Distribution, cloning, and characterization of porcine nucleosidetriphosphate diphosphohydrolase-1

Raf Lemmens1, Luc Vanduffel1, Agnes Kittel2, Adrien R. Beaudoin3, Ouhida Benrezzak3 and Jean Se vigny4

1Department of Medische Basiswetenschappen, Limburgs Universitair Centrum, Diepenbeek, Belgium; 2Institute of Experimental Medicine,

Budapest, Hungary; 3DeÂp. de Biologie, Universite de Sherbrooke, QueÂbec, Canada; 4Department of Medicine, Beth Israel Deaconess Medical

Center, Harvard Medical School, Boston, MA, USA

In this study, we have investigated the distribution of the enzyme nucleoside triphosphate diphosphohydrolase-1

(NTPDase1; EC 3.6.1.5) in a subset of pig tissues by biochemical activity and Western blotting with antibodies

against porcine NTPDase1. The highest expression of this enzyme was found in vascular endothelium, smooth

muscle, spleen and lung.

The complete cDNA of NTPDase1 from aorta endothelial cells was sequenced using primer walking. The

protein consists of 510 amino acids, with a calculated molecular mass of 57 756 Da. The amino-acid sequence

indicated seven putative N-glycosylation sites and one potential intracellular cGMP- and cAMP-dependent

protein kinase phosphorylation site. As expected, the protein has a very high homology to other known

mammalian ATPDases and CD39 molecules, and includes all five apyrase conserved regions.

Expression of the complete cDNA in COS-7 cells confirmed that NTPDase1 codes for a transmembrane

glycoprotein with ecto-ATPase and ecto-ADPase activities. Two proteolytic products of NTPDase1, with

molecular mass of 54 and 27 kDa, respectively, were consistently present in proteins from transfected COS-7

cells and in particulate fractions from different tissues. A trypsin cleavage site, giving rise to these two cleavage

products, was identified. In order to remain enzymatically active, the two cleavage products have to interact by

non±covalent interactions.

Keywords: NTPDase1; CD39; ATPDase; cloning; distribution.

E-type ATPases are a family of enzymes, hydrolyzing a widerange of extracellular nucleoside tri- and diphosphates. Theseenzymes are dependent on divalent cations for their activity.They were initially subdivided in two groups: the ecto-ATPases(EC 3.6.1.15) and the ecto-ATP diphosphohydrolases (ATPDasesor apyrases: EC 3.6.1.5). The former hydrolyze mainly nucleo-side triphosphates while the latter are capable of hydrolyzingboth nucleoside tri- and diphosphates [1].

In recent years, it has been demonstrated that mammalianmembrane associated ATPDase was homologous to humanCD39, a lymphoid cell activation antigen [2±6]. Furthercharacterization of cloned members of proteins related toCD39, allowed the suggestion of a unifying nomenclature. Allmembers of the CD39-ATP diphosphohydrolase family belongto the E-NTPDase family [7]. Several functions for theseectonucleotidases have been proposed which are mainly relatedto the regulation of P2-signalling systems by the combinedaction of E-NTPDases and ecto-5 0-nucleotidase [1,8].

The cDNA of human NTPDase1 encodes a 510 amino-acidintegral membrane protein of 57 kDa, with six potential

N-linked glycosylation sites, 11 cysteine residues and twomembrane spanning regions: one from amino acid 14±36 andthe other from amino acid 478±500 [2]. The protein has shortintracellular N- and C-terminal ends and a large extracellularloop. E-NTPDases share five apyrase conserved regions(ACR1±5) [9±11].

The type-I ATPDase, described by SeÂvigny et al. [12] inporcine pancreatic tissue, is a truncated form of NTPDase1(CD39), starting just before ACR4 [4,13]. However, as itappears that ACR1, 4 and 5 are essential for enzymatic activity[11,14], the NTPDase activity of type I ATPDase is intriguing.One goal of the present study was to further characterize thistype-I ATPDase and other modifications of porcine CD39. Wecompared the N-terminal amino-acid sequences of type-IATPDase (54 kDa) [12] and CD39 from kidney (78 kDa)[15] with the full length cDNA sequence. Our analysis alsoinvolved the use of specific antibodies directed against differentparts of porcine CD39.

Another goal of the present study was to examine thedistribution of porcine NTPDase1 in a variety of tissues. Thecomparison of the biochemical ATPase and ADPase activitieswith the expression of NTPDase1 revealed the presence ofother related enzymes in different tissues.

M A T E R I A L S A N D M E T H O D S

Materials

Salts, buffers and nucleotides were obtained from Sigma(Bornem, Belgium). Oligomycin, levamisole and ouabain(octahydrate) were from Janssen Chimica (Beerse, Belgium).

