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phosphodiesterase of the hematophagus sand lutzomyia fly, Lutzomyia longipalpis
Insect Biochemistry and Molecular Biology 30 (2000) 279–285 www.elsevier.com/locate/ibmb
The salivary 5⬘-nucleotidase/phosphodiesterase of the hematophagus sand lutzomyia fly, Lutzomyia longipalpis Jose´ M.C. Ribeiro a
a,*
, Edgar D. Rowton b, Rosane Charlab
a
Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4, Room 126, 4 Center Drive, MSC-0425, NIH, Bethesda, MD 20892-0425, USA b Department of Entomology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA Received 5 May 1999; received in revised form 11 November 1999; accepted 13 November 1999
Abstract Salivary gland homogenates from adult female Lutzomyia longipalpis sand flies contain large amounts of 5⬘-nucleotidase and phosphodiesterase activities. Phosphodiesterase activity was found to be associated with 5⬘-nucleotidase in several independent experiments: (i) it coelutes with 5⬘-nucleotidase on a molecular sieving column, (ii) it coelutes with 5⬘-nucleotidase on a chromatofocusing column, and (iii) it has the same thermal inactivation kinetics as the 5⬘-nucleotidase activity. Additionally, both activities are independent of divalent cations, and both are decreased following a blood meal, suggesting that they reside in the same molecule. The role of salivary nucleotidases and purine nucleotides in blood-feeding by sand flies is discussed. Published by Elsevier Science Ltd. Keywords: Saliva; Sand fly; Nucleotidase; Apyrase; Hematophagy; Purinergic; Platelet; Adenosine; UDPG; Bis-p-nitrophenylphosphate; ADP
1. Introduction Nucleotides are released by injured cells when hematophagous arthropods probe their host’s skin for blood (Ribeiro, 1987). Intracellular concentrations of ATP and ADP are on the millimolar range, whereas extracellular concentrations are below the micromolar range. Extracellular ATP is pro-inflammatory, inducing neutrophil aggregation and superoxide release (Ford-Hutchinson, 1982; Kuroki and Minakami, 1989), and ADP is a potent inducer of platelet aggregation. Perhaps for these reasons, most blood-sucking arthropods have relatively large amounts of enzymes degrading these two nucleotides in their salivary glands (Ribeiro 1987, 1995). Apyrases are enzymes that hydrolyze pyrophosphate bonds from ATP and ADP to release AMP and orthophosphate. This enzyme is commonly found in the saliva of blood-sucking arthropods (Ribeiro 1987, 1995). A strictly calcium-dependent salivary apyrase activity has
* Corresponding author. Tel.: +1-301-496-3066; fax: +1-301-4024941. E-mail address: [email protected] (J.M.C. Ribeiro). 0965-1748/00/$ - see front matter. Published by Elsevier Science Ltd. PII: S 0 9 6 5 - 1 7 4 8 ( 9 9 ) 0 0 1 2 3 - X
been found in Old World sand flies from the genus Phlebotomus (Ribeiro et al., 1989), as well as in the New World fly, Lutzomyia longipalpis (Ribeiro et al., 1986). While sequencing salivary gland specific cDNA clones from the fly L. longipalpis (Charlab et al., 1999), a cDNA with substantial similarity to 5⬘-nucleotidases (GenBank accession AF 132510) was found, including nucleotidases with phosphodiesterase activity (Volknandt et al., 1991). These nucleotidases, which normally hydrolyze monophosphoesther bonds, could potentially further hydrolyze AMP, produced by salivary apyrases, to adenosine, if they were secreted enzymes. Adenosine has vasodilatory and anti-platelet activities through its activity on the adenosine receptors that increase intracellular cyclic-AMP (Collis, 1989; Edlund et al., 1987). Accordingly, we have demonstrated that salivary gland homogenates of L. longipalpis contain a divalent cation insensitive 5⬘-nucleotidase activity, and that this activity is lost following a blood meal, suggesting that it is secreted (Charlab et al., 1999). In this paper we further characterize the salivary 5⬘nucleotidase/phosphodiesterase activity of L. longipalpis.
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2. Materials and methods Organic compounds were obtained from Sigma Chemical Co. (St Louis, MO). All water used was of 18 M⍀ quality and was produced by a MilliQ apparatus from Millipore (Bedford, MA). Sand flies were reared at the Walter Reed Army Medical Research Institute on a fermented mixture of rabbit chow and rabbit feces, as described previously (Modi and Tesh, 1983). Adult sand flies were maintained with free access to a 20% solution of sucrose unless otherwise specified. Salivary glands from 3- to 10-day-old adult flies were dissected and transferred to 10 mM Hepes pH 7.0, 0.15 M NaCl (Hepes saline) in 1.5 ml polypropylene vials, usually in groups of 20 pairs of glands in 20 µl of Hepes saline, or individually in 10 µl of Hepes saline. Salivary glands were stored at ⫺75°C until needed, when they were disrupted by sonication using a Branson Sonifier 450 homogenizer (Danbury, CT). Salivary homogenates were centrifuged at 10,000g×2 min and the supernatants used for the experiments. Orthophosphate released from nucleotides was detected by the Fiske and Subbarow (1925) technique, as adapted to a microtiter plate (Marinotti et al., 1990). Phosphodiesterase activity was determined either with the colorimetric substrate bis-p-nitrophenylphosphate (2 mM final concentration, product released monitored at 405 nm), or with uridinediphosphateglucose (UDPG) (0.1 mM final concentration, product released monitored by anion exchange HPLC). Assays were done in the presence of 10 or 50 mM Hepes buffer pH 7.0 and 100 mM NaCl. Other additions (divalent cations, EDTA) were done as indicated in the Results section. All colorimetric assays were done in a ThermoMax plate reader from Molecular Devices (Menlo Park, CA). HPLC based detection of nucleotides and nucleotide sugars were done using an anion exchange TSK-DEAE column (4.5×150 mm), obtained through BioRad (Hercules, CA), perfused isocratically with the indicated concentration of sodium phosphate adjusted to pH 3.5 with phosphoric acid. A CM 4100 pump and SM4100 dual wavelength detector from Thermo Separation Products were used (Rivera Beach, FL). Molecular sieving chromatography was done with a TSK 2000SW column (0.8×75 cm) perfused at 1 ml/min for 25 min with 150 mM NaCl, 10 mM Hepes buffer pH 7.0, followed by an increase in NaCl to 1 M in a 10-min gradient. Note that this protocol exploits a cation-exchange mode for this column (Anspach et al., 1988). Fractions were collected at 0.4min intervals, from which aliquots were used for indicated assays. Chromatofocusing was run with a MonoP (0.5×5 cm) column from Pharmacia (Upsala, Sweden) using 75 mM Tris–acetate pH 9.3 as starting buffer and 10% Pharmalyte 96, adjusted to pH 6.0 with acetic acid. The column was eluted at 0.5 ml/min and fractions were collected at 0.5-min intervals. The pH was individually
