Regulatory Role of Arginine 204 in the Catalytic Activity of Rat Alloantigens ART2a and ART2b*
Linda A. Stevens
,
Christelle Bourgeois,
Rita Bortell
and
Joel Moss
From the
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1590 and the
Diabetes Division, University of Massachusetts Medical Center, Worcester, Massachusetts 01605
Received for publication, October 9, 2002
, and in revised form, March 12, 2003.
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ABSTRACT
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ART2a (RT6.1) and ART2b (RT6.2) are NAD glycohydrolases (NADases) that are linked to T lymphocytes by glycosylphosphatidylinositol anchors. Although both mature proteins possess three conserved regions (I, II, III) that form the NAD-binding site and differ by only ten amino acids, only ART2b is auto-ADP-ribosylated and only ART2a is glycosylated. To investigate the structural basis for these differences, wild-type and mutant ART2a and ART2b were expressed in rat mammary adenocarcinoma (NMU) cells and released with phosphatidylinositol-specific phospholipase C. All mutants were immunoreactive NADases. Arginine 204 (Arg204), NH2-terminal to essential glutamate 209 in Region III, is found in ART2b, but not ART2a. Replacement of Arg204 in ART2b with lysine, tyrosine, or glutamate abolished auto-ADP-ribosylation. Unlike wild-type ART2a, ART2a(Y204R) was auto-ADP-ribosylated. The tryptophan mutant ART2b(R204W) was auto-ADP-ribosylated and exhibited enhanced NADase activity. Incubation with NAD and auto-ADP-ribosylation decreased the NADase activities of wild-type ART2b and ART2b (R204W), whereas activity of ART2b(R204K), which is not auto-modified, was unchanged by NAD. Facilitation of auto-ADP-ribosylation by tryptophan 204 suggests that the hydrophobic amino acid mimics an ADP-ribosylated arginine. Thus, Arg204 in ART2b serves as a regulatory switch whose presence is required for additional auto-ADP-ribosylation and regulation of catalytic activity.
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INTRODUCTION
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Mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to a specific amino acid in an acceptor protein. In place of an amino acid, some of these enzymes utilize water as an acceptor, generating ADP-ribose and nicotinamide from NAD (NAD glycohydrolase activity). The properties of these enzymes have been most studied with bacterial toxins (e.g. cholera toxin, an arginine-specific ADP-ribosyltransferase (ART))1 that use ADP-ribosylation to modify proteins and thereby alter activity of critical metabolic or regulatory pathways in mammalian cells (1).
The amino acid sequences of the mammalian ARTs differ significantly from those of the toxins and each other. Analysis of the crystallographic structure of toxin ADP-ribosyltransferases identified three regions involved in formation of the catalytic site, NAD binding, and activation of the ribosyl-nicotinamide bond, which is required for ADP-ribose transfer (2, 3). These regions appear to be present also in the mammalian transferases (4). Region I is defined by an arginine or histidine, Region II, by a sequence rich in hydrophobic amino acids, or by serine X serine, (where X represents threonine, serine, or alanine), and Region III by glutamate (Glu). In the bacterial toxins, ART1, and ART2 (mouse and rat), site-specific mutagenesis of Region III verified the importance of glutamate in catalysis (5, 6, 7, 8). Recently, by comparative analysis of crystallographic structures, Han and Tainer (9) extended the significance of the Region III sequences by identifying an ARTT motif (ADP-ribosylating turn-turn motif) that could be implicated in specificity and recognition of the substrate.
Rat ART2a (RT6.1) and ART2b (RT6.2) (for review, see Ref. 10) are encoded by two alleles of a single copy gene (11); the human counterpart has stop signals in the coding region (12). To date, only post-thymic peripheral and intestinal intraepithelial T lymphocytes are known to express ART2 proteins (13). Although their biological functions are unknown, the absence, depletion, or reduction of ART2-expressing T lymphocytes is associated with autoimmune diabetes (14, 15).
