Abstract
Murine T cells express the GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) on the cell surface. In response to T cell activation or extracellular NAD+ or ATP-mediated gating of the P2X7 ion channel ARTC2.2 is shed from the cell surface as a soluble enzyme. Shedding alters the target specificity of ARTC2.2 from cell surface proteins to secreted proteins. Here we demonstrate that shed ARTC2.2 potently ADP-ribosylates IFN-γ in addition to other cytokines. Using mass spectrometry, we identify arginine 128 as the target site of ADP-ribosylation. This residue has been implicated to play a key role in binding of IFN-γ to the interferon receptor 1 (IFNR1). Indeed, binding of IFN-γ to IFNR1 blocks ADP-ribosylation of IFN-γ. Moreover, ADP-ribosylation of IFN-γ inhibits the capacity of IFN-γ to induce STAT1 phosphorylation in macrophages and upregulation of the proteasomal subunit ß5i and the proteasomal activator PA28-α in podocytes. Our results show that ADP-ribosylation inhibits the signaling functions of IFN-γ and point to a new regulatory mechanism for controlling signaling by IFN-γ.
Highlights
ARTC2.2 is a toxin-related, GPI-anchored ecto-ADP-ribosyltransferase expressed on the surface of murine T cells [1, 2]
Analysis of IFN-γ under reducing conditions reveals a discernible shift in migration of the IFN-γ band in Coomassie-stained SDS-PAGE after incubation with shed ARTC2.2 and NAD+ (Figures 1D,E, lane 2 vs. lane 1), suggesting that ARTC2.2 ADP-ribosylates both molecules of the IFN-γ homodimer, since the presence of ADP-ribosylated and non-ADP-ribosylated IFN-γ should result in two bands after size fractionation by reducing SDS-PAGE
To identify possible soluble targets we performed a comparative radio-ADP-ribosylation assay with different cytokines and recombinant soluble ARTC2.2 and 32P-NAD+
Summary
ARTC2.2 is a toxin-related, GPI-anchored ecto-ADP-ribosyltransferase expressed on the surface of murine T cells [1, 2]. ARTC2.2 catalyzes arginine-specific ADP-ribosylation of P2X7 and other cell surface proteins in response to NAD+ released from damaged cells [3, 4]. ADP-ribosylation is a reversible posttranslational modification that regulates the function of target proteins [5, 6]. The targets of ADP-ribosylation are mainly ecto-domains of membrane proteins like P2X7, integrins or Fc-gamma-receptors [4, 7]. ADP-ribosylation of soluble proteins has been described previously, for example, ARTC1-mediated ADP-ribosylation of HNP-1 reduces the antimicrobial activity of HNP-1 [8]
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