Mono-ADP-Ribosylation Catalyzed by Arginine-Specific ADP-Ribosyltransferases

  • Linda A. Stevens
  • Joel MossEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1813)


Methods are described for determination of arginine-specific mono-ADP-ribosyltransferase activity of purified proteins and intact cells by monitoring the transfer of ADP-ribose from NAD+ to a model substrate, e.g., arginine, agmatine, and peptide (human neutrophil peptide-1 [HNP1]), and for the nonenzymatic hydrolysis of ADP-ribose-arginine to ornithine, a noncoded amino acid. In addition, preparation of purified ADP-ribosylarginine is included as a control substrate for ADP-ribosylation reactions.

Key words

ADP-ribosylarginine HNP-1 NAD+ Mono-ADP-ribosylation ADP-ribose Defensins 



Funding: The study was funded by the Intramural Research Program, NIH, NHLBI.


  1. 1.
    Moss J, Balducci E, Cavanaugh E, Kim HJ, Konczalik P, Lesma EA, Okazaki IJ, Park M, Shoemaker M, Stevens LA, Zolkiewska A (1999) Characterization of NAD:arginine ADP-ribosyltransferases. Mol Cell Biochem 193(1–2):109–113CrossRefGoogle Scholar
  2. 2.
    Spangler BD (1992) Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol Rev 56(4):622–647PubMedPubMedCentralGoogle Scholar
  3. 3.
    Freissmuth M, Gilman AG (1989) Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity. J Biol Chem 264(36):21907–21914PubMedGoogle Scholar
  4. 4.
    Moss J, Vaughan M (1978) Isolation of an avian erythrocyte protein possessing ADP-ribosyltransferase activity and capable of activating adenylate cyclase. Proc Natl Acad Sci U S A 75(8):3621–3624CrossRefGoogle Scholar
  5. 5.
    Osborne JC Jr, Stanley SJ, Moss J (1985) Kinetic mechanisms of two NAD:arginine ADP-ribosyltransferases: the soluble, salt-stimulated transferase from Turkey erythrocytes and choleragen, a toxin from Vibrio cholerae. Biochemistry 24(19):5235–5240CrossRefGoogle Scholar
  6. 6.
    Zolkiewska A, Nightingale MS, Moss J (1992) Molecular characterization of NAD:arginine ADP-ribosyltransferase from rabbit skeletal muscle. Proc Natl Acad Sci U S A 89(23):11352–11356CrossRefGoogle Scholar
  7. 7.
    Glowacki G, Braren R, Firner K, Nissen M, Kuhl M, Reche P, Bazan F, Cetkovic-Cvrlje M, Leiter E, Haag F, Koch-Nolte F (2002) The family of toxin-related ecto-ADP-ribosyltransferases in humans and the mouse. Protein Sci 11(7):1657–1670. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Peterson JE, Larew JS, Graves DJ (1990) Purification and partial characterization of arginine-specific ADP-ribosyltransferase from skeletal muscle microsomal membranes. J Biol Chem 265(28):17062–17069PubMedGoogle Scholar
  9. 9.
    Okazaki IJ, Zolkiewska A, Nightingale MS, Moss J (1994) Immunological and structural conservation of mammalian skeletal muscle glycosylphosphatidylinositol-linked ADP-ribosyltransferases. Biochemistry 33(43):12828–12836CrossRefGoogle Scholar
  10. 10.
    Koch F, Haag F, Thiele HG (1990) Nucleotide and deduced amino acid sequence for the mouse homologue of the rat T-cell differentiation marker RT6. Nucleic Acids Res 18(12):3636CrossRefGoogle Scholar
  11. 11.
