Ecto-(Ca
,Mg
)-apyrases
(ecto-ATPases) or E-type ATPases (reviewed in (1) ) are
glycoproteins that hydrolyze extracellular nucleotide tri- and/or
diphosphates. These enzymes have a high specificity activity and are
insensitive to inhibitors of P-type, F-type, and V-type
ATPases(2, 3, 4, 5) .
Progress in
the study of ecto-apyrases has been impeded by the lack of a specific
inhibitor of enzymatic activity and by their low protein abundance.
Moreover, most ecto-apyrases are inactivated by the detergents normally
used to solubilize membrane-bound proteins. Recently, Handa and
Guidotti (6) reported that a potato tuber (Solanum
tuberosum) apyrase was similar in amino acid sequence to CD39, a
human and mouse lymphoid cell antigen(7) , and to several other
newly identified NTPases, (
)including yeast guanosine
diphosphatase(8) , garden pea nucleotide triphosphatase
(NTPase), (
)and Toxoplasma gondii NTPase(10, 11) . CD39, a membrane glycoprotein,
was originally identified as the major surface marker of
EBV-transformed B lymphoblastoid cells(12) . Later it was found
primarily on activated immune
cells(13, 14, 15) . Although the function of
CD39 is largely unknown, the similarity between CD39 and the NTPases
suggested that CD39 might be an ecto-apyrase.
In this report, we
show that EBV-transformed B lymphocytes have ecto-apyrase activity,
whereas non-EBV-transformed B lymphocytes do not. Ecto-apyrase
activities were also found associated with other immunocompetent cells,
including cytotoxic T cells (16) and natural killer (NK)
cells(4) , but not with resting thymocytes and lymphomas (4, 17, 18) . Both in our studies and those
of other investigators, there is a coincidence between expression of
CD39 and ecto-apyrase activity. All these clues lead to the hypothesis
that CD39 may encode an ecto-apyrase. Finally, by transfection of COS-7
cells with CD39 cDNA, we show that CD39 indeed has ecto-apyrase
activity.
MATERIALS AND METHODS
Reagents
Nucleotides, N-ethylmaleimide,
ouabain, activated charcoal, DEAE-dextran, chloroquine, horseradish
peroxidase-conjugated anti-mouse antibody, and nitrocellulose membrane
were purchased from Sigma. [
-
P]ATP
(triethylammonium salt) was purchased from DuPont NEN Research
Products. RPMI 1640, penicillin/streptomycin, DMEM, and L-glutamine were purchased from Life Technologies, Inc.
Anti-CD39 monoclonal antibody AC-2 (7, 12) was
purchased from AMAC, Inc. (Westbrook, ME). Chemiluminescent reagents
were purchased from Kirkegard & Perry Laboratories (Gaithersburg,
MD).
Cell Cultures
EBV-transformed B-lymphoblasts from
a patient with cystic fibrosis (GM7227A) were from the Coriel Institute
for Medical Research (Camden, NJ)(19) . Human T leukemia Jurkat
cells, EBV-transformed B lymphoblast LG2 cells, murine T lymphoma
BW5147 cells, and B lymphoma A20 and Daudi cells were kindly provided
by Dr. Basya Ryalov (Dept. of Molecular and Cellular Biology, Harvard
University). All immune cells were maintained in medium RPMI 1640.
COS-7 cells were kept at 50-70% confluence in DMEM. All cultures
were supplemented with 10% fetal bovine serum, 10 mM glutamine, penicillin (100 units/ml), and streptomycin (100
µg/ml).
Assay of Apyrase Activity
Apyrase activity was
determined by measuring the amount of [
P]P
released from [
-
P]ATP after
precipitation of nucleotides with activated
charcoal(4, 20) . Intact cells (1
10
) or cell homogenates were added to buffer containing 20
mM HEPES-Tris (pH 7.4), 120 mM NaCl, 5 mM KCl, 1 mM EGTA, 0.3 mM ATP, and 0.8 µCi of
[
-
P]ATP; the final volume was 200 µl.
Cell extracts were obtained by homogenizing cells in the buffer with a
Dounce homogenizer. The assay mixture was incubated at 37 °C for 20
min, and then the reaction was stopped by the addition of 0.5 ml of
cold 20% (w/v) activated charcoal in 1.0 M HCl. The assay
tubes were incubated on ice for 10 min and centrifuged at 10,000
g for 10 min to pellet the charcoal. Aliquots (140
µl) of the supernatant containing the released
[
P]P
were transferred to
scintillation fluid, and radioactivity was measured by using a Beckman
LS5801 liquid scintillation spectrophotometer. Alternatively, the
apyrase activity was determined by measuring the inorganic phosphate
released as described by Ames(21) , except that the time for
color development was 20 min at 37 °C. All assays were performed in
triplicate and reported as the mean and standard deviation. The
Ca
- or Mg
-stimulated apyrase was
determined by subtracting values obtained with EGTA alone from those
with 1.5 mM CaCl
or MgCl
plus
chelator.
