu14 to be determined. Met7 was found to be oxidized in the native form, consistent with the fact that its side chain protrudes into the solvent, occupying 7.5% of the molecular surface. vhl-1 shows anti-HIV activity with an EC50 value of 0.87 μm. Cyclotides are a recently characterized family of naturally occurring circular mini-proteins of 28 to 37 amino acid residues isolated from plants of the Rubiaceae and Violaceae families (1Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar, 2Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Their head-to-tail backbone and six conserved cysteine residues make up a topologically unique structure designated as a cyclic cystine knot in which two disulfide bonds and their connecting backbone segments form an embedded ring that is penetrated by a third disulfide bond (3Craik D.J. Daly N.L. Waine C. Toxicon. 2001; 39: 43-60Crossref PubMed Scopus (417) Google Scholar). Fig. 1 shows the structure of kalata B1, the first example characterized in the cyclotide family, and its sequence is presented in Table I. The fact that cyclotides are exceptionally resistant to thermal and proteolytic degradation has led to the suggestion that the cyclic cystine knot motif contributes greatly to their stability. By contrast, the amino acid sequences between the six cysteines, which form six loops that present on the surface of the molecules, affect the surface characteristics and biological activities of the cyclotides. The cyclotides have been divided into two subfamilies, Möbius and bracelet cyclotides (1Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar, 4Craik D.J. Toxicon. 2001; 38: 1809-1813Crossref Scopus (101) Google Scholar) based on the presence or absence, respectively, of a cis-Pro peptide bond in the circular peptide backbone.Table INew cyclotides characterized in this work and selected examples of known cyclotides Conserved cysteine residues are boxed and presented in bold.View Large Image Figure ViewerDownload Hi-res image Download (PPT)a All masses are provided as mono-isotopic massb V. hederaceae leaf cyclotide (vhl)c Non-tissue-specific expressed cyclotide. Note that cyclotides that are specific to a particular tissue are named accordingly, while those present in a range of tissues are designated using previously described nomenclature (1) Open table in a new tab a All masses are provided as mono-isotopic mass b V. hederaceae leaf cyclotide (vhl) c Non-tissue-specific expressed cyclotide. Note that cyclotides that are specific to a particular tissue are named accordingly, while those present in a range of tissues are designated using previously described nomenclature (1) The cyclotides show a diverse range of biological activities, including uterotonic activity of kalata B1 from Oldenlandia affinis DC (5Gran L. Lloydia (Cinci.). 1973; 36: 207-208PubMed Google Scholar, 6Saether O. Craik D.J. Campbell I.D. Sletten K. Juul J. Norman D.G. Biochemistry. 1995; 34: 4147-4158Crossref PubMed Scopus (373) Google Scholar), the HIV 1The abbreviations used are: HIV, human immunodeficiency virus; HPLC, high performance liquid chromatography; RP, reverse phase; LC, liquid chromatography; MS, mass spectrometry; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; NOESY, nuclear Overhauser enhancement spectroscopy; TOCSY, total correlation spectroscopy; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; ESI, electrospray ionization. inhibitory activity of circulins from Chassalia parvifolia Schum, cycloviolins from Leonia cymosa Mart., and palicourein from Palicourea condensata Standl (7Gustafson K.R. Sowder II, R.C. Henderson L.E. Parsons I.C. Kashman Y. Cardellina II, J.H. McMahon J.B. Buckheit Jr., R.W. Pannell L.K. Boyd M.R. J. Am. Chem. Soc. 1994; 116: 9337-9338Crossref Scopus (259) Google Scholar, 8Hallock Y.F. Sowder II, R.C. Pannell L.K. Hughes C.B. Johnson D.G. Gulakowski R. Cardellina II, J.H. Boyd M.R. J. Org. Chem. 2000; 65: 124-128Crossref PubMed Scopus (106) Google Scholar, 9Bokesch H.R. Pannell L.K. Cochran P.K. Sowder II, R.C. McKee T.C. Boyd M.R. J. Nat. Prod. 2001; 64: 249-250Crossref PubMed Scopus (130) Google Scholar), the antimicrobial activity of kalata B1, circulins A and B, and cyclopsychotride A from Psychotria longipes Muell. Arg (10Tam J.P. Lu Y.A. Yang J.L. Chiu K.