Cloning and characterization of mouse nucleoside triphosphate diphosphohydrolase-3

Article abstract of DOI:10.1016/j.bcp.2004.02.012

We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent cations (Ca ²⁺>Mg²⁺) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4) and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH 5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP according to Michaelis-Menten kinetics with apparent K_ms of 11, 10, 19 and 27 μM, respectively. In agreement with the K_m values, the pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.

Full text of DOI:10.1016/j.bcp.2004.02.012

Biochemical Pharmacology 67 (2004) 1917–1926
Cloning and characterization of mouse nucleoside triphosphate
diphosphohydrolase-3
*
´
Elise G. Lavoie, Filip Kukulski, Sebastien A. Levesque, Joanna Lecka, Jean Sevigny
´
´
´
Department of Anatomy and Physiology, Centre de recherche en Rhumatologie et Immunologie,
´ ´
Universite Laval, Sainte-Foy, Que., Canada G1V 4G2
Received 2 December 2003; accepted 12 February 2004
Abstract
We have cloned and characterized the nucleoside triphosphate diphosphohydrolase-3 (NTPDase3) from mouse spleen. Analysis
of cDNA shows an open reading frame of 1587 base pairs encoding a protein of 529 amino acids with a predicted molecular mass
of 58 953 Da and an estimated isoelectric point of 5.78. The translated amino acid sequence shows the presence of two transmembrane
domains, eight potential N-glycosylation sites and the five apyrase conserved regions. The genomic sequence is located on
chromosome 9F4 and is comprised of 11 exons. Intact COS-7 cells transfected with an expression vector containing the coding
sequence for mouse NTPDase3 hydrolyzed P2 receptor agonists (ATP, UTP, ADP and UDP) but not AMP. NTPDase3 required divalent
cations (Ca2þ > Mg2þ) for enzymatic activity. Interestingly, the enzyme had two optimum pHs for ATPase activity (pH 5.0 and 7.4)
and one for ADPase activity (pH 8.0). Consequently, the ATP/ADP and UTP/UDP hydrolysis ratios were two to four folds higher at pH
5.0 than at pH 7.4, for both, intact cells and protein extracts. At pH 7.4 mouse NTPDase3 hydrolyzed ATP, UTP, ADP and UDP
according to Michaelis–Menten kinetics with apparent Kms of 11, 10, 19 and 27 mM, respectively. In agreement with the Km values, the
pattern of triphosphonucleoside hydrolysis showed a transient accumulation of the corresponding diphosphonucleoside and similar
affinity for uracil and adenine nucleotides. NTPDase3 hydrolyzes nucleotides in a distinct manner than other plasma membrane bound
NTPDases that may be relevant for the fine tuning of the concentration of P2 receptor agonists.
# 2004 Elsevier Inc. All rights reserved.
Keywords: NTPDase3; ecto-ATPase; CD39L3; P2 receptors; ATP; UTP
1. Introduction
the extracellular milieu. These enzymes hydrolyze nucleo-
side triphosphates (e.g. ATP) and diphosphates (e.g. ADP)
with different ability. NTPDase1 (CD39) [5,6] hydrolyzes
both ATP and ADP equally well whereas NTPDase2
(CD39L1) [7,8] prefers triphosphonucleosides. NTPDase3
(also named CD39L3 and HB6 [9,10]) and NTPDase8 [11]
slightly prefer ATP over ADP by a ratio of about 3 and 2,
respectively. Other members of the E-NTPDase family are
associated with membranes of intracellular organelles
(NTPDases 4–7) and have different particularities regard-
ing, for example, substrate specificity and membrane
topology. NTPDase4 (hLALP70) [12–14] prefers UDP
as substrate and is anchored by two transmembrane
domains in the Golgi apparatus. NTPDase5 (CD39L4)
[15] and NTPDase6 (CD39L2) [16–18] have a preference
for nucleoside diphosphates and possess a single trans-
membrane domain near the N-terminus of the protein. The
first is bound to the endoplasmic reticulum and the second
to the Golgi apparatus. A soluble form of the two latter
Ecto-nucleoside triphosphate diphosphohydrolase
(E-NTPDase) designates a family of transmembrane pro-
teins that catalyze the hydrolysis of g and/or b phosphate
residues of nucleotides [1]. Members of this enzyme
family possess five characteristic apyrase conserved
regions (ACR) [2–4], one or two transmembrane domains
and they require divalent cations, such as Ca2þ or Mg2þ
for their activity [1].
