The adenosine transporter of Toxoplasma gondii. Identification by insertional mutagenesis, cloning, and recombinant expression

Chi Wu Chiang, Nicola Carter, William J. Sullivan, Robert G.K. Donald, David S. Roos, Fardos N.M. Naguib, Mahmoud H. El Kouni, Buddy Ullman, Craig M. Wilson

研究成果: Article同行評審

71 引文 斯高帕斯(Scopus)

摘要

Purine transport into the protozoan parasite Toxoplasma gondii plays an indispensable nutritional function for this pathogen. To facilitate genetic and biochemical characterization of the adenosine transporter of the parasite, T. gondii tachyzoites were transfected with an insertional mutagenesis vector, and clonal mutants were selected for resistance to the cytotoxic adenosine analog adenine arabinoside (Ara-A). Whereas some Ara-A- resistant clones exhibited disruption of the adenosine kinase (AK) locus, others displayed normal AK activity, suggesting that a second locus had been tagged by the insertional mutagenesis plasmid. These Ara-A(r) AK+ mutants displayed reduced adenosine uptake capability, implying a defect in adenosine transport. Sequences flanking the transgene integration point in one mutant were rescued from a genomic library of Ara-A(r) AK+ DNA, and Southern blot analysis revealed that all Ara-A(r) AK+ mutants were disrupted at the same locus. Probes derived from this locus, designated TgAT, were employed to isolate genomic and cDNA clones from wild-type libraries. Conceptual translation of the TgAT cDNA open reading frame predicts a 462 amino acid protein containing 11 transmembrane domains, a primary structure and membrane topology similar to members of the mammalian equilibrative nucleoside transporter family. Expression of TgAT cRNA in Xenopus laevis oocytes increased adenosine uptake capacity in a saturable manner, with an apparent K(m) value of 114 μM. Uptake was inhibited by various nucleosides, nucleoside analogs, hypoxanthine, guanine, and dipyridamole. The combination of genetic and biochemical studies demonstrates that TgAT is the sole functional adenosine transporter in T. gondii and a rational target for therapeutic intervention.

原文English
頁(從 - 到)35255-35261
頁數7
期刊Journal of Biological Chemistry
274
發行號49
DOIs
出版狀態Published - 1999 12月 3

All Science Journal Classification (ASJC) codes

  • 生物化學
  • 分子生物學
  • 細胞生物學

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