Cloning, purification, and identification of the liver canalicular ecto-ATPase as NTPDase8

Abstract

Extracellular nucleotides regulate critical liver functions via the activation of specific transmembrane receptors. The hepatic levels of extracellular nucleotides, and therefore the related downstream signaling cascades, are modulated by cell-surface enzymes called ectonucleotidases, including nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39), NTPDase2/CD39L1, and ecto-5'-nucleotidase/CD73. The goal of this study was to determine the molecular identity of the canalicular ecto-ATPase/ATPDase that we hypothesized to correspond to the recently cloned NTPDase8. Human and rat NTPDase8 cDNAs were cloned, and the genes were located on chromosome loci 9q34 and 3p13, respectively. The recombinant proteins, expressed in COS-7 and HEK293T cells, were biochemically characterized. NTPDase8 was also purified from rat liver by Triton X-100 solubilization, followed by DEAE, Affigel Blue, and concanavalin A chromatographies. Importantly, NTPDase8 was responsible for the major ectonucleotidase activity in liver. The ion requirement, apparent K(m) values, nucleotide hydrolysis profile, and preference as well as the resistance to azide were similar for recombinant NTPDase8s and both purified rat NTPDase8 and porcine canalicular ecto-ATPase/ATPDase. The partial NH(2)-terminal amino acid sequences of all NTPDase8s share high identity with the purified liver canalicular ecto-ATPase/ATPDase. Histochemical analysis showed high ectonucleotidase activities in bile canaliculi and large blood vessels of rat liver, in agreement with the immunolocalization of NTPDase1, 2, and 8 with antibodies developed for this study. No NTPDase3 expression could be detected in liver. In conclusion, NTPDase8 is the canalicular ecto-ATPase/ATPDase and is responsible for the main hepatic NTPDase activity. The canalicular localization of this enzyme suggests its involvement in the regulation of bile secretion and/or nucleoside salvage.

High dose of extracellular ATP switched autophagy to apoptosis in anchorage-dependent and anchorage-independent hepatoma cells

“…To further verify that eATP, not its derivates, was responsible for these effects on hepatoma cells, we used a non-hydrolysable ATP analogue, BzATP, to investigate its effect on hepatoma cells and confirmed that these effects were mediated by eATP. Though both literature [36] and our data showed that hepatoma cells had no detectable CD39 expression (data not shown), another two ectonucleotidases, NTPDase2 and NTPDase8, were expressed on hepatoma cells [36,37], which might cause degradation of eATP. Furthermore, CD39/ENTPD1 expressed on non-parenchymal cells, including sinusoidal immune and endothelial cells, could hydrolyze eATP in a paracrine manner.…”

Cellular function and molecular structure of ecto-nucleotidases

“…Since coexpression of both NTPDase3α and NTPDase3β reduced the amount of NTPDase3α targeted to the plasma membrane, the authors speculated that NTPDase3β could function as a possible modulator of nucleotidase activity and purinergic signaling [105]. NTPDase8 (495 aa) was cloned from liver cDNA libraries, heterologously expressed and functionally characterized [71,72].…”

Expression of mediators of purinergic signaling in human liver cell lines

“…NTPDase8 has been identified as a hepatocyte canalicular marker [57]; however, expression was detected in HSC and Mz-ChA-1 cells as well. In contrast, CD73 is expressed in hepatocytes in normal liver; however, expression is dramatically redistributed to HSC [58] in the cirrhotic liver.…”