(Received for publication, November 20, 1995; and in revised form, January 17, 1996)
From the
Mono ADP-ribosylation is a posttranslational protein
modification that has been implicated in the regulation of key
biological functions in bacteria as well as in animals. Recently, the
first cDNAs for eucaryotic mono(ADPribosyl)transferases were cloned and
found to exhibit significant sequence similarity only to one other
known protein, the T cell differentiation antigen Rt6. In this paper we
describe secondary structure analyses of Rt6 and related proteins and
show conserved structure motifs and amino acid residues consistent with
a common ancestry of these eucaryotic proteins and bacterial
ADP-ribosyltransferases. Moreover, we have expressed soluble mouse
Rt6-1 and Rt6-2 gene products in which C-terminal tags
(FLAG-His) replace the native glycosylphosphatidylinositol
anchor signal sequences. Purified recombinant Rt6-2, but not
Rt6-1, shows NAD
glycohydrolase activity, which
is inhibited by the arginine analogue agmatine. Immunoprecipitation of
recombinant Rt6-1 and Rt6-2 with anti-FLAG M2 antibody
followed by incubation with [
P]NAD
leads to rapid and covalent incorporation of radioactivity into
the light chain of the M2 antibody. The bound label is resistant to
treatment with HgCl
but sensitive to NH
OH,
characteristic of arginine-linked ADP-ribosylation. These results
demonstrate that Rt6-1 and Rt6-2 possess the enzymatic
activities typical for NAD
-dependent arginine/protein
mono(ADPribosyl)transferases (EC 2.4.2.31). They are the first such
enzymes to be molecularly characterized in the immune system.
Mono ADP-ribosylation is a posttranslational protein
modification in which the ADP-ribose (ADPR) ()moiety of
NAD
is transferred from NAD
to a
specific amino acid residue in a target protein, while the nicotinamide
moiety is released(1, 2) . The reaction is catalyzed
by a family of amino acid-specific ADP-ribosyltransferases, which
includes some of the most potent bacterial toxins such as diphtheria
and cholera toxins. These toxins interfere with cellular functions by
catalyzing mono ADP-ribosylation of key cellular target proteins in
their human hosts, such as elongation factor EF2 and the
subunit
of heterotrimeric G-proteins. The crystal structure has been determined
for four of the bacterial toxins, revealing a highly conserved core
surrounding the presumptive active site
crevice(3, 4, 5, 6) . In case of
diphtheria toxin, the co-crystallized NAD
analogue
ApUp was observed to bind in this crevice(7) . These findings
support the concept that all bacterial toxins with
ADP-ribosyltransferase activity have a common fold of the catalytic
site in spite of highly divergent amino acid sequences(8) . In vitro, many of the bacterial toxins can modify proteins
other than their physiologic targets and, in the absence of target
protein, some toxins can use water as an alternative acceptor resulting
in the hydrolysis of NAD
to nicotinamide and ADPR,
which can be measured as NAD
glycohydrolase activity.
Ample biochemical evidence has shown that endogenous mono ADP-ribosylation reactions occur also in animal tissues(9, 10, 11, 12) . Recent findings suggest that this posttranslational protein modification may be used to control important endogenous physiological functions such as the induction of long term potentiation in the brain, terminal muscle cell differentiation, and the cytotoxic activity of killer T cells (13, 14, 15, 16) . Recently, the first eucaryotic ADP-ribosyltransferases were purified and sequenced from rabbit skeletal muscle and chicken bone marrow(17, 18) . The primary sequences of these eucaryotic proteins encode glycosylphosphatidylinositol (GPI)-anchored membrane proteins with entirely extracellular polypeptide chains. Homology searches (17, 18) revealed significant sequence similarity of the muscle and bone marrow enzymes to a similarly GPI-anchored T cell membrane protein RT6 (19, 20) . Limited amino acid sequence identities to bacterial toxins have also been noted(21, 22) .
