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A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites
International Journal for Parasitology 30 (2000) 19±27
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A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites Alasdair J. Nisbet, Peter F. Billingsley * Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK Received 24 August 1999; received in revised form 25 October 1999; accepted 25 October 1999
Abstract Extracts of ectoparasitic mites of birds (Dermanyssus gallinae), sheep (Psoroptes ovis) and plants (Tetranychus urticae) and of free-living mites (Acarus siro) contained acid and alkaline phosphatase, C4 and C8 esterases, lipase, leucine and valine aminopeptidases and a range of glycosidase activities. Dermanyssus gallinae and P. ovis, species highly adapted to an animal parasitic lifestyle, had very similar pro®les and contained low activities of glycosidases. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only b-galactosidase and b-glucuronidase. All extracts hydrolysed haemoglobin with optima below pH 6, and this hydrolysis was associated with an aspartic proteinase and variable cysteine proteinase activity dependent on species. Inhibitor-labelling with biotinyl-Phe-Ala-FMK revealed the presence of cysteine proteinases with molecular masses of 25± 33.5 kDa. Each mite species contains the enzymes necessary to complete digestion of the diet in the intracellular lysosomal compartment. The absolute and relative activities of each enzyme varied, and are discussed according to phylogeny and dietary habit. # 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Acari; Aspartic proteinase; Cathepsin B; Cathepsin L; Cysteine proteinase
1. Introduction The digestive systems of invertebrate parasites and pests are excellent potential targets for control agents, particularly through the inhibition of digestive enzymes or food absorption. This has been the basis for vaccines produced against animal parasites and the production of pest-resistant transgenic crop plants in recent years [1, 2]. However, the utilisation of a common food source by more than one species of pest or parasite does not necessarily imply the same digestive physiology and biochemistry. Rather, dietary utilisation usually superimposes onto a phylogenetic template of gut structure and function [3]. A detailed knowledge of the pest's digestive system is therefore required to exploit the novel control approaches and * Corresponding author. Fax: +44-1224-272396 E-mail address: [email protected] (P.F. Billingsley)
to integrate them with the control of other parasites and pests of the same host. Within the sub-class Acari, much of the detailed information on digestion has been obtained on members of the order Ixodida (ticks) [4]; however, recent studies on the allergenicity of dust mites (order Astigmata) have identi®ed putative digestive enzymes in faecal pellets as potent allergens. These enzymes include cysteine proteinases [5±8], serine proteinases [6, 9, 10], a lysozyme-like enzyme [11], and amylase [12, 13]. The structure and cellular nature of the digestive systems of a number of mites have also been determined, and appear to be similar to that of ticks. The main sites of digestion are the ventriculus and caecae, and most enzymic hydrolysis takes place within digestive cells after phagocytosis and pinocytosis of the gut contents [4]. Digestive cells have been identi®ed in Psoroptes ovis (Mathieson BF, PhD thesis, University of Bangor 1995) and these have been related to the high activities of cathepsin D-like aspartic proteinase
0020-7519/00/$20.00 # 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 1 6 9 - 1
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in extracts of this mite and the conspeci®c Psoroptes cuniculi [14, 15]. The aim of this study was to compare the hydrolytic enzymes present in extracts of parasitic or pest mites from three dierent phylogenetic groups within the sub-class Acari. The mites have dierent life-histories and are adapted to dierent food sources: Dermanyssus gallinae (order Mesostigmata) has three obligate parasitic stages, periodically visiting avian hosts to feed on blood; Psoroptes ovis (order Astigmata) causes psoroptic mange and spends the entire life-cycle on a mammalian host (sheep, goat or cattle) feeding principally on semi-liquid in¯ammatory exudates; Acarus siro (order Astigmata) is an important pest, feeding on stored produce, e.g. ¯our and grain; and Tetranychus urticae (order Prostigmata) is a polyphagous pest of plants feeding on the cell contents of pallisade and spongy mesophyll of living leaves. 2. Materials and methods 2.1. Mites Dermanyssus gallinae were collected live from polythene bags which had contained the carcasses of wild and domestic birds (crossbill, Loxia sp.; tawny owl, Strix aluco; and canary, Serinus canaria) obtained from the Scottish Agricultural College, Auchincruive. Psoroptes ovis were maintained on sheep at the Central Veterinary Laboratory. Acarus siro were purchased from the Central Science Laboratory and maintained on 3:1 dried brewer's yeast (``Yeastamin'', English Grains Healthcare) and wheat germ (Westmill Foods Ltd.) at room temperature in darkness. Tetranychus urticae were obtained from the Scottish Crop Research Institute and from the Horticultural Research Institute and were maintained at room temperature on sweet pepper plants (Capsicum annuum). After harvesting, mites were frozen immediately or maintained live for a maximum of 2 days (D. gallinae, P. ovis) before transfer to a freezer at ÿ808C. Thawed mites were washed in several volumes of 1% Triton-X 100 in PBS (T-PBS) on ice. Where necessary, remnants of insoluble host material were separated from the mites using a ®ne-gauge hypodermic needle followed by dierential sedimentation in the detergent solution and several more washes. The mites were then crushed in 100 ml T-PBS in a glass:glass Dounce homogeniser on ice (20 strokes) and transferred to a clean vial. The suspension was kept on ice for 10 min and mixed brie¯y every minute for ca. 10 s using a vortex mixer. The suspension was then divided into ``soluble'' and ``insoluble'' fractions by centrifugation. For initial experiments (API ZYM) the soluble fraction was that which formed the supernatant after centrifugation at 700 g for 1 min at 48C. For all other experiments the
