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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Menzel, Koudelka, Rissiek, Haag, Meyer-Schwesinger, Tholey and Koch-Nolte.)

Mouse T cells express the toxin-related ecto-ADP-ribosyltransferase ARTC2 that catalyzes the posttranslational ADP-ribosylation of cell surface proteins by transferring the ADP-ribose group of its substrate nicotinamide adenine dinucleotide (NAD + ) to arginine residues of its target proteins. One well known target of ARTC2 is the ATP-gated P2X7 ion channel. ADP-ribosylation of P2X7 induces gating of the channel, calcium influx, ecto-domain shedding, phosphatidylserine externalization, and finally cell death. Previous studies have shown that the ARTC2 substrate NAD + is released during T cell preparation. Since P2X7 is differentially expressed among T cell subpopulations, preparation-related ADP-ribosylation has a strong impact on the vitality of T cells that express high levels of P2X7. With this chapter we provide a protocol to monitor the consequences of preparation-related P2X7 ADP-ribosylation on T cells using regulatory T cells as generic T cell subpopulation known to express high levels of P2X7. However, this protocol could be easily adapted to other T cell populations.

Mammalian ARTCs are expressed as glycosylphosphatidylinositol (GPI)-anchored ectoenzymes (ARTC1-ARTC4) or secretory proteins (ARTC5) by different cell types. The ARTC2 enzymes catalyze mono-ADP-ribosylation of arginine residues in the extracellular domain of membrane proteins or secretory proteins. In this chapter we provide protocols to monitor the expression and activity of ARTCs on the cell membrane of living cells and in soluble form in biological fluids.

Mammalian ecto-ADP-ribosyltransferases (ecto-ARTs or also ARTCs) catalyze the ADP-ribosylation of cell surface proteins using extracellular nicotinamide adenine dinucleotide (NAD + ) as substrate. By this post-translational protein modification, ecto-ARTs modulate the function of various target proteins. A functional role of ARTC2 has been demonstrated for peripheral immune cells such as T cells and macrophages. Yet, little is known about the role of ecto-ARTs in the central nervous system and on microglia. Here, we identified ARTC2.1 as the major ecto-ART expressed on murine microglia. ARTC2.1 expression was strongly upregulated on microglia upon co-stimulation with LPS and an ERK1/2 inhibitor or upon IFNβ stimulation. We identified several target proteins modified by ARTC2.1 on microglia with a recently developed mass spectrometry approach, including two receptors for immunoglobulin G (IgG), FcγR1 and FcγR2B. Both proteins were verified as targets of ARTC2.1 in vitro using a radiolabeling assay with 32 P-NAD + as substrate. Moreover, ADP-ribosylation of both targets strongly inhibited their capacity to bind IgG. In concordance, ARTC2.1 induction in WT microglia and subsequent cell surface ADP-ribosylation significantly reduced the phagocytosis of IgG-coated latex beads, which was unimpaired in NAD + /DTT treated microglia from ARTC2.1 -/- mice. Hence, induction of ARTC2.1 expression under inflammatory conditions, and subsequent ADP-ribosylation of cell surface target proteins could represent a hitherto unnoticed mechanism to regulate the immune response of murine microglia.

ARTC2.2 is a toxin-related, GPI-anchored ADP-ribosyltransferase expressed by murine T cells. In response to NAD(+) released from damaged cells during inflammation, ARTC2.2 ADP-ribosylates and thereby gates the P2X7 ion channel. This induces ectodomain shedding of metalloprotease-sensitive cell surface proteins. In this study, we show that ARTC2.2 itself is a target for P2X7-triggered ectodomain shedding. We identify the metalloprotease cleavage site 3 aa upstream of the predicted GPI anchor attachment site of ARTC2.2. Intravenous injection of NAD(+) increased the level of enzymatically active ARTC2.2 in serum, indicating that this mechanism is operative also under inflammatory conditions in vivo. Radio-ADP-ribosylation assays reveal that shedding refocuses the target specificity of ARTC2.2 from membrane proteins to secretory proteins. Our results uncover nucleotide-induced membrane-proximal proteolysis as a regulatory mechanism to control the substrate specificity of ARTC2.2., (Copyright © 2015 by The American Association of Immunologists, Inc.)

The spore-forming gut bacterium Clostridium difficile is the leading cause of antibiotic-associated diarrhea in hospitalized patients. The major virulence factors are two large glucosylating cytotoxins. Hypervirulent strains (e.g. ribotype 027) with higher morbidity and mortality additionally produce the binary CDT toxin (Clostridium difficile transferase) that ADP-ribosylates actin and induces microtubule-based cell protrusions. Nanobodies are robust single domain antibodies derived from camelid heavy chain antibodies. Here we report the generation of functional nanobodies against the enzymatic CDTa and the heptameric receptor binding subunit CDTb. The nanobodies were obtained from a variable-domain repertoire library isolated from llamas immunized with recombinant CDTa or CDTb. Five CDTa-specific nanobodies blocked CDTa-mediated ADP-ribosylation of actin. Three CDTa-specific and two CDTb-specific nanobodies neutralized the cytotoxicity of CDTa+b. These nanobodies hold promise as new tools for research, diagnosis and therapy of C. difficile associated disease.

