title: 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.
authors.affiliation: Centre de Recherche en Rhumatologie et Immunologie, 2705 Boulevard Laurier, local T1-49, G1V 4G2 Québec, QC, Canada.
authors.forename: Michel
authors.initials: M
authors.lastname: Fausther
authors.forename: Joanna
authors.initials: J
authors.lastname: Lecka
authors.forename: Filip
authors.initials: F
authors.lastname: Kukulski
authors.forename: Sébastien A
authors.initials: SA
authors.lastname: Lévesque
authors.forename: Julie
authors.initials: J
authors.lastname: Pelletier
authors.forename: Herbert
authors.initials: H
authors.lastname: Zimmermann
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Dranoff
authors.forename: Jean
authors.initials: J
authors.lastname: Sévigny
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001081:Apyrase / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002384:Catalysis / Q000187:drug effects; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003840:Deoxycholic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019810:Sodium Azide / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D015703:Antigens, CD; D004791:Enzyme Inhibitors; D009711:Nucleotides; D011994:Recombinant Proteins; D003840:Deoxycholic Acid; D019810:Sodium Azide; D000251:Adenosine Triphosphatases; C027201:ectoATPase; C518948:nucleoside triphosphate diphosphohydrolase 8, human; C518947:nucleoside triphosphate diphosphohydrolase 8, rat; D001081:Apyrase; C067869:CD39 antigen