Mutagenesis of Lysine 62, Asparagine 64, and Conserved Region 1 Reduces the Activity of Human Ecto-ATPase (NTPDase 2)

Abstract

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.

The GDA1_CD39 superfamily: NTPDases with diverse functions

“…Mutation of K62 to alanine resulted in loss of protein expression. Substitution of K62 by arginine restored expression and some activity, which was stimulated by ConA [43]. The corresponding lysine in human NTPDase3 is K79 and similar results were obtained with the K79A and K79R mutants.…”

“…NTPDases 1-3 and 8 have similar molecular sizes (∼500 amino acid) but with variable extent of glycosylation. The glycosylation of these proteins is important for correct protein folding, membrane targeting, and activity [40][41][42][43].…”

“…Basu et al suggested that a positively charged amino acid in the vicinity of the conserved glycosylation site (N81) of human NTPDase3 is necessary for ConA binding [198]. The effect of mutating this conserved asparagine was studied in three enzymes: rat NTPDase1 [42], human NTPDase2 [43], and human NTPDase3 [40]. Mutation of N73 in rat NTPDase1 to serine resulted in loss of only 50% activity.…”

“…Mutation of N81 in NTPDase3 to aspartate or glutamate resulted in loss of activity and the activity could not be stimulated by ConA, suggesting that the oligosaccharide attached to N81 in the wild-type NTPDase3 provides the ConA binding site [41]. The N64Q mutant of human NTPDase2 lost 95% of activity, but almost ∼60% wild-type enzyme activity was obtained after the membrane was preincubated with ConA [43], indicating that N64 is not the exclusive ConA binding site in human NTPDase2. We postulated that lack of glycosylation at N64 perturbed the protein structure and the active site of NTPDase2, which is corrected upon binding ConA.…”

Cellular function and molecular structure of ecto-nucleotidases

“…Numerous mutations have been created to depict amino acid residues particularly within the ACRs responsible for determining catalytic properties [83,84,[113][114][115][116][117]. In addition, five chimeric cDNAs were constructed in which N-terminal domains of increasing length of rat NTPDase1 were replaced by the corresponding sequences of NTPDase2 and vice versa and expressed in Chinese hamster ovary (CHO) cells [118].…”