Eur. J. Biochem. 267, 4106±4114 (2000) q FEBS 2000

Correspondence to L. Vanduffel, Limburgs Universitair Centrum,

Department of MBW, Universitaire Campus; Building D, B-3590

Diepenbeek, Belgium. Fax: 1 32 11 268599, Tel.: 1 32 11 268556,

E-mail: luc.vanduffel@luc.ac.be

Abbreviations: NTPDase, nucleoside triphosphate diphosphohydrolase;

ATPDase, ATP diphosphohydrolase; ACR, apyrase conserved region;

AMV, avian myeloblastoma virus; DAB, 3,3 0-diaminobenzidine; DMEM,

Dulbecco's modified Eagle medium; HRP, horseradish peroxidase;

PNGase F, peptide N-glycosidase F.

Enzymes: NTPDase1, ecto-ATP oliphosphohydrolases, ATPDases or

apyrases (EC 3.6.1.5).

(Received 7 April 2000, accepted 3 May 2000)

Immobilon-P membranes were purchased from Millipore(Bedford, MA, USA). Cell culture media, Lipofectaminereagent and G418 (geneticin) were bought from Life Tech-nologies Inc. (Merelbeke, Belgium). Chromatography mediawere from Pharmacia (Roosendaal, the Netherlands). Reagentsfor SDS/PAGE and immunoblotting were obtained fromBio-Rad Laboratories (Nazareth, Belgium). Glutaraldehyde andDurcupan were from Fluka Chemie AG (Buchs, Switzerland).Goat anti-(rabbit IgG) Ig or goat anti-(mouse IgG) Igconjugated to horseradish peroxidase were bought from Pierce(Rockford, IL, USA). Biotin-labeled horse anti-mouse secondaryIgs, diaminobenzidine (DAB) and the avidin-biotin kit camefrom Vector Laboratories (Burlingame, CA, USA), horseradishperoxidase ABC complex was purchased from Dako (Glostrup,Denmark) and the Renaissance Chemiluminescence reagentplus was bought from NEN (Boston, MA, USA). All PCR-reagents were from Perkin Elmer (Zaventem, Belgium). PCRprimers were synthesized by Life Technologies Inc. (Merel-beke, Belgium). Vectors and cloning kits were bought fromInvitrogen (Leek, the Netherlands). Restriction enzymes andthe 5 0-RACE kit were purchased from Boehringer-Mannheim(Mannheim, Germany). The RNEasy total RNA extraction kitwas obtained from Qiagen (Leusden, the Netherlands), andAMV reverse transcriptase was from Promega (Leiden, theNetherlands).

Preparation of particulate fractions

Particulate fractions from young pigs were prepared aspreviously described [16]. Briefly, homogenates were filteredthrough cheesecloth, centrifuged for 15 min at 700 g and thesupernatants were centrifuged for 1 h at 100 000 g in a SW41Beckman rotor. The pellets were resuspended, loaded on a 40%(w/v) sucrose cushion and centrifuged for 90 min at 100 000 gin a Beckman SW41. The fluffy layers were recovered andwashed twice with 1 mm NaHCO3, 0.1 mm PMSF. Eachwashing was followed by a 50-min centrifugation at 150 000 g.The final pellets were resuspended in 7.5% glycerin (v/v)and 5 mm Tris/HCl, pH 8.0 at a concentration of2±5 (mg protein)´ml21. Enzyme activity was measured thesame day.

Determination of NTPDase1 activity

Enzyme assays on homogenate and particulate fractions werecarried out at 37 8C in 1 mL of 8 mm CaCl2, 5 mm tetramisole,50 mm imidazole, 50 mm Tris/HCl (pH 7.5). Tetramisole wasused as an inhibitor of alkaline phosphatase. Reaction wasstarted by adding 0.2 mm of the substrate (ATP or ADP) andstopped with 0.25 mL of malachite green reagent. Inorganicphosphate was estimated according to Baykov et al. [17].Enzyme activity was expressed as nmoles of inorganic phos-phate released per min per mg of protein, which corresponds tomU [18]. Protein was estimated by the method of Bradford [19]using bovine serum albumin as a standard.

Enzyme activity measurements of transfected cells in96-well microtiter plates were carried out as follows. Cells wereinitially washed three times with iso-osmotic Tris-bufferedsaline (150 mm NaCl in 0.1 m Tris, pH 7.5). The NTPDase1activity was determined by adding to each well 50 mLiso-osmotic reaction mixture, containing 1 mm ouabain,0.1 mg´mL21 Concanavalin A, 1 mm N-ethylmaleimide,5 mg´mL21 oligomycin, 1 mm levamisole, 100 mm sodiumvanadate, 100 mm NaCl, 5 mm KCl, 2 mm ATP, 2 mm CaCl2and 0.1 mm EDTA in 30 mm Tris/HCl pH 7.5. In some

experiments, CaCl2 and EDTA were replaced by MgCl2 andEGTA, and ATP by any other NTP or NDP. After 30 minincubation at 37 8C, the reaction mixtures were transferred toanother 96-well microtiter plate containing 150 mL of 0.25%(w/v) CuSO4, 1% (w/v) SDS in 4.6% (w/v) sodium acetatepH 4.0. Inorganic phosphate, produced during the incubation,was measured according to the method of LeBel et al. [20].

