(Received for publication, September 17, 1996)
From the Department of Pharmacology and Cell Biophysics, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267-0575
The ecto-ATPase from chicken gizzard (smooth
muscle) was solubilized, and the 66-kDa cell membrane ecto-ATPase
protein was purified. The protein was then subjected to both enzymatic
and chemical cleavage, and the resultant peptides were purified by reverse phase high pressure liquid chromatography and sequenced. Several of these internal peptide sequences were used to design oligonucleotides to screen a chicken muscle library to identify the
cDNA encoding the ecto-ATPase. Two overlapping partial clones were
sequenced, yielding the complete coding region and a long 3-untranslated sequence. The deduced amino acid sequence is in agreement with the N-terminal and peptide sequences obtained from the
purified protein. The chicken muscle ecto-ATPase is a slightly basic
(predicted pI = 7.93) 494-amino acid protein (54.4 kDa), containing a single transmembrane domain at each end of the protein. The majority of the protein is predicted to be extracellular, making it
a Type Ia plasma membrane protein. There are four putative N-glycosylation sites, a single potential
cAMP/cGMP-dependent protein kinase phosphorylation site, as
well as a single putative tyrosine kinase phosphorylation site.
Analysis of the sequence using the BLAST programs demonstrated homology
with other ecto-ATPases and ecto-apyrases, including those from the
parasitic protozoan Toxoplasma gondii, potato tubers, and
garden pea, as well as a guanosine diphosphohydrolase from yeast.
However, the most striking homology observed was to the human and mouse
lymphoid cell activation antigen 39 (CD39), a molecule now known to
have apyrase activity. The chicken ecto-ATPase showed considerable
amino acid sequence homology with CD39 over the entire length of the
sequence, excluding about 30-40 amino acids at the extreme ends of the
protein (which include the two membrane-spanning helices). The sequence
homology between the gizzard ecto-ATPase and CD39 was confirmed by
Western blots demonstrating immunocross-reactivity between mono- and
polyclonal antibodies raised against the chicken ecto-ATPase and two
commercially available monoclonal antibodies against the human CD39
protein. The results suggest that the muscle ecto-ATPase may be
involved in cell adhesion, since the highly homologous CD39 protein is involved in homotypic adhesion of activated B lymphocytes.
Cell membrane ecto-ATPases are millimolar divalent
cation-dependent, low specificity enzymes that hydrolyze
all nucleoside triphosphates (NTPases). They are integral membrane
glycoproteins that can be distinguished from ecto-apyrases (ecto-ATP
Diphosphohydrolases or ecto-ATPDases) by their inability to hydrolyze
ADP and other nucleoside diphosphates at rates that are more than
1-2% that of their ATP hydrolysis rates. A recent review
summarizes the properties and postulated functions of the ecto-ATPases
and ecto-apyrases (1). From the results of work on the single-celled
parasitic protozoan Toxoplasma gondii, it appears that the
ecto-ATPase and ecto-apyrase enzymes from that source are closely
related as judged by sequence analysis (2, 3). However, significant
differences in enzymology and susceptibility to inactivation by
detergents exist between ecto-ATPases and ecto-apyrases from a variety
of sources (for a review, see Ref. 1), suggesting that they may not be
as closely related to each other as is suggested by the sequence
homology of the T. gondii enzymes.
The physiological functions of the ecto-ATPases and ecto-apyrases are not known. However, many functions have been hypothesized, including roles in cellular adhesion, termination of purinergic signaling, and purine recycling (for a review, see Ref. 1), as well as secretion (4) and vesicle trafficking (5). The goal of this study was to clone and sequence the cDNA encoding the chicken muscle ecto-ATPase to gain information about the structure and physiological function of the whole class of ecto-ATPase enzymes by analysis of sequence homologies with proteins of known function. The sequence reported here represents the first vertebrate ecto-ATPase to be cloned and sequenced. (The rat liver cellular adhesion molecule of 105 kDa "ecto-ATPase" sequence (6) apparently does not encode the ATPase (7), and the rat liver enzyme is classified as an ecto-apyrase since it hydrolyzes ADP as well as ATP.) The results reported here suggest that at least one physiological function of the ecto-ATPase is involvement in the process of cell adhesion, since it is highly homologous with the lymphoid cell activation antigen (CD39),1 which is known to be involved in homotypic activated B-cell adhesion (8). This conclusion is consistent with several previous reports obtained from several different species and tissues that suggested by indirect methods that the ecto-ATPase may be involved in cellular adhesion in rat liver (6), chicken muscle (9, 10), and rat brain (11), as well as the recent finding that the CD39 protein, which is involved in homotypic lymphocyte adhesion (8), has ecto-apyrase activity (12).