RT6, originally discovered in the rat as a T cell
alloantigen(23) , is a T cell differentiation and activation
antigen(24, 25) . Its expression is restricted to
peripheral T cells and intraepithelial lymphocytes of the
gut(24, 26) . Molecular cloning showed that RT6
antigens are encoded by a single copy gene in the rat with two known,
remarkably divergent alleles (designated RT6 and RT6
) (27, 20) and by two closely linked genes in the mouse
(designated Rt6-1 and Rt6-2)(28) . Rt6-1 and Rt6-2 have conserved open reading frames, and the
deduced amino acid sequences are 78.5% identical(28) . A defect
in the development of RT6/Rt6-expressing cells coincides with increased
susceptibility for autoimmune disease in different animal
models(29, 30) . In the first study of RT6 enzyme
activity, Takada et al.(21) reported that rat RT6
displays NAD
glycohydrolase activity but, in contrast
to the skeletal muscle enzyme, does not modify arginine analogues. This
led to the provisional classification of RT6 as a NAD
glycohydrolase rather than an
ADP-ribosyltransferase(21, 22) . More recently, Haag et al.(31) and Maehama et al.(32) showed that rat RT6 is capable of arginine-linked
automodification, although modification of heterologous target proteins
by rat RT6 was not demonstrated.
The mono(ADPribosyl)transferases
share their NAD glycohydrolase activity with another
family of NAD
metabolizing enzymes, the
ADP-ribosylcyclases, which include the lymphocyte surface proteins CD38 (33) and BST-1/BP-3(34, 35) . These enzymes
catalyze the conversion of NAD
into cyclic ADP-ribose
(cADPR) and nicotinamide. They differ somewhat in their relative
NAD
glycohydrolase versus ADPR cyclase
activities. The physiological function of the ADP-ribosylcyclases is at
present still unclear.
To characterize further the relationship of Rt6 and known mono(ADPribosyl)transferases we have performed secondary structure prediction analyses of Rt6 and related vertebrate proteins. Moreover, we have expressed soluble versions of mouse Rt6-1 and Rt6-2 and report here the molecular characterization and enzymatic properties of these recombinant proteins. The results show that these proteins, indeed, should be classified as arginine/protein mono(ADPribosyl)transferases (EC 2.4.2.31).
Recombinant protein was purified by affinity chromatography either on M2-antibody Sepharose (Kodak/IBI) or nickel nitrilotriacetic acid-agarose (Qiagen) columns according to the manufacturers' instructions. Columns were washed extensively with TBS, 0.1% Triton X-100, and bound protein was eluted with 50 mM glycine-HCl, pH 2.5 (M2 column), or 100 mM imidazol (nickel nitrilotriacetic acid column). Acid-eluted material was immediately neutralized with 1 M Tris, pH 8.0. Protein concentration was estimated by comparison of Coomassie-stained bands in SDS-polyacrylamide gels using a dilution series of lysozyme as a standard.
For
silver staining, blots were submersed in freshly prepared staining
solution (0.1 ml of 20% (w/v) silver nitrate added dropwise to a
solution of 0.5 ml of 40% (w/v) sodium citrate, 0.4 ml of 20% (w/v)
ferric sulfate, and 9 ml of HO). Blots were washed in water
after bands became visible (usually after 0.5-5 min).
For
immunostaining, blots were blocked with 10% goat serum in TBS and
incubated for 2-16 h at 4 °C with primary antibody at
appropriate dilutions (K48 serum at 1:2000, affinity-purified K48
antibodies at 1:10, M2 antibody at 1 µg/ml) in TBS, 0.5% Tween-20
(TBST), 10% goat serum and washed extensively in TBST. Secondary
reagents for detection of bound K48 were biotinylated goat anti-rabbit
Ig (1:2000, Amersham Corp.) and streptavidin peroxidase (1:5,000,
Amersham Corp.); for detection of bound M2-antibody peroxidase-labeled
donkey anti-mouse Ig (1:5,000, Amersham Corp.). After washing in TBST,
bound antibody was detected with the ECL system (Amersham Corp.)
according to the manufacturer's instructions and by exposure to
Amersham ECL films. For detection of bound P, the blots
were subjected to autoradiography by exposure to a Kodak X-Omat AR film
for 12-16 h at -80 °C.