Both ART2a and ART2b are linked to the cell surface by GPI anchors, but only ART2a is glycosylated (16, 17). In their mature, processed forms, ART2b and ART2a differ by 10 amino acids. ART2 catalyzes the hydrolysis of NAD to ADP-ribose and nicotinamide but, in contrast to ART1, does not transfer ADP-ribose to arginine or other small guanidino compounds (5). The proteins differ significantly in their abilities to catalyze auto-modification, with ART2b, but not ART2a, capable of auto-ADP-ribosylation (18, 19). In general, the role of GPI-linked NAD metabolizing proteins in T-cell signaling is not clear. Association of ART2 with T-cell src tyrosine kinases was increased by T-cell pretreatment with phorbol 12-myristate 13-acetate (20). NAD and the product of NAD hydrolysis, ADP-ribose, inhibited antigen-induced T-cell proliferation in rats, suggesting that ART2 enzymatic activity may mediate an NAD-dependent immunomodulatory signal (21). The allelic differences between ART2a and ART2b genes result in variations in amino acid sequence and enzymatic properties but the differences in the signaling properties of the isoenzymes is unknown.
To determine the molecular basis for the catalytic diversity of the ART2 proteins and the structural requirements for auto-ADP-ribosylation and its effects on ART2 activity, we compared the amino acid sequences, paying particular attention to the critical catalytic Region III to identify crucial conserved and non-conserved amino acids. Here we report that auto-modification of ART2b is abolished by the replacement of arginine 204, which is located just proximal to the catalytic glutamate and is part of the ARTT motif, and that substitution of arginine for tyrosine (Y204R) in ART2a enhanced auto-ADP-ribosylation transfer to arginine in the glycosylated and non-glycosylated isoforms. Surprisingly, the mutant generated by replacement of arginine 204 in ART2b with tryptophan, ART2b (R204W), retained auto-ADP-ribosylation activity but not on arginine residues, consistent with the conclusion that the hydrophobic tryptophan may partially replace ADP-ribose-arginine in modifying protein function. These data add support to the significance of aromatic residues at position 204 in the ARTT motif. Auto-modification of ART2b and replacement of arginine with tryptophan (ART2b(R204W)) modulated NAD glycohydrolase activity, suggesting that ART2a and ART2b signaling activity may be differentially regulated by their substrate NAD.
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MATERIALS AND METHODS
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Construction of Wild-type ART2a (RT6.1) and ART2b (RT6.2) Expression PlasmidsThe rat ART2a open reading frame was amplified by PCR using an ART2a-pCRII.1 plasmid as a template and cloned in the pMAMneo mammalian expression vector (Clontech, Palo Alto, CA) as an NheI/XhoI fragment, carrying a Kozak consensus region (GCCACG) upstream of the first codon. Construction of the rat ART2b-pMAMneo mammalian expression vector was described earlier (5). To improve the level of expression of recombinant ART2b in mammalian cells, a Kozak consensus sequence was placed upstream of the first ATG, using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), according to the manufacturer's instructions with a pair of complementary mutant primers corresponding to the following sequence: CGGACTCACCATAGGGACCAAGCTAGCCGCCATGCCATCAAATATTTGCAAGTTCTTCC. Plasmid construct sequences were verified by DNA sequencing of the entire open reading frame.
Site-directed Mutagenesis of Wild-type ART2a and ART2bPoint mutations were introduced in ART2a and ART2b cDNAs using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Sequences of sense strands from which pairs of complementary mutation primers were synthesized to produce the indicated changes in amino acids are listed in Table I. All clones were screened by restriction digestion and confirmed by DNA sequencing (both strands) of the entire open reading frames.
Cell Culture and Protein ExpressionNMU cells, grown in Eagle's minimal essential medium with 10% fetal calf serum (Invitrogen) at 37 °Cin5%CO2, were transfected with the pMAMneo vector (Clontech) containing the indicated ART2a or ART2b constructs using the Lipo-fectomine Plus Reagent (Invitrogen) according to manufacturer's instructions. Transfected cells were selected with Geneticin (G418; Invitrogen), 0.5 mg/ml.
