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Functional analysis of expression of human ecto-nucleoside triphosphate diphosphohydrolase-1 and/or ecto-5'-nucleotidase in pig endothelial cells. 2014

M De Giorgi, and I Pelikant-Malecka, and A Sielicka, and E M Slominska, and R Giovannoni, and A Cinti, and M G Cerrito, and M Lavitrano, and R T Smolenski
a Department of Surgery and Translational Medicine , University of Milano-Bicocca , Milano , Italy.

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.

UI MeSH Term Description Entries
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000227 Adenine Nucleotides Adenine Nucleotide,Adenosine Phosphate,Adenosine Phosphates,Nucleotide, Adenine,Nucleotides, Adenine,Phosphate, Adenosine,Phosphates, Adenosine
D000241 Adenosine A nucleoside that is composed of ADENINE and D-RIBOSE. Adenosine or adenosine derivatives play many important biological roles in addition to being components of DNA and RNA. Adenosine itself is a neurotransmitter. Adenocard,Adenoscan
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001081 Apyrase A calcium-activated enzyme that catalyzes the hydrolysis of ATP to yield AMP and orthophosphate. It can also act on ADP and other nucleoside triphosphates and diphosphates. EC 3.6.1.5. ADPase,ATP-Diphosphatase,Adenosine Diphosphatase,ADP Phosphohydrolase,ATP Diphosphohydrolase,ATP-ADPase,ATP ADPase,ATP Diphosphatase,Diphosphatase, Adenosine,Diphosphohydrolase, ATP,Phosphohydrolase, ADP
D013552 Swine Any of various animals that constitute the family Suidae and comprise stout-bodied, short-legged omnivorous mammals with thick skin, usually covered with coarse bristles, a rather long mobile snout, and small tail. Included are the genera Babyrousa, Phacochoerus (wart hogs), and Sus, the latter containing the domestic pig (see SUS SCROFA). Phacochoerus,Pigs,Suidae,Warthogs,Wart Hogs,Hog, Wart,Hogs, Wart,Wart Hog
D014162 Transfection The uptake of naked or purified DNA by CELLS, usually meaning the process as it occurs in eukaryotic cells. It is analogous to bacterial transformation (TRANSFORMATION, BACTERIAL) and both are routinely employed in GENE TRANSFER TECHNIQUES. Transfections
D015703 Antigens, CD Differentiation antigens residing on mammalian leukocytes. CD stands for cluster of differentiation, which refers to groups of monoclonal antibodies that show similar reactivity with certain subpopulations of antigens of a particular lineage or differentiation stage. The subpopulations of antigens are also known by the same CD designation. CD Antigen,Cluster of Differentiation Antigen,Cluster of Differentiation Marker,Differentiation Antigens, Leukocyte, Human,Leukocyte Differentiation Antigens, Human,Cluster of Differentiation Antigens,Cluster of Differentiation Markers,Antigen Cluster, Differentiation,Antigen, CD,CD Antigens,Differentiation Antigen Cluster,Differentiation Marker Cluster,Marker Cluster, Differentiation
D015720 5'-Nucleotidase A glycoprotein enzyme present in various organs and in many cells. The enzyme catalyzes the hydrolysis of a 5'-ribonucleotide to a ribonucleoside and orthophosphate in the presence of water. It is cation-dependent and exists in a membrane-bound and soluble form. EC 3.1.3.5. 5'-AMP Nucleotidase,AMP Phosphatase,Adenylate Phosphatase,Antigens, CD73,CD73 Antigens,Cytidylate Phosphatase,Ecto-5'-Nucleotidase,IMP Nucleotidase,IMP Phosphatase,Inosinate Phosphatase,Pyrimidine 5'-Nucleotidase,Thymidine Phosphatase,Uridylate 5'-Nucleotidase,5'-Nucleotidase Phosphoribolase,Antigen, CD73,IMPase,5' AMP Nucleotidase,5' Nucleotidase,5' Nucleotidase Phosphoribolase,CD73 Antigen,Ecto 5' Nucleotidase,Pyrimidine 5' Nucleotidase,Uridylate 5' Nucleotidase
D015870 Gene Expression The phenotypic manifestation of a gene or genes by the processes of GENETIC TRANSCRIPTION and GENETIC TRANSLATION. Expression, Gene,Expressions, Gene,Gene Expressions

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