For analysis of ADP-ribosyltransferase
activity, purified Rt6 or Rt6 precipitated from Sf9 cell supernatants
with M2-antibody affinity matrix was suspended in 50 µl of PBS (0.2
µg Rt6/ml) containing 2 µCi of
[P]NAD
(5000 Ci/mol, Amersham
Corp.) or 2 µCi of [
P]ADPR. The latter was
prepared by incubating 2 µCi of
[
P]NAD
in 50 µl of PBS with
0.1 milliunit of N. crassa NAD
glycohydrolase
(Sigma) for 30 min at 37 °C; complete conversion of
[
P]NAD
to
[
P]ADPR was verified by thin-layer
chromatography. Labeling reactions were carried out for 30 min at 37
°C. After labeling, affinity matrix-associated protein was
precipitated by centrifugation. Soluble protein was precipitated with
Strataclean-Resin (Stratagene) (10 µl/reactions) according to the
manufacturer's instructions. Precipitates were washed extensively
with PBS and then suspended in either PBS, 10 mM NAD
, 10 mM HgCl
, 1 M NaCl, or 1 M NH
OH, and incubated on a tumbler
overnight at room temperature. Precipitates were then again pelleted by
centrifugation and subjected to SDS-PAGE analyses as described above.
Figure 1:
Conserved amino acid residues and
secondary structure motifs in Rt6-1 and Rt6-2 and related
eucaryotic proteins. A, multiple sequence alignment was
performed with the MaxHom program, and the resulting alignment was used
as input for secondary structure prediction analyses with the PHDsec
program(38, 37) . Note that amino acid insertions that
occur in the other proteins relative to Rt6 are omitted, and
neighboring amino acid residues are indicated by lower case
lettering. Only residues with >72% average accuracy for the
three states, helix, strand, and loop, are indicated by H, E, and L, respectively; residues with >90%
accuracy are underlined(38, 37) . Secondary
structure motifs resembling those in the highly conserved presumptive
catalytic core of the four bacterial ADP-ribosylating toxins of known
structure are indicated below the alignment using the nomenclature for E. coli heat labile enterotoxin (see Fig. 1B).
Highly conserved arginine, serine, and glutamic acid residues are
marked by arrows above the alignment, four conserved cysteine
residues are marked by asterisks. N- and C-terminal signal
sequences are indicated by brackets. Sequences were compiled
from GenBank data base accession numbers X52991, X87616, M30311 (mouse
Rt6-1, Rt6-2, and rat RT6.2, respectively); M98764 and
S74683 (rabbit and human skeletal muscle mono(ADPribosyl)transferase,
respectively); and X82397 (mono(ADPribosyl)transferase from chicken
erythroblasts) and from reference (18) (chicken bone marrow
mono(ADPribosyl)transferases 1 and 2). B, schematic diagram of
the secondary structure units forming the presumptive catalytic core of
ADP-ribosylating bacterial toxins. The nomenclature used is that for E. coli heat labile enterotoxin(4) . 44 amino acids
corresponding roughly to the illustrated strands and
helix
can be superimposed with an root-mean-square difference of 1.6 Å
in the known structures of E. coli heat labile enterotoxin,
pertussis toxin, diphtheria toxin, and pseudomonas exotoxin
A(3, 4, 5, 8, 6) . The
presumptive active site crevice is lined by
1,
3-
3, and
6. The conserved catalytic glutamic acid residue in
6, which
can be cross-linked to NAD
by photoaffinity labeling
in all four toxins, is marked by E. The conserved arginine
residue in
1 and the conserved serine residue in
3, which
interact via a hydrogen bond in the E. coli and pertussis
toxins are marked by R and S (note that these
residues are histidine and tyrosine in pseudomonas and diphtheria
toxins, see C). C, alignment of residues lining the
active site crevice in bacterial toxins of known three-dimensional
structure with similar sequences in other ADP-ribosyltransferases.
Nomenclature of conserved secondary structure units is as in A and B. The four bacterial toxins with known
three-dimensional structures are marked by
on the right.
Distinct subfamilies are separated by horizontal lines.
