Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: Requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment
Jhf. Drosopoulos et al., Site-directed mutagenesis of human endothelial cell ecto-ADPase/soluble CD39: Requirement of glutamate 174 and serine 218 for enzyme activity and inhibition of platelet recruitment, BIOCHEM, 39(23), 2000, pp. 6936-6943
Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasi
s. It rapidly metabolizes ADP released from stimulated platelets, thereby p
reventing further platelet activation and recruitment. We recently develope
d a recombinant, soluble form of human CD39, solCD39, with enzymatic and bi
ological properties identical to CD39. To identify amino acids essential fo
r enzymatic/biological activity, we performed site-directed mutagenesis wit
hin the four highly conserved apyrase regions of solCD39. Mutation of gluta
mate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in c
omplete and similar to 90% loss of solCD39 enzymatic activity, respectively
. Furthermore, compared to wildtype, S57A exhibited a 2-fold increase in AD
Pase activity without change in ATPase activity, while the tyrosine 127 to
alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The
ADPase activity of wild-type solCD39 and each mutant, except for R135A, was
greater with calcium as the required divalent cation than with magnesium,
but for ATPase activity generally no such preference was observed. Y127A de
monstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold hi
gher than that of wild-type, even though its enzyme activity was greatly re
duced. SolCD39 mutants were further characterized by correlating enzymatic
with biological activity in an in vitro platelet aggregation system. Each s
olCD39 mutant was similar to wild-type in reversing platelet aggregation, e
xcept for E174A and S218A. E174A, completely devoid of enzymatic activity,
failed to inhibit platelet responsiveness, as anticipated. S218A, with 91%
loss of ADPase activity, could still reverse platelet aggregation, albeit m
uch less effectively than wild-type solCD39. Thus, glutamate 174 and serine
218 are essential for both the enzymatic and biological activity of solCD3
9.