©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Conservation of a Common Motif in Enzymes Catalyzing ADP-ribose Transfer
IDENTIFICATION OF DOMAINS IN MAMMALIAN TRANSFERASES (*)

(Received for publication, September 22, 1994; and in revised form, October 26, 1994)

Tatsuyuki Takada (§) Keiko Iida Joel Moss

From the Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1434

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Bacterial toxin ADP-ribosyltransferases, e.g. diphtheria toxin (DT) and pertussis toxin, have in common consensus sequences involved in catalytic activity, which are localized to three regions. Region I is notable for a histidine or arginine; region II, 50-75 amino acids downstream, is rich in aromatic/hydrophobic amino acids; and region III, further downstream, has a glutamate and other acidic amino acids. A similar motif was observed in the sequence of the glycosylphosphatidylinositol-linked muscle ADP-ribosyltransferase. Site-directed mutagenesis was performed to verify the role of this motif. Proteins were expressed in rat adenocarcinoma cells, released from the cell with phosphatidylinositol-specific phospholipase C, and quantified with polyclonal antibodies. Transferase His in region I aligned with His of DT; as with DT, the H114N mutant was active. Aromatic/hydrophobic amino acids (region II) were found 30-50 amino acids downstream of this histidine. Although transferase has a Glu-Tyr-Ile sequence characteristic of region III in DT, Glu was not critical for activity. In an alternative region III containing Glu-Glu-Glu, Glu and Glu but not Glu were critical. Glu aligned with critical glutamates in DT, Pseudomonas exotoxin, and C3 transferase. Thus, the mammalian ADP-ribosyltransferases have motifs similar to toxin ADP-ribosyltransferases, suggesting that these sequences are important in ADP-ribose transfer reactions.


INTRODUCTION

Mono-ADP-ribosylation is a reversible post-translational modification of proteins in which NAD:arginine ADP-ribosyltransferases catalyze the addition of an ADP-ribose moiety, and ADP-ribosylarginine hydrolases release ADP-ribose from the modified protein. An NAD:arginine ADP-ribosyltransferase was purified from rabbit skeletal muscle (1, 2) and the gene cloned from human and rabbit muscle(2, 3) . This glycosylphosphatidylinositol-anchored protein is believed to modify integrin alpha7 and thus may affect cell adhesion(4) . ADP-ribosylarginine hydrolases were purified from rat brain and cloned from rat, mouse, and human sources; the protein exhibits considerable cross-species immunoreactivity and sequence conservation(5, 6) . In animal cells, it appears that these two enzymes could work in concert to generate an ADP-ribosylation cycle, similar to that used to regulate nitrogen fixation in Rhodospirillum rubrum(7) .

Mono-ADP-ribosylation is critical to the mechanism of action of several bacterial toxins (e.g. diphtheria toxin, cholera toxin). Structure/function relationships of bacterial toxin ADP-ribosyltransferases have been studied by x-ray crystallography, photocross-linking, and site-directed mutagenesis(8, 9, 10, 11, 12, 13, 14) . The results suggest that the toxin ADP-ribosyltransferases possess a common motif consisting of three regions: Region I contains a histidine or arginine and is followed in 50-75 amino acids by an aromatic and/or hydrophobic amino acid-rich region II. Region III is further downstream and is characterized by a critical glutamate and usually other acidic residues. In the case of the region III glutamate, even a conservative replacement with aspartate drastically reduced activity(8, 9, 10, 11, 12, 13, 14) . This result suggests that a precise configuration is required for the ADP-ribose transfer reaction. Although there is no significant overall sequence similarity among the muscle and toxin ADP-ribosyltransferases, as described here, the muscle ADP-ribosyltransferase appears to have three regions with consensus sequences similar to those in the toxins. In region III, although multiple glutamate residues could be aligned with the cognate domains in the toxins, we identified, by in vitro mutagenesis, one which aligns with that found in the toxin transferases and is critical for activity.


EXPERIMENTAL PROCEDURES

Materials

[adenine-U-^14C]NAD (269 mCi/mmol) and enhanced chemiluminescence Western blotting reagents were from Amersham Corp., beta-NAD was from Sigma, AG1-X2 and nitrocellulose membranes were from Bio-Rad, [adenylate-P]NAD (30 Ci/mmol) was from DuPont NEN, and PI-PLC (^1)was from ICN (Aurora, OH).

