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A Novel Phospholipase A2Activity in Saliva of the Lone Star Tick,Amblyomma americanum(L.)
EXPERIMENTAL PARASITOLOGY ARTICLE NO. PR974201
87, 121–132 (1997)
A Novel Phospholipase A2 Activity in Saliva of the Lone Star Tick, Amblyomma americanum (L.) A. S. Bowman, C. L. Gengler, M. R. Surdick, K. Zhu, R. C. Essenberg,* J. R. Sauer, and J. W. Dillwith Department of Entomology and *Department of Biochemistry and Molecular Biology, Oklahoma State University, 127 Noble Research Center, Stillwater, Oklahoma 74048-0464, U.S.A. BOWMAN, A. S., GENGLER, C. L., SURDICK, M. R., ZHU, K., ESSENBERG, R. C., SAUER, J. R., AND DILLWITH, J. W. 1997. A novel phospholipase A2 activity in saliva of the lone star tick, Amblyomma americanum (L.). Experimental Parasitology 87, 121–132. Saliva from female lone star ticks, Amblyomma americanum, contained a novel phospholipase A2 (PLA2) activity that hydrolyzed 14Carachidonate from 14C-arachidonyl phosphatidylcholine. The tick saliva PLA2 (ts-PLA2) was active over a broad pH range (4.5–11.5) with two distinct pH optima of pH 5.5 and 9.5. Though extracellular PLA2s are reported to be activated by millimolar Ca2+, ts-PLA2 was sensitive to submicromolar Ca2+ and was half-maximally activated by 3.5mM Ca2+. Tick saliva contains >500mM Ca2+and the feeding lesion in the host is expected to contain millimolar Ca2+. Saliva exhibited a single peak of PLA2 activity corresponding to a molecular weight of 55.7 ± 1.3 kDa by size exclusion chromatography. The ts-PLA2 was unaffected by a variety of compounds known to inhibit either secreted or cytosolic PLA2s from other sources. However, ts-PLA2 was inhibited by the substrate analog, oleyloxyethyl phosphorylcholine (IC50 4 1.4 mM), and the end product, arachidonic acid (IC50 4 38 mM). Low concentrations of dithiothreitol did not greatly affect ts-PLA2, but activity was reduced at higher concentrations. The PLA2 activity found in A. americanum salivary glands showed many similarities to ts-PLA2, but also some distinct differences. Secreted at the tick–host interface, ts-PLA2 is thought to play an important, but unknown, role during the prolonged tick feeding. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: Amblyomma americanum; phospholipase A2 (E.C. 3.1.14); saliva; salivary gland; tick; AA, arachidonic acid; 14C-PC; L-a-1-palmitoyl-2-[114 C]arachidonyl phosphatidylcholine; ACA, N-(p-amylcinnamoyl) anthranilic acid; 4-BPB, pbromophenacyl bromide; DEDA, 7,7-dimethyleicosadienoic acid; DTT, dithiothreitol; ETYA, 5,8,11,14-eicosatetraynoic acid; OBAA, 3(-4-octadecyl)-benzoylacrylic acid; ODYA, 17octadecynoic acid; ONO-RS-082, 2(p-amylcinnamoyl)amino-4-chlorobenzoic acid; OPC, oleyloxyethyl phosphorylcholine; thioetheramide-PC, thioetheramide-phosphorylcholine; ts-PLA2, tick salivary PLA2; sg-PLA2, salivary gland PLA2.
INTRODUCTION Phospholipase A2 (PLA2; EC 3.1.14) catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield free fatty acids and lysophospholipids (for reviews see Kudo et al., 1993; Mayer and Marshall, 1993; Dennis, 1994; Mukherjee et al., 1994). PLA2s are ubiquitously found throughout the animal kingdom reflecting the universal occurrence of phospholipids. PLA2s play important roles in phospholipid digestion, rearrangement of cellular membrane phospholipid structures, inflammatory responses, defense and predation mechanisms, and signal transduction. Perhaps the best studied PLA2s are the small secreted enzymes of
bee venom, snake venoms, and mammalian pancreatic secretions. Additionally, larger PLA2s have been identified that are not secreted and show a high preference for arachidonatecontaining phospholipids, thus releasing arachidonic acid for the conversion to eicosanoid lipid mediators. More recently, there have been a number of PLA2s reported exhibiting a variety of characteristics that do not fit into the standard PLA2 classification scheme. Phospholipase A2 activity in parasites has received little attention, but one might expect that PLA2 is present in all parasites. Phospholipase A2 activity has been reported in Rickettsia rickettsii (Walker et al., 1983), Trypanosoma cruzi (Connelly and Kierszenbaum, 1984), Toxo-
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plasma gondii (Saffer and Schwartzman, 1991; Gomez-Marin et al., 1996), Entamoeba histolytica (Ravdin et al., 1985), and Schistosoma japonicum (Rogers et al., 1991). Recently, we found a PLA2 activity in the salivary glands of the lone star tick (Amblyomma americanum) that exhibits some unusual characteristics (Bowman et al., 1998). In the present study we investigated the presence of a PLA2 activity in the saliva collected from A. americanum and compared it to the PLA2 found in the salivary gland. MATERIALS AND METHODS Saliva and salivary gland collection. Mated adult female lone star ticks, A. americanum (L.), were reared on sheep at the Oklahoma State University Central Tick Rearing Facility according to the methods of Patrick and Hair (1975). Ticks were removed from the sheep during the ‘‘slowfeeding’’ phase (7–10 days on the sheep), their mouthparts were cleaned of any adhering host tissue, and they were induced to salivate within 2 hr. The ticks were affixed on adhesive tape ventral side up and induced to salivate by 10ml intrahemocoelic injections at 15-min intervals up to 60 min with buffered tick saline containing 1 mM dopamine, 1 mM theophylline, and 3% (v/v) dimethyl sulfoxide. The composition of buffered tick saline is given in full elsewhere (Needham and Sauer, 1979). Saliva was collected into 25-ml glass capillary tubes positioned over the mouthparts and was dispelled at 15-min intervals into microcentrifuge tubes held on ice. Before assay or storage the saliva was centrifuged at 11,500g for 5 min to sediment any debris. For storage, saliva was diluted with an equal volume of 0.8% NaCl containing 1 mg/ml essentially fatty acid-free BSA and frozen at −70°C. Adult ticks, as above, were dissected while submerged in ice-cold 0.1 M Mops, 20 mM EGTA, pH 6.8, and cleaned of any adhering trachea or other tissue. Glands were rinsed in 100 mM Tris, pH 9.0, homogenized in a glass/glass homogenizer in the same buffer, and then centrifuged at 11,500g for 10 min at 4°C to remove cellular debris. Homogenate not immediately used was stored at −70°C. Phospholipase A2 activity assay. PLA2 activity was assessed by the amount of radioactive arachidonic acid released from phosphatidylcholine in a nonmixed micellar assay based upon the method of Van den Bosch et al. (1991) with some modifications . Substrate, phosphatidylcholine, 14 14 L-a-1-palmitoyl-2-[1- C]arachidonyl ( C-PC), was dried under nitrogen and then resuspended as micelles by sonication (3 × 45 sec burst, 20 kilocycles/sec, 90 W, Artek 300, Farmingdale, NY) in assay buffer (100 mM Tris–HCl, 5 mM CaCl2, pH 9.0). The micellar suspension was aliquoted into 12 × 75 mm borosilicate test tubes and prewarmed to 37°C in a water bath for 5 min, and the assay was initiated
by the addition of the enzyme source and 15 sec vigorous vortexing. Tubes were incubated at 37°C in a moderately fast shaking water bath (80 cycles/min). Typically, the assay consisted of 80 ml substrate suspension (1 nmole 14C-PC, 0.05 mCi) and 20 ml enzyme source to give a 4 mM final concentration of CaCl2 and the reaction proceeded for 5 min. The reaction was terminated by the addition of 750-ml ice-cold Dole’s reagent (700:60, 2-propanol: 1 mM HCl) and 10 ml of 0.18 mg/ml unlabeled arachidonic acid to act as carrier during the extraction. Then 700 ml heptane and 400 ml water were added and the phases allowed to partition following vortexing. A 500-ml aliquot of the upper phase was removed to a 12 × 75 mm borosilicate glass test tube containing a slurry of 200 ml heptane and 50 mg activated silicic acid (BioSilA, 100–200 mesh; Bio-Rad, Richmond, CA) and vortexed. The silicic acid was allowed to settle for 15 min or centrifuged down at 3375g for 5 min. The radioactivity was determined in a 500-ml aliquot of the supernatant containing liberated 14C-arachidonic acid using BioCount scintillation cocktail (Research Products International Corp., Mount Prospect, IL) and a Beckman LS6000SC scintillation counter (Fullerton, CA) employing an automatic quench correction curve. For each assay a blank was run with no enzyme source to account for any nonenzymatic hydrolysis of substrate. Assays were performed in triplicate. For the pH optima studies, micelles of the labeled substrate were generated in distilled water (1 nmole/40 ml). A series of buffers, 200 mM containing 10 mM CaCl2, was used for the appropriate pH: acetate, pH 4.0–5.5; Tris– maleate, pH 6.0–7.0; Tris–HCl, pH 7.5–9.0; and glycine, pH 9.5–11.0. Equal volumes (40 ml) of buffer and substrate were mixed to yield 100 mM buffer and 5 mM CaCl2 and the reaction was initiated by the addition of 20 ml saliva. Calcium studies. Saliva was collected by 10 ml intrahemocoelic injections of 1 mM dopamine, 1 mM theophylline, and 3% (v/v) DMSO dissolved either in buffered tick saline (see Needham and Sauer, 1979) that contains 3.52 mM CaCl2 or in 0.8% NaCl. The calcium content of the saliva was determined using a kit (Catalog No. 587, Sigma, St. Louis, MO) essentially as described by the manufacturer’s instructions, except adapted for microplate assay and read at 545nm. For the Ca2+ requirement of the PLA2, assays were performed in 80 mM glycine, pH 9.5, containing 20 mM of chelator nitrilotriacetic acid and the Ca2+ level was amended by the addition of CaCl2 solution. Free Ca2+ concentrations accounted for the amount of Ca2+ present in the saliva sample and were calculated using the program COMICS (Perrin and Sayce, 1967). Size exclusion chromatography. Fresh saliva (∼1 ml) was concentrated to 200 ml using an ultrafiltration unit (10-kDa cut-off; Amicon Inc., Beverley, MA) and subjected to size exclusion chromatography over a Superose 6 HR 10/30 FPLC column (Pharmacia, Piscataway, NJ) connected to a Waters HPLC system. Typically, the elution buffer was 10 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 9.0, run-
TICK SALIVARY PHOSPHOLIPASE A2 ning at 0.5 ml/min, and 0.2- to 0.5-ml fractions were collected. PLA2 activity was assayed as described above, except employing a 10-min incubation period. The column was calibrated daily with nine protein molecular weight markers (1350–670,000 Da) obtained from Bio-Rad and Sigma. Inhibition studies. The PLA2 inhibitors ACA; DEDA; ETYA; OBAA; 17-octadecynoic acid ODYA; ONO-RS082; thioetheramide-PC; and OPC were obtained from BioMol Research Laboratories (Plymouth Meeting, PA). Quinacrine, 4-BPB, indomethacin and arachidonic acid were from Sigma. Ethanol was used as the carrier for arachidonic acid, OPC, thioetheramide-PC, ETYA, DEDA, 17ODYA, 4-BPB, ACA, indomethacin, and quinacrine. Dimethyl sulfoxide was used as the carrier for aristolochic acid, OBAA, and ONO-RS-082. Assays consisted of 80 ml 100 mM Tris–HCl, 5 mM CaCl2, pH 9.0, containing 1 nmole 14C-PC micelles to which 10 ml of inhibitor in the appropriate carrier was added and the reaction initiated by the addition of 20 ml enzyme source, either saliva or salivary gland homogenate. The presence of ∼9% ethanol or dimethyl sulfoxide alone had a stimulatory effect on the PLA2 activity presumably by altering the physicochemical parameters of the 14C-PC micelles. For all inhibitors tested, a control was simultaneously performed to account for the presence of the carrier. Alternate PLA2 substrates. The catalytic activity of the tick saliva and salivary gland PLA2 was also measured against membranous substrates. Escherichia coli (Strains 9637, 23848, and 254040; American Type Culture Collection, Rockville, MD) were labeled with 3H-oleic acid by co-incubation, essentially as described by Elsbach and Weiss (1991). Such labeled E. coli were resuspended in 100 mM Tris–HCl, 5 mM CaCl2, pH 9.0, and autoclaved for 15 min at 120°C and 2.7 kg/cm2. This procedure inactivates the bacterial PLA2 and renders the envelope phospholipids as a suitable PLA2 substrate. 3H-Oleic acid labeled membranes were similarly prepared from Candida albicans (Strains ATCC 36232 and 11006). For the tick saliva and salivary gland PLA2 activity assay, an autoclaved suspension of labeled E. coli or C. albicans was substituted for the 14C-PC micellar substrate. Naja naja snake venom PLA2 (Sigma) activity was also determined against the membranous substrates.
