Purification, Cloning, and Expression of an Apyrase from the Bed Bug Cimex lectularius

An enzyme that hydrolyzes the phosphodiester bonds of nucleoside tri- and diphosphates, but not monophosphates, thus displaying apyrase (EC 3.6.1.5) activity, was purified from salivary glands of the bed bug, Cimex lectularius. The purifiedC. lectularius apyrase was an acidic protein with a pI of 5.1 and molecular mass of ∼40 kDa that inhibited ADP-induced platelet aggregation and hydrolyzed platelet agonist ADP with specific activity of 379 units/mg protein. Amplification of C. lectulariuscDNA corresponding to the N-terminal sequence of purified apyrase produced a probe that allowed identification of a 1.3 kilobase pair cDNA clone coding for a protein of 364 amino acid residues, the first 35 of which constituted the signal peptide. The processed form of the protein was predicted to have a molecular mass of 37.5 kDa and pI of 4.95. The identity of the product of the cDNA clone with nativeC. lectularius apyrase was proved by immunological testing and by expressing the gene in a heterologous host. Immune serum made against a synthetic peptide with sequence corresponding to the C-terminal region of the predicted cDNA clone recognized bothC. lectularius apyrase fractions eluted from a molecular sieving high pressure liquid chromatography and the apyrase active band from chromatofocusing gels. Furthermore, transfected COS-7 cells secreted a Ca2+-dependent apyrase with a pI of 5.1 and immunoreactive material detected by the anti-apyrase serum.C. lectularius apyrase has no significant sequence similarity to any other known apyrases, but homologous sequences have been found in the genome of the nematode C. elegansand in mouse and human expressed sequence tags from fetal and tumor EST libraries.

Vertebrates protect themselves against excessive blood loss by activating blood clotting mechanisms that are induced by platelet aggregation at the site of injury. To feed effectively, blood-sucking arthropods have developed various mechanisms that counteract these host responses (1,2). Since ADP released by injured cells and by aggregating platelets is one of the most important physiological mediators of platelet aggregation (3), saliva of most blood-feeding arthropods contains large amounts of apyrase (ATP:diphosphohydrolase, EC 3.6.1.5) that hydrolyzes ATP and ADP into AMP and P i and thus inhibits platelet aggregation (4 -6). After some initial controversy over whether apyrase activity resides in specialized enzymes or just represents a combination of enzymes (discussed in Refs. 1 and 7), apyrase enzymes from various plant and animal sources have been purified and characterized in sufficient detail (5, 8 -18).
These studies revealed that, despite their common ability to hydrolyze ATP and ADP with approximately the same activity, apyrase preparations from different sources show wide variance in absolute activities, pH optima, and divalent cation requirements (4). Moreover, the first two apyrases to be sequenced, those from salivary glands of the mosquito Aedes aegypti (5) and from the parasitic protozoan Toxoplasma gondii (8) turned out to belong to completely different enzyme families. A. aegypti salivary apyrase is a member of the 5Ј-nucleotidase family of enzymes; unlike vertebrate 5Ј-nucleotidases, however, this apyrase is a soluble protein and does not hydrolyze the ester linkage of AMP (5). In contrast, T. gondii apyrase is almost identical to an NTPase (EC 3.6.1.15) from the same organism (8) and belongs to a large family of ecto-ATPases, also referred to as E-ATPases (7). In addition to recently characterized apyrases from potatoes (9), chickens (10,11), rats (12,13), and humans (14,15), this family includes more specialized enzymes, such as chicken gizzard ATPase (16) and yeast GDPase (19). Despite the lack of sequence similarity, apyrases from both families are active with either Mg 2ϩ or Ca 2ϩ and are commonly referred to as (Ca 2ϩ , Mg 2ϩ )-apyrases (20). Recently, we have found that the apyrase activity from the bed bug Cimex lectularius is strictly dependent upon Ca 2ϩ ions and, unlike any previously described apyrase, cannot be activated by Mg 2ϩ , Mn 2ϩ , or Zn 2ϩ (21).
