Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly - The N-glycosylation state of CD39 correlates with surface activity and localization
Xt. Zhong et al., Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly - The N-glycosylation state of CD39 correlates with surface activity and localization, J BIOL CHEM, 276(44), 2001, pp. 41518-41525
CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosp
hohydrolase family. The active site for native CD39 is located on the outer
surface of the cellular plasma membrane; however, it is not yet known at w
hat stage this enzyme becomes active along the secretory pathway to the pla
sma membrane. In this study, sucrose density fractionations performed on CD
39-transfected COS-7 cell membranes suggest that CD39 activity resides prim
arily in the plasma membrane. Furthermore, we have created recombinant, sol
uble versions of CD39, one that is secreted and others that are retained in
the endoplasmic reticulum, to demonstrate that CD39 is not active until it
reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the s
ecreted active soluble CD39 in COS-7 cells is found to receive a higher deg
ree of N-glycan addition than the inactive form retained intracellularly. W
hen COS-7 cells were treated with tunicamycin to prevent N-glycosylation, s
oluble CD39 was not detected in the extracellular medium and remained inact
ive intracellularly. Surface biotinylation analysis also revealed that surf
ace-expressed wild type CD39 receives a higher degree of N-glycosylation th
an intracellular forms and that inhibition of N-glycosylation prevents its
plasma membrane localization. In addition, both intact and digitonin-permea
blized COS-7 cells transfected with CD39 possess similar ecto-ATPase activi
ties, further supporting the conclusion that only surface-expressed CD39 is
enzymatically active. All of these data suggest that intracellular CD39 is
inactive and that only a fully glycosylated CD39 has apyrase activity and
is localized at the cell surface.