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Preliminary characterisation of esterase and platelet-activating
factor (PAF)-acetylhydrolase activities from cat flea
(Ctenocephalides felis) salivary glands
M.T. Cheeseman
a,*, P.A. Bates
b, J.M. Crampton
caDepartment of Veterinary Pathology, University of Liverpool, Liverpool, UK bLiverpool School of Tropical Medicine, University of Liverpool, Liverpool, UK
cSchool of Biological Sciences, University of Liverpool, Liverpool, UK
Received 19 January 2000; received in revised form 1 June 2000; accepted 6 June 2000
Abstract
Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters α-naphthyl acetate (|210 pmol/min/gland pair; 10.0µmol/min/mg specific activity; Km|59µM) and β-naphthyl acetate (|110 pmol/min/gland pair; 5.2µmol/min/mg specific
activity; Km|132µM). Salivary gland extracts have PAF-acetylhydrolase activity (|5 pmol/min/gland pair; 0.24µmol/min/mg
spe-cific activity) but do not have detectable acetylcholinesterase activity. Native–PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native–PAGE. Renaturation of esterase activity after SDS–PAGE gave |56 kDa,|57 kDa and |58 kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of|59 kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain α- and β-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.2001 Elsevier Science Ltd. All rights reserved.
Keywords: Cat flea (Ctenocephalides felis); Salivary gland; Saliva; Esterase; Glycoprotein; Platelet-activating factor (PAF); PAF-acetylhydrolase
1. Introduction
The cat flea (Ctenocephalides felis) is the most important ectoparasite of domestic dogs and cats chiefly because flea bites cause flea allergy dermatitis (FAD), the most common canine skin disease world-wide, and miliary dermatitis in cats (Rust and Dryden, 1997; Gross et al., 1992). Cat flea salivary glands contain a mixture of allergens whose function in blood feeding is unknown (McCall et al., 1997; Frank et al., 1998; Lee et al., 1997,
* Corresponding author. Current Corresponding address: University of London, The Royal Veterinary College, Dept Pathology & Infec-tious Disease, Hawkshead Lane, North Mymms, Hatfield, Herts AL9 7TA. Tel.:+44-1707-666208; fax:+44-1707-661464.
E-mail address: [email protected] (M.T. Cheeseman).
0965-1748/01/$ - see front matter2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 ( 0 0 ) 0 0 1 1 3 - 2
Lin-coln, 1976) and blackflies (Wirtz, 1990) and tick phos-pholipase A2 (Bowman et al., 1997; Zhu et al., 1998),
are not so clear because naturally occurring ester and phospholipid substrates have not been identified.
In this study we describe the heterogeneity and glyco-sylation of cat flea salivary gland naphthyl esterases and document salivary gland platelet-activating factor (PAF)-acetylhydrolase activity. The potential anti-inflammatory and anti-allergic role of salivary gland PAF-acetylhydrolase activity in fleas and other blood-feeding arthropods is discussed.
2. Methods and materials
2.1. Animals
Professor D. Jacobs of The Department of Pathology and Infectious Diseases, The Royal Veterinary College, University of London, supplied the cat fleas. One to two weeks old, unfed male and female cat fleas (Ctenocephalides felis, strain RVC 003) were used in these experiments.
2.2. Chemicals
Chemicals were purchased from Sigma unless other-wise indicated. PAF (1-O-hexadecyl-2-O-acetyl-sn-gly-cero-3-phosphocholine) was purchased from Bachem, and [3H]PAF (1-O-hexadecyl-2-[3
H]acetyl-sn-glyceryl-3-phosphorylcholine), 13.5 Ci/mmol, was purchased from NEN Life Sciences Inc.
2.3. Sample preparation
Cold immobilised fleas were dissected in ice-cold ster-ile phosphate-buffered saline (PBS). Salivary glands (SG) and the remainder of the carcass minus the salivary glands (hereafter referred to as carcasses) were each transferred to an aliquot of appropriate ice-cold buffer (ca. one salivary gland pair per µl; one carcass per 5 or 10µl). Samples were stored frozen at 270°C for up to
8 weeks before thawing and homogenisation on ice. Sali-vary gland preparations were adjusted to 0.5% Triton X-100 and homogenised with a micropestle in a 1.5 ml polypropylene tube. Carcass samples were homogenised with short bursts of an electric dispersal probe (Ultra-Turrax, IKA Labotechnic). The homogenates were fil-tered through a 0.22µm microcentrifuge filter and the filtrate stored on ice until use (within 1 h).