Four members of the family are tightly bound to the
plasma membrane via two transmembrane domains, and
have a large extracellular region with an active site facing
,
Abbreviations: ACR, apyrase conserved region; EST, expressed
sequence tag; NTPDase, nucleoside triphosphate diphosphohydrolase;
RT, reverse transcription
* Corresponding author. Tel.: þ1-418-654-2772; fax: þ1-418-654-2765.
´
E-mail address: Jean.Sevigny@crchul.ulaval.ca (J. Sevigny).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.02.012

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1918
enzymes can be secreted after a proteolytic cleavage.
Finally, NTPDase7 (LALP1) [19], prefers nucleoside tri-
phosphates as substrates and is located in intracellular
vesicles.
2.2. RT-PCR cloning
Total RNA was isolated from mouse spleen with Trizol
reagent (Invitrogen). cDNA was synthesized with Super-
Script II (Invitrogen) from 500 ng of total RNA with oligo
(dT)18 as the primer, in accordance with manufacturer’s
instructions (Invitrogen). For amplification, 10% of the
reverse transcription (RT) reaction was used as template in
a final volume of 50 ml reaction mixture containing 0.6 mM
primer, 400 mM dNTP and 3.5 U Expand High Fidelity
PCR System (Roche). The following set of primers were
designed based on the 50 and 30 ends of EST sequences
(GenBank accession numbers BB644796 and W46136,
respectively): forward 50CTT-TTC-AGC-AAC-CCG-
CAG-C30, reverse 50TTC-CTG-CCA-GAG-CAC-CTC-
C30. Amplification was started with 2 min 30 s at 94 8C
followed by 35 cycles of 30 s denaturation at 94 8C, 1 min
annealing at 62 8C, 2 min primer extension at 72 8C and
ended by 7 min incubation at 72 8C. The PCR product of
approximately 1.8 kb was purified on agarose gel using the
QIAEX II gel extraction kit (Qiagen) and ligated into the
expression vector pcDNA3.1/V5-His (Invitrogen). Plasmid
DNAwas purified with QIAprep Spin Miniprep kit (Qiagen)
and orientation of the insert verified by restriction mapping.
Two independent fragments cloned from different PCR
reactions were amplified and fully sequenced in one direc-
tion. The sequences of both clones were identical.
Our laboratory studies the function of plasma membrane
bound NTPDases (members 1–3 and 8) using mouse
models. NTPDases 1, 2 and 8 have been cloned in human
and mouse while NTPDase3 has been cloned in human [9]
and recently in rat (personal communication from Dr. H.
Zimmermann). NTPDase3 (HB6) cDNA was originally
cloned from a human brain library [9]. Northern Blots
show strong expression of human NTPDase3 in brain,
pancreas, spleen and prostate, and weak expression in a
few other tissues [10]. Rat NTPDase3 mRNAwas found by
RT-PCR in PC12, a rat neural cell line [20]. The human
form has a molecular mass of 79 kDa with seven potential
N-glycosylation sites that are important for enzymatic
activity [21], especially the site near ACR1 [22]. Dr.
Kirley’s group in Cincinnati has demonstrated by directed
mutagenesis that the substitution of various amino acid
residues of human NTPDase3 abrogated phosphohydro-
lase activity and/or affected substrate specificity [23–26].
They have documented the relationship between the active
site of NTPDases with the nucleotide binding site of actin/
heat shock 70/sugar kinase superfamily [27]. They have
also shown that the transmembrane domains of human
NTPDase3 play a role in the formation of the quaternary
structure of the protein, which would be constituted by an
asymmetric dimer of dimers linked by a disulfide bond
between intracellular cysteine residues 10 [28]. In human
NTPDase1, the transmembrane domains also regulates
enzymatic activity [29] and substrate specificity [30].
This sums up most of the actual knowledge about human
NTPDase3. Orthologues from other species have not yet
been reported except for data based on ESTs and RT-PCR
of a fragment that would be expected to be the homologue
of human NTPDase3. These experiments suggested the
expression of NTPDase3 in rat [20] and mouse [10]. In this
work we have cloned NTPDase3 from mouse spleen and
have characterized its biochemical properties related to the
regulation of the concentration of P2 receptor agonists.