soluble fraction was the supernatant after centrifugation at 10 000 g for 20 min at 48C. Extracts were stored at ÿ808C prior to use in enzyme assay. 2.2. Enzyme activities of mite extracts A semi-quantitative kit (API ZYM, bioMeÂrieux SA) was used to identify the range of enzyme activities in the mite extracts. Suspensions of each fraction in TPBS were diluted to 2 ml with distilled water and incubated on API ZYM strips for 4 h at 378C before colour development was assessed visually. The quantity of hydrolysed substrate was scored and compared with a semi-quantitative standard reading scale. Controls were incubated with T-PBS diluted accordingly. 2.3. Haemoglobin hydrolysis assay Hydrolysis of acid-denatured haemoglobin was measured using a previously described method [16]. Enzyme activity was measured in the presence of inhibitors of cysteine proteinases (E-64), metallo-proteinases (EDTA) and aspartic proteinases (pepstatin A). Soluble mite extracts were incubated with each inhibitor, or carrier solvent control in 0.2 M glycine buer (pH 3) for 30 min before the addition of haemoglobin and incubation for 5 h at 378C. For subsequent experiments, the speci®c aspartic proteinase substrate H-Pro-Thr-Glu-Phe-Phe(NO2)Arg-Leu-OH [17] or the cysteine proteinase substrate Z-Phe-Arg-pNA [18] was employed. 2.4. Relative activity and inhibition of aspartic proteinase in mite extracts Ten microlitres of soluble mite extract (7.5 mg protein) were combined with 0.9 ml of 0.1 M sodium formate buer (pH 3) and 100 ml H-Pro-Thr-Glu-PhePhe(NO2)-Arg-Leu-OH substrate (1 mg ml ÿ 1 in ddH2O) on ice. The reaction mixture was then transferred to a quartz cuvette and incubated at 308C for 5 min. The reduction in absorbance at 300 nm was recorded continually for 30 min thereafter. Rate of hydrolysis was compared with reagent control incubations. Inhibition of hydrolysis was measured by preincubating the mite extract for 30 min at 308C with sodium formate buer containing 20±200 pM pepstatin A. This was followed by chilling on ice, the addition of substrate and incubation at 308C for a further 2 h prior to the measurement of reduction in absorbance. 2.5. Cysteine proteinase assay Cysteine proteinase activity was measured using an adaptation of a previously described method [19]. Five
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
microlitres of mite extract were added to 113.75 ml 50 mM sodium acetate buer (pH 3.5 or 4.5) containing 5 mM cysteine and 1 mM EDTA. To measure inhibition of the enzyme activity E-64 (1 mM) or ZPhe-Ala-DMK (75 mM) were included in the buer. Z-Phe-Ala-DMK was added from a stock solution in methanol (®nal concentration 4.75%) and appropriate control reactions were performed in the presence of 4.75% methanol. Following incubation at 308C for 20 min, 6.25 ml of 10 mM Z-Phe-Arg-pNA in DMSO were added and the increase in absorbance at 405 nm was measured continually for 30 min. Rates of increase were corrected using reagent blanks. Cysteine proteinases were visualised on nitrocellulose blots using an adaptation of a previously described method [20]. Brie¯y, mite extracts (0.75 mg ml ÿ 1 protein) were incubated with 50 mM sodium acetate (pH 5.0) containing 5 mM cysteine and 1 mM EDTA for 20 min at 378C, then split into two fractions. ZPhe-Ala-DMK in methanol was then added to one fraction to a ®nal concentration of 1.5 mM, and the same volume of methanol without inhibitor was added to the other fraction to give a methanol concentration of 4.75%. After a further incubation for 20 min at 378C, biotinylPhe-Ala-FMK in methanol was added to both fractions to a ®nal concentration of 0.075 mM and a ®nal methanol concentration of 9%. Following incubation at 378C for a further 20 min, samples were boiled in reducing loading buer and separated on a 12.5% polyacrylamide minigel at 180 V (BioRad Miniprotean System) prior to overnight transfer to nitrocellulose membrane at 100 mA. Subsequent detection of the enzyme:biotinylated inhibitor complexes and biotinylated mol. wt markers on the nitrocellulose membranes was performed as previously described [20]. 2.6. Analysis of data Each experiment was performed in triplicate using appropriate controls to adjust for spontaneous hydrolysis of the substrates. The IC50 values of enzyme inhibitors were calculated from the rectilinear portions of their inhibition curves. Reaction rates were calculated using the analytical programs DeltaSoft II (BioMetallics Inc.) and SOFTMaxPro 2.1.1 (Molecular Devices). Comparison of means was achieved using ANOVA after arcsine transformations where appropriate. 2.7. Chemicals Triton-X 100, acid-denatured haemoglobin, trichloroacetic acid (TCA), L-trans-epoxysuccinyl-leucylamide(4-guanidino)-butane (E-64), ethylenediaminetetra-
21
acetic acid (EDTA), pepstatin A and 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) alkaline phosphatase substrate were purchased from Sigma Chemical Co. H-Pro-Thr-Glu-PhePhe(NO2)-Arg-Leu-OH was purchased from Calbiochem-Novabiochem. Z-Phe-Arg-pNA and ZPhe-Ala-DMK were purchased from Bachem. BiotinylPhe-Ala-FMK was purchased from Enzyme Systems Products, Livermore. 3. Results 3.1. Enzyme activities in mite extracts Acarus siro displayed the widest range of enzyme activities (18); D. gallinae and P. ovis both contained 12 of the 19 activities tested for and T. urticae contained 11. Soluble extracts of each species, with the exception of P. ovis, exhibited high acid and alkaline phosphatase activities. Psoroptes ovis contained only low alkaline phosphatase activity (Table 1). All three esterase types were detected, although lipase activity was weak. Leucine aminopeptidase was present in all four species, but was low in P.ovis compared with the other species. Valine aminopeptidase was also present in all species except T. urticae, and cysteine aminopeptidase was present only in A. siro. The only serine endopeptidase activity detected was a weak chymotrypsin activity in A. siro extracts. Glycosidase activity was more variable among the species; only b-glucuronidase, b-glucosidase and N-acetyl-b-glucosaminidase were detected in all species. Dermanyssus gallinae and P.ovis, species highly adapted to a parasitic lifestyle, had very similar pro®les of glycosidases and contained low activities of this class of enzymes in general. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only b-galactosidase and b-glucuronidase. No additional enzyme activities were detected in the insoluble fraction of mite extracts (data not shown). 3.2. Haemoglobin hydrolysis assay Haemoglobin was hydrolysed by all of the mite extracts in a pH-dependent fashion (Fig. 1). The optimum pH for hydrolysis lay between pH 3 and 5, and declined outside this range. The acid hydrolysis of haemoglobin by T. urticae extract was enhanced by ETDA (Fig. 2), but the other extracts were not signi®cantly aected by its presence. Haemoglobin hydrolysis by P. ovis extract was also unaected by E-64 which caused a small, but statistically signi®cant (P < 0.05), inhibition of activity in D. gallinae extracts and variable, but signi®cant
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Table 1 Enzyme activities in Triton-X 100 soluble fractions of four mite species Enzyme Phosphatase Acid Alkaline Phospho-amidase Esterase Esterase (C4) Esterase lipase (C8) Lipase (C14) Aminopeptidase Leucine Valine Cystine Serine proteinase Trypsin Chymotrypsin Glycosidase a-Galactosidase b-Galactosidase b-Glucuronidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase a-Fucosidase
D. gallinae
P. ovis
A. siro
T. urticae
+++++ +++ ++++
++++ + +++
+++++ +++++ ++++
+++++ +++++ +++++
++ +++ ++
+++ +++ +
++ ++++ +
++ +++ +
++++ ++++ ÿ
+ + ÿ
+++++ +++ +
++ ÿ ÿ
ÿ ÿ
ÿ ÿ
ÿ +
ÿ ÿ
ÿ ÿ ++ ÿ + ++++ ÿ +
ÿ + + ÿ + ++ ÿ ÿ
++ ++++ +++++ ++ +++++ +++++ +++++ ++++
ÿ ++++ ++++ ÿ + + ÿ ÿ
The quantity of hydrolysed substrate was scored and compared with the semi-quantitative standard reading scale. `` ÿ'' represents 0 nmol, `` + '' 5 nmol, `` ++ '' 10 nmol, `` +++ '' 20 nmol, `` ++++ '' 30 nmol and `` +++++ '' 50 nmol hydrolysed substrate. The assay was performed in triplicate for each suspension (protein content = 0.5 mg ml ÿ 1), controls were incubated with PBS containing 1% Triton-X 100 diluted accordingly.