ADP-ribosyltransferases comprise a family of enzymes originally discovered as bacterial toxins and later characterised also in mammals. In mice, the ADP-ribosyltransferase ARTC2.2 is expressed at the surface of T lymphocytes and has been studied extensively. In the presence of extracellular NAD(+), ARTC2.2 ADP-ribosylates several cell surface target proteins and thereby regulates their function. P2X7, an ATP-gated cation channel, has been discovered as a prominent ARTC2.2 target at the surface of mouse T cells. ADP-ribosylation of P2X7 in the presence of low micromolar extracellular NAD(+) induces long-lasting P2X7 activation and triggers cell death. Regulatory T cell subsets (Tregs and NKT cells) are remarkably sensitive to NAD(+)-induced cell death (NICD). Thus, liberation of endogenous NAD(+) by stressed cells is now viewed as a danger signal promoting immune responses by hindering regulatory T cells. This review will highlight the recent discoveries on the in vivo role of the ARTC2.2/P2X7 pathway triggered by the endogenous release of extracellular NAD(+), the relative sensitivity of lymphocytes subsets to this regulatory pathway and its pharmacological manipulation using camelid-derived ARTC2.2-blocking nanobodies.

In 1901, the first Nobel Prize in Physiology or Medicine was awarded to Emil von Behring for his ground-breaking discovery of serum therapy: serum from horses vaccinated with toxin-containing culture medium of Corynebacterium diphtheriae contained life-saving 'antitoxins'. The molecular nature of the ADP-ribosylating toxin and the neutralizing antibodies were unraveled only 50 years later. Today, von Behring's antibody therapy is being refined with a new generation of recombinant antibodies and antibody fragments. Nanobodies, which are single-domain antibodies derived from the peculiar heavy-chain antibodies of llamas and other camelids, are emerging as a promising new class of highly specific enzyme inhibitors. In this review, we illustrate the potential of nanobodies as tools to block extracellular and intracellular ADP-ribosyltransferases (ARTs), using the toxin-related membrane-bound mammalian ecto-enzyme ARTC2 and the actin-ADP-ribosylating Salmonella virulence plasmid factor B toxin of Salmonella enterica as examples., (© 2013 FEBS.)

T cells constitutively express low amounts of a toxin-related ADP-ribosylating ecto-enzyme, ART2.2. In inflammatory settings, cells release NAD, the substrate for ART2.2. The ART2.2 catalyzed ADP-ribosylation of cell surface proteins induces cell death. However, the low expression levels of ART2.2 have hampered analysis of ART2.2 in physiological settings. Here we report the generation of transgenic mice over-expressing ART2.2 under the control of the H2K promoter and Igμ enhancer. ART2.2 transgenic mice were healthy and fertile and exhibited normal development of the major lymphocyte subsets. Most T cells and a small subpopulation of B cells from transgenic mice showed more than 10-fold higher levels of ART2.2 expression than their wild-type counterparts. Exposure of ART2.2-transgenic T cells to low, submicromolar concentrations of NAD caused cell membrane alterations including uptake of propidium iodide, externalization of phosphatidylserine, and shedding of CD62L, while ART2.2-transgenic B cells were resistant to NAD. The ART2.2-overexpressing animals described here confirm that ART2.2 is an essential component for the regulation of T-cell functions by extracellular NAD and provide a useful tool to further elucidate the function of ART2.2 in vivo., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The mammalian mono-ADP-ribosyltransferases are a family of enzymes related to bacterial toxins that can catalyse both intracellular and extracellular mono-ADP-ribosylation of target proteins involved in different cellular processes, such as cell migration, signalling and inflammation. Here, we report the molecular cloning and functional characterisation of a novel glycosylphosphatidylinositol (GPI)-anchored mono-ADP-ribosyltransferase isoform from Chinese hamster ovary (CHO) cells (cARTC2.1) that has both NAD-glycohydrolase and arginine-specific ADP-ribosyltransferase activities. cARTC2.1 has the R-S-EXE active-site motif that is typical of arginine-specific ADP-ribosyltransferases, with Glu209 as the predicted catalytic amino acid. When over-expressed in CHO cells, the E209G single point mutant of cARTC2.1 cannot hydrolyse NAD(+), although it retains low arginine-specific ADP-ribosyltransferase activity. This ADP-ribosyltransferase activity was abolished only with an additional mutation in the R-S-EXE active-site motif, with both of the glutamate residues of the EKE sequence of cARTC2.1 mutated to glycine (E207/209G). These glutamate-mutated proteins localise to the plasma membrane, as does wild-type cARTC2.1. Thus, the partial or total loss of enzymatic activity of cARTC2.1 that arises from these mutations does not affect its cellular localisation. Importantly, an endogenous ADP-ribosyltransferase is indeed expressed and active in a subset of CHO cells, while a similar activity cannot be detected in ovarian cancer cells. With respect to this endogenous ecto-ART activity, we have identified two cell populations: ART-positive and ART-negative CHO cells. The subset of ART-positive cells, which represented 5% of the total cells, is tightly maintained in the CHO cell population., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Arginine adenosine-5'-diphosphoribosylation (ADP-ribosylation) is an enzyme-catalyzed, potentially reversible posttranslational modification, in which the ADP-ribose moiety is transferred from NAD(+) to the guanidino moiety of arginine. At 540 Da, ADP-ribose has the size of approximately five amino acid residues. In contrast to arginine, which, at neutral pH, is positively charged, ADP-ribose carries two negatively charged phosphate moieties. Arginine ADP-ribosylation, thus, causes a notable change in size and chemical property at the ADP-ribosylation site of the target protein. Often, this causes steric interference of the interaction of the target protein with binding partners, e.g. toxin-catalyzed ADP-ribosylation of actin at R177 sterically blocks actin polymerization. In case of the nucleotide-gated P2X7 ion channel, ADP-ribosylation at R125 in the vicinity of the ligand-binding site causes channel gating. Arginine-specific ADP-ribosyltransferases (ARTs) carry a characteristic R-S-EXE motif that distinguishes these enzymes from structurally related enzymes which catalyze ADP-ribosylation of other amino acid side chains, DNA, or small molecules. Arginine-specific ADP-ribosylation can be inhibited by small molecule arginine analogues such as agmatine or meta-iodobenzylguanidine (MIBG), which themselves can serve as targets for arginine-specific ARTs. ADP-ribosylarginine specific hydrolases (ARHs) can restore target protein function by hydrolytic removal of the entire ADP-ribose moiety. In some cases, ADP-ribosylarginine is processed into secondary posttranslational modifications, e.g. phosphoribosylarginine or ornithine. This review summarizes current knowledge on arginine-specific ADP-ribosylation, focussing on the methods available for its detection, its biological consequences, and the enzymes responsible for this modification and its reversal, and discusses future perspectives for research in this field.

