An ecto-nucleoside triphosphate diphosphohydrolase (ecto-NTPDase) has been cloned from human liver RNA by RT-PCR. The 1.5 kb cDNA codes for a protein of 495 amino acids. Sequence analysis indicated that it is most closely related to a chicken ecto-ATPDase previously cloned in our laboratory [Knowles et al. (2002) Eur. J. Biochem. 269, 2373-2382] and a mouse homologue that has been designated as E-NTPDase 8 [Bigonnesses et al. (2004) Biochemistry 43, 5511-5519]. The human E-NTPDase 8 has similar topology as the avian and mouse E-NTPDase 8 but has fewer potential N-glycosylation sites and only two amino acid residues in the cytoplasm at its C-terminus. Despite 52% identity in primary structures, enzymatic properties of human E-NTPDase 8 expressed in HEK293 cells differ from that of the chicken E-NTPDase 8. In contrast to the chicken E-NTPDase 8, the human E-NTPDase 8 hydrolyzes MgADP poorly and is inhibited by several detergents as well as benzyl alcohol; the latter attribute may be related to weaker interaction of the transmembranous domains of the human E-NTPDase 8. To demonstrate that inhibition by detergents is mediated by the transmembranous domains, a recombinant pSecTag2 plasmid containing the extracellular domain (ECD) of the human E-NTPDase 8 was constructed. The soluble human E-NTPDase 8 which was secreted into the culture media of transfected HEK293 cells was purified by ammonium sulfate fractionation and nickel affinity chromatography. Besides becoming resistant to detergent inhibition, the soluble human E-NTPDase 8 ECD displays greater activity with Ca nucleotide substrates, an increased affinity for ATP, different pH dependence, and a decreased sensitivity to azide inhibition when compared to the membrane-bound enzyme. These differences may result from the different conformations that the ECD assume without or with constraints exerted by the transmembranous domains. These results indicate that the transmembranous domains are important in regulating enzyme activity as well as in determining the structure of human E-NTPDase 8.

The human ecto-ATPase (NTPDase 2) contains conserved motifs including five apyrase conserved regions (ACRs) and four conserved regions (CRs) as well as conserved lysine and arginine residues that are also present in other cell surface E-NTPDases. Some of the positively charged amino acids may be involved in ATP binding. The protein also contains six potential N-linked glycosylation sites. Results obtained with seven lysine and six arginine mutants indicate the importance of K62 that is located in CR1, K182, which is downstream of ACR3, and R155, which immediately follows CR3. Mutation of asparagine at the six potential N-linked glycosylation sites individually to glutamine established the importance of N64 in CR1 and N443 in ACR5 in protein function and expression. Mutation of N64, which is conserved in all cell surface NTPDases, results in the expression of an unstable protein, the activity of which is only manifested in the presence of concanavalin A. Both K62 and N64 reside in CR1 that is conserved in all cell surface NTPDases. In the sequence of the CR1 of human ecto-ATPase, 58WPADKENDTGIV69, 65DTG67 is similar to the phosphate-binding motif (DXG) in ACR1 and 4. The D65A and G67A mutants have reduced protein expression and activity. Mutations of other residues in CR1 to alanine led to partial to complete loss of protein expression and activity except for P59. The alanine mutants of the three acidic amino acid residues, D61, E63, and D65, all have decreased affinity for divalent ions. D61 can be substituted by glutamate, but E63 appears to be invariable. Taken together, these results indicate that CR1, which follows ACR1 in the cell surface NTPDases, is an essential structural element in these enzymes.