Here we report purification, cloning, and heterologous expression of the salivary apyrase from C. lectularius. This enzyme is clearly not homologous to the salivary apyrase from A. aegypti (5). This means that, just as blood feeding has independently evolved in different groups of insects, salivary apyrase activity that usually accompanies blood sucking has been independently acquired by these two different insects. Moreover, we found no sequence similarity between C. lectularius apyrase and any previously characterized nucleotidebinding enzymes, which indicates that it belongs to a novel type of ATPases.

EXPERIMENTAL PROCEDURES
Insect Rearing-C. lectularius colonies were maintained at 27°C and 65% humidity. Insects were fed every 10 days by exposing them to the shaved abdomen of an anesthetized rabbit. Salivary glands of insects of 8 -10 days after feeding were dissected and stored in Hepes saline (10 mM Hepes at pH 7.0 in 150 mM NaCl) at Ϫ75°C until needed. Before use, salivary glands were thawed and disrupted with a pestle, and the * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. homogenate was cleared by centrifugation at 14,000 rpm for 5 min at 4°C.
Apyrase Activity-Apyrase activity of the fractions from different stages of purification was assayed by measuring the release of P i from ATP or ADP in 50 mM Tris-Cl buffer (pH 8.5), containing 0.1 M NaCl, 5 mM CaCl 2 , and 2 mM nucleotides, as described previously (21). One unit of enzyme activity was defined as the amount of enzyme that released 1 mol of orthophosphate/min at 37°C.
ADP hydrolysis was also monitored by a platelet aggregation assay that was performed on a Thermomax microplate reader (Molecular Devices, Menlo Park, CA) with a kinetic module (22) with further modifications. Briefly, apyrase preparations were incubated in a 96well flat-bottom plate (Falcon 3912, Becton Dickinson, Oxnard, CA) in the presence of 5 M ADP in 50 l of 10 mM Hepes (pH 7.4), containing 150 mM NaCl. Aggregation was started by the addition of 50 l of human citrated (0.38%) platelet-rich plasma (23). The plate was then stirred continuously at room temperature, and absorbance readings at 650 nm were taken at 60-s intervals. A rapid and sharp decrease in absorbance after a lag phase indicated aggregation of platelets. With this procedure, all of the fractions resulting from a whole chromatographic procedure could be analyzed quickly and simultaneously for anti-platelet activity.
Purification and Sequencing of C. lectularius Salivary Apyrase-Salivary gland homogenate proteins were separated by either chromatofocusing HPLC 1 or strong cation exchange HPLC, followed in each case by reverse-phase HPLC. Chromatographic protocols were performed using a CM4100 pump and a SM4100 dual wavelength detector (Thermo Separation Products, Riviera Beach, FL). The absorbance at 280 nm was recorded using an 80386-based microcomputer.
The chromatofocusing step was performed with a Mono P column (20 ϫ 0.5 cm) (Amersham Pharmacia Biotech, Uppsala, Sweden) in the range of pH 6.3-4.0 according to the manufacturer's protocol. After equilibration of the column in 25 mM Bis-Tris, pH 6.3, homogenate of 300 salivary gland pairs was adjusted to pH 6.3 and injected into the column. The proteins were eluted with Polybuffer 74 (Amersham Pharmacia Biotech) diluted 1:10 in water (pH 4.0) with a flow rate of 0.5 ml/min, and collected at 1-min intervals.
Strong cation exchange HPLC was performed in an Alltech SCX macrosphere column (25 cm ϫ 4.3 mm) (Alltech Associates, Deerfield, IL) using a linear gradient from 0 to 1 M NaCl in 20 mM sodium acetate buffer (pH 5.0) in 60 min with a flow rate of 0.5 ml/min; fractions were collected at 1-min intervals.
Reverse-phase HPLC chromatography was performed with a Hamilton C18 PRP-infinity column (10 cm ϫ 4.3 mm) (Alltech) using a linear gradient from 10 to 60% acetonitrile in 0.1% trifluoroacetic acid in 60 min with a flow rate of 0.5 ml/min. Eluted fractions were collected at 1-min intervals. An aliquot of each fraction was dried in the presence of 10 l of BSA (1 mg/ml), resuspended in 50 l of 100 mM Hepes saline buffer (pH 7.0), and tested for apyrase activity.