    Lindskog C, Linne J, Fagerberg L, Hallstrom BM, Sundberg CJ, Lindholm M, Huss M, Kampf C, Choi H, Liem DA, Ping P, Varemo L, Mardinoglu A, Nielsen J, Larsson E, Ponten F, Uhlen M (2015) The human cardiac and skeletal muscle proteomes defined by transcriptomics and antibody-based profiling. BMC Genomics 16:475. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Koch-Nolte F, Haag F, Braren R, Kuhl M, Hoovers J, Balasubramanian S, Bazan F, Thiele HG (1997) Two novel human members of an emerging mammalian gene family related to mono-ADP-ribosylating bacterial toxins. Genomics 39(3):370–376. CrossRefPubMedGoogle Scholar
  13. 13.
    Corda D, Di Girolamo M (2002) Mono-ADP-ribosylation: a tool for modulating immune response and cell signaling. Sci STKE 2002(163):PE53PubMedGoogle Scholar
  14. 14.
    Zolkiewska A (2005) Ecto-ADP-ribose transferases: cell-surface response to local tissue injury. Physiology (Bethesda) 20:374–381. CrossRefGoogle Scholar
  15. 15.
    Okazaki IJ, Kim HJ, Moss J (1996a) Cloning and characterization of a novel membrane-associated lymphocyte NAD:arginine ADP-ribosyltransferase. J Biol Chem 271(36):22052–22057CrossRefGoogle Scholar
  16. 16.
    Domenighini M, Rappuoli R (1996) Three conserved consensus sequences identify the NAD-binding site of ADP-ribosylating enzymes, expressed by eukaryotes, bacteria and T-even bacteriophages. Mol Microbiol 21(4):667–674CrossRefGoogle Scholar
  17. 17.
    Han S, Tainer JA (2002) The ARTT motif and a unified structural understanding of substrate recognition in ADP-ribosylating bacterial toxins and eukaryotic ADP-ribosyltransferases. Int J Med Microbiol 291(6–7):523–529PubMedGoogle Scholar
  18. 18.
    Hottiger MO, Hassa PO, Luscher B, Schuler H, Koch-Nolte F (2010) Toward a unified nomenclature for mammalian ADP-ribosyltransferases. Trends Biochem Sci 35(4):208–219. CrossRefPubMedGoogle Scholar
  19. 19.
    Grahnert A, Friedrich M, Pfister M, Haag F, Koch-Nolte F, Hauschildt S (2002) Mono-ADP-ribosyltransferases in human monocytes: regulation by lipopolysaccharide. Biochem J 362(Pt 3):717–723CrossRefGoogle Scholar
  20. 20.
    Parusel I, Kahl S, Braasch F, Glowacki G, Halverson GR, Reid ME, Schawalder A, Ortolan E, Funaro A, Malavasi F, Hardie D, Halder S, Buckley CD, Haag F, Koch-Nolte F (2005) A panel of monoclonal antibodies recognizing GPI-anchored ADP-ribosyltransferase ART4, the carrier of the Dombrock blood group antigens. Cell Immunol 236(1–2):59–65. CrossRefPubMedGoogle Scholar
  21. 21.
    Heine H, Ulmer AJ, Flad HD, Hauschildt S (1995) Lipopolysaccharide-induced change of phosphorylation of two cytosolic proteins in human monocytes is prevented by inhibitors of ADP-ribosylation. J Immunol 155(10):4899–4908PubMedGoogle Scholar
  22. 22.
    Saxty BA, Kefalas P, Yadollahi-Farsani M, MacDermot J (1998) Arginine-specific ADP-ribosyltransferases in leukocytes. J Leukoc Biol 63(1):15–21CrossRefGoogle Scholar
  23. 23.
    Donnelly LE, Rendell NB, Murray S, Allport JR, Lo G, Kefalas P, Taylor GW, MacDermot J (1996) Arginine-specific mono(ADP-ribosyl)transferase activity on the surface of human polymorphonuclear neutrophil leucocytes. Biochem J 315(Pt 2):635–641CrossRefGoogle Scholar
  24. 24.