(P < 0.05) inhibition in A. siro and T. urticae extracts. Only pepstatin A signi®cantly inhibited haemoglobin hydrolysis in every mite extract. 3.3. Activity and inhibition of aspartic proteinase in mite extracts The rates of hydrolysis of H-Pro-Thr-Glu-PhePhe(NO2)-Arg-Leu-OH by all mite extracts were linear over 30 min. Dermanyssus gallinae and A. siro extracts hydrolysed the substrate at approximately twice the rate measured for P. ovis extracts (Table 2). This higher aspartic proteinase activity in D.gallinae and A. siro extracts is re¯ected in the IC50 values measured for pepstatin A inhibition (Fig. 3, Table 2), being approximately double that of P. ovis extracts. Tetranychus urticae extracts displayed very low aspartic proteinase activity, and the rate of hydrolysis of HPro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH by T. urticae extract was too low for accurate measurement of the IC50 of pepstatin A. 3.4. Cysteine proteinase activity Hydrolysis of Z-Phe-Arg-pNA by all mite extracts indicated the presence of cysteine proteinase activity
(Table 3). Activity increased from pH 3.5 to pH 4.5, except in D. gallinae where it remained constant. Psoroptes ovis extracts contained the lowest cysteine proteinase activity. Cysteine proteinases were detected on nitrocellulose following labelling of the mite extracts with biotinylPhe-Ala-FMK (Fig. 4). Dermanyssus gallinae, P. ovis and T. urticae extracts contained cysteine proteinases of 26.5, 28 and 25 kDa, respectively. Acarus siro extracts contained two distinct bands of 31.5 and 33.5 kDa. The presence of 1.5 mM Z-Phe-Ala-DMK prevented formation of the detectable enzyme:biotinylated inhibitor complexes, con®rming the speci®city of the interaction. 4. Discussion In engorged mites, the ventriculus and associated diverticulae and caecae, which are the principal areas of digestion, occupy the majority of the body cavity [4] (Mathieson BRF, PhD thesis, University of Bangor 1995). Many of the enzymes that we have identi®ed are known to be involved in the hydrolysis of food within the digestive tract of invertebrates. Because of the small size of the mites used in this study (ca. 0.75±
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
Fig. 1. Hydrolysis of haemoglobin by Triton-X 100 soluble fraction of mite extract. Mite extract was incubated with haemoglobin solution in a buer of the appropriate pH. The soluble polypeptides were then precipitated using trichloroacetic acid and removed by centrifugation before the absorbance of the supernatant was measured at 280 nm. Data shown are the means of three replicates in the same experiment (2S.E.).
1.5 mm), it was not possible to dissect guts to de®ne the enzyme activities which we have identi®ed as purely gut-derived. However, comparison of the spectrum of enzymes in the four species we examined with those previously found in house-dust-mite faeces and tick midguts shows strong similarities, e.g. acid and alkaline phosphatases, esterase, lipase, aminopeptidases, a range of glycosidases and a 25 kDa cysteine proteinase (major allergen Der p 1) [5, 13, 21]. Whole-body extracts of the four species of mites from three phylogenetic orders and with diering dietary habits showed considerable uniformity in their range of phosphatase, esterase and aminopeptidase enzymes. These types of enzymes are present in extracts from the tick Boophilus microplus [21], where they are associated with digestion within the midgut. Acid phosphatase activity was particularly strong in all four species and has previously been localised associated with periphery of membranes surrounding the mesenteron wall and phagolysosomes within the digestive tract of Oribatid mites [22]. Non-speci®c esterases have also previously been demonstrated in mite extracts [23]. Lipase activity was absent from extracts of P. cuniculi [14] but was present, albeit at low levels, in extracts of each of the species tested in the present study. There is strong genotypic evidence to suggest
23
Fig. 2. Inhibition of haemoglobin hydrolysis by Triton-X 100 soluble fraction of mite extract. Mite extract was pre-incubated with 1 mM E-64, 5 mM EDTA or 1 nM pepstatin A prior to the addition of haemoglobin solution and further incubation. The soluble polypeptides were then precipitated using trichloroacetic acid and removed by centrifugation before the absorbance of the supernatant was measured at 280 nm. Percentage activity was calculated using the hydrolysis of a comparative control sample containing carrier solvent (methanol) at a concentration of 1% in the case of pepstatin A. Data shown are the means (2S.E.) of three replicates in three repetitions of the experiment (n = 9). Each repetition of the experiment used dierent extracts of each mite species. *Value signi®cantly dierent from 100% activity observed in controls (P < 0.05).