measured in the tubes after aliquots were taken for enzymatic assays. For enzyme assays following the chromatofocusing column, 20 µl of each fraction were mixed with 80 µl of 100 mM Hepes buffer pH 7.4, containing bis-p-nitrophenylphosphate or AMP, to give 2 mM in a final 100-µl assay. Phosphodiesterase assay was followed on a plate reader using the 405-nm filter at 30°C and the results are expressed in milliabsorbance units (1 milliabsorbance unit=1 mOD/min). For AMPase assay, fractions were incubated for 2 h at 30°C, then 10 µl of each reaction medium were added to 90 µl water and the orthophosphate was measured. This dilution was done because the ampholytes used in the chromatofocusing chromatography caused a precipitate to form with the molybdate/reducer used in the Fiske and Subbarow reagent. When used at the above concentration, this precipitate did not interfere with the orthophosphate determination. To investigate whether the phosphodiesterase activity was associated with the gland cells or was in the gland lumen, salivary glands of adult female flies were collected and transferred to flat bottom 96 well plates (containing 50 µl of 5 mM Hepes pH 7.4 and 150 mM NaCl). The glands were punctured with fine needles to let the contents escape. The gland ghosts (glands without lumen contents) were transferred with fine needles to conical tubes containing 20 µl of the same saline, where they were sonicated. Gland ghosts and gland lumen (0.2 pair equivalents of glands per assay) were thus assayed for hydrolysis of UDPG (15 min incubation, 30°C). Statistical analysis was done with SigmaStat from Jandel Software (San Rafael, CA).
3. Results When salivary gland homogenates of adult female L. longipalpis were incubated with either AMP, ADP or ATP in the presence of CaCl2 and MgCl2, we observed that hydrolysis of AMP actually proceeded faster than hydrolysis of either ADP or ATP (Fig. 1). We have previously shown that AMP hydrolysis, but not hydrolysis of ADP or ATP, could proceed even in the absence of added divalent cations (Charlab et al., 1999). Because a sequence found in a cDNA library of L. longipalpis had similarity to 5⬘-nucleotidases and phosphodiesterases having UDPG hydrolase activity, we investigated whether salivary gland homogenates of this sand fly could hydrolyze UDPG. Indeed, salivary homogenates converted UDPG to uridine, as indicated in Fig. 2. UDPG hydrolysis proceeded equally well in the presence of Ca2+, Mg2+, Mn2+, Co2+ or in the absence of divalent cations and in the presence of EDTA. Zinc sulfate, however, inhibited hydrolysis of UDPG (Fig. 3). To investigate whether the phosphodiesterase activity was intracellular or secreted into the gland lumen, we
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Fig. 1. Hydrolysis of AMP, ADP and ATP by salivary gland homogenates of adult female L. longipalpis sand flies. Reaction media consisted of 50 mM TrisCl buffer pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.4 mM nucleotide and one pair of salivary gland homogenates (approximately 1 µg of protein) per ml of reaction. At indicated times 0.1-ml aliquots of each nucleotide reaction media were taken to measure orthophosphate. Results are expressed as the ratio of moles of Pi/moles of nucleotide (NTP) released.
measured UDPGase activity on the gland lumen and on homogenized gland ghost contents (Fig. 4). The lumen hydrolyzed 97±2.96% of the substrate, while the ghosts hydrolyzed 11.67±3.73% (mean±SE, n=3), a highly significant difference when analyzed by the t-test. Similarly, salivary gland homogenates from starving flies were compared with homogenates of flies recently blood fed for their ability to hydrolyze UDPG. In this experiment, we used 0.1 pairs of gland equivalent per assay, in a 10-min incubation time. We also found a significantly higher amount of enzyme activity in starved flies (57.4±5.1 and 10.4±2.2% hydrolysis, mean±SE, n=9, P⬍0.001, t-test). Both results indicate that most of the phosphodiesterase activity of adult female sand flies is secreted into the gland lumen. In an experiment attempting to characterize the salivary nucleotidase activity of L. longipalpis, we submitted 20 pairs of homogenized salivary glands from adult female L. longipalpis to molecular sieving chromatography using a TSK2000 SW (silica based) column, using an elution protocol that exploits its cation exchange and molecular sieving modes (Fig. 5). The eluate was analyzed for ADPase activity (not AMPase activity, see below) as well as phosphodiesterase activity using the colorimetric substrate, bis-p-nitrophenylphophate. This colorimetric phosphodiesterase substrate allowed for a more convenient monitoring of column eluates when compared to the UDPG substrate. The
Fig. 2. Hydrolysis of UDPG by salivary gland homogenates of adult female L. longipalpis sand flies. Reaction media (40 µl) consisted of 50 mM TrisCl pH 7.3, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.1 mM UDPG, and 0.2 pairs of homogenized salivary glands, and were incubated at 37°C. At indicated times the aliquots were acidified with 10 µl of 0.12 M HCl, stored in dry ice powder, and transferred to a ⫺75°C freezer until analyzed by anion exchange chromatography, run isocratically with 0.1 M sodium phosphate pH 3.5. (A) Chromatographic runs of the reaction at 0, 8 and 15 min. (B) Superimposed chromatographic runs of the standards uridine, UMP and UDPG. Glucose is not detected at the wavelength used (254 nm). (C) Time course of UDPG substrate disappearance, and of UMP and uridine generation, as detected by integrating the areas of chromatograms in (A), and other similar chromatograms at different time points.