Antibodies

The preparation of monoclonal antibodies has previously beendescribed [21]. Briefly, mice were immunized with a partiallypurified protein fraction from porcine kidney NTPDase1 andthe hybridomas were screened using an ATPDase capture assay.A number of different clones were generated and furthercharacterized by Western blot for their interaction withNTPDase1 (78 kDa protein) under both reducing andnonreducing conditions. Monoclonal antibody 4D11 onlyworks under nonreducing conditions and reacts with a fragmentof NTPDase1 corresponding to the 54-kDa type-I ATPDasesuggesting the presence of an epitope between amino acids202±510 in porcine CD39. The monoclonal antibody 1H12detects the N-fragment of the protein corresponding to the27-kDa fragment (amino acids 1±201) under both reducing ornonreducing conditions. These antibodies have been describedpreviously [15]. The rabbit polyclonal antibody raised against a16-amino-acid peptide derived from the N-terminus of theporcine pancreatic type-I ATPDase (KSDTQETYGALDLG-GA) has been characterized before [22]. The latter antibodyworks under reducing and nonreducing conditions. Apart fromreacting with the 78-kDa band, all antibodies also interactedwith either a 54-kDa or a 27-kDa protein band.

Electrophoresis and immunoblotting

Polyacrylamide gel electrophoresis was carried out with orwithout 1% (v/v) 2-mercaptoethanol according to Laemmli [23](reducing or nonreducing conditions, respectively) and theproteins were transferred to poly(vinylidene difluoride)membranes by electroblotting [24].

cDNA synthesis and sequencing

Porcine aortas were obtained from a local slaughterhouse.Endothelial cells were collected by scraping the intima andincubated in M199 medium with 0.2% (w/v) collagenase for20 min at 37 8C. After a first passage the cells were trypsinized,total RNA was extracted and reverse transcribed. The cDNAwas precipitated with 3 m sodium acetate in ice cold ethanoland resuspended in 35 mL of RNAse free water.

Based on the N-terminal amino-acid sequence and thehomology to bovine and human CD39, forward (5 0-AAA-CAAAAGCTGCYMSTTATGGAAG-3 0) (position 218 to 17)and reverse (5 0-GCGAGTTTRCTCACTCCAGCCAG-3 0)(position 1535±1557) primers were designed. PCR amplifica-tion was performed with 0.2 pmol of both primers, 0.7 mLcDNA, 2.5 mL 10 � PCR buffer, 0.25 mL 100 � dNTP mixand 0.175 mL Taq polymerase (5 U´mL21 ampliTaq DNApolymerase, Perkin Elmer) in a total volume of 25 mL. Theobtained PCR amplicons were analyzed on a 0.8% agarose gel.The band with the correct length was purified on a Sephadex-G50M column. The DNA sequences were evaluated on a 6%polyacrylamide gel using the 373 ABI DNA sequencer (Perkin-Elmer) and total sequence analysis was performed by primerwalking.

q FEBS 2000 Porcine NTPDase1 (Eur. J. Biochem. 267) 4107

To obtain the sequence of the 5 0-end of the porcine CD39cDNA, a 5 0-RACE kit was used. PCR amplicons were ligated inthe pCR2.1 TOPO vector (Invitrogen) and transformed by heatshock into competent Escherichia coli TOP10. Plasmid DNAwas prepared from 9 recombinant clones and inserts of thecorrect length were identified.

Preparation of NTPDase1 constructs

cDNA fragments were PCR-amplified, using a forward primerlinked to a HindIII restriction site and a reverse primer linked toa BamHI site. A first set of primers was designed to amplify thecomplete coding cDNA sequence for NTPDase1. In a secondset a reverse primer was designed with an in-frame stop codonafter amino acid 201. This resulted in a cDNA expressing onlythe first 201 amino acids and thus containing ACR1±3. For theexpression of the last 309 amino acids a forward primer wasdesigned with an in-frame start codon just before amino acid202. This corresponds to the coding sequence of type-IATPDase. The obtained PCR fragments were cloned intothe pCR2.1 TOPO vector according to the manufacturer'sinstructions (Invitrogen).

COS-7 cell transfection and membrane preparation

COS-7 cells were grown in complete DMEM and weretransfected as previously described [4]. Briefly, cells wereincubated for 5 h at 37 8C with 2 mg plasmid DNA,preincubated for 45 min with 6 mL Lipofectamine reagent.The incubation was stopped by adding fetal bovine serum andafter a further 24 h incubation, the medium was replaced and72 h later the cells were either tested or trypsinized. Thetrypsinized cells were plated on new plates, and 400 mg´mL21

G418 (geneticin) was added for several weeks to obtain a stableNTPDase1-expressing cell line.

To prepare membrane proteins, cells were sonicated 42 hafter transfection, in about 10 volumes of 95 mm NaCl, 0.1 mmPMSF, 10 mg´mL21 aprotinin in 45 mm Tris, pH 7.6 at 4 8Cand the homogenate was centrifuged for 5 min at 300 g. Parti-culate fractions were prepared by centrifuging aliquots of thesupernatant, containing 50 mg of protein, for 1 h at 150 000 g.