    Kefalas P, Allport JR, Donnelly LE, Rendell NB, Murray S, Taylor GW, Lo G, Yadollahi-Farsani M, MacDermot J (1997) Arginine-specific mono(ADP-ribosyl)transferase activity in human neutrophil polymorphs. A possible link with the assembly of filamentous actin and chemotaxis. Adv Exp Med Biol 419:241–244CrossRefGoogle Scholar
  25. 25.
    Wang J, Nemoto E, Dennert G (1997) Regulation of cytotoxic T cell functions by a GPI-anchored ecto-ADP-ribosyltransferase. Adv Exp Med Biol 419:191–201CrossRefGoogle Scholar
  26. 26.
    Wang J, Nemoto E, Kots AY, Kaslow HR, Dennert G (1994) Regulation of cytotoxic T cells by ecto-nicotinamide adenine dinucleotide (NAD) correlates with cell surface GPI-anchored/arginine ADP-ribosyltransferase. J Immunol 153(9):4048–4058PubMedGoogle Scholar
  27. 27.
    Liu ZX, Yu Y, Dennert G (1999) A cell surface ADP-ribosyltransferase modulates T cell receptor association and signaling. J Biol Chem 274(25):17399–17401CrossRefGoogle Scholar
  28. 28.
    Mehta K, Shahid U, Malavasi F (1996) Human CD38, a cell-surface protein with multiple functions. FASEB J 10(12):1408–1417CrossRefGoogle Scholar
  29. 29.
    Han MK, Cho YS, Kim YS, Yim CY, Kim UH (2000) Interaction of two classes of ADP-ribose transfer reactions in immune signaling. J Biol Chem 275(27):20799–20805. CrossRefPubMedGoogle Scholar
  30. 30.
    Tonnesen MG, Feng X, Clark RA (2000) Angiogenesis in wound healing. J Investig Dermatol Symp Proc 5(1):40–46. CrossRefPubMedGoogle Scholar
  31. 31.
    Boulle N, Jones EM, Auguste P, Baird A (1995) Adenosine diphosphate ribosylation of fibroblast growth factor-2. Mol Endocrinol 9(6):767–775. CrossRefPubMedGoogle Scholar
  32. 32.
    Jones EM, Baird A (1997) Cell-surface ADP-ribosylation of fibroblast growth factor-2 by an arginine-specific ADP-ribosyltransferase. Biochem J 323(Pt 1):173–177CrossRefGoogle Scholar
  33. 33.
    Saxty BA, Yadollahi-Farsani M, Upton PD, Johnstone SR, MacDermot J (2001) Inactivation of platelet-derived growth factor-BB following modification by ADP-ribosyltransferase. Br J Pharmacol 133(8):1219–1226. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    van Wetering S, Tjabringa GS, Hiemstra PS (2005) Interactions between neutrophil-derived antimicrobial peptides and airway epithelial cells. J Leukoc Biol 77(4):444–450CrossRefGoogle Scholar
  35. 35.
    Lehrer RI, Ganz T (1992) Defensins: endogenous antibiotic peptides from human leukocytes. Ciba Found Symp 171:276–290; discussion 290–3PubMedGoogle Scholar
  36. 36.
    Balducci E, Horiba K, Usuki J, Park M, Ferrans VJ, Moss J (1999) Selective expression of RT6 superfamily in human bronchial epithelial cells. Am J Respir Cell Mol Biol 21(3):337–346CrossRefGoogle Scholar
  37. 37.
    Okazaki IJ, Moss J (1999) Characterization of glycosylphosphatidylinositiol-anchored, secreted, and intracellular vertebrate mono-ADP-ribosyltransferases. Annu Rev Nutr 19:485–509CrossRefGoogle Scholar
  38. 38.
    Paone G, Wada A, Stevens LA, Matin A, Hirayama T, Levine RL, Moss J (2002) ADP ribosylation of human neutrophil peptide-1 regulates its biological properties. Proc Natl Acad Sci U S A 99(12):8231–8235. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Paone G, Stevens LA, Levine RL, Bourgeois C, Steagall WK, Gochuico BR, Moss J (2006a) ADP-ribosyltransferase-specific modification of human neutrophil peptide-1. J Biol Chem 281(25):17054–17060CrossRefGoogle Scholar
  40. 40.