chromatographic fractions showed an early eluting peak of ADPase activity, whereas the majority of the ADPase activity eluted at the end of the salt gradient (Fig. 5B). The early eluting ADPase peak coeluted with phosphodiesterase activity (Fig. 5C). No phosphodiesterase activity eluted with the late eluting ADPase containing peak (Fig. 5C). Further investigation of the early-eluting ADPase/phosphodiesterase peak, by combining the three most active fractions and testing their activity against different phosphorylated substrates, indicated it to have a potent AMPase activity (Fig. 6A), followed by UDPGase activity (Fig. 6B), some ADPase activity (Fig. 6(C)), and virtually no ATPase activity (Fig. 6D). Note
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Fig. 3. Effect of divalent cations, or EDTA, on UDPG hydrolysis by salivary phosphodiesterase activity of adult female L. longipalpis. Reaction mixture (20 µl) consisted of 0.1 mM UDPG, 50 mM TrisCl pH 7.4, 100 mM NaCl, and 2 mM indicated divalent cations or 0.1 mM EDTA. Reaction was started by adding salivary gland homogenate equivalent to 0.1 pairs of glands, and incubated for 10 min at 30°C. Reaction was stopped by adding 5 µl 0.12 M HCl, and the vial was frozen in dry ice powder until analyzed by DEAE chromatography as in the legend of Fig. 2. Results express the average±SE (n=3) concentration of products formed after 10 min incubation with retention times of UMP (white bars) or uridine (black bars). In the presence of EDTA the production of UMP was not monitored due to the interference of EDTA in the chromatogram.
that the time axis for AMPase activity is 10 times smaller than for the other graph axis. The same results were confirmed in a different chromatographic run. This phosphodiesterase peak was also able to hydrolyze pnitrophenyl phenylphosphonate and p-nitrophenylphosphoryl-choline (relatively unspecific phosphodiesterase substrates), but did not hydrolyze thymidine 5⬘-monophosphate p-nitrophenyl ester, a substrate for phosphodiesterase I (E.C. 3.1.4.1) enzymes (Ito et al., 1987), or APSA (di-adenosine pentaphosphate), a substrate for pyrophosphohydrolases (E.C. 3.6.1.29, 3.6.1.17 and 3.6.1.41) (Guranowski et al., 1994) (not shown). The Lutzomyia salivary gland cDNA with similarity to the 5⬘-nucleotidase encodes for a putative protein with a predicted pH of 6.94. Following submission of 20 pairs of homogenized salivary glands from adult female L. longipalpis to chromatofocusing on a 9–6 pH range indicated both AMPase (Fig. 7A) and phosphodiesterase (Fig. 7B) activities to elute in the same pH range of 7.59–7.37. Thermal sensitivity of AMPase and phosphodiesterase activities of the salivary gland homogenates were investigated by incubating homogenates for 10 min at different temperatures, followed by determination of residual
Fig. 4. Salivary phosphodiesterase activity of adult female L. longipalpis sand flies is mostly a secretory product. (A) Hydrolysis of UDPG by salivary gland lumen contents, or homogenized salivary gland ghosts. Assays were performed as in Fig. 2, using 0.2 gland pair equivalents per assay, incubated for a single time point of 15 min. (B) Hydrolysis of UDPG by salivary gland homogenates obtained from flies before or after a blood meal. 0.1 pair equivalents were used per assay, incubated for 10 min. Other conditions were as in the legend to Fig. 2.
hydrolytic activity against AMP or bis-p-nitrophenylphosphate. Both activities had a similar inactivation curve, resisting temperatures up to 60°C, being completely inactivated when incubated at 80°C (Fig. 8A). Similarly, when incubated for different time periods at 70°C, both activities showed similar inactivation kinetics (Fig. 8B).
4. Discussion In this paper we characterized the presence of a secreted phosphodiesterase activity in the salivary glands of adult female L. longipalpis. Three independent assays (molecular sieving, isoelectric chromatofocusing, and thermal sensitivity) indicated that the phosphodiesterase and AMPase activities reside in the same enzyme. Additionally, both activities were divalent cation independent. It is thus possible that the activities herein described derive from either the recently isolated cDNA (GenBank accession AFl32510, Charlab et al., 1999) or a closely related sequence(s), coding for a 5⬘-
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Fig. 5. Molecular sieving chromatography of 20 pairs of homogenized salivary glands of adult female L. longipalpis sand flies. The UV absorption at 280 nm is shown in (A). The column was eluted with 0.15 M NaCl and 5 mM Hepes buffer pH 7.0 for 25 min, when a 10min linear gradient to 1 M NaCl was applied. Fractions were collected at 0.4-min intervals. Column aliquots were assayed for ADPase activity (B), after incubation at 37°C with 2 mM ADP, 5 mM CaCl2, 50 mM Hepes pH 7.4 and 100 mM NaCl, and for phosphodiesterase activity (C) following incubation of 2 mM bis-p-nitrophenyl phosphate in 50 mM Hepes pH 7.4 and 100 mM NaCl, at 37°C.
