(Received for publication, March 20, 1995; and in revised form, July 10, 1995)
From the
Rat T lymphocyte alloantigen 6.1 (RT6.1), which was synthesized
as the fusion protein with a maltose-binding protein in Escherichia
coli, displayed NAD-dependent
auto-ADP-ribosylation in addition to an enzyme activity of
NAD
glycohydrolase. Such ADP-ribosylation of RT6.1 was
also observed in lymphocytes isolated from rat tissues as follows. When
intact rat lymphocytes expressing RT6.1 mRNA were incubated with
[
-
P]NAD
, its radioactivity
was incorporated into a cell surface protein with the M
of 31,000. The radiolabeled 31-kDa protein was released from the
cell surface by treatment of the cells with
phosphatidylinositol-specific phospholipase C and immunoprecipitated
with anti-RT6.1 antiserum. The radioactivity incorporated into the
31-kDa protein was recovered as 5`-[
P]AMP upon
incubation with snake venom phosphodiesterase and also removed by
NH
OH treatment. These results suggested that the
NAD
-dependent modification of the 31-kDa protein was
due to ADP-ribosylation of glycosylphosphatidylinositol-anchored RT6.1
at an arginine residue. When intact lymphocytes, in which RT6.1 had
been once modified by [
P]ADP-ribosylation, were
further incubated in the absence of NAD
, there was
reduction of the radioactivity in the
[
P]ADP-ribosylated RT6.1. The reduced
radioactivity was recovered from the incubation medium as
[
P]ADP-ribose. This reduction was effectively
inhibited by the addition of ADP-ribose to the reaction mixture.
Moreover, readdition of NAD
caused the
ADP-ribosylation of RT6.1 again. Thus, the ADP-ribosylation of RT6.1
appeared to proceed reversibly in intact rat lymphocytes.
ADP-ribosylation is one of the post-translational modifications
of cellular proteins, in which the ADP-ribose moiety of NAD is transferred to specific amino acid residues of mostly
GTP-binding proteins. This unique modification has been found in enzyme
reactions catalyzed by bacterial toxins such as diphtheria, cholera,
and pertussis toxins(1, 2, 3) . Enzyme
activities of bacterial ADP-ribosyltransferases have widely been
utilized to identify and characterize the substrate proteins, because
the protein functions are profoundly affected by ADP-ribosylation.
Besides these bacterial toxins, activities of ADP-ribosyltransferases
appeared to be present in several mammalian
cells(4, 5, 6, 7, 8) . One
of the mammalian enzymes, NAD
:arginine
ADP-ribosyltransferase, of which ADP-ribose acceptor was initially
identified as the guanidino group of arginine or its related compounds,
was purified from rabbit skeletal muscle(5) . Zolkiewska et
al.(6) have recently cloned a cDNA encoding the enzyme
protein with a possible structure of glycosylphosphatidylinositol
(GPI(
))-anchored protein. An ecto-enzyme activity of
NAD
:arginine ADP-ribosyltransferase was also found in
myogenically differentiated C2C12 cells, and its substrate was
identified as a cell surface adhesion molecule, integrin
7(7) . The NAD
-dependent ADP-ribosylation
of integrin
7 was markedly reduced after treatment of the cells
with phosphatidylinositol-specific phospholipase C, indicating that the
enzyme was indeed anchored in the cell surface via GPI
linkage(7) . Based on a homology search with the amino acid
sequences of this type of mammalian enzymes, RT6 alloantigen was
expected to have a similar enzyme
activity(6, 8, 9, 10) .
RT6
alloantigen is specifically expressed in the cell surface of T
lymphocytes(11) , although it is not detected in thymocytes,
bone marrow cells, or B lymphocytes(11) , suggesting that its
expression is restricted to the final stages of post-thymic T
lymphocyte development. Although the physiological role of RT6 in a
specific cell function is still unknown, its defect in lymphocytes has
been implicated in disorders of diabetes and mercury-induced renal
autoimmunity in animal models(12, 13, 14) .
Recent biochemical analysis reveals that there are at least two types
of RT6 alloantigen, RT6.1 and RT6.2, and both are covalently anchored
in cell surface membranes via GPI linkage(15, 16) .
Takada et al.(9) have recently reported that RT6.2
exogenously expressed in rat adenocarcinoma cells is capable of
catalyzing the hydrolysis of NAD to ADP-ribose and
nicotinamide. Although intrinsic activity of NAD
glycohydrolase was thus proven to be present in the molecule of
RT6.2, there is no report showing that RT6 alloantigen has an enzyme
activity of ADP-ribosyltransferase. We report here that a recombinant
RT6.1 fused with MBP, which was expressed in and purified from Escherichia coli, catalyzed not only NAD
glycohydrolysis but also auto-ADP-ribosylation reaction.
Moreover, such ADP-ribosylation of RT6.1 effectively occurred in the
cell surface of intact rat lymphocytes in the presence of
NAD
. The ADP-ribosylation reaction appeared to proceed
reversibly in intact rat lymphocytes.