Electron microscopical enzyme histochemistry

Transfected COS-7 cells were grown on eight-chamber slides.Sixty hours after transfection, the cells were washed with

Table 1. Distribution of ATPase and ADPase activities in porcine tissues. The results are expressed as average ^ standard error of the mean. Each

experiment was performed in triplicate and the variation between triplicate measurements was less than 5%. A low level of activity of less then

3 (nmol Pi)´min21´mg21 for both ATP and ADP as substrate was detected on the membrane fraction of red blood cells. The number of experiments on each

particular tissue is shown in parentheses.

Activity [(nmol Pi)´min21´mg21]

Homogenate Particulate fraction

% inhibition by

10 mm sodium azide

% inhibition by

10 mm sodium azide

Tissue ATP ADP ATP ADP ATP ADP ATP ADP

Circulatory system

Aortic endothelial cells 106 ^ 27 102 �^ 26 55 �^ 2 69 �^ 1 1962 �^ 643 1553 �^ 530 54 �^ 1 70 �^ 2

Aorta (3) 41 ^ 17 14 �^ 3 41 �̂ 2 45 �^ 15 295 �^ 79 182 �^ 43 31 �^ 4 40 �^ 9

Vena cava (3) 26 ^ 6 17 �^ 3 48 �̂ 2 62 �^ 3 86 �^ 15 59 �^ 5 50 �̂ 1 59 �^ 1

Heart (4) 96 ^ 18 12 �^ 2 24 �̂ 16 28 �^ 8 180 �^ 42 14 �^ 4 91 �̂ 2 70 �^ 3

Respiratory system

Tracheal smooth musclea 75 ^ 5 51 �^ 9 54 �̂ 5 63 �^ 1 621 �^ 295 454 �^ 209 49 �^ 2 61 �^ 2

Lung (3) 46 ^ 6 31 �^ 5 54 �̂ 3 59 �^ 9 245 �^ 64 192 �^ 53 46 �^ 1 62 �^ 2

Digestive system

Liver (3) 87 ^ 4 61 �^ 4 24 �̂ 4 15 �^ 2 506 �^ 65 405 �^ 68 13 �^ 5 5 �̂ 1

Stomach (3)b 63 ^ 7 32 �^ 1 55 �̂ 3 53 �^ 6 301 �^ 4 173 �^ 23 49 �^ 2 64 �^ 3

Duodenum (3)b 38 ^ 1 16 �^ 1 59 �̂ 2 28 �^ 6 164 �^ 26 114 �^ 9 41 �̂ 3 49 �^ 3

Parotid (3)b 32 ^ 7 11 �^ 2 47 �̂ 4 29 �^ 1 135 �^ 20 63 �^ 10 27 �^ 1 29 �^ 2

Pancreas (3)b 19 ^ 1 8 �̂ 1 61 �̂ 2 42 �^ 12 67 �^ 8 44 �^ 5 48 �̂ 1 52 �^ 4

Immune system

Spleen (6) 56 ^ 4 39 �^ 3 58 �̂ 3 72 �^ 1 473 �^ 57 330 �^ 37 53 �^ 3 68 �^ 2

Lymph nodes (4) 31 ^ 3 19 �^ 2 53 �̂ 3 50 �^ 3 140 �^ 25 104 �^ 29 50 �^ 2 65 �^ 4

Thymus (6) 22 ^ 2 9 �̂ 1 52 �̂ 6 50 �^ 6 113 �^ 9 59 �^ 9 50 �̂ 6 63 �^ 2

Others

Kidney (4) 56 ^ 12 28 �^ 5 55 �̂ 4 43 �^ 6 205 �^ 65 142 �^ 46 46 �^ 2 45 �^ 3

Brain (3) 31 ^ 9 12 �^ 4 55 �̂ 3 31 �^ 9 43 �^ 1 24 �^ 2 64 �̂ 6 71 �^ 10

Skeletal muscle (3) 10 ^ 28 11 �^ 2 7 �^ 8 5 �^ 10 106 �^ 15 14 �^ 2 47 �̂ 4 18 �^ 4

a Experiment carried out twice with fresh tissues from a pool of 10 individual animals. b These data have been published previously [16] and are included

here as a comparison.

