Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Two Blast-independent tools, CyPerl and CyExcel, for harvesting hundreds of novel cyclotides and analogues from plant genomes and protein databases


Main conclusion

Two high-throughput tools harvest hundreds of novel cyclotides and analogues in plants.

Cyclotides are gene-encoded backbone-cyclized polypeptides displaying a diverse range of bioactivities associated with plant defense. However, genome-scale or database-scale evaluations of cyclotides have been rare so far. Here, a novel time-efficient Perl program, CyPerl, was developed for searching cyclotides from predicted ORFs of 34 available plant genomes and existing plant protein sequences from Genbank databases. CyPerl-isolated sequences were further analyzed by removing repeats, evaluating their cysteine-distributed regions (CDRs) and comparing with CyBase-collected cyclotides in a user-friendly Excel (Microsoft Office) template, CyExcel. After genome-screening, 186 ORFs containing 145 unique cyclotide analogues were identified by CyPerl and CyExcel from 30 plant genomes tested from 10 plant families. Phaseolus vulgaris and Zea mays were the richest two species containing cyclotide analogues in the plants tested. After screening protein databases, 266 unique cyclotides and analogues were identified from seven plant families. By merging with 288 unique CyBase-listed cyclotides, 510 unique cyclotides and analogues were obtained from 13 plant families. In total, seven novel plant families containing cyclotide analogues and 202 novel cyclotide analogues were identified in this study. This study has established two Blast-independent tools for screening cyclotides from plant genomes and protein databases, and has also significantly widened the plant distribution and sequence diversity of cyclotides and their analogues.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6



Cysteine-distributed regions of cyclotides and analogues


Endoplasmic reticulum


N-terminal repeat


  1. Aboye TL, Ha H, Majumder S, Christ F, Debyser Z, Shekhtman A, Neamati N, Camarero JA (2012) Design of a novel cyclotide-based CXCR4 antagonist with anti-human immunodeficiency virus (HIV)-1 activity. J Med Chem 55:10729–10734

  2. Avrutina O, Schmoldt H, Gabrijelcic-Geiger D, Le Nguyen D, Sommerhoff CP, Diederichsen U, Kolmar H (2005) Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II. Biol Chem 386:1301–1306

  3. Barbeta BL, Marshall AT, Gillon AD, Craik DJ, Anderson MA (2008) Plant cyclotides disrupt epithelial cells in the midgut of Lepidopteran larvae. Proc Natl Acad Sci USA 105:1221–1225

  4. Bokesch HR, Pannell LK, Cochran PK, Sowder RC 2nd, McKee TC, Boyd MR (2001) A novel anti-HIV macrocyclic peptide from Palicourea condensata. J Nat Prod 64:249–250

  5. Burman R, Gruber CW, Rizzardi K, Herrmann A, Craik DJ, Gupta MP, Göransson U (2010) Cyclotide proteins and precursors from the genus Gloeospermum: filling a blank spot in the cyclotide map of Violaceae. Phytochemistry 71:13–20

  6. Burman R, Gunasekera S, Strömstedt AA, Göransson U (2014) Chemistry and biology of cyclotides: circular plant peptides outside the box. J Nat Prod 77:724–736

  7. Chen B, Colgrave ML, Daly NL, Rosengren KJ, Gustafson KR, Craik DJ (2005) Isolation and characterization of novel cyclotides from Viola hederacea: solution structure and anti-HIV activity of vhl-1, a leaf-specific expressed cyclotide. J Biol Chem 28:22395–22405

  8. Colgrave ML, Kotze AC, Huang YH, O’Grady J, Simonsen SM, Craik DJ (2008) Cyclotides: natural, circular plant peptides that possess significant activity against gastrointestinal nematode parasites of sheep. Biochemistry 47:5581–5589

  9. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676

  10. Craik DJ (2012) Host-defense activities of cyclotides. Toxins (Basel) 4:139–156

  11. Craik DJ, Malik U (2013) Cyclotide biosynthesis. Curr Opin Chem Biol 17:546–554

  12. Craik DJ, Daly NL, Bond T, Waine C (1999) Plant cyclotides: a unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif. J Mol Biol 94:1327–1336

