Glutamic acid 207 in rodent T-cell RT6 antigens is essential for arginine-specific ADP-ribosylation.

A rat T-cell antigen RT6.1 catalyzes NAD glycohydrolysis but not ADP-ribose transfer, even though the antigen has significant amino acid identity with eucaryotic arginine-specific ADP-ribosyltransferases. Since a highly conserved Glu in the catalytic region of these transferases is substituted with Gln at position 207 in RT6.1, we replaced the Gln with Glu, Asp, or Ala, by site-directed mutagenesis. The Glu-207 mutant produced ADP-ribosylarginine during incubation with NAD and L-arginine. The Asp-207 mutant but not the Ala-207 mutant produced ADP-ribosylarginine, but at a lower rate. In contrast, these mutations affected NAD glycohydrolase activity of RT6.1 to a much lesser extent. Kinetic studies of transferase reaction revealed that kcat of the Glu-207 mutant increased compared to findings with the Asp-207 mutant. Moreover, the mouse homologue of rat RT6 lost arginine-specific ADP-ribosyltransferase activity when Glu-207 was replaced with Gln. Thus, Glu-207 in rodent T-cell RT6 antigens is essential for transfer reaction of ADP-ribose to arginine.

Arginine-specific ADP-ribosyltransferase catalyzes transfer of the ADP-ribose moiety of NAD to simple guanidino compounds such as arginine or an arginine residue of a target protein, forming ADP-ribose-acceptor adducts (1,2). Molecular cloning has revealed the primary structures of ADP-ribosyltransferases in eucaryotes, including rabbit (3) and human skeletal muscles (4) and chicken bone marrow cells (5). Homology searches revealed highly conserved regions in the deduced amino acid sequences of chicken and skeletal muscle transferases (3)(4)(5). A gene with overall sequence similarity to these transferases was cloned from chicken erythroblasts; the functional expression was not described (6).
The only known protein to which these arginine-specific ADP-ribosyltransferases show significant homology is the rat (7,8) and mouse (9) T-cell antigenic system RT6 (3,5). It has been reported that RT6-specific antisera activate T-cells (10) and that defects in RT6 expression are associated with the pathogenesis of autoimmune insulin-dependent diabetes in diabetes-prone BB rats (11). Based on sequence similarity, Takada et al. (12) examined enzyme activities of the rat RT6.2 expressed in mammary adenocarcinoma cells and found that cells transfected with the RT6.2 gene exhibited NAD glycohy-drolase (NADase), but not ADP-ribosyltransferase, activity. In contrast, Haag et al. (13) detected an arginine-specific auto-ADP-ribosylation of RT6.2 but no modification on RT6.1. More recently, Maehama et al. (14) have reported that RT6.1 was auto-ADP-ribosylated at arginine residues. Thus, whether rat RT6 antigens are indeed arginine-specific ADP-ribosyltransferases has remained controversial.
We report here that the mutant RT6.1 in which Gln-207 was replaced with glutamic acid exhibited arginine-specific ADPribosyltransferase activity, while wild-type RT6.1 exhibited only NADase activity. Furthermore, the mouse homologue of rat RT6 (MRT6H), a recently characterized arginine-specific ADP-ribosyltransferase (15), lost activity upon substitution of Glu-207 with glutamine.
Preparation of Other Proteins-A rat recombinant ADP-ribosylarginine hydrolase (AAH) was prepared as a glutathione S-transferase (GST)-fusion protein (GST-AAH), as described previously (16). Whole histones were purified from liver nuclei of the chicken (17).