4108 R. Lemmens et al. (Eur. J. Biochem. 267) q FEBS 2000

0.05 m cacodylate buffer pH 7.4, and fixed with 3% para-formaldehyde, 1% glutaraldehyde, 0.25 m sucrose and 2 mmCaCl2 in 0.05 m cacodylate buffer, pH 7.4 for 1 h at 4 8C. Next,the cells were washed three times with 0.05 m Tris/maleatebuffer, pH 7.4, containing 0.2 m sucrose. The NTPDase1reaction was performed by incubating for 45 min, at roomtemperature, in 0.05 m Tris/maleate buffer, containing 2 mmPbNO3 as a phosphate capturing agent, 5 mm KCl, 1 mmlevamisole, 1 mm ouabain, 1.5 mm CaCl2 and 1 mm ATP. Thereaction was stopped by rinsing thoroughly with buffer, and thePb3(PO4)2 was converted to PbS by incubating the sections with1% (NH4)2S for 1 min

Fixation of these transfected cells was carried out with 1%osmium tetroxide in the cacodylate buffer for 30 min in thedark. After washing in 30% and 50% ethanol, the cells wereincubated for 30 min with 2% uranyl acetate in 70% ethanol inthe dark. Following further dehydration with 90% ethanol andtwice with 100% ethanol, the cells were transferred for 30 minat 37 8C to a 1 : 1 (v/v) absolute ethanol/Durcupan resinmixture. This was followed by 30 min impregnation withpure Durcupan at 60 8C and overnight embedding at 60 8C.Ultrathin sections were cut and examined in a Philips EM208S transmission electron microscope.

Computional sequence analysis

The amino-acid sequence, deduced from the obtained cDNAsequence was analyzed by a Prosite Pattern scan using`Network Protein Sequence @nalysis' at the PoÃle Bio-Informatique Lyonnais, Lyon, France [25]. The proteinstructure was predicted by SOSUI [26]. The amino-acidsequence was compared to the nonredundant sequence databaseusing the gapped blastp program at the National Center forBiotechnology Information [27]. A Fasta3 Database Searchwas performed against the SWALL Non-Redundant Proteinsequence database (Swissprot 1 Trembl 1 TremblNew) [28].A search for homologous nucleotide sequences was performedusing the blastn program against different databases, includ-ing the yeast (Saccharomyces cerevisiae) genomic database,

and the nonredundant database of GenBank EST (expressedsequence tags) Division. Phylogenetic analysis of the nucleotidesequences was performed with treecon for windows [29].Unless otherwise indicated, all computer programs wereexecuted using the default values for all variables.

R E S U L T S

Distribution of NTPDase1 in porcine tissues

Table 1 shows that for a same porcine tissue or organ thespecific ATPase and ADPase activities are generally compar-able, except for the heart and skeletal muscle, where theATPase activity markedly exceeds the activity with ADP assubstrate in both homogenate and particulate fraction. Theinhibition of both activities by 10 mm sodium azide iscomparable in most tissues with a somewhat more pronouncedinhibition of ADPase than of ATPase activity.

Some interesting exceptions can be observed. ATPaseactivity of heart particulate fraction was decreased by about90% in the presence of 10 mm sodium azide, compared to 24%reduction in the homogenate. In the skeletal muscle homo-genate, the ATPase activity is quite resistant to azide. Anotherstriking difference is found in the liver. Although the ratio ofhydrolysis of ATP/ADP in liver particulate fraction is com-parable to that in many other tissues, the inhibition by sodiumazide is minimal (only 13% with ATP as substrate and 5% withADP).

The particulate fractions of the tissues in Table 1 weresubjected to PAGE under both reducing (not shown) andnonreducing conditions followed by Western blotting. Theresults obtained under nonreducing conditions with a specificmonoclonal antibody (4D11) against porcine NTPDase1, whichbinds an epitope between amino acids 202±510 [15], are shownin Fig. 1. Western blots showed very high expression in aorticendothelial cells (not shown), tracheal smooth muscle, lung andspleen. High expression was also found in stomach, duodenum,kidney, lymph node and aorta but no signal could be detected inred blood cells. The latter is in agreement with the background

Fig. 1. Western blot analysis of the particulate fraction of porcine tissues. Samples of 8 mg protein (4 mg for pancreatic zymogen granule membranes

(ZGM)) were fractionated on a 10% acrylamide SDS/PAGE under nonreducing conditions, transferred to an Immobilon-P membrane and incubated with the

monoclonal anti-NTPDase1 antibody, 4D11, as described in Materials and methods.

q FEBS 2000 Porcine NTPDase1 (Eur. J. Biochem. 267) 4109

level of ATPase and ADPase activities measured in theseelements.

In most cases, the ADPase activity shown in Table 1correlates well with the intensity of the bands on theimmunoblots, with one important exception being the liver,where the high activity is only reflected by a rather weak bandon the Western blot with 4D11. These Western blots alsoshowed the presence of a 54-kDa band. In the pancreas and itszymogen granules, the 54-kDa band was abundant while the78-kDa protein could barely be detected. In trachea and lungthis monoclonal antibody also detected higher molecularmass bands after SDS/PAGE under nonreducing conditions,possibly attributable to the presence of oligomers linked bydisulphide bridges that might have arisen nonspecificallyduring the preparation of these fractions. With the additionof 2-mercaptoethanol in the sample buffer only the 78- and54-kDa band could be detected using a polyclonal antibody (notshown). With another monoclonal antibody (1H12), whichreacts with an epitope in the first 201 amino acids ofNTPDase1, a 27-kDa band was detected together with the78-kDa NTPDase1 under both reducing and nonreducingconditions (results not shown). It is noteworthy that this27-kDa band was detected in all tissues which also showed the54-kDa band as seen with the antibody 4D11 under nonreduc-ing conditions as well as with a polyclonal antibody underreducing conditions (not shown for the latter).

