Acta Parasitologica

, Volume 60, Issue 1, pp 54–64 | Cite as

Cloning and expression of apyrase gene from Ancylostoma caninum in Escherechia coli

  • Qiao Ying 
  • Theerakamol PengsakulEmail author


Apyrase encoding metal-ions activated plasma membrane protease is present in animal and plant tissues. This enzyme can hydrolyze ADP and ATP pyrophosphate bond, resulting in AMP and free phosphate groups, and plays an important role for insects and parasites to evade host immune system. However localization and function of apyrase in the canine hookworm, Ancylostoma caninum, remains unknown. To analyze apyrase gene in A. caninum (a eukaryotic parasitic hookworm), a pair of primers was designed according to the previous EST data. The full-length cDNA of apyrase gene was amplified from A. caninum by RT-PCR. The partial cDNA of apyrase encodes 249 amino acid protein was expressed in Escherechia coli. The recombinant protein was induced to express under proper conditions and the molecular size was as expected. The recombinant protein was purified. The transcripts of apyrase in different stages of A. caninum were analyzed by the Real-time PCR assay, and Immuno-localization assays were used to research the protein expression in different stages of A. caninum.


Ancylostoma caninum apyrase clone real-time PCR expression Immuno-localization 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Barros F.S., de Menezes L.F., Pinheiro A.A.S. et al. 2000. Ectonucleotide Diphosphohydrolase Activities in Entamoeba histolytica [J]. Arch Biochem Biophys, 375, 304–314, DOI: 10.1006/abbi.1999.1592CrossRefGoogle Scholar
  2. Berredo-Pinho M., Peres-Sampaio C.E., Chrispim P.P. et al. 2001. A Mg-dependent ecto-ATPase in Leishmania amazonensis and its possible role in adenosine acquisition and virulence [J]. Arch Biochem Biophys, 391, 16–24, DOI: 10.1006/abbi.2001.2384CrossRefGoogle Scholar
  3. Bisaggio D.F., Peres-Sampaio C.E., Meyer-Fernandes J.R. et al. 2003. Ecto-ATPase activity on the surface of Trypanosoma cruzi and its possible role in the parasite-host cell interaction [J]. Parasitology research, 91, 273–282, DOI: 10.1007/s00436-003-0965-8CrossRefGoogle Scholar
  4. Bours M.J., Swennen E.L., Di Virgilio F. et al. 2006. Adenosine 5′- triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation [J]. Pharmacology and Therapeutics, 112, 358–404, DOI: 10.1016/j.pharmthera.2005.04.013CrossRefGoogle Scholar
  5. Champagne D.E., Smartt C.T., Ribeiro J.M. et al. 1995. The salivary gland-specific apyrase of the mosquito Aedes aegypti is a member of the 5’-nucleotidase family [J]. Proc Natl Acad Sci U S A, 92, 694–698, DOI: 10.1073/pnas.92.3.694CrossRefGoogle Scholar
  6. Charlab R., Valenzuela J.G., Rowton E.D. et al. 1999. Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis [J]. Proc Natl Acad Sci U S A, 96, 15155–15160, DOI: 10.1073/pnas.96.26.15155CrossRefGoogle Scholar
  7. Cross M.L., Cupp M.S., Cupp E.W. et al. 1993. Modulation of murine immunological responses by salivary gland extract of Simulium vittatum (Diptera: Simuliidae) [J]. J Med Entomol, 30, 928–935CrossRefGoogle Scholar
  8. D. LeBel G.G.P., S. Phaneuf P. St-Jean, 1980. Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas [J]. Journal of Biological Chemistry, 255, 1227–1233Google Scholar