Amino acid composition analysis, tryptic digestion, and internal and N-terminal sequencing of the purified apyrase were performed at the Harvard University microchemistry facility (Cambridge, MA) under the direction of Dr. William S. Lane.
Salivary Gland cDNA Library-C. lectularius salivary gland mRNA was isolated from 500 gland pairs using the Micro-FastTrack mRNA isolation kit (Invitrogen, San Diego, CA) yielding a total of 2.8 mg of poly(A) ϩ mRNA. The cDNA library was made following the instructions for the ZAP-cDNA synthesis kit (Stratagene, La Jolla, CA). The unamplified library has a complexity of 3.5 ϫ 10 5 recombinants.
Cloning of Partial cDNA Sequence of Cimex Apyrase-For PCR experiments, mRNA from 100 pairs of C. lectularius salivary glands was isolated using the Micro-FastTrack mRNA isolation kit (Invitrogen). The mRNA was then reverse transcribed to cDNA using Superscript II RNase H Ϫ reverse transcriptase (Life Technologies, Inc.) and the primer 5Ј-GGGGAGGCTCGAGTTTTTTTTTTTTTTTT-3Ј following the manufacturer's protocol with the exception that no radioactive nucleotides were used for the reaction. The cDNA obtained was then used as a template for the PCR reactions described below.
One forward and one reverse (sense and antisense) degenerate oligonucleotide primers were designed from the N-terminal sequence of the purified salivary apyrase. The forward primer was 5Ј-TAYGARIT-NGGNCAYGCNISIGGNGARAC-3Ј (where I represents inosine), and the reverse primer was 5Ј-TCRTCYTTISINACNGCRTTYTT-3Ј. The PCR conditions were as follows: 75°C for 5 min and then 94°C for 2 min; 30 cycles of 1 min at 94°C, 1.5 min at 40°C, 1 min at 72°C; and finally 5 min at 72°C in an Ericomp PowerBlock system (San Diego, CA). The reaction mixture included 2.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris, pH 8.3, 0.01% gelatin, 0.2 mM of each dNTP, and 1.2 units of Ampli-Taq polymerase (Life Technologies, Inc.).
PCR products were separated on a 1% agarose gel, purified using the Sephaglas Bandprep Kit (Amersham Pharmacia Biotech), and cloned into a PCRII vector using the TA cloning system (Invitrogen). Competent cells were transformed following the manufacturer's protocol, and white colonies were isolated and grown overnight in LB plus ampicillin (100 g/ml) at 37°C. Plasmids from four independent clones were isolated using the Wizard Miniprep kit (Promega, Madison, WI), and the inserts were sequenced using dye terminator reactions with an automated ABI 377 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, CA), according to the manufacturer's instructions.
cDNA Library Screening and cDNA Clone Isolation-After confirming that the PCR product contained the sequence coding for the Nterminal region of the Cimex apyrase, the PCR insert was excised from the plasmid with EcoRI and cleaned as described above. The PCR insert was then labeled with dUTP-digoxigenin using now specific forward and reverse primers with the following PCR conditions: 75°C for 5 min; 94°C for 2 min; 25 cycles of 1 min at 94°C, 1.5 min at 42°C, 1 min at 72°C; and finally 5 min at 72°C. The reaction mixture included the PCR insert as a template, 2.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris, pH 8.3, 0.01% gelatin, 0.2 mM of each dNTP, DNA labeling mix (Genius system; Boehringer Mannheim), and 2 units of Ampli-Taq polymerase (Life Technologies). The 150-base pair PCR clone labeled with dUTPdigoxigenin was used to screen a C. lectularius salivary gland cDNA library. Phage plaques were lifted with a Hybond-N nylon membrane (Amersham Pharmacia Biotech) and hybridized with the digoxigeninlabeled PCR probe using the plaque hybridization protocol of the Genius system (Boehringer Mannheim). Positive plaques were picked and plated again for a secondary screening. Well isolated positive plaques were selected, and the phagemid carrying the apyrase clone was isolated from the phage using the in vivo excision protocol from the UNI-ZAP vector manual (Stratagene).
cDNA Sequence-White colonies that originated from the phagemid excision protocol were isolated and grown overnight in Luria broth plus ampicillin (100 g/ml) at 37°C. Plasmid isolation was performed using the Wizard Miniprep kit (Promega). The inserts of four isolated plasmids were sequenced as described above using the T3 and T7 primers and then by custom primers constructed from the internal sequence of the apyrase clone.
