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J. Biol. Chem., Vol. 281, Issue 44, 33363-33372, November 3, 2006

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ART2, a T Cell Surface Mono-ADP-ribosyltransferase, Generates Extracellular Poly(ADP-ribose)^*

Alan R. Morrison¹, Joel Moss, Linda A. Stevens, James E. Evans^¶, Caitlin Farrell, Eric Merithew^¶, David G. Lambright^¶, Dale L. Greiner, John P. Mordes, Aldo A. Rossini, and Rita Bortell²

From the Departments of Medicine and ^¶Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 and the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590

NAD functions in multiple aspects of cellular metabolism and signaling through enzymes that covalently transfer ADP-ribose from NAD to acceptor proteins, thereby altering their function. NAD is a substrate for two enzyme families, mono-ADP-ribosyltransferases (mARTs) and poly(ADP-ribose) polymerases (PARPs), that covalently transfer an ADP-ribose monomer or polymer, respectively, to acceptor proteins. ART2, a mART, is a phenotypic marker of immunoregulatory cells found on the surface of T lymphocytes, including intestinal intraepithelial lymphocytes (IELs). We have shown that the auto-ADP-ribosylation of the ART2.2 allelic protein is multimeric. Our backbone structural alignment of ART2 (two alleles of the rat art2 gene have been reported, for simplicity, the ART2.2 protein investigated in this study will be referred to as ART2) and PARP suggested that multimeric auto-ADP-ribosylation of ART2 may represent an ADP-ribose polymer, rather than multiple sites of mono-ADP-ribosylation. To investigate this, we used highly purified recombinant ART2 and demonstrated that ART2 catalyzes the formation of an ADP-ribose polymer by sequencing gel and by HPLC and MS/MS mass spectrometry identification of PR-AMP, a breakdown product specific to poly(ADP-ribose). Furthermore, we identified the site of ADP-ribose polymer attachment on ART2 as Arg-185, an arginine in a crucial loop of its catalytic core. We found that endogenous ART2 on IELs undergoes multimeric auto-ADP-ribosylation more efficiently than ART2 on peripheral T cells, suggesting that these distinct lymphocyte populations differ in their ART2 surface topology. Furthermore, ART2.2 IELs are more resistant to NAD-induced cell death than ART2.1 IELs that do not have multimeric auto-ADP-ribosylation activity. The data suggest that capability of polymerizing ADP-ribose may not be unique to PARPs and that poly(ADP-ribosylation), an established nuclear activity, may occur extracellularly and modulate cell function.

Received for publication, July 31, 2006

^* This work was supported in part by University of Massachusetts Center Grant DK32520 and National Institutes of Health Research Grants DK49106 (to D. L. G. and J. P. M.), DK36024 (to D. L. G.), and DK25306 (to A. A. R. and J. P. M.), Juvenile Diabetes Research Foundation Grant 1-2002-394 (to R. B.), and the Intramural Research Program, National Institutes of Health, NHLBI. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of an American Diabetes Association Physician-Scientist Training Award.

² To whom correspondence should be addressed: Diabetes Division, Suite 218, 373 Plantation St., Worcester, MA 01605. Tel.: 508-856-3788; Fax: 508-856-4093; E-mail: rita.bortell{at}umassmed.edu.

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