The Salivary Adenosine Deaminase from the Sand Fly Lutzomyia longipalpis

Experimental Parasitology 95, 45–53 (2000) doi:10.1006/expr.2000.4503, available online at http://www.idealibrary.com on

The Salivary Adenosine Deaminase from the Sand Fly Lutzomyia longipalpis

Rosane Charlab,*,1 Edgar D. Rowton,† and Jose´ M. C. Ribeiro* *Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4, Room 126, 4 Center Drive, MSC-0425, National Institutes of Health, Bethesda, Maryland 20892-0425; and †Department of Entomology, Walter Reed Army Institute of Research, Washington, DC

Charlab, R., Rowton, E. D., and Ribeiro J. M. C. 2000. The salivary adenosine deaminase from the sand fly Lutzomyia longipalpis. Experimental Parasitology 95, 45–53. In the process of sequencing a subtracted cDNA library from the salivary glands of the sand fly Lutzomyia longipalpis, we identified a cDNA with similarities to gene products of the adenosine deaminase family. Prompted by this cDNA finding, we detected adenosine deaminase activity at levels of 1 U/mg protein in salivary gland homogenates. The activity was significantly reduced following a blood meal indicating its apparent secretory fate. The native enzyme has a Km of ,10 mM, an isoelectric pH between 4.5 and 5.5, and an apparent molecular weight of 52 kDa by size exclusion chromatography. The possible role of this enzyme, which converts adenosine to inosine, in the feeding physiology of L. longipalpis is discussed. Index Descriptors and Abbreviations: Lutzomyia longipalpis; Phlebotomine sand flies; blood-feeding insects; salivary glands; adenosine deaminase (EC 3.5.4.4); adenosine; inosine.

vasodilators continuously stimulates the search for and isolation of novel and potent pharmaceuticals from such animals (Champagne, 1994; Ribeiro, 1987, 1995). However, the majority of the compounds found so far were discovered after using a particular bioassay sensitive to the presence of antihemostatic activities in the salivary gland extracts, followed by purification of the active component(s). Discovery of bioactive salivary gland molecules has been thus limited and bound by the question asked or the bioassay used. Mass sequencing of salivary gland cDNA libraries, on the other hand, may lead to finding sequences that may code for unexpected products (Charlab et al., 1999). We report in this paper the sequence of a salivary gland cDNA with similarities to adenosine deaminases. Based on this finding, we confirmed that the salivary glands of Lutzomyia longipalpis express large amounts of this enzyme, which is significantly reduced following a blood meal, indicating its secretory fate. Adenosine deaminase converts the vasodilatory, antiplatelet, and immunosuppressive molecule adenosine (Dionisotti et al., 1992; Edlund et al., 1987; Seegmiller et al., 1977; Urquhart and Broadley, 1991; Webster, 1984) into inosine, which also has multiple antiinflammatory effects (Hasko et al., 2000). Adenosine can be produced at sites of feeding by the combined action of salivary apyrase (which hydrolyzes ATP and ADP to AMP) (Ribeiro et al., 1989; Charlab et al., 1999) and salivary 58-nucleotidase (Charlab et. al., 1999; Ribeiro et al., 2000). The possible role of salivary adenosine deaminase in feeding is discussed.

INTRODUCTION

Salivary antihemostatic and immunosuppressive compounds have evolved independently for almost every family of blood-sucking arthropods (Ribeiro, 1995). The encountered diversity in salivary anticoagulants, antiplatelets, and 1

To whom correspondence should be addressed. FAX: (301) 4024941; E-mail: [email protected].