RESULTS PLA2 Activity as a Function of pH, Time, Temperature and Protein Tick saliva contained a catalytic activity that hydrolyzed arachidonic acid from the sn-2 position of 14C-PC (Fig. 1). The PLA2 activity was assessed as a function of incubation buffer pH (Fig. 1A) and exhibited two distinct pH optima at pH 5.5 and 9.5 and was markedly decreased at less than pH 5.0. The activity was linear with
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time to 5 min after which activity was somewhat reduced (Fig. 1B). PLA2 activity was dependent upon the amount of saliva present in the assay (Fig. 1C) and showed no noticeable deviation from linearity up to ∼2 mg protein/assay tube. All experiments in the present report used 1–2 mg/assay. The PLA2 activity exhibited a broad temperature range optimum 37–55°C with noticeably less activity above 55°C (Fig. 1D). It should be noted that the interassay variation was quite large primarily due to differences in 14C-PC substrate micelle preparation. However, intraassay variation was small, typically <10%, thus allowing within-experiment comparisons. Most experiments were performed at both pH 5.5 and 9.0 with similar results, but for clarity, subsequent results are reported only for pH 9.0. PLA2 Stability to Freezing and High Temperatures Tick saliva PLA2 activity undergoing one freeze–thaw cycle retained only 63 ± 9% (n 4 5) activity compared to the fresh saliva. However, ts-PLA2 activity could be stabilized by the addition of an equal volume of 1 mg/ml BSA before freezing, resulting in 90 ± 4% (n 4 5) activity being retained after a single freeze– thaw cycle. All subsequent experiments involved either fresh or pooled saliva that was aliquoted and frozen in the presence of 0.5 mg/ ml BSA. The ts-PLA2 was relatively resistant to preincubation at 90°C prior to being incubated with the substrate at 37°C (Fig. 2). More than 60% of ts-PLA2 activity was still detected after 5 min preincubation at 90°C, and discernable activity was still evident after 25 min. A thermal inactivation plot shows a uniformly logarithmic decline (r2 4 0.990) in ts-PLA2 activity with increasing preincubation time at 90°C (Fig. 2, inset). Calcium Requirement for the PLA2 Activity The presence of the Ca2+ chelator EGTA (10 mM) in the incubation buffer reduced the tsPLA2 activity to essentially zero (data not shown). This dependence of the ts-PLA2 upon
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FIG. 1. Tick saliva PLA2 activity determined by its ability to hydrolyze 14C-arachidonate from 14Cphosphatidylcholine as a function of (A) incubation buffer pH; (B) incubation time; (C) enzyme source protein concentration; and (D) incubation temperature. The standard assay constituted ø2.0 mg protein, 5 min incubation at 37°C, and pH 9.0. Data presented are means ± SD (n 4 3) of representative experiments. Each experiment was performed at least three times with similar results.
free Ca2+ was further investigated over a controlled concentration range (Fig. 3). ts-PLA2 activity was sensitive to as little as 0.05 mM free Ca2+ and half-maximally activated by 3.5 ± 0.8 mM free Ca2+. Millimolar Ca2+ did not enhance ts-PLA2 activity compared to micromolar Ca2+ (data not shown). The total Ca2+ concentration was determined for the saliva collected from ticks by injection with the secretagogue dopamine/theophylline mixture either dissolved in a Ca2+-free or Ca2+containing solution. The presence of Ca2+ in the injectate resulted in higher Ca2+ levels in the saliva. With either collection technique, the total Ca2+ concentration of the saliva was never less than 500 mM.
Size Exclusion Chromatography Saliva subjected to size exclusion chromatography on a Superose 6 FPLC column with 10 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 9.0, elution buffer was fractionated as a single peak of PLA2 activity with an apparent molecular weight of 55,700 ± 1300 Da (Fig. 4). Separations using the above elution buffer without any NaCl revealed two peaks of PLA2: the major peak had an apparent molecular weight of 66,100 ± 2,500 Da and the minor peak (∼25% of the activity) was 130,000 ± 2500 Da (data not shown). Saliva was also eluted using 10 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 7.4, and the fractions were assayed
TICK SALIVARY PHOSPHOLIPASE A2
FIG. 2. Stability of ts-PLA2 activity to a 90°C preincubation treatment for various periods of time. After the heat treatment, the saliva sample was allowed to cool and then assayed at 37°C per standard protocol. Data are means ± SD (n 4 3) of one experiment repeated twice with similar results. Inset shows a log transformed plot of the data.
for PLA2 activity either in 100 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2, pH 9.0, or in 100 mM Na-acetate, 150 mM NaCl, 10 mM CaCl2, pH 5.5. Under either assay condition a single
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FIG. 4. Size exclusion chromatography of crude saliva on an FPLC Superose 6 column eluted with 10 mM Tris, 150 mM NaCl, 1 mM EGTA, pH 9.0. A single peak of PLA2 activity eluted corresponding to 55,700 ± 1300 Da (mean ± SD, n 4 3) and no activity was detected in the region of 14 kDa, the molecular weight of secreted PLA2s in other animals.
peak of activity (∼56 kDa) was detected (data not shown). Substrate Specificity PLA2 in tick saliva was active against 1palmitoyl, 2-arachidonyl-PC and was used in most of the studies. Additionally, 1-palmitoyl, 2-linoleoyl-PC and dipalmitoyl-PC proved to be good substrates for ts-PLA2 (data not shown). However, membranous substrates in the form of radiolabeled and autoclaved E. coli or C. albicans were unsuitable substrates with essentially no apparent PLA2 activity detectable (data not shown). The N. naja venom PLA2 standard showed excellent activity against these prepared substrates. Effective Inhibitors of Saliva PLA2
FIG. 3. ts-PLA2 sensitivity to free calcium. Free Ca2+ concentrations were calculated with COMICS (Perrin and Sayce, 1967) and constructed using 5 mM nitrilotriacetic acid and various amounts of CaCl2. The contribution of Ca2+ present in the enzyme source, tick saliva, was taken into account. Data are the means ± SD of one representative experiment performed three times with similar results.
The substrate analog oleyloxyethyl phosphorylcholine (OPC) and the end product arachidonic acid proved to be effective inhibitors of ts-PLA2 (Fig. 5). OPC was a potent inhibitor exhibiting an IC50 4 1.4 mM and IC90 4 26 mM. Arachidonic acid exhibited an IC50 4 38 mM and would not give complete inhibition of
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FIG. 5. Dose-dependent inhibition of ts-PLA2 activity by the substrate analog oleyloxyethyl phosphorylcholine (OPC) and the end product arachidonic acid (AA). For OPC the calculated IC50 4 1.4 mM and IC90 4 26 mM, and for AA IC50 4 38 mM. Data are means ± SD (n 4 3) of one representative experiment repeated three times with similar results.
ts-PLA2 at any concentration. Higher concentrations of arachidonic acid are likely to be affecting the physicochemical characteristics of the 14C-PC substrate micelles as much as interacting with the enzyme.
FIG. 6. Heat stability of ts-PLA2 and sg-PLA2 activity. Tick saliva (solid circles) or salivary gland homogenate (open circles) were preincubated for 15 min at different temperatures, allowed to cool, and then assayed for PLA2 activity at 37°C per standard protocol. Data are means ± SD (n 4 3) of one representative experiment repeated three times with similar results.
enate at 30–50°C enhanced the sg-PLA2 activity. The PLA2 activity in the saliva and the salivary gland were both inhibited by DTT in a similar dose-dependent manner (Fig. 7). About one-third of the ts-PLA2 and sg-PLA2 was in-
Comparative Aspects of PLA2 in the Saliva and the Salivary Gland The PLA2 present in the saliva or salivary gland exhibited different responses to a 15-min preincubation period at higher temperatures before being assayed for activity at 37°C (Fig. 6). Preincubation of the saliva at temperatures between 30 and 60°C gave a marked increase in the measured ts-PLA2 activity with a 1.5-fold increase at 50°C compared to no preincubation. Higher preincubation temperatures reduced the activity in the saliva, but over 80% of the activity was still present after 15 min at 80°C. In contrast, only 20% of the PLA2 activity present in the salivary gland homogenate remained after the 80°C preincubation and the activity decreased at preincubations above 50°C. Like the ts-PLA2, preincubation of the gland homog-
FIG. 7. Effect of the reducing agent dithiothreitol (DTT) on the activity of ts-PLA2 and sg-PLA2. Tick saliva (dashed line) and salivary gland homogenate (solid line) were assayed in the presence of DTT. Assays were performed in triplicate, error bars are omitted for clarity but were <12%.