  13. Daly NL, Gustafson KR, Craik DJ (2004) The role of the cyclic peptide backbone in the anti-HIV activity of the cyclotide kalata B1. FEBS Lett 574:69–72

  14. Gerlach SL, Göransson U, Kaas Q, Craik DJ, Mondal D, Gruber CW (2013) A systematic approach to document cyclotide distribution in plant species from genomic, transcriptomic, and peptidomic analysis. Biopolymers 100:433–437

  15. Göransson U, Sjogren M, Svangard E, Claeson P, Bohlin L (2004) Reversible antifouling effect of the cyclotide cycloviolacin O2 against barnacles. J Nat Prod 67:1287–1290

  16. Gruber CW, Elliott AG, Ireland DC, Delprete PG, Dessein S, Göransson U, Trabi M, Wang CK, Kinghorn AB, Robbrecht E, Craik DJ (2008) Distribution and evolution of circular miniproteins in flowering plants. Plant Cell 20:2471–2483

  17. Gründemann C, Thell K, Lengen K, Garcia-Käufer M, Huang YH, Huber R, Craik DJ, Schabbauer G, Gruber CW (2013) Cyclotides suppress human T-lymphocyte proliferation by an Interleukin 2-dependent mechanism. PLoS One 8:e68016

  18. Gustafson KR, Sowder RC, Henderson LE, Parsons IC, Kashman Y, Cardellina JH, McMahon JB, Buckheit RB, Pannell LK, Boyd MR (1994) Circulins A and B. Novel human immunodeficiency virus (HIV)-inhibitory macrocyclic peptides from the tropical tree Chassalia parvifolia. J Am Chem Soc 116:9337–9338

  19. Hellinger R, Koehbach J, Fedchuk H, Sauer B, Huber R, Gruber CW, Gründemann C (2014) Immunosuppressive activity of an aqueous Viola tricolor herbal extract. J Ethnopharmacol 151:299–306

  20. Herrmann A, Burman R, Mylne JS, Karlsson G, Gullbo J, Craik DJ, Clark RJ, Göransson U (2008) The alpine violet, Viola biflora, is a rich source of cyclotides with potent cytotoxicity. Phytochemistry 69:939–952

  21. Ireland DC, Colgrave ML, Nguyencong P, Daly NL, Craik DJ (2006) Discovery and characterization of a linear cyclotide from Viola odorata: implications for the processing of circular proteins. J Mol Biol 357:1522–1535

  22. Jennings C, West J, Waine C, Craik D, Anderson M (2001) Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proc Natl Acad Sci USA 98:10614–10619

  23. Ji Y, Majumder S, Millard M, Borra R, Bi T, Elnagar AY, Neamati N, Shekhtman A, Camarero JA (2013) In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide. J Am Chem Soc 135:11623–11633

  24. Kaas Q, Craik DJ (2010) Analysis and classification of circular proteins in CyBase. Biopolymers 94:584–591

  25. Kedarisetti P, Mizianty MJ, Kaas Q, Craik DJ, Kurgan L (2014) Prediction and characterization of cyclic proteins from sequences in three domains of life. Biochim Biophys Acta 1844:181–190

  26. Koehbach J, Gruber CW (2013) From ethnopharmacology to drug design. Commun Integr Biol 6:e27583

  27. Koehbach J, Attah AF, Berger A, Hellinger R, Kutchan TM, Carpenter EJ, Rolf M, Sonibare MA, Moody JO, Wong GK, Dessein S, Greger H, Gruber CW (2013a) Cyclotide discovery in Gentianales revisited-identification and characterization of cyclic cystine-knot peptides and their phylogenetic distribution in Rubiaceae plants. Biopolymers 100:438–452

