Asparagine 81, an invariant glycosylation site near apyrase conserved region 1, is essential for full enzymatic activity of ecto-nucleoside triphosphate diphosphohydrolase 3

Abstract

N-linked glycosylation is important for the function, cellular localization, and oligomerization of membrane-bound ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases). NTPDase3 is a prototypical cell membrane-associated eNTPDase, which is equally related and enzymatically intermediate to the other two cell surface membrane NTPDases (NTPDase1 and 2). The protein sequence of NTPDase3 contains seven putative N-glycosylation sites located in the ecto-domain. Only one of these putative glycosylation sites, asparagine 81 in NTPDase3, which is located near apyrase conserved region 1 (ACR1), is invariant in all the cell surface membrane eNTPDases. Using site-directed mutagenesis, mutants were constructed to eliminate this highly conserved N-glycosylation site in NTPDase3. The results indicate that glycosylation at this position is essential for full enzymatic activity, with mutant ATPase activity decreased more than ADPase activity. Enzymatic deglycosylation of this site is shown to be responsible for the inactivation of the wild-type enzyme by treatment with peptide N-glycosidase-F. In addition, glycosylation of this conserved site is necessary for the stabilization/stimulation of nucleotidase activity upon treatment with the lectin concanavalin A. However, lack of glycosylation at this site did not result in large changes in tertiary or quaternary structure, as measured by Cibacron blue binding, chemical cross-linking, and native gel electrophoretic analysis. Since this N-glycosylation site is invariant in cell membrane eNTPDases, it is postulated that glycosylation of this residue near ACR1 is crucial for full enzymatic activity of the cell membrane NTPDases.