Polyclonal Antibody to C. lectularius Salivary Apyrase-A synthetic peptide (KVLIEETKIDDHKYEGVDFV) based on the predicted C-terminal region of the apyrase clone was produced at the Laboratory of Molecular Structure, NIAID, National Institutes of Health (Twinbrook Facility, Rockville, MD). Five milligrams of the synthetic peptide were conjugated to 7 mg of keyhole limpet hemocyanin at the molar coupling ratio of peptide to carrier of 122 and used to immunize rabbits at Spring Valley Laboratories (Woodbine, MD). Preimmune sample was taken before the first injection, and immune serum samples were taken after two and three injections, respectively. Antibody specificity was verified by ELISA of crude salivary homogenate and prepurified apyrase with immune and preimmune sera.
Isoelectric Focusing Gel Electrophoresis-Salivary apyrase was separated from other salivary proteins by isoelectric focusing using the PHAST system (Amersham Pharmacia Biotech) as described previously (21). Briefly, the sample eluted from a molecular sieving HPLC column was added to an applicator designed to hold 4 ml of sample. The applicator delivered the samples to a minigel with a pH range from 3 to 9, and the gel was run as described in the manufacturer's specifications.
Apyrase activity was detected by submerging the gel into a solution containing 50 mM Tris, pH 9.0, 20 mM CaCl 2 , 150 mM NaCl, and 5 mM of ADP, ATP, or AMP. The presence of a white precipitate indicated the presence of released phosphate (apyrase activity), which was complexed with calcium in the solution to form calcium phosphate precipitate in the surface of the gel.
ELISA of C. lectularius Apyrase-Salivary gland proteins eluted from a molecular sieving HPLC column were assayed with preimmune and immune serum (anti-apyrase) using conventional ELISA protocols. Dilutions of preimmune and immune serum were 1:500, and dilutions for the secondary antibody (anti-rabbit IgG peroxidase conjugate; Sigma) were 1:2000.
Western Blotting-Proteins separated by isoelectric focusing gel electrophoresis were transferred to a nitrocellulose membrane (Schleicher & Schuell) by diffusion for 40 min at 37°C, followed by blocking with 6% BSA in 20 mM Tris, pH 8.0, and 500 mM NaCl for 1 h. Primary antibody to C. lectularius salivary apyrase peptide was used at a dilution of 1:200 in 20 mM Tris, 500 mM NaCl and was incubated for 2 h at room temperature; preimmune serum was run as a control. After three washes with 20 mM Tris, pH 8.0, and 500 mM NaCl, the membrane was incubated with an anti-rabbit IgG peroxidase conjugate (1:10,000) for 1 h at room temperature. The membrane was then washed three times as described above. Bands were visualized using the 3,3Ј,5,5Ј-tetramethyl benzidine membrane peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD).
Construction of the Expression Plasmid-The expression plasmid pCI-neo (Promega, Madison, WI) was used to express C. lectularius recombinant apyrase. Recombinant protein was made without the removal of the signal peptide to allow secretion of the produced protein into the medium. C. lectularius apyrase gene was synthesized with unique XhoI and NotI restriction sites at the 5Ј-and 3Ј-ends, respectively by PCR using the following oligonucleotides: sense primer, 5Ј-GCTAGCCTCGAGATGAGGTCATCTTACAGGGTAGGGAAC; antisense primer, 5Ј-AAGGGAAGCGGCCGCTTATACAAAGTCAACACCT-TCATA. After amplification, the PCR product was cloned directly into pCRScript (Stratagene, La Jolla, CA) and sequenced. After confirmation of the sequence, the apyrase gene was isolated from the pCRScript construct by restriction with XhoI and NotI, gel-purified, and ligated into the XhoI/NotI-restricted pCI-neo (Promega).
