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 NAD-mediated 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.

F.K.-N. and N.K., who designed and supervised this study, share senior authorship.

We thank Martina Matthes, Maren Kühl, Marion Nissen, Vivienne Welge, Katrin Bartels, Dunja Freese, Felix Scheuplein, UKE, Hamburg; Christiane Steeg, BNI, Hamburg; and Jason Dietrich, Ling Ling Wang, and Leonid Pravoverov, University of California at San Francisco, for technical assistance. F.K.-N. and W.O. thank the laboratory staffs of the Litmann and Killeen laboratories for generous hospitality during their stays as visiting scientists at the University of California at San Francisco. F.K.-N. cloned the targeting constructs and isolated the targeted ES cells, N.K. performed the blastocyst injections, J.L. performed the histological examinations, and W.O. performed the RT-PCR, fluorescence-activated cell sorting analyses, and proliferation assays. F.H., J.L., M.S., and D.L. made essential contributions to the study design. F.K.-N. wrote the paper. Parts of the work described in this study represent the partial fulfillment of the requirements for the graduate thesis of W.O. We thank Bernhard Fleischer, Hamburg, and Edward Leiter, Bar Harbor, Maine, for helpful discussions and critical reading of the manuscript.

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 545 B9 to F.K.-N. and F.H.).