that P. cuniculi and P. ovis are conspeci®c [24] but phenotypically adapted to rabbit or sheep hosts, respectively. The absence of detectable lipase activity in the former may be a re¯ection of the diet utilised on the rabbit. Whole-blood is consumed on the rabbit compared with a more sebaceous diet of epidermis and serous exudate on the sheep [25]. Similarly, P. cuniculi extracts digest haemoglobin more eectively than P. ovis extracts [15]. The two parasitic species from dierent orders, D. gallinae and P. ovis, had very similar pro®les of glycosidases and contained low activities of this enzyme class, whereas the polyphagous species A. siro contained a wide range of glycosidase activities. The phytophagous species, T. urticae, only displayed high activities of b-galactosidase and b-glucuronidase. bGalactosidase activity has previously been described in T. urticae [26] and also in phytophagous Oribatid mites [22]. The natural substrates for aryl b-galactosi-
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Table 2 Rates of hydrolysis of aspartic proteinase substrate by mite extracts and its inhibition by pepstatin A Species
Hydrolysis rate (mOD unit min-1)
IC50 (pM)
D. gallinae P. ovis A. siro T. urticae
0.4112 0.004a 0.2142 0.014b 0.4392 0.020a 0.0582 0.003c
80.522.4 51.425.5 82.521.6 ±
Values shown are means of three replicates in a single repetition of the experiment (2S.E.). The experiment was repeated using dierent extracts of each mite species with similar results.Values annotated with the same letter are not signi®cantly dierent (P < 0.05)
dases in phytophagous insects are thought to be galactolipids which are particularly abundant in photosynthetic tissues [27], and this may explain the high activity of this enzyme class in T. urticae. Contrary to a previous study [26], b-glucosidase activity was demonstrated in T. urticae whereas a-galactosidase and a-glucosidase were absent. This may result from rearing a polyphagous species of mite on a dierent host plant or it may re¯ect the presence of dierent gut
Fig. 3. Inhibition of hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)Arg-Leu-OH by mite extracts using pepstatin A. Ten microlitres of soluble mite extract were combined with 0.9 ml of 0.1 M sodium formate buer (pH 3) containing a range of concentrations of pepstatin A for 30 min at 308C. This was followed by the addition of substrate and incubation at 308C for a further 2 h prior to the measurement of reduction in absorbance at 300 nm. Values shown are means of three replicates in a single experiment (2S.E.). The experiment was repeated using dierent extracts of each mite species with similar results.
symbionts in dierent populations of T. urticae. bGlucosidase activity was found in all of the mite extracts and may be responsible for hemicellulose digestion or the cleavage of carbohydrate moieties from glycoproteins dependent on the diet of each species [27]. N-Acetyl-b-glucosaminidase was also present in each species. This enzyme is involved in the moulting process in invertebrates and, as mixed stages of mites were used for the extractions, may be expected to be present. Trypsin activity was absent from all mite species, but A. siro extracts displayed a low chymotrypsin activity. Chymotrypsin-like enzymes constitute the group 6 allergens characterised from pyroglyphid dust mites [28] with which A. siro has allergenic crossreactivity [29]. Tetranychus urticae extracts are also responsible for occupational allergic disease in agricultural workers, but do not display antigenic crossreactivity with dust-mite extracts [30]. The acidic pH optimum for haemoglobin and the lack of hydrolysis above pH 6 suggests the absence, or very low activity, of detectable serine proteinases in the mite extracts. Only pepstatin A, a speci®c aspartic proteinase inhibitor, signi®cantly reduced haemoglobin hydrolysis by every mite extract, although E-64, a cysteine proteinase inhibitor, reduced the proteolysis by ca. 50% in A. siro and T. urticae extracts. Enhancement of haemoglobin hydrolysis in the presence of EDTA was also suggestive of cysteine proteinase activity in T. urticae. Overall, the dierences in the eects of dierent enzyme inhibitors on the hydrolysis of haemoglobin can be largely attributed to the relative amounts of aspartic and cysteine proteinases present in the extracts. Thus, hydrolysis of haemoglobin by P. ovis extract, which was low in cysteine proteinase activity, was not signi®cantly aected by a cysteine proteinase inhibitor. In contrast, haemoglobin hydrolysis in the remaining species was signi®cantly reduced by the presence of a cysteine proteinase inhibitor, and each of these species had signi®cantly higher cysteine proteinase activity than P. ovis. The cysteine proteinase substrate Z-Phe-Arg-pNA and the inhibitors used (Z-Phe-Ala-DMK and biotinyl-Phe-Ala-FMK) are speci®c to the plant enzyme papain and cathepsins B and L which are lysosomal exo- and endopeptidases, respectively [31]. Cysteine proteinases with sequence homology to cathepsin Blike molecules comprise the group 1 allergens of Dermatophagoides spp. [5], and their presence in faecal pellets suggests a digestive role. Aspartic proteinases were also present in each of the extracts, along with a leucine aminopeptidase. The presence of these enzymes alongside histological evidence from a variety of tick and mite species [32±36] supports their role in lysoso-
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
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Fig. 4. Detection of mite cysteine proteinase activity using biotinyl-Phe-Ala-FMK. Mite extracts were pre-incubated with 50 mM sodium acetate (pH 5.0) containing 5 mM cysteine and 1 mM EDTA for 20 min at 378C, then split into two fractions. Z-Phe-Ala-DMK was then added to one fraction (+lanes) to a ®nal concentration of 1.5 mM and the same volume of methanol without inhibitor was added to the other fraction (ÿ lanes). After a further incubation, biotinyl-Phe-Ala-FMK in was added to both fractions to a ®nal concentration of 0.075 mM. Following incubation, samples were boiled in reducing loading buer and fractions containing 3.5 mg protein separated on a 12.5% polyacrylamide minigel prior to overnight transfer to nitrocellulose membrane. Subsequent detection of the enzyme:biotinylated inhibitor complexes and biotinylated molecular mass markers on the nitrocellulose membranes was performed as previously described [20]. Molecular mass markers, in kDa, are indicated on each blot.
mal, intracellular digestion of food in each of the species. This involves the internalisation of solid and liquid food items by pinocytosis and phagocytosis by specialised digestive cells in the gut, lysosomal proteol-
ysis by endopeptidases (cathepsins D and L) followed by intra- or extracellular digestion by cytosolic or membrane-bound exopeptidases (cathepsin B or membrane-bound aminopeptidase) [37].