Expression of Cimex Apyrase in COS-7 Cells-COS-7 cells (ATCC CRL 1651) were cultivated in a humidified 37°C incubator with 5% CO 2 in Dulbecco's modified Eagle's medium supplemented with 4 mM glutamine, 100 units/ml penicillin, 100 g/ml streptomycin, and 10% heatinactivated fetal calf serum (complete medium). Subconfluent COS-7 cells were transfected with cDNA for Cimex apyrase or with the pCI-neo vector-alone construct. Transfections were performed with the Bio-Rad electroporation system, using 200 V and 1000 millifarads in a 0.4-cm gap cuvette. Briefly, 50 g of DNA were used for each transfection at a cell density of 10 7 /ml. After electroporation, the cells were allowed to sit for 10 min at room temperature and then placed into 10 ml of complete medium in a 75-cm 2 culture flask at 37°C and 5% CO 2 . After a 16-h incubation, the supernatants were collected, and 10 ml of serum-free Dulbecco's modified Eagle's medium were added to the cultures. This procedure was repeated daily up to 3 days after transfection. The resultant supernatants were centrifuged at 10,000 ϫ g 5 min after collection and kept at Ϫ20°C for Western blotting and enzymatic assays. Control cells were transfected with the same pCI-neo plasmid lacking the insert, and supernatants were obtained as for the CL-Apytransfected cells.
Characterization of Recombinant Apyrase-COS-7 cell supernatant was concentrated using a Centricon Plus-20 filter (Millipore, Bedford, MA) and dialyzed against Hepes-buffered saline. Concentrated supernatant was assayed for apyrase activity as indicated above, with the modifications indicated in the figure legends. For immunoblotting, concentrated proteins of COS-7 supernatant were separated on 4 -20% SDS-polyacrylamide gel electrophoresis (Novex) and electroblotted to nitrocellulose using the Novex Xcell Blot module. Membranes were blocked with 5% dried skim milk in PBS, 0.05% Tween 20 for 1 h at room temperature. After blocking, membranes were incubated in primary polyclonal antiserum at 1:2000 dilution in 5% dried skim milk, PBS, 0.05% Tween 20 for 1 h at room temperature. Blots were incubated in secondary antibody (anti-rabbit IgG peroxidase conjugate; Sigma) at 1:10,000 dilution in 5% dried skim milk, PBS, 0.05% Tween 20 for 1 h at room temperature. Bands were visualized using the 3,3Ј,5,5Ј-tetramethyl benzidine membrane peroxidase substrate (Kirkegaard and Perry).

RESULTS
Purification of C. lectularius Salivary Apyrase-The salivary apyrase activity of C. lectularius was previously found (21) to be exclusively dependent on Ca 2ϩ , to have an optimum pH of 8.5, and to hydrolyze ATP and ADP but not AMP. Additionally, the pI of the apyrase activity was determined to be 5.1. Based on this result, C. lectularius salivary homogenates were chromatofocused on a column eluting with a pH gradient from 6.3 to 4.0. A well isolated peak with apyrase activity eluted at pH 5.1, as expected (Fig. 1). The active fractions hydrolyzed ATP (Fig.  1B) and ADP (Fig. 1C) but not AMP (Fig. 1D). The active fractions were further injected into a reverse-phase column, resulting in the separation of two UV-absorbing peaks ( Fig.   2A). Aliquots from the reverse-phase fractions, dried in the presence of 10 g BSA and resuspended in buffer, allowed apyrase activity to be detected in the small peak eluting at 45 min (Fig. 2B). An aliquot of the apyrase-containing peak was subjected to SDS-polyacrylamide gel electrophoresis and resulted in the presence of only one silver-stained band of approximately 40 kDa (not shown). The remaining material was enzymatically cleaved with trypsin, and three internal peptides with the following sequences were obtained: 1) TVEADDTTE-TYFTAFDLEGK, 2) KFETQGANVII, and 3) ALSQEAYDSK. No N-terminal sequence was attempted during this purification step.
