Functional Analysis of Expression of Human Ecto-Nucleoside Triphosphate Diphosphohydrolase-1 and/or Ecto-5′-Nucleotidase in Pig Endothelial Cells

Abstract

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5′-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 μM ATP or 50 μM ADP or 50 μM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2 ± 1.1 and 24.5 ± 3.4 μM respectively while it remained below 1 μM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7 ± 0.1 and 5.7 ± 2.2 μM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.

Keywords:

• Ecto-5′-Nucleotidase
• ecto-nucleoside triphosphate diphosphohydrolase-1
• endothelial cells
• Adenosine
• Nucleotides
• xenotransplantation
• 2A