Sequencing, functional expression and characterization of rat NTPDase6, a nucleoside diphosphatase and novel member of the ecto-nucleoside triphosphate diphosphohydrolase family

We have isolated and characterized the cDNA encoding nucleoside triphosphat e diphosphohydrolase 6 (NTPDase6), a novel member of the ecto-nucleoside tr iphosphate diphosphohydrolase family. The rat-brain-derived cDNA has an ope n reading frame of 1365 bp encoding a protein of 455 amino acid residues, a calculated molecular mass of 49971 Da and a predicted N-terminal hydrophob ic sequence. It shares 86% sequence identity with the human CD39L2 sequence and 48% acid 51% identity respectively with sequences of the two related h uman and murine nucleoside diphosphatases (CD39L4, NTPDase5/ER-UDPase). The mRNA was expressed in all tissues investigated, revealing two major transc ripts with differing abundances. PCR analysis suggests a single open readin g frame. A Myc-His-tagged NTPDase6 was expressed in Chinese hamster ovary ( CHO) and PC12 cells for immunological analysis and protein isolation. The p rotein was contained in membrane fractions of transfected CHO cells and occ urred in a soluble form in the cell culture supernatants. NTPDase6 preferen tially hydrolysed nucleoside 5'-diphosphates. With different substrates the order of activity was GDP > IDP >> UDP, CDP >> ADP. Nucleoside 5'-triphosp hates were hydrolysed only to a minor extent and no hydrolysis of nucleosid e 5'-monophosphates was observed. The enzyme was strongly and equally activ ated by Ca2+ and Mg2+ and had a K-m for GDP of 211 muM. The immunohistochem ical analysis of transfected CHO and PC12 cells suggests that NTPDase6 is a ssociated with the Golgi apparatus and to a small extent also with the plas ma membrane. The enzyme might support glycosylation reactions in the Golgi apparatus and, when released from cells, might catalyse the hydrolysis of e xtracellular nucleotides.