To determine the specific activity of whole body ester-ases, groups of 80–100 frozen fleas were weighed, then homogenised in 1 ml of PBS with 0.5% Triton X-100. Aliquots were assayed for protein (Peterson, 1977) using bovine serum albumin (BSA) as a standard and for ester-ase activity (see below).
2.4. Esterase assay
Enzyme extracts were mixed with a 200µl aliquot of substrate solution containing 0.83 mMα-naphthyl acet-ate (αNA) orβ-naphthyl acetate (βNA) in 0.1 M sodium phosphate buffer with 0.5% Triton X-100 pH 6.5 (TP buffer) (4% v/v 20 mM NA stock solution in ethanol diluted in TP buffer). In addition, a series of naphthyl ester substrates —α- andβ-naphthyl propionate,α- and β-naphthyl butyrate and α-naphthyl caprylate — were used to assay partially purified esterase (see below). The mixture was incubated at 25°C for 15–120 min and the reaction stopped by the addition of 800µl of 0.19 mg/ml Fast Garnet in TP buffer. The absorbance was read 10 min later at A600 and A490 for the α- andβ-naphthol
reaction products, respectively, and converted to nmol of naphthol by interpolation with the appropriate standard curve. Values for enzyme reactions were corrected for spontaneous breakdown of substrate by subtraction of control values from reactions in which buffer alone was incubated with substrate solution. Boiling the enzyme preparation abolished activity. Esterase activities are expressed in pmol of substrate/min at 25°C; assays per-formed in triplicate have a standard error (SE) of 2–3% of the mean. Specific activity values for SG preparations were calculated using an estimate of|21 ng protein per salivary gland pair (data from Cheeseman, 1998). Michaelis constants (Km) were determined with
Lineweaver–Burk plots using 0.1 mM to 0.8 mM αNA andβNA substrates. The acetylcholinesterase activity of salivary gland extracts was tested using the substrate acetylthiocholine iodide (Baker et al., 1998).
2.5. Detection of secreted esterase
Unfed adult fleas, 1–2 week old, were allowed 15 min to probe nitrocellulose membrane wetted with 2 mM adenosine triphosphate (ATP) solution. Nitrocellulose membranes were air dried, rinsed with distilled water, and stained with αNA/Fast Blue BB substrate/stain sol-ution (cytochemistry kit from Sigma Chemical Company). This staining procedure was also employed for IEF, Native–PAGE and SDS–PAGE gels (see below).
2.6. Native–PAGE of esterases
2.7. IEF
Samples were prepared in 50% v/v glycerol with 0.5% Triton X-100. Aliquots (2µl) of extract containing ca. two SG pairs or |0.33 carcass equivalents were focused on a 5% acrylamide gel with pH 3–10 ampholytes according to the manufacturer’s instructions (Mini IEF cell, Biorad) alongside coloured IEF markers (Sigma). The pI of the stained esterase bands was calculated by interpolation with a standard curve.
2.8. Renaturation of esterase activity after SDS–PAGE
A sample of 75 SG pairs in 75µl SDS–PAGE sample buffer with 50 mM dithiothreitol was heated at 37°C for 60 min without homogenisation. Aliquots (25µl) of the filtrate were electrophoresed on a discontinuous SDS– PAGE (4% stacking and 12% resolving) gel alongside coloured molecular-weight markers (Biorad). The ester-ase activity was renatured by incubating the gel for 60 min at 37°C in 2.5% Triton X-100, rinsing three times with distilled water, and then incubating for 30 min in 0.5 M Tris–HCl pH 7.6, 0.5 M NaCl, 0.375 M CaCl2and
0.67% Brij 35. The gel was stained for esterase activity as outlined above. The molecular weight of esterase bands was interpolated by reference to the standard curve of marker proteins.
2.9. Concanavalin A lectin chromatography of salivary gland esterases
A sample of 100 SG pairs prepared in 20 mM Tris pH 7.0, 50 mM NaCl, 1 mM CaCl2and 0.1% Triton
X-100 was layered on to a 0.5 ml Concanavalin A agarose gel (Vector Laboratories) equilibrated in the same buffer and allowed to stand for 10 min. The column was washed with four bed volumes at 4.8 ml/h and 0.4 ml fractions collected. Bound material was eluted with an 8 ml gradient containing 0–300 mMα-d-methyl
manno-pyranoside in the same buffer. Chromatography was per-formed at room temperature. Fractions were collected on ice and then assayed for esterase activity against αNA.