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hibited by 1 mM DTT, but even at 125 mM DTT the activities were still only inhibited by ∼70%. A battery of agents known to inhibit PLA2s from other sources were tested against ts-PLA2 and sg-PLA2 (Table I) at concentrations that had been reported to be effective against the other PLA2s. The degree of inhibition ranged from absent (aristolochic acid) to very low (17-ODYA, OBAA, ONO-RS-082), to lower than expected (4-BPB, thioetheramidePC, quinacrine). The arachidonic acid analogs, DEDA and ETYA, gave partial inhibition of both ts-PLA2 and sg-PLA2. In no case was inhibition of the tick PLA2 greater than that reported for PLA2s from other sources. The inhibitors had similar efficacies against ts-PLA2 and sg-PLA2. DISCUSSION Compared to other ectoparasites adult ticks remain attached to the host for an unusually long time. For example, the adult female A. americanum remains attached to the host for 10–14 days. The salivary glands are the organs of osmoregulation (Sauer et al., 1995) and excess water and ions in the bloodmeal are continuously being returned to the host via the saliva, thus concentrating the nutrients for future
egg production. The saliva contains an array of pharmacologically active profeeding compounds to counter the host’s hemostatic, vasoconstrictive, inflammatory, and immune responses (Bowman et al., 1997). Here we report upon another active compound secreted at the host–parasite interface, a PLA2 with unique characteristics. The saliva used in this study was collected by intrahemocoelic injection of dopamine, the neurotransmitter regulating the control of salivation in ticks (Sauer et al., 1995), a common and accepted technique for investigating the contents of tick saliva (Bowman et al., 1995; Zhu et al., 1997a,b; Kaufman and Nuttall, 1996). We have recently found that ‘‘natural saliva’’ (collected without dopamine injection) also contains ts-PLA2 activity (Zhu et al., 1998), but limited collectable quantities prevent natural saliva from being used in studies. The ts-PLA2 exhibited activity over a broad pH range, but demonstrated two distinct pH optima of pH 5.5 and 9.5, indicating the possibility that two different PLA2s may be present in the saliva. However, this is unlikely because experiments where ts-PLA2 activity was determined at both pH 5.5 and pH 9.0 yielded similar results. The thermal inactivation plot at 90°C of tsPLA2 showed a uniform decline in activity (Fig.
TABLE I The Effects of Some Known PLA2 Inhibitors upon the PLA2 Activity Found in Tick Saliva (ts-PLA2) and Salivary Gland Homogenate (sg-PLA2) Inhibition of PLA2 activity (%)
Inhibitor
Concentrationa (mM)
sg-PLA2
ts-PLA2
Thioetheramide-PC ETYA DEDA 17-ODYA Aristolochic acid 4-BPB Quinacrine Indomethacin OBAA ACA ONO-RS-082
2 10 15 10 5 50 250 400 40 10 1
8 36 21 8 0 22 13 25 0 21 0
11 24 26 0 0 10 0 35 0 10 10
Note. Determinations were performed in triplicate. a Concentrations (>>IC50) at which PLA2s from other sources have been reported to be inhibited.
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2, inset); if two different PLA2 enzymes were responsible for the activity one would expect two rates of deactivation each with its own inactivation constant. Additionally, only one peak of coeluting PLA2 activity was observed when the saliva was subjected to size exclusion chromatography and the eluate assayed at both pHs. Tick saliva is ∼pH 9.5 (Bowman et al., 1995). The small (∼14 kDa) secreted PLA2s from bee and snake venoms and mammalian pancreatic or inflammatory fluid also exhibit alkaline optima (∼pH 8–10). Though tick saliva is pH 9.5, the pH at the feeding lesion is unknown but is likely to be more similar to host interstitial fluid (∼pH 7.8). At this more neutral pH ts-PLA2 is still active. A distinguishing feature between secreted and cytosolic PLA2s is their Ca2+ sensitivities. Secreted or extracellular PLA2s are active only in the presence of millimolar Ca2+, the concentration found in interstitial fluids and gut lumens. We are aware of only one extracellular PLA2 that is active at micromolar Ca2+ that is found in the venom of a marine snail (McIntosh et al., 1995). In contrast, cytosolic or intracellular PLA2s are sensitive to submicromolar Ca2+ with sharp increases in activity as the Ca2+ concentration increases from 10−7 to 10−6 M (Kudo et al., 1993). The free Ca2+ concentration in the cytosol generally increases to around the micromolar level upon cell stimulation (Rink et al., 1982). The ts-PLA2’s Ca2+ requirement is most intriguing. Being present in the saliva, the ts-PLA2 is obviously a secreted extracellular enzyme, but it is sensitive to 0.05 mM free Ca2+ and is half-maximally activated by 3.5 mM free Ca2+. In this respect, the ts-PLA2 is like an intracellular PLA2. The total Ca2+ concentration in the tick saliva was >500 mM and the Ca2+ concentration in the feeding lesion would be in the millimolar range; thus, the ts-PLA2 would be fully activated from the time of secretion. We found that the presence of Ca2+ in the secretagogue mixture injected into the tick resulted in higher Ca2+ levels in the saliva. The finding that the composition of the injection media affects the composition of the tick saliva should be noted by researchers in this area.
The ts-PLA2 activity was stable to high incubation temperatures both prior to and during the assay incubation period and to freezing. The necessity of the presence of 0.5 mg/ml BSA during the freezing was probably because of the dilute nature of tick saliva (∼0.2 mg protein/ml). If the protein content of the saliva was increased by ultrafiltration, ts-PLA2 was stable to freezing without the addition of any BSA (unpublished observations). ts-PLA2 activity was enhanced by 15-min preincubation temperatures of 40– 60°C and the activity was still present at 80°C. Cytosolic PLA2s are inactivated by temperatures above 57°C (Mayer and Marshall, 1993). In contrast, secreted PLA2s are resistant to higher temperatures (Mayer and Marshall, 1993). The physical stability of secreted PLA2s is afforded by the numerous, typically five to seven, disulfide bonds which are completely absent in the cytosolic PLA2s (Dennis, 1994). The presence of the disulfide bonds leads secreted PLA2s to be inactivated by reducing agents such as DTT, whereas cytosolic PLA2s are unaffected (Mayer and Marshall, 1993). The sensitivity of ts-PLA2 to DTT was intermediate between that of cytosolic and secreted PLA2s. Cytosolic PLA2 from human monocytic cell line U937 is resistant to DTT (Clark et al., 1990) and cytosolic PLA2 from mouse macrophages is 2-mercaptoethanol resistant (Leslie et al.,1988). Rat inflammatory exudate PLA2 activity is inhibited by 60% in the presence of 1 mM DTT and 80% in 3 mM (Chang et al., 1987), while human synovial fluid PLA2 activity is abolished in 3 mM DTT (Hara et al., 1989). The ts-PLA2 activity was reduced by ∼35% in 1 mM DTT and ∼70% in 125 mM DTT. It would seem that reduction of the disulfide linkages in ts-PLA2 brings about some conformational structure change of the enzyme that reduces its catalytic activity, but these changes appear not to be near the active site since catalytic activity is evident even at very high DTT levels. The high physical stability of ts-PLA2 would be suited to a protein originating in one location but targeted to function in a foreign environment. Tick saliva PLA 2 readily hydrolyzed 1palmitoyl-2-arachidonyl-phosphatidylcholine
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substrate when presented as sonicated micelles without any additional lipids or detergents required. However, membranous substrates in the form of 3H-oleic acid labeled E. coli or C. albicans could not serve as a substrate for tsPLA2 even though we verified their suitability for N. naja snake venom PLA2. The lack of activity of ts-PLA2 against the labeled membrane substrate is unusual since secreted PLA2s almost universally hydrolyze this form of substrate (Elsbach and Weiss, 1991). The cytosolic PLA2s are not assayed using such micellar substrates. Secreted PLA2s are uniformly ∼14 kDa and calcium-dependent cytosolic PLA2s are 85 kDa though there are also reports of Ca 2 + independent cytosolic PLA2s of 40 kDa (Dennis, 1994; Mayer and Marshall, 1993). Size exclusion chromatography of tick saliva revealed a single peak of PLA2 activity corresponding to a molecular weight of 56 kDa. Interestingly, PLA2 activity recovered from the column was consistently greater (∼175%) than that applied to the column. The phenomenon of increased PLA2 activity following size exclusion chromatography has been reported for several PLA2s, and it has been suggested that the removal of some endogenous inhibitory factors is responsible (Hara et al., 1991a). An alternative explanation is that crude saliva contains some endogenous phospholipid substrate and greater activity is recorded when the ts-PLA2 is assayed in the absence of any competing substrate. The PLA2 activity from both the salivary gland and in the saliva was not readily inhibited by a variety of PLA2 inhibitors that are known to inhibit either one or both of the secreted and the cytosolic PLA2s. Aristolochic acid inhibits secreted PLA 2 s in both snake venom (Vishwanath et al., 1987) and human neutrophils (Rosenthal et al., 1989) by apparently binding to a nonactive site region of the enzyme and causing a conformational change (Vishwanath et al., 1987). Aristolochic acid was ineffective against tick PLA2. BPB inactivates secreted PLA2s by covalently alkylating histidine-48 in the enzyme’s active site (Kudo et al., 1993) but is largely ineffective against cytosolic
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PLA2 (Mayer and Marshall, 1993). ts-PLA2 was only marginally affected by BPB, indicating that its catalytic mechanism is different from that of the secreted PLA2s. Quinacrine inhibits some PLA2s, notably secreted PLA2s, by forming complexes with the phospholipid and perturbing the interaction between the enzyme and substrate (Hirata, 1989). Neither source of tick PLA2 was inhibited by quinacrine. Indomethacin, a potent nonsteroidal inhibitor of cyclooxygenase, inhibits PLA2 activity at higher concentrations through antagonism against Ca2+ (Hirata, 1989). High concentrations of indomethacin caused only partial inhibition of both sg-PLA2 and ts-PLA2. The PLA2 substrate analog thioetheramide-PC is a potent inhibitor of secreted PLA2s, but is ineffective against cytosolic PLA2 (Hope et al., 1993; Mayer and Marshall, 1993). Thioetheramide-PC had no effect upon tick PLA2. However, another PLA2 substrate analog, OPC, was an effective inhibitor of both ts-PLA2 and sg-PLA2. Though OPC inhibits secreted PLA2s, it does not inhibit cytosolic PLA2s (Hope et al., 1993). Arachidonic acid inhibited both ts-PLA2 and sg-PLA2, and the arachidonic acid analogs DEDA and ETYA acid were also inhibitory toward the tick PLA2s, but another analog, 17-ODYA, was largely ineffective. Arachidonic acid analogs inhibit secreted PLA2s (Welton et al., 1984; Hope et al., 1993) but are ineffective against cytosolic PLA2 (Hope et al., 1993). Overall, the inhibitor profile was similar for ts-PLA2 and sg-PLA2, but both showed clear differences from both the cytosolic and the secreted PLA2s, indicating that the tick PLA2 has a different catalytic mechanism or substrate binding site. We had previously found a PLA2 activity present in the salivary glands of A. americanum (Bowman et al., 1998). Finding a PLA2 activity in the saliva that shares many of the properties of sg-PLA2 poses the question of whether they are the same enzyme. The apparent molecular weights (56 kDa, ts-PLA2; and 54 kDa, sgPLA2) and the submicromolar Ca2+ activation are clear similarities. In the present report, we show that the DTT sensitivity of both ts-PLA2 and sg-PLA2 are similar, as are the responses to
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the inhibitors tested. There are, however, some differences. Whereas size exclusion chromatography of saliva yields greater than 100% recovery of ts-PLA2 activity, greatly reduced activity was consistently observed in fractionating the salivary gland homogenate. Additionally, the sg-PLA2 activity was susceptible to high preincubation temperatures, but ts-PLA2 activity was enhanced. Both of these differences between sgPLA2 and ts-PLA2 could possibly be explained by differences in the matrices, that is, a dilute saliva and a tissue homogenate. Further work is required to demonstrate whether ts-PLA2 and sg-PLA2 are the same or different enzymes. What, however, is the significance to the tick of a salivary PLA2 secreted into the feeding lesion of the host? Secreted PLA2s from T. cruzi (Connelly and Kiersenbaum, 1984), T. gondii (Saffer and Schwartzman, 1991; Gomez-Marin et al., 1996), and R. rickettsii (Walker et al., 1983) have been implicated in host cell invasion by these intracellular parasites. While ticktransmitted pathogens present in the saliva may utilize the ts-PLA2 to enhance their invasion of host cells, this would serve no obvious benefit to the tick. We have shown that ts-PLA2 is not involved in the anticoagulant activity of A. americanum saliva (Zhu et al., 1997a) and has limited hemolytic activity of sheep erythrocytes as a type of extra-corporeal digestion (Zhu et al., 1997b). Tick saliva contains extremely high levels of prostaglandins, including PGE2, believed to aid tick feeding due to their antihemostatic, vasodilatory, anti-inflammatory and immunosuppressive properties (Bowman et al., 1996). The ectoparasitic larval stage of the cattle grub (Hypoderma lineatum) secretes a factor that induces host peripheral blood mononuclear cells to synthesize high levels of PGE2 which subsequently downregulate the immune response (Nicolas-Gaulard et al., 1995). Exogenous PLA2s have been shown to stimulate granulocytes to produce PGE2 (Hara et al., 1991b). In such a way, ts-PLA2 could cause host cells to produce PGE2 that would augment the PGE2 already present in the tick saliva. In preliminary studies, size exclusion fractions of tick saliva containing the ts-PLA2 activity
stimulated prostacylin (PGI2) production by cultured endothelial cells (unpublished observations). Prostacyclin would benefit tick feeding by preventing platelet aggregation, inducing vasodilation, and countering vasoconstriction (Bowman et al., 1996). In summation, tick saliva contains an unusual secreted PLA2 that is active at Ca2+ concentrations that might be found inside a cell. The molecular weight, Ca2+ activation level, and sensitivity to several PLA2 inhibitors preclude tsPLA2 from being placed in any of the present PLA2 classification categories. The PLA2 activities found in the tick saliva and salivary gland show many similarities, but also exhibit some distinct differences. Currently, we do not know the teleologic function of ts-PLA2, but its presence in tick saliva indicates that it must play some role at the tick–host interface. ACKNOWLEDGMENTS The authors express their thanks to Jerry Bowman, Central Tick Rearing Facility, OSU, for supplying the ticks. We are grateful to Drs. Steve P. White and Doug Bergman for reviewing an earlier draft of the manuscript. This work was approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported by NIH Grant AI-31460.