Later, we found that using strong cation exchange HPLC at the first step resulted in even more effective purification of C. lectularius salivary apyrase. After cation exchange chromatography, both apyrase and anti-platelet activities co-eluted (Fig.  3). Additionally, this fraction inhibited collagen-induced platelet aggregation when using a low dose (5 g/ml) of collagen (not shown). The specific activity for ADP hydrolysis at this step was 379 units/mg protein (Fig. 3). The active apyrase fraction was submitted to reverse-phase chromatography. Aliquots of the fractions were dried in the presence of 10 g of BSA and tested for apyrase activity. A well isolated peak with the same retention time as in the first purification strategy showed apyrase activity (data not shown). Edman degradation of this protein revealed the sequence YELGHASGETNANSKYPLT-TPVEENLKV?FKIGVISDDDKNAVSKDESNT.
Isolation of C. lectularius Apyrase cDNA Clone-The N-terminal amino acid sequence of the purified apyrase was used to design oligonucleotide primers for a PCR reaction using as a template cDNA of C. lectularius salivary glands. A single PCR product of 150 base pairs was obtained, and its sequence was found to correspond to the N-terminal region of the native C. lectularius apyrase. This PCR product was then labeled by digoxigenin and used to screen a C. lectularius salivary gland cDNA library. Two positive clones of approximately 1.3 kilobase pairs in length were obtained and sequenced. Their sequences turned out to be identical, and each contained a single open reading frame of 1092 base pairs.
Analysis of C. lectularius Apyrase cDNA Sequence-We sequenced 1354 base pairs of the isolated apyrase clone, which contained an open reading frame coding for 364 amino acids (Fig. 4). The sequences of three internal tryptic peptides of C. lectularius apyrase were all represented in the deduced protein, and its sequence from Tyr-36 to Thr-85 coincided with the N-terminal sequence of purified C. lectularius apyrase obtained by Edman degradation (Fig. 4, underlined). The first 35 amino acids of the predicted protein were thus considered to form the signal peptide and the remaining 329 amino acids to constitute the mature protein. Indeed, sequence analysis of the unprocessed protein using the SignalP program (24)  Search of the nonredundant protein data base at the National Center for Biotechnology Information (Bethesda, MD) using the gapped BLASTP program (26) did not find any statistically significant similarity between the predicted sequence of the C. lectularius salivary apyrase and sequences of any characterized enzymes, including other apyrases (5, 14 -16). Direct comparison of the C. lectularius salivary apyrase with other apyrases, such as CD39 (12,15), potato apyrase (9), or A. aegypti salivary apyrase (5) using the MACAW (27), BLAST 2.0 (26), 2 and Lalign (28) programs also did not reveal any significant similarity or conserved sequence motifs.
Anti-apyrase Antibody Recognized C. lectularius Salivary Apyrase-Because the sequence of the C. lectularius apyrase was not similar to other apyrases, we made antibodies against the predicted sequence of the putative carboxyl-terminal region (KVLIEETKIDDHKYEGVDFV) of the C. lectularius apyrase to investigate whether antigenic activity would co-elute with apyrase activity in different protein separation protocols. Accordingly, when salivary gland homogenate of C. lectularius was subjected to molecular sieving HPLC column, a chromatographic procedure different from those used above to purify the protein, the apyrase activity co-eluted with immunoreactive activity (Fig. 5). Western blot analysis also confirmed recognition by the anti-serum of antigen co-localizing with apyrase activity in isoelectric focusing gels (Fig. 6). An antiserum against the C-terminal region predicted by CL-Apy cDNA thus recognized an antigen co-eluting with apyrase activity by two independent protein separation methods.