Table 3 Cysteine proteinase activity in mite extracts Treatment
pH 3.5
D. gallinae P. ovis A. siro T. urticae a
4.5
Control
Methanola
E-64
Z-Phe-Ala-DMK
Control
Methanol
E-64
Z-Phe-Ala-DMK
2.0620.05b 0.0620.06 1.2220.04 0.6620.02
2.342 0.06 0 1.242 0.02 0.692 0.01
0.0720.07 0 0.0420.02 0.0420.02
0.0320.02 0 0 0
2.032 0.05 0.312 0.01 1.882 0.05 0.802 0.01
2.20 20.09 0.24 20.04 1.72 20.05 0.91 20.05
0 0 0.0520.01 0
0 0.05 20.04 0.02 20.01 0
Control reaction for Z-Phe-Ala-DMK inhibition containing methanol at a concentration of 4.75%. Figures shown are mean reaction rates (mOD units min ÿ 1) 2S.E.M. (n = 3).
b
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A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
The cysteine proteinases detected may also be responsible for inducing allergic-type responses; Der p 1, the house-dust-mite cysteine proteinase, possesses several properties related to its enzymatic activities which make it a potent allergen [38, 39]. Determination of the mechanistic class of proteolytic allergens from mite species is important, because desensitisation to the allergen may be achieved by exposure of the host or atopic individual to enzyme:inhibitor complexes [40].
Acknowledgements We are very grateful to Dr M. Taylor and Mr B. Groves, CVL, Surrey, UK; Dr M. Solomon, HRI, Kent, UK; Dr J.A.T. Woodford, SCRI, Dundee, UK; and Mr T. Pennycott, SAC, Auchincruive, UK for provision of mites; Professor J.H. McKerrow, University of California, San Francisco, CA, USA and Dr A.J. Walker, University of Manchester, UK for advice on cysteine proteinase detection; and to the Ministry of Agriculture, Fisheries and Food, UK for funding this research under MAFF Open Contract: CSA 3402.
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[12] Lake FR, Ward LD, Simpson RJ, Thompson PJ, Stewart GA. Allergenicity and physicochemical characterization of house dust mite derived amylase. Int Arch Allergy Appl Immunol 1991;94:357±8. [13] Stewart GA, Lake FR, Thompson PJ. Faecally derived hydrolytic enzymes from Dermatophagoides pteronyssinus: physicochemical characterisation of potential allergens. Int Arch Allergy Appl Immunol 1991;95:248±56. [14] Nisbet AJ, Billingsley PF. Hydrolytic enzymes of Psoroptes cuniculi (Delafond). Insect Biochem Molec Biol 1999;29:25±32. [15] Nisbet AJ, Billingsley PF. Immunological control of scab mites: digestive enzymes as candidate compounds. Vet Parasitol 1999;83:231±9. [16] Houseman JG, Downe AER. Characterisation of an acidic proteinase from the posterior midgut of Rhodnius prolixus StaÊl. Insect Biochem 1982;12:651±5. [17] Dunn BM, Jiminez M, Parten BF, Valler MJ, Rolph CE, Kay J. A systematic series of synthetic chromophoric substrates for aspartic proteinases. Biochem J 1986;237:899±906. [18] Tchoupe JR, Moreau T, Gauthier F, Bieth JG. Photometric or ¯uorometric assay of cathepsin-B, cathepsin-L and cathepsin-H and papain using substrates with an aminotri¯uoromethylcoumarin leaving group. Biochim Biophys Acta 1991;1076:149±51. [19] Walker AJ, Glen DM, Shewry PR. Puri®cation and characterisation of a digestive cysteine proteinase from the ®eld slug (Deroceras reticulatum): a potential target for slug control. J Agric Food Chem 1998;46:2873±81. [20] Walker B, Cullen BM, Kay G, Halliday IM, McGinty A, Nelson J. The synthesis, kinetic characterisation and application of a novel biotinylated anity label for cathepsin B. Biochem J 1992;283:449±53. [21] Kerlin RL, Hughes S. Enzymes in saliva from four parasitic arthropods. Med Vet Entomol 1992;6:121±6. [22] Dinsdale D. The digestive activity of a phthiracarid mite mesenteron. J Insect Physiol 1974;20:2247±60. [23] Bowman CE, Lessiter MJ. Amylase and esterase polymorphisms in economically important stored product mites (Acari: Astigmata). Comp Biochem Physiol 1985;81B:353±60. [24] Zahler M, Essig A, Gothe R, Rinder H. Genetic evidence suggests that Psoroptes isolates of dierent phenotypes, hosts and geographic origins are conspeci®c. Int J Parasitol 1998;28:1713±9. [25] Raerty DE, Gray JS. The feeding behaviour of Psoroptes spp. on rabbits and sheep. J Parasitol 1987;73:901±6. [26] Ehrhardt P, Voss G. Die Carbohydrasen der Spinnmilbe Tetranychus urticae Koch (Acari, Trombidiformes, Tetranychidae). Experientia 1961;17:307. [27] Terra WR, Ferreira C, JordaÄo BP, Dillon RJ. Digestive enzymes. In: Lehane MJ, Billingsley PF, editors. Biology of the insect midgut. London: Chapman & Hall, 1996;153±94. [28] Yasueda H, Mita H, Yui Y, Shida T. Isolation and characterisation of two allergens from Dermatophagoides farinae. Int Arch Allerg Appl Immunol 1986;81:214±23. [29] Johansson E, Johansson SGO, Vanhagehamsten M. Allergenic characterisation of Acarus siro and Tyrophagus putrescentiae and their cross reactivity with Lepidoglyphus destructor and Dermatophagoides pteronyssinus. Clin Exp Allergy 1994;24:743± 51. [30] Delgado J, Orta JC, Navarro AM, Conde J, Martinez J, Palacios R. Occupatonal allergy in greenhouse workers: sensitisation to Tetranychus urticae. Clin Exp Allergy 1997;27:640±5. [31] Barrett AJ, Kirschke H. Cathepsin B, cathepsin H and cathepsin L. Methods Enzymol 1981;80:535±60. [32] Hughes TE. Some histological changes which occur in the gut epithelium of Ixodes ricinus females during gorging and up to oviposition. Ann Trop Med Parasitol 1954;48:397±404.
