Amino Acids

, Volume 43, Issue 1, pp 365–378 | Cite as

Binding activity, structure, and immunogenicity of synthetic peptides derived from Plasmodium falciparum CelTOS and TRSP proteins

  • Hernando CurtidorEmail author
  • Gabriela Arévalo-Pinzón
  • Adriana Bermudez
  • Dayana Calderon
  • Magnolia Vanegas
  • Liliana C. Patiño
  • Manuel A. Patarroyo
  • Manuel E. Patarroyo
Original Article


Several sporozoite proteins have been associated with Plasmodium falciparum cell traversal and hepatocyte invasion, including the cell-traversal protein for ookinetes and sporozoites (CelTOS), and thrombospondin-related sporozoite protein (TRSP). CelTOS and TRSP amino acid sequences have been finely mapped to identify regions specifically binding to HeLa and HepG2 cells, respectively. Three high-activity binding peptides (HABPs) were found in CelTOS and one HABP was found in TRSP, all of them having high α-helical structure content. These HABPs’ specific binding was sensitive to HeLa and HepG2 cells’ pre-treatment with heparinase I and chondroitinase ABC. Despite their similarity at three-dimensional (3D) structural level, TRSP and TRAP HABPs located in the TSR domain did not compete for the same binding sites. CelTOS and TRSP HABPs were used as a template for designing modified sequences to then be assessed in the Aotus monkey experimental model. Antibodies directed against these modified HABPs were able to recognize both the native parasite protein by immunofluorescence assay and the recombinant protein (expressed in Escherichia coli) by Western blot and ELISA assays. The results suggested that these modified HABPs could be promising targets in designing a fully effective, antimalarial vaccine.


Plasmodium falciparum Sporozoite CelTOS TRSP Peptide Vaccine 



We would like to thank Jason Garry for translating this manuscript.

Conflict of interest

The authors declare no conflict of interest. The authors alone are responsible for the content and writing of this manuscript.