Protein expression was induced with 1 µM dexamethasone (Sigma) for 24 h. Trypsinized confluent cells were sedimented by centrifugation (1000 x g), washed with DPBS, and incubated (1 h, 37 °C) with 0.05 unit of phosphatidylinositol-specific phospholipase C (PI-PLC) (ICN, Costa Mesa, CA) in 500 µl of DPBS to cleave the GPI anchor and release protein from the cell surface. Cells were sedimented by centrifugation (1000 x g), and supernatant containing PI-PLC-releasable protein linked to the COOH-terminal oligosaccharide was collected.
NAD Glycohydrolase and ADP-ribosyltransferase AssaysNADase activity (standard assay) was measured in DPBS containing 0.1 mM [carbonyl-14C]NAD (0.05 µCi) for 1 h at 30 °C, (total volume = 150 µl). Samples (50 µl) were applied to AG1-X2 (Bio-Rad) columns (0.4 x 4 cm), equilibrated, and eluted with water for liquid scintillation counting as described previously (22). Transferase activity was assayed similarly with or without 20 mM agmatine as ADP-ribose acceptor and with [adenine-14C]NAD substituted for [carbonyl-14C]NAD.
Auto-ADP-ribosylation ActivitySamples of medium from PI-PLC-treated cells (protein determined by NADase activity measurement) were incubated with 1 mM ADP-ribose, 10 µM [32P]NAD (10 µCi/assay reaction) in DPBS, for 1 h at 30 °C. Where indicated, unlabeled NAD was added and incubation continued for 1 h at 30 °C before termination with the addition of an equal volume of 20% cold trichloroacetic acid. Reaction mixtures were centrifuged (16,000 x g for 25 min) and supernatants discarded. Proteins were dissolved in SDS-sample buffer, separated by SDS-PAGE in 12% gels, and transferred to nitrocellulose. Blots were exposed to film (XAR-2, Eastman Kodak Co.) or analyzed by PhosphorImager (Amersham Biosciences). Immunoreactivity was quantified by incubation with rabbit antipeptide antiserum 1126 (Immunogen; amino-terminal ART2a and ART2b) or NAD2 (Immunogen; Region III) (14) and detected by chemiluminescence (ECL, Amersham Biosciences).
Chemical Stability of the ADP-ribose-Amino Acid LinkageAfter trichloroacetic acid precipitation and centrifugation, proteins that had been auto-ADP-ribosylated as described above were neutralized, dissolved in 0.1 M Tris-HCl, pH 7.5, and incubated in 0.1 M Tris-HCl, pH 7.5, 0.2 M HCl, 10 mM HgCl2 (Sigma), 2 M NH20H (Sigma) (neutralized with NH4OH), or 2 M NaCl for 2 h at 37 °C. Reactions were stopped by addition of equal volumes of 20% trichloroacetic acid and the proteins separated by SDS-PAGE in 12% gels (23).
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RESULTS AND DISCUSSION
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The sequences of ART2a and ART2b differ by 14 amino acids, 10 of which are located NH2-terminal to the region excised during addition of the GPI anchor (Fig. 1). In ART2b, but not other ARTs, an arginine is present at position 204 (Arg204) as part of the ARTT motif, located at the amino end of catalytic Region III. A putative consensus N-glycosylation signal is present at positions 5860 in ART2a but not in ART2b. Both ART2a and ART2b have NADase activity, but only ART2b is significantly auto-ADP-ribosylated. To define the structural basis for these differences in catalytic function, we employed site-specific mutagenesis with synthesis of recombinant ART2a and ART2b proteins in NMU cells using a dexamethasone-sensitive promoter. The GPI-anchored proteins released from cells using PI-PLC to cleave the GPI anchor were incubated with [32P]NAD to assess auto-ADP-ribosylation (Fig. 2A). All recombinant proteins had NADase activity (Fig. 3) and all reacted with ART2 antisera, exhibiting the expected size of 29 kDa on immunoblots (Fig. 2B). ART2a, the glycosylated isoform, had an additional band at
33 kDa, consistent with its single consensus sequence for N-glycosylation.