Reverse Transcription-PCR and Construction of Expression
Plasmid
Total RNA was isolated from approximately 10
human EBV-transformed B lymphocytes (LG2) by the acid guanidinium
thiocyanate method(22) . One microgram of total RNA served as
template for cDNA synthesis by avian myeloblastosis virus reverse
transcriptase (Life Technologies, Inc.) for 60 min at 42 °C in the
presence of 25 pmol of a specific 30-base oligonucleotide as primer in
a total reaction of 20 µl(23) . The reverse transcription
primer was the same one as the antisense primer to be used in the
subsequent PCR reaction. The whole reverse transcription mixture was
then used as template in 35 cycles of amplification. The sense primer
(5`-GCGAATTCTTATGGAAGATACAAAGGAGTC), with an EcoRI site at the
5` end, contains a sequence identical with nucleotides 68-87 of
human CD39(7) . The antisense primer
(5`-GCTGAATTCGCTATACCATATCTTTCCAGA) is complementary to nucleotides
1581-1600 of the CD39 coding sequence, except that an EcoRI site is present. After amplification, the PCR product
(1.6 kilobases) was subcloned into pCI-neo (Promega).
Expression of CD39 in COS-7 Cells and ATPase
Assay
COS-7 cells were transfected with cDNA for CD39 or with
the pCI-neo vector alone by the DEAE-dextran method(24) . The
cells were seeded for 2 days before transfection at a density of 1700
cells per cm
. On the day of transfection, the cells were
washed twice with DMEM. Then, DMEM containing 10% Nuserum, 0.4 mg/ml
DEAE-dextran, and chloroquine (100 µM) was added. DNA was
added at a concentration of 1.25 µg/ml, and the cells were
incubated for 4 h at 37 °C with 5% CO
. Afterward, the
cells were washed once with DMEM, shocked for 2 min in 10% dimethyl
sulfoxide in phosphate-buffered saline and washed twice with
phosphate-buffered saline. The cells were incubated in DMEM containing
10% fetal bovine serum at 37 °C with 5% CO
. After 2 to
3 days, cells were detached by adding 10 mM EDTA and then
incubated at 37 °C for 20 min. The detached cells were pooled,
centrifuged, washed twice with assay buffer, and assayed for
calcium-dependent ecto-apyrase activity.
Immunoblots
Cells extracts (10 or 50 µg) were
resolved by 9% SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose membranes for 2 h at 500 mA. Membranes were washed in
rinse buffer (phosphate-buffered saline with 3% Tween 20) at room
temperature for 15 min and incubated overnight at 4 °C with AC2
anti-CD39 mAb (0.2 µg/ml) in rinse buffer. The membranes were
washed three times with rinse buffer at room temperature and were
incubated with a 1:2000 dilution of horseradish peroxidase-conjugated
anti-mouse antibody in rinse buffer for 1.5 h at room temperature.
After three washes, membranes were developed with chemiluminescent
reagents, and the emitted light was recorded by x-ray film.
RESULTS
Ca
and
Mg
-stimulated Apyrase Activity of EBV-transformed B
Lymphocytes
Human EVB-transformed B lymphocytes LG2 and GM7227A
were incubated in the standard reaction buffer with
[
-
P]ATP in the presence of 1 mM NaN
and 0.5 mM Na
VO
for 20 min. After charcoal precipitation of the nucleotides, the
released [
-
P]P
in the
supernatant represents the apyrase activity. The hydrolysis of
[
-
P]ATP was linear for at least 25 min, and
no radioactivity was released above background levels in assays
containing no cells. Fig. 1A (left panel)
shows the time course of Ca
- and
Mg
-stimulated apyrase activity of intact LG2 cells.
In the absence of a divalent cation, almost no hydrolysis of ATP was
detected, whereas both Ca
and Mg
stimulated the activity. At the end of the incubation, cells were
intact (>90%) as demonstrated by exclusion of trypan blue.