REFERENCES Bowman, A. S., Coons, L. B., Needham, G. R., and Sauer, J. R. 1997. Tick salivary secretions at the tick–host interface: Recent advances and implications for vector competence. Medical and Veterinary Entomology, in press. Bowman, A. S., Dillwith, J. W., and Sauer, J. R. 1996. Tick salivary prostaglandins: Presence, origin and significance. Parasitology Today 12, 388–396. Bowman, A. S., Sauer, J. R., Zhu, K., and Dillwith, J. W. 1995. Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum. Insect Biochemistry and Molecular Biology 25, 735–741. Bowman, A. S., Surdick, M. R., Gengler, C. L., Zhu, K., Essenberg, R. C., Sauer, J. R., and Dillwith, J. W. 1998. Phospholipase A2 activity in salivary glands of the lone star tick, Amblyomma americanum. [Submitted for publication] Chang, H. W., Kudo, I., Tomita, M., and Inoue, K., 1987. Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rat. Journal of Biochemistry 102, 147–154. Clark, J. D., Milona, N., and Knopf, J. L. 1990. Purification of a 110-kilodalton cytosolic phospholipase A2 from the
TICK SALIVARY PHOSPHOLIPASE A2 human monocytic cell line U9337. Proceedings of the National Academy of Sciences USA 87, 7708–7712. Connelly, M. C., and Kierszenbaum, F. 1984. Modulation of macrophage interaction with Trypanosoma cruzi by phospholipase A2-sensitive components of the parasite membrane. Biochemical and Biophysical Research Communications 121, 931–939. Dennis, E. A. 1994. Diversity of group types, regulation, and function of phospholipase A2. Journal of Biological Chemistry 269, 13057–13060. Elsbach, P., and Weiss, J. 1991. Utilization of labeled Escherichia coli as phospholipase substrate. Methods: A Companion to Methods in Enzymology 197, 24–31. Gomez Marin, J. E., Bonhomme, A., Guenounou, M., and Pinon, J. M. 1996. Role of interferon-g against invasion by Toxoplasma gondii in a human monocytic cell line (THP1): Involvement of the parasite’s secretory phospholipase A2. Cellular Immunology 169, 218–225. Hara, S., Kudo, I., Chang, H. W., Matsuta, K., Miyamoto, T., and Inoue, K. 1989. Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis. Journal of Biochemistry 105, 395–399. Hara, S. Chang, H. W., Horigome, K., Kudo, I., and Inoue, K. 1991a. Purification of mammalian nonpancreatic extracellular phospholipases A2. Methods: A Companion to Methods in Enzymology 197, 381–389. Hara, S., Kudo, I., and Inoue, K. 1991b. Augmentation of prostaglandin E2 production by mammalian phospholipase A2 added exogenously. Journal of Biochemistry 110, 163–165. Hirata, F. 1989. Drugs that inhibit the activities or activation of phospholipases and other acylhydrolases. In ‘‘Handbook of Eicosanoids: Prostaglandins and Related Lipids. Vol II. Drugs Acting via the Eicosanoids’’ (A. L. Willis, Ed.), pp. 47–58. CRC Press, Boca Raton, FL. Hope, W. C., Chen, T., and Morgan, D. W. 1993. Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2. Agents and Actions 39, C39–C42. Kaufman, W. R., and Nuttall, P. A. 1996. Amblyomma variegatum (Acari:Ixodidae): Mechanism and control of arbovirus secretion in tick saliva. Experimental Parasitology 82, 316–323. Kudo, I., Murakami, M., Hara, S., and Inoue, K. 1993. Mammalian non-pancreatic phospholipases A 2. Biochimica et Biophysica Acta 117, 217–231. Leslie, C. C., Voelker, D. R., Channon, J. Y., Wall, M. M., and Zelarney, P. T. 1988. Properties and purification of an arachidonyl-hyrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. Biochimica et Biophysica Acta 963, 476–492. Mayer, R. J., and Marshall, L. A. 1993. New insights on mammalian phospholipase A2(s); comparison of arachidonyl-selective and -nonselective enzymes. FASEB Journal 7, 339–348. McIntosh, M. J., Ghomashchi, F., Gelb, M. H., Dooley,
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D. J., Stoehr, S. J., Giordani, A. B., Naisbitt, S. R., and Olivera, B. M. 1995. Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail, Conus magus. Journal of Biological Chemistry 270, 3518–3525. Mukherjee, A. B., Miele, L., and Pattabiraman, N. 1994. Phospholipase A2 enzymes: Regulation and physiological role. Biochemical Pharmacology 48, 1–10. Needham, G. R., and Sauer, J. R. 1979. Involvement of calcium and cyclic AMP in controlling Ixodid tick salivary fluid secretion. Journal of Parasitology 65, 531–542. Nicolas-Gaulard, I., Moire, N., and Boulard, C. 1995. Effect of the parasite enzyme, hypodermin A, on bovine lymphocyte proliferation and interleukin-2 production via the prostaglandin pathway. Immunology 84, 160–165. Patrick, C. D., and Hair, J. A. 1975. Laboratory rearing procedures and equipment for multihost ticks (Acarina: Ixodidae). Journal of Medical Entomology 12, 389–390. Perrin, D. D., and Sayce, I. G. 1967. Computer calculation of equilibrium concentrations in mixtures of metal ions and complexing species. Talanta 14, 833–842. Ravdin, J., Murphy, C. F., Guerrant, R. L., and Long-Krug, S. A. 1985. Effect of antagonists of calcium amd phospholipase A on the cytopathogenicity of Entamoeba histoytica. Journal of Infectious Diseases 152, 542–549. Rink, T. J., Smith, S. W., and Tsien, R. Y. 1982. Cytoplasmic free Ca2+ in human platelets: Ca2+thresholds and Ca-independent activation for shape-change and secretion. FEBS Letters 148, 21–26. Rogers, M. V., Henkle, K. J., Herrmann, V., McLaren, D. J., and Mitchell, G. F. 1991. Evidence that a 16kilodalton integral membrane protein antigen from Schistosoma japonicum adult worms is a Type A2 phospholipase. Infection and Immunity 59, 1442–1447. Rosenthal, M. D., Vishwanath, B. S., and Franson, R. C. 1989. Effects of aristolochic acid on phospholipase A2 activity and arachidonate metabolism of human neutrophils. Biochimica et Biophysica Acta 1001, 1–8. Saffer, L. D., and Schwartzman, J. D. 1991. A soluble phospholipase of Toxoplasma gondii associated with host cell penetration. Journal of Protozoology 38, 454–460. Sauer, J. R., McSwain, J. L., Bowman, A. S., and Essenberg, R. C. 1995. Tick salivary gland physiology. Annual Review of Entomology 40, 245–267. van den Bosch, H., De Jong, J. G. N., and Aarsman, A. J. 1991. Phospholipase A2 from rat liver mitochondria. Methods: A Companion to Methods in Enzymology 197, 365–373. Vishwanath, B. S., Rao, A. G. A., and Gowda, V. 1987. Interaction of phospholipase A2 from Vipera russelli venom with aristolochic acid: A circular dichroism study. Toxicon 25, 939–946. Walker, D. H., Firth, W. T., Ballard, J. G., and Hegarty, B. C. 1983. Role of phospholipase-associated penetration mechanism in cell injury by Rickettsia rickettsii. Infection and Immunity 40, 840–842.
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Welton, A. F., Hope, W. C., Fielder-Nagy, C., and BatulaBernardo, C. 1984. Analogs of arachidonic acid methylated at C-7 and C-10 inhibit leukotriene biosynthesis and phospholipase A2 activity. Prostaglandins 28, 649. Zhu, K., Sauer, J. R., Bowman, A. S., and Dillwith, J. W. 1997a. Identification and characterization of anticoagulant activities in the saliva of the lone star tick, Amblyomma americanum (L.). Journal of Parasitology 83, 38– 43. Zhu, K., Dillwith, J. W., Bowman, A. S., and Sauer, J. R.