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27 [33] Brody AR, McGrath JC, Wharton GW. Dermatophagoides farinae: the digestive system. J NY Entomol Soc 1972;80:152± 77. [34] Woodring JP, Galbraith CA. The anatomy of the adult uropodid Fuscuropoda agitans (Arachnida: Acari), with comparative observations on other Acari. J Morphol 1976;150:19±58. [35] Coons LB. Fine structure of the digestive system of Macrocheles muscaedomesticae (Scopoli) (Acarina: Mesostigmata). Int J Insect Morph Embryol 1978;7:137±53. [36] Mothes U, Seitz KA. Functional microscopic anatomy of the digestive system of Tetranychus urticae. Acarologia 1981;22:257±70.
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www.elsevier.nl/locate/ijpara
A comparative survey of the hydrolytic enzymes of ectoparasitic and free-living mites Alasdair J. Nisbet, Peter F. Billingsley * Department of Zoology, University of Aberdeen, Tillydrone Avenue, Aberdeen AB24 2TZ, Scotland, UK Received 24 August 1999; received in revised form 25 October 1999; accepted 25 October 1999
Abstract Extracts of ectoparasitic mites of birds (Dermanyssus gallinae), sheep (Psoroptes ovis) and plants (Tetranychus urticae) and of free-living mites (Acarus siro) contained acid and alkaline phosphatase, C4 and C8 esterases, lipase, leucine and valine aminopeptidases and a range of glycosidase activities. Dermanyssus gallinae and P. ovis, species highly adapted to an animal parasitic lifestyle, had very similar pro®les and contained low activities of glycosidases. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only b-galactosidase and b-glucuronidase. All extracts hydrolysed haemoglobin with optima below pH 6, and this hydrolysis was associated with an aspartic proteinase and variable cysteine proteinase activity dependent on species. Inhibitor-labelling with biotinyl-Phe-Ala-FMK revealed the presence of cysteine proteinases with molecular masses of 25± 33.5 kDa. Each mite species contains the enzymes necessary to complete digestion of the diet in the intracellular lysosomal compartment. The absolute and relative activities of each enzyme varied, and are discussed according to phylogeny and dietary habit. # 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. Keywords: Acari; Aspartic proteinase; Cathepsin B; Cathepsin L; Cysteine proteinase
1. Introduction The digestive systems of invertebrate parasites and pests are excellent potential targets for control agents, particularly through the inhibition of digestive enzymes or food absorption. This has been the basis for vaccines produced against animal parasites and the production of pest-resistant transgenic crop plants in recent years [1, 2]. However, the utilisation of a common food source by more than one species of pest or parasite does not necessarily imply the same digestive physiology and biochemistry. Rather, dietary utilisation usually superimposes onto a phylogenetic template of gut structure and function [3]. A detailed knowledge of the pest's digestive system is therefore required to exploit the novel control approaches and * Corresponding author. Fax: +44-1224-272396 E-mail address: [email protected] (P.F. Billingsley)
to integrate them with the control of other parasites and pests of the same host. Within the sub-class Acari, much of the detailed information on digestion has been obtained on members of the order Ixodida (ticks) [4]; however, recent studies on the allergenicity of dust mites (order Astigmata) have identi®ed putative digestive enzymes in faecal pellets as potent allergens. These enzymes include cysteine proteinases [5±8], serine proteinases [6, 9, 10], a lysozyme-like enzyme [11], and amylase [12, 13]. The structure and cellular nature of the digestive systems of a number of mites have also been determined, and appear to be similar to that of ticks. The main sites of digestion are the ventriculus and caecae, and most enzymic hydrolysis takes place within digestive cells after phagocytosis and pinocytosis of the gut contents [4]. Digestive cells have been identi®ed in Psoroptes ovis (Mathieson BF, PhD thesis, University of Bangor 1995) and these have been related to the high activities of cathepsin D-like aspartic proteinase
0020-7519/00/$20.00 # 2000 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 0 2 0 - 7 5 1 9 ( 9 9 ) 0 0 1 6 9 - 1
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A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
in extracts of this mite and the conspeci®c Psoroptes cuniculi [14, 15]. The aim of this study was to compare the hydrolytic enzymes present in extracts of parasitic or pest mites from three dierent phylogenetic groups within the sub-class Acari. The mites have dierent life-histories and are adapted to dierent food sources: Dermanyssus gallinae (order Mesostigmata) has three obligate parasitic stages, periodically visiting avian hosts to feed on blood; Psoroptes ovis (order Astigmata) causes psoroptic mange and spends the entire life-cycle on a mammalian host (sheep, goat or cattle) feeding principally on semi-liquid in¯ammatory exudates; Acarus siro (order Astigmata) is an important pest, feeding on stored produce, e.g. ¯our and grain; and Tetranychus urticae (order Prostigmata) is a polyphagous pest of plants feeding on the cell contents of pallisade and spongy mesophyll of living leaves. 