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

Abstract

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-ADPribosylation. 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.

Structure and mechanism of a canonical poly(ADP-ribose) glycohydrolase

“…The target analyses were based on the accurate mass feature of the instrument, applying a mass window of 50 mDa. Information on the electrospray ionization mass spectra for oligomeric ADP-ribose species is only scarcely available in the literature 27 . Therefore, the recorded total ion current chromatograms were systematically examined using specific masses of possible mono-and multiply charged ions of individual oligomers.…”

Extracellular Poly(ADP-Ribose) Is a Pro-inflammatory Signal for Macrophages

“…PAR could also be made extracellularly. ART2, an enzyme found on the surface of T-cells, was recently shown to synthesize PAR (Morrison et al, 2006). …”

Rapid Degradation of Poly(ADP-ribose) after Injection into the Mouse Bloodstream

“…The resulting 32 P-oligononucleotide DNA was treated with bacterial alkaline phosphatase (Toyobo, Japan) at 60°C for 1 h and the released 32 P-ortho-phosphate thereby recovered. Thin-Layer Chromatography (TLC) Two-dimensional TlC was carried out with solvent buffers of isobutyric acid-25% NH 4 OH-H 2 O (50 : 1.1 : 28.9) in the first dimension and 0.1 m sodium-phosphate (pH 6.3)-ammonium sulfate-npropanol (100 : 60 : 2) in the second dimension, as described elsewhere.…”

“…3) PAR is mostly synthesized in the nuclei by PARP1 and PARP2 after DNA damage but also on the extracellular surface by ADP-ribosyl transferase 2, 4) while its degradation is catalyzed by the enzymes poly(ADP-ribose) glycohydrolase (PARG), [5][6][7][8] ADP-ribosyl hydrolase-3 (ARH3), 9) and phosphodiesterase.…”