  9. de Jesus J.B., Podlyska T.M., Hampshire A. et al. 2002. Characterization of an ecto-phosphatase activity in the human parasite Trichomonas vaginalis [J]. Parasitol Research, 88, 991–997, DOI: 10.1007/s00436-001-0583-2CrossRefGoogle Scholar
  10. Deirdre M. Murphy V.V.I., Terence L. Kirley. 2003. Bacterial Expression and Characterization of a Novel, Soluble, Calcium-Binding, and Calcium-Activated Human Nucleotidase [J]. Biochemistry, 42, 2412–2421, DOI: 10.1021/bi026763bCrossRefGoogle Scholar
  11. Devader C., Webb R.J., Thomas G.M.H. et al. 2006. Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development [J]. Gene, 367, 135–141, DOI: 10.1016/j.gene.2005.10.014CrossRefGoogle Scholar
  12. Failer B.U., Braun N., Zimmermann H. 2002. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase [J]. Journal of Biological Chemistry, 277, 36978–36986, DOI: 10.1074/jbc.M201656200CrossRefGoogle Scholar
  13. Gounaris K., Selkirk M.E. 2005. Parasite nucleotide-metabolizing enzymes and host purinergic signalling [J]. Trends in Parasitology, 21, 17–21, DOI: 10.1016/ Scholar
  14. Gounaris K., Selkirk M.E., Sadeghi S.J. 2004. A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis [J]. Mol Biochem Parasitol, 136, 257–264, DOI: 10.1016/j.molbiopara.2004.04.008CrossRefGoogle Scholar
  15. Guan SH L.H., Yang D.L., Lu M.J., Roggendorf M., Schlaak J. 2005. Establishment of a new low-density cDNA macroarray and the application in the activity of IFN against HBV [J]. Chinese journal of experimental and clinical virology, 19, 236–239PubMedGoogle Scholar
  16. Knowles A.F., Isler R.E., Reece J.F. 1983. The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes [J]. Biochim Biophys Acta, 731, 88–96, DOI: 10.1016/0005-2736(83)90401-7CrossRefGoogle Scholar
  17. Komoszynski M., Wojtczak A. 1996. Apyrases (ATP diphosphohydrolases, EC function and relationship to ATPases [J]. Biochimica et Biophysica Acta (BBA) — Molecular Cell Research, 1310, 233–241, DOI: 10.1016/0167-4889(95)00135-2CrossRefGoogle Scholar
  18. Law J.H., Ribeiro J.M., Wells M.A. 1992. Biochemical insights derived from insect diversity [J]. Annual review of biochemistry, 61, 87–111, DOI: 10.1146/ Scholar
  19. Lemos A.P., Peres-Sampaio C.E., Guimaraes-Motta H. et al. 2000. Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase [J]. ZEITSCHRIFT FUR NATURFORSCHUNG C, 55, 392–398CrossRefGoogle Scholar
  20. Lewis-Carl S., Kirley T.L. 1997. Immunolocalization of the Ecto-ATPase and Ecto-apyrase in Chicken Gizzard and Stomach: PURIFICATION AND N-TERMINAL SEQUENCE OF THE STOMACH ECTO-APYRASE [J]. Journal of Biological Chemistry, 272, 23645–23652, DOI: 10.1074/jbc.272.38.23645CrossRefGoogle Scholar
  21. Marcus A.J., Safier L.B. 1993. Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis [J]. The FASEB Journal, 7, 516–522CrossRefGoogle Scholar
  22. Meyer-Fernandes J.R., Dutra P.M., Rodrigues C.O. et al. 1997. Mg-dependent ecto-ATPase activity in Leishmania tropica [J]. Arch Biochem Biophys, 341, 40–46, DOI: 10.1006/abbi.1997.9933CrossRefGoogle Scholar
  23. Nakaar V., Beckers C.J., Polotsky V. et al. 1998. Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase [J]. Mol Biochem Parasitol, 97, 209–220, DOI: 10.1016/S0166-6851(98)00153-4CrossRefGoogle Scholar