nucleotidase/phosphodiesterase enzyme. Further evidence that the sequenced cDNA codes for the activities described in this paper requires purification and partial sequence information of the pure enzyme, possibly using sensitive tandem mass spectrometry/mass spectrometry of pure enzyme digests, due to the scarcity of the biological material. L. longipalpis salivary 5⬘-nucleotidase cDNA has similarities to both microbial and eukariote 5⬘-nucleotidase sequences (for example, 54% similarity and 46% aminoacid identity with mouse 5⬘-nucleotidase), as well as to bacterial UDPG hydrolase (Charlab et al., 1999). These enzymes were grouped as a family by Volknandt et al. (1991) when analyzing the 5⬘-nucleotidase from the electric ray electric lobe, and comparing it to mammalian and prokariotic enzymes. Prompted by the primary sequence similarity between these enzymes, Volknandt et al., 1991 measured the UDPG hydrolase activity of the ray 5⬘-nucleotidase, finding it to be 8% of the hydrolytic activity against AMP. The ray 5⬘-nucleotidase also hydrolyzed ADP at 4% of the rate of AMP hydrolysis (Grondal and Zimmermann, 1987), and had no
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activity against ATP. Accordingly, Lutzomyia salivary hydrolytic activities against AMP, ADP and UDPG observed on the first activity peak of the size exclusion column chromatography could result from a single gene product, most probably related to the recently isolated cDNA coding for a 5⬘-nucleotidase (Charlab et al., 1999). Our results also help to generalize the idea that 5⬘-nucleotidases, which are phosphomonoesterases, may also display phosphodiesterase function, as proposed by Volknandt et al. (1991). In the molecular sieving column experiment (Fig. 5), two peaks were resolved displaying ADPase activity. One peak co-eluted with phosphodiesterase activity, and the other with apyrase activity. Apyrase activity eluted from the TSK-2000 column disappears after 1 h at room temperature, or if frozen and thawed (results not shown). The 5⬘-nucleotidase/phosphodiesterase activity is more stable, resisting several cycles of freezing and thawing. The phosphodiesterase is divalent cation-independent and has substrate preference of the type AMP⬎UDPG⬎ADP⬎⬎⬎ATP, while apyrase, as previously reported, has calcium-dependent activity (Ribeiro et al., 1986), and no phosphodiesterase activity (Fig. 5). There are thus in Lutzomyia two different salivary enzymes hydrolyzing ADP. Most of the salivary ADPase, however, is exerted by the apyrase enzyme. Bacterial 5⬘-nucleotidases have broad substrate specificities, hydrolyzing ATP, ADP and AMP (Zimmermann, 1992). On the other hand, eukariote 5⬘nucleotidases have specificity towards AMP, and do not hydrolyze ATP (Zimmermann and Braun, 1996). Exceptionally, the salivary apyrase of the mosquito Aedes aegypti is a member of the 5⬘-nucleotidase family, requires either calcium or magnesium ions to hydrolyze either ADP or ATP, but it does not hydrolyze AMP (Champagne et al., 1995). Like Lutzomyia salivary 5⬘nucleotidase cDNA, Aedes apyrase cDNA lacks the hydrophobic carboxyterminal region normally found in membrane bound 5⬘-nucleotidases. This terminal amino acid stretch contains a consensus serine residue that is linked to an inositol phosphate anchor (Ogata et al., 1990). Interestingly, the Lutzomyia cDNA coding for the 5⬘-nucleotidase/phosphodiesterase does not code for the characteristic hydrophobic carboxyterminal of membrane bound 5⬘-nucleotidases, nor the consensus serine to which the inositol phosphate anchor is linked in such membrane bound enzymes (Charlab et al., 1999), suggesting that this clone codes for a secreted, rather then membrane bound, enzyme. Our results indicate that Lutzomyia salivary 5⬘-nucleotidase cDNA appear to code for a ‘real’ 5⬘-nucleotidase, not for an apyrase enzyme, as is the case with Aedes. Salivary 5⬘-nucleotidase of Lutzomyia could confer a selective advantage in converting AMP to adenosine, a substance with potent anti-platelet and vasodilatory properties. If the salivary phosphodiesterase activity of
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Fig. 6. Hydrolysis of AMP (A), UDPG (B), ADP (C) and ATP (D) by the ADPase/phosphodiesterase peak fractions from Fig. 5. Reaction media as in Fig. 2, except that 4 µl of the pooled fractions were used per time point assay in a final reaction volume of 20 µl. All nucleotide concentrations were 100 µM. Reactions were stopped with 5 µl of 0.125 M HCl.
L. longipalpis is a by-product of its AMPase activity, it may not be related to blood feeding, but rather to be an epi-phenomenon of the 5⬘-nucleotidase activity. It cannot be discounted, however, that some unknown physiologically relevant substrate is cleaved by the phosphodiesterase capability of Lutzomyia sand fly saliva. Additionally, we have recently found (Charlab, Rowton and Ribeiro, unpublished) that Lutzomyia salivary homogenates have a powerful salivary adenosine deaminase activity, which further converts adenosine to inosine, which is relatively inactive against platelets and vascular smooth muscle. The full implications of purinergic catabolic enzymes and their role in blood feeding in Lutzomyia remain to be discovered.
References Fig. 7. Isoelectric focusing of the salivary AMPase and phosphodiesterase activities of adult female L. longipalpis sand flies. (A) Phosphodieseterase activity. (B) AMPase activity shown in circles, and fraction pH in continuous line. For more information, see Materials and methods.
Anspach, B., Gierlich, H.U., Unger, K.K., 1988. Comparative study of Zorbax Bio Series GF 250 and GF 450 and TSK-Gel3000 SW and SWXL columns in size-exclusion chromatography of proteins. J. Chromutogr. 443, 45–54. Champagne, D.E., Smartt, C.T., Ribeiro, J.M., James, A.A., 1995. The salivary gland-specific apyrase of the mosquito Aedes aegypti is a
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Fig. 8. Thermal sensitivity of AMPase and phosphodiesterase activities of adult female L. longipalpis salivary glands. (A) Salivary homogenates were incubated for 10 min at indicated temperatures, when vials were transferred to wet ice, and assayed for residual activity against AMP (squares) or bis-p-nitrophenylphosphate (circles). (B) Salivary homogenates were incubated for indicated times at 70°C, and processed as in (A). Results are the mean±SE of a triplicate experiment (in most cases error bars are smaller than symbols). member of the 5⬘-nucleotidase family. Proc. Natl. Acad. Sci. USA 92, 694–698. Charlab, R., Valenzuela, J.G., Rowton, E., Ribeiro, J.M.C., 1999. Towards an understanding of the biochemical and pharmacologic complexity of a blood feeding sand fly, Lutzomyia longipalpis. Proc. Natl. Acad. Sci. USA 96, 15155–15160. Collis, M.G., 1989. The vasodilator role of adenosine. Phamac. Ther. 41, 143–162. Edlund, A., Siden, A., Sollevi, A., 1987. Evidence for an anti-aggregatory effect of adenosine at physiological concentrations and for its role in the action of dipyridamole. Thromb. Res. 45, 183–190. Fiske, C.H., Subbarow, Y., 1925. The colorimetric determination of phosphorus. J. Biol. Chem. 66, 375–400. Ford-Hutchinson, A.W., 1982. Aggregation of rat neutrophils by nucleotide triphosphates. Br. J. Pharmacol. 76, 367–371. Grondal, E.J., Zimmermann, H., 1987. Purification, characterization and cellular localization of 5⬘-nucleotidase from Torpedo electric organ. Biochem. J. 245, 805–810. Guranowski, A., Brown, P., Ashton, P.A., Blackbum, G.M., 1994. Regiospecificity of the hydrolysis of diadenosine polyphosphates catalyzed by three specific pyrophosphohydrolases. Biochemistry 33, 235–240. Ito, K., Yamamoto, T., Minamiura, N., 1987. PhosphodiesteraseI in human urine: purification and characterization of the enzyme. J. Biochem. (Tokyo) 102, 357–359. Kuroki, M., Minakami, S., 1989. Extracellular ATP triggers superoxide production in human neutrophils. Biochem. Biophys. Res. Contm. 162, 377–380.
