MonoADP-ribosylation is a post-translational modification, catalyzed by ADP-ribosyltransferase (ADPRT), that transfers the ADP-ribose moiety from NAD+ to a specific amino acid residue of target protein and releases the nicotinamide moiety. To date, four amino acid-specific ADPRT have been reported, specific for arginine, cysteine, diphthamide (a modified form of histidine), and asparagine residues. These transferases were originally discovered as the cholera, pertussis, and diphtheria toxins and the Clostridium botulinum C3 enzyme (1, 2), in the order of amino acid-specificity described above. The native form of such ADP-ribosylating toxins is a heteromultimer, and one of the subunits has ADP-ribosyltransferase activity (1). The catalytic subunit penetrates the host cell with the assistance of the other subunits. The prokaryotic ADPRT modify target proteins, including a variety of GTP-binding proteins, in eukaryotic cells (Fig. 1). This modification leads to alterations in the target protein, and consequently in cell functions (1). The bacterial toxins and C3 enzyme have proven extremely useful in studies on signal transduction pathways in various cell types of eukaryotes. MonoADP-ribosylation must be distinguished from poly-ADP ribosylation.
Figure 1. Target protein and acceptor site for ADP-ribosylation in eukaryotic cells by prokaryotic toxins and C3 enzyme.
The reversible Arg-specific ADP-ribosylation (Fig. 2), like protein phosphorylation, was initially documented as a regulatory mechanism for control of nitrogen fixation in the photosynthetic bacteria Rhodospirillum rubrum and Azospirillum braziliense (3). Endogenous dinitrogenase reductase-ADPRT modifies Arg101 on the target protein, dinitrogenase reductase, resulting in inactivation of the reductase. Recovery from the inactive form is achieved through cleavage of the Arg-ADP-ribose linkage by dinitrogenase reductase-activating glycohydrolase. This is the only example of metabolic regulation through endogenous ADP-ribosylation. Purification, characterization, and molecular cloning of eukaryotic Arg-specific ADP-ribosyltransferase (4-7) and of ADP-ribosyl-Arg hydrolase (8) were reported. By analogy to prokaryotes, metabolic regulation through reversible ADP-ribosylation in eukaryotes has been postulated.
Among the eukaryotic ADPRTs detected, the best-defined one is Arg-specific. Purification and characterization of the ADPRT from turkey erythrocytes (4), rabbit skeletal muscle (9), and chicken peripheral heterophils (polymorphonuclear leukocytes) (5) revealed that these enzymes exhibit different physical and regulatory properties, kinetics, and intracellular localization. Guanidino compounds, such as arginine and agmatine, function in vitro as acceptors for Arg-specific ADPRT (4). With b-NAD+ and arginine as substrate, a-ADP-ribosylated arginine is formed by Arg-specific ADP-ribosyltransferase; and the a anomer, but not the b anomer, is utilized as substrate by ADP-ribosyl-Arg hydrolase (10).
Figure 2. Arg-specific ADP-ribosylation and de-ADP-ribosylation reactions.
Some of the ADPRTs catalyze NAD glycohydrolysis or auto-Arg-specific ADP-ribosylation, when either water or the enzyme itself serve as acceptor for ADP-ribose (1, 2). Upon incubation of intact cells or cell lysates with [ P]NAD , it would appear that the ADP-ribose released from NAD by cellular NAD glycohydrolase is attached to some proteins nonenzymatically (11, 12). Thus, enzymatic and non-enzymatic ADP-ribosylations should be differentiated.
Molecular cloning and expression studies of complementary DNA for Arg-specific ADPRT revealed that the rabbit and human skeletal muscle transferases are glycosylphosphatidylinositol-anchored (GPI-anchored) (6) and that two forms of transferases from chicken bone marrow cells are secreted (7).
The previously known rat and mouse T-cell marker RT6 have significant sequence Homology to the ADPRT. It is now accepted that these proteins are GPI-anchored and possess transferase and/or NAD glycohydrolase activities (13-15).
A common feature of the vertebrate Arg-specific ADP-ribosyltransferase is their resemblance to the transferase from cholera toxin and to Escherichia coli heat-labile enterotoxin. Strictly conserved regions around the active site containing two Glu residues (E207 and E209, indicated with an asterisk in Fig. 3) are seen in all Arg-specific ADPRT detected in vertebrates, bacteria, and viruses. On the other hand, RT6.1 and RT6.2 (rat), which have NAD glycohydrolase activity, but not Arg-specific ADPRT, contain Gln in place of Glu207. The recombinant protein of a mutant Gln207Glu-RT6.1 possesses Arg-specific ADPRT activity (16, 17), but alterations in the kinetic parameters of the NAD glycohydrolase reaction are slight (17). Furthermore, the mouse homologues of rat RT6, Rt6.1, and Rt6.2 have Glu207 and ADPRT activity. When the Glu is replaced with Gln, Rt6.1 loses the activity (17).
Figure 3. Comparison of the amino acid sequences of a Glu-rich motif in eukaryotic, prokaryotic, and viral Arg-specific ADP-ribosyltransferases and T-cell antigen RT6. References are cited by the numbers in parentheses. ADPRT: arginine-specific ADP-ribosyltransferase.
It has been reported that defects in RT6 expression are associated in various animal models with the pathogenesis of autoimmune insulin-dependent diabetes and systemic lupus erythematosus (18, 19), although a possible mechanism for RT6-mediated protection from these autoimmune diseases is unknown. Definite physiological functions of monoADP-ribosylation in eukaryotes must be established by further investigation.