Chicken membranes were isolated as described
previously from gizzards obtained from a local slaughterhouse (13).
Endoproteinase-Lys-C and endoproteinase-Glu-C were obtained from
Promega and Boehringer Mannheim, respectively. Cyanogen bromide was
from Pierce, and the reverse phase HPLC columns were from Vydac. The
chicken gizzard phage cDNA library was obtained from Clontech, and
a chicken muscle lambda Zap® phage cDNA library and
Epicurian Coli ultracompetent bacteria were purchased from Stratagene.
The GeneTrapperTM cDNA positive selection system kit was purchased
from Life Technologies, Inc. Synthetic oligonucleotides were obtained
from the DNA core facility at the University of Cincinnati. Sequenase
version 2 kits were from U. S. Biochemical Corp. The 3-oligo labeling
system and the ECL detection reagents were from Amersham.
The 66-kDa ecto-ATPase protein was purified either using a column chromatography scheme (13) or by immunoprecipitation with monoclonal antibody 6 (10). Using either methodology, a final step of preparative SDS-PAGE was employed to obtain a pure 66-kDa ecto-ATPase protein. The protein was then blotted onto a polyvinylidene difluoride membrane (14), the membrane was blocked, and the protein bound to the membrane was cleaved enzymatically with endoproteinase-Glu-C, endoproteinase-Lys-C, or a combination of the two enzymes as described (15, 16). Alternatively, the ecto-ATPase was subjected to CNBr cleavage subsequent to electroelution from the preparative SDS-PAGE gel and acetone precipitation. Approximately a 100-fold excess of CNBr over methionine residues was added, and cleavage was performed in 70% formic acid for 24 h at 22 °C and was light-protected and under N2.
Purification and Sequencing of PeptidesThe soluble peptides resulting from the enzymatic and CNBr cleavage reactions were injected onto a reverse phase HPLC column and eluted using a gradient of acetonitrile with 0.1% trifluoroacetic acid as the ion pairing reagent, essentially as described previously (17). In some cases the peptides were repurified using a more shallow solvent gradient or a reverse phase HPLC column of different selectivity, or by using 6 mM HCl as the ion-pairing reagent. The pure peptides were then dried and sequenced on a Porton 2090E protein sequencer in the Protein Service facility of the Department of Pharmacology and Cell Biophysics at the University of Cincinnati.
Western Blot AnalysisFor Western blot analysis of various protein preparations, the proteins were boiled for 5 min in SDS-PAGE sample buffer prior to electrophoresis (18). Reductant (dithiothreitol) was omitted, as it was found that the reactivity of most of the anti-ecto-ATPase monoclonal antibodies was drastically reduced or eliminated by reduction (19). Monoclonal antibodies against CD39 were obtained commercially from Immunotech (clone AC2, "CD39 Ab 1") and Zymed (clone A1, "CD39 Ab 2"). Mouse 7gg7 (B-cell hybridoma) cells were obtained from Dr. J. W. Thomas (Vanderbilt) and prepared by Dr. Ken Dombrowski (Texas Tech University). Visualization of all Western blots was accomplished using a horseradish peroxidase-conjugated secondary antibody and subsequent ECL detection.