2.10. Glycopeptidase F deglycosylation of salivary esterases
Aliquots (11µl) of PBS extract containing |40 SG pairs were incubated at 25°C for 2 h with a 1µl aliquot of glycopeptidase F containing either 0.65 U or 0.13 U of activity (Sigma). Controls reactions included omission of glycopeptidase F or SG extract. At the end of the incubation period each reaction mixture was mixed with 24µl of 2×-concentrated Native–PAGE buffer with 1% Triton X-100, electrophoresed on a native gel and stained as above.
2.11. Gel filtration
Samples of 175 to 275 SG pairs were prepared in 50 mM Tris pH 7.0, 300 mM NaCl and 0.01% Triton X-100, and passed through a 120 cm×1 cm diameter col-umn packed with Ultrogel ACA 44 (Sigma). The colcol-umn was eluted at 4 ml/h and 1 ml fractions collected. Frac-tions were assayed for esterase activity with αNA and PAF-acetylhydrolase (see below). Chromatography was performed at 5°C, and the column calibrated with mol-ecular-weight markers from Sigma (alcohol dehydrogen-ase, 150 kDa; albumin, 66 kDa; carbonic anhydrdehydrogen-ase, 29 kDa; cytochrome C, 12.4 kDa; and the void volume determined with Blue Dextran, 2000 kDa). The molecu-lar weights of enzymes were determined by interpolation with a standard curve of Ve/V0 against log molecular
weight. A pool of fractions containing esterase activity was frozen at 270°C until assayed for activity against
a series of α- andβ-naphthyl esters.
2.12. PAF-acetylhydrolase assay
PAF-acetylhydrolase activity in whole salivary gland extracts or gel filtration fractions was assayed using [3H]PAF as described by Tselepis et al. (1991) and
Kitsi-ouli et al. (1999). Aliquots of salivary extract prepared in 1 mg/ml BSA in PBS pH 7.4, or gel filtration fractions diluted with an equal volume of 2 mg/ml BSA in PBS, were added to 150µl of substrate solution containing 40µM PAF and 148 nM [3H]PAF (
|0.3µCi per assay) in 1 mg/ml BSA/PBS and incubated at 37°C for 30 min or 60 min. The reaction was stopped by adding an equal volume of ice-cold 20% trichloroacetic acid (TCA). The mixture was held on ice for 5 min and then centrifuged at 14,000g for 4 min. An aliquot of supernatant contain-ing the [3H]acetate reaction product was mixed with an
equal volume of Microscint 40 (Packard) and counted on a Packard Topcount Microscintillation counter. Assays performed in duplicate have an SE of 7% of the mean. Control reactions included incubation of column buffer or boiled enzyme preparation with the substrate solution. The total amount of non-isotopically labelled PAF hydrolysed in each reaction was extrapolated from the proportion of [3H]acetate released in each reaction from
the total [3H]PAF added (calculated from counting an
aliquot of [3H]PAF substrate solution). After TCA
pre-cipitation, less than 0.15% of [3H] appeared in the
super-natant of control reactions incubated with buffer alone, boiling abolished enzyme activity and release of super-natant [3H] in control reactions without enzyme was
,0.15% in 60 mins. Triton X-100 in gel filtration frac-tions (50µl aliquots containing 0.01% detergent) did not interfere with TCA precipitation of unhydrolysed [3
3. Results
Homogenates of salivary glands had esterase activity against αNA (|210 pmol/min/gland pair, 10.0µmol/min/mg specific activity) and βNA (|110 pmol/min/gland pair, 5.2µmol/min/mg specific activity). The Km for SG homogenates are 59±6µM
αNA (mean±SE, n=2) and 132±12µMβNA. When fleas were allowed to probe nitrocellulose membranes wetted with a phagostimulant (2 mM ATP) solution, they deposited clusters of naphthyl-esterase-positive saliva spots that were approximately the diameter of the mouthparts (Fig. 1). Control membranes that had been exposed to flea probing but developed in solution omit-ting αNA did not give a positive reaction. Fleas weigh on average|0.5 mg and whole flea homogenates yielded 26±2µg of soluble protein per flea (n=4) and 3.9±0.7 nmol/min of αNA esterase activity per flea (|0.15±0.01µmol/min/mg esterase specific activity).
Native–PAGE resolves SG esterases into three major bands of low relative mobility; one band is retained in the stacking gel. The fastest migrating band has a similar mobility to the slowest migrating of the seven carcass esterase bands (some faint carcass esterase bands do not photograph well enough to be seen in Fig. 2). The same patterns of SG and carcass esterases occur when βNA is used as the substrate (data not shown). IEF of SG esterases resolves four sharply focused bands of pI|6.4, |6.2, |5.9 and |5.7, and two less distinct bands of pI|8.3 and |7.5 (average of two separate experiments). Carcass esterases are resolved into a separate pattern of four bands at pI|5.4,|5.2,|5.1 and|4.9 (Fig. 3). Rena-turation of enzyme activity after SDS–PAGE gave sharp, closely grouped |56 kDa,|57 kDa and|58 kDa naph-thyl-esterase-positive bands (Fig. 4).