Enzyme Assays-MBP-RT6.1, the RT6.1 mutants, MBP-MRT6H, or E207Q-MRT6H were incubated in 50 mM Tris-Cl Ϫ (pH 7.5) and 5 mM NAD in the presence or absence of 0.1 M L-arginine in a final volume of 0.1 ml at 37°C, for the indicated times. The reactions were terminated by 10-fold dilution with 0.1% trifluoroacetic acid. ADP-ribosylarginine was separated on a Cosmosil 5C-18 MS column (4.6 ϫ 150 mm, Nacalai Tesque) with 0.1% trifluoroacetic acid as a mobile phase, at a flow rate of 0.3 ml/min and detected at 254 nm (18). For the kinetic experiments of ADP-ribosyltransferase activity, purified MBP-RT6.1 or the RT6.1 mutants were incubated in 50 mM Tris-Cl Ϫ (pH 7.5), 10 g of bovine serum albumin, and the specified concentrations of [ 32 P]NAD and arginine-rich histone (Sigma, type VIII-S) in a final volume of 0.1 ml at 37°C for the indicated time. The reactions were terminated by adding 10% trichloroacetic acid, and radioactivity of the acid-insoluble material collected on a glass filter (Whatman GF/A) was counted. For NADase assay, purified MBP-RT6.1 or the RT6.1 mutants were incubated in 50 mM Tris-Cl Ϫ (pH 7.5), 20 g of BSA, and varying concentrations of [carbonyl-14 C]NAD (0.39 kBq/nmol) in a final volume of 0.2 ml at 37°C for 10 min. The reactions were terminated by adding 0.2 ml of 5 N KCN and 0.4 ml of water-saturated ethyl acetate. The amount of radioactivity extracted into the organic phase was counted. Kinetic parameters were determined by analysis of a Lineweaver-Burk plot of initial rates of ADP-ribosylation and NAD hydrolysis.
Zymographic in Situ Gel Assay-MBP-MRT6H and E207Q-MRT6H were fractionated by SDS-PAGE on a 12.5% gel under nonreducing conditions. The proteins were renatured by incubating the gel in 2.5% Triton X-100 and then in distilled water. The gel was incubated with 10 M [adenylate-32 P]NAD (6.72 kBq/nmol) and 0.2 mg/ml poly-L-arginine for 14 h at 25°C. After the incubation, the gel was fixed with 10% trichloroacetic acid, washed to remove unreacted NAD, dried, and exposed to the x-ray film.
In Fig. 2, the amino acid sequences of rodent T-cell RT6 antigens were compared with those of the eucaryotic argininespecific ADP-ribosyltransferases in the strictly conserved region containing the two catalytic glutamic acid residues (21). In RT6.1 and RT6.2, the preceding glutamic acid residue is re- placed with glutamine. Assuming that Gln-207 in RT6.1 and the corresponding glutamic acid in the eucaryotic transferases may account for differences in their enzymatic activities, we introduced site-directed mutations into RT6.1 cDNA to replace Gln-207 with glutamic acid (Q207E-RT6.1), as well as with aspartic acid (Q207D-RT6.1) or alanine (Q207A-RT6.1). The resultant cDNAs were expressed in E. coli, and the RT6.1 mutants were partially purified with amylose resin. We then incubated wild-type RT6.1 and the RT6.1 mutants with 5 mM NAD and 0.1 M L-arginine at 37°C and analyzed the reaction products by reversed-phase HPLC. Fig. 3 shows time courses of changes in the amount of ADP-ribosylarginine. Q207E-RT6.1 rapidly formed ADP-ribosylarginine. Q207D-RT6.1 also formed ADP-ribosylarginine, but at a rate equivalent to about 3% that of Q207E-RT6.1 (Fig. 3). Wild-type RT6.1 (as described above) and Q207A-RT6.1 did not catalyze ADP-ribosylation (Fig. 3). Therefore, whether or not the amino acid residue 207 in RT6.1 has a carboxyl group in its side chain seems to determine if the protein can catalyze arginine-specific ADP-ribosylation.
To confirm the significance of Glu-207 of RT6.1 in arginine-specific ADP-ribosylation, we made use of the fact that MRT6H, a recently characterized arginine-specific ADP-ribosyltransferase (15), has intrinsic Glu-207 (9) (Fig. 2). We expressed MRT6H in E. coli as an MBP-fusion protein and searched for ADP-ribosyltransferase activity. As shown in Fig.  4, zymographic in situ assay revealed that arginine-specific ADP-ribosyltransferase activity was associated with a 70-kDa protein, consistent with the molecular mass of MBP-MRT6H. HPLC analysis detected ADP-ribosylarginine formation during incubation of MBP-MRT6H with NAD and L-arginine (0.74 Ϯ 0.07 nmol/g/h, mean Ϯ S.D. of three separate experiments). We then substituted Glu-207 of MRT6H with glutamine (E207Q-MRT6H). Neither zymographic in situ assay (Fig. 4, lane 2) nor HPLC analysis of ADP-ribosylarginine formation detected arginine-specific ADP-ribosyltransferase activity of E207Q-MRT6H.