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46 MATERIALS AND METHODS

Chemicals were purchased from Sigma Chemical Corporation (St. Louis, MO). All water used was of 18 MV quality and was produced by a MilliQ apparatus from Millipore (Bedford, MA). Lutzomyia longipalpis (Jacobina strain) sand flies were reared at the Walter Reed Army Institute of Research on a fermented mixture of rabbit chow and rabbit feces as described previously (Modi and Tesh, 1983). Adult sand flies were kept with free access to a 10% solution of sucrose unless otherwise specified. For bioassays, salivary glands from 3- to 10-day-old adult flies were dissected and transferred to 10 or 20 ml 10 mM Hepes, pH 7.0, 1 0.15 M NaCl in 1.5-ml polypropylene vials, usually in groups of 20 pairs of glands in 20 ml Hepes saline or individually in 10 ml Hepes saline. To detect enzyme activity distribution between the lumen and the cellular portion of the glands, salivary gland pairs were transferred to individual wells of a 96-well polystyrene microplate containing 100 ml Hepes saline. Each sacular gland was punctured with fine needles and the contents were allowed to evacuate. The empty sacs were transferred to a polypropylene conical tube containing 100 ml Hepes saline, and the remaining solution, containing luminal contents, was transferred to another tube. Salivary glands were kept at 2758C until needed, when they were disrupted by sonication using a Branson Sonifier 450 homogenizer (Danbury, CT). Salivary homogenates were centrifuged at 10,000g 3 2 min and the supernatants were used for the experiments. For mRNA isolation, salivary glands and carcasses were dissected from female flies at 0–1 day after emergence, transferred to 20 ml sterile Dulbecco’s phosphate-buffered saline (GIBCO) or Hepes saline in RNase/DNase-free 1.5-ml polypropylene tubes, and kept at 2758C until thawed and homogenized in lysis buffer (Micro-FastTrack 2.0 mRNA isolation kit, Invitrogen, San Diego, CA). For manufacture of a subtracted cDNA library, mRNA was isolated from 123 pairs of salivary glands and 54 carcasses of L. longipalpis sand flies using the Micro-FastTrack mRNA isolation kit according to manufacturer’s instructions (Invitrogen, San Diego, CA). The PCRSelect cDNA Subtraction Kit (Clontech, Palo Alto, CA) was used to generate a subtracted cDNA library enriched for salivary gland-specific sequences. Briefly, tester and driver cDNAs were prepared from salivary gland mRNA and carcass mRNA, respectively, using the SMART PCR cDNA synthesis methodology (Clontech). Then, tester and driver cDNAs were hybridized, and the unhybridized tester sequences were selectively amplified by PCR. The subtracted cDNAs were cloned into pPCR-Script cloning vector from Stratagene (La Jolla, CA). Competent cells were transformed following the manufacturer’s instructions, and white colonies were isolated and grown overnight in LB plus 100 mg/ ml ampicillin at 378C. Plasmids 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) following the manufacturer’s instructions. The cDNA insert with similarity to adenosine deaminase was used as a probe to isolate the correspondent fulllength clone from a L. longipalpis cDNA salivary gland library as previously described (Charlab et al., 1999, Ribeiro et al., 2000). Two full-length clones were sequenced as previously described (Valenzuela et al., 1998). Analysis of the predicted protein sequence was done using the BLAST programs with BLOSUM-62 matrix (http://www.ncbi.nlm. gov/BLAST/) (Altschul et al., 1997), the sequence analysis services programs (http://molbio.info.nih.gov) at the computational molecular