Methods

Construction of Wild-type and Mutant Rabbit Muscle ADP-ribosyltransferase Expression Vectors

Wild-type transferase cDNA was amplified from the lambda ZAP clone previously isolated (2) (with forward and reverse primers 5`-ACGTACGTACGTGCTAGCATGTGGGTTCCTGCCGTGGCGAAT-3` and 5`-ACGTACGTACGTCTCGAGTCAGAAGAGGCCTGGGCTTCCTGG-3`, respectively), digested with NheI and XhoI, and then cloned into a pMAMneo expression vector (Clontech, Palo Alto, CA), resulting in the rWT plasmid. Mutants were prepared by polymerase chain reaction(15, 16) . The following oligonucleotides and their inverse complementary oligonucleotides (not shown) were used to introduce mutations into the transferase. The modified codons are underlined. The mutation is identified in parentheses. 5`-GATTTCGGGATGAAAACGGGGTGGCCCTG-3` (H114N), 5`-CTTTTTCCCTGGGGACGAGGAGGTTCTG-3` (E238D), 5`-CTTTTTCCCTGGGCAGGAGGAGGTTCTG-3` (E238Q), 5`-TTCCCTGGGGAGGACGAGGTTCTGATC-3` (E239D), 5`-CCTGGGGAGGAGGACGTTCTGATCCCC-3` (E240D), 5`-CCTGGGGAGGAGGCGGTTCTGATCCCC-3` (E240A), 5`-TCATACAACTGCGACTACATCAAAGAA-3` (E278D), 5`-TCATACAACTGCGCGTACATCAAAGAA-3` (E278A), 5`-GAGTACATCAAAGACATGCAGTGCAAG-3` (E282D), 5`-GAGTACATCAAAGCAATGCAGTGCAAG-3` (E282A). For mutants E238D, E238Q, E239D, E240D, E240A, E278D, E278A, E282D, and E282A, the BstXI-XhoI fragment of the rWT plasmid was replaced with the amplified fragment carrying the mutation. For the H114N mutant, the Eco47III-SacII region was replaced with a mutated fragment. Polymerase chain reaction-derived sequences were verified using an automated sequencer 370A (Applied Biosystems) with a PRISM Ready Reaction DyeDeoxy Terminator cycle sequencing kit (Applied Biosystems).

Expression of Recombinant Proteins in Rat NMU Cells

Rat NMU cells from the American Type Culture Collection were grown at 37 °C in a 5% CO(2) incubator in Eagle's minimal essential medium (Biofluids, Rockville, MD) supplemented with 10% fetal calf serum. Cells were transfected with 16 µg of pMAMneo vector or constructs using the calcium phosphate precipitation method(17) ; transformants were selected using Geneticin (Life Technologies, Inc.). Expression of recombinant proteins was induced by addition of 1 µM dexamethasone for 24 h. Recombinant wild-type and mutant transferases were released from intact cells with PI-PLC(16) .

Immunodetection of Wild-type and Mutant Transferases

PI-PLC-released proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) in 12% Tris glycine gels and transferred to nitrocellulose, which was incubated with anti-serum against recombinant rabbit muscle ADP-ribosyltransferase (3) followed by anti-rabbit IgG-horseradish peroxidase conjugate. Chemiluminescence was used for detection and was quantified by densitometry.

Labeling with [P]NAD

PI-PLC-released proteins from cells expressing wild-type and mutant transferases were precipitated with trichloroacetic acid, resolved by SDS-PAGE, and transferred to nitrocellulose, which was incubated with binding buffer (25 mM Tris, pH 7.5, 100 mM NaCl, 0.3% bovine serum albumin, 0.3% Tween 20) at room temperature for 1 h, and then transferred to fresh binding buffer containing [P]NAD (4 µCi/ml) for 5 h. After washing three times with binding buffer, a PhosphoImager (Molecular Dynamics) was used for quantification.

ADP-ribosyltransferase Assay

Reaction was carried out in 150 µl of 50 mM potassium phosphate, pH 7.5, with 20 mM agmatine and 0.1 mM [adenine-U-^14C]NAD (1.7 mCi/mmol) using PI-PLC-released protein fractions from intact cells expressing wild-type and mutant transferases. After 30 min at 30 °C, a sample (100 µl) was applied to a column (0.5 times 4 cm) of Dowex AG1-X2, and [^14C]ADP-ribosylagmatine was eluted with 5 ml of H(2)0 for radioassay in a liquid scintillation counter.


