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) withi n a large extracellular region. To study the structure of this ectoenzyme, human CD39 was modified by directed mutations within these ACRs or by seque ntial deletions at both termini, ATPDase activity was well preserved with F LAG 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 bioche mical activity. Native and mutant forms of CD39 lacking TMR were observed t o undergo multimerization, associated with the formation of intermolecular disulfide bonds. Limited tryptic cleavage of intact CD39 resulted in two no ncovalently membrane-associated fragments (56 and 27 kDa) that substantiall y augmented ATPDase activity. Glycosylation variation accounted for minor h eterogeneity in native and mutant forms of CD39 but did not influence ATPDa se function. Enzymatic activity of ATPDase may be influenced by certain pos ttranslational modifications that are relevant to vascular inflammation.