1997b. Identification of hemolytic activity in the saliva of the lone star tick (Acari:Ixodidae). Journal of Medical Entomology 34, 160–166. Zhu, K., Bowman, A. S., Dillwith, J. W., and Sauer, J. R. 1997c. Phospholipase A2 activity in the salivary glands and saliva of the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidiae) during tick feeding. [Submitted for publication] Received 2 June 1997; accepted with revision 28 July 1997
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A Novel Phospholipase A2 Activity in Saliva of the Lone Star Tick, Amblyomma americanum (L.) A. S. Bowman, C. L. Gengler, M. R. Surdick, K. Zhu, R. C. Essenberg,* J. R. Sauer, and J. W. Dillwith Department of Entomology and *Department of Biochemistry and Molecular Biology, Oklahoma State University, 127 Noble Research Center, Stillwater, Oklahoma 74048-0464, U.S.A. BOWMAN, A. S., GENGLER, C. L., SURDICK, M. R., ZHU, K., ESSENBERG, R. C., SAUER, J. R., AND DILLWITH, J. W. 1997. A novel phospholipase A2 activity in saliva of the lone star tick, Amblyomma americanum (L.). Experimental Parasitology 87, 121–132. Saliva from female lone star ticks, Amblyomma americanum, contained a novel phospholipase A2 (PLA2) activity that hydrolyzed 14Carachidonate from 14C-arachidonyl phosphatidylcholine. The tick saliva PLA2 (ts-PLA2) was active over a broad pH range (4.5–11.5) with two distinct pH optima of pH 5.5 and 9.5. Though extracellular PLA2s are reported to be activated by millimolar Ca2+, ts-PLA2 was sensitive to submicromolar Ca2+ and was half-maximally activated by 3.5mM Ca2+. Tick saliva contains >500mM Ca2+and the feeding lesion in the host is expected to contain millimolar Ca2+. Saliva exhibited a single peak of PLA2 activity corresponding to a molecular weight of 55.7 ± 1.3 kDa by size exclusion chromatography. The ts-PLA2 was unaffected by a variety of compounds known to inhibit either secreted or cytosolic PLA2s from other sources. However, ts-PLA2 was inhibited by the substrate analog, oleyloxyethyl phosphorylcholine (IC50 4 1.4 mM), and the end product, arachidonic acid (IC50 4 38 mM). Low concentrations of dithiothreitol did not greatly affect ts-PLA2, but activity was reduced at higher concentrations. The PLA2 activity found in A. americanum salivary glands showed many similarities to ts-PLA2, but also some distinct differences. Secreted at the tick–host interface, ts-PLA2 is thought to play an important, but unknown, role during the prolonged tick feeding. © 1997 Academic Press INDEX DESCRIPTORS AND ABBREVIATIONS: Amblyomma americanum; phospholipase A2 (E.C. 3.1.14); saliva; salivary gland; tick; AA, arachidonic acid; 14C-PC; L-a-1-palmitoyl-2-[114 C]arachidonyl phosphatidylcholine; ACA, N-(p-amylcinnamoyl) anthranilic acid; 4-BPB, pbromophenacyl bromide; DEDA, 7,7-dimethyleicosadienoic acid; DTT, dithiothreitol; ETYA, 5,8,11,14-eicosatetraynoic acid; OBAA, 3(-4-octadecyl)-benzoylacrylic acid; ODYA, 17octadecynoic acid; ONO-RS-082, 2(p-amylcinnamoyl)amino-4-chlorobenzoic acid; OPC, oleyloxyethyl phosphorylcholine; thioetheramide-PC, thioetheramide-phosphorylcholine; ts-PLA2, tick salivary PLA2; sg-PLA2, salivary gland PLA2.
INTRODUCTION Phospholipase A2 (PLA2; EC 3.1.14) catalyzes the hydrolysis of the sn-2 fatty acyl bond of phospholipids to yield free fatty acids and lysophospholipids (for reviews see Kudo et al., 1993; Mayer and Marshall, 1993; Dennis, 1994; Mukherjee et al., 1994). PLA2s are ubiquitously found throughout the animal kingdom reflecting the universal occurrence of phospholipids. PLA2s play important roles in phospholipid digestion, rearrangement of cellular membrane phospholipid structures, inflammatory responses, defense and predation mechanisms, and signal transduction. Perhaps the best studied PLA2s are the small secreted enzymes of
bee venom, snake venoms, and mammalian pancreatic secretions. Additionally, larger PLA2s have been identified that are not secreted and show a high preference for arachidonatecontaining phospholipids, thus releasing arachidonic acid for the conversion to eicosanoid lipid mediators. More recently, there have been a number of PLA2s reported exhibiting a variety of characteristics that do not fit into the standard PLA2 classification scheme. Phospholipase A2 activity in parasites has received little attention, but one might expect that PLA2 is present in all parasites. Phospholipase A2 activity has been reported in Rickettsia rickettsii (Walker et al., 1983), Trypanosoma cruzi (Connelly and Kierszenbaum, 1984), Toxo-
121 0014-4894/97 $25.00 Copyright © 1997 by Academic Press All rights of reproduction in any form reserved.
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plasma gondii (Saffer and Schwartzman, 1991; Gomez-Marin et al., 1996), Entamoeba histolytica (Ravdin et al., 1985), and Schistosoma japonicum (Rogers et al., 1991). Recently, we found a PLA2 activity in the salivary glands of the lone star tick (Amblyomma americanum) that exhibits some unusual characteristics (Bowman et al., 1998). In the present study we investigated the presence of a PLA2 activity in the saliva collected from A. americanum and compared it to the PLA2 found in the salivary gland. MATERIALS AND METHODS Saliva and salivary gland collection. Mated adult female lone star ticks, A. americanum (L.), were reared on sheep at the Oklahoma State University Central Tick Rearing Facility according to the methods of Patrick and Hair (1975). Ticks were removed from the sheep during the ‘‘slowfeeding’’ phase (7–10 days on the sheep), their mouthparts were cleaned of any adhering host tissue, and they were induced to salivate within 2 hr. The ticks were affixed on adhesive tape ventral side up and induced to salivate by 10ml intrahemocoelic injections at 15-min intervals up to 60 min with buffered tick saline containing 1 mM dopamine, 1 mM theophylline, and 3% (v/v) dimethyl sulfoxide. The composition of buffered tick saline is given in full elsewhere (Needham and Sauer, 1979). Saliva was collected into 25-ml glass capillary tubes positioned over the mouthparts and was dispelled at 15-min intervals into microcentrifuge tubes held on ice. Before assay or storage the saliva was centrifuged at 11,500g for 5 min to sediment any debris. For storage, saliva was diluted with an equal volume of 0.8% NaCl containing 1 mg/ml essentially fatty acid-free BSA and frozen at −70°C. Adult ticks, as above, were dissected while submerged in ice-cold 0.1 M Mops, 20 mM EGTA, pH 6.8, and cleaned of any adhering trachea or other tissue. Glands were rinsed in 100 mM Tris, pH 9.0, homogenized in a glass/glass homogenizer in the same buffer, and then centrifuged at 11,500g for 10 min at 4°C to remove cellular debris. Homogenate not immediately used was stored at −70°C. Phospholipase A2 activity assay. PLA2 activity was assessed by the amount of radioactive arachidonic acid released from phosphatidylcholine in a nonmixed micellar assay based upon the method of Van den Bosch et al. (1991) with some modifications . Substrate, phosphatidylcholine, 14 14 L-a-1-palmitoyl-2-[1- C]arachidonyl ( C-PC), was dried under nitrogen and then resuspended as micelles by sonication (3 × 45 sec burst, 20 kilocycles/sec, 90 W, Artek 300, Farmingdale, NY) in assay buffer (100 mM Tris–HCl, 5 mM CaCl2, pH 9.0). The micellar suspension was aliquoted into 12 × 75 mm borosilicate test tubes and prewarmed to 37°C in a water bath for 5 min, and the assay was initiated
by the addition of the enzyme source and 15 sec vigorous vortexing. Tubes were incubated at 37°C in a moderately fast shaking water bath (80 cycles/min). Typically, the assay consisted of 80 ml substrate suspension (1 nmole 14C-PC, 0.05 mCi) and 20 ml enzyme source to give a 4 mM final concentration of CaCl2 and the reaction proceeded for 5 min. The reaction was terminated by the addition of 750-ml ice-cold Dole’s reagent (700:60, 2-propanol: 1 mM HCl) and 10 ml of 0.18 mg/ml unlabeled arachidonic acid to act as carrier during the extraction. Then 700 ml heptane and 400 ml water were added and the phases allowed to partition following vortexing. A 500-ml aliquot of the upper phase was removed to a 12 × 75 mm borosilicate glass test tube containing a slurry of 200 ml heptane and 50 mg activated silicic acid (BioSilA, 100–200 mesh; Bio-Rad, Richmond, CA) and vortexed. The silicic acid was allowed to settle for 15 min or centrifuged down at 3375g for 5 min. The radioactivity was determined in a 500-ml aliquot of the supernatant containing liberated 14C-arachidonic acid using BioCount scintillation cocktail (Research Products International Corp., Mount Prospect, IL) and a Beckman LS6000SC scintillation counter (Fullerton, CA) employing an automatic quench correction curve. For each assay a blank was run with no enzyme source to account for any nonenzymatic hydrolysis of substrate. Assays were performed in triplicate. For the pH optima studies, micelles of the labeled substrate were generated in distilled water (1 nmole/40 ml). A series of buffers, 200 mM containing 10 mM CaCl2, was used for the appropriate pH: acetate, pH 4.0–5.5; Tris– maleate, pH 6.0–7.0; Tris–HCl, pH 7.5–9.0; and glycine, pH 9.5–11.0. Equal volumes (40 ml) of buffer and substrate were mixed to yield 100 mM buffer and 5 mM CaCl2 and the reaction was initiated by the addition of 20 ml saliva. Calcium studies. Saliva was collected by 10 ml intrahemocoelic injections of 1 mM dopamine, 1 mM theophylline, and 3% (v/v) DMSO dissolved either in buffered tick saline (see Needham and Sauer, 1979) that contains 3.52 mM CaCl2 or in 0.8% NaCl. The calcium content of the saliva was determined using a kit (Catalog No. 587, Sigma, St. Louis, MO) essentially as described by the manufacturer’s instructions, except adapted for microplate assay and read at 545nm. For the Ca2+ requirement of the PLA2, assays were performed in 80 mM glycine, pH 9.5, containing 20 mM of chelator nitrilotriacetic acid and the Ca2+ level was amended by the addition of CaCl2 solution. Free Ca2+ concentrations accounted for the amount of Ca2+ present in the saliva sample and were calculated using the program COMICS (Perrin and Sayce, 1967). Size exclusion chromatography. Fresh saliva (∼1 ml) was concentrated to 200 ml using an ultrafiltration unit (10-kDa cut-off; Amicon Inc., Beverley, MA) and subjected to size exclusion chromatography over a Superose 6 HR 10/30 FPLC column (Pharmacia, Piscataway, NJ) connected to a Waters HPLC system. Typically, the elution buffer was 10 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 9.0, run-
TICK SALIVARY PHOSPHOLIPASE A2 ning at 0.5 ml/min, and 0.2- to 0.5-ml fractions were collected. PLA2 activity was assayed as described above, except employing a 10-min incubation period. The column was calibrated daily with nine protein molecular weight markers (1350–670,000 Da) obtained from Bio-Rad and Sigma. Inhibition studies. The PLA2 inhibitors ACA; DEDA; ETYA; OBAA; 17-octadecynoic acid ODYA; ONO-RS082; thioetheramide-PC; and OPC were obtained from BioMol Research Laboratories (Plymouth Meeting, PA). Quinacrine, 4-BPB, indomethacin and arachidonic acid were from Sigma. Ethanol was used as the carrier for arachidonic acid, OPC, thioetheramide-PC, ETYA, DEDA, 17ODYA, 4-BPB, ACA, indomethacin, and quinacrine. Dimethyl sulfoxide was used as the carrier for aristolochic acid, OBAA, and ONO-RS-082. Assays consisted of 80 ml 100 mM Tris–HCl, 5 mM CaCl2, pH 9.0, containing 1 nmole 14C-PC micelles to which 10 ml of inhibitor in the appropriate carrier was added and the reaction initiated by the addition of 20 ml enzyme source, either saliva or salivary gland homogenate. The presence of ∼9% ethanol or dimethyl sulfoxide alone had a stimulatory effect on the PLA2 activity presumably by altering the physicochemical parameters of the 14C-PC micelles. For all inhibitors tested, a control was simultaneously performed to account for the presence of the carrier. Alternate PLA2 substrates. The catalytic activity of the tick saliva and salivary gland PLA2 was also measured against membranous substrates. Escherichia coli (Strains 9637, 23848, and 254040; American Type Culture Collection, Rockville, MD) were labeled with 3H-oleic acid by co-incubation, essentially as described by Elsbach and Weiss (1991). Such labeled E. coli were resuspended in 100 mM Tris–HCl, 5 mM CaCl2, pH 9.0, and autoclaved for 15 min at 120°C and 2.7 kg/cm2. This procedure inactivates the bacterial PLA2 and renders the envelope phospholipids as a suitable PLA2 substrate. 3H-Oleic acid labeled membranes were similarly prepared from Candida albicans (Strains ATCC 36232 and 11006). For the tick saliva and salivary gland PLA2 activity assay, an autoclaved suspension of labeled E. coli or C. albicans was substituted for the 14C-PC micellar substrate. Naja naja snake venom PLA2 (Sigma) activity was also determined against the membranous substrates.