RESULTS AND DISCUSSION

Structural analysis of toxin ADP-ribosyltransferases using primary sequence alignments, x-ray crystallography, and photocross-linking have demonstrated that, despite different primary amino acid sequences, some characteristic regions common to all defined the NAD-binding site(8, 14) . Region I has a specific histidine or arginine, which plays a critical role in hydrogen bonding. Region II contains aromatic and/or hydrophobic residues, which are important in positioning the nicotinamide and adenine moieties. Region III contains a critical glutamate, which plays a key role in the ADP-ribosyltransferase reaction, with even a conservative substitution (e.g. aspartate) diminishing its activity. Moreover, this glutamate appears to be common to all toxin ADP-ribosyltransferases and, in some cases (e.g. diphtheria toxin), is followed by Tyr-Ile. In DT, ETA, PT, and LT, the distances between regions I and II and regions I and III are similar, corresponding to 50-75 and 105-130 amino acids, respectively. Similar consensus sequences were found in the rabbit muscle transferase (Fig. 1) with His denoting region I (Fig. 1A) and Tyr-Gly serving as the aromatic/hydrophobic domain for region II (Fig. 1B). The acidic region was less well defined with multiple downstream glutamates in region III (Fig. 1C). In this model, the distances between I and II (approximately 30-45 amino acids) and I and III (127-165 amino acids) were in good agreement with the motif mentioned above. To determine which glutamates in the muscle ADP-ribosyltransferase participate in the catalytic site, a series of mutants was prepared.


Figure 1: Comparison of critical amino acid sequences of rabbit muscle and toxin ADP-ribosyltransferases, and RT6.2 NAD glycohydrolase. A, region I containing a histidine (down triangle). B, region II with hydrophobic residues underlined. C, region III with alignment based on the similarity to C3 ADP-ribosyltransferase (C1) or poly(ADP-ribose) synthetase (C2). Sequences are in the single letter code with position of the first amino acid indicated by the number following the protein name. An identical residue is indicated by an asterisk, a gap by a hyphen, and a possible critical residue by down triangle. Conservative substitutions are shown in uppercase; non-conservative substitutions are given in lowercase. All comparisons are relative to the sequence present in the muscle transferase. RMT, rabbit muscle ADP-ribosyltransferase(2) ; RT6.2, an NAD glycohydrolase(27) ; DT, diphtheria toxin(28) ; PT, pertussis toxin(29) ; C3, C. botulinum C3 ADP-ribosyltransferase(30) ; LT, heat-labile enterotoxin(31) ; ETA, Pseudomonas exotoxin A(32) ; PARS, poly(ADP-ribose) synthetase(33, 34) .



To demonstrate that both wild-type and mutant proteins were present and structurally intact on the cell surface, the GPI-linked protein was quantified by Western blotting after release of the GPI-linked protein from intact cells by PI-PLC. Anti-serum against recombinant transferase specifically reacted with 36-38-kDa bands derived from cells transformed with wild-type and mutant genes, consistent with the conclusion that the proteins were properly processed, expressed on the cell surface, and released by PI-PLC treatment. There were at least two bands that reacted with antiserum in all constructs (Fig. 2). These might reflect different degrees of glycosylation. All of the active mutants had activities in a range similar to that of the wild type (Table 1).


Figure 2: Immunodetection of recombinant rabbit muscle ADP-ribosyltransferases. Indicated amount of activity as noted below or 400 µl of PI-PLC-released fractions (Vec, E238D, E238Q, E240D, E240A) were subjected to SDS-PAGE in 12% gels, transferred to nitrocellulose, and reacted with antibody against rabbit muscle ADP-ribosyltransferase as described under ``Experimental Procedures'' (A-C represent different SDS-polyacrylamide gels). The activities (pmol/min) applied were Vec (vector), 5.5 ± 2; H114N, 646 ± 13; E238D, 5.4 ± 5.8; E238Q, 1.0 ± 4.5; E239D, 565 ± 26; E240D, 4.8 ± 1.6; E240A, 3.4 ± 3.9; E278D, 720 ± 37; E278A, 579 ± 17; E282D, 570 ± 14; E282A, 552 ± 5.3. Wild-type (WT) activities in A-C were 604 ± 11, 404 ± 10, and 611 ± 36, respectively. Data are means of triplicate measurements ± S.D. Positions of protein standards (kDa) are indicated on the left.





