Structural and functional characterization of engineered bifunctional fusion proteins of CD39 and CD73 ectonucleotidases

Am J Physiol Cell Physiol. 2021 Jan 1;320(1):C15-C29. doi: 10.1152/ajpcell.00430.2020. Epub 2020 Oct 14.


Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.

Keywords: bifunctional fusion; ectonucleotidase; inflammation; platelet aggregation; purinergic signaling.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • 5'-Nucleotidase / chemistry
  • 5'-Nucleotidase / genetics
  • 5'-Nucleotidase / metabolism
  • 5'-Nucleotidase / pharmacology*
  • Adenine Nucleotides / metabolism
  • Anti-Inflammatory Agents / chemistry
  • Anti-Inflammatory Agents / metabolism
  • Anti-Inflammatory Agents / pharmacology*
  • Apyrase / chemistry
  • Apyrase / genetics
  • Apyrase / metabolism
  • Apyrase / pharmacology*
  • GPI-Linked Proteins / chemistry
  • GPI-Linked Proteins / genetics
  • GPI-Linked Proteins / metabolism
  • GPI-Linked Proteins / pharmacology
  • HEK293 Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Hydrolysis
  • Kinetics
  • Platelet Aggregation / drug effects*
  • Platelet Aggregation Inhibitors / chemistry
  • Platelet Aggregation Inhibitors / metabolism
  • Platelet Aggregation Inhibitors / pharmacology*
  • Protein Conformation
  • Protein Engineering*
  • Recombinant Fusion Proteins / metabolism
  • Signal Transduction
  • Structure-Activity Relationship
  • Substrate Specificity


  • Adenine Nucleotides
  • Anti-Inflammatory Agents
  • GPI-Linked Proteins
  • Platelet Aggregation Inhibitors
  • Recombinant Fusion Proteins
  • 5'-Nucleotidase
  • NT5E protein, human
  • Apyrase
  • ENTPD1 protein, human