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FIG. 1. Deduced amino acid sequences of rat ART2a (RT6.1) (GenBankTM accession number CAA36301
[GenBank]
) and rat ART2b (RT6.2) (GenBankTM accession number AAA42085
[GenBank]
). Identical amino acids are shaded. Arginines 204 (1) and 81 (2) are specific to ART2b. In ART2a, Asn58 (3) and 58NKSE61 (4) are in a putative consensus glycosylation signal not present in ART2b. Regions I, II, and III, believed to participate in formation of the catalytic site in the bacterial toxin and mammalian ADP-ribosyltransferases, are indicated by over-lines and the putative catalytic amino acids by an asterisk. Consensus sequence of the ARTT motif involved in substrate specificity and recognition is shown under the corresponding region in rat ART2 proteins ( , hydrophobic amino acid; X, any amino acid). Underlines indicate signal sequences, which are excised during the export into the endoplasmic reticulum (amino terminus) and attachment of the GPI anchor (carboxyl terminus).
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FIG. 2. Auto-[32P]ADP-ribosylation (A) and immunoblot (B) of the supernatants from NMU cells transfected with vector alone (C), ART2b wild-type (WT), R81K, R204K, ART2a wild-type (WT), M81R, and Y204R. After dexamethasone induction for 24 h, cells were lifted from the plate by incubation with trypsin-EDTA (0.025%, 0.02%), sedimented, and then the supernatant was incubated with PI-PLC (0.05 unit) for 1 h at 37 °C in DPBS to release GPI-anchored proteins. The supernatants were incubated without or with 10 µM [32P]NAD and 1 mM ADP-ribose for 1 h at 30 °C before precipitation with 10% trichloroacetic acid; the pellets were dissolved in sample buffer and separated by SDS-PAGE in 12% gels. Blots were used for autoradiography, followed by reaction with rabbit peptide antiserum NAD2 and detection by chemiluminescence. Reduction of immunoreactivity on Western blots, or inability to distinguish individual bands on autoradiograms, is due to the spreading of proteins by multiple modifications. The amount of ART2a(Y204R) protein is less than wild type or ART2a(M81R). Data shown are from one experiment representative of eight.
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FIG. 3. Auto-ADP-ribosylation (A), immunoreactivity (B), and NAD glycohydrolase activity (C) of ART2b, ART2a, and mutants. Samples of ART2b contained the same amounts of NADase activity (1.4 nmol/h) but very different amounts of ART2b proteins. Samples of proteins were incubated with [32P]NAD as described under "Materials and Methods," separated by SDS-PAGE in 12% gels, transferred to nitrocellulose, and analyzed by PhosphorImager (A). The same ART2b and ART2a blots were then reacted with antisera 1126 and NAD2, respectively, and detected by chemiluminescence (B). Shown are data for ART2b wild-type (WT), R204K, R81K, R204Y, R204E, R204W, R81K,R204K, and ART2a wild-type (WT); M81R; Y204R; M81R,Y204R; N58A,Y204R; or 59NMA61,Y204R. Data shown are one experiment representative of five.
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Multiple species of auto-ADP-ribosylated wild-type ART2b were observed by SDS-PAGE (Fig. 2A). The molecular mass is predicted to increase by 542 Da per addition of each ADP-ribose moiety. The autoradiograms demonstrate the increasing molecular weight of [32P]ADP-ribose-modified proteins. Replacement of arginine 204 by a conservative lysine (R204K) abolished auto-ADP-ribosylation, whereas substitution of lysine for arginine 81 had no effect, consistent with arginine 204 controlling auto-ADP-ribosylation activity (Fig. 2). In contrast, wild-type ART2a with tyrosine in position 204 was not significantly auto-modified. However, auto-ADP-ribosylation of the mutants ART2a(Y204R) or ART2a(M81R,Y204R) was observed (Figs. 2 and 3). Replacement of methionine with arginine at position 81 (ART2a(M81R)) had no effect. Therefore, a single amino acid, Arg204, was responsible for auto-ADP-ribosylation of ART2b or ART2a(Y204R).