Determination of Region I (Analysis of His)

Region I of the consensus motif consists of histidine/arginine, with His and His being present in DT and ETA, respectively, and Arg^9 and Arg^7 in PT and LT, respectively. These residues may be involved in hydrogen bonding, perhaps positioning the substrate, rather than electron transfer(12, 13) ; in agreement with this hypothesis, His in DT can be replaced by asparagine, whereas the glutamine mutant is inactive(12, 13) . A similar histidine is present in RT6.2, an NAD glycohydrolase (Fig. 1A). In most of the bacterial ADP-ribosyltransferases, this histidine/arginine is preceded by a tyrosine or phenylalanine. A phenylalanine is present in RT6.2. The corresponding region of muscle ADP-ribosyltransferase also has a histidine at this position, which can be aligned with DT and ETA (Fig. 1A); it is, however, preceded by a glutamate rather than an aromatic residue. Since the H114N mutant is active and its activity, relative to immunoreactivity, was similar to wild type, transferase His would appear to be equivalent to His of DT. With DT, H21Q is inactive, consistent with hydrogen bonding via the -nitrogen at the imidazole ring. Unfortunately, an immunoreactive H114Q mutant was not found in the PI-PLC-released protein, perhaps due to defects in protein transport/translocation. In the case of PT, there is a critical histidine (His) between regions I and II. The H35N mutant of PT, however, was inactive (18) and may have a function different from His in the transferase.

Determination of Region III (Analysis of the Glu, Glu, GluAcidic Region)

Multiple glutamate residues near the carboxyl terminus are candidates for the critical moiety (region III) involved in NAD binding and activation. Selection of the target glutamates was achieved by multiple amino acid alignments. Given these constraints, two acidic regions were identified in the muscle transferase with similarities to those in the toxin transferases. One region contains Glu, Glu, and Glu. The acidic amino acids in this region were aligned with the bacterial toxin ADP-ribosyltransferases. Photocross-linking of NAD to the C3-like ADP-ribosyltransferase from Clostridium limosum(19) identified a radiolabeled glutamate and adjacent amino acids that were identical to a sequence in Clostridium botulinum C3 transferase, suggesting that Glu of the C. botulinum C3 transferase (equivalent to Glu in the sequence reported by Popoff et al.(20) ) is involved in NAD binding. Amino acids in proximity to Glu of C3 transferase were easily aligned with a corresponding region in the muscle transferase, suggesting that Glu in C3 transferase was equivalent to Glu of the muscle transferase ( Fig. 1(C1)). Based on this sequence analysis, Glu appeared to correspond to the conserved critical glutamate ( Fig. 1(C1)). In agreement, both E240D and E240A were inactive. Both proteins were expressed on the surface of NMU cells and released with PI-PLC. A sequence similar to that containing Glu is also present in the RT6.2 NAD glycohydrolase.

Glutamate 239 is not conserved among toxin ADP-ribosyltransferases and, as expected, the E239D mutant was active (Table 1). Both the E238D and E238Q mutants were inactive, although these proteins were also expressed on the surface and released by PI-PLC. These results are in good agreement with studies of LT, in which the E110D and E112D mutations caused a drastic decrease in activity(11) . This region of LT can be easily aligned with the corresponding region of the transferase ( Fig. 1(C1)). In LT, Glu contributes to a salt bridge with Arg(21) . Although a glutamate in similar context is not present in DT and ETA, other acidic residues (e.g. Glu (DT), Glu (ETA)) are present in the vicinity or adjacent to the critical glutamate (e.g. Glu (DT), Glu (ETA)). It has been proposed that they may play a role in hydrogen bonding with the 2`-OH of ribose in NAD(22) . Thus, although Glu is also critical for activity, based on sequence alignment, this residue is not always present among transferases; the glutamate or glutamine in this position, however, may contribute to hydrogen bonding or salt bridge formation (e.g. LT).

