(Received for publication, September 22, 1994; and in revised form, October 26, 1994)
From the
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.
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 7 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.
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
(). 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
. 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.
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).
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.
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 S2-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.