CHARLAB, ROWTON, AND RIBEIRO

biology Internet site at the National Institutes of Health, the SMARTSimple Modular Architecture Research tool (Schultz et al., 1998) and Pfam database for domain identification (Bateman et al., 2000), and the Compute pI/Mw tool (http://www.expasy.ch/tools/pi tool.html). Measurement of adenosine deaminase activity was done in quartz microcuvettes holding 60-ml samples (Starna Cells, Atascadero, CA). Indicated concentrations of adenosine in 10 mM Hepes 1 150 mM NaCl (HS) (or as otherwise indicated buffer) were added to the cuvette, followed by addition of salivary gland homogenates (usually equivalent to 5–10% of one pair of glands, or 50–100 ng protein). After mixing the solution by several cycles of aspiration (30 ml) and deposition of the sample, the absorbance at 265 nm or the absorbance at both 241 and 265 nm was read at 5-s or 15-s intervals, respectively, using a Lambda 18 spectrophotometer from Perkin–Elmer (Norwalk, CT). The following buffers were used for pH dependence of the adenosine deaminase activity: acetate, pH 4 and 5.0; MES, pH 6.0; Hepes, pH 7.0; and Tris, pH 8.0 and 9.0. Chloride or sodium salts of the buffers were used. All buffers were used at a final concentration of 50 mM in a medium additionally containing 0.1 M NaCl plus the indicated concentration of substrate and enzyme source. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the deamination of 1 mmole adenosine per minute (2DA 5 8.6 min21 ml21 at 265 nm) (Agarwal and Parks, 1978). Gel permeation chromatography experiments were performed with Super TSK-2000SW (4 mm 3 25 cm; TosoHaas, Montgomeryville, PA) isocratically perfused with 10 mM Hepes, pH 7.0, 1 0.6 M NaCl at a flow rate of 0.2 ml/min. A ThermoSeparation product (Riviera Beach, FL) CM4100 pump and SM 4100 UV detector were used. Fractions were collected at 30-s intervals. Ten microliters of each fraction was used to determine adenosine deaminase activity in the assay described above. The column was calibrated with cytochrome C, myoglobin, carbonic anhydrase, ovalbumin, and the monomer and dimer of bovine serum albumin. Isoelectric focusing electrophoresis was done in a Pharmacia Phast System (Pharmacia, Uppsala, Sweden) using a 3–9 pH gradient gel according to the manufacturer’s recommendation. Standards provided by Pharmacia were used to calibrate the gels. One pair of glands in 4 mL was applied to the gel. After the run, 2.5 3 8-mm portions of one of the gel lanes were excised and transferred to 1.5-ml conical polypropylene tubes. To each tube, 50 ml HS was added, and the tubes were agitated at room temperature for 60 min. Following centrifugation at 10,000g 3 2 min, 30 ml of the supernatant was transferred to a quartz microcuvette containing 30 ml of 100 mM adenosine in 100 mM Hepes buffer, pH 7.2, and the decrease in absorbance at 265 nm was measured for 5 min.

RESULTS While sequencing a cDNA library from salivary glands of adult female L. longipalpis sand flies that was subtracted from common body sequences (Charlab et al., 1999), we identified a 368-bp cDNA fragment coding for an open reading frame of 122 amino acids with significant similarity to insect-derived growth factor (IDGF) from Sarcophaga peregrina flesh fly (GenBank D83125), to salivary growth

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SALIVARY ADA OF L. Longipalpis

