Molecular Evidence of Adenosine Deaminase Linking Adenosine A2A Receptor and CD26 Proteins

Abstract

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits.

Therapeutic Perspectives of Adenosine Deaminase Inhibition in Cardiovascular Diseases

“…[ 4 ] ADA1 constitutes the majority of ADA activity and it is present in virtually all tissues, with the highest levels in lymphoid cells, red blood cells and endothelium [ 4 , 5 ]. In addition to cytosolic form, ADA1 can be found as an ecto-enzyme binding to the cell surface by CD26 protein or adenosine receptors [ 6 ]. The second iso-enzyme (ADA2) belongs to the adenosine deaminase growth factor family and it is found with ADA1 mainly in monocytes/macrophages [ 7 ].…”

“…Except its enzymatic function, cell-surface eADA plays a significant extra-enzymatic role in the interactions between cells that expressed ADA-anchoring proteins on their surfaces ( Figure 1 ). Indeed, there is an evidence of the formation of trimeric complexes dipeptidyl peptidase IV (CD26)-ADA-A 2A R involving two cells [ 6 ]. This co-stimulatory and cell-to-cell connecting actions of eADA along with its activity regulate many cellular processes related to proliferation and differentiation, which affect pathological conditions associated with cardiovascular diseases such as endothelial activation and dysfunction, inflammation, myocardial ischemia-reperfusion injury or coagulation disorders.…”

Correlation of adenosine deaminase operating under nitro-oxidative stress with tumor and vascularization in patients with advanced gallbladder carcinoma

“…By driving tumor escape from the immune control and down regulating tissue-destructive effects of activated immune cells, Ado finely tunes the growth and dissemination of tumor mass through the engagement of adenosinergic receptors expressed on neoplastic cells (Moreno et al, 2018;Öztürk et al, 2015). Ado also mediates the inhibition of effector immune cells, including CD8 cytotoxic T lymphocytes, natural killer cells, dendritic cells, macrophages, and promotes CD4 + cells differentiation into T regulatory cells (Chaudhary and Elkord, 2016).…”

Purinergic System Signaling in Metainflammation-Associated Osteoarthritis

“…Separately, ecto-5'-nucleotidase (CD73) transforms AMP into adenosine ( 13 – 16 ). Additionally, one of the main regulators of the purinergic system in the extracellular space is adenosine deaminase (ADA), which anchores to the plasma membrane through the dipeptidyl peptidase IV (CD26) ( 17 ) and metabolizes adenosine to inosine ( 7 , 18 ). Adenosine kinase (AK) converts adenosine back to AMP ( 19 ).…”