Functional expression of a cDNA encoding a human ecto-ATPase

Abstract

1 The metabolism of extracellular nucleotides plays an important role in nucleotide signalling mediated by P2 receptors. The nucleotide sequence encoding a putative human ecto-ATPase named CD39L1 was reported recently. However, the biological activity of this protein has not been established. 2 Based on the sequence of CD39L1 we isolated from mRNA from human ECV-304 cells a sequence encoding a 495 amino acid protein that is identical to CD39L1, with the exception that this sequence contains a 23 amino acid stretch in the putative extracellular loop that is missing in CD39L1. Partial sequence of a genomic DNA clone indicates that the CD39L1 gene corresponds to an alternative spliced form of the human ecto-ATPase. 3 Stable expression of isolated sequence in NIH-3T3 mouse fibroblasts conferred a marked nucleotide hydrolytic activity consistent with the activity of an ecto-ATPase. 4 The human ecto-ATPase hydrolyzed all naturally occurring nucleoside triphosphates in a Ca(2+)- or Mg(2+)-dependent manner. Nucleoside diphosphates were hydrolyzed at a rate approximately 5% of that of the corresponding triphosphates. The apparent Km and Vmax values were: 394+/-62 microM and 107+/-7 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ATP, and 102+/-33 microM and 4+/-0.4 nmol Pi min-1 10(6) cells-1 for the hydrolysis of ADP, respectively. 5 In conclusion, we report here the cloning and functional expression of a human ecto-ATPase. The study of the biochemical properties and the regulatory mechanisms of ecto-ATPases of defined sequence will be valuable in the definition of their role in nucleotide signalling.

Figure 1

DNA sequence and predicted protein sequence of the human ecto-ATPase. Underlined with solid lines are the two putative transmembrane domains. ▪, Potential N-glycosylation sites; ^*, potential protein kinase A phosphorylation sites; ▴, potential protein kinase C phosphorylation sites. Boxed sequences correspond to apyrase conserved regions. This sequence has been submitted to the GeneBank database with accession number AF144748.

Figure 2

Alignment of deduced amino acid sequences of the human ecto-ATPase, CD39L1, and human CD39. Shaded areas indicate amino acid identity to the human ecto-ATPase described here. Gaps (−) were introduced to improve alignment. GeneBank accession numbers are: U91510 for CD39L1 and S73813 or U87967 for CD39.

Figure 3

Rate of hydrolysis of ATP, ADP and AMP by NIH-3T3 cells expressing the human ecto-ATPase. NIH-3T3 cells expressing the empty pLXPIH vector (a) or the vector containing the ecto-ATPase gene (b) were incubated at 37°C with 100 μM ATP, ADP or AMP as described in Methods. At indicated times the reaction was stopped and the cell-free supernatant was removed from the cells. Nucleotide hydrolysis was estimated as the release of inorganic phosphate. Data shown are the mean±s.e.mean of triplicate assays from a representative experiment repeated at least five times using confluent monolayers of cells seeded in 48-well culture plates.

Figure 4

Substrate dependence for the hydrolysis of ATP and ADP by the human ecto-ATPase. NIH-3T3 cells stably expressing the ecto-ATPase were incubated in the presence of the indicated concentrations of ATP and ADP for 3 min. The rates of hydrolysis were measured as the release of inorganic phosphate and expressed as nmol Pi min⁻¹ 10⁶ cells⁻¹ and were obtained from incubations in which less than 10% of the substrate was hydrolyzed. The data shown are the mean±s.d.mean from five experiments performed with triplicate samples.