Marinotti, O., James, A., Ribeiro, I.M.C., 1990. Diet and salivation in female Aedes aegypti mosquitoes. J. Insect Physiol. 36, 545–548. Modi, G.B., Tesh, R.B., 1983. A simple technique for mass rearing Lutzomyia longipalpis and Phlebotonmus papatasi (Diptera: Psychodidae) in the laboratory. J. Med. Ent. 20, 568. Ogata, S., Hayashi, Y., Misumi, Y., Ikchara, Y., 1990. Membraneanchoring domain of rat liver 5⬘-nuclcotidase: identification of the COOH-terminal serine-523 covalently attached with a glycolipid. Biochemistry 29, 7923–7927. Ribeiro, J.M.C., 1987. Role of arthropod saliva in blood feeding. Ann. Rev. Entomol. 32, 463–478. Ribeiro, J.M.C., 1995. Blood-feeding arthropods: live syringes or invertebrate pharmacologists? Infect. Agents Dis. 4, 143–152. Ribeiro, J.M.C., Modi, G.B., Resh, R.B., 1989. Salivary apyrase activity of some old world phlebotomine sand flies. Insect Biochem. 19, 409–412. Ribeiro, J.M.C., Rossignol, P.A., Spielman, A., 1986. Blood finding strategy of a capillary feeding sand fly Lutzowyia longipalpis. Camp. Biochem. Physiol. 83A (15), 683–686. Volknandt, W., Vogel, M., Pevsner, J., Misumi, Y., Ikehara, Y., Zimmermann, H., 1991. 5⬘-nucleotidase from the electric ray electric lobe. Primary structure and relation to mammalian and prokaryotic enzymes. Eur. J. Biochem. 202, 855–861. Zimmermann, H., 1992. 5⬘-Nucleotidase: molecular structure and functional aspects. Biochem. J. 285, 345–365. Zimmermann, H., Braun, N., 1996. Extracellular metabolism of nucleotides in the nervous system. J. Auton. Pharmacol. 16, 397– 400.
The salivary 5⬘-nucleotidase/phosphodiesterase of the hematophagus sand lutzomyia fly, Lutzomyia longipalpis Jose´ M.C. Ribeiro a
a,*
, Edgar D. Rowton b, Rosane Charlab
a
Section of Medical Entomology, Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Building 4, Room 126, 4 Center Drive, MSC-0425, NIH, Bethesda, MD 20892-0425, USA b Department of Entomology, Walter Reed Army Institute of Research, Washington, DC 20307-5100, USA Received 5 May 1999; received in revised form 11 November 1999; accepted 13 November 1999
Abstract Salivary gland homogenates from adult female Lutzomyia longipalpis sand flies contain large amounts of 5⬘-nucleotidase and phosphodiesterase activities. Phosphodiesterase activity was found to be associated with 5⬘-nucleotidase in several independent experiments: (i) it coelutes with 5⬘-nucleotidase on a molecular sieving column, (ii) it coelutes with 5⬘-nucleotidase on a chromatofocusing column, and (iii) it has the same thermal inactivation kinetics as the 5⬘-nucleotidase activity. Additionally, both activities are independent of divalent cations, and both are decreased following a blood meal, suggesting that they reside in the same molecule. The role of salivary nucleotidases and purine nucleotides in blood-feeding by sand flies is discussed. Published by Elsevier Science Ltd. Keywords: Saliva; Sand fly; Nucleotidase; Apyrase; Hematophagy; Purinergic; Platelet; Adenosine; UDPG; Bis-p-nitrophenylphosphate; ADP
1. Introduction Nucleotides are released by injured cells when hematophagous arthropods probe their host’s skin for blood (Ribeiro, 1987). Intracellular concentrations of ATP and ADP are on the millimolar range, whereas extracellular concentrations are below the micromolar range. Extracellular ATP is pro-inflammatory, inducing neutrophil aggregation and superoxide release (Ford-Hutchinson, 1982; Kuroki and Minakami, 1989), and ADP is a potent inducer of platelet aggregation. Perhaps for these reasons, most blood-sucking arthropods have relatively large amounts of enzymes degrading these two nucleotides in their salivary glands (Ribeiro 1987, 1995). Apyrases are enzymes that hydrolyze pyrophosphate bonds from ATP and ADP to release AMP and orthophosphate. This enzyme is commonly found in the saliva of blood-sucking arthropods (Ribeiro 1987, 1995). A strictly calcium-dependent salivary apyrase activity has
* Corresponding author. Tel.: +1-301-496-3066; fax: +1-301-4024941. E-mail address: [email protected] (J.M.C. Ribeiro). 0965-1748/00/$ - see front matter. Published by Elsevier Science Ltd. PII: S 0 9 6 5 - 1 7 4 8 ( 9 9 ) 0 0 1 2 3 - X
been found in Old World sand flies from the genus Phlebotomus (Ribeiro et al., 1989), as well as in the New World fly, Lutzomyia longipalpis (Ribeiro et al., 1986). While sequencing salivary gland specific cDNA clones from the fly L. longipalpis (Charlab et al., 1999), a cDNA with substantial similarity to 5⬘-nucleotidases (GenBank accession AF 132510) was found, including nucleotidases with phosphodiesterase activity (Volknandt et al., 1991). These nucleotidases, which normally hydrolyze monophosphoesther bonds, could potentially further hydrolyze AMP, produced by salivary apyrases, to adenosine, if they were secreted enzymes. Adenosine has vasodilatory and anti-platelet activities through its activity on the adenosine receptors that increase intracellular cyclic-AMP (Collis, 1989; Edlund et al., 1987). Accordingly, we have demonstrated that salivary gland homogenates of L. longipalpis contain a divalent cation insensitive 5⬘-nucleotidase activity, and that this activity is lost following a blood meal, suggesting that it is secreted (Charlab et al., 1999). In this paper we further characterize the salivary 5⬘nucleotidase/phosphodiesterase activity of L. longipalpis.