  28. Koehbach J, O’Brien M, Muttenthaler M, Miazzo M, Akcan M, Elliott AG, Daly NL, Harvey PJ, Arrowsmith S, Gunasekera S, Smith TJ, Wray S, Göransson U, Dawson PE, Craik DJ, Freissmuth M, Gruber CW (2013b) Oxytocic plant cyclotides as templates for peptide G protein-coupled receptor ligand design. Proc Natl Acad Sci USA 110:21183–21188

  29. Lindholm P, Goransson U, Johansson S, Claeson P, Gullbo J, Larsson R, Bohlin L, Backlund A (2002) Cyclotides: a novel type of cytotoxic agents. Mol Cancer Ther 1:365–369

  30. Mulvenna JP, Wang C, Craik DJ (2006) CyBase: a database of cyclic protein sequence and structure. Nucleic Acids Res 34:D192–D194

  31. Mylne JS, Wang CK, van der Weerden NL, Craik DJ (2010) Cyclotides are a component of the innate defense of Oldenlandia affinis. Biopolymers 94:635–646

  32. Mylne JS, Chan LY, Chanson AH, Daly NL, Schaefer H, Bailey TL, Nguyencong P, Cascales L, Craik DJ (2012) Cyclic peptides arising by evolutionary parallelism via asparaginyl-endopeptidase-mediated biosynthesis. Plant Cell 24:2765–2778

  33. Nguyen GK, Zhang S, Nguyen NT, Nguyen PQ, Chiu MS, Hardjojo A, Tam JP (2011) Discovery and characterization of novel cyclotides originated from chimeric precursors consisting of albumin-1 chain a and cyclotide domains in the Fabaceae family. J Biol Chem 286:24275–24287

  34. Nguyen GK, Lim WH, Nguyen PQ, Tam JP (2012) Novel cyclotides and uncyclotides with highly shortened precursors from Chassalia chartacea and effects of methionine oxidation on bioactivities. J Biol Chem 287:17598–17607

  35. Nguyen GK, Lian Y, Pang EW, Nguyen PQ, Tran TD, Tam JP (2013) Discovery of linear cyclotides in monocot plant Panicum laxum of Poaceae family provides new insights into evolution and distribution of cyclotides in plants. J Biol Chem 288:3370–3380

  36. Park S, Strömstedt AA, Göransson U (2014) Cyclotide structure-activity relationships: qualitative and quantitative approaches linking cytotoxic and anthelmintic activity to the clustering of physicochemical forces. PLoS One 9:e91430

  37. Poth AG, Colgrave ML, Lyons RE, Daly NL, Craik DJ (2011a) Discovery of an unusual biosynthetic origin for circular proteins in legumes. Proc Natl Acad Sci USA 108:10127–10132

  38. Poth AG, Colgrave ML, Philip R, Kerenga B, Daly NL, Anderson MA, Craik DJ (2011b) Discovery of cyclotides in the Fabaceae plant family provides new insights into the cyclization, evolution, and distribution of circular proteins. ACS Chem Biol 6:345–355

  39. Poth AG, Mylne JS, Grassl J, Lyons RE, Millar AH, Colgrave ML, Craik DJ (2012) Cyclotides associate with leaf vasculature and are the products of a novel precursor in petunia (Solanaceae). J Biol Chem 287:27033–27046

  40. Poth AG, Chan LY, Craik DJ (2013) Cyclotides as grafting frameworks for protein engineering and drug design applications. Biopolymers 100:480–491

  41. Qin Q, McCallum EJ, Kaas Q, Suda J, Saska I, Craik DJ, Mylne JS (2010) Identification of candidates for cyclotide biosynthesis and cyclisation by expressed sequence tag analysis of Oldenlandia affinis. BMC Genom 11:111

  42. Schroeder CI, Swedberg JE, Craik DJ (2013) Recent progress towards pharmaceutical applications of disulfide-rich cyclic peptides. Curr Protein Pept Sci 14:532–542