  24. Nisbet A.J., Zarlenga D.S., Knox D.P. et al. 2011. A calcium-activated apyrase from Teladorsagia circumcincta: an excretory/secretory antigen capable of modulating host immune responses? [J]. Parasite Immunol, 33, 236–243, DOI: 10.1111/j.1365-3024.2011.01278.xCrossRefGoogle Scholar
  25. Peres-Sampaio C.E., Palumbo S.T., Meyer-Fernandes J. 2001. An ecto-ATPase activity present in Leishmania tropica stimulated by dextran sulfate [J]. ZEITSCHRIFT FUR NATURFORSCHUNG C, 56, 820–825CrossRefGoogle Scholar
  26. Plesner L. 1995. Ecto-ATPases: identities and functions [J]. International review of cytology, 158, 141–214, DOI: 10.1016/S0074-7696(08)62487-0CrossRefGoogle Scholar
  27. Ribeiro J. 1987. Role of saliva in blood-feeding by arthropods [J]. Annual review of entomology, 32, 463–478, DOI: 10.1146/annurev.en.32.010187.002335CrossRefGoogle Scholar
  28. Smith T.M., Hicks-Berger C.A., Kim S. et al. 2002. Cloning, expression, and characterization of a soluble calcium-activated nucleotidase, a human enzyme belonging to a new family of extracellular nucleotidases [J]. Arch Biochem Biophys, 406, 105–115, DOI: 10.1016/S0003-9861(02)00420-4CrossRefGoogle Scholar
  29. Strobel R.S., Nagy A.K., Knowles A.F. et al. 1996. Chicken Oviductal Ecto-ATP-Diphosphohydrolase: PURIFICATION AND CHARACTERIZATION [J]. Journal of Biological Chemistry, 271, 16323–16331, DOI: 10.1074/jbc.271.27.16323CrossRefGoogle Scholar
  30. Ting-Fang Wang G.G. 1996. CD39 Is an Ecto-(Ca2+,Mg2+)-apyrase [J]. Journal of Biological Chemistry, 271, 9898–9901, DOI: 10.1074/jbc.271.17.9898CrossRefGoogle Scholar
  31. Trautmann A. 2009. Extracellular ATP in the immune system: more than just a” danger signal” [J]. Science signaling, 2, pe6, DOI: 10.1126/scisignal.256pe6CrossRefGoogle Scholar
  32. Uccelletti D., Pascoli A., Farina F. et al. 2008. APY-1, a novel Caenorhabditis elegans apyrase involved in unfolded protein response signalling and stress responses [J]. Molecular Biology of the Cell, 19, 1337–1345, DOI: 10.1091/mbc.E07-06-0547CrossRefGoogle Scholar
  33. Valenzuela J.G., Belkaid Y., Rowton E. et al. 2001. The salivary apyrase of the blood-sucking sand fly Phlebotomus papatasi belongs to the novel Cimex family of apyrases [J]. The Journal of Experimental Biology, 204, 229–237PubMedGoogle Scholar
  34. Williamson A.L., Brindley P.J., Abbenante G. et al. 2002. Cleavage of hemoglobin by hookworm cathepsin D aspartic proteases and its potential contribution to host specificity [J]. The FASEB Journal, 16, 1458–1460, DOI: 10.1096/fj.02-0181fjeCrossRefGoogle Scholar
  35. Yoshida M., Amano T. 1995. A common topology of proteins catalyzing ATP-triggered reactions [J]. FEBS Lett, 359, 1–5, DOI: 0014-5793(94)01438-7CrossRefGoogle Scholar
  36. Zimmermann H., Braun N. 1996. Extracellular metabolism of nucleotides in the nervous system [J]. Journal of autonomic pharmacology, 16, 397–400, DOI: 10.1111/j.1474-8673.1996.tb00062.xCrossRefGoogle Scholar

Copyright information

© W. Stefański Institute of Parasitology, PAS 2015

Authors and Affiliations

  1. 1.College of Ocean and Earth SciencesXiamen UniversityXiamen Fujian ProvincePeople’s Republic of China
  2. 2.Faculty of Medical TechnologyPrince of Songkla UniversityHat Yai, SongkhlaThailand

Personalised recommendations