2.3. Genomic characterization of Entpd3
The obtained mouse NTPDase3 cDNA sequence was
used to search the National Center for Biotechnology Infor-
mation (NCBI) mouse genome database. The sequence
identified as NT_039482 revealed perfect identity. The
exon/intron junctions and their characteristics were ana-
lyzed with NCBI BLAST programs.
2.4. COS-7 cell transfection and protein preparation
COS-7 cells were transfected in 10 cm plates using
Lipofectamine (Invitrogen), as previously described [6].
Briefly, 60–80% confluent cells were incubated for 5 h at
37 8C in DMEM in absence of fetal bovine serum (FBS)
with 6 mg of plasmid DNA and 24 ml of Lipofectamine
reagent. Then, an equal volume of DMEM containing 20%
FBS was added and 40–44 h later cells were collected for
analysis. For protein preparation, transfected cells were
washed three times with Tris–saline buffer at 4 8C, har-
vested by scraping in 95 mM NaCl, 0.1 mM PMSF and
45 mM Tris, pH 7.5, and washed twice by centrifugation at
300 ꢀ g for 5 min at 4 8C. Cells were resuspended in the
harvesting buffer containing 10 mg/ml aprotinin and soni-
cated. Nuclear and cellular debris were discarded after
another centrifugation as described above, and resulting
supernatant was kept at ꢁ80 8C until use. Protein concen-
tration was estimated by the Bradford microplate assay,
2. Materials and methods
2.1. Materials
Agarose, aprotinin, ethylenediaminetetraacetic acid
(EDTA), ethylene glycol-bis(2-aminoethylether)-N-N-N0-
N0-tetraacetic acid (EGTA), sodium acetate, 2-(4-morpho-
lino)-ethane sulfonic acid (MES), nucleotides, phenyl-
methylsulfonyl fluoride (PMSF), sodium acetate,
tetrabutylammonium hydrogen sulphate(TBA)andTris(hy-
droxymethyl) aminomethane (Tris) were purchased from
Sigma-Aldrich. All cell culture media were obtained from
Invitrogen and 24-well plates from VWR Canlab.

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using bovine serum albumin as a standard of reference
[31].
for the first 20 min and then 1 ml/min up to 90 min. The
nucleotides were detected by UVabsorption at 260 nm and
identified by comparison with retention time and UV
spectrum of the appropriate standards.
2.5. NTPDase activity measurement
The protein extract’s enzyme activity was measured at
37 8C in 0.5 ml of the following incubation medium: 5 mM
CaCl2, 80 mM Tris, pH 7.4 as described previously [32].
Enzyme preparation was added to the incubation mixture
and pre-incubated for 3 min. Unless indicated otherwise,
reaction was started with 0.5 mM substrate (ATP, ADP,
UTP, UDP or AMP), stopped after 20 min by the addition
of 0.125 ml of malachite green reagent, and the inorganic
phosphate released during the hydrolysis of the exogenous
nucleotide measured [33]. The biochemical activities at the
surface of intact COS-7 cells transiently transfected with
NTPDase3 expression vector were evaluated in 24-well
plates. Activity assays were carried out in similar condi-
tions with the addition of 145 mM NaCl to the incubation
medium and stopped by sampling an aliquot of 0.2 ml and
rapid mixing with 50 ml of malachite green reagent. Opti-
mum pH was determined using the following buffers:
100 mM acetate for pH 4.0–5.5, 100 mM MES for pH
5.5–7.0 or 100 mM Tris for pH 7.0–9.0, in the presence of
5 mM CaCl2. In the indicated experiments MgCl2 replaced
CaCl2, and for controls, 1 mM EDTA plus 1 mM EGTA
were added instead of divalent cation to chelate residual
Mg2þ and Ca2þ. All experiments were performed in
triplicate with appropriate controls.