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2. Materials and methods Organic compounds were obtained from Sigma Chemical Co. (St Louis, MO). All water used was of 18 M⍀ quality and was produced by a MilliQ apparatus from Millipore (Bedford, MA). Sand flies were reared at the Walter Reed Army Medical Research Institute on a fermented mixture of rabbit chow and rabbit feces, as described previously (Modi and Tesh, 1983). Adult sand flies were maintained with free access to a 20% solution of sucrose unless otherwise specified. Salivary glands from 3- to 10-day-old adult flies were dissected and transferred to 10 mM Hepes pH 7.0, 0.15 M NaCl (Hepes saline) in 1.5 ml polypropylene vials, usually in groups of 20 pairs of glands in 20 µl of Hepes saline, or individually in 10 µl of Hepes saline. Salivary glands were stored at ⫺75°C until needed, when they were disrupted by sonication using a Branson Sonifier 450 homogenizer (Danbury, CT). Salivary homogenates were centrifuged at 10,000g×2 min and the supernatants used for the experiments. Orthophosphate released from nucleotides was detected by the Fiske and Subbarow (1925) technique, as adapted to a microtiter plate (Marinotti et al., 1990). Phosphodiesterase activity was determined either with the colorimetric substrate bis-p-nitrophenylphosphate (2 mM final concentration, product released monitored at 405 nm), or with uridinediphosphateglucose (UDPG) (0.1 mM final concentration, product released monitored by anion exchange HPLC). Assays were done in the presence of 10 or 50 mM Hepes buffer pH 7.0 and 100 mM NaCl. Other additions (divalent cations, EDTA) were done as indicated in the Results section. All colorimetric assays were done in a ThermoMax plate reader from Molecular Devices (Menlo Park, CA). HPLC based detection of nucleotides and nucleotide sugars were done using an anion exchange TSK-DEAE column (4.5×150 mm), obtained through BioRad (Hercules, CA), perfused isocratically with the indicated concentration of sodium phosphate adjusted to pH 3.5 with phosphoric acid. A CM 4100 pump and SM4100 dual wavelength detector from Thermo Separation Products were used (Rivera Beach, FL). Molecular sieving chromatography was done with a TSK 2000SW column (0.8×75 cm) perfused at 1 ml/min for 25 min with 150 mM NaCl, 10 mM Hepes buffer pH 7.0, followed by an increase in NaCl to 1 M in a 10-min gradient. Note that this protocol exploits a cation-exchange mode for this column (Anspach et al., 1988). Fractions were collected at 0.4min intervals, from which aliquots were used for indicated assays. Chromatofocusing was run with a MonoP (0.5×5 cm) column from Pharmacia (Upsala, Sweden) using 75 mM Tris–acetate pH 9.3 as starting buffer and 10% Pharmalyte 96, adjusted to pH 6.0 with acetic acid. The column was eluted at 0.5 ml/min and fractions were collected at 0.5-min intervals. The pH was individually
measured in the tubes after aliquots were taken for enzymatic assays. For enzyme assays following the chromatofocusing column, 20 µl of each fraction were mixed with 80 µl of 100 mM Hepes buffer pH 7.4, containing bis-p-nitrophenylphosphate or AMP, to give 2 mM in a final 100-µl assay. Phosphodiesterase assay was followed on a plate reader using the 405-nm filter at 30°C and the results are expressed in milliabsorbance units (1 milliabsorbance unit=1 mOD/min). For AMPase assay, fractions were incubated for 2 h at 30°C, then 10 µl of each reaction medium were added to 90 µl water and the orthophosphate was measured. This dilution was done because the ampholytes used in the chromatofocusing chromatography caused a precipitate to form with the molybdate/reducer used in the Fiske and Subbarow reagent. When used at the above concentration, this precipitate did not interfere with the orthophosphate determination. To investigate whether the phosphodiesterase activity was associated with the gland cells or was in the gland lumen, salivary glands of adult female flies were collected and transferred to flat bottom 96 well plates (containing 50 µl of 5 mM Hepes pH 7.4 and 150 mM NaCl). The glands were punctured with fine needles to let the contents escape. The gland ghosts (glands without lumen contents) were transferred with fine needles to conical tubes containing 20 µl of the same saline, where they were sonicated. Gland ghosts and gland lumen (0.2 pair equivalents of glands per assay) were thus assayed for hydrolysis of UDPG (15 min incubation, 30°C). Statistical analysis was done with SigmaStat from Jandel Software (San Rafael, CA).
3. Results When salivary gland homogenates of adult female L. longipalpis were incubated with either AMP, ADP or ATP in the presence of CaCl2 and MgCl2, we observed that hydrolysis of AMP actually proceeded faster than hydrolysis of either ADP or ATP (Fig. 1). We have previously shown that AMP hydrolysis, but not hydrolysis of ADP or ATP, could proceed even in the absence of added divalent cations (Charlab et al., 1999). Because a sequence found in a cDNA library of L. longipalpis had similarity to 5⬘-nucleotidases and phosphodiesterases having UDPG hydrolase activity, we investigated whether salivary gland homogenates of this sand fly could hydrolyze UDPG. Indeed, salivary homogenates converted UDPG to uridine, as indicated in Fig. 2. UDPG hydrolysis proceeded equally well in the presence of Ca2+, Mg2+, Mn2+, Co2+ or in the absence of divalent cations and in the presence of EDTA. Zinc sulfate, however, inhibited hydrolysis of UDPG (Fig. 3). To investigate whether the phosphodiesterase activity was intracellular or secreted into the gland lumen, we
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Fig. 1. Hydrolysis of AMP, ADP and ATP by salivary gland homogenates of adult female L. longipalpis sand flies. Reaction media consisted of 50 mM TrisCl buffer pH 7.5, 100 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 0.4 mM nucleotide and one pair of salivary gland homogenates (approximately 1 µg of protein) per ml of reaction. At indicated times 0.1-ml aliquots of each nucleotide reaction media were taken to measure orthophosphate. Results are expressed as the ratio of moles of Pi/moles of nucleotide (NTP) released.