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8913-8918Crossref PubMed Scopus (406) Google Scholar), the cytotoxic activity of cycloviolacin O2 from Viola odorata L. and vitri A from Viola tricolor L (11Lindholm P. Göransson U. Johansson S. Claeson P. Gulbo J. Larsson R. Bohlin L. Backlund A. Mol. Cancer Ther. 2002; 1: 365-369Crossref PubMed Scopus (43) Google Scholar, 12Svangard E. Göransson U. Hocaoglu Z. Gullbo J. Larsson R. Claeson P. Bohlin L. J. Nat. Prod. 2004; 67: 144-147Crossref PubMed Scopus (163) Google Scholar)., the neurotensin antagonistic activity of cyclopsychotride A (13Witherup K.M. Bogusky M.J. Anderson P.S. Ramjit H. Ransom R.W. Wood T. Sardana M. J. Nat. Prod. 1994; 57: 1619-1625Crossref PubMed Scopus (228) Google Scholar), the hemolytic activity of violapeptide I from Viola tricolor L (14Schöpke T. Hasan Agha M.I. Kraft R. Otto A. Hiller K. Sci. Pharm. 1993; 61: 145-153Google Scholar), the trypsin inhibitory activity of MCoTI-I and II from Momordica cochinchinensis (15Hernandez J.F. Gagnon J. Chiche L. Nguyen T.M. Andrieu J.P. Heitz A. Trinh Hong T. Pham T.T. Le Nguyen D. Biochemistry. 2000; 39: 5722-5730Crossref PubMed Scopus (297) Google Scholar), and the insecticidal activity of kalata B1 and kalata B2 (16Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (405) Google Scholar, 17Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry. 2005; 44: 851-860Crossref PubMed Scopus (195) Google Scholar). All of these cyclotides were originally found through screening programs for biological activities or, in the case of the uterotonic activity, from native medicine usage (5Gran L. Lloydia (Cinci.). 1973; 36: 207-208PubMed Google Scholar). Some cyclotides, for example, kalata B1, circulins, and cyclopsychotride A, effectively show several different inhibitory activities, suggesting that the loops of one cyclotide molecule may have various biological functions and can potentially bind to different target sites. Perhaps most interesting is the finding that kalata B1 and kalata B2 inhibit the growth and development of Helicoverpa punctigera larvae, leading to the suggestion that cyclotides play an important role in plant defense against pests or pathogens (16Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (405) Google Scholar, 17Jennings C.V. Rosengren K.J. Daly N.L. Plan M. Stevens J. Scanlon M. Waine C. Norman D.G. Anderson M.A. Craik D.J. Biochemistry. 2005; 44: 851-860Crossref PubMed Scopus (195) Google Scholar). The diverse bioactivities, along with their unique structural characteristics, render the cyclotides extremely important in terms of potential agro-chemical or pharmaceutical applications (4Craik D.J. Toxicon. 2001; 38: 1809-1813Crossref Scopus (101) Google Scholar, 16Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (405) Google Scholar, 18Craik D.J. Simonsen S. Daly N.L. Curr. Opin. Drug Discov. Devel. 2002; 5: 251-260PubMed Google Scholar). The cyclotides, like many other defense proteins, are derived from precursor polypeptides, which undergo a process of post-translational modification. Recently we reported the isolation and characterization of cyclotide cDNA clones from V. odorata (19Dutton J.L. Renda R.F. Waine C. Clark R.J. Daly N.L. Jennings C.V. Anderson M.A. Craik D.J. J. Biol. Chem. 2004; 279: 46858-46867Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). A single full-length cDNA transcript encodes one or several cyclotides, which are separated by conserved peptide fragments termed N-terminal repeats that may function in the cyclization and folding of cyclotides (19Dutton J.L. Renda R.F. Waine C. Clark R.J. Daly N.L. Jennings C.V. Anderson M.A. Craik D.J. J. Biol. Chem. 2004; 279: 46858-46867Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). We also synthesized the N-terminal repeats based on the cDNA clones and elucidated their α-helical solution structures by NMR spectroscopy. Interestingly the N-terminal repeat sequences were not as conserved as those predicted from cDNA clones from O. affinis (16Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (405) Google Scholar). This might result from the fact that the cDNAs from V. odorata encode both subfamilies, Möbius and bracelet cyclotides, instead of representing mainly one subfamily of cyclotides, as was the case for the cDNAs isolated from O. affinis (16Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (405) Google Scholar, 19Dutton J.L. Renda R.F. Waine C. Clark R.J. Daly N.L. Jennings C.V. Anderson M.A. Craik D.J. J. Biol. Chem. 2004; 279: 46858-46867Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). To date, ∼50 cyclotides have been reported from a number of species of the Rubiaceae and Violaceae plant families, and all have molecular masses ranging