3. Results
3.1. Cloning and nucleic acid analysis of mouse
NTPDase3 cDNA
Mouse NTPDase3 cDNA was cloned by RT-PCR with
oligonucleotide primers designed from potential EST
sequences selected from a homology search with the
human cDNA sequence. The nucleic acid sequences of
the two independent clones obtained were identical. They
showed an open reading frame (ORF) of 1587 bp encoding
a protein of 529 amino acid residues with a predicted
molecular mass of 58 953.5 Da and an isoelectric point of
5.78. The deduced amino acid sequence reveals the five
ACRs, eight potential N-glycosylation sites, putative phos-
phorylation sites on the extracellular loop and one protein
kinase C consensus phosphorylation site in the intracellular
C-terminal segment at residue 511 (Fig. 1 and data not
shown). Hydropathicity analysis of mouse NTPDase3
amino acid sequence predicted the presence of two trans-
membrane domains, one at the N terminus between the
amino acid residues 21 and 43 and one at the C terminus
between residues 486 and 508 (Fig. 2). The amino acid
sequence alignment of 18 members of the E-NTPDase
family and related proteins with mouse NTPDase3 was
generated with GeneBee software (http://www.genebee.m-
su.su/). As expected, the highest homology was found with
rat NTPDase3 (94.3%), recently added to the data base,
and human NTPDase3 (81.3%; Fig. 3).
2.6. Separation and quantification of nucleotides by
HPLC
Enzymatic reaction was carried out as described above.
Aliquots of 20 ml from the reaction mixture were taken at
specific time points and transferred to a fresh tube contain-
ing an equal volume of ice-cold 1 M perchloric acid. The
samples were centrifuged for 5 min at 1000 ꢀ g. Super-
natants were neutralized with 1 M KOH (4 8C), centri-
fuged for 5 min at 1000 ꢀ g and then lipids were removed
by liquid-liquid extraction with n-heptane (5:1, v/v). The
nucleotide content of the samples was determined in 20 ml
aliquots separated by HPLC-RP under isocratic conditions.
Adenine nucleotides (ATP, ADP and AMP) were sepa-
rated on a 15 cm ꢀ 4:6 mm, 3 mm SUPELCOSILTM LC-
18-T column (Supelco) with a mobile phase composed of
25 mM TBA, 5 mM EDTA, 100 mM KH2PO4/K2HPO4,
pH 7.0 and 2% (v/v) methanol, at a flow-rate of 1 ml/min.
Uracil nucleotides (UTP, UDP and UMP) were resolved
using SUPELCOSILTM LC-18-T column (25 cm ꢀ
4:6 mm, 5 mm, Supelco) as described above with the
difference that the mobile phase did not contain methanol.
Combined adenine and uracil nucleotide samples were
analyzed with the latter column and a mobile phase
composed of 16.7 mM TBA, 3.3 mM EDTA, 66.7 mM
KH2PO4/K2HPO4, pH 7.0. The flow rate was 0.5 ml/min
3.2. Genomic characterization of Entpd3
A homology search in the Mouse Sequence Data
Base (www.ncbi.nih.gov/genome/seq/MmBlast.html) with
NTPDase3 cDNA located the gene to chromosome 9F4
(entry NT_039482). Alignment of the cDNA sequence
with the mouse genomic sequence reveals that the gene
covers over 26.9 kb and is organized into 11 exons and 10
introns, all in agreement with the donor/acceptor rule. The
precise length of the first (non-coding) exon as well as three
introns can not be measured at the moment (Table 1). Fig. 4
and Table 1 summarize the genomic structure of mouse
Entpd3.
3.3. Biochemical characterization of mouse NTPDase3
The biochemical characteristics of mouse NTPDase3
were determined with crude protein extracts, and/or with
intact COS-7 cells transiently transfected with pcDNA3.1/
V5-His containing mouse NTPDase3 cDNA. The time

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Fig. 1. Nucleotide and predicted amino acid sequence of mouse NTPDase3 cDNA. The initiation codon and stop codon (indicated by ꢂ) are bold face,
potential N-glycosylation sites are represented by ‘‘N’’ surrounded by an hexagon, two hydrophobic regions are double underlined and the five apyrase
conserved regions (ACR 1–5) are boxed. The two oligonucleotide primers used for PCR are indicated by dotted lines and the 50 (1–2) and 30 (1810–1965)
non-coding sections presented were obtained from mouse EST sequences, GeneBank accession number BB644796 and W46136, respectively.

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1921
Fig. 2. Hydropathicity analysis of mouse NTPDase3. The deduced amino acid sequence was analyzed according to the method of Kyte and Doolittle [39].