To evaluate the effects of replacement of Gln-207 with glutamic acid, aspartic acid, or alanine, we purified wild-type RT6.1 and the RT6.1 mutants to homogeneity (data not shown) and determined the kinetic parameters (K m , k cat , and k cat /K m ) for NADase and ADP-ribosyltransferase reactions. For NADase reaction, none of the mutants showed major alterations in K m for NAD, except for Q207E-RT6.1 which showed a slightly higher K m (2-fold) than the wild-type RT6.1 (Table I). k cat and k cat /K m were unchanged by Q207E mutation, while the NAD hydrolysis catalyzed by Q207D and Q207A mutants was much slower (7-fold lower k cat ) and less efficient (5-fold lower k cat /K m ) than that by the wild-type RT6.1. Kinetic analysis of the ADP-ribosyltransferase reaction was carried out with arginine-rich histone as a substrate. Q207E-RT6.1 and Q207D-RT6.1 could ADP-ribosylate the histone, although the   wild-type RT6.1 and Q207A-RT6.1 were completely inactive (Table II). Radioactivity incorporated from [ 32 P]NAD into the histone was removed by GST-AAH (data not shown). Q207E-RT6.1 has a higher affinity for the histone (5-fold lower K m ) and a much higher rate (18-fold higher k cat ), thus a much higher efficiency (86-fold higher k cat /K m ) than Q207D-RT6.1 (Table II). K m for NAD was 5.3 M for Q207E-RT6.1 and 2.4 M for Q207D-RT6.1 (Table II).
In the present study, we found that automodification of the rat recombinant RT6.1 is not due to auto-ADP-ribosylation at an arginine residue, and that the RT6.1 mutants in which Gln-207 in RT6.1 was replaced with glutamic acid or aspartic acid residue exhibited arginine-specific ADP-ribosyltransferase activity. As shown in Fig. 2, eucaryotic arginine-specific ADP-ribosyltransferases have catalytic glutamic acid residues at positions corresponding to Gln-207 in RT6.1, and this is the case for bacterial or viral arginine-specific transferases. van Damme et al. (22) have recently reported that in Clostridium perfringens -toxin, Glu-419, which corresponds to residue 207 in RT6.1, was photoaffinity-labeled with NAD. Furthermore, mutation of Glu-207 in MRT6H to glutamine residue abolished transferase activity (Fig. 4). Therefore, it seems likely that Glu-207 in rodent T-cell RT6 antigens is essential for ADPribose transfer reaction.
Replacement of the uncharged amide of Gln-207 with a carboxyl group, as in Q207E-RT6.1, led to arginine-specific ADPribosyltransferase activity. Withdrawal of the carboxyl group of glutamic acid at position 207 by 1 methylene unit, as in Q207D-RT6.1, reduced the k cat by 18-fold. On the other hand, substitutions at position 207 in RT6.1 affected NADase activity to a much lesser extent than ADP-ribosyltransferase activity. Thus, the presence and precise spatial location of a carboxyl group at position 207 in the mutants seem to be responsible for argininespecific ADP-ribosyltransferase activity, but relatively unimportant for NADase activity.
Koch-Nolte et al. (15) have just reported that NADase activity of mouse Rt6 is inhibited by the arginine analogue agmatine. We also observed that Q207E-RT6.1 exhibited no NADase activity in the presence of 0.1 M L-arginine (data not shown). Since Q207E-RT6.1 displayed a 10-fold lower K m for NAD in ADP-ribosyltransferase reaction than in NAD hydrolysis (Table II), Q207E-RT6.1 might catalyze ADP-ribosylation more efficiently than NAD hydrolysis in the presence of ADP-ribose acceptors.
Wang et al. (23) discovered on the surface of mouse cytotoxic T-cells glycosylphosphatidylinositol-anchored arginine-specific ADP-ribosyltransferase with a molecular mass of 35 kDa. Soman et al. (24) reported the presence of a guanidine groupspecific ADP-ribosyltransferase (30 -33 kDa) on mouse T lymphomas. We also detected arginine-specific ADP-ribosylation of 34-and 32-kDa proteins in mouse spleen lymphocytes (16). Assuming that RT6s are glycosylphosphatidylinositol-anchored membrane proteins (25-35 kDa) (25,26), MRT6H may perhaps represent the transferases present in mouse lymphocytes. ADP-ribosyltransferases in rat lymphocytes have not apparently been demonstrated to modify exogenous substrates such as agmatine.