from 2.8 to 3.5 kDa. A recent study by HPLC and LC-MS on cyclotides from Viola hederaceae revealed that up to 66 different masses, most likely corresponding to the same number of cyclotides, were discerned in extracts of various parts of the plant (20Trabi M. Craik D.J. Plant Cell. 2004; 16: 2204-2216Crossref PubMed Scopus (96) Google Scholar). This suggests that the cyclotides are a very diverse family of proteins and potentially are much more common in plants than had previously been realized. To expand our knowledge of structure-activity relationships of the cyclotides, we introduce a new strategy for determining the sequence of cyclic disulfide rich peptides, report the isolation and characterization of four novel cyclotides from V. hederaceae, and present the solution structure of vhl-1, one of the new cyclotides. Isolation and Purification of vhl-1—Fresh whole plant of V. hederaceae was collected from a garden in Brisbane, Australia and separated into different parts: leaves, petioles, flowers, pedicels, roots, bulbs, as well as above and below ground runners. The leaves were homogenized using a blender (Moulinex) and extracted with dichloromethane:methanol (1:1) overnight. Plant debris was removed using a cotton plug. The filtrate was repeatedly partitioned with dichloromethane and water. The organic soluble fraction was discarded, and the methanol/water layer was concentrated on a rotary evaporator (Bücchi) prior to lyophilization. Then the mixture was diluted with distilled water to a final methanol concentration <20% and lyophilized on a freeze-drier (Speed-Vac). The dried material was redissolved in a minimal amount of buffer A (0.05% trifluoroacetic acid prepared in distilled water). The solution was then passed through a solid phase filter (Sartorius) before purification using preparative RP-HPLC on an Agilent 1100 series system with variable wavelength detector and Phenomenex Jupiter C18 column (250 × 22 mm, 5 μm, 300 Å). Gradients of buffer A (0.05% aqueous trifluoroacetic acid) and buffer B (90% acetonitrile, 0.05% trifluoroacetic acid) were employed with a flow rate of 8 ml/min and a gradient of 1% buffer B per minute. Further purification was performed using semipreparative RP-HPLC on a Phenomenex Jupiter C18 column (250 × 10 mm, 5 μm, 300 Å) and using an analytical Phenomenex Jupiter C18 column (250 × 4.6 mm, 5 μm, 300 Å). The final purity was examined with analytical RP-HPLC on a Grom column (150 × 2 mm, 3 μm, equipped with a security-guard column) with a flow rate of 300 μl/min. Masses were analyzed on a Micromass LCT mass spectrometer equipped with an electrospray ionization source. Aminoethylation of Cysteines—The reduction and alkylation of the disulfide bonds was performed according to the method described in the literature (21Göransson U. Broussalis A.M. Claeson P. Anal. Biochem. 2003; 318: 107-117Crossref PubMed Scopus (57) Google Scholar) with minor modification. 5 nmol of vhl-1 was reduced with 0.4 μmol of dithiothreitol in 200 μl 0.25 m Tris-HCl, containing 1 mm EDTA and 8 m guanidine HCl (pH 8.5, 37 °C) under N2. After 2 h, 20 μmol of bromoethylamine dissolved in 20 μl of 0.25 m Tris-HCl, containing 1 mm EDTA and 8 m guanidine HCl (pH 8.5) was added. The reaction was incubated in a water bath at 37 °C in the dark under N2 overnight and terminated by injection onto RP-HPLC and eluted with a linear gradient of 0–80% buffer B in 80 min. The molecular masses of the collected fractions were confirmed by LCT-ESI-MS prior to lyophilization and storage at -20 °C. Reduction of vhl-1 and MALDI-MS Analysis—To ∼6 nmol of vhl-1 in 20 μlof0.1 m NH4HCO3 (pH 8.0), 1 μlof0.1 m TCEP was added, and the solution was incubated at 65 °C for 10 min. The reduction was confirmed by MALDI-TOF-MS after desalting using Ziptips (Millipore), which involved several washing steps followed by elution in 10 μlof80% acetonitrile (0.5% formic acid). The desalted samples were mixed in a 1:1 ratio with matrix consisting of a saturated solution of α-cyano-4-hydroxycinnamic acid in 50% acetonitrile (0.5% formic acid). The instrument used was a Voyager DE-STR mass spectrometer (Applied Biosystems). 