  1. Akdi RR, Sharma A, Malhotra P, Sharma A (2008) Role of Plasmodium falciparum thrombospondin-related anonymous protein in host-cell interactions. Malar J 7:63CrossRefGoogle Scholar
  2. Attie A, Raines R (1995) Analysis of receptor-ligand interactions. J Chem Educ 72:119–124CrossRefGoogle Scholar
  3. Bergmann-Leitner ES, Mease RM, De La Vega P, Savranskaya T, Polhemus M, Ockenhouse C, Angov E (2010) Immunization with pre-erythrocytic antigen CelTOS from Plasmodium falciparum elicits cross-species pro1tection against heterologous challenge with Plasmodium berghei. PLoS ONE 5 (8):e12294. doi: 10.1371/journal.pone.0012294
  4. Bermudez A, Vanegas M, Patarroyo ME (2008) Structural and immunological analysis of circumsporozoite protein peptides: a further step in the identification of potential components of a minimal subunit-based, chemically synthesised antimalarial vaccine. Vaccine 26(52):6908–6918PubMedCrossRefGoogle Scholar
  5. Bongfen SE, Ntsama PM, Offner S, Smith T, Felger I, Tanner M, Alonso P, Nebie I, Romero JF, Silvie O, Torgler R, Corradin G (2009) The N-terminal domain of Plasmodium falciparum circumsporozoite protein represents a target of protective immunity. Vaccine 27(2):328–335PubMedCrossRefGoogle Scholar
  6. Cifuentes G, Bermudez A, Rodriguez R, Patarroyo MA, Patarroyo ME (2008) Shifting the polarity of some critical residues in malarial peptides’ binding to host cells is a key factor in breaking conserved antigens’ code of silence. Med Chem 4(3):278–292PubMedCrossRefGoogle Scholar
  7. Cifuentes G, Vanegas M, Martinez NL, Pirajan C, Patarroyo ME (2009) Structural characteristics of immunogenic liver-stage antigens derived from P. falciparum malarial proteins. Biochem Biophys Res Commun 384(4):455–460PubMedCrossRefGoogle Scholar
  8. Combet C, Blanchet C, Geourjon C, Deleage G (2000) NPS@: network protein sequence analysis. Trends Biochem Sci 25(3):147–150PubMedCrossRefGoogle Scholar
  9. Cowman AF, Crabb BS (2006) Invasion of red blood cells by malaria parasites. Cell 124(4):755–766. doi: S0092-8674(06)00181-4[pii]10.1016/j.cell.2006.02.006 PubMedCrossRefGoogle Scholar
  10. Cowman AF, Baldi DL, Duraisingh M, Healer J, Mills KE, O’Donnell RA, Thompson J, Triglia T, Wickham ME, Crabb BS (2002) Functional analysis of Plasmodium falciparum merozoite antigens: implications for erythrocyte invasion and vaccine development. Philos Trans R Soc Lond B Biol Sci 357(1417):25–33. doi: 10.1098/rstb.2001.1010 PubMedCrossRefGoogle Scholar
  11. Curtidor H, Torres MH, Alba MP, Patarroyo ME (2007) Structural modifications to a high-activity binding peptide located within the PfEMP1 NTS domain induce protection against P. falciparum malaria in Aotus monkeys. Biol Chem 388(1):25–36. doi: 10.1515/BC.2007.003 PubMedCrossRefGoogle Scholar
  12. Curtidor H, Arevalo G, Vanegas M, Vizcaino C, Patarroyo MA, Forero M, Patarroyo ME (2008a) Characterization of Plasmodium falciparum integral membrane protein Pf25-IMP and identification of its red blood cell binding sequences inhibiting merozoite invasion in vitro. Protein Sci 17(9):1494–1504. doi: ps.036251.108[pii]10.1110/ps.036251.108 PubMedCrossRefGoogle Scholar
  13. Curtidor H, Garcia J, Vanegas M, Puentes F, Forero M, Patarroyo ME (2008b) Identification of peptides with high red blood cell and hepatocyte binding activity in the Plasmodium falciparum multi-stage invasion proteins: PfSPATR and MCP-1. Biochimie 90(11–12):1750–1759. doi: 10.1016/j.biochi.2008.08.003 PubMedCrossRefGoogle Scholar
  14. Florens L, Washburn MP, Raine JD, Anthony RM, Grainger M, Haynes JD, Moch JK, Muster N, Sacci JB, Tabb DL, Witney AA, Wolters D, Wu Y, Gardner MJ, Holder AA, Sinden RE, Yates JR, Carucci DJ (2002) A proteomic view of the Plasmodium falciparum life cycle. Nature 419(6906):520–526PubMedCrossRefGoogle Scholar
  15. Garcia JE, Puentes A, Patarroyo ME (2006) Developmental biology of sporozoite–host interactions in Plasmodium falciparum malaria: implications for vaccine design. Clin Microbiol Rev 19(4):686–707PubMedCrossRefGoogle Scholar
  16. Gardner MJ, Hall N, Fung E, White O, Berriman M, Hyman RW, Carlton JM, Pain A, Nelson KE, Bowman S, Paulsen IT, James K, Eisen JA, Rutherford K, Salzberg SL, Craig A, Kyes S, Chan MS, Nene V, Shallom SJ, Suh B, Peterson J, Angiuoli S, Pertea M, Allen J, Selengut J, Haft D, Mather MW, Vaidya AB, Martin DM, Fairlamb AH, Fraunholz MJ, Roos DS, Ralph SA, McFadden GI, Cummings LM, Subramanian GM, Mungall C, Venter JC, Carucci DJ, Hoffman SL, Newbold C, Davis RW, Fraser CM, Barrell B (2002) Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419(6906):498–511PubMedCrossRefGoogle Scholar