RESULTS PLA2 Activity as a Function of pH, Time, Temperature and Protein Tick saliva contained a catalytic activity that hydrolyzed arachidonic acid from the sn-2 position of 14C-PC (Fig. 1). The PLA2 activity was assessed as a function of incubation buffer pH (Fig. 1A) and exhibited two distinct pH optima at pH 5.5 and 9.5 and was markedly decreased at less than pH 5.0. The activity was linear with
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time to 5 min after which activity was somewhat reduced (Fig. 1B). PLA2 activity was dependent upon the amount of saliva present in the assay (Fig. 1C) and showed no noticeable deviation from linearity up to ∼2 mg protein/assay tube. All experiments in the present report used 1–2 mg/assay. The PLA2 activity exhibited a broad temperature range optimum 37–55°C with noticeably less activity above 55°C (Fig. 1D). It should be noted that the interassay variation was quite large primarily due to differences in 14C-PC substrate micelle preparation. However, intraassay variation was small, typically <10%, thus allowing within-experiment comparisons. Most experiments were performed at both pH 5.5 and 9.0 with similar results, but for clarity, subsequent results are reported only for pH 9.0. PLA2 Stability to Freezing and High Temperatures Tick saliva PLA2 activity undergoing one freeze–thaw cycle retained only 63 ± 9% (n 4 5) activity compared to the fresh saliva. However, ts-PLA2 activity could be stabilized by the addition of an equal volume of 1 mg/ml BSA before freezing, resulting in 90 ± 4% (n 4 5) activity being retained after a single freeze– thaw cycle. All subsequent experiments involved either fresh or pooled saliva that was aliquoted and frozen in the presence of 0.5 mg/ ml BSA. The ts-PLA2 was relatively resistant to preincubation at 90°C prior to being incubated with the substrate at 37°C (Fig. 2). More than 60% of ts-PLA2 activity was still detected after 5 min preincubation at 90°C, and discernable activity was still evident after 25 min. A thermal inactivation plot shows a uniformly logarithmic decline (r2 4 0.990) in ts-PLA2 activity with increasing preincubation time at 90°C (Fig. 2, inset). Calcium Requirement for the PLA2 Activity The presence of the Ca2+ chelator EGTA (10 mM) in the incubation buffer reduced the tsPLA2 activity to essentially zero (data not shown). This dependence of the ts-PLA2 upon
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FIG. 1. Tick saliva PLA2 activity determined by its ability to hydrolyze 14C-arachidonate from 14Cphosphatidylcholine as a function of (A) incubation buffer pH; (B) incubation time; (C) enzyme source protein concentration; and (D) incubation temperature. The standard assay constituted ø2.0 mg protein, 5 min incubation at 37°C, and pH 9.0. Data presented are means ± SD (n 4 3) of representative experiments. Each experiment was performed at least three times with similar results.
free Ca2+ was further investigated over a controlled concentration range (Fig. 3). ts-PLA2 activity was sensitive to as little as 0.05 mM free Ca2+ and half-maximally activated by 3.5 ± 0.8 mM free Ca2+. Millimolar Ca2+ did not enhance ts-PLA2 activity compared to micromolar Ca2+ (data not shown). The total Ca2+ concentration was determined for the saliva collected from ticks by injection with the secretagogue dopamine/theophylline mixture either dissolved in a Ca2+-free or Ca2+containing solution. The presence of Ca2+ in the injectate resulted in higher Ca2+ levels in the saliva. With either collection technique, the total Ca2+ concentration of the saliva was never less than 500 mM.
Size Exclusion Chromatography Saliva subjected to size exclusion chromatography on a Superose 6 FPLC column with 10 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 9.0, elution buffer was fractionated as a single peak of PLA2 activity with an apparent molecular weight of 55,700 ± 1300 Da (Fig. 4). Separations using the above elution buffer without any NaCl revealed two peaks of PLA2: the major peak had an apparent molecular weight of 66,100 ± 2,500 Da and the minor peak (∼25% of the activity) was 130,000 ± 2500 Da (data not shown). Saliva was also eluted using 10 mM Tris–HCl, 150 mM NaCl, 1 mM EGTA, pH 7.4, and the fractions were assayed
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FIG. 2. Stability of ts-PLA2 activity to a 90°C preincubation treatment for various periods of time. After the heat treatment, the saliva sample was allowed to cool and then assayed at 37°C per standard protocol. Data are means ± SD (n 4 3) of one experiment repeated twice with similar results. Inset shows a log transformed plot of the data.
for PLA2 activity either in 100 mM Tris–HCl, 150 mM NaCl, 10 mM CaCl2, pH 9.0, or in 100 mM Na-acetate, 150 mM NaCl, 10 mM CaCl2, pH 5.5. Under either assay condition a single
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FIG. 4. Size exclusion chromatography of crude saliva on an FPLC Superose 6 column eluted with 10 mM Tris, 150 mM NaCl, 1 mM EGTA, pH 9.0. A single peak of PLA2 activity eluted corresponding to 55,700 ± 1300 Da (mean ± SD, n 4 3) and no activity was detected in the region of 14 kDa, the molecular weight of secreted PLA2s in other animals.
peak of activity (∼56 kDa) was detected (data not shown). Substrate Specificity PLA2 in tick saliva was active against 1palmitoyl, 2-arachidonyl-PC and was used in most of the studies. Additionally, 1-palmitoyl, 2-linoleoyl-PC and dipalmitoyl-PC proved to be good substrates for ts-PLA2 (data not shown). However, membranous substrates in the form of radiolabeled and autoclaved E. coli or C. albicans were unsuitable substrates with essentially no apparent PLA2 activity detectable (data not shown). The N. naja venom PLA2 standard showed excellent activity against these prepared substrates. Effective Inhibitors of Saliva PLA2
FIG. 3. ts-PLA2 sensitivity to free calcium. Free Ca2+ concentrations were calculated with COMICS (Perrin and Sayce, 1967) and constructed using 5 mM nitrilotriacetic acid and various amounts of CaCl2. The contribution of Ca2+ present in the enzyme source, tick saliva, was taken into account. Data are the means ± SD of one representative experiment performed three times with similar results.
The substrate analog oleyloxyethyl phosphorylcholine (OPC) and the end product arachidonic acid proved to be effective inhibitors of ts-PLA2 (Fig. 5). OPC was a potent inhibitor exhibiting an IC50 4 1.4 mM and IC90 4 26 mM. Arachidonic acid exhibited an IC50 4 38 mM and would not give complete inhibition of
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FIG. 5. Dose-dependent inhibition of ts-PLA2 activity by the substrate analog oleyloxyethyl phosphorylcholine (OPC) and the end product arachidonic acid (AA). For OPC the calculated IC50 4 1.4 mM and IC90 4 26 mM, and for AA IC50 4 38 mM. Data are means ± SD (n 4 3) of one representative experiment repeated three times with similar results.
ts-PLA2 at any concentration. Higher concentrations of arachidonic acid are likely to be affecting the physicochemical characteristics of the 14C-PC substrate micelles as much as interacting with the enzyme.
FIG. 6. Heat stability of ts-PLA2 and sg-PLA2 activity. Tick saliva (solid circles) or salivary gland homogenate (open circles) were preincubated for 15 min at different temperatures, allowed to cool, and then assayed for PLA2 activity at 37°C per standard protocol. Data are means ± SD (n 4 3) of one representative experiment repeated three times with similar results.
enate at 30–50°C enhanced the sg-PLA2 activity. The PLA2 activity in the saliva and the salivary gland were both inhibited by DTT in a similar dose-dependent manner (Fig. 7). About one-third of the ts-PLA2 and sg-PLA2 was in-
Comparative Aspects of PLA2 in the Saliva and the Salivary Gland The PLA2 present in the saliva or salivary gland exhibited different responses to a 15-min preincubation period at higher temperatures before being assayed for activity at 37°C (Fig. 6). Preincubation of the saliva at temperatures between 30 and 60°C gave a marked increase in the measured ts-PLA2 activity with a 1.5-fold increase at 50°C compared to no preincubation. Higher preincubation temperatures reduced the activity in the saliva, but over 80% of the activity was still present after 15 min at 80°C. In contrast, only 20% of the PLA2 activity present in the salivary gland homogenate remained after the 80°C preincubation and the activity decreased at preincubations above 50°C. Like the ts-PLA2, preincubation of the gland homog-
FIG. 7. Effect of the reducing agent dithiothreitol (DTT) on the activity of ts-PLA2 and sg-PLA2. Tick saliva (dashed line) and salivary gland homogenate (solid line) were assayed in the presence of DTT. Assays were performed in triplicate, error bars are omitted for clarity but were <12%.
