Multiple steps determine CD73 shedding from RPE: lipid raft localization, ARA1 interaction, and MMP-9 up-regulation

Abstract

Physiologically, retinal pigment epithelium (RPE) expresses high levels of CD73 in their membrane, converting AMP to immune suppressive adenosine, mediates an anti-inflammatory effect. However, after being exposed to inflammatory factors, RPE rapidly becomes CD73-negative cells, which render RPE's immune suppressive function and accelerate local inflammation. Here, we investigated the mechanism leading to the loss of membrane CD73 in RPE. We found the controversy that when membrane CD73 was significantly diminished in inflammatory RPE, Cd73 mRNA levels were not changed at all. It was further verified that, matrix metalloproteinase-9 (MMP-9) mediated the shedding of CD73 from the cell membrane of inflammatory RPE by catalyzing its K547/F548 site. However, MMP-9 could not catalyze uncomplexed CD73, the interaction of CD73 with adenosine receptor A1 subtype (ARA1) is necessary for being catalyzed by MMP-9. After being treated by LPS and TNF-α, the formation of CD73/ARA1 complex in RPE was verified by co-immunoprecipitation and FRET-based assays. It was also revealed that CD73 need to be localized in lipid rafts to be capable of interacting with ARA1, since CD73/ARA1 interaction and CD73 shedding were completely blocked by the addition of lipid raft synthesis inhibitor. As a conclusion, multiple steps are involved in CD73 shedding in RPE, including up-regulation of MMP-9 activity, localization of CD73 in lipid rafts, and the formation of CD73/ARA1 complex. Lipid rafts committed CD73 with high mobility, shuttled CD73 to ARA1 to form a complex, which was capable of being recognized and catalyzed by MMP-9.

Defining the CD39/CD73 Axis in SARS-CoV-2 Infection: The CD73- Phenotype Identifies Polyfunctional Cytotoxic Lymphocytes

“…In humans, CD73 is generally expressed on endothelial cells, B cells, naïve CD8 + T cells, and a subset of memory CD4 + T cells [20]. Recent studies show that upon activation, CD73 may be shed from the cell surface resulting in a disturbance of ATP/ADO balance in the extracellular matrix (ECM) [20,21]. In fact, cells that lose CD73 could be protected by pericellular ADO, thus maintaining their effector function [20].…”

Clustering of adenosine A2Breceptors with ectonucleotidases in caveolin‐rich lipid rafts underlies immunomodulation byLeishmania amazonensis

“…Thus, CD39 and CD73 have been independently described to be associated with lipid rafts. [40][41][42][43] In addition, a single study using transfected cells also indicates that adenosine receptors are present in lipid rafts at the cell membrane. 44 Although the possible association of the two enzymes together with adenosine receptors in lipid rafts was suggested in a review, 45 a study using FRET-microscopy failed to demonstrate the association of CD39 with A 2A R. 46 In addition, to the best of our knowledge, such multi-molecular organization has not been demonstrated with A 2B R.…”

Generation and Function of Non-cell-bound CD73 in Inflammation

“…Matrix metalloproteinase 9 (MMP-9) has been shown to cleave lipid raft-associated CD73 from the membrane of activated mouse retinal pigment epithelial cells. The generated sCD73, however, was enzymatically inactive (60). In contrast, an active form of sCD73 generated by proteolytic cleavage and lacking the GPI-anchor was found in bull seminal plasma (53).…”