  17. Havel TF, Wuthrich K (1985) An evaluation of the combined use of nuclear magnetic resonance and distance geometry for the determination of protein conformations in solution. J Mol Biol 182(2):281–294PubMedCrossRefGoogle Scholar
  18. Hay SI, Okiro EA, Gething PW, Patil AP, Tatem AJ, Guerra CA, Snow RW (2010) Estimating the global clinical burden of Plasmodium falciparum malaria in 2007. PLoS Med 7 (6):e1000290. doi: 10.1371/journal.pmed.1000290
  19. Hisaeda H, Yasutomo K, Himeno K (2005) Malaria: immune evasion by parasites. Int J Biochem Cell Biol 37(4):700–706. doi: 10.1016/j.biocel.2004.10.009 PubMedCrossRefGoogle Scholar
  20. Houghten RA (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen–antibody interaction at the level of individual amino acids. Proc Natl Acad Sci USA 82(15):5131–5135PubMedCrossRefGoogle Scholar
  21. Ishino T, Yano K, Chinzei Y, Yuda M (2004) Cell-passage activity is required for the malarial parasite to cross the liver sinusoidal cell layer. PLoS Biol 2(1):E4PubMedCrossRefGoogle Scholar
  22. Ishino T, Chinzei Y, Yuda M (2005) A Plasmodium sporozoite protein with a membrane attack complex domain is required for breaching the liver sinusoidal cell layer prior to hepatocyte infection. Cell Microbiol 7(2):199–208PubMedCrossRefGoogle Scholar
  23. Kaiser K, Camargo N, Coppens I, Morrisey JM, Vaidya AB, Kappe SH (2004a) A member of a conserved Plasmodium protein family with membrane-attack complex/perforin (MACPF)-like domains localizes to the micronemes of sporozoites. Mol Biochem Parasitol 133(1):15–26PubMedCrossRefGoogle Scholar
  24. Kaiser K, Matuschewski K, Camargo N, Ross J, Kappe SH (2004b) Differential transcriptome profiling identifies Plasmodium genes encoding pre-erythrocytic stage-specific proteins. Mol Microbiol 51(5):1221–1232PubMedCrossRefGoogle Scholar
  25. Kappe SH, Buscaglia CA, Nussenzweig V (2004) Plasmodium sporozoite molecular cell biology. Annu Rev Cell Dev Biol 20:29–59PubMedCrossRefGoogle Scholar
  26. Kariu T, Ishino T, Yano K, Chinzei Y, Yuda M (2006) CelTOS, a novel malarial protein that mediates transmission to mosquito and vertebrate hosts. Mol Microbiol 59(5):1369–1379PubMedCrossRefGoogle Scholar
  27. Khusmith S, Charoenvit Y, Kumar S, Sedegah M, Beaudoin RL, Hoffman SL (1991) Protection against malaria by vaccination with sporozoite surface protein 2 plus CS protein. Science 252(5006):715–718PubMedCrossRefGoogle Scholar
  28. Kumar KA, Sano G, Boscardin S, Nussenzweig RS, Nussenzweig MC, Zavala F, Nussenzweig V (2006) The circumsporozoite protein is an immunodominant protective antigen in irradiated sporozoites. Nature 444(7121):937–940. doi: nature05361[pii]10.1038/nature05361 PubMedCrossRefGoogle Scholar
  29. Labaied M, Camargo N, Kappe SH (2007) Depletion of the Plasmodium berghei thrombospondin-related sporozoite protein reveals a role in host cell entry by sporozoites. Mol Biochem Parasitol 153(2):158–166PubMedCrossRefGoogle Scholar
  30. Lambros C, Vanderberg JP (1979) Synchronization of Plasmodium falciparum erythrocytic stages in culture. J Parasitol 65(3):418–420PubMedCrossRefGoogle Scholar
  31. Lopez R, Curtidor H, Urquiza M, Garcia J, Puentes A, Suarez J, Ocampo M, Vera R, Rodriguez LE, Castillo F, Cifuentes G, Patarroyo ME (2001) Plasmodium falciparum: binding studies of peptide derived from the sporozoite surface protein 2 to Hep G2 cells. J Pept Res 58(4):285–292PubMedCrossRefGoogle Scholar
  32. Mayer DC, Mu JB, Kaneko O, Duan J, Su XZ, Miller LH (2004) Polymorphism in the Plasmodium falciparum erythrocyte-binding ligand JESEBL/EBA-181 alters its receptor specificity. Proc Natl Acad Sci USA 101(8):2518–2523PubMedCrossRefGoogle Scholar
  33. Merrifield RB (1963) Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J Am Chem Soc 85:2149–2154CrossRefGoogle Scholar
  34. Mongui A, Angel DI, Moreno-Perez DA, Villarreal-Gonzalez S, Almonacid H, Vanegas M, Patarroyo MA (2010) Identification and characterization of the Plasmodium vivax thrombospondin-related apical merozoite protein. Malar J 9:283. doi: 10.1186/1475-2875-9-283 PubMedCrossRefGoogle Scholar