ADP-ribosylation of host cell proteins is a common mode of cell intoxication by pathogenic bacterial toxins. Antibodies induced by immunization with inactivated ADP-ribosylating toxins provide efficient protection in case of some secreted toxins, e.g., diphtheria and pertussis toxins. However, other ADP-ribosylating toxins, such as Salmonella SpvB toxin, are secreted directly from the Salmonella-containing vacuole into the cytosol of target cells via the SPI-2 encoded bacterial type III secretion system, and thus are inaccessible to conventional antibodies. Small-molecule ADP-ribosylation inhibitors are fraught with potential side effects caused by inhibition of endogenous ADP-ribosyltransferases. Here, we report the development of a single-domain antibody from an immunized llama that blocks the capacity of SpvB to ADP-ribosylate actin at a molar ratio of 1:1. The single-domain antibody, when expressed as an intrabody, effectively protected cells from the cytotoxic activity of a translocation-competent chimeric C2IN-C/SpvB toxin. Transfected cells were also protected against cytoskeletal alterations induced by wild-type SpvB-expressing strains of Salmonella. This proof of principle paves the way for developing new antidotes against intracellular toxins.

CD4(+)CD25(+)FoxP3(+) regulatory T cells (T reg cells) play a major role in the control of immune responses but the factors controlling their homeostasis and function remain poorly characterized. Nicotinamide adenine dinucleotide (NAD(+)) released during cell damage or inflammation results in ART2.2-mediated ADP-ribosylation of the cytolytic P2X7 receptor on T cells. We show that T reg cells express the ART2.2 enzyme and high levels of P2X7 and that T reg cells can be depleted by intravenous injection of NAD(+). Moreover, lower T reg cell numbers are found in mice deficient for the NAD-hydrolase CD38 than in wild-type, P2X7-deficient, or ART2-deficient mice, indicating a role for extracellular NAD(+) in T reg cell homeostasis. Even routine cell preparation leads to release of NAD(+) in sufficient quantities to profoundly affect T reg cell viability, phenotype, and function. We demonstrate that T reg cells can be protected from the deleterious effects of NAD(+) by an inhibitory ART2.2-specific single domain antibody. Furthermore, selective depletion of T reg cells by systemic administration of NAD(+) can be used to promote an antitumor response in several mouse tumor models. Collectively, our data demonstrate that NAD(+) influences survival, phenotype, and function of T reg cells and provide proof of principle that acting on the ART2-P2X7 pathway represents a new strategy to manipulate T reg cells in vivo.

The objective of this study was to quantify enzymatic activity on the surface of T cells by magnetic resonance imaging (MRI) using R2 and R2* relaxometry. Lymphoma cells expressing adenosine diphosphate (ADP)-ribosyltransferase 2 (ART2) were incubated with increasing doses of its substrate etheno-nicotinamide adenine dinucleotide (NAD), resulting in increasing amounts of surface protein ADP-ribosylation. Etheno-ADP-ribosylated proteins were detected with monoclonal antibody 1G4 and superparamagnetic iron oxide conjugated secondary antibodies (Ab-SPIO). Labeling efficiency was determined with R2 and R2* relaxometry on a clinical 3.0 T scanner. Parallel aliquots of cells were analyzed by flow cytometry. Cell-bound SPIO conjugates were detected by immunofluorescence and electron microscopy and quantified by atomic absorption spectroscopy. To mimic an inflammatory site in vivo, Ab-SPIO-labeled cells were injected subcutaneously in mice and analyzed by MRI. Immunofluorescence and electron microscopy confirmed cell-surface localization of Ab-SPIO. MRI of Ab-SPIO-labeled cells showed a corresponding signal reduction. Increases in R2 and R2* determined by magnetic resonance relaxometry correlated linearly with the expression level of ART2 and the concentration of the ART2 substrate etheno-NAD. R2 and R2* increases correlated linearly with the results from flow cytometry and atomic absorption spectroscopy analyses. Quantitative R2 and R2* mapping enable noninvasive determination of enzymatic activity on T cells and holds promise for characterization of inflammatory sites in vivo by MRI.