Bars indicate the two predicted transmembrane domains (TM) corresponding to amino acids 21–43 and 486–508.
course study with protein extracts revealed that the reaction
was linear for the first 60 min with either ATP or ADP as
substrate (Fig. 5A). The following assays were carried out
for 15–20 min. Murine NTPDase3 required divalent
cations for enzymatic activity, Ca2þ being more effective
than Mg2þ as tested for ATP and ADP (Fig. 5B). In the
presence of 1 mM EDTA and 1 mM EGTA, to remove
traces of divalent cations, no activity could be detected
(Fig. 5B). Fig. 5C shows that ATPase activity of NTPDase3
had two maxima, one at pH 5.0 and a second at pH 7.4. In
contrast, using the plasmids kindly provided by Dr. Kirley
and Dr. Zimmerman, a single maximum was observed at
physiological pH for both the human and rat orthologues
(data not shown). With ADP as substrate, the biochemical
activity was relatively similar between pH 5.5 and 9.0 with
a maximal activity at pH 8.0 that was statistically sig-
Fig. 3. Phylogenetic analysis of amino acid sequences of selected NTPDases and related proteins. The percentage of amino-acid identity of these proteins
with mouse NTPDase3 was determined by pairwise alignment using ALIGNp (http://www.infobiogen.fr/services/analyseq/cgi-bin/alignp_in.pl). The
GeneBank accession numbers of the sequences are as follows: mouse NTPDase3, AY376710; rat CD39L3, NM_178106; human HB6, AF034840; chicken
ecto-ATPDase, AF041355; mouse NTPDase8, AY364442; mouse NTPDase2, AY376711; rat ecto-ATPase, Y11835; human CD39L1, NM_001246; chicken
ecto-ATPase, U74467; mouse CD39, AF037366; human CD39, S73813; human LALP1, AF269255; human UDPase, AF016032; pea apyrase, Z32743;
potato apyrase, U58597; human CD39L4, AF039918; rat NTPDase6, AJ277748; human CD39L2, AF039916; Toxoplasma gondii apyrase, U96965.

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Table 1
Summary of the genomic structure of mouse Entpd3
Exon Position in
NT_039482.2
Splice acceptor
Length of
Splice donor
Length
of intron
(bases)
Nucleotide position of
important sequences
in current exon
exon (bases)
1
10888830–10888855
na
>66
AGCGCCCAG/gtaaggtct
768
Promoter ꢁ227
to ꢁ150
2
3
10889624–10889675
10893020–10893147
10903396–10903513
10904817–10904967
10906564–10906723
10907508–10907741
10909653–10909925
10910093–10910203
10911071–10911208
10915478–10915711
cccctgcag/CCCGGAGAG
tcattgcag/GCTTCAGGG
tccctgcag/TATGGCGTC
gctcttcag/GCTCTGGGA
cttgcatag/GTTGCAGAA
actatgcag/AAGAACCTG
tacctccag/AGCCCTTCC
cgctcacag/GCGTTCGCA
cctttccag/CTCCCAGCC
cctccacag/GTGGGGAAC
52 (from Met 40)
AACAGGCAG/gtaagtgtt
GGGCTGAAG/gtaagccgg
GTGTGAAAG/gtaaagggg
CTTGCTGAG/gtaagggct
TTCCTGGAG/gtgtgtgga
CTTCTACAG/gtaccaggg
CCATTTGTG/gtaagaggc
TGGAGCGAG/gtcagtatt
GAAAAAGAA/gtaagtgca
TGA
ꢄ3344
ꢄ10 248
ꢄ1303
1596
784
Met 13–15
128
4
118
ACR1* 16–39
ACR2 126–143
ACR3 98–136
ACR4 58–81
5
151
6
160
7
234
1911
167
8
273
9
111
867
10
11
138
4269
Up to TGA 234
ACR5 22–33
TGA 235-237
polyA 1187 bp
Introns þ
exons >26 922 after TGA
Sizes and junctions of the exons and introns of mouse Entpd3 are presented according to the sequence of accession No. NT_039482.2. Intron sequences are given in small
characters and exons in capitals. As the gene has not been fully sequenced yet the length of the introns between exons 2–3, 3–4 and 4–5 can not be measured with precision at
this point.
*
ACR: Apyrase conserved region; na: not available.
nificant (P < 0:01). The specificity of substrate for calcium
dependent ecto-nucleotidase activity on intact transiently
transfected COS-7 cells is illustrated on Fig. 5D for the two
pHs that showed the highest ATPase activity (Fig. 5C).