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hibited by 1 mM DTT, but even at 125 mM DTT the activities were still only inhibited by ∼70%. A battery of agents known to inhibit PLA2s from other sources were tested against ts-PLA2 and sg-PLA2 (Table I) at concentrations that had been reported to be effective against the other PLA2s. The degree of inhibition ranged from absent (aristolochic acid) to very low (17-ODYA, OBAA, ONO-RS-082), to lower than expected (4-BPB, thioetheramidePC, quinacrine). The arachidonic acid analogs, DEDA and ETYA, gave partial inhibition of both ts-PLA2 and sg-PLA2. In no case was inhibition of the tick PLA2 greater than that reported for PLA2s from other sources. The inhibitors had similar efficacies against ts-PLA2 and sg-PLA2. DISCUSSION Compared to other ectoparasites adult ticks remain attached to the host for an unusually long time. For example, the adult female A. americanum remains attached to the host for 10–14 days. The salivary glands are the organs of osmoregulation (Sauer et al., 1995) and excess water and ions in the bloodmeal are continuously being returned to the host via the saliva, thus concentrating the nutrients for future
egg production. The saliva contains an array of pharmacologically active profeeding compounds to counter the host’s hemostatic, vasoconstrictive, inflammatory, and immune responses (Bowman et al., 1997). Here we report upon another active compound secreted at the host–parasite interface, a PLA2 with unique characteristics. The saliva used in this study was collected by intrahemocoelic injection of dopamine, the neurotransmitter regulating the control of salivation in ticks (Sauer et al., 1995), a common and accepted technique for investigating the contents of tick saliva (Bowman et al., 1995; Zhu et al., 1997a,b; Kaufman and Nuttall, 1996). We have recently found that ‘‘natural saliva’’ (collected without dopamine injection) also contains ts-PLA2 activity (Zhu et al., 1998), but limited collectable quantities prevent natural saliva from being used in studies. The ts-PLA2 exhibited activity over a broad pH range, but demonstrated two distinct pH optima of pH 5.5 and 9.5, indicating the possibility that two different PLA2s may be present in the saliva. However, this is unlikely because experiments where ts-PLA2 activity was determined at both pH 5.5 and pH 9.0 yielded similar results. The thermal inactivation plot at 90°C of tsPLA2 showed a uniform decline in activity (Fig.
TABLE I The Effects of Some Known PLA2 Inhibitors upon the PLA2 Activity Found in Tick Saliva (ts-PLA2) and Salivary Gland Homogenate (sg-PLA2) Inhibition of PLA2 activity (%)
Inhibitor
Concentrationa (mM)
sg-PLA2
ts-PLA2
Thioetheramide-PC ETYA DEDA 17-ODYA Aristolochic acid 4-BPB Quinacrine Indomethacin OBAA ACA ONO-RS-082
2 10 15 10 5 50 250 400 40 10 1
8 36 21 8 0 22 13 25 0 21 0
11 24 26 0 0 10 0 35 0 10 10
Note. Determinations were performed in triplicate. a Concentrations (>>IC50) at which PLA2s from other sources have been reported to be inhibited.
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2, inset); if two different PLA2 enzymes were responsible for the activity one would expect two rates of deactivation each with its own inactivation constant. Additionally, only one peak of coeluting PLA2 activity was observed when the saliva was subjected to size exclusion chromatography and the eluate assayed at both pHs. Tick saliva is ∼pH 9.5 (Bowman et al., 1995). The small (∼14 kDa) secreted PLA2s from bee and snake venoms and mammalian pancreatic or inflammatory fluid also exhibit alkaline optima (∼pH 8–10). Though tick saliva is pH 9.5, the pH at the feeding lesion is unknown but is likely to be more similar to host interstitial fluid (∼pH 7.8). At this more neutral pH ts-PLA2 is still active. A distinguishing feature between secreted and cytosolic PLA2s is their Ca2+ sensitivities. Secreted or extracellular PLA2s are active only in the presence of millimolar Ca2+, the concentration found in interstitial fluids and gut lumens. We are aware of only one extracellular PLA2 that is active at micromolar Ca2+ that is found in the venom of a marine snail (McIntosh et al., 1995). In contrast, cytosolic or intracellular PLA2s are sensitive to submicromolar Ca2+ with sharp increases in activity as the Ca2+ concentration increases from 10−7 to 10−6 M (Kudo et al., 1993). The free Ca2+ concentration in the cytosol generally increases to around the micromolar level upon cell stimulation (Rink et al., 1982). The ts-PLA2’s Ca2+ requirement is most intriguing. Being present in the saliva, the ts-PLA2 is obviously a secreted extracellular enzyme, but it is sensitive to 0.05 mM free Ca2+ and is half-maximally activated by 3.5 mM free Ca2+. In this respect, the ts-PLA2 is like an intracellular PLA2. The total Ca2+ concentration in the tick saliva was >500 mM and the Ca2+ concentration in the feeding lesion would be in the millimolar range; thus, the ts-PLA2 would be fully activated from the time of secretion. We found that the presence of Ca2+ in the secretagogue mixture injected into the tick resulted in higher Ca2+ levels in the saliva. The finding that the composition of the injection media affects the composition of the tick saliva should be noted by researchers in this area.
The ts-PLA2 activity was stable to high incubation temperatures both prior to and during the assay incubation period and to freezing. The necessity of the presence of 0.5 mg/ml BSA during the freezing was probably because of the dilute nature of tick saliva (∼0.2 mg protein/ml). If the protein content of the saliva was increased by ultrafiltration, ts-PLA2 was stable to freezing without the addition of any BSA (unpublished observations). ts-PLA2 activity was enhanced by 15-min preincubation temperatures of 40– 60°C and the activity was still present at 80°C. Cytosolic PLA2s are inactivated by temperatures above 57°C (Mayer and Marshall, 1993). In contrast, secreted PLA2s are resistant to higher temperatures (Mayer and Marshall, 1993). The physical stability of secreted PLA2s is afforded by the numerous, typically five to seven, disulfide bonds which are completely absent in the cytosolic PLA2s (Dennis, 1994). The presence of the disulfide bonds leads secreted PLA2s to be inactivated by reducing agents such as DTT, whereas cytosolic PLA2s are unaffected (Mayer and Marshall, 1993). The sensitivity of ts-PLA2 to DTT was intermediate between that of cytosolic and secreted PLA2s. Cytosolic PLA2 from human monocytic cell line U937 is resistant to DTT (Clark et al., 1990) and cytosolic PLA2 from mouse macrophages is 2-mercaptoethanol resistant (Leslie et al.,1988). Rat inflammatory exudate PLA2 activity is inhibited by 60% in the presence of 1 mM DTT and 80% in 3 mM (Chang et al., 1987), while human synovial fluid PLA2 activity is abolished in 3 mM DTT (Hara et al., 1989). The ts-PLA2 activity was reduced by ∼35% in 1 mM DTT and ∼70% in 125 mM DTT. It would seem that reduction of the disulfide linkages in ts-PLA2 brings about some conformational structure change of the enzyme that reduces its catalytic activity, but these changes appear not to be near the active site since catalytic activity is evident even at very high DTT levels. The high physical stability of ts-PLA2 would be suited to a protein originating in one location but targeted to function in a foreign environment. Tick saliva PLA 2 readily hydrolyzed 1palmitoyl-2-arachidonyl-phosphatidylcholine
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substrate when presented as sonicated micelles without any additional lipids or detergents required. However, membranous substrates in the form of 3H-oleic acid labeled E. coli or C. albicans could not serve as a substrate for tsPLA2 even though we verified their suitability for N. naja snake venom PLA2. The lack of activity of ts-PLA2 against the labeled membrane substrate is unusual since secreted PLA2s almost universally hydrolyze this form of substrate (Elsbach and Weiss, 1991). The cytosolic PLA2s are not assayed using such micellar substrates. Secreted PLA2s are uniformly ∼14 kDa and calcium-dependent cytosolic PLA2s are 85 kDa though there are also reports of Ca 2 + independent cytosolic PLA2s of 40 kDa (Dennis, 1994; Mayer and Marshall, 1993). Size exclusion chromatography of tick saliva revealed a single peak of PLA2 activity corresponding to a molecular weight of 56 kDa. Interestingly, PLA2 activity recovered from the column was consistently greater (∼175%) than that applied to the column. The phenomenon of increased PLA2 activity following size exclusion chromatography has been reported for several PLA2s, and it has been suggested that the removal of some endogenous inhibitory factors is responsible (Hara et al., 1991a). An alternative explanation is that crude saliva contains some endogenous phospholipid substrate and greater activity is recorded when the ts-PLA2 is assayed in the absence of any competing substrate. The PLA2 activity from both the salivary gland and in the saliva was not readily inhibited by a variety of PLA2 inhibitors that are known to inhibit either one or both of the secreted and the cytosolic PLA2s. Aristolochic acid inhibits secreted PLA 2 s in both snake venom (Vishwanath et al., 1987) and human neutrophils (Rosenthal et al., 1989) by apparently binding to a nonactive site region of the enzyme and causing a conformational change (Vishwanath et al., 1987). Aristolochic acid was ineffective against tick PLA2. BPB inactivates secreted PLA2s by covalently alkylating histidine-48 in the enzyme’s active site (Kudo et al., 1993) but is largely ineffective against cytosolic
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PLA2 (Mayer and Marshall, 1993). ts-PLA2 was only marginally affected by BPB, indicating that its catalytic mechanism is different from that of the secreted PLA2s. Quinacrine inhibits some PLA2s, notably secreted PLA2s, by forming complexes with the phospholipid and perturbing the interaction between the enzyme and substrate (Hirata, 1989). Neither source of tick PLA2 was inhibited by quinacrine. Indomethacin, a potent nonsteroidal inhibitor of cyclooxygenase, inhibits PLA2 activity at higher concentrations through antagonism against Ca2+ (Hirata, 1989). High concentrations of indomethacin caused only partial inhibition of both sg-PLA2 and ts-PLA2. The PLA2 substrate analog thioetheramide-PC is a potent inhibitor of secreted PLA2s, but is ineffective against cytosolic PLA2 (Hope et al., 1993; Mayer and Marshall, 1993). Thioetheramide-PC had no effect upon tick PLA2. However, another PLA2 substrate analog, OPC, was an effective inhibitor of both ts-PLA2 and sg-PLA2. Though OPC inhibits secreted PLA2s, it does not inhibit cytosolic PLA2s (Hope et al., 1993). Arachidonic acid inhibited both ts-PLA2 and sg-PLA2, and the arachidonic acid analogs DEDA and ETYA acid were also inhibitory toward the tick PLA2s, but another analog, 17-ODYA, was largely ineffective. Arachidonic acid analogs inhibit secreted PLA2s (Welton et al., 1984; Hope et al., 1993) but are ineffective against cytosolic PLA2 (Hope et al., 1993). Overall, the inhibitor profile was similar for ts-PLA2 and sg-PLA2, but both showed clear differences from both the cytosolic and the secreted PLA2s, indicating that the tick PLA2 has a different catalytic mechanism or substrate binding site. We had previously found a PLA2 activity present in the salivary glands of A. americanum (Bowman et al., 1998). Finding a PLA2 activity in the saliva that shares many of the properties of sg-PLA2 poses the question of whether they are the same enzyme. The apparent molecular weights (56 kDa, ts-PLA2; and 54 kDa, sgPLA2) and the submicromolar Ca2+ activation are clear similarities. In the present report, we show that the DTT sensitivity of both ts-PLA2 and sg-PLA2 are similar, as are the responses to
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the inhibitors tested. There are, however, some differences. Whereas size exclusion chromatography of saliva yields greater than 100% recovery of ts-PLA2 activity, greatly reduced activity was consistently observed in fractionating the salivary gland homogenate. Additionally, the sg-PLA2 activity was susceptible to high preincubation temperatures, but ts-PLA2 activity was enhanced. Both of these differences between sgPLA2 and ts-PLA2 could possibly be explained by differences in the matrices, that is, a dilute saliva and a tissue homogenate. Further work is required to demonstrate whether ts-PLA2 and sg-PLA2 are the same or different enzymes. What, however, is the significance to the tick of a salivary PLA2 secreted into the feeding lesion of the host? Secreted PLA2s from T. cruzi (Connelly and Kiersenbaum, 1984), T. gondii (Saffer and Schwartzman, 1991; Gomez-Marin et al., 1996), and R. rickettsii (Walker et al., 1983) have been implicated in host cell invasion by these intracellular parasites. While ticktransmitted pathogens present in the saliva may utilize the ts-PLA2 to enhance their invasion of host cells, this would serve no obvious benefit to the tick. We have shown that ts-PLA2 is not involved in the anticoagulant activity of A. americanum saliva (Zhu et al., 1997a) and has limited hemolytic activity of sheep erythrocytes as a type of extra-corporeal digestion (Zhu et al., 1997b). Tick saliva contains extremely high levels of prostaglandins, including PGE2, believed to aid tick feeding due to their antihemostatic, vasodilatory, anti-inflammatory and immunosuppressive properties (Bowman et al., 1996). The ectoparasitic larval stage of the cattle grub (Hypoderma lineatum) secretes a factor that induces host peripheral blood mononuclear cells to synthesize high levels of PGE2 which subsequently downregulate the immune response (Nicolas-Gaulard et al., 1995). Exogenous PLA2s have been shown to stimulate granulocytes to produce PGE2 (Hara et al., 1991b). In such a way, ts-PLA2 could cause host cells to produce PGE2 that would augment the PGE2 already present in the tick saliva. In preliminary studies, size exclusion fractions of tick saliva containing the ts-PLA2 activity
stimulated prostacylin (PGI2) production by cultured endothelial cells (unpublished observations). Prostacyclin would benefit tick feeding by preventing platelet aggregation, inducing vasodilation, and countering vasoconstriction (Bowman et al., 1996). In summation, tick saliva contains an unusual secreted PLA2 that is active at Ca2+ concentrations that might be found inside a cell. The molecular weight, Ca2+ activation level, and sensitivity to several PLA2 inhibitors preclude tsPLA2 from being placed in any of the present PLA2 classification categories. The PLA2 activities found in the tick saliva and salivary gland show many similarities, but also exhibit some distinct differences. Currently, we do not know the teleologic function of ts-PLA2, but its presence in tick saliva indicates that it must play some role at the tick–host interface. ACKNOWLEDGMENTS The authors express their thanks to Jerry Bowman, Central Tick Rearing Facility, OSU, for supplying the ticks. We are grateful to Drs. Steve P. White and Doug Bergman for reviewing an earlier draft of the manuscript. This work was approved for publication by the Director, Oklahoma Agricultural Experiment Station. This research was supported by NIH Grant AI-31460.