Gluand GluRegion

A second region in the muscle transferase that aligns with region III of toxins contains Glu. The muscle transferase Glu-Tyr-Ile sequence is similar to that in DT. Both the E278D and E278A mutants were as active as wild type. Thus, although Glu and the adjacent Tyr, Ile can be easily aligned with a critical glutamate and adjacent residues ( Fig. 1(C2)) in DT ADP-ribosyltransferase (23) and poly(ADP-ribose) synthetase(24) , these glutamates do not play a critical role in activity of the muscle transferase. This result is consistent with the fact that aside from DT, amino acids in the vicinity of Glu did not readily align with those in the toxin ADP-ribosyltransferases.

Glu can be aligned with Asp of poly(ADP-ribose) synthetase, which was active with a glutamate replacement, but not with an alanine substitution(25) . Both the E282D and E282A mutants of the transferase, however, were as active as wild type, suggesting Glu is neither critical for activity nor equivalent to Asp of poly(ADP-ribose) synthetase.

It appears that the NAD binding motif consisting of three regions, which is found in bacterial toxin ADP-ribosyltransferases, is also present in mammalian ADP-ribosyltransferases. In particular, we found that Glu of the muscle transferase corresponds to the critical glutamate, which is strictly conserved among ADP-ribosyltransferases, consistent with the importance of this motif in ADP-ribose transfer reactions.

NAD Labeling

To examine further the activity of the transferase proteins in a possibly more sensitive assay system, the PI-PLC-released proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes. Incubation of the membranes with [P]NAD resulted in labeling of 37-kDa bands that colocalized with the transferase (Fig. 3). Labeling was detected in wild-type and active mutants (e.g. H114N, E239D, E278D, and E282D). Inactive mutants (e.g. E238D and E240D), although exhibiting an immunoreactive protein of the correct size, did not incorporate radioactivity (Table 1). These studies were in complete agreement with earlier data on the relative lack of activity exhibited by certain motif mutants.


Figure 3: Labeling of recombinant rabbit muscle ADP-ribosyltransferase in the presence of [adenylate-P]NAD. Indicated amount of activity as noted below or 400 µl of PI-PLC-released fractions (Vec, E238D, E240D) were subjected to SDS-PAGE in 12% gels, transferred to nitrocellulose, and then incubated with [P]NAD as described under ``Experimental Procedures.'' The activities (pmol/min) applied were Vec (vector), 5.5 ± 2; wild type (WT), 527 ± 15; H114N, 1292 ± 26; E238D, 5.4 ± 5.8; E239D, 565 ± 26; E240D, 4.8 ± 1.6; E278D, 1070 ± 29; E282D, 570 ± 14. Data are means of triplicate measurements ± S.D. Positions of protein standards (kDa) are indicated on the left. Presence of recombinant transferase was verified by immunoblotting.



The mammalian and bacterial ADP-ribosyltransferases are believed to act via an S(N)2-like mechanism, with nucleophilic attack on NAD followed by displacement of nicotinamide and inversion of configuration. Since a three-part segmented motif is present in RT6.2, an NAD glycohydrolase exposed on post-thymic lymphocytes, the muscle ADP-ribosyltransferase, as well as in toxin ADP-ribosyltransferases, which use different ADP-ribose acceptors, the consensus motif described here may be related to NAD binding and ribosyl nicotinamide bond activation and not to structure of the attacking nucleophile. It is unclear if a similar binding motif is present in other enzymes that utilize NAD, such as ADP-ribosyl cyclase(26) . Further structural studies are needed to define the active site in that family of proteins.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Rm. 5N-307, Bldg. 10, National Institutes of Health, Bethesda, MD 20892-1434. Tel.: 301-496-1254; Fax: 301-402-1610.

(^1)
The abbreviations used are: PI-PLC, phosphatidylinositol-specific phospholipase C; DT, diphtheria toxin; ETA, Pseudomonas exotoxin A; PT, pertussis toxin; LT, heat-labile enterotoxin; PAGE, polyacrylamide gel electrophoresis; C3, C. botulinum C3 ADP-ribosyltransferase.


ACKNOWLEDGEMENTS

We thank Dr. Martha Vaughan for helpful discussions and critical review of the manuscript and Carol Kosh for expert secretarial assistance.


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