cDNA Cloning and SequencingThe lambda Zap® chicken
muscle cDNA library was mass-excised into a double-stranded plasmid
cDNA library as described by the manufacturer. The double-stranded
plasmid library was then converted to a single-stranded library using
Gene II and exonuclease III enzymes, and the resulting library was
screened using the GeneTrapper cDNA positive selection system as
described by the manufacturer using a degenerate inosine-containing
oligonucleotide antisense probe, CG14 (5-GCI GG(C/T) TC(C/T) TGI GCI
GGI ATC AT-3
) designed from the peptide sequence of a CNBr fragment of
the ecto-ATPase ((M)IPAQEPA). Rather than using electroporated
Escherichia coli for the transformation as is recommended by
the GeneTrapper manual, Epicurian Coli ultracompetent E. coli were transformed and plated on LB/ampicillin plates and
screened by colony hybridization with a number of oligonucleotide
probes designed from ecto-ATPase internal protein sequences. All colony
lifts and Southern blots were screened using oligonucleotides labeled
and detected with the 3
-fluorescein-dUTP labeling system (Amersham),
with the final detection being enhanced chemiluminescence of the
horseradish peroxidase-conjugated anti-fluorescein antibody. DNA
sequencing was performed using Sequenase version 2. The entire sequence
of the clones was obtained by "oligo walking" (designing synthetic
oligonucleotide primers based on the 3
-end of the obtained sequence).
The clones were sequenced in this manner from both ends until
significant overlap of the sequences was obtained near the middle of
the clones.
After determining that the initial clones did not contain the sequence
encoding the N terminus of the protein, the same cDNA library was
rescreened using a non-degenerate antisense oligonucleotide (5-CAC ATG
CTG TGC TCG CTG ACC-3
) designed from a sequence near the 5
-end of the
first partial clone (19(2nd)). Positive clones were purified,
sequenced, and analyzed as above. From the second screening, a partial
clone (CS-Q) that encoded the known N terminus and overlapped
approximately 610 bases with the first clone (19(2nd)) was isolated and
sequenced.
Homologies of the chicken muscle ecto-ATPase deduced amino acid sequences were found using internet access to the BLASTX and BLASTP programs2 (20) from the National Institutes of Health and the BLASTP/BEAUTY (21) programs.3
Several attempts to isolate the chicken gizzard ecto-ATPase
cDNA from the Clontech gizzard cDNA library using conventional phage plating and screening with various radioactively labeled degenerate oligonucleotide probes proved to be unsuccessful. However, the desired clones were obtained by using a chicken muscle lambda Zap® phage cDNA library. This phage library was
mass-excised and converted into a plasmid cDNA library, which is
the required starting material for the GeneTrapper cDNA
positive selection system kit. The GeneTrapper system then converts the
double-stranded plasmid library into a single stranded library and
enriches the cDNA encoding the ecto-ATPase by affinity purification
using, in this case, a biotinylated degenerate probe designed from a
cyanogen bromide peptide. (A chicken skeletal muscle library was used
in the GeneTrapper system since no chicken gizzard (smooth muscle)
cDNA library was commercially available that could be readily
converted to the plasmid cDNA library required as the starting
material for the GeneTrapper cDNA positive selection system.) A
2.1-kilobase pair clone (designated 19(2nd)), which hybridized to
several degenerate probes designed from internal protein sequences of
the 66-kDa ecto-ATPase, was sequenced. This clone was found to have
approximately 750 bases of 3-untranslated sequence and did not encode
the known N-terminal sequence of the protein (13). However, the deduced
amino acid sequence did agree with many internal peptide sequences
obtained from the purified gizzard protein. A non-degenerate probe was
designed from near the 5
-end of the first clone (19(2nd)), and after
rescreening the same library in the same manner, a second overlapping
partial clone was found (CS-Q, 0.83 kilobase pairs). Except for
approximately 20 bases at the extreme 5
-end of clone 19(2nd), the
sequences in the approximately 610-base pair region of overlap between
the two clones were identical, and the deduced amino acid sequence of
the overlapping cDNA sequence agreed with the sequences determined from the purified protein (see Fig. 1).
There is one peptide sequence (the protein N terminus) encoded by the
non-overlapping 5-end of clone CS-Q, seven peptide sequences encoded
in cDNA overlap sequence between the two partial clones, and six
peptide sequences encoded by the non-overlapping coding region of clone
19(2nd) (see Fig. 1). Thus, there are a total of 14 peptide sequences
(including the N terminus and the membrane-spanning region very close
to the predicted C terminus of the protein) that agree with the protein
sequence deduced from the cDNA sequence that was spliced together
from the two partial clones.
