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Licensed Unlicensed Requires Authentication Published by De Gruyter December 30, 2014

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

Ying Qiao and Theerakamol Pengsakul
From the journal Acta Parasitologica


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


Barros F.S., De Menezes L.F., Pinheiro A.A.S. et al. 2000. Ectonucleotide Diphosphohydrolase Activities in Entamoeba histolytica. Archives of Biochemistry and Biophysics, 375, 304-314. DOI: 10.1006/abbi.1999.1592Search in Google Scholar

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. Archives of Biochemistry and Biophysics, 391, 16-24, DOI: 10.1006/abbi.2001.2384Search in Google Scholar

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. Parasitology research, 91, 273-282. DOI: 10.1007/s00436-003-0965-8Search in Google Scholar

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. Pharmacology and Therapeutics, 112, 358-404. DOI: 10.1016/j.pharmthera.2005. 04.013Search in Google Scholar

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. Proceeding of the National Academy of Science USA, 92, 694-698. DOI: 10.1073/pnas.92.3.694Search in Google Scholar

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. Proceeding of the National Academy of Science USA, 96, 15155-15160. DOI: 10.1073/pnas. 96.26.15155Search in Google Scholar

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). Journal of Medical Entomology, 30, 928-935Search in Google Scholar

D LeBel G.G.P., S Phaneuf P. St-Jean, 1980. Characterization and purification of a calcium-sensitive ATP diphosphohydrolase from pig pancreas. Journal of Biological Chemistry, 255, 1227-1233 Search in Google Scholar

de Jesus J.B., Podlyska T.M., Hampshire A. et al. 2002. Characterization of an ecto-phosphatase activity in the human parasite Trichomonas vaginalis. Parasitology Research, 88, 991-997. DOI: 10.1007/s00436-001-0583-2Search in Google Scholar

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. Biochemistry, 42, 2412-2421. DOI: 10.1021/bi026763b Search in Google Scholar

Devader C., Webb R.J., Thomas G.M.H. et al. 2006. Xenopus apyrase (xapy), a secreted nucleotidase that is expressed during early development. Gene, 367, 135-141. DOI: 10. 1016/j.gene.2005.10.014Search in Google Scholar

Failer B.U., Braun N., Zimmermann H. 2002. Cloning, expression, and functional characterization of a Ca(2+)-dependent endoplasmic reticulum nucleoside diphosphatase. Journal of Biological Chemistry, 277, 36978-36986. DOI: 10.1074/jbc. M201656200Search in Google Scholar

Gounaris K., Selkirk M.E. 2005. Parasite nucleotide-metabolizing enzymes and host purinergic signalling. Trends in Parasitology, 21, 17-21, DOI: 10.1016/ in Google Scholar

Gounaris K., Selkirk M.E., Sadeghi S.J. 2004. A nucleotidase with unique catalytic properties is secreted by Trichinella spiralis. Molecular and Biochemical Parasitology, 136, 257-264. DOI: 10.1016/j.molbiopara.2004.04.008Search in Google Scholar

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. Chinese journal of experimental and clinical virology, 19, 236-239Search in Google Scholar

Knowles A.F., Isler R.E., Reece J.F. 1983. The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes. Biochimica et Biophysica Acta, 731, 88-96. DOI: 10. 1016/0005-2736(83)90401-7Search in Google Scholar

Komoszynski M., Wojtczak A. 1996. Apyrases (ATP diphosphohydrolases, EC function and relationship to ATPases. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1310, 233-241, DOI: 10.1016/0167-4889(95)00 135-2Search in Google Scholar

Law J.H., Ribeiro J.M., Wells M.A. 1992. Biochemical insights derived from insect diversity. Annual review of biochemistry, 61, 87-111, DOI: 10.1146/ in Google Scholar

Lemos A.P., Peres-Sampaio C.E., Guimaraes-Motta H. et al. 2000. Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase. Journal Zeitschrift für Naturforschung C, 55, 392-398Search in Google Scholar

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. Journal of Biological Chemistry, 272, 23645-23652. DOI: 10.1074/jbc.272.38.23645Search in Google Scholar

Marcus A.J., Safier L.B. 1993. Thromboregulation: multicellular modulation of platelet reactivity in hemostasis and thrombosis. The FASEB Journal, 7, 516-522Search in Google Scholar

Meyer-Fernandes J.R., Dutra P.M., Rodrigues C.O. et al. 1997. Mg-dependent ecto-ATPase activity in Leishmania tropica. Archives of Biochemistry and Biophysics, 341, 40-46. DOI: 10.1006/abbi.1997.9933Search in Google Scholar

Nakaar V., Beckers C.J., Polotsky V. et al. 1998. Basis for substrate specificity of the Toxoplasma gondii nucleoside triphosphate hydrolase. Molecular and Biochemical Parasitology, 97, 209-220. DOI: 10.1016/S0166-6851(98)00153-4Search in Google Scholar

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? Parasite Immunology, 33, 236-243. DOI: j.1365-10.1111/3024.2011.01278.x Search in Google Scholar

Peres-Sampaio C.E., Palumbo S.T., Meyer-Fernandes J. 2001. An ecto-ATPase activity present in Leishmania tropica stimulated by dextran sulfate. Journal Zeitschrift für Naturforschung C, 56, 820-825Search in Google Scholar

Plesner L. 1995. Ecto-ATPases: identities and functions. International review of cytology, 158, 141-214. DOI: 10.1016/ S0074-7696(08)62487-0Search in Google Scholar

Ribeiro J. 1987. Role of saliva in blood-feeding by arthropods. Annual review of entomology, 32, 463-478. DOI: 10.1146/ annurev.en.32.010187.002335Search in Google Scholar

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. Archives of Biochemistry and Biophysics, 406, 105-115. DOI: 10.1016/S0003-9861(02) 00420-4Search in Google Scholar

Strobel R.S., Nagy A.K., Knowles A.F. et al. 1996. Chicken Oviductal Ecto-ATP-Diphosphohydrolase: PURIFICATION AND CHARACTERIZATION. Journal of Biological Chemistry, 271, 16323-16331. DOI: 10.1074/jbc.271.27.16323Search in Google Scholar

Ting-Fang Wang G.G. 1996. CD39 Is an Ecto-(Ca2+,Mg2+)-apyrase. Journal of Biological Chemistry, 271, 9898-9901. DOI: 10.1074/jbc.271.17.9898Search in Google Scholar

Trautmann A. 2009. Extracellular ATP in the immune system: more than just a” danger signal”. Science Signaling, 2, pe6. DOI: 10.1126/scisignal.256pe6Search in Google Scholar

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. Molecular Biology of the Cell, 19, 1337-1345. DOI: 10.1091/mbc.E07-06-0547Search in Google Scholar

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. The Journal of Experimental Biology, 204, 229-237Search in Google Scholar

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. The FASEB Journal, 16, 1458-1460. DOI: 10.1096/fj.02-0181fje Search in Google Scholar

Yoshida M., Amano T. 1995. A common topology of proteins catalyzing ATP-triggered reactions. FEBS Letters, 359, 1-5. DOI: 0014-5793(94)01438-7 Search in Google Scholar

Zimmermann H., Braun N. 1996. Extracellular metabolism of nucleotides in the nervous system. Journal of Autonomic Pharmacology, 16, 397-400, DOI: 10.1111/j.1474-8673.1996. tb00062.x Search in Google Scholar

Received: 2014-3-24
Revised: 2014-9-9
Accepted: 2014-9-9
Published Online: 2014-12-30
Published in Print: 2014-3-1

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