    Stevens LA, Levine RL, Gochuico BR, Moss J (2009) ADP-ribosylation of human defensin HNP-1 results in the replacement of the modified arginine with the noncoded amino acid ornithine. Proc Natl Acad Sci U S A 106(47):19796–19800. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Stevens LA, Barbieri JT, Piszczek G, Otuonye AN, Levine RL, Zheng G, Moss J (2014) Nonenzymatic conversion of ADP-ribosylated arginines to ornithine alters the biological activities of human neutrophil peptide-1. J Immunol 193(12):6144–6151. CrossRefPubMedGoogle Scholar
  42. 42.
    Zolkiewska A, Moss J (1993) Integrin alpha 7 as substrate for a glycosylphosphatidylinositol-anchored ADP-ribosyltransferase on the surface of skeletal muscle cells. J Biol Chem 268(34):25273–25276PubMedGoogle Scholar
  43. 43.
    Zolkiewska A, Thompson WC, Moss J (1998) Interaction of integrin alpha 7 beta 1 in C2C12 myotubes and in solution with laminin. Exp Cell Res 240(1):86–94. CrossRefPubMedGoogle Scholar
  44. 44.
    Burkin DJ, Kaufman SJ (1999) The alpha7beta1 integrin in muscle development and disease. Cell Tissue Res 296(1):183–190CrossRefGoogle Scholar
  45. 45.
    Soman G, Mickelson JR, Louis CF, Graves DJ (1984) NAD: guanidino group specific mono ADP-ribosyltransferase activity in skeletal muscle. Biochem Biophys Res Commun 120(3):973–980CrossRefGoogle Scholar
  46. 46.
    Kefalas P, Saxty B, Yadollahi-Farsani M, MacDermot J (1999) Chemotaxin-dependent translocation of immunoreactive ADP-ribosyltransferase-1 to the surface of human neutrophil polymorphs. Eur J Biochem 259(3):866–871CrossRefGoogle Scholar
  47. 47.
    Little E, Ramakrishnan M, Roy B, Gazit G, Lee AS (1994) The glucose-regulated proteins (GRP78 and GRP94): functions, gene regulation, and applications. Crit Rev Eukaryot Gene Expr 4(1):1–18CrossRefGoogle Scholar
  48. 48.
    Chambers JE, Petrova K, Tomba G, Vendruscolo M, Ron D (2012) ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load. J Cell Biol 198(3):371–385. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Leno GH, Ledford BE (1989) ADP-ribosylation of the 78-kDa glucose-regulated protein during nutritional stress. Eur J Biochem 186(1–2):205–211CrossRefGoogle Scholar
  50. 50.
    Fabrizio G, Di Paola S, Stilla A, Giannotta M, Ruggiero C, Menzel S, Koch-Nolte F, Sallese M, Di Girolamo M (2015) ARTC1-mediated ADP-ribosylation of GRP78/BiP: a new player in endoplasmic-reticulum stress responses. Cell Mol Life Sci 72(6):1209–1225. CrossRefPubMedGoogle Scholar
  51. 51.
    Cassel D, Selinger Z (1977) Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site. Proc Natl Acad Sci U S A 74(8):3307–3311CrossRefGoogle Scholar
  52. 52.
    Lupi R, Corda D, Di Girolamo M (2000) Endogenous ADP-ribosylation of the G protein beta subunit prevents the inhibition of type 1 adenylyl cyclase. J Biol Chem 275(13):9418–9424CrossRefGoogle Scholar
  53. 53.