200 shots per spectra were acquired in positive ion reflector mode. The laser intensity was set to 1800, the accelerating voltage was set to 20,000 V; the grid voltage was set to 64% of the accelerating voltage, and the delay time was 165 ns. The low mass gate was set to 500 Da. Data were collected between 500 and 5000 Da. Calibration was undertaken using a peptide mixture obtained from Sigma Aldrich (MSCal1). Enzymatic Digestion and Nanospray MS-MS Sequencing—To the reduced peptide, trypsin, endoproteinase GluC (endo-GluC), or a combination of both were added to give a final peptide-to-enzyme ratio of 50:1. The trypsin incubation was allowed to proceed for 1 h, the endo-GluC was over 3 h, while for the combined digestion trypsin was added initially for 1 h followed by the addition of endo-GluC for a further 3 h. The digestions were quenched by the addition of an equal volume of 0.5% formic acid and desalted using Ziptips (Millipore). Samples were stored at 4 °C prior to analysis. The fragments resulting from the digestion were examined first by MALDI-TOF-MS followed by sequencing by nanospray MS-MS on a QStar mass spectrometer. A capillary voltage of 900 V was applied and spectra were acquired between m/z 60–2000 for both TOF spectra and product ion spectra. The collision energy for peptide fragmentation was varied between 10 and 50 V, depending on the size and charge of the ion. The Analyst software program was used for data acquisition and processing. The MS-MS spectra were examined and sequenced based on the presence of both b and y series of ions present (N- and C-terminal fragments). The same procedure was utilized for the sequencing of the three other novel cyclotides isolated from V. hederaceae. Chymotrypsin digests using the same conditions as for trypsin were also conducted to confirm the results obtained for each of the peptide sequences. NMR Experiments—The sample for NMR spectroscopy was prepared by dissolving vhl-1 in 70% H2O, 25% CD3CN, and 5% D2O to a final concentration of 1.4 mm, since aggregation of the peptide was observed in 100% water. All spectra were recorded on Bruker ARX 500 or Bruker ARX 600 spectrometers equipped with a shielded gradient unit, with sample temperature in the range 283–330 K. All spectra were acquired in phase-sensitive mode using time proportional phase incrementation (22Marion D. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3524) Google Scholar). For TOCSY (23Braunschweiler L. Ernst R.R. J. Magn. Reson. 1983; 53: 521-528Crossref Scopus (3108) Google Scholar), using MLEV-17 (24Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar) with a mixing time of 80 ms, and NOESY (25Jeener J. Meier B.H. Bachmann P. Ernst R.R. J. Chem. Phys. 1979; 71: 4546-4553Crossref Scopus (4838) Google Scholar) with mixing times of 100, 200 and 250 ms, water suppression was achieved using a WATERGATE (water suppression by gradient-tailored excitation) (26Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3527) Google Scholar) sequence. Double quantum-filtered COSY (27Rance M. Sørensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wüthrich K.O. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2597) Google Scholar) and E-COSY (28Griesinger C. Sørensen O.W. Ernst R.R. J. Magn. Reson. 1987; 75: 474-492Google Scholar) were also recorded. Slowly exchanging amide protons were identified by recording a series of one-dimensional spectra and two-dimensional TOCSY spectra at 298 K over a period of 20 h immediately after dissolution of a sample in 25% CD3CN and 75% D2O. The pH dependence was monitored for vhl-1 at 298 K by altering the sample pH from 2.0 to 6.0 by adding HCl or NaOH. The pKa of the titrating Glu3 and Glu14 residues was determined for vhl-1 by nonlinear curve fitting of the data points. The 3JHN-Hα coupling constants were obtained from a high resolution one-dimensional spectrum and from line shape analysis of the anti-phase cross-signal splitting in a high resolution DQF-COSY spectrum. All two-dimensional spectra were collected over 4096 data points in the f2 dimension and 512 increments in the f1 and processed using XWINNMR (Bruker) on a Silicon Graphics Octane work station. The f1 dimension was generally zero-filled to 2048 real data points, with the f1 and f2 dimensions being multiplied by a sine-squared function prior to Fourier transformation. Chemical shifts were internally referenced to sodium 2,2-dimethyl-2silapentane-5-sulfonate. Structural Restraints—Distance restraints were derived primarily from cross-peaks in a 250-ms mixing time NOESY spectrum recorded at 298 K. The cross-peaks were analyzed and resonance assignments were achieved using the program SPARKY. 2Goddard, T. D. and Kneller, D. G., SPARKY 3 program, University of California, San Francisco. 