Figure 5:
Time course of
[P]ADP-ribosylation of RT6.1 in rat lymphocytes.
Lymphocytes (2
10
cells/ml) were incubated with
0.27 (open circles) or 1.1 (closed circles)
µM [
-
P]NAD
as
described under ``Experimental Procedures.'' At the indicated
times, the cells were lysed and subjected to SDS-PAGE. The extent of
[
P]ADP-ribosylated RT6.1 was measured by an
imaging analyzer, and the data are normalized and expressed as
percentages of the maximum value in the case of 1.1 µM NAD
. Results are the average ± the range
of duplicate determination.
Figure 1:
Recombinant RT6.1 purified as a
MBP-fusion protein and NAD-dependent
auto-ADP-ribosylation of the purified protein. Lane 1, the
recombinant RT6.1 purified as a MBP fusion protein (MBP-trRT6.1) was separated by SDS-PAGE and then stained with
Coomassie Brilliant Blue R-250. Lane 2, the purified protein
was incubated with [
-
P]NAD
and subjected to SDS-PAGE and autoradiography as described under
``Experimental Procedures.'' The position of the recombinant
RT6.1 is indicated by an arrow.
Figure 2:
Radiolabeling of 31-kDa protein by
[-
P]NAD
in rat lymphocytes
and identification of the 31-kDa protein as RT6.1. Lymphocytes (1
10
cells) were incubated with
[
-
P]NAD
for 1 h and
subjected to the following treatments. A, cell lysates
obtained from the radiolabeled cells were mixed with Laemmli buffer
containing 2-mercaptoethanol or the buffer alone and subjected to
SDS-PAGE under reducing (lane 1) or non-reducing (lane
2) conditions. Autoradiography was obtained as described under
``Experimental Procedures.'' B, radiolabeled cells
were lysed in 40 µl of Laemmli buffer (lane 1) or
resuspended in 30 µl of HBSS containing 1 mM ADP-ribose.
The resuspended cells were further incubated with 2 µl of
phosphatidylinositol-specific phospholipase C (18.2 units/ml) at 37
°C for 20 min, and the reaction mixture was separated from the
cells by a rapid centrifugation. The cells in the pellet were lysed in
40 µl of Laemmli buffer (lane 2), and the supernatant
containing the reaction mixture was mixed with 10 µl of 4-fold
concentrated Laemmli buffer (lane 3). 20 µl of each of the
samples was subjected to SDS-PAGE and autoradiography. C, the
lysate of radiolabeled cells was subjected to immunoprecipitation with
preimmune serum (lane 1) or anti-RT6.1 antiserum (lane
2) as described under ``Experimental
Procedures.''
Figure 3:
Release of
5`-[P]AMP by treatment of the radiolabeled RT6.1
with snake venom phosphodiesterase. Lymphocytes (2
10
cells) were incubated with
[
-
P]NAD
for 1 h, washed,
and solubilized with 130 µl of lysis buffer. After centrifugation,
56 µl of 90% trichloroacetic acid was added to the lysate, followed
by standing on ice for 10 min. After centrifugation, the precipitate
was washed with 4% trichloroacetic acid and dissolved in 15 µl of
0.2 M Tris-HCl (pH 9.0). This sample was further incubated
with (PDE) or without (Cont) 0.5 units of snake venom
phosphodiesterase (Boehringer Mannheim) in the presence of 6 mM MgCl
at 37 °C for 30 min. The reaction mixture (5
µl) was applied on a polyethyleneimine cellulose plate (Schleicher
& Schuell) and developed with 0.5 M formic acid/0.1 M lithium chloride (A) or 0.5 M guanidine
hydrochloride (B). Autoradiography was obtained as described
under ``Experimental
Procedures.''
Figure 4:
Treatment of
[P]ADP-ribosylated RT6.1 with hydroxylamine or
mercury chloride. [
P]ADP-ribosylated RT6.1 (RT6.1) in rat lymphocytes (3
10
cells),
together with the
-subunits of G
(420 ng;
) and G
(215 ng;
), which had been
[
P]ADP-ribosylated by cholera and pertussis
toxins, respectively, was subjected to SDS-PAGE, and then the separated
proteins were transferred to a polyvinylidene difluoride filter as
described under ``Experimental Procedures.'' The filters were
incubated with 1 M NaCl (A), 1 M neutralized
NH
OH (B), or 10 mM HgCl
(C) at 45 °C for 2 h and then subjected to
autoradiography.
Figure 6:
Release of
[P]ADP-ribose from
[
P]ADP-ribosylated RT6.1 in rat lymphocytes.
Lymphocytes (5
10
cells) were incubated with 0.27
µM [
P]NAD
for 1 h
as described under ``Experimental Procedures.'' The cells,
after being washed, were suspended in HBSS containing 2 mM ADP-ribose in order to remove radioactive material that
nonspecifically bound to the cell surface. After incubation at 37
°C for 10 min, the cells were resuspended in 200 µl of HBSS and
immediately lysed (lane 1) or further incubated at 37 °C
for 20 min with (lane 3) or without (lane 2) 2 mM ADP-ribose. A, the reaction mixture was analyzed by thin
layer chromatography as described in Fig. 4A. B, the cells were lysed and then subjected to SDS-PAGE and
autoradiography.