factors 1 (GenBank AF140521) and 2 (GenBank AF140522) from Glossina morsitans morsitans, and to mollusk-derived growth factor (MDGF) from Aplysia californica (GenBank AF117336). Significant sequence similarity was also found between the Lutzomyia partial cDNA clone and adenosine deaminase from Streptomyces. IDGF, Glossina salivary growth factors 1 and 2, and MDGF also showed similarity to adenosine deaminases, when their sequences were compared to GenBank deposited sequences using the BLAST program (Altschul et al., 1997). Using the partial clone sequence obtained in the subtracted library as a probe, we screened a full-length salivary gland cDNA library for the L. longipalpis salivary adenosine deaminase. We isolated a 1789-bp full-length cDNA clone (GenBank AF234182) with sequence similarity to adenosine deaminases that included the partial cDNA clone sequence. The open reading frame encodes a sequence of 508 amino acid residues. Sequence analysis of the predicted protein identified a potential signal peptide with a cleavage site between residues 18 and 19 (Nielsen, et al., 1997). The mature protein has a molecular weight of 56,428 Da and a pI of 5.58. A Pfam Adenosine/ AMP deaminase (A deaminase) domain with an E value of 6.3 e-79 was identified in the predicted protein sequence starting at residue 101 and ending at residue 494 (Fig. 1). Adenosine has antihemostatic activities (Dionisotti et al., 1992; Edlund et al., 1987; Urquhart and Broadley, 1991). Furthermore, ecto-adenosine deaminase has a costimulatory functional role in lymphocytes (De Meester et al., 1994; Franco et al., 1998) and modulates adenosine receptor affinity to adenosine (Saura et al., 1996). Given these facts, we decided to search for ADA activity in Lutzomyia salivary gland extracts. Accordingly, salivary homogenates were incubated with adenosine and scanned at regular intervals in the UV range of 220–300 nm. Incubation of 0.1 pairs of salivary glands in 60 ml of the reaction mixture led to change of the spectra from a maximum of 258 nm to a maximum at 248 nm (Fig. 2A), with differential spectra showing a maximum at 241 and a minimum at 265 nm (Fig. 2B), as expected for the conversion of adenosine to inosine (Agarwal et al., 1975). Conversion of adenosine proceeded linearly for more than 50% of the reaction, which achieved near completion in 1 h (Fig. 2C). To rule out the possibility that the adenosine deaminase activity found in the salivary gland homogenate could be due to a housekeeping product of low significance to the feeding physiology of the fly, we measured the amounts of enzyme in the salivary glands before and after feeding and also the distribution of the enzyme in the lumen and in the cellular gland contents. Results indicate that the enzyme is significantly lost after a blood meal (Fig. 3A) and that ,80%

of the activity is found in the gland lumen (Fig 3B). Both results indicate that salivary ADA is of a secretory fate. To further characterize the salivary adenosine deaminase activity, we determined the enzyme Km and its activity over a pH range. The substrate dependence followed a hyperbolic function, yielding a straight line on double reciprocal plots (Fig. 4). The measured Km , determined by double reciprocal plots, was 8.4 6 0.96 with a Vmax of 1.45 6 0.003 U/mg protein (mean 6 SE, n 5 3). The activity had a broad pH optimum from pH 5 to 8 (Fig. 5). Size exclusion chromatography of salivary homogenates yielded a single peak of activity indicating a molecular weight of 52 kDa with a range of 47–57 kDa based on the maximum and minimum retention times of the most active fraction, as deducted from the calibration of the column as shown in the inset of Fig. 6. Isoelectric focusing electrophoresis experiments indicate that the salivary adenosine deaminase has a pI between 4.5 and 5.5 (Fig. 7).

DISCUSSION

In this paper we report for the first time the presence of an adenosine deaminase activity in the salivary glands of a blood-sucking insect. The secretory nature of this enzyme is indicated by the reduction of the enzyme activity following a blood meal and by its predominance in the gland lumen (Fig. 3). Considering that individual salivary glands have ca. 1 mg of protein (Ribeiro et al., 1986) and that the activity per pair of glands is on the order of 1 mU, we estimate the salivary-specific activity to be on the order of 1 U/mg crude protein. For comparison, red blood cell hemolysates contain 0.00023 U/mg of protein (Agarwal and Parks, 1978), Escherichia coli homogenates have 0.142 U/mg protein (Nygaard, 1978), and the crude powder of calf intestinal mucosa as sold by Sigma Chemical Company has 1–5 U/mg of protein. The amounts of adenosine deaminase in the salivary glands of L. longipalpis thus appear to be similar to those in cells or tissues adapted to an active purine catabolism. Our search for this enzyme activity in salivary homogenates was prompted by the finding of a cDNA clone with similarity to adenosine deaminase. The estimated molecular weight (52 kDa—fig. 6), Km (Fig. 4), and broad pH optima (Fig. 5) found for the native Lutzomyia salivary ADA are typical of this family of enzymes (Maguire and Sim, 1971; Wolfenden et al., 1967) and are in agreement with the MW (56,428 Da) and pI (5.58) of the predicted Lutzomyia salivary protein with similarity to adenosine deaminases.