2. Materials and methods 2.1. Mites Dermanyssus gallinae were collected live from polythene bags which had contained the carcasses of wild and domestic birds (crossbill, Loxia sp.; tawny owl, Strix aluco; and canary, Serinus canaria) obtained from the Scottish Agricultural College, Auchincruive. Psoroptes ovis were maintained on sheep at the Central Veterinary Laboratory. Acarus siro were purchased from the Central Science Laboratory and maintained on 3:1 dried brewer's yeast (``Yeastamin'', English Grains Healthcare) and wheat germ (Westmill Foods Ltd.) at room temperature in darkness. Tetranychus urticae were obtained from the Scottish Crop Research Institute and from the Horticultural Research Institute and were maintained at room temperature on sweet pepper plants (Capsicum annuum). After harvesting, mites were frozen immediately or maintained live for a maximum of 2 days (D. gallinae, P. ovis) before transfer to a freezer at ÿ808C. Thawed mites were washed in several volumes of 1% Triton-X 100 in PBS (T-PBS) on ice. Where necessary, remnants of insoluble host material were separated from the mites using a ®ne-gauge hypodermic needle followed by dierential sedimentation in the detergent solution and several more washes. The mites were then crushed in 100 ml T-PBS in a glass:glass Dounce homogeniser on ice (20 strokes) and transferred to a clean vial. The suspension was kept on ice for 10 min and mixed brie¯y every minute for ca. 10 s using a vortex mixer. The suspension was then divided into ``soluble'' and ``insoluble'' fractions by centrifugation. For initial experiments (API ZYM) the soluble fraction was that which formed the supernatant after centrifugation at 700 g for 1 min at 48C. For all other experiments the
soluble fraction was the supernatant after centrifugation at 10 000 g for 20 min at 48C. Extracts were stored at ÿ808C prior to use in enzyme assay. 2.2. Enzyme activities of mite extracts A semi-quantitative kit (API ZYM, bioMeÂrieux SA) was used to identify the range of enzyme activities in the mite extracts. Suspensions of each fraction in TPBS were diluted to 2 ml with distilled water and incubated on API ZYM strips for 4 h at 378C before colour development was assessed visually. The quantity of hydrolysed substrate was scored and compared with a semi-quantitative standard reading scale. Controls were incubated with T-PBS diluted accordingly. 2.3. Haemoglobin hydrolysis assay Hydrolysis of acid-denatured haemoglobin was measured using a previously described method [16]. Enzyme activity was measured in the presence of inhibitors of cysteine proteinases (E-64), metallo-proteinases (EDTA) and aspartic proteinases (pepstatin A). Soluble mite extracts were incubated with each inhibitor, or carrier solvent control in 0.2 M glycine buer (pH 3) for 30 min before the addition of haemoglobin and incubation for 5 h at 378C. For subsequent experiments, the speci®c aspartic proteinase substrate H-Pro-Thr-Glu-Phe-Phe(NO2)Arg-Leu-OH [17] or the cysteine proteinase substrate Z-Phe-Arg-pNA [18] was employed. 2.4. Relative activity and inhibition of aspartic proteinase in mite extracts Ten microlitres of soluble mite extract (7.5 mg protein) were combined with 0.9 ml of 0.1 M sodium formate buer (pH 3) and 100 ml H-Pro-Thr-Glu-PhePhe(NO2)-Arg-Leu-OH substrate (1 mg ml ÿ 1 in ddH2O) on ice. The reaction mixture was then transferred to a quartz cuvette and incubated at 308C for 5 min. The reduction in absorbance at 300 nm was recorded continually for 30 min thereafter. Rate of hydrolysis was compared with reagent control incubations. Inhibition of hydrolysis was measured by preincubating the mite extract for 30 min at 308C with sodium formate buer containing 20±200 pM pepstatin A. This was followed by chilling on ice, the addition of substrate and incubation at 308C for a further 2 h prior to the measurement of reduction in absorbance. 2.5. Cysteine proteinase assay Cysteine proteinase activity was measured using an adaptation of a previously described method [19]. Five
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
microlitres of mite extract were added to 113.75 ml 50 mM sodium acetate buer (pH 3.5 or 4.5) containing 5 mM cysteine and 1 mM EDTA. To measure inhibition of the enzyme activity E-64 (1 mM) or ZPhe-Ala-DMK (75 mM) were included in the buer. Z-Phe-Ala-DMK was added from a stock solution in methanol (®nal concentration 4.75%) and appropriate control reactions were performed in the presence of 4.75% methanol. Following incubation at 308C for 20 min, 6.25 ml of 10 mM Z-Phe-Arg-pNA in DMSO were added and the increase in absorbance at 405 nm was measured continually for 30 min. Rates of increase were corrected using reagent blanks. Cysteine proteinases were visualised on nitrocellulose blots using an adaptation of a previously described method [20]. Brie¯y, mite extracts (0.75 mg ml ÿ 1 protein) were incubated with 50 mM sodium acetate (pH 5.0) containing 5 mM cysteine and 1 mM EDTA for 20 min at 378C, then split into two fractions. ZPhe-Ala-DMK in methanol was then added to one fraction to a ®nal concentration of 1.5 mM, and the same volume of methanol without inhibitor was added to the other fraction to give a methanol concentration of 4.75%. After a further incubation for 20 min at 378C, biotinylPhe-Ala-FMK in methanol was added to both fractions to a ®nal concentration of 0.075 mM and a ®nal methanol concentration of 9%. Following incubation at 378C for a further 20 min, samples were boiled in reducing loading buer and separated on a 12.5% polyacrylamide minigel at 180 V (BioRad Miniprotean System) prior to overnight transfer to nitrocellulose membrane at 100 mA. Subsequent detection of the enzyme:biotinylated inhibitor complexes and biotinylated mol. wt markers on the nitrocellulose membranes was performed as previously described [20]. 2.6. Analysis of data Each experiment was performed in triplicate using appropriate controls to adjust for spontaneous hydrolysis of the substrates. The IC50 values of enzyme inhibitors were calculated from the rectilinear portions of their inhibition curves. Reaction rates were calculated using the analytical programs DeltaSoft II (BioMetallics Inc.) and SOFTMaxPro 2.1.1 (Molecular Devices). Comparison of means was achieved using ANOVA after arcsine transformations where appropriate. 2.7. Chemicals Triton-X 100, acid-denatured haemoglobin, trichloroacetic acid (TCA), L-trans-epoxysuccinyl-leucylamide(4-guanidino)-butane (E-64), ethylenediaminetetra-
21