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.

ADP-ribosyltransferases (ARTs) and ADP-ribosylhydrolases (ARHs) catalyze opposing reactions, which are termed ADP-ribosylation and de-ADP-ribosylation. ARTs transfer the ADP-ribose unit from NAD (nicotinamide adenine dinucleotide) onto an acceptor, while ARHs release the ADP-ribose from the target. Like protein phosphorylation, ADP-ribosylation is a posttranslational modification regulating protein function. In many cases, ADP-ribosylation inactivates the target protein. Numerous bacterial toxins intoxicate cells by attaching an ADP-ribose moiety to a functionally important amino acid residue, thereby blocking the interaction of the target protein with other proteins. In other cases, ADP-ribosylation activates protein function. On the surface of T cells, ART2.2 ADP-ribosylates the P2X7 purinoceptor on arginine 125, thereby gating the P2X7 ion channel by presenting a ligand to its nucleotide-binding site. ADP-ribosylation is not limited to protein targets and ARTs have been described that ADP-ribosylate DNA, RNA, and small molecules. Mammalian cells express distinct families of ARTs and ARHs. Recently, molecular cloning, site directed mutagenesis and three-dimensional structural analyses of prototype mammalian ARTs and ARHs have shed fresh insight into the structure and function of these intriguing enzymes.

ADP-ribosylation is a post-translational modification regulating protein function in which amino acid-specific ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD onto specific target proteins. Attachment of the bulky ADP-ribose usually inactivates the target by sterically blocking its interaction with other proteins. P2X7, an ATP-gated ion channel with important roles in inflammation and cell death, in contrast, is activated by ADP-ribosylation. Here, we report the structural basis for this gating and present the first molecular model for the activation of a target protein by ADP-ribosylation. We demonstrate that the ecto-enzyme ART2.2 ADP-ribosylates P2X7 at arginine 125 in a prominent, cysteine-rich region at the interface of 2 receptor subunits. ADP-ribose shares an adenine-ribonucleotide moiety with ATP. Our results indicate that ADP-ribosylation of R125 positions this common chemical framework to fit into the nucleotide-binding site of P2X7 and thereby gates the channel.

The purpose of our study was to develop a tool for blocking the function of a specific leukocyte ecto-enzyme in vivo. ART2.2 is a toxin-related ecto-enzyme that transfers the ADP-ribose moiety from NAD onto other cell surface proteins. ART2.2 induces T cell death by activating the cytolytic P2x7 purinoceptor via ADP-ribosylation. Here, we report the generation of ART2.2-blocking single domain antibodies from an immunized llama. The variable domain of heavy-chain antibodies (VHH domain) represents the smallest known antigen-binding unit generated by adaptive immune responses. Their long CDR3 endows VHH domains with the extraordinary capacity to extend into and block molecular clefts. Following intravenous injection, the ART2.2-specific VHH domains effectively shut off the enzymatic and cytotoxic activities of ART2.2 in lymphatic organs. This blockade was highly specific (blocking ART2.2 but not the related enzymes ART1 or ART2.1), rapid (within 15 min after injection), and reversible (24 h after injection). Our findings constitute a proof of principle that opens up a new avenue for targeting leukocyte ecto-enzymes in vivo and that can serve as a model also for developing new antidotes against ADP-ribosylating toxins.

Nicotinamide adenosine dinucleotide (NAD) can act as a modulator of multiple immune and inflammatory responses when released into extracellular compartments. These actions of extracellular NAD are largely mediated by a family of mammalian ecto-ADP-ribosyltransferases (ARTs) that covalently modify target extracellular or cell surface proteins by transferring ADP-ribose to arginine or cysteine residues. In this study, we report that bone marrow-derived macrophages (BMDM) from BALB/c mice lack constitutive expression of any of the six murine ecto-ART subtypes, but selectively up-regulate ART2.1 in response to multiple proinflammatory mediators including agonists for TLR and type I and type II IFN. Stimulation of BMDM with LPS, IFN-beta, or IFN-gamma induced high expression of ART2.1, but not ART2.2, as a GPI-anchored cell surface ectoenzyme. ART2.1 expression in response to LPS was potentiated by inhibition of ERK1/2 signaling, but inhibited by blockade of the NF-kappaB, PI3K, and JAK-STAT pathways or the presence of neutralizing anti-IFN-beta. The catalytic function of the induced cell surface ART2.1 was strictly dependent on the presence of extracellular thiol-reducing cofactors, suggesting that in vivo activity of ART2.1-expressing macrophages may be potentiated in hypoxic or ischemic compartments. Consistent with the mutated art2a gene in C57BL/6 mice, LPS- or IFN-stimulated BMDM from this strain lacked expression of cell surface ART2 activity in the presence or absence of extracellular thiol reductants. Collectively, these studies identify ART2.1 as a new candidate for linking autocrine/paracrine activation of inflammatory macrophages to the release of NAD, a critical intracellular metabolite.