Members of each subfamily show significant sequence similarities
(>20% overall sequence identity). Residues conserved in at least
three distinct subfamilies are boxed. The presumptive
catalytic arginine, serine, and glutamic acid residues are marked by arrows on the bottom. Residues in the eucaryotic enzymes that
occur also in at least three of the six subfamilies of bacterial
enzymes are marked by an asterisk on the bottom. ETA, Pseudomonas exotoxin A; DT, diphtheria toxin; DRR, Rhodospirillum rubrum dinitrogenase reductase
ADP-ribosyltransferase; DAB, Azosporillium brasiliense ADP-ribosyltransferase; DRC, Rhodospirillum
capsulatus ADP-ribosyltransferase; LT, E. coli heat labile enterotoxin; PT, pertussis toxin; CT, cholera toxin; MTX, Bacillus sphaericus mosquitocidal toxin; ETS, pseudomonas extoxin S; C3C and C3D, Clostridium botulinum type C and type D
phage exoenyzmes C3; EDIN, epidermal cell differentiation
inhibitor from Staphylococcus aureus; T2 and T4, gpALT ADP-ribosyltransferase from E. coli bacteriophages T2 and T4; rRT6, rat T cell marker; RT6, mRt6-1, and mRt6-2,
mouse T cell markers Rt6-1 and Rt6-2; rMAT and hMAT, rabbit and human skeletal muscle
ADP-ribosyltransferases; chBMAT1 and chBMA2, chicken
bone marrow ADP-ribosyltransferases 1 and 2. Sequences for vertebrate
proteins are as in A; sequences for bacterial and
bacteriophage proteins were compiled from Genbank accession numbers:
K01995 (ETA), P00588 (DT), P14299 (DRR),
M87319 (DAB), X71131 (DRC), P06717 (LT),
P04977 (PT), P01555 (CT), S27514 (MTX),
L27629 (ETS), Q00901 (C3C), P15879 (C3D),
P24121 (EDIN), X69893 (T2), and X15811 (T4).
Figure 2:
Schematic diagram of native Rt6 and
expression constructs. Native Rt6-1 and Rt6-2 coding
sequences (top) are compared with those of recombinant
constructs for expressing tagged protein in the baculovirus system
(bottom). L, posttranslationally cleaved N-terminal leader
signal sequence for translocation to the endoplasmic reticulum; T, posttranslationally cleaved C-terminal tail signal sequence
for GPI-anchor attachment; F, FLAG tag-binding site for
monoclonal antibody M2; H, His tag-binding site
for metal chelating resin; N, N terminus of native Rt6
generated by cleavage of L; CC, potential C termini of native
Rt6 generated by cleavage of T. The precise position of the cleavage
site in mouse Rt6 is not known. At the position homologous to the
cleavage site determined in rat RT6.2 by sequencing of the C-terminal
peptide(54) , mouse Rt6 contains insertions of 7 (Rt6-1)
and 9 (Rt6-2) amino acid residues. C, C terminus of
recombinant Rt6 resulting from a stop codon following F and H. Forks indicate potential N-linked glycosylation
sites; boldface lines indicate four cysteine residues
conserved in the known eucaryotic
transferases.
Figure 3:
Western blot analysis of recombinant Rt6
in insect cell supernatants. A, supernatants (20 µl/lane)
from Sf9 cells infected with six individual plaque-purified
Rt6-1- and Rt6-2-encoding baculoviruses (lanes
1-6 and 7-12, respectively) were subjected to
SDS-PAGE and Western blot analyses. The blot was immunostained with
FLAG tag-specific mouse monoclonal antibody M2, peroxidase-labeled
secondary antibody, and the ECL detection system (Amersham Corp.). The
blot was exposed for 1 s to Kodak X-Omat AR film. B, Sf9 cell
supernatants (2 µl/lane) were subjected to SDS-PAGE and Western
blot analysis as in A. The blot was immunostained with Rt6
peptide-specific rabbit serum K48 in the absence (lanes
1-3) or presence (lanes 4-6) of cognate
peptide 48 (10 µg/ml), peroxidase-labeled secondary antibody, and
the ECL detection system (Amersham Corp.). Lanes 1 and 4, Rt6-1-containing supernatant; lanes 2 and 5, Rt6-2-containing supernatant; lanes 3 and 6, rainbow M marker (Amersham Corp.).
Note that K48, which was raised against Rt6 peptide coupled to
ovalbumin, reacts with ovalbumin both in the absence (lane 3)
and presence (lane 6) of peptide 48. Arrows indicate
bands corresponding to recombinant Rt6
proteins.
Figure 4:
Comparative Western blot analyses of
recombinant Rt6-1 and Rt6-2 from Sf9 cells and native
Rt6-1 and Rt6-2 released from mouse spleen cells by PI-PLC.
Spleen cells from BALBc/ByJ (lane 1), C57BL/6J (lane
2), and NZW/LacJ (lane 3) mice were treated with PI-PLC.