Disruption of LG2 cells by homogenization in a Dounce homogenizer did
not increase either Ca
- or
Mg
-apyrase activity (right panel),
suggesting that the (Ca
,Mg
)-apyrase
activity was probably associated with the external surface of the
plasma membrane. The release of P
from EBV-transformed LG2
and GM7227A cells was 349 ± 8 and 218 ± 10 nmol per
million cells per 20 min, respectively. To examine whether this
hydrolytic activity was due to secreted enzymes or an intracellular
enzyme released through ``leaky'' membrane, cells were
incubated for 30 min in assay buffer without ATP and pelleted. Apyrase
activity in the supernatant was less than 5% of the total apyrase
activity, whereas the cell pellet contained more than 90% of the total
apyrase activity. These results indicated the presence of a
membrane-associated ecto-apyrase in EBV-transformed B lymphocytes.
Figure 1:
Ecto-(Ca
,
Mg
)-apyrase activity in EBV-transformed B cells. A, time course of Ca
- and
Mg
-stimulated apyrase activities of EBV- transformed
B cell, LG2. Intact cells (left panel) or homogenate (right panel) were assayed at a concentration of 5
10
cells/ml in the presence of 1 mM NaN
, 0.5 mM Na
VO
, and
1 mM EGTA without added Ca
or Mg
(
), or with 1.5 mM Ca
(
), or with 1.5 mM Mg
(
). B, ecto-(Ca
)-apyrase activities of
EBV-transformed B cells (LG2 and GM7227A), non-EBV-transformed B cells
(A20 and Daudi), and T lymphomas (Jurkat and BW5147), assayed as
described in A.
To further characterize the observed apyrase activity, assays were
done with human T leukemia Jurkat cells
(ecto-apyrase-deficient(18) ), African Burkitt lymphoma Daudi
cells, murine T lymphoma BW5147 cells, and murine B lymphoma A20 cells.
The apyrase activities of these cells were not statistically elevated
above the background level (Fig. 1B).
Characterization of Ecto-apyrase Activity
Table 1shows that inhibitors of P-type plasma membrane
ATPases (ouabain and vanadate), V-type vacuolar ATPase
(N-ethylmaleimide), and F-type mitochondrial ATPase (azide), did not
significantly inhibit the EBV-transformed LG2 ecto-apyrase activity.
Furthermore, 10 mM fluoride (a phosphatase inhibitor) was not
inhibitory. The nucleotide specificity of the LG2
(Ca
,Mg
)-apyrase activity is shown
in Table 2. The relative hydrolysis rates of the nucleotide
triphosphates is about the same, while the rate with ADP is about 60%
of that with ATP. No phosphate was released from AMP (substrate of
5`-nucleotidase) and p-nitrophenyl phosphate (substrate of
alkaline phosphatase). These results suggest that the
(Ca
,Mg
)-apyrase activity of
EBV-transformed LG2 cells is typical of an E-type ATPase.
Coincidence of CD39 Expression Pattern and Ecto-apyrase
Activity in Immunocompetent Cells
-CD39 was originally
identified as a surface antigen of EBV-transformed lymphoblastoid
cells(12) . CD39 is not encoded by the EBV genome, but is a
host gene. Epitope-tagging and topologic analysis indicated that CD39
has a large extracellular loop between short intracellular N and C
termini. The large extracellular loop was similar in amino acid
sequence to yeast guanosine phosphatase, indicating that CD39 may
encode an ecto-enzyme(7) . Later, CD39 was found primarily on
activated immune cells, but was absent from resting thymocytes,
lymphocytes, and lymphomas. In both our studies and those of other
investigators, there is a coincidence between the expression pattern of
CD39 and ecto-apyrase activity (Table 3). CD39 was only found in
cells with ecto-apyrase activity, suggesting that CD39 may encode an
ecto-apyrase.
Demonstration of CD39 as an Ecto-apyrase
Since
detergent solubilization is a major problem in purification of many
ecto-apyrases and LG2 cells lost more than 90% of the ecto-apyrase
activity after solubilization with C
E
or
Triton X-100, we decided to test whether CD39 encoded an ecto-apyrase
by expression. Human CD39 cDNA was amplified by reverse
transcription-PCR and subcloned into the mammalian expression vector
pCI-neo. COS-7 cells transfected with CD39 recombinant cDNA had about
5.4-fold higher Ca
-ATPase activity (309 ± 27.6
nmol/mg/h) than cells transfected with vector alone (57.6 ± 4.8
nmol/mg/h) (Fig. 2A). This activity was not inhibited
by P- or V-type ATPase inhibitors, since 1 mM NaN
and 0.5 mM Na
VO
were included in
the reaction buffer. Cells extracts (50 µg) of both preparations
were examined for the presence of CD39 by Western analysis (Fig. 2B). Cells transfected with recombinant CD39 cDNA
had 5-fold more CD39 protein (lane 2) as compared with cells
transfected with vector alone (lane 1), estimated by
comparison with the amount of CD39 protein in 10 µg (lane
3) and 50 µg (lane 4) of protein from extracts of LG2
cells.