Peptide N-glycosidase F (PNGase F) treatment of particulatefractions from pancreas zymogen granule membranes, kidney,

spleen, duodenum, lymph nodes and thymus showed a shiftfrom the 78-kDa band to a core protein of about 57 kDa, asreported for NTPDase1 of other species [11,22,30±32].Deglycosylation of the 54-kDa protein in the same tissuesshifted its molecular mass to 35 kDa, as described previouslyfor porcine pancreas and bovine aorta type I ATPDases [12,22],while the 27-kDa band shifted to 24 kDa (not shown).

Nucleic acid sequencing and analysis

Endothelial cells from aorta were used to isolate total RNA.With reverse transcriptase and an oligo(dT) primer, poly(A1)RNA was transcribed into cDNA, which was used as atemplate for PCR. Using primer walking, the complete cDNAsequence of porcine vascular NTPDase1 was obtained (notshown). This nucleotide sequence has been submitted to theNucleotide Sequence Database at EMBL and has been assignedaccession No AJ133746.

The sequence includes an ORF of 1530 nucleotides which istranslated into a protein of 510 amino acids with a calculatedmolecular mass of 57 756 Da. Figure 2 shows 12 cysteineresidues, the five apyrase conserved regions, as well as sevenN-glycosylation sites in the extracellular part of the molecule asrevealed by a Prosite Pattern scan. A consensus intracellularcGMP- and cAMP-dependent protein kinase phosphorylationsite is present in the N-terminal region. This site is also seen inbovine NTPDase1, but not in mouse, human, rat and chickenNTPDase1 molecules.

Fig. 2. Porcine NTPDase1 amino-acid

sequence. Underlined are ACR1 (dotted), ACR2

(single), ACR3 (double), ACR4 (wave), ACR5

(thick). The vertical line (|) between amino

acids 201 and 202 marks the beginning of type-I

ATPDase. Potential N-glycosylation sites are

indicated in bold, and the two hydrophobic

regions are shaded. The boxed sequence

represents a potential intracellular cAMP-

and cGMP-dependent protein kinase

phosphorylation site.

Fig. 3. Surface probability and possible

tryptic cleavage sites of porcine NTPDase1.

The arrows indicate the high surface probability

and trypsin cleavage site between amino acids

201 (arginine) and 202 (lysine).

4110 R. Lemmens et al. (Eur. J. Biochem. 267) q FEBS 2000

A hydrophobicity plot of the amino-acid sequence [33]pointed towards two hydrophobic areas, indicating the positionof the two a-helical membrane-spanning regions (amino acids14±36 and amino acids 478±500), a 13-amino-acid longintracellular N-terminus and an intracellular C-terminal regionof 10 amino acids (data not shown). This was confirmed by theSOSUI analysis (not shown). The surface probability and thepresence of possible tryptic cleavage sites of porcine CD39 areshown in Fig. 3.

A gapped blastp analysis showed a high amino-acid homo-logy of porcine NTPDase1 with other sequenced vertebrateE-type ATPases. Additionally, a distance estimation of thenucleotide sequences of vertebrate E-type ATPases was per-formed with treecon, showing the phylogenetic relationshipbetween these proteins (Fig. 4).

Expression of NTPDase1 and characterization of isoforms

Three constructs were developed in pcDNA3; a eukaryoticexpression vector for both transient and stable transfection,

using primers linked to HindIII and BamHI restriction sites forcloning. A first construct consisted of the complete codingsequence for porcine NTPDase1. A second construct consistedof a 927-nucleotide sequence encoding the protein formerlydescribed as type-I ATPDase [12], i.e. amino acids 202±510,with the addition of a start codon. This construct containsACR4 and ACR5 and is indicated as the 54-kDa construct,referring to the molecular mass of type-I ATPDase. Theremaining 603 nucleotide coding sequence, expressing aminoacids 1±201 with the addition of a stop codon, were also clonedin pcDNA3 and will consequently be named the 27-kDaconstruct. This sequence contains ACR1 to ACR3.

The plasmids containing these constructs were transfectedinto COS-7 cells with Lipofectamine reagent. Protein expressionwas determined by Western blots using different anti-NTPDase1antibodies, and by biochemical and enzyme-histochemicalassays for ATPase and ADPase activity. Western blot analysisof the membrane proteins clearly shows the presence of a78-kDa protein in cells transfected with NTPDase1 (Fig. 5).Both antibodies also detect a protein band at approximately54 kDa in these cells. Antibody 1H12 detects a 27-kDa proteinand as expected, this 27-kDa band is also seen in proteinextracts of COS-7 cells transfected with the 27-kDa construct.However, in cells transfected with the 54-kDa construct, noexpression was observed neither with monoclonal antibody4D11 under nonreducing conditions nor with a polyclonalantibody under reducing conditions (data not shown for thelatter). A plasma membrane fraction of the transfected cells hasbeen tested as well as the homogenate. Absence of immuno-reactivity on the cell cultured medium supernatant, showed thatthe protein was not secreted either. Plasma membranes andhomogenates of mock transfected cells were used as controlsand did not show any immunoreactivity (Fig. 5).

