Mutagenesis of lysine 62, asparagine 64, and conserved region 1 reduces the activity of human ecto-ATPase (NTPDase 2)

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, (58)WPADKENDTGIV(69), (65)DTG(67) 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.