Generation and Characterization of Ecto-ADP-Ribosyltransferase ART2.1/ART2.2-Deficient Mice

Abstract

This is the first study reporting the inactivation of a member of the mouse gene family of toxin-related ecto-ADP-ribosyltransferases (ARTs). Transfer of the ADP-ribose moiety from NAD onto extracellular arginine residues on T-cell membrane proteins is mediated by glycosylphosphatidylinositol-linked cell surface ARTs. Exposure of T cells to ecto-NAD blocks T-cell activation and induces T-cell apoptosis. To determine a possible role of ecto-ART2.1 and ART2.2 in these processes, we generated ART2.1/ART2.2 double-knockout mice. ART2-deficient mice were healthy and fertile and showed normal development of lymphoid organs. ART2-deficient T cells showed a dramatically reduced capacity to ADP-ribosylate cell surface proteins, indicating that most if not all ART activity on the T-cell surface can be attributed to the ART2s. Moreover, ART2-deficient T cells were completely resistant to NAD-induced apoptosis and partially resistant to NADmediated suppression of proliferation. These results demonstrate that the ART2 ectoenzymes are an essential component in the regulation of T-cell functions by extracellular NAD, e.g., following release of NAD upon lysis of cells in tissue injury and inflammation.Posttranslational modification of proteins provides an important mechanism for regulating cellular functions. Protein phosphorylation mediated by protein kinases is well established as an essential component of intracellular signal transduction. Mounting evidence suggests that protein ADP-ribosylation mediated by ADP-ribosyltransferases (ARTs) may serve a similar regulatory function in the extracellular environment (10, 18, 32).We have previously reported the molecular cloning and biochemical characterization of a family of glycosylphosphatidylinositol (GPI)-anchored and secretory mammalian cell surface mono(ADP-ribosyl)transferases (ART1 to ART5) which are related in structure and function to bacterial ADP-ribosylating toxins (20,21). These enzymes catalyze the transfer of the ADP-ribose moiety of NAD onto a specific amino acid residue of a target protein, while the nicotinamide moiety is released.In the context of the immune system, treatment of lymphocytes with ART inhibitors or the ART substrate NAD profoundly affects cellular functions, including clustering of T-cell receptors, proliferation, cytotoxicity, and in vivo homing (26,29,36). Recently, we and others have shown that extracellular NAD can induce apoptosis of naïve T cells in vitro and in vivo (1, 25). NAD-induced T-cell apoptosis shares certain features with the classic death receptor-induced pathway of apoptosis, including exposure of phosphatidylserine on the outer leaflet of the plasma membrane, followed by fragmentation of DNA and, ultimately, failure to exclude the DNA-staining dye propidium iodide (1). However, in contrast to death receptorinduced apoptosis, NAD-induced apoptosis cannot be blocked with caspase inhibitors and affects naïve cells more severely than activated T cells. Thus, exposure to NAD may constitute an alternative or complementary mechanism t...

NAD+ and ATP Released from Injured Cells Induce P2X7-Dependent Shedding of CD62L and Externalization of Phosphatidylserine by Murine T Cells

“…CD38-deficient mice (21) and ART2-deficient mice (17) were backcrossed onto the BALB/c background for 12 generations. The ART2 Ϫ/Ϫ lines are deficient in both ART2.1 and ART2.2 (17). P2X 7 -deficient mice were from the twelfth backcross generation to C57BL/6 mice (3).…”

“…NAD-mediated but not ATP-mediated activation of P2X 7 requires functional ART2. Hence, T cells from ART2-deficient mice (17) were sensitive to direct activation of P2X 7 with the soluble ligand ATP (Fig. 1, A and B, panel 9), but were resistant to NAD-induced activation ofNAD ϩ and ATP released from lysed erythrocytes activate P2X 7 on murine T cells…”

“…T cell activation induces the metalloprotease-mediated shedding of a soluble, enzymatically active isoform of ART2.2 (16). ART2-deficient mice (17) exhibit reduced sensitivities to Con-A-induced hepatitis (18).…”

NAD+-Consuming Enzymes in the Regulation of Lung Immune Responses

“…In another study, a deficiency in a novel immunoregulatory population of ART2+ intestinal intraepithelial lymphocytes has been associated with the development of autoimmune diabetes [41]. On the other hand, mice lacking ART2 exhibit increased survival in a model of natural killer T cell-mediated autoimmune hepatitis [37,42]. Hence other studies are necessary to clarify the role of ART2 protein in autoimmune diseases.…”

“…Indeed two crucial findings confirmed ART2 as a necessary mediator of NICD. First, cells derived from ART2-deficient mice were resistant to NICD [37]. Second, pre-incubation of cells with ART2-specific antibodies effectively protected cells from NICD as well [6,31,36].…”