25 backbone dihedral restraints were added on the basis of 3JHN-Hα coupling constants derived from the splitting of the amide and α protons and were constrained to -120 (±30)° for 3JHN-Hα in the range 8.5 ± 0.5 Hz (residues 1, 3, 4, 5, 7, 8, 10, 19, 21, 24, and 30), -120 (±15)° for 3JHN-Hα greater than 9.5 Hz (residues 26, 27), and -65 (±30)° for 3JHN-Hα less than 5.8 Hz (residues 6, 9, 11, 14, 15, 16, and 31). One additional constraint of -100 (±80)° (residue 18) was applied where the 3JHN-Hα coupling constant is ∼7.00 Hz, and the intraresidue Hαi-HNi NOE is weaker than the sequential Hαi-HNi NOE. Residues 22, 23, 28, and 29 with intense Hαi-HNi NOEs and 3JHN-Hα coupling constants of ∼7 Hz were restrained to 50 (±40)°. 3JHα-Hβ coupling constants derived from an E-COSY spectrum, together with NOE intensity patterns from the NOESY spectrum with a mixing time of 100 ms, were used to determine the stereospecific assignments of β-methylene protons and 17 χ1 dihedral angle restraints. Ten hydrogen bonds were determined based on slow exchange data and preliminary structure calculations. 20 restraints of 1.7–2.2 and 2.7–3.2 Å for these hydrogen bonds were added and used in the final structure calculation. Structure Calculation—Initial structures were calculated using DYANA (30Guntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2555) Google Scholar) based on NOE data output from SPARKY. After an iterative process in which preliminary structures were used to resolve ambiguities, sets of 50 structures were calculated using a torsion angle simulated annealing protocol within the program CNS (31Brünger A.T. Adams P.D. Rice L.M. Structure. 1997; 5: 325-336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). This protocol involved a high temperature phase comprising 4000 steps of 0.015 ps of torsion angle dynamics, a cooling phase with 4000 steps of 0.015 ps of torsion angle dynamics during which the temperature was lowered to 0 K, and finally an energy minimization phase comprising 500 steps of Powell minimization. The resultant structures were subjected to further molecular dynamics and energy minimization in a water shell (32Linge J.P. Nilges M. J. Biomol. NMR. 1999; 13: 51-59Crossref PubMed Scopus (239) Google Scholar). A set of 20 structures with the lowest overall energy that had no violations of distance restraints greater than 0.2 Å or dihedral angle restraints greater than 3° was chosen to represent the structure of vhl-1. Structures were visualized using the program MOLMOL (33Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 29-32Crossref Scopus (6489) Google Scholar) and analyzed with PROMOTIF_NMR (34Hutchinson E.G. Thornton J.M. Protein Sci. 1996; 5: 212-220Crossref PubMed Scopus (997) Google Scholar) and PROCHECK_NMR (35Laskowski R.A. Rullmannn J.A. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4428) Google Scholar). The 20 final structures of vhl-1 and associated restraints have been deposited in the Protein Data Bank (ID code: 1ZA8). Isolation and Purification of Cyclotides—Cyclotides show several defining characteristics, including late elution on HPLC and a mass range from 2.8 to 3.5 kDa. Based on these characteristics, we have established an effective and efficient procedure to isolate and purify cyclotides from various components of the crude plant extracts (16Jennings C. West J. Waine C. Craik D. Anderson M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10614-10619Crossref PubMed Scopus (405) Google Scholar). In the current study, we divided fresh collected plant material into eight parts (leaves, petioles, flowers, pedicels, above ground runners, below ground runners, bulbs, and roots) and used the extraction protocol to obtain crude cyclotide extracts. RP-HPLC profiles of different parts of V. hederaceae are shown in Fig. 2. Repeated RP-HPLC resulted in the separation and purification of four novel cyclotides corresponding to chromatographic peaks 4, 5 (two peptides), and 6. The molecular masses of these cyclotides determined as shown in Table I by electrospray ionization (ESI) mass spectrometry revealed they were new when compared with the molecular masses of known cyclotides. The cyclotide corresponding to chromatographic peak 3 is expressed only in leaves, but its mass of 3117.8 matches a reported cyclotide cycloviolacin O10 isolated from V. odorata (1Craik D.J. Daly N.L. Bond T. Waine C. J. Mol. Biol. 1999; 294: 1327-1336Crossref PubMed Scopus (648) Google Scholar). Chromatographic peak 7 corresponds to a cyclotide named vhr1, whose molecular characterization was reported recently (20Trabi M. Craik D.J. Plant Cell. 2004; 16: 