In ART2a(Y204R), modification of the putative consensus glycosylation site, replacing Asn58 with Ala, or changing 59KSE61 to 59MNA61, prevented glycosylation, resulting in a single immunoreactive band by SDS-PAGE (Fig. 3). Both non-glycosylated species were auto-ADP-ribosylated. The glycosylated ART2a(Y204R), concentrated on concanavalin A-Sepharose, was also auto-ADP-ribosylated (Fig. 4). The glycosylation site did not affect auto-ADP-ribosylation or the regulatory control by arginine 204.

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FIG. 4. Auto-ADP-ribosylation of ART2b, ART2a, and mutant proteins. Samples of supernatants from cells expressing ART2b wild-type (WT), R81K, ART2a(Y204R), and 59NMA61,Y204R were examined. ART2b proteins were incubated with 10 µM [32P]NAD for 1 h at 30 °C, followed by addition of 20% trichloroacetic acid or further incubation with 5 mM NAD for 1 h at 30 °C, before addition of 20% trichloroacetic acid. To concentrate glycosylated proteins before incubation with NAD, supernatants from cells expressing ART2a(Y204R) were incubated with concanavalin A-Sepharose for 1 h on ice. After washing the resin with DPBS, bound proteins were eluted with 0.3 M -methyl-o-mannopyranoside in DPBS and incubated for 1 h at 30 °C without or with 5 mM NAD and separated by SDS-PAGE on 12% gels, transferred to nitrocellulose, and quantified with a PhosphorImager (ART2b) or immunoblotting with antibody NAD2 (ART2a) and detection by chemiluminescence. Data are from one experiment representative of four experiments.
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Replacement of Arg204 in ART2b with tyrosine or glutamate abolished auto-ADP-ribosylation as well, although all the mutants retained NADase activity (Fig. 3), demonstrating that the absence of auto-modification was not due to the inability of the proteins to hydrolyze NAD. Surprisingly, ART2b(R204W) was also capable of auto-modification (Figs. 3 and 5). The mutant, like the wild type, however, was unable to transfer ADP-ribose to agmatine (data not shown). The auto-modification activity of ART2b(R204W), but not of ART2b(R204Y) or ART2a, suggests that the indolyl side chain of tryptophan in position 204 can promote auto-modification, whereas the phenol group of tyrosine cannot. These data support the hypothesis that the heterocyclic structure of the tryptophan side chain may mimic the purinyl group of ADP-ribosyl-arginine so that the auto-modification of ART2b requires ADP-ribosylation of Arg204.

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FIG. 5. Chemical stability of ADP-ribose-protein bonds in auto-ADP-ribosylated wild-type ART2b, ART2b(R204W), and ART2a(Y204R). Supernatants from NMU cells expressing wild-type ART2b, ART2b(R204W), and ART2a(Y204R) were auto-ADP-ribosylated with 10 µM [32P]NAD as described under "Materials and Methods" followed by addition of 20% trichloroacetic acid (I) or further incubation with 5 mM NAD at 30 °C for 1 h before precipitation with 20% trichloroacetic acid (II). Neutralized samples were incubated in 0.1 M Tris-HCl, pH 7.5 (lane 1), 0.2 M HCl (lane 2), 10 mM HgCl2 (lane 3), 2 M NH2OH (lane 4), or 0.2 M NaCl (lane 5) for 2 h at 37 °C before proteins were trichloroacetic acid-precipitated, separated by SDS-PAGE in 12% gels, and transferred to nitrocellulose. The blots were analyzed by autoradiography (A and C), reacted with antibodies 1126 or NAD2 (ART2a(Y204R)) (B and D) and detection by chemiluminescence. Variation in the amount of protein on immunoblots and autoradiograms reflects the response of the protein to different chemical treatments. The autoradiogram (A or C) data should be compared with immunoblots (B or D) under equal treatment conditions.