We further investigated whether RT6.1 once modified and
de-ADP-ribosylated was still capable of being
[P]ADP-ribosylated. After the first
ADP-ribosylation by incubation with NAD
, the cells
were washed and incubated with or without ADP-ribose. By this
incubation without NAD
, RT6.1 once modified was
expected to be de-ADP-ribosylated, and ADP-ribose inhibited the
de-ADP-ribosylation (see Fig. 6). Then the cells were washed and
subjected to [
P]ADP-ribosylation (Fig. 7). RT6.1 on the cells that had been incubated without
ADP-ribose at the second incubation was still capable of being
[
P]ADP-ribosylated (Fig. 7). However,
[
P]ADP-ribosylation of RT6.1 on the cells that
had been incubated with ADP-ribose at the second incubation was not
observed (Fig. 7). The second incubation with ADP-ribose did not
affect following [
P]ADP-ribosylation (data not
shown). Thus, the ADP-ribosylation of RT6.1 appeared to proceed
reversibly in intact rat lymphocytes if NAD
was
supplied to the extracellular environment.
Figure 7:
ADP-ribosylation of RT6.1 reversibly
proceeding in rat lymphocytes. Lymphocytes (5 10
cells) were first incubated with 1.1 µM
non-radiolabeled NAD
for 20 min as described under
``Experimental Procedures.'' The cells, after being washed,
were incubated at 37 °C for 20 min in 200 µl of HBSS in the
presence (lane 2) or absence (lane 1) of 2 mM ADP-ribose. The cells, after being washed, were incubated with
0.27 µM [
P]NAD
at
37 °C for 20 min. The cells were lysed and then subjected to
SDS-PAGE and autoradiography.
In the present study, we demonstrated that
NAD-dependent ADP-ribosylation of RT6.1 occurred in
intact lymphocytes. This ADP-ribosylation appeared to be catalyzed by
RT6.1 itself, because a recombinant RT6.1 that was expressed in E.
coli as a fusion protein with MBP also exhibited the same
modification upon incubation with NAD
. However,
ADP-ribosyltransferase activity of this fusion protein was extremely
low. Moreover, such an ADP-ribosylation was not apparently observed
when the membrane fraction instead of intact lymphocytes was incubated
with [
P]NAD
(data not shown).
Zolkiewska and Moss (7) have recently reported that integrin
7 is ADP-ribosylated by a GPI-anchored ADP-ribosyltransferase in
differentiated C2C12 cells. They have showed that the ADP-ribosylation
of integrin occurs only in the intact cells and not in the membrane
fraction. Takada et al. (9) have reported that RT6.2
exogenously expressed in adenocarcinoma cells exhibits only
NAD
glycohydrolase activity; the evidence for an
ADP-ribosylation of RT6.2 was not described in their report. These
results suggest that enzyme reactions catalyzed by these
ADP-ribosyltransferases proceed only when their substrates take certain
forms under physiological conditions.
In this report, we could
observe reversible ADP-ribosylation of RT6.1 in intact rat lymphocytes.
The reaction mixture of the ADP-ribosylation used in the present study
contained several nucleotides, such as ADP-ribose, FAD, and ATP, beside
the substrate of NAD. These compounds were very
effective in inhibiting the degradation of NAD
added
and/or the reversal reaction of ADP-ribosylated RT6.1. Especially the
existence of ATP in the reaction mixture was essentially required for a
significant level of the ADP-ribosylation of RT6.1 in the cells. In the
previous study(20) , Maehama et al. indicate that ATP
could inhibit activity of a rat ADP-ribosylarginine glycohydrolase, of
which substrates included ADP-ribosylated GTP-binding proteins modified
by cholera and botulinum C
toxins. Although the enzyme
responsible for the reversal reaction of modified RT6.1 has not been
extensively investigated in the present study, it can be assumed that
there is an enzyme(s) similar to the rat ADP-ribosylarginine
glycohydrolase in the cell surface. We observed that the
ADP-ribosylation of RT6.1 occurred in the presence of submicromolar
concentrations (0.1-0.2 µM) of NAD
,
suggesting that this modification may be considerable under the
physiological conditions.
Recently, Wang et al. (23) have reported that an enzyme of GPI-anchored
NAD:arginine ADP-ribosyltransferase is present in
cultured cytotoxic T cells. Incubation of the T cells with
NAD
caused ADP-ribosylation of the cell surface
proteins and suppressed the cell ability to lyse target cells. This
suppression appeared to be resultant from the failure of the cytotoxic
T cells to form specific conjugates with the target cells. It is thus
tempting to speculate that the ADP-ribosylation of RT6.1 similarly
exerts its influence on a cell function(s) of rat lymphocytes. Further
study on the possible cell function(s) linked to this unique
modification is currently under investigation in our laboratory.