Mouse NTPDase3 hydrolyzed all triphospho- and dipho-
sphonucleosides tested with a preference for triphospho-
nucleosides. As with all other NTPDases, this enzyme did
not hydrolyze AMP (Fig. 5D). Interestingly, the NTP/NDP
hydrolysis ratio changed with pH. At pH 7.4 the hydrolysis
ratios measured with intact transfected cells were 2:7 ꢃ 0:8
(mean ꢃ S:D:) and 5:4 ꢃ 1:6 for ATP/ADP and UTP/UDP,
respectively. These ratios were significantly increased to
3:6 ꢃ 0:9 and 14:5 ꢃ 5:4 at pH 5.0 with P values <0.001.
With protein extracts, the ATPase and UTPase activities
were two fold higher at pH 5.0 compared to pH 7.4, making
a slightly more pronounced change between the hydrolysis
ratios at these two pHs (Fig. 5C and data not shown). These
higher ATPase and UTPase activities at pH 5.0 with protein
extracts, when compared to the activity measured on intact
transiently tranfected cells, were observed in all experi-
ments. This may be attributable to the contribution of
newly synthesized and immature NTPDase3 in the protein
extracts.
To confirm the tight association of mouse NTPDase3 to
a membrane fraction, we performed an ultracentrifugation
of the protein extract at 100 000 ꢀ g for 1 h. Over 87% of
ATPase and ADPase activities were found in the pellet
(data not shown). The above observation, together with the
fact that biochemical activity was detected at the cell
surface of NTPDase3 transfected COS-7 cells, confirmed
that mouse NTPDase3 is an ectoenzyme tightly bound to
the plasma membrane.
3.4. Kinetics of mouse NTPDase3
The kinetic parameters for the substrates that are also the
agonists of P2 receptors were evaluated. Plotting the initial
velocities as a function of substrate concentration resulted
in a typical hyperbolic curve of the Michaelis–Menten
model (Fig. 6A for UTP and data not shown for UDP, ATP
and ADP). The apparent kinetic constants were evaluated
using the method of Woolf–Augustinsson–Hofstee and are
summarized in Table 2. The comparison of apparent Km
values showed that ATP and UTP had two times higher
affinity for the enzyme compared to their respective dipho-
sphonucleoside (Table 2 and Fig. 6A).
Fig. 4. Schematic representation of the genomic organization of mouse Entpd3. The gene contains 11 exons spanning over 26.9 kb on mouse chromosome 9
band F4. Start and stop codons, ACRs 1–5 as well as polyA signal are indicated on the cDNA. Exons are presented by black boxes on the genomic DNA
(gDNA). Further information is given in Table 1.

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Fig. 5. Biochemical characterization of mouse NTPDase3. Enzyme activity assays of protein extracts from NTPDase3 transfected COS-7 cells were carried
out in 5 mM CaCl2, 0.5 mM nucleotides, 100 mM Tris, pH 7.4 unless stated otherwise (panels A and C). For intact cells 145 mM NaCl was added to the
incubation medium (panel D). In these experiments, less than 5% of the substrate was hydrolyzed. ATPase and ADPase activities were assayed. For panel A, a
representative experiment out of two independent experiments is shown and for panels B–D, results are expressed as the mean ꢃ S:D: of two to five
independent experiments. Each individual experiment was performed in triplicate. (A) Time course of ATP and ADP hydrolysis by NTPDase3. Linear
regression gave a similar r2 of 0.98 for both ATP and ADP. (B) Effect of calcium (Ca2þ) and magnesium (Mg2þ) ions. NTPDase3 was more active in the
presence of Ca2þ (solid symbols and full lines) than in presence of Mg2þ (open symbols and dotted lines) with the ATPase (~) activity being higher then
ADPase (&) activity for both cations. (C) Effect of pH. Three different buffers were used: acetate: pH 4.0–5.5 (&); MES: pH 5.5–7.0 (~); Tris: pH 7.0–9.0
(*), solid symbols and full lines for ATP and open symbols and dotted lines for ADP. Activities for both ATP (at pHs 5.0 and 7.4) and ADP (at pH 8.0) were
significantly higher than the activities measured at the surrounding pHs (P < 0:01). (D) Substrate specificity of NTPDase3 expressed at the surface of intact
transiently transfected cells at pH 5.0 (filled bars) and 7.4 (open bars).
HPLC analysis of ATP and UTP hydrolysis by protein
extracts from NTPDase3 expressing COS-7 cells showed a
transient accumulation of the corresponding diphosphonu-
cleoside (ADP or UDP; Fig. 6B and C). Similar data were
observed with intact transfected cells (data not shown).