Mono ADP-ribosyltransferase 2 (ART2) is an ectoenzyme expressed on mouse T lymphocytes, which catalyze the transfer of ADP-ribose groups from NAD(+) onto several target proteins. In vitro, ADP-ribosylation by ART2 activates the P2X7 ATP receptor and is responsible for NAD(+)-induced T cell death (NICD). Yet, the origin of extracellular NAD(+) and the role of NICD in vivo remain elusive. In a model of acute inflammation induced by polyacrylamide beads, we demonstrate release of NAD(+) into exudates during the early phase of the inflammatory response. This leads to T cell depletion in the draining lymph nodes from wild-type and, more severely, from mice lacking the CD38 NAD(+) glycohydrolase, whereas no effect is observed in ART2-deficient animals. Intravenous injection of NAD(+) used to exacerbate NICD in vivo results in fast and dramatic ART2- and P2X7-dependent depletion of CD4+ and CD8+ T lymphocytes, which can affect up to 80% of peripheral T cells in CD38(-/-) mice. This affects mainly naive T cells as most cells surviving in vivo NAD+ treatment exhibit the phenotype of recently activated/memory cells. Consistently, treatment with NAD(+) abolishes primary Ab response to a T-dependent Ag in NICD-susceptible CD38(-/-) mice but has no effect on the secondary response when given several days after priming. Unexpectedly NAD+ treatment improves the response in their wild-type BALB/c counterparts. We propose that NAD(+) released during early inflammation facilitates the expansion of primed T cells, through ART2-driven death of resting cells, thus contributing to the dynamic regulation of T cell homeostasis.

Ubiquitously expressed CD38 and T cell-expressed ADP-ribosyltransferase 2 (ART2) are ectoenzymes competing for NAD substrate. CD38 exerts pleiotropic actions in hemopoietic and nonhemopoietic compartments via effects on calcium mobilization. ART2 is an ADP-ribosyltransferase on naive CD4+ and CD8+ T cells. ART2-catalyzed ADP-ribosylation of the P2X7 purinoreceptor elicits apoptosis. Transfer of a genetically disrupted CD38 allele into the autoimmune diabetes-prone NOD/Lt background accelerated diabetes onset in both sexes, whereas transfer of a disrupted ART2 complex had no effect. However, the fact that the accelerated pathogenesis mediated by CD38 deficiency required ART2 activity was demonstrated by combining both ART2 and CD38 deficiencies. Reciprocal bone marrow reconstitution studies demonstrated accelerated diabetes only when CD38-deficient bone marrow was transferred into CD38-deficient recipients. Neither decreases in beta cell function nor viability were indicated. Rather, the balance between T-effectors and T-regulatory cells was disturbed in CD38-deficient but ART2-intact NOD mice. In these mice, significant reductions in total viable CD8+ T cells were observed. This was accompanied by an age-dependent increase in a diabetogenic CD8 clonotype. This in turn correlated with impaired T-regulatory development (10-fold reduction in Foxp3 mRNA expression). These changes were corrected when CD38 deficiency was combined with ART2 deficiency. Both ART2-deficient and CD38/ART2 combined deficient T cells were resistant to NAD-induced killing in vitro, whereas CD38-deficient but ART2-intact T cells showed increased sensitivity, particularly the CD4+ CD25+ subset. Unexpectedly, diabetes development in the combined CD38/ART2 stock was strongly suppressed, possibly through epistatic interactions between genes linked to the targeted CD38 on Chromosome 5 and the ART2 complex on Chromosome 7.

Many bacterial toxins kill animal cells by adenosine diphosphate (ADP)-ribosylating intracellular target proteins. Mammalian cells express toxin-related cell surface ADP-ribosyltransferases (ARTs) that transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto arginine residues of other membrane proteins. The association of these glycosylphosphatidylinositol (GPI)-anchored ectoenzymes with glycolipid rafts focuses them onto components of the signal transduction machinery. Exposing murine T cells to NAD, the ART substrate, induces a cascade of reactions that culminates in cell death by apoptosis. This mechanism, dubbed 'NAD-induced cell death' or NICD, is initiated when ART2 ADP-ribosylates the cytolytic P2X7 purinergic receptor, inducing formation of a cation channel, opening of a nonselective pore, shedding of CD62L from the cell surface, exposure of phosphatidylserine on the outer leaflet of the plasma membrane, breakdown of the mitochondrial membrane potential, and DNA-fragmentation. The ART substrate NAD is produced in large amounts inside the cell and can be released from damaged cells during inflammation and tissue injury. In the extracellular environment, the signaling function of NAD is terminated by NAD-degrading ectoenzymes such as CD38. We propose that ART2-catalyzed ADP-ribosylation of P2X7 represents the paradigm of a regulatory mechanism by which ART-expressing cells can sense and respond to the release of NAD from damaged cells.