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FIG. 1. Nucleotide and deduced amino acid sequences of the L. longipalpis salivary gland full-length cDNA clone (GenBank Accession No. AF234182) with similarity to adenosine deaminases. The signal peptide predicted by the SignalP program (Nielsen et al., 1997) is underlined. The A deaminase Pfam (Bateman et al., 2000) domain is highlighted.

SALIVARY ADA OF L. Longipalpis

49 molecule (Dionisotti et al., 1992; Edlund et al., 1987; Seegmiller et al., 1977; Urquhart and Broadley, 1991; Webster, 1984), converting it to inosine, which is also an immunosuppressive purine (Hasko et al., 2000), but it is much less

FIG. 2. Lutzomyia longipalpis salivary homogenate displays adenosine deaminase activity. (A) Superimposed UV scans taken at 4-min intervals following addition of 20 mM adenosine to a reaction mixture containing the equivalent of 0.1 pairs of homogenized glands, 10 mM Hepes buffer pH 7.0 1 150 mM NaCl. The baseline of the apparatus was zeroed before addition of adenosine. (B) Differential scanning of the data in A. (C) Time dependence of the increase in absorbance at 241 nm minus the decrease in absorbance at 265 nm.

Lutzomyia longipalpis salivary glands contain large amounts of apyrase and 58-nucleotidase enzymes (Ribeiro et al., 1989, 1986; Charlab et al., 1999; Ribeiro et al., 2000). Apyrase converts ATP and ADP, released by injured cells at the feeding site, to AMP. AMP is converted to adenosine by 58-nucleotidase. Adenosine deaminase would convert adenosine to inosine. The presence of adenosine deaminase in the saliva of blood-sucking animals may appear paradoxical at a first glance. ADA destroys adenosine, an immunosuppressive, vasodilatory, and platelet aggregation inhibitory

FIG. 3. Comparison of L. longipalpis salivary gland adenosine deaminase activity in pre- and post-blood-fed flies (A) and in the lumen and cellular contents of the salivary glands (B). Cuvettes containing adenosine (20 mM) in 10 mM Hepes pH 7.0 1 150 mM NaCl were added to 1/4 pairs of homogenized salivary glands and the absorbance at 265 nm was read at 5-s intervals for 5 min to determine the enzyme activity.

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FIG. 4. Dependence on the substrate concentration of the salivary adenosine deaminase activity of L. longipalpis. Symbols and bars represent the means 6 SE of a triplicate experiment using a pool of five homogenized pairs of salivary glands. (Inset) Double reciprocal plot of the data.

active in terms of vasodilatation (Chinellato et al., 1994). However, in addition to the immunosuppressive effect of inosine, L. longipalpis saliva contains the immunosuppressive vasodilator maxadilan (Lerner et al., 1991; Qureshi et al., 1996) and the platelet inhibitor apyrase (Ribeiro et al., 1986) that could contribute to these effects. On the other hand, adenosine is a peripheral nociceptive agent (Burnstock and Wood, 1996) (a pain inducer in peripheral nerve receptors), and its removal from the biting site could prevent or attenuate perception of the host to the fly bite. Of particular interest is to determine whether salivary adenosine deaminase is playing some other role besides its immediate enzymatic activity. Adenosine deaminase has been the target of intense research since it was discovered that lack of this enzyme causes a genetic form of immunodeficiency in both mice and humans. Indeed, adenosine deaminase deficiency is the first known cause of severe combined immunodeficiency disease (SCID) (Hershfield, 1998). It is postulated that lack of this enzyme causes apoptosis of T cells due to accumulation of adenosine in the immediate vicinity of blasting lymphocytes (Hershfield, 1998). However, other roles for ADA have been identified (Franco et al., 1998). ADA binds to CD26, a surface lymphocyte protein (De Meester et al., 1994), and exercises a costimulatory function independent of its enzymatic activity (Martin et al., 1995). Perhaps Lutzomyia salivary ADA is interfering with lymphocyte function.