Finally, when ATP and UTP were added together to the
incubation medium, similar rates of hydrolysis were
observed for both adenine and uracil nucleotides (Fig. 6D).
reveals the presence of 529 amino acid residues as for
human (HB6) [9] and rat (sequence recently added to the
database) orthologues (Fig. 1 and data not shown). The
amino acid identity of mouse NTPDase3 with these two
proteins is over 80% (Fig. 3). Various sites are conserved
between these three enzymes including N-glycosylation
and putative phosphorylation sites. The potential N-gly-
cosylation sites are all conserved with the exception that
mouse NTPDase3 has an additional potential site on
residue 318 (Fig. 1). Protein glycosylation is important
for the catalytic activity of human NTPDase3 [21], espe-
cially on asparagine 81 near ACR1 [22]. The analysis of
the amino acid sequence of these proteins reveals a putative
protein kinase C phosphorylation site in the C-terminal
segment of mouse NTPDase3 (serine 511), and on HB6
(serine 512) [9]. The rat orthologue does not possess any
phosphorylation site in the intracellular portion of the
protein. A protein kinase C phosphorylation site is also
present in the intracellular domain of other plasma mem-
brane bound NTPDases: human [7] and mouse NTPDase2
4. Discussion
This work describes the cloning and biochemical char-
acterization of mouse NTPDase3. The genomic localiza-
tion of mouse Entpd3 on locus 9F4 confirmed that
the protein expressed is distinct from other NTPDases.
The human orthologue is located on chromosome 3 [10],
the homologous region to mouse chromosome 9. Mouse
Entpd3, as for ENTPD3, is comprised of 11 exons (Fig. 4
and data not shown). The open reading frame of its cDNA

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E.G. Lavoie et al. / Biochemical Pharmacology 67 (2004) 1917–1926
1924
Fig. 6. Kinetics and profile of nucleotides hydrolysis by mouse NTPDase3. For panel A, reaction was carried out for 12 min in the presence of 5 mM CaCl2
and 100 mM Tris pH 7.4. The average of three separate experiments each performed in triplicate is presented. Graphs were produced using GraphPad Prism
software (GraphPad Software Inc.). For the analysis of ATP and UTP hydrolysis products (panels B and C), the reaction was started by the addition of 32 mg
of NTPDase3 protein extract to the reaction mixture containing 0.5 mM substrate. For the analysis of the combined dephosphorylation of ATP and UTP
(panel D), the reaction was initiated with 78 mg of protein extract. Aliquots were taken at different time points and reaction stopped by the addition of an
equal volume of ice-cold 1 M perchloric acid. These samples were deproteinated, lipids removed and the nucleotide content analyzed by HPLC as described
in Section 2. (A) Michaelis–Menten representation of the hydrolysis of UTP with concentrations ranging from 5 to 200 mM. Inset, Woolf–Augustinsson–
Hofstee plot was used to evaluate the apparent Km and Vmax using the points in the linear portion of the curve in the Michaelis–Menten representation
(5–50 mM). The kinetic parameters are presented in Table 2. (B) Nucleotide content of samples collected at different time points of ATP hydrolysis by murine
NTPDase3 protein extracts: ATP (~), ADP (&), AMP (*). (C) UTP hydrolysis by NTPDase3 protein extracts: UTP (~), UDP (&), UMP (*).
(D) Simultaneous hydrolysis of ATP and UTP by NTPDase3: ATP (~), ADP (&), AMP (*), UTP (~), UDP (&), UMP (*).
have one in the C-terminal portion (serine 488), whereas
human and mouse NTPDase8 possess one in the N-term-
inal portion (serine 4) [11]. Whether phosphorylation plays
a role in NTPDase functions is not known.