  43. Shenkarev ZO, Nadezhdin KD, Lyukmanova EN, Sobol VA, Skjeldal L, Arseniev AS (2008) Divalent cation coordination and mode of membrane interaction in cyclotides: NMR spatial structure of ternary complex Kalata B7/Mn2 +/DPC micelle. J Inorg Biochem 102:1246–1256

  44. Simonsen SM, Sando L, Ireland DC, Colgrave ML, Bharathi R, Göransson U, Craik DJ (2005) A continent of plant defense peptide diversity: cyclotides in Australian Hybanthus (Violaceae). Plant Cell 17:3176–3189

  45. Skjeldal L, Gran L, Sletten K, Volkman BF (2002) Refined structure and metal binding site of the kalata B1 peptide. Arch Biochem Biophys 399:142–148

  46. Svangard E, Goransson U, Hocaoglu Z, Gullbo J, Larsson R, Claeson P, Bohlin L (2004) Cytotoxic cyclotides from Viola tricolor. J Nat Prod 67:144–147

  47. Tam JP, Lu YA, Yang JL, Chiu KW (1999) An unusual structural motif of antimicrobial peptides containing end-to-end macrocycle and cystine-knot disulfides. Proc Natl Acad Sci USA 96:8913–8918

  48. The Angiosperm Phylogeny Group (2009) An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Bot J Lin Soc 161:105–121

  49. Trabi M, Craik DJ (2004) Tissue-specific expression of head-to-tail cyclized miniproteins in Violaceae and structure determination of the root cyclotide Viola hederacea root cyclotide1. Plant Cell 16:2204–2216

  50. Trabi M, Svangård E, Herrmann A, Göransson U, Claeson P, Craik DJ, Bohlin L (2004) Variations in cyclotide expression in Viola species. J Nat Prod 67:806–810

  51. Wang CK, Colgrave ML, Gustafson KR, Ireland DC, Göransson U, Craik DJ (2007) Anti-HIV cyclotides from the Chinese medicinal herb Viola yedoensis. J Nat Prod 71:47–52

  52. Wang CK, Kaas Q, Chiche L, Craik DJ (2008) CyBase, a database of cyclic protein sequences and structures, with applications in protein discovery and engineering. Nucleic Acids Res 36:D206–D210

  53. Zarrabi M, Dalirfardouei R, Sepehrizade Z, Kermanshahi RK (2013) Comparison of the antimicrobial effects of semipurified cyclotides from Iranian Viola odorata against some of plant and human pathogenic bacteria. J Appl Microbiol 115:367–375

  54. Zhang J, Hu M, Li JT, Guan JP, Yang B, Shu WS, Liao B (2009a) A transcriptional profile of metallophyte Viola baoshanensis involved in general and species-specific cadmium-defense mechanisms. J Plant Physiol 166:862–870

  55. Zhang J, Liao B, Craik DJ, Li JT, Hu M, Shu WS (2009b) Identification of two suites of cyclotide precursor genes from metallophyte Viola baoshanensis: cDNA sequence variation, alternative RNA splicing and potential cyclotide diversity. Gene 431:23–32

Download references


The authors would like to thank the CyBase administrators, Dr. CK Wang and Dr. Q Kaas, for assisting cyclotide structure analysis. This work was funded by the National Natural Science Foundation of China (Grant No. 31100198, 31170480 and U1201233) and by the Open Project of the State Key Laboratory of Biocontrol (SKLBC2010K04). DJC is funded by a National Health and Medical Research Council (Australia) Senior Principal Research Fellowship (APP1026501). We also thank Prof. AJM Baker (Universities of Melbourne and Queensland, Australia) for help in improving the final version of the manuscript.

Conflict of interests

The authors declare that they have no conflict of interest.

Author information

Correspondence to Bin Liao.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Zhang, J., Hua, Z., Huang, Z. et al. Two Blast-independent tools, CyPerl and CyExcel, for harvesting hundreds of novel cyclotides and analogues from plant genomes and protein databases. Planta 241, 929–940 (2015).

Download citation


  • CyPerl
  • CyExcel
  • Cyclotides
  • Genomes
  • Protein databases
  • Plant family