measured UDPGase activity on the gland lumen and on homogenized gland ghost contents (Fig. 4). The lumen hydrolyzed 97±2.96% of the substrate, while the ghosts hydrolyzed 11.67±3.73% (mean±SE, n=3), a highly significant difference when analyzed by the t-test. Similarly, salivary gland homogenates from starving flies were compared with homogenates of flies recently blood fed for their ability to hydrolyze UDPG. In this experiment, we used 0.1 pairs of gland equivalent per assay, in a 10-min incubation time. We also found a significantly higher amount of enzyme activity in starved flies (57.4±5.1 and 10.4±2.2% hydrolysis, mean±SE, n=9, P⬍0.001, t-test). Both results indicate that most of the phosphodiesterase activity of adult female sand flies is secreted into the gland lumen. In an experiment attempting to characterize the salivary nucleotidase activity of L. longipalpis, we submitted 20 pairs of homogenized salivary glands from adult female L. longipalpis to molecular sieving chromatography using a TSK2000 SW (silica based) column, using an elution protocol that exploits its cation exchange and molecular sieving modes (Fig. 5). The eluate was analyzed for ADPase activity (not AMPase activity, see below) as well as phosphodiesterase activity using the colorimetric substrate, bis-p-nitrophenylphophate. This colorimetric phosphodiesterase substrate allowed for a more convenient monitoring of column eluates when compared to the UDPG substrate. The
Fig. 2. Hydrolysis of UDPG by salivary gland homogenates of adult female L. longipalpis sand flies. Reaction media (40 µl) consisted of 50 mM TrisCl pH 7.3, 150 mM NaCl, 2 mM CaCl2, 2 mM MgCl2, 0.1 mM UDPG, and 0.2 pairs of homogenized salivary glands, and were incubated at 37°C. At indicated times the aliquots were acidified with 10 µl of 0.12 M HCl, stored in dry ice powder, and transferred to a ⫺75°C freezer until analyzed by anion exchange chromatography, run isocratically with 0.1 M sodium phosphate pH 3.5. (A) Chromatographic runs of the reaction at 0, 8 and 15 min. (B) Superimposed chromatographic runs of the standards uridine, UMP and UDPG. Glucose is not detected at the wavelength used (254 nm). (C) Time course of UDPG substrate disappearance, and of UMP and uridine generation, as detected by integrating the areas of chromatograms in (A), and other similar chromatograms at different time points.
chromatographic fractions showed an early eluting peak of ADPase activity, whereas the majority of the ADPase activity eluted at the end of the salt gradient (Fig. 5B). The early eluting ADPase peak coeluted with phosphodiesterase activity (Fig. 5C). No phosphodiesterase activity eluted with the late eluting ADPase containing peak (Fig. 5C). Further investigation of the early-eluting ADPase/phosphodiesterase peak, by combining the three most active fractions and testing their activity against different phosphorylated substrates, indicated it to have a potent AMPase activity (Fig. 6A), followed by UDPGase activity (Fig. 6B), some ADPase activity (Fig. 6(C)), and virtually no ATPase activity (Fig. 6D). Note
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Fig. 3. Effect of divalent cations, or EDTA, on UDPG hydrolysis by salivary phosphodiesterase activity of adult female L. longipalpis. Reaction mixture (20 µl) consisted of 0.1 mM UDPG, 50 mM TrisCl pH 7.4, 100 mM NaCl, and 2 mM indicated divalent cations or 0.1 mM EDTA. Reaction was started by adding salivary gland homogenate equivalent to 0.1 pairs of glands, and incubated for 10 min at 30°C. Reaction was stopped by adding 5 µl 0.12 M HCl, and the vial was frozen in dry ice powder until analyzed by DEAE chromatography as in the legend of Fig. 2. Results express the average±SE (n=3) concentration of products formed after 10 min incubation with retention times of UMP (white bars) or uridine (black bars). In the presence of EDTA the production of UMP was not monitored due to the interference of EDTA in the chromatogram.
that the time axis for AMPase activity is 10 times smaller than for the other graph axis. The same results were confirmed in a different chromatographic run. This phosphodiesterase peak was also able to hydrolyze pnitrophenyl phenylphosphonate and p-nitrophenylphosphoryl-choline (relatively unspecific phosphodiesterase substrates), but did not hydrolyze thymidine 5⬘-monophosphate p-nitrophenyl ester, a substrate for phosphodiesterase I (E.C. 3.1.4.1) enzymes (Ito et al., 1987), or APSA (di-adenosine pentaphosphate), a substrate for pyrophosphohydrolases (E.C. 3.6.1.29, 3.6.1.17 and 3.6.1.41) (Guranowski et al., 1994) (not shown). The Lutzomyia salivary gland cDNA with similarity to the 5⬘-nucleotidase encodes for a putative protein with a predicted pH of 6.94. Following submission of 20 pairs of homogenized salivary glands from adult female L. longipalpis to chromatofocusing on a 9–6 pH range indicated both AMPase (Fig. 7A) and phosphodiesterase (Fig. 7B) activities to elute in the same pH range of 7.59–7.37. Thermal sensitivity of AMPase and phosphodiesterase activities of the salivary gland homogenates were investigated by incubating homogenates for 10 min at different temperatures, followed by determination of residual
Fig. 4. Salivary phosphodiesterase activity of adult female L. longipalpis sand flies is mostly a secretory product. (A) Hydrolysis of UDPG by salivary gland lumen contents, or homogenized salivary gland ghosts. Assays were performed as in Fig. 2, using 0.2 gland pair equivalents per assay, incubated for a single time point of 15 min. (B) Hydrolysis of UDPG by salivary gland homogenates obtained from flies before or after a blood meal. 0.1 pair equivalents were used per assay, incubated for 10 min. Other conditions were as in the legend to Fig. 2.
hydrolytic activity against AMP or bis-p-nitrophenylphosphate. Both activities had a similar inactivation curve, resisting temperatures up to 60°C, being completely inactivated when incubated at 80°C (Fig. 8A). Similarly, when incubated for different time periods at 70°C, both activities showed similar inactivation kinetics (Fig. 8B).