ART2 (RT6) belongs to the family of mono-ADP-ribosyltransferases (ARTs). ART2 is a T-cell differentiation marker expressed by the majority of mature peripheral T cells in the rat. The two known ART2 allotypes display approximately 95% amino acid identity. We sequenced the ART2 coding regions from 18 inbred rat strains and found two additional alleles, termed Art2 ( a2 ) and Art2 ( b2 ). Monoclonal antibody Gy12/61 specifically reacted with Art2 ( a2 ) but not Art2 ( a1 ) lymph node cells. Expression of ART2 allotypes in Jurkat cells confirmed this specificity. A polymerase chain reaction (PCR) assay using restriction fragment length polymorphisms is described, which allows the easy discrimination of Art2 alleles. All four laboratory rat alleles, as well as an additional sequence variant, were found amongst 18 wild rat DNA samples. PCR analysis confirmed the selective presence of a rodent identifier (ID) element in the Art2 ( a ) but not the Art2 ( b ) alleles in all rats studied. Analysis of Art2 ( a1 ) and Art2 ( b2 ) genes showed greater divergence in coding than in non-coding regions. Together with the finding of a high number of non-synonymous mutations leading mostly to non-conservative amino acid substitutions clustered on the side facing away from the cell surface, this suggests that the Art2 polymorphism has been subject to selection.

ART4 (CD297) is a member of the family of toxin-related ADP-ribosyltransferases (ARTs) and is the carrier of the Dombrock blood group alloantigens (Do). Two mouse monoclonal antibodies (MIMA-52 and MIMA-53), and two rat monoclonal antibodies (N0NI-B4 and NONI-B63) were obtained following immunization of mice with human Do/ART4-transfected cells and of rats with human Do/ART4 cDNA, respectively. All four mAbs recognize Do/ART4-transfected Jurkat cells but not untransfected cells by FACS analysis. Staining of Do/ART4-transfected cells by these mAbs was reduced following treatment of cells with PI-PLC, confirming that Do/ART4 is anchored in the cell membrane by linkage to glycosylphosphatidylinositol as predicted from its amino acid sequence. The four mAbs did not react with Gy(a-) (Dombrock null) erythrocytes but agglutinated other red blood cells. By flow cytometric analysis, all mAbs reacted prominently with erythrocytes, and weakly with peripheral blood monocytes and splenic macrophages, but not with B-lymphocytes or T-lymphocytes. The mAbs reacted weakly also with human umbilical vein endothelial cells and the basophilic leukemia KU-812. Immunohistology revealed staining of epithelia and endothelia on sections of tonsils. In FACS analyses NONI-B4 competed with MIMA-52 for binding to Do/ART4-transfected cells and erythrocytes, whereas NONI-B63 competed with MIMA-53. Neither of the mAbs reacted with mouse ART4-transfected cells, but NONI-B63 and MIMA-53 did react with a mouse/human ART4 chimera, indicating that the epitope recognized by these mAbs lies in the C-terminal half of the protein.

ADP-ribosyltransferases (ARTs) transfer ADP-ribose from NAD to arginine, asparagine, or cysteine residues in target proteins. This post-translational protein modification is the mechanism by which cholera-toxin and other bacterial toxins cause pathology in human host cells. Molecular cloning has identified five toxin-related GPI-anchored cell surface ARTs in the mouse (ART1, ART2.1, ART2.2, ART3, and ART4) and three in the human (ART1, ART3, and ART4). ART2-which has sparked interest because of its ability to activate the cytolytic P2X7 purinergic receptor by ADP-ribosylation-is encoded by two functional gene copies in the mouse genome while the human genome carries two inactivated ART2 pseudogenes. We generated stable transfectants for FLAG-tagged versions of each of the functional human and mouse ARTs. Using genetic immunization we raised monoclonal antibodies that recognize the native human ARTs on the surface of living cells. Some of these mAbs recognize an epitope shared with the mouse ART orthologue but not with more distant ART paralogues. Screening of primary cells and established cell lines by FACS revealed expression of ART1 by monocytes, neutrophils and myeloid leukemia cell lines but not by cell lines derived from solid tumors. ART1 and ART4 have been assigned the designations: CD296, and CD297, respectively.

Adenosine diphosphate (ADP)-ribosyl-transferases (ARTs) transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD) onto target proteins. T cells express ART2.2, a toxin-related, glycosylphosphatidylinositol (GPI)-anchored ecto-enzyme. After the release of NAD from cells, ART2.2 ADP-ribosylates the P2X7 purinoceptor, lymphocyte function-associated antigen (LFA-1), and other membrane. Using lymphoma transfectants expressing either ART2.2 with its native GPI anchor (ART2.2-GPI) or ART2.2 with a grafted transmembrane anchor (ART2.2-Tm), we demonstrated that ART2.2-GPI but not ART2.2-Tm associated with glycosphingolipid-enriched microdomains (lipid rafts). At limiting substrate concentrations, ART2.2-GPI exhibited more than 10-fold higher activity than ART2.2-Tm. On intact cells, ART2.2-GPI ADP-ribosylated a small number of distinct target proteins. Strikingly, the disruption of lipid rafts by cyclodextrin or membrane solubilization by Triton X-100 increased the spectrum of modified target proteins. However, ART2.2 itself was a prominent target for ADP-ribosylation only when GPI anchored. Furthermore, cholesterol depletion or detergent solubilization abolished the auto-ADP-ribosylation of ART2.2. These findings imply that ART2.2-GPI, but not ART2.2-Tm, molecules are closely associated on the plasma membrane and lend support to the hypothesis that lipid rafts exist on living cells as platforms to which certain proteins are admitted and others are excluded. Our results further suggest that raft association focuses ART2.2 on specific targets that constitutively or inducibly associate with lipid rafts.