Cells transiently transfected with the complete NTPDase1coding sequence showed a significant increase in both ATPase(12.4-fold; 42 ^ 2 nmol Pi per 104 cells per h) and ADPase(11.5-fold; 20 ^ 1 nmol Pi per 104 cells per h) activities,compared to cells mock-transfected with pcDNA3 only. Noincrease of activity was observed after transfection with the54-kDa or the 27-kDa construct, either separately ortogether.

The electron micrographs in Fig. 6 confirm the highmembrane-bound ATPase and ADPase activity of the cellstransfected with the 78-kDa construct. Control cells trans-fected with the vector only did not display any ATP orADP hydrolysis when examined by histochemical electronmicroscopy.

Fig. 4. Phylogenetic analysis of the

nucleotide sequences of vertebrate E-type

ATPases. Amino-acid identity of different

E-type ATPases with porcine NTPDase1 is

indicated in percentage between brackets after

the corresponding protein.

Fig. 5. Western blot analysis of membrane preparations of COS-7 cells,

transfected with NTPDase1 or with NTPDase1-constructs. Membranes

of transfected cells were prepared as described in Materials and methods

and fractionated on SDS/PAGE under either nonreducing (antibody 4D11)

or reducing (antibody 1H12) conditions. Proteins were transferred to an

Immobilon-P membrane and incubated with monoclonal antibodies 4D11

or 1H12.

q FEBS 2000 Porcine NTPDase1 (Eur. J. Biochem. 267) 4111

D I S C U S S I O N

In the present study, the distribution of NTPDase1 has beenevaluated in a variety of porcine tissues. For this purpose wecompared the immunoreactivity of NTPDase1 on Westernblots (Fig. 1) with the enzyme activities in homogenates andparticulate fractions (Table 1). Our results indicate the presenceof NTPDase activity, which in most tissues hydrolyzes bothATP and ADP to a similar extent. However, in heart andskeletal muscle, the ATPase activity considerably exceeds theADPase activity. The hydrolysis of ADP in these tissues wouldbe attributable to the action of NTPDase1, while the higherATPase activity could partially be due to other nucleotidasessuch as ecto-ATPase/CD39L1/NTPDase2.

To date, no specific inhibitor for E-NTPDases has been fullycharacterized. The most frequently used inhibitor is sodiumazide, which partially inhibits both ATPase and ADPaseactivities of mammalian NTPDase1 [1,8] and chicken oviductecto-ATPDase [34]. Therefore, we investigated the influence ofsodium azide on ATPase and ADPase activities in porcinetissues. In most porcine tissues the degree of inhibition of bothenzyme activities was similar, ranging from 30 to 70%. Theinhibition of ADPase was slightly more pronounced than ofATPase, in agreement with previous studies [1,8,15,35±39].In the particulate fraction from heart tissue, ATP hydrolysiswas strongly affected by sodium azide (Table 1). This doesnot seem to be due to a contamination by mitochondrialATPase as the particulate fractions were purified on a sucrosecushion, which allows the exclusion of dense material such asmitochondrial membranes. Another interesting observation wasthat the liver particulate fraction appeared quite resistant toazide. Further analysis of this nucleotidase activity revealed thepresence of a novel NTPDase that we recently characterized[40].

Western blot analysis showed a 78-kDa band in most tissues,as expected for NTPDase1, but also a lesser 54-kDa band intissues in which the 78-kDa protein is abundant. In the pancreasthe 78-kDa band could barely be detected, whereas a veryprominent 54-kDa band was present. This could be due to thehigh proteolytic activity in this tissue. A 54-kDa protein wasobserved in all mammalian species we tested so far: swine,beef, human and mouse [11,12,15,22,41].

The 54-kDa protein is the C-terminal part of the 78-kDaprotein, and contains only two of the five ACRs [11,13]. Allevidence points to the fact that the 54-kDa polypeptide derivesfrom the 78-kDa protein by proteolytic digestion. Using aspecific antibody against the first 201 amino acids of porcineNTPDase1 (27-kDa fragment) and antibodies against theC-terminal part of NTPDase1 (54-kDa fragment), we observed

that the 27-kDa protein band was present in all tissues in whichthe 54-kDa fragment could be detected.