4. Discussion In this paper we characterized the presence of a secreted phosphodiesterase activity in the salivary glands of adult female L. longipalpis. Three independent assays (molecular sieving, isoelectric chromatofocusing, and thermal sensitivity) indicated that the phosphodiesterase and AMPase activities reside in the same enzyme. Additionally, both activities were divalent cation independent. It is thus possible that the activities herein described derive from either the recently isolated cDNA (GenBank accession AFl32510, Charlab et al., 1999) or a closely related sequence(s), coding for a 5⬘-
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Fig. 5. Molecular sieving chromatography of 20 pairs of homogenized salivary glands of adult female L. longipalpis sand flies. The UV absorption at 280 nm is shown in (A). The column was eluted with 0.15 M NaCl and 5 mM Hepes buffer pH 7.0 for 25 min, when a 10min linear gradient to 1 M NaCl was applied. Fractions were collected at 0.4-min intervals. Column aliquots were assayed for ADPase activity (B), after incubation at 37°C with 2 mM ADP, 5 mM CaCl2, 50 mM Hepes pH 7.4 and 100 mM NaCl, and for phosphodiesterase activity (C) following incubation of 2 mM bis-p-nitrophenyl phosphate in 50 mM Hepes pH 7.4 and 100 mM NaCl, at 37°C.
nucleotidase/phosphodiesterase enzyme. Further evidence that the sequenced cDNA codes for the activities described in this paper requires purification and partial sequence information of the pure enzyme, possibly using sensitive tandem mass spectrometry/mass spectrometry of pure enzyme digests, due to the scarcity of the biological material. L. longipalpis salivary 5⬘-nucleotidase cDNA has similarities to both microbial and eukariote 5⬘-nucleotidase sequences (for example, 54% similarity and 46% aminoacid identity with mouse 5⬘-nucleotidase), as well as to bacterial UDPG hydrolase (Charlab et al., 1999). These enzymes were grouped as a family by Volknandt et al. (1991) when analyzing the 5⬘-nucleotidase from the electric ray electric lobe, and comparing it to mammalian and prokariotic enzymes. Prompted by the primary sequence similarity between these enzymes, Volknandt et al., 1991 measured the UDPG hydrolase activity of the ray 5⬘-nucleotidase, finding it to be 8% of the hydrolytic activity against AMP. The ray 5⬘-nucleotidase also hydrolyzed ADP at 4% of the rate of AMP hydrolysis (Grondal and Zimmermann, 1987), and had no
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activity against ATP. Accordingly, Lutzomyia salivary hydrolytic activities against AMP, ADP and UDPG observed on the first activity peak of the size exclusion column chromatography could result from a single gene product, most probably related to the recently isolated cDNA coding for a 5⬘-nucleotidase (Charlab et al., 1999). Our results also help to generalize the idea that 5⬘-nucleotidases, which are phosphomonoesterases, may also display phosphodiesterase function, as proposed by Volknandt et al. (1991). In the molecular sieving column experiment (Fig. 5), two peaks were resolved displaying ADPase activity. One peak co-eluted with phosphodiesterase activity, and the other with apyrase activity. Apyrase activity eluted from the TSK-2000 column disappears after 1 h at room temperature, or if frozen and thawed (results not shown). The 5⬘-nucleotidase/phosphodiesterase activity is more stable, resisting several cycles of freezing and thawing. The phosphodiesterase is divalent cation-independent and has substrate preference of the type AMP⬎UDPG⬎ADP⬎⬎⬎ATP, while apyrase, as previously reported, has calcium-dependent activity (Ribeiro et al., 1986), and no phosphodiesterase activity (Fig. 5). There are thus in Lutzomyia two different salivary enzymes hydrolyzing ADP. Most of the salivary ADPase, however, is exerted by the apyrase enzyme. Bacterial 5⬘-nucleotidases have broad substrate specificities, hydrolyzing ATP, ADP and AMP (Zimmermann, 1992). On the other hand, eukariote 5⬘nucleotidases have specificity towards AMP, and do not hydrolyze ATP (Zimmermann and Braun, 1996). Exceptionally, the salivary apyrase of the mosquito Aedes aegypti is a member of the 5⬘-nucleotidase family, requires either calcium or magnesium ions to hydrolyze either ADP or ATP, but it does not hydrolyze AMP (Champagne et al., 1995). Like Lutzomyia salivary 5⬘nucleotidase cDNA, Aedes apyrase cDNA lacks the hydrophobic carboxyterminal region normally found in membrane bound 5⬘-nucleotidases. This terminal amino acid stretch contains a consensus serine residue that is linked to an inositol phosphate anchor (Ogata et al., 1990). Interestingly, the Lutzomyia cDNA coding for the 5⬘-nucleotidase/phosphodiesterase does not code for the characteristic hydrophobic carboxyterminal of membrane bound 5⬘-nucleotidases, nor the consensus serine to which the inositol phosphate anchor is linked in such membrane bound enzymes (Charlab et al., 1999), suggesting that this clone codes for a secreted, rather then membrane bound, enzyme. Our results indicate that Lutzomyia salivary 5⬘-nucleotidase cDNA appear to code for a ‘real’ 5⬘-nucleotidase, not for an apyrase enzyme, as is the case with Aedes. Salivary 5⬘-nucleotidase of Lutzomyia could confer a selective advantage in converting AMP to adenosine, a substance with potent anti-platelet and vasodilatory properties. If the salivary phosphodiesterase activity of
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Fig. 6. Hydrolysis of AMP (A), UDPG (B), ADP (C) and ATP (D) by the ADPase/phosphodiesterase peak fractions from Fig. 5. Reaction media as in Fig. 2, except that 4 µl of the pooled fractions were used per time point assay in a final reaction volume of 20 µl. All nucleotide concentrations were 100 µM. Reactions were stopped with 5 µl of 0.125 M HCl.
L. longipalpis is a by-product of its AMPase activity, it may not be related to blood feeding, but rather to be an epi-phenomenon of the 5⬘-nucleotidase activity. It cannot be discounted, however, that some unknown physiologically relevant substrate is cleaved by the phosphodiesterase capability of Lutzomyia sand fly saliva. Additionally, we have recently found (Charlab, Rowton and Ribeiro, unpublished) that Lutzomyia salivary homogenates have a powerful salivary adenosine deaminase activity, which further converts adenosine to inosine, which is relatively inactive against platelets and vascular smooth muscle. The full implications of purinergic catabolic enzymes and their role in blood feeding in Lutzomyia remain to be discovered.
References Fig. 7. Isoelectric focusing of the salivary AMPase and phosphodiesterase activities of adult female L. longipalpis sand flies. (A) Phosphodieseterase activity. (B) AMPase activity shown in circles, and fraction pH in continuous line. For more information, see Materials and methods.
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