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Structural elements and limited proteolysis of CD39 influence ATP diphosphohydrolase activity
J Schulte am Esch1, J Sévigny, E Kaczmarek
1Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Biochemistry
|February 25, 1999
Summary
Specific regions within CD39 (adenosine triphosphate diphosphohydrolase) are crucial for its enzymatic activity. Maintaining intact apyrase conserved regions 1, 4, and 5 is essential for CD39 function.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- CD39 (adenosine triphosphate diphosphohydrolase, ATPDase) is an ectoenzyme with a complex structure.
- It possesses two transmembrane domains and five apyrase conserved regions (ACRs) in its extracellular domain.
Purpose of the Study:
- To investigate the structural requirements of human CD39 for its ectoenzyme activity.
- To identify the specific regions within CD39 essential for maintaining its biochemical function.
Main Methods:
- Site-directed mutagenesis was used to modify ACRs within human CD39.
- Sequential deletions were performed at both the N- and C-termini.
- FLAG tagging was employed to facilitate protein manipulation and analysis.
- Limited tryptic cleavage was used to assess fragment activity.
Main Results:
- ATPDase activity was preserved after FLAG tagging and removal of transmembrane regions.
- Deletions within ACR-1, ACR-4, or truncation mutants including ACR-1, -4, or -5 significantly reduced biochemical activity.
- Intact ACR-1, -4, and -5 are necessary for CD39's biochemical activity.
- CD39 forms multimers via intermolecular disulfide bonds when transmembrane regions are absent.
- Tryptic cleavage generated fragments that augmented ATPDase activity.
- Glycosylation variations did not impact ATPDase function.
Conclusions:
- The study highlights the critical role of intact ACR-1, -4, and -5 in maintaining CD39's ATPDase activity.
- CD39's enzymatic function can be modulated by posttranslational modifications, potentially relevant to vascular inflammation.
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