Jessica Robinson

Jessica Robinson

PI: Richard Gumina, MD, PhD , Division of Cardiovascular Medicine

The Impact of Single Nucleotide Polymorphisms on Ecto-nucleotidase Activities

Ecto-nucleotidases are cell surface molecules that metabolize extracellular nucleotides, and play an important role in vascular physiology and purine receptor signaling pathways. Purinergic signaling is critical for blood vessel homeostasis, atherosclerosis progression, and repair following myocardial infarction (MI). These processes are regulated through hydrolysis of adenosine triphosphate (ATP) to adenosine, known for its anti-inflammatory properties. A critical enzyme in this pathway is ecto-5’-nucleotidase (CD73), which is responsible for catalyzing the final step in this reaction by hydrolyzing the alpha phosphate group from adenosine monophosphate (AMP) to produce adenosine. Previous studies have shown that CD73 is anti-atherosclerotic, anti-thrombotic, and cardio-protective. We are examining a single nucleotide polymorphism (SNP) identified through PheWAS analysis of >24,000 patients associated with atherosclerosis. The most commonly observed SNP results in a missense mutation of a conserved active site arginine to a cysteine. We hypothesize that the substitution of cysteine for the active site arginine decreases the expression and/or activity of CD73. Therefore, the goal of this project is to determine how changes in the coding sequence of CD73 affect its expression and ecto-nucleotidase activity by using site-directed mutagenesis to generate the two isoforms of CD73. Expression of the CD73 mutants in human embryonic kidney (HEK-293) cells was employed to analyze the expression of CD73 by flow cytometry, western blot analysis, and immunohistochemistry. CD73 ecto-nucleotidase activity was assessed using the malachite green assay. The results of this project will define the molecular consequences of CD73 SNPs resulting in an active site missense arginine to a cysteine mutation.