Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity.
CD39, the mammalian ATP diphosphohydrolase (ATPDase), is thought to contain two transmembrane domains and five "apyrase conserved regions" (ACR) within a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by sequential deletions at both termini. ATPDase activity was well preserved with FLAG tagging, followed by the removal of either of the demonstrated C- or N-transmembrane regions. However, deletions within ACR-1 (aa 54-61) or -4 (aa 212-220), as well as truncation mutants that included ACR-1, -4, or -5 (aa 447-454), resulted in substantive loss of biochemical activity. Intact ACR-1, -4, and -5 within CD39 are therefore required for maintenance of biochemical activity. Native and mutant forms of CD39 lacking TMR were observed to undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two noncovalently membrane-associated fragments (56 and 27 kDa) that substantially augmented ATPDase activity. Glycosylation variation accounted for minor heterogeneity in native and mutant forms of CD39 but did not influence ATPDase function. Enzymatic activity of ATPDase may be influenced by certain posttranslational modifications that are relevant to vascular inflammation.
Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
J
J
Schulte am Esch
J
J
Sévigny
E
E
Kaczmarek
J B
JB
Siegel
M
M
Imai
K
K
Koziak
A R
AR
Beaudoin
S C
SC
Robson
D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D001081:Apyrase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D010450:Endopeptidases / Q000378:metabolism*; D004730:Endothelium, Vascular; D004789:Enzyme Activation / Q000235:genetics; D005434:Flow Cytometry; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000235:genetics; D015202:Protein Engineering; D017384:Sequence Deletion; D013552:Swine; D014471:Umbilical Veins
D015703:Antigens, CD; D007527:Isoenzymes; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; C085706:FLAG peptide; D010450:Endopeptidases; D000251:Adenosine Triphosphatases; D001081:Apyrase; C067869:CD39 antigen