Mono-ADP-ribosylation, in which the ADP-ribose moiety of NAD is transferred to a target protein, is catalyzed by a family of bacterial toxins and mammalian enzymes. Some toxin mono-ADP-ribosyltransferases (e.g., cholera toxin, diphtheria toxin) are responsible for symptoms of the diseases caused by the bacterium. Mammalian cells contain enzymes that catalyze reactions similar to the bacterial toxins. Mammalian mono-ADP-ribosyltransferases (ARTs) can be located within the cell and on the cell surface, sometimes linked through a glycosylphosphatidylinositol (GPI) anchor (ART1). Others, ART5, appear to be secreted. Several of the mammalian mono-ADP-ribosyltransferases have been cloned in the laboratory; they display some structural similarities to the toxins, with amino acid identities in the catalytic site. A product of transferase-catalyzed reactions, ADP-ribose-(arginine)protein, is cleaved by a 39-kDa ADP-ribosylarginine hydrolase (ARH1)to regenerate unmodified protein. Thus, transferases and hydrolases can catalyze opposing reactions to constitute an ADP-ribosylation cycle.? ? In addition to mono-ADP-ribosyltransferases, mammalian cells contain enzymes involved in poly(ADP-ribosylation); these proteins participate in several critical physiological processes, including DNA repair, cellular differentiation, and carcinogenesis. Multiple poly(ADP-ribose) polymerases have been identified in the human genome, but there is only one known poly(ADP-ribose) glycohydrolase (PARG), a 111-kDa protein that degrades the (ADP-ribose) polymer to ADP-ribose. Two other proteins in the mouse and human gene databases, the 39-kDa ARH2 and ARH3, appear to resemble ARH1. In the present study, we observed that the ARH1-like protein, termed poly(ADP-ribose) hydrolase or ARH3, exhibited PARG activity, generating ADP-ribose from poly-(ADP-ribose), but did not hydrolyze ADP-ribose-arginine, -cysteine, -diphthamide, or -asparagine bonds. The 39-kDa ARH3 shares amino acid sequence identity with both ARH1 and the catalytic domain of PARG. ARH3 activity, like that of ARH1, was enhanced by Mg(2+). Thiols, which enhance the activity of ARH1 from some species, were not required for the activity of murine ARH3. Critical vicinal acidic amino acids in ARH3, identified by mutagenesis (Asp(77) and Asp(78)), are located in a region similar to that required for activity in ARH1 (Asp (60) and Asp (61)) but different from the location of the critical vicinal glutamates in the PARG catalytic site. All findings are consistent with the conclusion that ARH3 has PARG activity but is structurally unrelated to PARG, except for regions in the catalytic domain. This new member of the PARG family might have different function(s) from the previously studied enzymes and could play a specific role(s) in the regulation of ADP-ribose metabolism.? ? Epithelial cells lining human airways and cells recruited to airways participate in the innate immune response in part by releasing human neutrophil peptides (HNP). We previously reported that arginine-specific mono-ADP-ribosyltransferases (ART) on the surface of these cells can catalyze the transfer of mono-ADP-ribose from NAD to proteins. In addition, we noted that ART1, a mammalian ADP-ribosyltransferase, present in epithelial cells lining the human airway, modified HNP-1, altering its function. ADP-ribosylated HNP-1 was identified in bronchoalveolar lavage fluid (BALF) from patients with asthma, idiopathic pulmonary fibrosis, or a history of smoking (and having two common polymorphic forms of ART1 that differ in activity), but not in healthy volunteers or patients with lymphangioleiomyomatosis (LAM). Modified HNP-1 was not found in the sputum of patients with cystic fibrosis or in leukocyte granules of healthy volunteers. The finding of ADP-ribosyl-HNP-1 in BALF but not in leukocyte granules suggests that the modification occurred in the airway. Most of the HNP-1 in the BALF from individuals with a history of smoking was, in fact, mono- or di-ADP-ribosylated. ART1 synthesized in Escherichia coli, glycosylphosphatidylinositol-anchored ART1 released with phosphatidylinositol-specific phospholipase C from transfected NMU cells, or ART1 expressed endogenously on C2C12 myotubes modified arginine 14 on HNP-1 with a secondary site on arginine 24. ADP-ribosylation of HNP-1 by ART1 was substantially greater than that by ART3, ART4, ART5, Pseudomonas aeruginosa exoenzyme S, or cholera toxin A subunit. Mouse ART2, which is an NAD:arginine ADP-ribosyltransferase, was able to modify HNP-1, but to a lesser extent than ART1. Although HNP-1 was not modified to a significant degree by ART5, it inhibited ART5 as well as ART1 activities. Human beta-defensin-1 (HBD1) was a poor transferase substrate. Reduction of the cysteine-rich defensins enhanced their ability to serve as ADP-ribose acceptors. Denaturation of the molecule may thus expose additional ADP-ribose acceptor sites. We conclude that ADP-ribosylation of HNP-1 appears to be primarily an activity of ART1 and occurs in inflammatory conditions and disease.? ? It is apparent from these and other studies that 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 ART2.2 allelic protein is multimeric and involves a critical arginine (185). Our backbone structural alignment of 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). Further, we identified the site of ADP-ribose polymer attachment on ART2 as R185, 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.
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