Expression of Cimex Apyrase cDNA in COS-7 Cells-Evidence presented so far strongly indicates that the 1.3-kilobase pair cDNA described in this work represents a salivary cDNA expressing a protein with apyrase activity. However, it could be derived from a co-purified molecule deprived of apyrase activity. In order to test whether the obtained CL-Apy cDNA sequence would translate into a protein with apyrase activity, we expressed it in COS-7 cells. While control COS-7 cells supernatants displayed (Ca 2ϩ , Mg 2ϩ )-dependent nucleotidase activity (Fig. 7, white bars), CL-Apy cDNA-transfected cells additionally produced an apyrase activity that was exclusively Ca 2ϩdependent (Fig. 7, dark bars) and with the same pI as the native enzyme (Fig. 8). Moreover, Western blots of transfected COS-7 cell supernatants yielded antigenic material recognized by the anti-apyrase antiserum generated against the C-terminal region of the putative apyrase gene product (Fig. 9). The immunoreactive band resulting from the recombinant protein had an apparent molecular weight slightly larger than that observed with the native antigen ( Fig. 9) and could be the result of protein modification (e.g. glycosylation, see below) by the host cells. We conclude that the CL-Apy cDNA depicted in Fig. 4 codes for a protein displaying apyrase activity.
Sequence Analysis of Cimex Apyrase-As noted above, sequence similarity searches of the National Center for Biotechnology Information protein data base did not show any statistically significant matches between C. lectularius salivary apyrase and any previously characterized enzymes. In fact, the only protein in the data base that showed any significant (p Ͻ 0.1) similarity to C. lectularius apyrase (Fig. 10) was an unknown protein from the nematode worm Caenorhabditis elegans (National Center for Biotechnology Information accession no. 868189), discovered in the course of the C. elegans sequencing project at Washington University (St. Louis, MO). Analysis of the GenBank TM expressed sequence tag (EST) data base, however, showed that proteins homologous to C. lectularius apyrase are also encoded in mouse and human genomes (Fig. 10) and are actually expressed in these organisms. Most of the human and mouse EST, however, corresponded only to the C-terminal part of the C. lectularius apyrase, which could be due to mRNA degradation or incomplete reverse transcription in the process of making these ESTs.
The alignment of the predicted protein sequences (Fig. 10) shows several highly conserved regions that could be related to the catalytic activity of C. lectularius apyrase. In particular, conserved Asp residues in the conserved motif DDRTG, located in a short loop between two ␤-strands, could play a role in binding Ca 2ϩ ions, which are required for C. lectularius apyrase activity (Fig. 6). Remarkably, none of the conserved regions highlighted in Fig. 10 corresponds to any previously FIG. 5. Immunodetection of C. lectularius salivary apyrase. A, salivary gland homogenate was separated by molecular sieving HPLC. B, the eluted fractions were tested for apyrase activity and immunoassayed by ELISA with anti-apyrase antibody (D) and control serum (C) and developed using peroxidase-conjugated secondary antibody.
FIG. 6. Western blot of native salivary apyrase separated by isoelectrofocusing gel electrophoresis. C. lectularius salivary gland proteins were separated by isoelectric focusing gel electrophoresis with a pH gradient from 3 to 9. Apyrase activity was detected by the formation of calcium phosphate precipitate (left panel, lanes 1 and 2). Proteins were transferred to nitrocellulose paper and incubated with preimmune (right panel, left side) or immune (right panel, right side) serum. pI standards are marked at the left.
described sequence motif. Conversely, scanning the C. lectularius apyrase sequence against the PROSITE and Pfam data bases (29,30) identified potential glycosylation, phosphorylation, and N-myristoylation sites but did not reveal any previously described nucleotide-binding sequence motifs. A direct comparison of the C. lectularius apyrase with several recently described generalized ATP-binding motifs, such as ATP-grasp (31) or P-ATPase (32), also failed to identify its nucleotidebinding motif. These observations indicate that C. lectularius apyrase and its homologs constitute a novel family of enzymes that contains new type of nucleotide-binding site. DISCUSSION We have purified, cloned, and expressed a soluble apyrase from the salivary glands of the bed bug, C. lectularius. Since the sequence of this enzyme is substantially different from that of any other reported apyrase, special efforts were undertaken to verify that the sequenced cDNA clone (Fig. 4) actually codes for C. lectularius apyrase.
The sequence obtained for C. lectularius apyrase was based on two independent purification protocols (chromatofocusing and reverse-phase HPLC or strong cation exchange and reverse-phase HPLC) that yielded different segments of the primary protein structure, all contained within the translated cDNA sequence. An antiserum against the putative carboxylterminal peptide, obtained from the information contained in the cDNA clone, recognized the apyrase-containing fractions in two different immunoassays (ELISA of samples from molecular sieving fractionation and the Western blot of salivary homogenate separated by isoelectric focusing gel electrophoresis). The final confirmation of the validity of the cDNA as coding for an apyrase was indicated by the expression and secretion by transfected COS-7 cells of a Ca 2ϩ -dependent apyrase with molecular weight, pI, and immunoreactivity similar to the native C. lectularius protein.