Figure 2:
CD39 encodes an ecto-apyrase. A,
Ca
-stimulated apyrase activity of COS-7 cells with
expression vector pCI-neo or CD39 recombinant DNA. B,
immunoblot analysis of CD39 in total cell extracts of COS-7 cells
transfected with vector (lane 1, 50 µg) or CD39
recombinant DNA (lane 2, 50 µg) and of LG2 Cells (lane
3, 10 µg; lane 4, 50 µg) as described under
``Materials and Methods.''
DISCUSSION
This paper reports that CD39 is an ecto-apyrase. Four lines
of evidence support this conclusion. First, the amino acid sequence of
CD39 is significantly homologous to several newly identified NTPases.
Second, the coincidence of the expression pattern of CD39 and
ecto-apyrase activities on immunocompetent cells is consistent with the
hypothesis that CD39 is an ecto-apyrase. Third, ecto-apyrase activities
were found on EBV-transformed B lymphocytes that express CD39 as a
major surface marker(12) . No ecto-apyrase activity was found
on non-EBV-transformed B lymphoma cell lines. Finally, expression of
CD39 cDNA in COS-7 cells increases their ecto-apyrase activity at least
5-fold.
CD39 is expressed on activated NK cells, B cells, and T cell
clones, but is not expressed by resting blood T, B, or NK cells,
neutrophils, or monocytes. CD39 expression in lymphoid tissue is
primarily limited to mantle zone and paracortical lymphocytes,
macrophages, and dendritic cells and is generally absent from germinal
centers(13) . Because CD39 encodes an ecto-apyrase, the
restriction of its expression to activated lymphoid cells and in
anatomical sites of ongoing B cell differentiation suggests that
ecto-apyrase activity may play an important role in immune responses.
One possible role of an ecto-apyrase in immune cells is to protect
them from potential lytic effects of extracellular ATP released by
their target cells(16, 25) . Extracellular ATP can
induce cell death in many immune cells and a few tumor cell lines.
ATP
binds to P
z purinergic receptors and
causes the opening of a nonselective membrane pore, which has a
molecular cut-off of approximately 0.9 kDa(26) . By measuring
ATP-induced uptake of extracellular markers of less than 0.9 kDa,
P
z receptors were found on most immune cells and some tumor
cell lines. Because of the relative high dose of extracellular ATP
needed for P
z receptor activation (half-maximal effective
concentration, EC
, varies from 100 µM to 1
mM), Di Virgilio (25) proposed that ``leakage
ATP'' from the cytoplasm of stressed or injured cells was the most
likely source for extracellular ATP and such an event was likely to
occur at the site of inflammatory or immune responses. Hydrolysis of
extracellular ATP by plasma membrane
ecto-(Ca
,Mg
)-apyrases leads to
closure of the P
z receptor pores(27) .
Interestingly, persistent treatment with low doses of interleukin-2 can
induce CD39 expression in T cell and NK cell in
vivo(15) ; in mouse splenocytes this treatment causes
resistance to extracellular ATP (28, 29, 30) .
Ecto-apyrase activities
have been found not only in the immune system, but also in many other
tissues. The broad distribution of ecto-apyrase activity suggests that
it may have essential functions. Unfortunately, the molecular structure
and function of ecto-apyrases in those tissues are largely unknown. At
least three hypotheses for the roles of ecto-apyrase have been
suggested. One function might be hydrolysis of ATP and other
nucleotides to terminates their roles as P
-purinergic
ligands (reviewed in (31) ). Secondly, ecto-apyrases may have a
principal role in the formation of AMP by hydrolysis of both ADP and
ATP(32) . Extracellular AMP can be converted by
ecto-5`-nucleotidase to adenosine, a ligand of P
receptors(33, 34) . Both ATP and adenosine
modulate a variety of key cellular processes in many tissues and
organs. A third possibility is the cooperation of ecto-apyrase and
5`-nucleotidase activities with a sodium-dependent adenosine
cotransporter in rat liver canalicular membranes to convert
extracellular ATP to intracellular adenosine leading to conservation of
the purine(9) . We wonder whether CD39 or similar genes may
correspond to ecto-apyrase activities in nonimmune tissues. Although
CD39 was also found in endothelium (13) and human placenta, (
)the expression pattern of CD39 in other nonimmune tissues
has not been established.
In summary, our results show the CD39
encodes an ecto-(Ca
,Mg
)-apyrase.
This is the first identification of a mammalian ecto-apyrase gene.
Further analysis of CD39 may help in the understanding of the structure
and function of other ecto-apyrases.