We observed that mouse NTPDase3 was tightly asso-
ciated with the plasma membrane with an active site facing
the extracellular milieu. The highest homology of this
protein was observed with the other NTPDases bearing
the same characteristics: mammalian NTPDases 1, 2 and 8
and the two related proteins from chicken described as an
ATPase [34] and an ATPDase [35] (Fig. 3). The biochem-
ical characteristics of NTPDases 1 and 2 have been largely
documented while NTPDase8 has been identified only
recently [11]. Similar to NTPDase1 and 8, but in contrast
to NTPDase2, NTPDase3 prefers Ca2þ over Mg2þ for both
ATPase and ADPase activities (Fig. 5B). One of the
particularities of mouse NTPDase3 is a biphasic curve
for ATPase activity as a function of pH with a first
maximum at pH 5.0 and a second at 7.4 (Fig. 5C). In
comparison, the optimal pH of human and rat orthologues
showed a single maximum at pH 7.5 for ATPase activity
(data not shown). Whether this reflects some variation in
the amino acid composition of the catalytic site remains to
be elucidated. Interestingly, mouse NTPDase3 had only
one peak of activity at pH 8 with ADP as substrate.
Consequently, this characteristic affected the ATP/ADP
and UTP/UDP hydrolysis ratios, which were increased by
two to four folds between physiological to acidic pHs. At
more alkaline pHs the ATP/ADP hydrolysis ratio was
close to 1 for protein extracts (Fig. 5C). This may affect
the control of extracellular nucleotide levels according to
the pH and location of the enzyme.
Table 2
Kinetic parameters of mouse NTPDase3
Substrate
Km (mM)
Vmax (U/mg of protein)
ATP
UTP
ADP
UDP
11 ꢃ 2
10 ꢃ 1
19 ꢃ 2
27 ꢃ 2
0.35 ꢃ 0.02
0.30 ꢃ 0.01
0.22 ꢃ 0.01
0.14 ꢃ 0.01
Km and Vmax values were estimated by regression analysis of Woolf–
Augustinsson–Hofstee plots. Results are expressed as the mean ꢃ S:E:M.
The curves drawn from these data gave an r2 of 0.97, 0.99, 0.98 and 0.99
for ATP, UTP, ADP and UDP, respectively.

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1925
The hydrolysis of ATP and UTP by mouse NTPDase3
showed a transient accumulation of the corresponding
diphosphonucleoside (ADP and/or UDP). When the level
of triphosphonucleoside(s) had sufficiently decreased, then
AMP or UMP accumulated in the medium (Fig. 6B and D).
This is in agreement with the measured kinetic constants of
mouse NTPDase3. The apparent Km for ATP and UTP
were half as much as the corresponding diphosphonucleo-
side (Table 2). In comparison, NTPDase1 hydrolyzes ATP
to AMP, one Pi at a time, without any accumulation of
ADP. In contrast, NTPDase2 hydrolyzes preferentially
ATP to ADP, the latter being a poor substrate of the
enzyme. The pattern of hydrolysis of NTPDase3 is some-
what in between the one of NTPDase1 and NTPDase2 [8].
For NTPDase1, the apparent Kms are similar for ATP and
ADP, in the order of 10 mM [36,37]. For human NTPDases
2 and 3 these constants are in the high micromolar range.
Hence, for intact NIH-3T3 cells stably transfected with
NTPDase2, the apparent Kms were around 400 mM for ATP
and 100 mM for ADP with apparent Vmaxs of 107 and of
4 nmol Pi/min per 106 cells, respectively [7]. The apparent
Kms of human NTPDase3 for ATP and ADP, evaluated
from protein extracts of transiently transfected COS-1
cells, were 128 and 96 mM, respectively, with apparent
Vmaxs of 2.0 and 0.5 mmol Pi/min/mg of protein [27].
The plasma membrane bound NTPDases 1–3 and 8
control the nucleotide levels in extracellular spaces, and
therefore, modulate P2 receptor signaling. The difference
in the biochemical and kinetic properties of these ectonu-
cleotidases may have profound physiological effects. The
affinity of the substrates toward the enzymes, the specific
activity and the products formed may determine distinct
roles for each plasma membrane bound NTPDase. For
example, we have recently shown that the different hydro-
lysis patterns of NTPDase1 and NTPDase2 dictated their
opposing roles in the control of platelet aggregation, as
demonstrated in vitro [38]. The AMP generated by
NTPDases, can then be further hydrolyzed by the ecto-
50-nucleotidase to adenosine. Thus, the hydrolysis products
of ATP (ADP and adenosine) react with different subtypes
of P2 and P1 receptors and trigger different physiological
actions.
Research (CIHR; MOP-49460 and M2C-50334) and from
´ ´
the ‘‘Fonds de la Recherche en Sante du Quebec’’ (FRSQ;
2822) to J.S.
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We thank Mrs. Julie Pelletier and Mr. Franc¸ois Bigon-
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