Co-expression of functional human Heme Oxygenase 1, Ecto-5'-Nucleotidase and ecto-nucleoside triphosphate diphosphohydrolase-1 by "self-cleaving" 2A peptide system

We developed an F2A-based multicistronic system to evaluate functional effects of co-expression of three proteins important for xenotransplantation: heme oxygenase 1 (HO1), ecto-5'-nucleotidase (E5NT) and ecto-nucleoside triphosphate diphosphohydrolase-1 (ENTPD1). The tricistronic p2A plasmid that we constructed was able to efficiently drive concurrent expression of HO1, E5NT and ENTPD1 in HEK293T cells. All three overexpressed proteins possessed relevant enzymatic activities, while addition of furin site interfered with protein expression and activity. We conclude that our tricistronic p2A construct is effective and optimal to test the combined protective effects of HO1, E5NT and ENTPD1 against xeno-rejection mechanisms

Functional analysis of expression of human ecto-nucleoside triphosphate diphosphohydrolase-1 and/or ecto-5′-nucleotidase in pig endothelial cells

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 M ATP or 50 M ADP or 50 M 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 M respectively while it remained below 1 M 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 M 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. © 2014 Taylor and Francis Group, LLC