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.

Mammalian ecto ADP-ribosyltransferases (ARTs) constitute a family of structurally related proteins expressed on the cell surface or secreted in the extracellular compartment. Using NAD+ as substrate, they transfer ADP-ribose groups onto target proteins. In contrast to intracellular poly(ADP-ribosyl)transferases (PARPs), these enzymes transfer a single ADPR and are thus mono-ARTs. Five paralogs (ART1-5) have been cloned but only four of them are expressed in human due to a defective ART2 gene, and six in the mouse as the result of ART2 gene duplication. The recent determination of the crystal structure of rat ART2 reveals homologies with bacterial ART toxins and provides a molecular basis for understanding the specificity of ARTs for their targets. A combination of different technological approaches reveals that ecto-ARTs are expressed in different tissues with privileged sites such as heart and skeletal muscles for ART1, T lymphocytes for ART2 or testis for ART5. It also indicates that ART expression is highly regulated. ADP-ribosylation of target proteins on cell surfaces or circulating in body fluids leads to reversible post-translational modifications which can inhibit the targets, as known for bacterial ARTs, or activate them, as in the crosstalk between mouse ART2 and the cytolytic P2X7 receptor on T lymphocytes. ART activity in the extracellular compartment provides sophisticated regulatory mechanisms for cell communication. This designates ecto-ARTs as new candidates for drug targeting.

Cytotoxicity induced by protein ADP-ribosylation is a common theme of certain bacterial toxins and of the mammalian ectoenzyme ART2. Exposure of T cells to NAD, the substrate for ART2-catalyzed ADP-ribosylation, induces exposure of phosphatidylserine, uptake of propidium iodide, and fragmentation of DNA. ART2-specific antibodies raised by gene gun immunization block NAD-induced apoptosis. ART2 catalyzed ADP-ribosylation of cell membrane proteins induces formation of cytolytic membrane pores by activating the P2X7 purinoceptor. This alternative pathway to T cell apoptosis could be triggered upon the release of NAD from intracellular stores, for example, during inflammatory tissue damage.

T cells express a toxin-related ADP-ribosylating ectoenzyme, ART2. Exposure of mature T cells to NAD, the substrate for ADP-ribosylation, induces cell death. ART2-catalyzed ADP-ribosylation activates the cytolytic P2X7 purinoceptor, causing calcium flux, pore formation, phosphatidylserine exposure, shedding of CD62L, cell shrinkage, and propidium iodide uptake. Interestingly, much lower NAD than ATP concentrations are required to activate P2X7. NAD-induced cell death (NICD) operates with endogenous sources of NAD released upon cell lysis. These findings identify P2X7 as a key effector of NICD and demonstrate that P2X7 can be activated by an endogenous ligand other than ATP. Our results delineate an alternative mechanism for inducing T cell death and set an interesting precedent for immunoregulation via crosstalk between NAD-dependent ADP-ribosyltransferases and purinoceptors.

NAD-dependent ADP-ribosylation is one of the posttranslational protein modifications. On mammalian cells, glycosylphosphatidylinositol-anchored cell surface ADP-ribosyltransferases (ARTs) ADP-ribosylate other cell surface proteins and thereby affect important cellular functions. Here we describe convenient flow-cytometric and immunoblot assays for monitoring ADP-ribosylation of cell surface proteins on living cells by exploiting the capacity of ARTs to utilize etheno-NAD as substrate. Etheno-ADP-ribosylation of cell surface proteins can be detected by flow cytometry with 1G4, a monoclonal antibody specific for ethenoadenosine. Labeling of cells with 1G4 is dependent on the expression of cell surface ARTs and occurs only after incubation of ART-expressing cells with etheno-NAD and not with etheno-ADP-ribose. Dose-response analyses show efficient 1G4 staining of ART-expressing cells at micromolar etheno-NAD concentrations. Half-maximal staining is obtained with 1-2 micro M etheno-NAD, saturation is reached at 5-20 micro M etheno-NAD. Immunoblot analyses confirm that ART-expressing cells incorporate ethenoadenosine covalently (i.e., SDS resistant) into several cell surface proteins. The flow-cytometric 1G4 staining assay can be used to identify subpopulations of cells expressing cell surface ART activity and to select ART(hi) cell variants. The immunoblot 1G4 staining assay can also be used to identify etheno-ADP-ribosylated target proteins. These new assays hold promise for many interesting applications in biochemistry and cell biology.

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.