    Lupi R, Dani N, Dietrich A, Marchegiani A, Turacchio S, Berrie CP, Moss J, Gierschik P, Corda D, Di Girolamo M (2002) Endogenous mono-ADP-ribosylation of the free Gbetagamma prevents stimulation of phosphoinositide 3-kinase-gamma and phospholipase C-beta2 and is activated by G-protein-coupled receptors. Biochem J 367(Pt 3):825–832. CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Kato J, Vekhter D, Heath J, Zhu J, Barbieri JT, Moss J (2015) Mutations of the functional ARH1 allele in tumors from ARH1 heterozygous mice and cells affect ARH1 catalytic activity, cell proliferation and tumorigenesis. Oncogene 4:e151. CrossRefGoogle Scholar
  55. 55.
    Kato J, Zhu J, Liu C, Stylianou M, Hoffmann V, Lizak MJ, Glasgow CG, Moss J (2011) ADP-ribosylarginine hydrolase regulates cell proliferation and tumorigenesis. Cancer Res 71(15):5327–5335. CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Karras GI, Kustatscher G, Buhecha HR, Allen MD, Pugieux C, Sait F, Bycroft M, Ladurner AG (2005) The macro domain is an ADP-ribose binding module. EMBO J 24(11):1911–1920. CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Dani N, Stilla A, Marchegiani A, Tamburro A, Till S, Ladurner AG, Corda D, Di Girolamo M (2009) Combining affinity purification by ADP-ribose-binding macro domains with mass spectrometry to define the mammalian ADP-ribosyl proteome. Proc Natl Acad Sci U S A 106(11):4243–4248. CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Martello R, Leutert M, Jungmichel S, Bilan V, Larsen SC, Young C, Hottiger MO, Nielsen ML (2016) Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue. Nat Commun 7:12917. CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Vivelo CA, Wat R, Agrawal C, Tee HY, Leung AK (2017) ADPriboDB: the database of ADP-ribosylated proteins. Nucleic Acids Res 45(D1):D204–D209. CrossRefPubMedGoogle Scholar
  60. 60.
    Moss J, Stevens LA, Cavanaugh E, Okazaki IJ, Bortell R, Kanaitsuka T, Mordes JP, Greiner DL, Rossini AA (1997) Characterization of mouse Rt6.1 NAD:arginine ADP-ribosyltransferase. J Biol Chem 272(7):4342–4346CrossRefGoogle Scholar
  61. 61.
    Oka S, Kato J, Moss J (2006) Identification and characterization of a mammalian 39-kDa poly(ADP-ribose) glycohydrolase. J Biol Chem 281(2):705–713. CrossRefPubMedGoogle Scholar
  62. 62.
    Moss J, Vaughan M (1977) Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor. J Biol Chem 252(7):2455–2457PubMedGoogle Scholar
  63. 63.
    Kato J, Zhu J, Liu C, Moss J (2007) Enhanced sensitivity to cholera toxin in ADP-ribosylarginine hydrolase-deficient mice. Mol Cell Biol 27(15):5534–5543CrossRefGoogle Scholar
  64. 64.
    Wernick NL, Chinnapen DJ, Cho JA, Lencer WI (2010) Cholera toxin: an intracellular journey into the cytosol by way of the endoplasmic reticulum. Toxins (Basel) 2(3):310–325. CrossRefGoogle Scholar
  65. 65.
    Stevens LA, Bourgeois C, Bortell R, Moss J (2003) Regulatory role of arginine 204 in the catalytic activity of rat alloantigens ART2a and ART2b. J Biol Chem 278(22):19591–19596. CrossRefPubMedGoogle Scholar
  66. 66.
    Konczalik P, Moss J (1999) Identification of critical, conserved vicinal aspartate residues in mammalian and bacterial ADP-ribosylarginine hydrolases. J Biol Chem 274(24):16736–16740CrossRefGoogle Scholar
  67. 67.
    Wisniewski JR, Zougman A, Nagaraj N, Mann M (2009) Universal sample preparation method for proteome analysis. Nat Methods 6(5):359–362. CrossRefPubMedGoogle Scholar
  68. 68.
    Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357CrossRefGoogle Scholar
  69. 69.