  35. Patarroyo ME, Patarroyo MA (2008) Emerging rules for subunit-based, multiantigenic, multistage chemically synthesized vaccines. Acc Chem Res 41(3):377–386PubMedCrossRefGoogle Scholar
  36. Patarroyo ME, Cifuentes G, Bermudez A, Patarroyo MA (2008a) Strategies for developing multi-epitope, subunit-based, chemically synthesized anti-malarial vaccines. J Cell Mol Med 12(5B):1915–1935PubMedCrossRefGoogle Scholar
  37. Patarroyo ME, Cifuentes G, Rodriguez R (2008b) Structural characterisation of sporozoite components for a multistage, multi-epitope, anti-malarial vaccine. Int J Biochem Cell Biol 40(3):543–557PubMedCrossRefGoogle Scholar
  38. Pradel G, Garapaty S, Frevert U (2002) Proteoglycans mediate malaria sporozoite targeting to the liver. Mol Microbiol 45(3):637–651PubMedCrossRefGoogle Scholar
  39. Rathore D, Hrstka SC, Sacci JB Jr, De la Vega P, Linhardt RJ, Kumar S, McCutchan TF (2003) Molecular mechanism of host specificity in Plasmodium falciparum infection: role of circumsporozoite protein. J Biol Chem 278(42):40905–40910PubMedCrossRefGoogle Scholar
  40. Reyes C, Patarroyo ME, Vargas LE, Rodriguez LE, Patarroyo MA (2007) Functional, structural, and immunological compartmentalisation of malaria invasive proteins. Biochem Biophys Res Commun 354(2):363–371PubMedCrossRefGoogle Scholar
  41. Roccatano D, Colombo G, Fioroni M, Mark AE (2002) Mechanism by which 2, 2, 2-trifluoroethanol/water mixtures stabilize secondary-structure formation in peptides: a molecular dynamics study. Proc Natl Acad Sci USA 99(19):12179–12184. doi: 10.1073/pnas.182199699 PubMedCrossRefGoogle Scholar
  42. Rodriguez LE, Curtidor H, Urquiza M, Cifuentes G, Reyes C, Patarroyo ME (2008) Intimate molecular interactions of P. falciparum merozoite proteins involved in invasion of red blood cells and their implications for vaccine design. Chem Rev 108(9):3656–3705PubMedCrossRefGoogle Scholar
  43. Sinnis P, Coppi A (2007) A long and winding road: the Plasmodium sporozoite’s journey in the mammalian host. Parasitol Int 56(3):171–178PubMedCrossRefGoogle Scholar
  44. Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI (2005) The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434(7030):214–217PubMedCrossRefGoogle Scholar
  45. Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287(2):252–260PubMedCrossRefGoogle Scholar
  46. Suarez JE, Urquiza M, Puentes A, Garcia JE, Curtidor H, Ocampo M, Lopez R, Rodriguez LE, Vera R, Cubillos M, Torres MH, Patarroyo ME (2001) Plasmodium falciparum circumsporozoite (CS) protein peptides specifically bind to HepG2 cells. Vaccine 19(31):4487–4495PubMedCrossRefGoogle Scholar
  47. Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24 (8):1596–1599. doi: 10.1093/molbev/msm092
  48. Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinformatics Chapter 2:Unit 2 3. doi: 10.1002/0471250953.bi0203s00
  49. Thompson J, Cooke RE, Moore S, Anderson LF, Janse CJ, Waters AP (2004) PTRAMP; a conserved Plasmodium thrombospondin-related apical merozoite protein. Mol Biochem Parasitol 134(2):225–232. doi: 10.1016/j.molbiopara.2003.12.003 PubMedCrossRefGoogle Scholar
  50. Tossavainen H, Pihlajamaa T, Huttunen TK, Raulo E, Rauvala H, Permi P, Kilpelainen I (2006) The layered fold of the TSR domain of P. falciparum TRAP contains a heparin binding site. Protein Sci 15(7):1760–1768. doi: 15/7/1760[pii]10.1110/ps.052068506 PubMedCrossRefGoogle Scholar
  51. Tucker RP (2004) The thrombospondin type 1 repeat superfamily. Int J Biochem Cell Biol 36(6):969–974PubMedCrossRefGoogle Scholar
  52. Wüthrich K (New York 1986) NMR of proteins and nucleic acids. WileyGoogle Scholar
  53. Yuda M, Ishino T (2004) Liver invasion by malarial parasites—how do malarial parasites break through the host barrier? Cell Microbiol 6(12):1119–1125PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hernando Curtidor
    • 1
    • 2
    Email author
  • Gabriela Arévalo-Pinzón
    • 1
    • 2
  • Adriana Bermudez
    • 1
    • 2
  • Dayana Calderon
    • 1
    • 2
  • Magnolia Vanegas
    • 1
    • 2
  • Liliana C. Patiño
    • 1
    • 2
  • Manuel A. Patarroyo
    • 1
    • 2
  • Manuel E. Patarroyo
    • 1
    • 3
  1. 1.Fundación Instituto de Inmunología de Colombia FIDICBogotáColombia
  2. 2.Universidad del RosarioBogotáColombia
  3. 3.Universidad Nacional de ColombiaBogotáColombia

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