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Interestingly, the highest significant similarity to the translated cDNA full-length clone reported in this paper went to IDGF (GenBank D83125) of Sarcophaga peregrina (E value, e-138), a homodimeric growth factor believed to participate in the embryogenesis of this fly (Homma et al., 1996). Highly significant matches, using the Blastp program at NCBI, to mollusk-derived growth factor (MDGF; GenBank AF117336) and atrial gland-specific antigen (AGSA; GenBank J05059), both from Aplysia californica (E values, 6e-96 and 8e-47, respectively), and to translation products from salivary glands of Glossina morsitans morsitans, named salivary gland growth factors 2 (GenBank AF140522) and 1 (GenBank AF140521) (E values, 2e-92 and 5e-74, respectively) were also observed. The above sequences, except for AGSA, also share significant similarities with several adenosine deaminases. They also contain an A deaminase Pfam domain and may represent new members of the adenosine deaminase multifunctional family. Although adenosine deaminases have not been assigned as classical growth factors so far, there are indications that these enzymes may mediate specific interactions between cells relevant to the development of both mammalian nervous and lymphoid systems, as indicated above. Additionally, they may exercise their growth factor capability by destroying the apoptotic molecule adenosine.

FIG. 5. pH dependence of the salivary adenosine deaminase activity of L. longipalpis. Symbols and bars represent the means 6 SE of a triplicate experiment using a pool of five homogenized pairs of salivary glands.

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FIG. 6. Size exclusion chromatography of 25 pairs of homogenized glands from L. longipalpis. Symbols indicate the adenosine deaminase activity, and the continuous line indicates the UV absorption at 280 nm. In the inset the arrow indicates the retention time of the enzymatic activity relative to the molecular weight markers used. For details see Materials and Methods.

ADA also binds to the adenosine receptor A1, increasing the receptor’s sensitivity toward adenosine (Saura et al., 1996). Because the receptor’s affinity is much higher than the enzyme’s Km , the final effect of the binding of ADA to the A1 receptor would result in a higher sensitivity to small

concentrations of adenosine. These extraenzymatic roles of ADA could contribute to the immunosuppressor role of Lutzomyia saliva or to the increased agonist sensitivity in some subtypes of adenosine receptor. These ideas remain to be tested.

FIG. 7. Isoelectric focusing electrophoresis of one pair of glands of L. longipalpis indicating a pI between 4.5 and 5.5 for the salivary adenosine deaminase activity (•). The pI of markers is indicated by the open symbols (C). For more details see text.

52 Finally, it is interesting to contrast the divergent purinergic salivary strategies found in the New World L. longipalpis sand fly with that of the Old World Phlebotomus papatasi. The Old World fly has pharmacological amounts of adenosine and AMP and no maxadilan (Ribeiro et al., 1999). Adenosine and AMP may help sand fly feeding by their vasodilatory and antiplatelet properties (Ribeiro et al., 1999; Dionisotti et al., 1992; Edlund et al., 1987; Seegmiller et al., 1977; Urquhart and Broadley, 1991; Webster, 1984). This is in contrast with the “zero tolerance” for adenosinebased agonists found in L. longipalpis, as described above. These sand fly genera diverged before separation of the continents and thus before mammal irradiation. Their salivary pharmacology thus reflects their independent evolution to solve problems posed by their new mammalian hosts. Because we proposed that ADA may remove nociceptive agonists from the site of feeding by Lutzomyia, and because P. papatasi actually has salivary adenosine, an experiment could be done to test the perceived pain induced by the bite of these two sand fly species. This idea also remains to be tested.