Rt6-1- and Rt6-2-containing (lanes 4 and 5) Sf9 cell supernatants were prepared as in Fig. 2C. Proteins were precipitated from cleared
supernatants with Strataclean resin and subjected to SDS-PAGE and
Western blot analysis. The blot was immunostained with
affinity-purified K48 antibodies, peroxidase-labeled secondary
antibody, and the ECL detection system (Amersham Corp.). PI-PLC
supernatants were from 0.5 10
cells (lanes 1 and 2) and 2
10
cells (lane
3); lanes 4 and 5 each contain 1 µl of
insect cell supernatant. The blot was exposed to Kodak X-Omat AR film
for 8 s. Control lanes with Rt6-1-containing Sf9 cell supernatant (lane 6) and C57BL/6 mouse spleen cell PI-PLC supernatant (lane 7) were silver-stained for total
protein.
Figure 5:
FPLC analyses of the reaction products
after incubation of purified recombinant Rt6-1 and Rt6-2
with NAD. Purified recombinant Rt6-1 (panel
D) and Rt6-2 (panels A-C) were incubated with
1 mM NAD
for 0-90 min at 37 °C.
Reaction products were analyzed by FPLC on a fast flow Source Q resin
(Pharmacia) column, and products were detected by absorption at 280 nm.
The elution points of markers N (NAD
), n (nicotinamide), and A (ADPR) are indicated on the bottom. A, Rt6-2, 0 min; B, Rt6-2, 30 min; C, Rt6-2, 90 min; D, Rt6-1, 90
min.
The hydrolysis of NAD by
Rt6-2 was not affected by the addition of 10 mM histidine or 10 mM asparagine but was inhibited in a
dose-dependent manner by the arginine analogue agmatine (Fig. 6). The addition of 10 mM agmatine almost
completely blocked the hydrolysis of NAD
by
Rt6-2 but not that of recombinant CD38 (an ADP-ribosylcyclase) (Fig. 6, D versus E). Note the appearance of an
additional peak of absorbance in the samples containing Rt6-2 and
agmatine (marked by asterisks in Fig. 6, C and D) but not in that containing CD38 and agmatine (Fig. 6E). The nature of this peak is not known but
possibly represents ADPR-agmatine.
Figure 6:
FPLC
analyses of the reaction products after incubation of purified
recombinant Rt6-2 and CD38 with NAD. Purified
recombinant Rt6-2 (panels A-D) and CD38 (panel
E) were incubated with 1 mM NAD
for 90
min at 37 °C in the presence of 10 mM asparagine (A), 10 mM histidine (B), 1 mM agmatine (C), or 10 mM agmatine (D and E). Supernatants were analyzed by FPLC as in Fig. 5.
The elution points of markers are indicated as in Fig. 5. A peak
of absorbance possibly representing ADPR-agmatine is marked with an asterisk.
Figure 7:
SDS-PAGE analyses of immunoprecipitated
recombinant Rt6-1 and Rt6-2 labeled with
[P] NAD
. Sf9 cell supernatant,
purified Rt6, M2 Sepharose, and Rt6/M2 immunoprecipitates were
incubated for 30 min at 37 °C in the absence (lanes 1 and 2) or presence (lanes 3-8) of
[
P]NAD
. Samples were subjected
to SDS-PAGE and immunoblot analysis with antiserum K48 and the ECL
system as in Fig. 3(top panel). For autoradiography,
the blots were then covered with a black sheet of paper to quench
remaining chemiluminescence and exposed to Kodak X-Omat AR film for 16
h at -80 °C (bottom panel). Lane 1, crude
Sf9 cell supernatant containing recombinant Rt6-1; lane
2, Rt6-1/M2 immunoprecipitate (no radiolabel); lanes 3 and 6, M2 Sepharose beads; lane 4,
Rt6-1/M2 immunoprecipitate obtained from crude Sf9 cell
supernatant; lane 5: purified Rt6-2; lane 7,
purified Rt6-2 reprecipitated with M2 beads; lane 8,
Rt6-2/M2 immunoprecipitate from crude Sf9 cell supernatant. Bands
corresponding to Rt6 and the light chain of the M2 antibody are marked
by arrows.
Figure 8:
Sensitivity of incorporated radiolabel to
hydroxylamine. The M2L chain in Rt6-1- and Rt6-2-containing
immunoprecipitates (panels A and B, respectively) was
radiolabeled by incubation of beads with
[P]NAD
as in Fig. 7.