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To assess the ability of ART2 mutants to be auto-ADP-ribosylated by multiple ADP-ribose moieties, ART2b proteins were incubated first with 10 µM [32P]NAD to label the protein with high specific activity ADP-ribose (for detection of proteins by radiography), followed by addition of 5 mM unlabeled NAD and further incubation (Fig. 4). ART2a(Y204R), ART2a (Y204R,59NMA61), and ART2a(Y204R) after incubation and elution from concanavalin A-Sepharose of the glycosylated species, were incubated with 5 mM NAD (and subsequently detected by immunoreactivity). Wild-type ART2b, ART2b(R81K), and mutants ART2a(Y204R), ART2a(59MNA61,Y204R), and the glycosylated isoforms of ART2a(Y204R) were multiply auto-ADP-ribosylated as evidenced by the bands of lower mobility than the unmodified forms on SDS-PAGE. All of these proteins, including ART2b(R204W) (Fig. 6, inset), were capable of auto-ADP-ribosylation and were modified by multiple ADP-ribose moieties. It is unlikely that auto-modification was due to non-enzymatic addition of [32P]ADP-ribose, since reactions were carried out in the presence of 1 mM unlabeled ADP-ribose, or due to NAD binding, since the radiolabeling was not decreased by the presence of 5 mM unlabeled NAD.

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FIG. 6. Time course of nicotinamide release by ART2b and mutants. Supernatants from cells expressing wild-type ART2b, ART2b(R204K), and ART2b(R204W) were collected as described under "Material and Methods." NADase activity was assayed at 30 °C with 0.5 mM [carbonyl-14C]NAD ( 2000 cpm/nmol) in a total volume of 800 µl (ART2b) and in 1500 µl (ART2b(R204K), ART2b(R204W)). At the indicated times, samples (50 µl) were applied to AG1-X2 columns to measure the nicotinamide released or were precipitated with 10% trichloroacetic acid before SDS-PAGE separation of proteins in 12% gels. Immunoblots with antiserum NAD2 are given in the insets. NADase activity of the proteins was unaffected by incubation at 30 °C in buffer for 4 h. Data are from a single experiment representative of at least two.
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A specific ADP-ribose-amino acid linkage can often be identified by its susceptibility to cleavage by acid, hydroxylamine, and mercuric chloride (Fig. 5). To characterize the multiple ADP-ribose bonds that resulted from incubation of wild-type ART2b, or ART2a(Y204R) with millimolar NAD, their chemical sensitivity was tested. Hydroxylamine completely released the [32P]ADP-ribose from auto-ADP-ribosylated ART2b and ART2a(Y204R) (Fig. 5, IA, IIA, and IIC, lanes 4), consistent with the chemical stability of an ADP-ribose-arginine linkage. [32P]ADP-ribose was not released from auto-ADP-ribosylated ART2b(R204W) by hydroxylamine, mercuric chloride, or acid (Fig. 5, IC), suggesting that the auto-ADP-ribosylated amino acid was not arginine, cysteine, or lysine, respectively. Thus, the tryptophan mutant, although exhibiting auto-ADP-ribosyltransferase activity with multiple modifications, differed from wild-type ART2b in the ADP-ribose acceptor site(s) and, therefore, is only the partial functional equivalent of ADP-ribosyl-arginine.
Comparison of the Vmax kinetic constants determined by Lineweaver-Burk analysis for ART2b(R204K) and ART2b (R204W) showed an increased maximal velocity but similar Km values, when position 204 was occupied by the tryptophan residue with an aromatic side chain (Table II). In agreement, replacement of Tyr204 by Arg in ART2a had little effect on Km values but decreased Vmax, consistent with the decreased NADase activity of ART2a(Y204R) observed in Fig. 3. These data support the ARTT motif model in which turn1 contains a conserved aromatic residue 204 thought to be involved in substrate specificity and recognition (9).