Concanavalin A affinity chromatography of SG
Fig. 1. Esterase activity against α-naphthyl acetate in blots of secreted saliva (×250). Unfed adult fleas, 1–2 week old, were allowed 15 min to probe nitrocellulose membrane wetted with 2 mM ATP sol-ution. Membranes were air dried, rinsed with distilled water and stained withα-naphthyl acetate/Fast Blue BB, substrate/stain solution. See text for details.
Fig. 2. Native–PAGE of cat flea esterases. Salivary glands have three major esterase bands with low relative mobility (and one retained in the stacking gel), compared with bands with higher relative mobility found in the remainder of the carcass. Lane 1, two carcasses; lane 2, one carcass; lane 3, 40 salivary gland pairs. Extracts were electrophor-esed on a discontinuous native gel (the boundary between the 4% stacking and 4% resolving gels is marked with an arrow) and stained withα-naphthyl acetate/Fast Blue BB. See text for details.
Fig. 4. Renaturation of cat flea salivary gland esterase activity after SDS–PAGE resolves closely grouped|56 kDa,|57 kDa and|58 kDa naphthyl-esterase-positive bands. Extract (25µl) was electrophoresed on a discontinuous SDS–PAGE (4% stacking and 12% resolving) gel and, after renaturation, stained for esterase activity. Molecular-weight markers in kDa are shown in the right-hand column. See text for details.
extracts gave two peaks of esterase activity. The first unbound smaller peak eluted in the preliminary wash; the second, accounting for |77% of recovered activity, eluted at |150 mM competing sugar (Fig. 5). Treatment of SG extracts with 0.65 U of glycopeptidase F to enzy-matically remove oligosaccharide moieties produced a
Fig. 5. Concanavilin A affinity chromatography of salivary gland esterases gives two peaks of activity; the first is eluted in the prelimi-nary wash, the second is eluted at 150 mM competing sugar. An extract of 100 salivary gland pairs was loaded on to a 0.5 ml Concanavilin agarose gel equilibrated with 20 mM Tris pH 7.0, 50 mM NaCl, 1 mM CaCl2and 0.1% Triton X-100, washed at 4.8 ml/h and 0.4 ml fractions
collected. After five fractions, an 8 ml gradient of 0–300 mM α -d-methyl mannopyranoside was applied. Fractions were assayed for activity againstα-naphthyl acetate. See text for details.
new, fast-migrating esterase band, while treatment with 0.13 U produced a smaller amount of this entity (Fig. 6). Salivary gland homogenates had PAF-acetylhydrolase activity in calcium-free buffer systems [5.0±0.6 pmol/min/gland pair (n=4 separate experiments); |0.24µmol/min/mg specific activity]. Production of [3H]acetate was linear up to 30–60 mins
with up to |1.5 gland pairs and is curvilinear thereafter (Fig. 7).
On gel filtration, salivary gland naphthyl esterase and PAF-acetylhydrolase co-elute with an apparent molecu-lar weight of|59 kDa (Fig. 8). Fractions containing this material were pooled and 100µl aliquots tested for activity against a series of substrates: α-naphthyl propi-onate (500±1 pmol/min), β-naphthyl propionate (413±3 pmol/min), β-naphthyl acetate (311±6 pmol/min), α-naphthyl acetate (280±1 pmol/min),α-naphthyl butyr-ate (134±1 pmol/min), α-naphthyl caprylate (102±1 pmol/min) and β-naphthyl butyrate (76±1 pmol/min) (n=3 for all determinations).
Salivary gland extracts did not have detectable acetyl-cholinesterase activity against the substrate acetylthioch-oline iodide.
4. Discussion
Cat flea salivary gland homogenates have esterase activity against α- and β-naphthyl ester substrates and PAF-acetylhydrolase activity.
Fig. 7. PAF-acetylhydrolase activity in salivary gland homogenates. Salivary gland preparation was incubated for 30 min with substrate solution containing a mixture of PAF and [3H]PAF and hydrolysis
measured by release of [3H]acetate. See text for details.