REFERENCES Bowman, A. S., Coons, L. B., Needham, G. R., and Sauer, J. R. 1997. Tick salivary secretions at the tick–host interface: Recent advances and implications for vector competence. Medical and Veterinary Entomology, in press. Bowman, A. S., Dillwith, J. W., and Sauer, J. R. 1996. Tick salivary prostaglandins: Presence, origin and significance. Parasitology Today 12, 388–396. Bowman, A. S., Sauer, J. R., Zhu, K., and Dillwith, J. W. 1995. Biosynthesis of salivary prostaglandins in the lone star tick, Amblyomma americanum. Insect Biochemistry and Molecular Biology 25, 735–741. Bowman, A. S., Surdick, M. R., Gengler, C. L., Zhu, K., Essenberg, R. C., Sauer, J. R., and Dillwith, J. W. 1998. Phospholipase A2 activity in salivary glands of the lone star tick, Amblyomma americanum. [Submitted for publication] Chang, H. W., Kudo, I., Tomita, M., and Inoue, K., 1987. Purification and characterization of extracellular phospholipase A2 from peritoneal cavity of caseinate-treated rat. Journal of Biochemistry 102, 147–154. Clark, J. D., Milona, N., and Knopf, J. L. 1990. Purification of a 110-kilodalton cytosolic phospholipase A2 from the
TICK SALIVARY PHOSPHOLIPASE A2 human monocytic cell line U9337. Proceedings of the National Academy of Sciences USA 87, 7708–7712. Connelly, M. C., and Kierszenbaum, F. 1984. Modulation of macrophage interaction with Trypanosoma cruzi by phospholipase A2-sensitive components of the parasite membrane. Biochemical and Biophysical Research Communications 121, 931–939. Dennis, E. A. 1994. Diversity of group types, regulation, and function of phospholipase A2. Journal of Biological Chemistry 269, 13057–13060. Elsbach, P., and Weiss, J. 1991. Utilization of labeled Escherichia coli as phospholipase substrate. Methods: A Companion to Methods in Enzymology 197, 24–31. Gomez Marin, J. E., Bonhomme, A., Guenounou, M., and Pinon, J. M. 1996. Role of interferon-g against invasion by Toxoplasma gondii in a human monocytic cell line (THP1): Involvement of the parasite’s secretory phospholipase A2. Cellular Immunology 169, 218–225. Hara, S., Kudo, I., Chang, H. W., Matsuta, K., Miyamoto, T., and Inoue, K. 1989. Purification and characterization of extracellular phospholipase A2 from human synovial fluid in rheumatoid arthritis. Journal of Biochemistry 105, 395–399. Hara, S. Chang, H. W., Horigome, K., Kudo, I., and Inoue, K. 1991a. Purification of mammalian nonpancreatic extracellular phospholipases A2. Methods: A Companion to Methods in Enzymology 197, 381–389. Hara, S., Kudo, I., and Inoue, K. 1991b. Augmentation of prostaglandin E2 production by mammalian phospholipase A2 added exogenously. Journal of Biochemistry 110, 163–165. Hirata, F. 1989. Drugs that inhibit the activities or activation of phospholipases and other acylhydrolases. In ‘‘Handbook of Eicosanoids: Prostaglandins and Related Lipids. Vol II. Drugs Acting via the Eicosanoids’’ (A. L. Willis, Ed.), pp. 47–58. CRC Press, Boca Raton, FL. Hope, W. C., Chen, T., and Morgan, D. W. 1993. Secretory phospholipase A2 inhibitors and calmodulin antagonists as inhibitors of cytosolic phospholipase A2. Agents and Actions 39, C39–C42. Kaufman, W. R., and Nuttall, P. A. 1996. Amblyomma variegatum (Acari:Ixodidae): Mechanism and control of arbovirus secretion in tick saliva. Experimental Parasitology 82, 316–323. Kudo, I., Murakami, M., Hara, S., and Inoue, K. 1993. Mammalian non-pancreatic phospholipases A 2. Biochimica et Biophysica Acta 117, 217–231. Leslie, C. C., Voelker, D. R., Channon, J. Y., Wall, M. M., and Zelarney, P. T. 1988. Properties and purification of an arachidonyl-hyrolyzing phospholipase A2 from a macrophage cell line, RAW 264.7. Biochimica et Biophysica Acta 963, 476–492. Mayer, R. J., and Marshall, L. A. 1993. New insights on mammalian phospholipase A2(s); comparison of arachidonyl-selective and -nonselective enzymes. FASEB Journal 7, 339–348. McIntosh, M. J., Ghomashchi, F., Gelb, M. H., Dooley,
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D. J., Stoehr, S. J., Giordani, A. B., Naisbitt, S. R., and Olivera, B. M. 1995. Conodipine-M, a novel phospholipase A2 isolated from the venom of the marine snail, Conus magus. Journal of Biological Chemistry 270, 3518–3525. Mukherjee, A. B., Miele, L., and Pattabiraman, N. 1994. Phospholipase A2 enzymes: Regulation and physiological role. Biochemical Pharmacology 48, 1–10. Needham, G. R., and Sauer, J. R. 1979. Involvement of calcium and cyclic AMP in controlling Ixodid tick salivary fluid secretion. Journal of Parasitology 65, 531–542. Nicolas-Gaulard, I., Moire, N., and Boulard, C. 1995. Effect of the parasite enzyme, hypodermin A, on bovine lymphocyte proliferation and interleukin-2 production via the prostaglandin pathway. Immunology 84, 160–165. Patrick, C. D., and Hair, J. A. 1975. Laboratory rearing procedures and equipment for multihost ticks (Acarina: Ixodidae). Journal of Medical Entomology 12, 389–390. Perrin, D. D., and Sayce, I. G. 1967. Computer calculation of equilibrium concentrations in mixtures of metal ions and complexing species. Talanta 14, 833–842. Ravdin, J., Murphy, C. F., Guerrant, R. L., and Long-Krug, S. A. 1985. Effect of antagonists of calcium amd phospholipase A on the cytopathogenicity of Entamoeba histoytica. Journal of Infectious Diseases 152, 542–549. Rink, T. J., Smith, S. W., and Tsien, R. Y. 1982. Cytoplasmic free Ca2+ in human platelets: Ca2+thresholds and Ca-independent activation for shape-change and secretion. FEBS Letters 148, 21–26. Rogers, M. V., Henkle, K. J., Herrmann, V., McLaren, D. J., and Mitchell, G. F. 1991. Evidence that a 16kilodalton integral membrane protein antigen from Schistosoma japonicum adult worms is a Type A2 phospholipase. Infection and Immunity 59, 1442–1447. Rosenthal, M. D., Vishwanath, B. S., and Franson, R. C. 1989. Effects of aristolochic acid on phospholipase A2 activity and arachidonate metabolism of human neutrophils. Biochimica et Biophysica Acta 1001, 1–8. Saffer, L. D., and Schwartzman, J. D. 1991. A soluble phospholipase of Toxoplasma gondii associated with host cell penetration. Journal of Protozoology 38, 454–460. Sauer, J. R., McSwain, J. L., Bowman, A. S., and Essenberg, R. C. 1995. Tick salivary gland physiology. Annual Review of Entomology 40, 245–267. van den Bosch, H., De Jong, J. G. N., and Aarsman, A. J. 1991. Phospholipase A2 from rat liver mitochondria. Methods: A Companion to Methods in Enzymology 197, 365–373. Vishwanath, B. S., Rao, A. G. A., and Gowda, V. 1987. Interaction of phospholipase A2 from Vipera russelli venom with aristolochic acid: A circular dichroism study. Toxicon 25, 939–946. Walker, D. H., Firth, W. T., Ballard, J. G., and Hegarty, B. C. 1983. Role of phospholipase-associated penetration mechanism in cell injury by Rickettsia rickettsii. Infection and Immunity 40, 840–842.
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Welton, A. F., Hope, W. C., Fielder-Nagy, C., and BatulaBernardo, C. 1984. Analogs of arachidonic acid methylated at C-7 and C-10 inhibit leukotriene biosynthesis and phospholipase A2 activity. Prostaglandins 28, 649. Zhu, K., Sauer, J. R., Bowman, A. S., and Dillwith, J. W. 1997a. Identification and characterization of anticoagulant activities in the saliva of the lone star tick, Amblyomma americanum (L.). Journal of Parasitology 83, 38– 43. Zhu, K., Dillwith, J. W., Bowman, A. S., and Sauer, J. R.
1997b. Identification of hemolytic activity in the saliva of the lone star tick (Acari:Ixodidae). Journal of Medical Entomology 34, 160–166. Zhu, K., Bowman, A. S., Dillwith, J. W., and Sauer, J. R. 1997c. Phospholipase A2 activity in the salivary glands and saliva of the lone star tick, Amblyomma americanum (L.) (Acari: Ixodidiae) during tick feeding. [Submitted for publication] Received 2 June 1997; accepted with revision 28 July 1997