After extensive washing, beads were suspended in PBS (lanes
1), 10 mM NAD
(lanes 2), 1 M NaCl (lanes 3), 1 M NH
OH (lanes 4), or 10 mM HgCl
(lanes
5) and incubated overnight at room temperature. Beads were
pelleted by centrifugation and boiled in SDS-PAGE sample buffer for 5
min before loading onto the gel. Immunoblot analyses with antiserum K48
and the ECL system (top panels) and autoradiography of the
same blots (bottom panels) were performed as in Fig. 7.
The findings presented in this paper demonstrate that mouse
Rt6-1 and Rt6-2 are GPI-anchored
NAD-dependent arginine-specific
mono(ADPribosyl)transferases. They are the first such enzymes to be
molecularly characterized in the immune system. Our results show that
these T cell membrane proteins exhibit predicted secondary structure
motifs and enzymatic activities similar to those of ADP-ribosylating
bacterial toxins. The data are compatible with a distant evolutionary
relationship of Rt6 and related eucaryotic membrane proteins and
ADP-ribosylating bacterial toxins. This raises some interesting
questions.
The results of our secondary structure prediction
analyses confirm alignments made previously between eucaryotic and
procaryotic transferases on the basis of amino acid sequence
similarities around Glu(21, 22) and
considerably extend the region of predicted structural homology. It is
of note that the predicted catalytic domain is encompassed entirely in
the
-sheet-rich C-terminal half of the eucaryotic proteins (Fig. 1). The prediction that Glu
plays a
catalytic role is supported by the finding that site-directed mutation
of this residue almost completely abolishes enzyme activities of mouse
Rt6 (
)as has also been observed in case of the muscle
enzyme(22) . The significance of the additional, mainly
helical, section in the N-terminal half of the eucaryotic proteins is
presently unknown and open for speculation. Interesting possibilities
include roles in ligand binding or translocation across the cell
membrane.
The enzymatic activities of recombinant soluble
Rt6-1 and Rt6-2 are characteristic for
NAD-dependent arginine-specific
mono(ADPribosyl)transferases. This includes their capacity to
ADP-ribosylate arginine in nonphysiological target proteins ( Fig. 7and Fig. 8) as well as their different
NAD
glycohydrolase activities ( Fig. 4and Fig. 5). In the case of arginine-specific bacterial toxins,
ADP-ribosylation of the physiological target is most efficient, but in
its absence, other proteins, such as arginine-rich histones, can also
be modified(1, 2) . This property is shared by the Rt6
proteins, which also readily ADP-ribosylate arginine-rich histones but
not bovine serum albumin or many other control proteins (not shown).
Interestingly, a His tag-specific antibody (Qiagen),
which is of the same isotype as the M2 antibody, does not serve as a
target for ADP-ribosylation by Rt6 (not shown). Presumably, the M2
light chain contains an arginine residue in a setting suitable for
modification by Rt6, which the His
tag-specific antibody
lacks. Moreover, it is possible that the high local concentration of
enzyme and target on the surface of the Sepharose beads is responsible
for the high efficiency of the reaction with the M2 antibody. In any
case, this system will provide a simple tool for analyzing Rt6 mutants, e.g. in identifying essential residues by site-directed
mutagenensis. Moreover, it will be interesting to see whether
FLAG-tagged versions of other transferases can also modify the M2 light
chain or whether this is a peculiar property of the tagged Rt6
proteins.
Considering that the two mouse Rt6 proteins are just
slightly more similar to one another than either is to rat RT6 (79 versus 71-73% sequence identity), it is intriguing that
these proteins show such striking differences in enzymatic activities.
Remarkably, neither arginine-rich histones nor the M2 antibody serve as
substrates for ADP-ribosylation by FLAG-tagged recombinant rat RT6.1 or
RT6.2 alloantigens (not shown). Moreover, the latter show much stronger
NAD glycohydrolase and, in case of RT6.2,
automodification activities than do the mouse Rt6 proteins (not shown).
The reason for these differences is unresolved. With the availability
of recombinant RT6/Rt6 proteins, a molecular dissection of the
structural domains and critical amino acid residues responsible for the
observed differences in NAD
glycohydrolase,
automodification, and arginine-ADP-ribosyltransferase activities should
now be possible.
Of course, it is obvious that neither the M2 light
chain nor histones are the physiological target proteins for Rt6. The
fact that Rt6 does efficiently modify these artificially presented
targets indicates that it may be difficult to distinguish
ADP-ribosylation of physiologically relevant targets from
ADP-ribosylation of irrelevant targets in vitro. Certainly,
caution is warranted when interpreting modifications observed in
vitro, e.g. upon incubation of intact cells or cell
lysates with radioactive NAD.