The mammalian extracellular ADP-ribosyl transferases ART1 through ART5 are sequence-related to each other. Among them ART2 is involved in immuno regulation. The variant ART2.2 was expressed in the periplasm of Escherichia coli and crystallized. Its structure was determined by X-ray diffraction at 1.7A resolution in one crystal form and at slightly lower resolutions in two others. The active center was indicated by a ligated nicotinamide analogue, which also revealed a small induced-fit. The centerpiece of the chainfold of ART2.2 agrees with those of all bacterial ADP-ribosyl transferases. This correspondence and the nicotinamide position were used to model the binding structure of the whole substrate NAD(+) at ART2.2. Two of the bacterial enzymes are structurally more closely related to ART2.2 while the others are more closely related to the eukaryotic poly(ADP-ribosyl)polymerase. This splits the ADP-ribosyl transferases into two distinct subfamilies. A special feature of ART2.2 is its long N-terminal extension and two disulfide bridges that are far away from the active center. They stabilize the protein against denaturation and presumably also against shearing forces parallel with the membrane where ART2.2 is anchored.

The two alleles of the rat T-cell differentiation alloantigen RT6 are highly divergent and their expression is distinctively regulated. While the majority of T cells of RT6a/RT6b heterozygous laboratory rats (Rattus norvegicus) expresses both alleles, a subpopulation expresses only RT6b. To identify cis-regulatory elements that potentially control monoallelic expression, we compared the sequences of both alleles. A striking difference is the presence or absence of a rodent identifier (ID) sequence in intron 7. All investigated inbred RT6a rat strains (n=7) had this integration, while it was absent in all investigated RT6b rats (n=9). An ID element was also identified at precisely the same integration site in one RT6 allele of the closely related species Rattus rattus. The ID elements of both species showed nucleotide substitutions characteristic of different subfamilies, and their flanking repeats differed in length, indicating that two independent integration events had occurred into the same site adjacent to a mammalianwide interspersed repeat. Analysis of the surrounding sequences did not disclose any motifs to explain preferential integration into one allele. Our data indicate that the RT6 alleles diverged about 1 myr ago and that the ID element integrated into the RT6a locus soon after this. We have previously shown that DNA methylation plays an important role in regulating monoallelic RT6 expression. The possibility that the ID element in the RT6a allele interferes with the required demethylation process and thus accounts for monoallelic expression is discussed.

ADP-ribosyltransferases including toxins secreted by Vibrio cholera, Pseudomonas aerurginosa, and other pathogenic bacteria inactivate the function of human target proteins by attaching ADP-ribose onto a critical amino acid residue. Cross-species polymerase chain reaction (PCR) and database mining identified the orthologs of these ADP-ribosylating toxins in humans and the mouse. The human genome contains four functional toxin-related ADP-ribosyltransferase genes (ARTs) and two related intron-containing pseudogenes; the mouse has six functional orthologs. The human and mouse ART genes map to chromosomal regions with conserved linkage synteny. The individual ART genes reveal highly restricted expression patterns, which are largely conserved in humans and the mouse. We confirmed the predicted extracellular location of the ART proteins by expressing recombinant ARTs in insect cells. Two human and four mouse ARTs contain the active site motif (R-S-EXE) typical of arginine-specific ADP-ribosyltransferases and exhibit the predicted enzyme activities. Two other human ARTs and their murine orthologues deviate in the active site motif and lack detectable enzyme activity. Conceivably, these ARTs may have acquired a new specificity or function. The position-sensitive iterative database search program PSI-BLAST connected the mammalian ARTs with most known bacterial ADP-ribosylating toxins. In contrast, no related open reading frames occur in the four completed genomes of lower eucaryotes (yeast, worm, fly, and mustard weed). Interestingly, these organisms also lack genes for ADP-ribosylhydrolases, the enzymes that reverse protein ADP-ribosylation. This suggests that the two enzyme families that catalyze reversible mono-ADP-ribosylation either were lost from the genomes of these nonchordata eucaryotes or were subject to horizontal gene transfer between kingdoms.

ADP-ribosyltransferases catalyze the transfer of the ADP-ribose moiety from NAD(+) onto proteins and other targets. These enzymes have been found in prokaryotes and in vertebrates; a eukaryotic enzyme structure is not yet known. The enzyme from Rattus norvegicus was expressed in the Escherichia coli periplasm at a level of about 0.2 mg per litre of culture, purified and crystallized. Native data sets were collected to 2.0 A resolution. A self-rotation function revealed a local twofold axis in crystal form A and a Patterson function showed a translational relationship in form B. Form C contains only one molecule in the asymmetric unit.

ADP-ribosyltransferase activity was shown to be present on the surface of human monocytes. Incubating the cells in the presence of BSA leads to an increase in enzyme activity. The acceptor amino acid mainly responsible for the ADP-ribose bond was identified as a cysteine residue. An increase in ADP-ribosyltransferase activity was observed when cells were treated for 16 h with bacterial lipopolysaccharide (LPS). Possible candidates for catalysing the reaction are mono-ADP-ribosyltransferases (ARTs). When measuring expression of the mRNA of ART1, 3, 4 and 5, only ART3 mRNA was detected in unstimulated monocytes. Upon stimulation for 16 h with LPS, lipoteichoic acid or peptidoglycan, ART4 mRNA was found to be expressed. No ART4 signal appeared after a 4 h exposure of the cells to LPS. Cell-surface proteins were labelled when incubating monocytes with [(32)P]NAD(+). Their molecular masses were 29, 33, 43, 45, 60 and 82 kDa. In response to LPS an additional protein of 31 kDa was found to be labelled. The bound label was resistant to treatment with NH(2)OH but sensitive to HgCl(2), characteristic of a cysteine-linked ADP-ribosylation.