The protein sequence deduced from the cDNA is a 494-amino acid protein with a calculated protein molecular mass of 54,402 Da, a pI of 7.93, and four potential N-glycosylation sites (Fig. 1). The Kozak consensus sequence for initiation in higher eukaryotes (22) is indicated by double underlining of the DNA sequence, and the initiation methionine is marked. There is a single potential cAMP/cGMP phosphorylation site and a single potential tyrosine kinase phosphorylation site (see Fig. 1).
The hydrophilicity plot analysis of the protein sequence is shown in
Fig. 2, indicating the likelihood of the following
membrane topology: a putative membrane-spanning region that appears to be an uncleaved signal sequence at the N terminus, a large
extracellular loop containing approximately 85% of the total protein
as well as all of the carbohydrates and including the active site(s), and a putative membrane-spanning region very close to the C terminus. Thus, the ecto-ATPase has the properties of a Type Ia membrane protein.
Only approximately 10 amino acids are predicted to be intracellular.
Also shown in Fig. 2 are the "antigenic index" and the "surface
probability" as predicted by the Mac Vector computer program. These
analyses reveal several potential antigenic "hot spots," suggesting
possible regions containing the epitopes recognized by existing
antibodies (10, 19), as well as indicating sequences that may be useful
for the design of anti-peptide antibodies.
The deduced protein sequence shows homology with several known
ecto-ATPases and apyrases, as shown in Fig. 3. The
greatest amino acid homology is with mouse CD39, which is homologous to the entire ecto-ATPase amino acid sequence with the exception of the N-
and C-terminal putative membrane-spanning regions (Figs. 3 and
4).
The close relationship of the chicken muscle ecto-ATPase with the human
CD39 protein suggested by the sequence analysis was confirmed by
demonstrating immunocross-reactivity of a gizzard ecto-ATPase
affinity-purified polyclonal antibody (19), a gizzard ecto-ATPase
monoclonal antibody (6 (10)), and two commercially available anti-human
CD39 monoclonal antibodies using two mammalian species (pig coronary
artery tissue and mouse 7gg7 (B-cell hybridoma) cells), as seen
in Fig. 5.
The GeneTrapper "affinity enrichment" technique of screening a
cDNA library proved successful in isolating chicken ecto-ATPase clones after several failed attempts using conventional phage plating
and screening techniques. Two overlapping partial clones were isolated
that encode the entire protein sequence, as judged by the agreement of
both clones with many peptide sequences obtained from the gizzard
ecto-ATPase purified protein (Fig. 1). The protein encoded by the
cDNA sequenced is consistent with the known molecular mass of the
isolated protein (54.4 kDa for the deduced sequence versus
53 kDa estimated by SDS-PAGE for the purified protein (13)). The
initiation methionine occurs directly before the N-terminal sequence of
the mature protein and is preceded by a Kozak consensus sequence for
initiation in higher eukaryotes (GCCGCC(A/G)CCG (22)).
The slightly basic pI (7.93) predicted is also consistent with the
behavior of the ecto-ATPases on ion exchange columns (13, 23) and with
the assertion that much of the net negative charge on the ecto-ATPase
at physiological pH is due to sialic residues on the glycan chains
(24). The presence of four putative glycosylation sites is consistent
with our unpublished observation of at least four N-linked
glycan chains as demonstrated by quantum decreases in molecular mass on
SDS-PAGE during time course studies of deglycosylation of the purified
enzyme by peptide N-glycosidase-F. The topological
prediction of only approximately 10 amino acids of the ecto-ATPase
being located intracellularly suggests that it is unlikely that the
ecto-ATPase is modulated by intracellular proteins or involved in
signal transduction pathways mediated by direct interactions of
ecto-ATPase with intracellular proteins.