Fig. 8. Gel filtration of salivary glands on Ultrogel ACA 44 gives a single coincident peak (Ve=52 ml) of naphthyl esterase and
PAF-acetylhydrolase activity with an apparent molecular weight of |59 kDa. An extract of 175 salivary gland pairs was passed through a 100 ×1 cm column packed with gel equilibrated and eluted with 50 mM Tris pH 7.0, 300 mM NaCl and 0.01% Triton X-100 at 4 ml/h, and 1 ml fractions collected. Fractions were assayed for activity against
α-naphthyl acetate (j, right axis calibrated in nmol) and PAF (h, left axis calibrated in pmol). See text for details.
Cat flea salivary glands are a relatively rich source of naphthyl esterase activity, containing |5% of the total body esterase activity and|66 times the specific esterase activity of whole body homogenates. Fleas disgorge sali-vary naphthyl esterase during exploratory probing of
nitrocellulose membrane wetted with a phagostimulant. Native–PAGE and IEF resolve SG into three and six naphthyl-esterase-positive bands, respectively, and both patterns are distinct from patterns of carcass esterases. Gel filtration experiments and renaturation of esterase after SDS–PAGE give molecular-weight estimates for salivary esterases in the 56 kDa to 59 kDa range, close to the expected conserved subunit size of 60–70 kDa that is typical of other esterases (Oakeshott et al., 1993).
The basis of the electrophoretic heterogeneity of sali-vary gland esterases is currently unclear. Evidence that enzymatic deglycosylation can affect Native–PAGE mobility indicates that the glycosylation state may be contributory to apparent esterase heterogeneity. The possibility that some esterase-positive bands are artefac-tual breakdown products cannot be excluded. Cat flea salivary glands may express tissue-specific esterases, or over-express minor esterases. Cat fleas would presum-ably have a large esterase gene family encoding a num-ber of esterases, in the same way that Drosophila has 30 soluble esterases separable by combined IEF and Native–PAGE that are encoded by 23 genes. Individual members of the esterase gene family have activity against diverse substrates including acetylcholine, car-boxylesters, lipids and xenobiotics such as organ-ophosphate insecticides (Oakeshott et al., 1993). The naturally occurring substrates for salivary gland esterase are not known.
Cat flea salivary gland PAF-acetylhydrolase activity co-elutes with naphthyl esterase activity and it is poss-ible that one or more esterase(s) in this fraction has activity against PAF. PAF-acetylhydrolases described in other species are a special class of phospholipase A2(EC
3.1.1.47) that are serine-dependent hydrolases, do not require Ca2+ for activity and are specific for the short
acyl chains in the sn-2 position of the phospholipid. In the case of PAF there is hydrolysis of the sn-2 ester bond, releasing acetate and biologically inactive lyso-PAF (Derewenda and Ho, 1999).
ovalbumin (Yukawa et al., 1992), so hydrolysis of pico-molar amounts of PAF may be significant.
Adult cat fleas live in continuous ectoparasitic associ-ation with their hosts (Rust and Dryden, 1997). There may be a selective advantage for fleas to secrete salivary PAF-acetylhydrolase activity if it limits localised inflammatory and immune reactions to salivary antihae-mostatic factors. The host might be alerted to the pres-ence of fleas by an inflammatory reaction to injected sal-iva and respond by grooming or scratching, thereby interrupting flea feeding. Mechanisms that downregulate allergic responses may be important because individual cats that develop FAD have lower populations of fleas because they groom more and surviving fleas have lower fecundity (McDonald et al., 1998). The reason that some dogs and cats develop FAD is at present poorly under-stood. The observation that cat fleas have salivary gland PAF-acetylhydrolase activity suggests that PAF has an important role in the pathogenesis of the disease.
It will be interesting to see if other blood-feeding arthropods have salivary gland PAF-acetylhydrolase activity and whether it is associated with the documented salivary esterase and phospholipase A2 activities. Other
salivary enzymes, such as apyrase, which share the same substrates (ADP and ATP), have arisen independently from different gene families in Cimex lectularius, Rhod-nius prolixus and Aedes aegypti (Valenzuela et al., 1998). If blood-feeding arthropod vectors of disease have a salivary PAF-acetylhydrolase, this might enhance parasite transmission by downregulating host inflamma-tory and immune reactions at the site of inoculation. Immunomodulatory salivary products such as sandfly maxadilan have been shown to have this action (Qureshi et al., 1996).
Acknowledgements
The University of Liverpool Research Development Fund and the Department of Veterinary Pathology funded this study. We thank Professor D.F. Kelly for his support, Mr R. Pearson for the photography, Dr D. Robertson and Dr A. Macintyre for technical help, and Professor D. Jacobs and Ms M. Hutchinson of the Department of Pathology and Infectious Diseases, Royal Veterinary College, for the supply of cat fleas.
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