title: 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.
abstract: Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase 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 demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except 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 much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.
authors.affiliation: Department of Medicine, Division of Hematology and Medical Oncology, VA New York Harbor Healthcare System, New York, New York 10010-5050, USA. jhfliess@mail.med.cornell.edu
authors.forename: J H
authors.initials: JH
authors.lastname: Drosopoulos
authors.forename: M J
authors.initials: MJ
authors.lastname: Broekman
authors.forename: N
authors.initials: N
authors.lastname: Islam
authors.forename: C R
authors.initials: CR
authors.lastname: Maliszewski
authors.forename: R B
authors.initials: RB
authors.lastname: Gayle
authors.forename: A J
authors.initials: AJ
authors.lastname: Marcus
mesh_terms: D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001081:Apyrase / Q000235:genetics* / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D017124:Conserved Sequence; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009391:Nephelometry and Turbidimetry; D010974:Platelet Aggregation / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins; D012995:Solubility
chemical_list: D015703:Antigens, CD; D002413:Cations, Divalent; D011994:Recombinant Proteins; D010450:Endopeptidases; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen