Assembly of Active High Molecular Weight CD39 Complex: Role of Proteolysis and Endocytosis

Abstract 2110

HUVECs, neutrophils, monocyte/macrophages and T cell subsets all express ectonucleoside triphosphate diphosphohydrolase 1 (CD39) on the cell surface. In addition, they all express both P2X and P2Y receptors and dose dependently respond to ATP. ATP enhances superoxide production in activated neutrophils and supports chemotaxis of macrophages responding to a chemoattractant. ATP also induces apoptosis of anti-inflammatory T regulatory cells and supports the differentiation of pro-inflammatory Th17 cells. Lastly, ADP drives thrombus formation by activation of platelet P2Y12 receptors. An increase in the activity of expressed CD39 would result in increased metabolism of these pro-thrombotic and pro-inflammatory nucleotides.

We examined the reported relationship between CD39 cleavage and cell surface enzymatic activity. We cloned N-terminal and C-terminal V5 and VP16 tagged CD39 into eukaryotic expression vectors as well as lentiviral genomic vectors for analysis of CD39 expression in 293 cells and HUVECs. Western blots of membrane fractions prepared from HUVECs and transiently and stably transfected 293 cells identified the previously described CD39 cleavage fragments. Streptavidin precipitation of biotinylated membrane proteins demonstrated that both fragments are present on the cell surface of both transduced HUVECs and transfected 293 cells.

We next separated membranes on a discontinuous sucrose gradient to yield ER, Golgi/plasma membrane, early endosome and late endosome enriched membrane fractions. Surprisingly, full length CD39 and the N- and C-terminal fragments appeared in both the Golgi/plasma membrane and early endosome fractions. In addition, the early endosome CD39 exhibited enzyme activity equal to that of the Golgi/plasma membrane CD39. We then prepared membranes from cells treated with either chloroquine or bafilomycin, reagents known to interfere with endosomal acidification and/or maturation. In each case, we observed a decrease in the fractional cleavage of full length CD39 and a proportional decrease in associated enzymatic activity. When sonicated membranes were resolved on a continuous sucrose gradient, the N- and C-terminal fragments and a fraction of full length CD39 as well as maximal enzymatic activity were found in the low density, “raft” fractions. These results suggest that the formation of an enzymatically active CD39 complex requires N- and C-terminal CD39 fragments as well as membrane cholesterol. When 293 cells that stably expressed CD39 were transiently transfected with dominant negative dynamin 2, we observed a decrease in fractional cleavage as well as a proportional decrease in enzymatic activity. This result suggested that the cleavage event occurs following endocytosis of plasma membrane expressed CD39. Finally, we treated both stably transfected 293 cells and HUVECs with the cell permeable cysteine protease inhibitor zLLY.fmk. Prepared membranes analyzed by Western blot showed a decrease in fractional cleavage of full length CD39. Apyrase assays showed a corresponding decrease in ATPase and ADPase activity.

We then examined the cholesterol dependence of CD39 activity by depleting membrane cholesterol with MβCD. As expected, ATPase activity decreased in a dose dependent manner. The predominant “active” species appeared as a 1.4 megadalton complex on a 3–12% BN gel of Digitonin solubilized membranes prepared from cultured cells treated with the cleavable cross-linker DTSSP. As a result of membrane cholesterol depletion, there was a proportional decrease in the amount of full length CD39 and N- and C-terminal fragments present in the DTSSP cross-linked HMW complex. Interestingly, there was a marked increase in the abundance of lower molecular weight complexes in cholesterol depleted cells.

In conclusion, we provide evidence that CD39 enzymatic activity resides in a megadalton complex formed by protein-protein interactions between full length CD39 and C- and N-terminal fragments generated by cleavage of the full length molecule. Assembly of the oligomeric protein complex requires membrane cholesterol and likely occurs on intracellular membranes. Moreover, approximately 50% of the active enzyme complex remains sequestered on intracellular membranes. These results suggest that up-regulation of CD39 metabolism of pro-thrombotic and pro-inflammatory nucleotides involves pathways independent of gene transcription.