    Allen MD, Buckle AM, Cordell SC, Lowe J, Bycroft M (2003) The crystal structure of AF1521 a protein from Archaeoglobus fulgidus with homology to the non-histone domain of macroH2A. J Mol Biol 330(3):503–511CrossRefGoogle Scholar
  70. 70.
    Moss J, Yost DA, Stanley SJ (1983) Amino acid-specific ADP-ribosylation. J Biol Chem 258(10):6466–6470PubMedGoogle Scholar
  71. 71.
    Payne DM, Jacobson EL, Moss J, Jacobson MK (1985) Modification of proteins by mono(ADP-ribosylation) in vivo. Biochemistry 24(26):7540–7549CrossRefGoogle Scholar
  72. 72.
    Weng B, Thompson WC, Kim HJ, Levine RL, Moss J (1999) Modification of the ADP-ribosyltransferase and NAD glycohydrolase activities of a mammalian transferase (ADP-ribosyltransferase 5) by auto-ADP-ribosylation. J Biol Chem 274(45):31797–31803CrossRefGoogle Scholar
  73. 73.
    Tsuchiya M, Tanigawa Y, Mishima K, Shimoyama M (1986) Determination of ADP-ribosyl arginine anomers by reverse-phase high-performance liquid chromatography. Anal Biochem 157(2):381–384CrossRefGoogle Scholar
  74. 74.
    Moss J, Oppenheimer NJ, West RE Jr, Stanley SJ (1986) Amino acid specific ADP-ribosylation: substrate specificity of an ADP-ribosylarginine hydrolase from Turkey erythrocytes. Biochemistry 25(19):5408–5414CrossRefGoogle Scholar
  75. 75.
    Moss J, Stanley SJ, Nightingale MS, Murtagh JJ Jr, Monaco L, Mishima K, Chen HC, Williamson KC, Tsai SC (1992) Molecular and immunological characterization of ADP-ribosylarginine hydrolases. J Biol Chem 267(15):10481–10488PubMedGoogle Scholar
  76. 76.
    McDonald LJ, Moss J (1994) Enzymatic and nonenzymatic ADP-ribosylation of cysteine. Mol Cell Biochem 138(1–2):221–226CrossRefGoogle Scholar
  77. 77.
    Cervantes-Laurean D, Minter DE, Jacobson EL, Jacobson MK (1993) Protein glycation by ADP-ribose: studies of model conjugates. Biochemistry 32(6):1528–1534CrossRefGoogle Scholar
  78. 78.
    Hsia JA, Tsai SC, Adamik R, Yost DA, Hewlett EL, Moss J (1985) Amino acid-specific ADP-ribosylation. Sensitivity to hydroxylamine of [cysteine(ADP-ribose)]protein and [arginine(ADP-ribose)]protein linkages. J Biol Chem 260(30):16187–16191PubMedGoogle Scholar
  79. 79.
    Okazaki IJ, Kim HJ, McElvaney NG, Lesma E, Moss J (1996b) Molecular characterization of a glycosylphosphatidylinositol-linked ADP-ribosyltransferase from lymphocytes. Blood 88(3):915–921PubMedGoogle Scholar
  80. 80.
    Rosenthal F, Feijs KL, Frugier E, Bonalli M, Forst AH, Imhof R, Winkler HC, Fischer D, Caflisch A, Hassa PO, Luscher B, Hottiger MO (2013) Macrodomain-containing proteins are new mono-ADP-ribosylhydrolases. Nat Struct Mol Biol 20(4):502–507. CrossRefPubMedGoogle Scholar
  81. 81.
    Kleine H, Poreba E, Lesniewicz K, Hassa PO, Hottiger MO, Litchfield DW, Shilton BH, Luscher B (2008) Substrate-assisted catalysis by PARP10 limits its activity to mono-ADP-ribosylation. Mol Cell 32(1):57–69. CrossRefPubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Pulmonary Branch, National Heart, Lung, and Blood InstituteNational Institutes of HealthBethesdaUSA

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