As is strongly suggested by the hydrophilicity analysis and the fact
that the chicken ecto-ATPase is indeed an ecto enzyme (9, 24, 25), the
majority of the protein (including the active site) is located on the
exterior of the cell membrane. This is confirmed by the location of the
putative N-glycosylation sites, at least some of which must
be glycosylated to account for purification (13) and lectin modulation
of activity data (9, 13, 26). It seems likely that most of the
cyst(e)ine residues in the large extracellular loop (11 or 12 cyst(e)ine residues depending on exactly where the N-terminal
membrane-spanning sequence exits the membrane) are disulfide-bonded,
since 1) they are exposed to the oxidizing extracellular media, 2)
cysteine-selective chemical modification reagents are not inhibitors of
chicken gizzard ecto-ATPase activity, and 3) dithiothreitol has a
significant inhibitory effect on activity even though there are no
intermolecular disulfide bonds in the chicken ecto-ATPase (19). The
presence of the intramolecular disulfide bonds, as well as the
glycosylation, would likely increase the stability of the protein and
probably contribute significantly to the resistance to proteases that
is characteristic of the ecto-ATPases. The N-terminal sequence has the
properties of an uncleaved signal sequence, and the assignment of
membrane-spanning regions (and therefore membrane topology), appears to
be straightforward. A linear model of the ecto-ATPase consistent with
the sequence-derived information described above is presented in Fig.
6.
The chicken muscle ecto-ATPase is homologous to ecto-ATPases and
ecto-apyrases from a variety of plants, lower organisms, and mammals
(Fig. 3). The deduced amino acid sequence contains the proposed
apyrase-conserved regions (ACR1-ACR4 (27)). ACR1 and ACR4 sequences
are similar to the actin-hsp70-hexokinase - and
-phosphate
binding motifs (27), but no Walker consensus ATP binding motif
sequences (28) are present. The only ecto-ATPase/apyrase cloned and
sequenced that the deduced chicken muscle ecto-ATPase amino acid
sequence does not share homology with is the mosquito saliva apyrase
(29). The mosquito apyrase also does not have homology with other
enzymes in the ecto-ATPase/apyrase family derived from many diverse
species (27), suggesting that either the mosquito enzyme is unrelated
to the others or that the mosquito clone sequenced does not represent
the mosquito apyrase protein.
The chicken muscle ecto-ATPase-deduced amino acid sequence is most homologous with the lymphoid cell activation antigen CD39 (Figs. 3 and 4). The homology encompasses the entire extracellular domain of the chicken ecto-ATPase, accounting for approximately 85% of the protein. CD39 is involved in homotypic activated lymphocyte cell adhesion (8). Thus, this suggests that the physiological function of the ecto-ATPase is related to that of CD39-cell adhesion. This is consistent with our finding that a monoclonal antibody that recognized integrin on Western blots is capable of immunoprecipitating gizzard ecto-ATPase (10), since integrin is a ligand for other known cell adhesion molecules. Also, there are several reports in the literature that the ecto-ATPases and ecto-apyrases from various sources are either associated with or identical to adhesion molecules (6, 9, 11). The hypothesis supported by the body of work generated by this laboratory over the last few years (10, 13, 19, 23, 30, 31) is that the ecto-ATPase is not an adhesion molecule itself but is in some (yet to be understood) way critical to cell adhesion or to the regulation of the adhesive process. This regulation may be by dephosphorylation of proteins phosphorylated by ecto-protein kinases or, more likely, by control of the concentrations of extracellular nucleotides serving as triggers for cell adhesion. Human ecto-apyrase (CD39) present in vascular endothelial cells has been speculated to serve such a function in the bloodstream, i.e. to keep the levels of ADP low during normal conditions so that platelet aggregation is not induced, which would lead to pathologies associated with vascular clot formation. Related to this is the recent finding that inhibition of the vascular ecto-ATPase/apyrase by free radical-induced oxidative damage is thought to be involved in triggering the clotting responsible for the acute cessation of blood supply to xenografts, resulting in death of the transplanted tissue (32). However, it seems unlikely that the chicken muscle ecto-ATPase described in this work could fulfill such a function in the circulatory system since the true ecto-ATPases, unlike the ecto-apyrases, do not hydrolyze ADP, and ADP is the nucleotide most important for the maintenance of circulatory homeostasis.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U74467[GenBank].
I thank Dr. Jim Stout for technical assistance in generating and purifying some of the peptides for amino acid sequencing, Dr. Sue-Hwa Lin for the training in molecular biological techniques that I received at the M. D. Anderson Cancer Center in Houston, and Dr. Lois Lane for assistance in the interpretation of the cloning results, as well as for critical reading of this manuscript.