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FIG. 7. Lineweaver-Burk analysis of ART2b and ART2b(R204K) by NAD glycohydrolase activity. NADase activity was measured for 1.5 h as described under "Materials and Methods" with ART2b and ART2b(R204K) with increasing amounts of NAD as indicated. Data are from one experiment representative of three (ART2b) or four (ART2b(R204K)).
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The influence of auto-modification on the ability of the ART2 proteins to hydrolyze NAD was investigated. The rate of hydrolysis by wild-type ART2b appeared to increase during the first hour of incubation with 0.5 mM NAD and then decreased in parallel with the increase in multiply ADP-ribosylated forms of the enzyme seen on SDS-PAGE (Fig. 6). The rate of increase in ART2b NADase during the first hour may reflect the initial transfer of ADP-ribose to arginine 204 and the initiation of additional modifications that inhibit ART2b catalysis. This kinetic pattern is consistent with the non-linear Lineweaver-Burk analysis of ART2b (Fig. 7). The curvature of the analysis suggests that the catalytic activity of the protein is changing during the 1.5 h of the assay due to activation by the initial auto-ADP-ribosylation and inhibition by subsequent additions of ADP-ribose. Thus, it was not possible to calculate the true kinetic constants. In agreement, the activity of ART2b(R204K), which is not auto-ADP-ribosylated, was constant for 4 h. The NADase activity of ART2b(R204W), which was constant for at least 2 h, i.e. with no initial stimulation, decreased like the wild-type ART2b with increasing auto-ADP-ribosylation. The Lineweaver-Burk analysis was linear similar to ART2b (R204K) (data not shown), since the extent of auto-ADP-ribosylation and inhibition is less. The absence of initial acceleration of NAD hydrolysis in the presence of ART2b(R204W) is consistent with the hypothesis that tryptophan can behave like ADP-ribosylarginine, initiating multiple modifications that influence catalytic activity. It has been reported that auto-ADP-ribosylation regulates NADase and transferase activities of other ARTs that contain aromatic residues at the position corresponding to Arg204 (24, 25). The arginine in ART2b, therefore, is an apparently unique specific regulatory site among ADP-ribosyltransferases.
Our data indicate that the degree of hydrophobicity of amino acid 204 can regulate the NAD glycohydrolase and auto-ADP-ribosylation activity of the ART2b protein. In the presence of millimolar amounts of NAD, the ADP-ribosylation of arginine can initiate the auto-modifications that also modulate the NADase activity. These data demonstrate the essential function of the amino acid sequence and of the critical role of position 204 in the ARTT motif of Region III. Our results suggest that allotype variations generated during rat ART2 gene evolution have resulted in two differentially regulated NADases that could influence their function in T-cell regulation.
The ability of tryptophan to replace, in part, the function of ADP-ribosylarginine could be of use in protein design. Although ADP-ribosylation has effects on protein function, the modification itself is unstable in biological systems. It can be cleaved by pyrophosphatases, with release of AMP, and the resulting phosphoribosyl protein can be further degraded by phosphatases, yielding ribosyl protein. Obviously, synthesis of an ADP-ribosylated protein requires an additional step(s) following production of a recombinant molecule. In contrast, a protein containing tryptophan can be produced by standard techniques and should have no unusual instability in biological systems. The extent to which ADP-ribose-arginine in protein can be replaced by tryptophan is currently under study.
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FOOTNOTES
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* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
To whom correspondence should be addressed. Fax: 301-402-1610; E-mail: stevensl{at}nhlbi.nih.gov.
1 The abbreviations used are: ART, mono-ADP-ribosyltransferase; ARTT, ADP-ribosylating turn-turn; GPI, glycosylphosphatidylinositol; NMU, rat mammary adenocarcinoma; NADase, NAD glycohydrolase; DPBS, Dulbecco's phosphate-buffered saline; PI-PLC, phosphatidylinositol-specific phospholipase C. 
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ACKNOWLEDGMENTS
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We thank Dr. Martha Vaughan and Dr. Vincent C. Manganiello for useful discussions and critical review of the manuscript.
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