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of rabbit thoracic aorta: common and different sites of action. Journal of Pharmacy and Pharmacology 46, 337–341. De Meester, I., Vanham, G., Kestens, L., Vanhoof, G., Bosmans, E., Gigase, P., and Scharpe, S. 1994. Binding of adenosine deaminase to the lymphocyte surface via CD26. European Journal of Immunology 24, 566–570. Dionisotti, S., Zocchi, C., Varani, K., Borea, P. A., and Ongini, E. 1992. Effects of adenosine derivatives on human and rabbit platelet aggregation. Correlation of adenosine receptor affinities and antiaggregatory activity. Naunyn Schmiedebergs Archives of Pharmacology 346, 673–676. Edlund, A., Siden, A., and Sollevi, A. 1987. Evidence for an antiaggregatory effect of adenosine at physiological concentrations and for its role in the action of dipyridamole. Thrombosis Research 45, 183–190. Franco, R., Valenzuela, A., Lluis, C., and Blanco, J. 1998. Enzymatic and extraenzymatic role of ecto-adenosine deaminase in lymphocytes. Immunology Review 161, 27–42. Hasko, G., Kuhel, D. G., Nemeth, Z. H., Mabley, J. G., Stachlewitz, R. F., Virag, L., Lohinai, Z., Southan, G. J., Salzman, A. L., and Szabo, C. 2000. Inosine inhibits inflammatory cytokine production by a posttranscriptional mechanism and protects against endotoxininduced shock. Journal of Immunology 164 (2), 1013–1019. Hershfield, M. S. 1998. Adenosine deaminase deficiency: Clinical expression, molecular basis, and therapy. Seminars in Hematology 35, 291–298.

REFERENCES

Agarwal, R. P., and Parks, R. E., Jr. 1978. Adenosine deaminase from human erythrocytes. Methods in Enzymology 51, 502–507. Agarwal, R. P., Sagar, S. M., and Parks, R. E., Jr. 1975. Adenosine deaminase from human erythrocytes: Purification and effects of adenosine analogs. Biochemistry and Pharmacology 24, 693–701.

Homma, K., Matsushita, T., and Natori, S. 1996. Purification, characterization, and cDNA cloning of a novel growth factor from the conditioned medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Journal of Biological Chemistry 271, 13,770– 13,775. Lerner, E. A., Ribeiro, J. M. C., Nelson, R. J., and Lerner, M. R. 1991. Isolation of maxadilan, a potent vasodilatory peptide from the salivary glands of the sand fly Lutzomyia longipalpis. Journal of Biological Chemistry 266, 11,234–11,236.

Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. 1997. Gapped BLAST and PSIBLAST: A new generation of protein database search programs. Nucleic Acids Research 25, 3389–402.

Maguire, M. H., and Sim, M. K. 1971. Studies on adenosine deaminase. 2. Specificity and mechanism of action of bovine placental adenosine deaminase. European Journal of Biochemistry 23, 22–29.

Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Howe, K. L., and Sonnhammer, E. L. 2000. The Pfam Protein Families Database. Nucleic Acids Research 28, 263–266.

Martin, M., Huguet, J., Centelles, J. J., and Franco, R. 1995. Expression of ectoadenosine deaminase and CD26 in human T cells triggered by the TCR-CD3 complex. Possible role of adenosine deaminase as costimulatory molecule. Journal of Immunology 155, 4630–4643.

Burnstock, G., and Wood, J. N. 1996. Purinergic receptors: Their role in nociception and primary afferent neurotransmission. Current Opinions in Neurobiology 6, 526–532. Champagne, D. E. 1994. The role of salivary vasodilators in bloodfeeding and parasite transmission. Parasitology Today 10, 430–433.

Modi, G. B., and Tesh, R. B. 1983. A simple technique for mass rearing Lutzomyia longipalpis and Phlebotomus papatasi (Diptera: Psychodidae) in the laboratory. Journal of Medical Entomology 20, 568–569. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. 1997. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6.