Although somewhat unexpected, the identification of a novel type of apyrase in C. lectularius is hardly surprising. The existence of enzymes that have arisen independently to have a common activity has been repeatedly observed before in different enzyme groups, e.g. proteases (33) and glycosidases (34) (see Refs. 35 and 36 for discussion). A comprehensive analysis of such analogous, as opposed to homologous, proteins shows that they are found in at least 5% of all of the enzyme classification nodes for which sequences are currently available (36). In many cases, such analogous enzymes seem to evolve by recruitment of enzymes acting on different but related substrates, i.e. by minor structural change of a protein that leads to a novel substrate specificity or even a new class of reactions  , lanes 1 and 2). Salivary gland homogenate was used to mark the location of the native enzyme (APY-glands). Proteins were transferred to nitrocellulose paper and incubated with immune serum. Molecular weight standards are marked at the right. (36,37).
The selective pressure to counteract host hemostasis mechanisms, coupled with independent adaptation to hematophagy in different groups of blood-sucking arthropods makes their salivary glands a place where the recruitment scenario could be particularly advantageous. Indeed, while apyrases from plants, vertebrates, and the protozoan parasite T. gondii are closely related to ATPases, the enzyme from the mosquito A. aegypti appears to have evolved from 5Ј-nucleotidases (5). Structurally, shifting from AMP to ADP, the substrate specificity of the enzyme that still catalyzes the same reaction (cleaving of the terminal phosphate) should not take too many amino acid substitutions. In the E-ATPase family, ecto-ATPases and ectoapyrases differ by only several amino acid residues (8,16). Recently sequenced apyrase from the blood-sucking insect Rhodnius prolixus (38) 3 did not show any sequence similarity to either mosquito apyrase or E-ATPases (36). Instead, it turned out to be closely related to inositol-1,4,5-triphosphate 5-phosphatase (EC 3.1.3.56). Again, the substrates of these enzymes are structurally similar, and some minor structural changes could have converted inositol triphosphatase into an apyrase.
The C. lectularius apyrase described in this work has no homology to any of the above-mentioned enzymes and thus belongs to yet another family of apyrases. Remarkably, it has homologs (Fig. 10) in worm and human genomes, which also code for apyrase members of the E-ATPase family (15,16). The functions of these proteins still remain obscure. It is interesting to note, however, that most of the ESTs that had coded for mouse and human homologs of C. lectularius apyrase have been prepared from fetal or tumor tissues (see legend to Fig.  10). Extracellular adenosine and AMP have been implicated in regulation of cell motility (39), and a major tumor cell motilitystimulating protein, ataxin, has recently been shown to have ATP pyrophosphatase and ATPase activities (40). While evolu-tionarily unrelated to the enzymes of the ataxin-PC-1 family (37), human and mouse homologs of C. lectularius apyrase could have a related role in cell progression. FIG. 10. Homologs of C. lectularius apyrase in worm, mouse, and human. The worm protein is the F08C6.6 gene product from C. elegans (GenBank TM accession number U29378); mouse and human homologs have been assembled from the translated expression sequences tags (ESTs) with GenBank TM accession numbers AA008851, AA125090, and AA396665 (mouse) and AA632390, AA337541, AA311735, AA075972, AA35-6674, AA337180, AA348005, AA112425, and AA074378 (human). Reverse shading indicates stretches of conserved amino acid residues that could be involved in catalytic activity; other conserved residues are shown in boldface type. Gray shading indicates uncharged amino acid residues (A, I, L, V, M, F, Y, or W) with a propensity to form ␤-strand. Secondary structure elements predicted by the PHD program (41) are indicated as H (␣-helices), E (␤-strands), and L (loop regions); positions of conserved Pro and small residues (G, A, or S) are indicated by asterisks.