The comparison of the N-terminal amino-acid sequence oftype-I ATPDase (54 kDa) from pig pancreas [4,13] with thefull-length cDNA sequence has indicated a tryptic cleavage sitewith a fragment starting at amino acid 202. Trypsin is cleavingproteins after lysine and/or arginine. Further analysis of theprotein showed that amino acids 199±207 displayed a highsurface probability. A potential trypsin cleavage site coincidingwith a high surface probability domain is also present inNTPDase1 from bovine (Lys202), rat and mouse NTPDase1(Lys204). In human NTPDase1, the peak of surface probabilityis localized a few amino acids before, with an arginine atposition 194. It is then expected that similar cleavage productswould be formed in other species also explaining the presenceof a 54 and 27-kDa protein fragments. This modification ofthe NTPDase1 by a serine protease could potentially be ofpathophysiological significance as it was recently shown thatincubation of recombinant human CD39 with trypsin convertedthe 78-kDa band to a 54 and a 27-kDa protein bands, which wasinterestingly concomitant with an increase in enzyme activity[11].

Analysis of the N-terminal sequence of pig NTPDase1(78 kDa) with the cDNA sequence suggests another post-translational modification. The 1530 nucleotide open readingframe of porcine NTPDase1 shows that the protein starts with amethionine and a glutamic acid residue (Fig. 2). By N-terminalamino-acid sequencing, however, we determined that theprotein purified from porcine kidney begins with the asparticacid found in position 3 of the original translation product [15].So far, this post-translational modification has not beendescribed for other mammalian NTPDase1 molecules. OtherNTPDase1 amino-acid sequences were derived from thecorresponding cDNA nucleotide sequences and all start withmethionine, glutamic acid and aspartic acid. The N-terminalamino-acid sequence of human placental NTPDase1 differsfrom the sequence of human CD39 in its first 11 amino acids.Nevertheless, all described internal sequences provide evidencethat the placental NTPDase1 and CD39 are almost identicalmolecules, with only distinct N-terminal endings [42].

The complete cDNA of NTPDase1 was functionally expressedin COS-7 cells and showed a marked increase in ATPase andADPase activities at the surface of the transfected cells.Transfection under the same conditions with either the 54-kDaor the 27-kDa construct gave no measurable increase in ATPaseor ADPase activity. Expression of the full-length cDNA and the27-kDa construct showed the expression of the expected proteinsize on a Western blot with 1H12. Other bands were alsoobserved in both of these constructs that correlate with non

Fig. 6. Enzyme histochemical

electronmicrograph of COS-7 cells transiently

transfected with porcine NTPDase1 cDNA.

COS-7 cells were transiently transfected with

the pcDNA3, containing porcine NTPDase1

cDNA. After 72 h, the cells were fixed, and

incubated with a reaction mixture containing

1 mm substrate as indicated, and 2 mm PbNO3

as a phosphate capturing agent. For control, cells

mock-transfected with the vector only were

assayed for ATPase activity. After 45 min

incubation, the reaction was stopped and the

released phosphate was visualized as described

in Materials and methods. Bar, 10 mm.

4112 R. Lemmens et al. (Eur. J. Biochem. 267) q FEBS 2000

glycosylated proteins. For example, for the 27-kDa construct, aweek 24-kDa protein band was observed next to the main27-kDa protein which is consistent with the presence of oneN-linked glycosylation site in the region corresponding to thefirst 201 amino acids of porcine NTPDase1 (Fig. 2). Indeed,PNGase F treatment of the 27-kDa fragment from pig pancreas,bovine aorta and human recombinant protein resulted in aconversion to a 24-kDa protein band (not shown).

In a previous study we purified a 54-kDa ATPDase andidentified it as type-I ATPDase [12]. The latter pancreaticNTPDase1 preparation, purified by about 3500-fold, stillshowed the presence of the 27-kDa protein as detected withmonoclonal antibody 1H12 (results not shown). The fact thatthe expression of porcine NTPDase1 in COS-7 cells alsoresulted in the presence of a 54- and 27-kDa protein (Fig. 5),confirms the results previously described for human CD39 [11].It is noteworthy that purification of the human recombinantCD39 with a different procedure also did not separate the54-kDa from the 27-kDa protein [11]. These data stronglysuggest that the two fragments of the complete 78-kDa proteinoccur together and stay together. The interaction between thesefragments does not involve disulfide linkages as 2-mercapto-ethanol had no effect on the protein pattern after SDS/PAGE.

In summary, porcine NTPDase1 cDNA was sequenced andexpressed in COS-7 cells. The complete cDNA sequenceallowed the identification of a serine protease cleavage site,resulting in the production of a 54-kDa type-I ATPDase and anassociated 27-kDa fragment. In order to remain enzymaticallyactive, the two cleavage products appear to stay together bynoncovalent interactions.

A C K N O W L E D G E M E N T S

We thank Agnes Roosen, Johanne Proulx and Julie Roy for their technical

assistance. We also thank Dr L. Michiels of the Dokter Willems Institute for

his valuable help with the sequencing. This work was supported by grants

from the Bilateral Scientific and Technological Cooperation; BIL97/32 and

Fonds pour la Formation de Chercheurs et l'Aide aÁ la Recherche du QueÂbec

(FCAR), and Natural Sciences and Engineering Research Council of

Canada (NSERC). J. SeÂvigny was a recipient of fellowships from the Heart

and Stroke Foundation of Canada and from the FCAR.

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