The physiological
target proteins of Rt6 and its eucaryotic relatives presently remain
unknown. The fact that the identified eucaryotic
ADP-ribosyltransferases are GPI-anchored membrane proteins raises the
intriguing and as yet unresolved question whether these enzymes have
extra- or intracellular targets. If they target intracellular proteins
as do their bacterial toxin cousins, the question arises as to their
mechanism of entry into the cytoplasm. If they have extracellular
targets as some evidence seems to suggest, how is access to the
required substrate NAD assured, considering that
NAD
is a classic intracellular metabolite and that
cell membranes are impermeable to NAD
? A dying cell is
one potential source for extracellular NAD
, as is a
hypothetical specific secretory mechanism akin to that recently
discovered for ATP, another ``classic intracellular
metabolite'' (49) . On the other hand, it is also
conceivable that there exists a mechanism for translocating the
eucaryotic ectoenzymes to the cytoplasm analogous to that used by
bacterial toxins.
In the case of the muscle cell membrane-associated
ADP-ribosyltransferase activity, in vitro studies have put
forward evidence for modifications of both extra- and intracellular
target proteins. Incubation of intact mouse muscle cells with
[P]NAD
results in
arginine-linked ADP-ribosylation of several protein bands, the most
prominent of which has been identified as the muscle cell-specific
7 integrin chain (14) . Incubation of the membrane
fraction from rabbit or canine muscle cells with
[
P]NAD
also leads to labeling
of serveral protein bands(50, 51) . In the canine
system, the intracellular G
subunit of the G
protein
regulating adenylate cyclase was identified as the most efficient
target for arginine-linked ADP-ribosylation(51) . G
proteins and
7 integrin are thought to play important roles
in signal transduction and cell-matrix interaction, respectively, and
it is conceivable although not yet established that the observed target
protein modifications alter physiologically relevant cellular
functions.
In the case of T cell membrane-associated enzyme
activity, modification of several distinct bands by arginine
ADP-ribosylation has been observed after incubation of lymphoma cells
and activated mouse cytotoxic T cells (CTLs) with exogenous
[P]NAD
(52, 16) .
Although these potential target proteins have not been defined in
molecular terms, interesting functional consequences of cell surface
ADP-ribosylation were observed in case of the CTLs. Thus, treatment of
activated CTLs with ecto-NAD
led to a dramatic
suppression of the ability of these cells to proliferate in response to
stimulator cells and to lyse target cells(16) . Moreover,
treatment of the CTLs with PI-PLC released the activity that catalyzes
ADP-ribosylation of cell surface proteins and rendered the cells
refractory to the suppressive effects of ecto NAD
. It
is quite possible that the GPI-anchored enzyme activity detected by
Wang et al.(16) corresponds to Rt6. If so, and if
suppression of CTL functions by Rt6 acting on extracellular
NAD
and cell surface proteins were physiologically
relevant, this would provide a basis for explaining the observed
coincidences between defects in Rt6 gene structure and/or expression
and enhanced susceptibility for autoimmune diseases in different animal
models(29, 30) . It is conceivable, for example, that
interference with Rt6-mediated regulation of CTL functions could lead
to enhanced T cell autoreactivity. In this context it is of interest to
note that treatment of NOD mice with antibodies specific for integrin
4, a T cell-specific relative of the most prominent target for
surface ADP-ribosylation in skeletal muscle cells, has recently been
shown to markedly suppress progression to autoimmune
disease(53) . It is tempting to speculate that the function of
4 integrin is affected similarly by these antibodies as by
hypothetical Rt6-catalyzed ADP-ribosylation.
These exciting observations have opened a new field of experimental investigation at the interface of enzymology and immunology. The soluble, enzymatically active recombinant Rt6 proteins described here provide valuable new experimental tools to address interesting questions, e.g. what are the physiological target protein(s) of the Rt6 proteins, what is the structural basis for their enzymatic activity, and how can we probe for the possible immunomodulating activities of these reagents? Thus, it may be expected that these reagents will help shed light on the biological significance of the endogenous relatives of ADP-ribosylating bacterial toxins in animal tissues. Appropriate experiments are underway in our laboratories.