2204-2216Crossref PubMed Scopus (96) Google Scholar). Among the four novel cyclotides, the two corresponding to peak 5 are expressed in several different tissues and were designated as cycloviolacins H2 and H3. The one with a relatively late retention time corresponding to peak 6 is only expressed in leaves and named as vhl-2 (V. hederaceae leaf cyclotide-2). Special emphasis was put on a cyclotide vhl-1 with a retention time of 32.0 min (peak 4) as it is relatively abundant and expressed specifically in leaves and not in any other parts of the plant. From the crude extract of V. hederaceae leaves vhl-1 was isolated and purified to homogeneity by RP-HPLC as shown in the last panel in Fig. 2. The yield of vhl-1 was about 1 mg/kg fresh plant material. Amino Acid Sequence Analysis—Amino acid analysis of vhl-1 indicated that it is composed of 31 amino acids: 1 Ala, 6 Cys, 2 Glu or Gln, 2 Phe, 2 Gly, 3 Ile, 2 Lys, 1 Leu, 1 Met, 2 Asp or Asn, 5 Ser, 1 Thr, 2 Val and 1 Tyr. It shows resistance to enzymatic cleavage by trypsin, consistent with the presence of a cyclic cystine knot motif characteristic of all cyclotides. After reduction and alkylation, each reduced and S-aminoethylated cysteine contributes to an increment of molecular mass by 44 Da. Nanospray MS and LC-ESI-MS analysis of the native (3330, mono-isotopic mass) and S-aminoethylated peptide (3594) demonstrated that, like other cyclotides, six cysteines are present in three intramolecular disulfide bonds. Tryptic cleavage was then carried out on the alkylated peptide in an attempt to use a recently proposed “loop sequencing” method for determining the primary structure of cyclotides (21Göransson U. Broussalis A.M. Claeson P. Anal. Biochem. 2003; 318: 107-117Crossref PubMed Scopus (57) Google Scholar, 36Göransson U. Craik D.J. J. Biol. Chem. 2003; 278: 48188-48196Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). However, this approach resulted in very complicated enzymatic fragments, implying that, besides the six alkylated cysteines, several other positively charged amino acid residues exist in the primary structure. All of the alkylated cysteines and positive residues provide cleavage sites for trypsin and, as a result, produce additional fragments, complicating the MS-MS data. An alternative strategy outlined in Fig. 3, which includes both enzymatic digestion and MS-MS fragmentation, was used to complete the sequence analysis. The native cyclotide was reduced by TCEP and subjected to enzymatic digestion directly, without alkylation of the cysteines. The reduction of the peptide was confirmed by the observation of peaks at m/z 11123+ and 8344+, which correspond to a molecular mass of 3336 Da due to the addition of six protons to the native peptide. Digestion of the reduced peptide with endo-GluC gave rise to two major peaks at m/z 627.72+ and 707.33+ corresponding to two fragments with masses of 1253.5 and 2118.9 and implying the presence of two glutamic acid residues. Several other peaks with relatively low intensities were also produced after digestion by trypsin or a combination of trypsin and endo-GluC. The study of the MS-MS fragmentation of the first fragment at m/z 627.72+ resulted in completion of its amino acid sequence as shown in Fig. 4A, suggesting that it consists of 11 amino acid residues with the sequence SCAF*ISFCFTE. The b2 ion of the fragment at m/z 191 could be either SC or CS. However, it was determined to be SC by comparison with known cyclotides and further confirmed by NMR spectroscopy. The second fragment at m/z 707.33+ was difficult to analyze by MS-MS fragmentation because of its length. However, two partial sequences from both N-terminal (VIGCSCKNKV-) and C-terminal (-LNSISCGE) were observed as highlighted in Fig. 4B and in the supplemental Table I. The b and y ions greater than ∼5% in relative intensity are labeled in Fig. 4, and a number of ions that allow full sequence elucidation are also present in" @default.
• W2076008842 created "2016-06-24" @default.
• W2076008842 creator A5000965397 @default.
• W2076008842 creator A5010005016 @default.
• W2076008842 creator A5034657959 @default.
• W2076008842 creator A5055209466 @default.
• W2076008842 creator A5079308758 @default.
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• W2076008842 date "2005-06-01" @default.
• W2076008842 modified "2023-10-11" @default.
• W2076008842 title "Isolation and Characterization of Novel Cyclotides from Viola hederaceae" @default.
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