Charlab, R., Valenzuela, J. G., Rowton, E. D., and Ribeiro, J. M. C. 1999. Toward an understanding of the biochemical and pharmacological complexity of the saliva of a hematophagous sand fly Lutzomyia longipalpis. Proceedings of the National Academy of Sciences USA 96, 15,155–15,160.

Nygaard, P. 1978. Adenosine deaminase from Escherichia coli. Methods in Enzymology 51, 508–512.

Chinellato, A., Ragazzi, E., Pandolfo, L., Froldi, G., Caparrotta, L., and Fassina, G. 1994. Purine- and nucleotide-mediated relaxation

Qureshi, A. A., Ohnuma, M. T. M., Granstein, R. D., and Lerner, E. A. 1996. Immunomodulatory properties of maxadilan, the vasodilator

SALIVARY ADA OF L. Longipalpis

53

peptide from sand fly salivary gland extracts. American Journal of Tropical Medicine and Hygiene 54, 665–671.

adenosine receptors in pig brain cortical membranes. Journal of Neurochemistry 66, 1675–1682.

Ribeiro, J. M. C. 1987. Role of arthropod saliva in blood feeding. Annual Review of Entomology 32, 463–478.

Seegmiller, J. E., Watanabe, T., and Schreier, M. H. 1977. The effect of adenosine on lymphoid cell proliferation and antibody formation. Ciba Foundation Symposium 48, 249–276.

Ribeiro, J. M. C. 1995. Blood-feeding arthropods: Live syringes or invertebrate pharmacologists? Infectious Agents and Diseases 4, 143–152. Ribeiro, J. M. C., Modi, G. B., and Tesh, R. B. 1989. Salivary apyrase activity of some old world phlebotomine sand flies. Insect Biochemistry 19, 409–412. Ribeiro, J. M. C., Rossignol, P. A., and Spielman, A. 1986. Blood finding strategy of a capillary feeding sandfly, Lutzomyia longipalpis. Comparative Biochemistry and Physiology 83A, 683–686. Ribeiro, J. M. C., Rowton, E. D., and Charlab, R. 2000. The salivary 58-nucleotidase/phosphodiesterase of the hematophagous sand fly Lutzomyia longipalpis. Insect Biochemistry and Molecular Biology 30(4), 279–285. Ribeiro, J. M. C., Katz, O., Pannell, L. K., Waitumbi, J., and Warburg, A. 1999. Salivary glands of the sand fly Phlebotomus papatasi contain pharmacologically active amounts of adenosine and 58-AMP. Journal of Experimental Biology 202, 1551–1559. Saura, C., Ciruela, F., Casado, V., Canela, E. I., Mallol, J., Lluis, C., and Franco, R. 1996. Adenosine deaminase interacts with A1

Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. 1998. SMART, a simple modular architecture research tool: Identification of signaling domains. Proceedings of the National Academy of Sciences USA 95, 5857–5864. Urquhart, R. A., and Broadley, K. J. 1991. Comparison of adenosine receptor agonists with other vasodilators on noradrenaline-, potassium- and phorbol ester-contracted rabbit aorta. European Journal of Pharmacology 200, 35–43. Valenzuela, J. G., Charlab, R., Galperin, M. Y., and Ribeiro, J. M. C. 1998. Purification, cloning, and expression of an Apyrase from the bed bug Cimex lectularius. A new type of nucleotide-binding enzyme. Journal of Biological Chemistry 273, 30,583–30,590. Webster, H. K. 1984. Role of purines in lymphocyte function. Asian Pacific Journal of Allergy Immunology 2, 311–317. Wolfenden, R., Sharpless, T. K., and Allan, R. 1967. Substrate binding by adenosine deaminase. Specificity, pH dependence, and competition by mercurials. Journal of Biological Chemistry 242, 977–983. Received 31 August 1999; accepted with revision 29 February 2000