acetic acid (EDTA), pepstatin A and 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT) alkaline phosphatase substrate were purchased from Sigma Chemical Co. H-Pro-Thr-Glu-PhePhe(NO2)-Arg-Leu-OH was purchased from Calbiochem-Novabiochem. Z-Phe-Arg-pNA and ZPhe-Ala-DMK were purchased from Bachem. BiotinylPhe-Ala-FMK was purchased from Enzyme Systems Products, Livermore. 3. Results 3.1. Enzyme activities in mite extracts Acarus siro displayed the widest range of enzyme activities (18); D. gallinae and P. ovis both contained 12 of the 19 activities tested for and T. urticae contained 11. Soluble extracts of each species, with the exception of P. ovis, exhibited high acid and alkaline phosphatase activities. Psoroptes ovis contained only low alkaline phosphatase activity (Table 1). All three esterase types were detected, although lipase activity was weak. Leucine aminopeptidase was present in all four species, but was low in P.ovis compared with the other species. Valine aminopeptidase was also present in all species except T. urticae, and cysteine aminopeptidase was present only in A. siro. The only serine endopeptidase activity detected was a weak chymotrypsin activity in A. siro extracts. Glycosidase activity was more variable among the species; only b-glucuronidase, b-glucosidase and N-acetyl-b-glucosaminidase were detected in all species. Dermanyssus gallinae and P.ovis, species highly adapted to a parasitic lifestyle, had very similar pro®les of glycosidases and contained low activities of this class of enzymes in general. In contrast, the polyphagous species A. siro contained moderate to high activities of every glycosidase examined, whereas the phytophagous species, T. urticae, displayed high activities of only b-galactosidase and b-glucuronidase. No additional enzyme activities were detected in the insoluble fraction of mite extracts (data not shown). 3.2. Haemoglobin hydrolysis assay Haemoglobin was hydrolysed by all of the mite extracts in a pH-dependent fashion (Fig. 1). The optimum pH for hydrolysis lay between pH 3 and 5, and declined outside this range. The acid hydrolysis of haemoglobin by T. urticae extract was enhanced by ETDA (Fig. 2), but the other extracts were not signi®cantly aected by its presence. Haemoglobin hydrolysis by P. ovis extract was also unaected by E-64 which caused a small, but statistically signi®cant (P < 0.05), inhibition of activity in D. gallinae extracts and variable, but signi®cant
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A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
Table 1 Enzyme activities in Triton-X 100 soluble fractions of four mite species Enzyme Phosphatase Acid Alkaline Phospho-amidase Esterase Esterase (C4) Esterase lipase (C8) Lipase (C14) Aminopeptidase Leucine Valine Cystine Serine proteinase Trypsin Chymotrypsin Glycosidase a-Galactosidase b-Galactosidase b-Glucuronidase a-Glucosidase b-Glucosidase N-Acetyl-b-glucosaminidase a-Mannosidase a-Fucosidase
D. gallinae
P. ovis
A. siro
T. urticae
+++++ +++ ++++
++++ + +++
+++++ +++++ ++++
+++++ +++++ +++++
++ +++ ++
+++ +++ +
++ ++++ +
++ +++ +
++++ ++++ ÿ
+ + ÿ
+++++ +++ +
++ ÿ ÿ
ÿ ÿ
ÿ ÿ
ÿ +
ÿ ÿ
ÿ ÿ ++ ÿ + ++++ ÿ +
ÿ + + ÿ + ++ ÿ ÿ
++ ++++ +++++ ++ +++++ +++++ +++++ ++++
ÿ ++++ ++++ ÿ + + ÿ ÿ
The quantity of hydrolysed substrate was scored and compared with the semi-quantitative standard reading scale. `` ÿ'' represents 0 nmol, `` + '' 5 nmol, `` ++ '' 10 nmol, `` +++ '' 20 nmol, `` ++++ '' 30 nmol and `` +++++ '' 50 nmol hydrolysed substrate. The assay was performed in triplicate for each suspension (protein content = 0.5 mg ml ÿ 1), controls were incubated with PBS containing 1% Triton-X 100 diluted accordingly.
(P < 0.05) inhibition in A. siro and T. urticae extracts. Only pepstatin A signi®cantly inhibited haemoglobin hydrolysis in every mite extract. 3.3. Activity and inhibition of aspartic proteinase in mite extracts The rates of hydrolysis of H-Pro-Thr-Glu-PhePhe(NO2)-Arg-Leu-OH by all mite extracts were linear over 30 min. Dermanyssus gallinae and A. siro extracts hydrolysed the substrate at approximately twice the rate measured for P. ovis extracts (Table 2). This higher aspartic proteinase activity in D.gallinae and A. siro extracts is re¯ected in the IC50 values measured for pepstatin A inhibition (Fig. 3, Table 2), being approximately double that of P. ovis extracts. Tetranychus urticae extracts displayed very low aspartic proteinase activity, and the rate of hydrolysis of HPro-Thr-Glu-Phe-Phe(NO2)-Arg-Leu-OH by T. urticae extract was too low for accurate measurement of the IC50 of pepstatin A. 3.4. Cysteine proteinase activity Hydrolysis of Z-Phe-Arg-pNA by all mite extracts indicated the presence of cysteine proteinase activity
(Table 3). Activity increased from pH 3.5 to pH 4.5, except in D. gallinae where it remained constant. Psoroptes ovis extracts contained the lowest cysteine proteinase activity. Cysteine proteinases were detected on nitrocellulose following labelling of the mite extracts with biotinylPhe-Ala-FMK (Fig. 4). Dermanyssus gallinae, P. ovis and T. urticae extracts contained cysteine proteinases of 26.5, 28 and 25 kDa, respectively. Acarus siro extracts contained two distinct bands of 31.5 and 33.5 kDa. The presence of 1.5 mM Z-Phe-Ala-DMK prevented formation of the detectable enzyme:biotinylated inhibitor complexes, con®rming the speci®city of the interaction. 4. Discussion In engorged mites, the ventriculus and associated diverticulae and caecae, which are the principal areas of digestion, occupy the majority of the body cavity [4] (Mathieson BRF, PhD thesis, University of Bangor 1995). Many of the enzymes that we have identi®ed are known to be involved in the hydrolysis of food within the digestive tract of invertebrates. Because of the small size of the mites used in this study (ca. 0.75±
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
Fig. 1. Hydrolysis of haemoglobin by Triton-X 100 soluble fraction of mite extract. Mite extract was incubated with haemoglobin solution in a buer of the appropriate pH. The soluble polypeptides were then precipitated using trichloroacetic acid and removed by centrifugation before the absorbance of the supernatant was measured at 280 nm. Data shown are the means of three replicates in the same experiment (2S.E.).
1.5 mm), it was not possible to dissect guts to de®ne the enzyme activities which we have identi®ed as purely gut-derived. However, comparison of the spectrum of enzymes in the four species we examined with those previously found in house-dust-mite faeces and tick midguts shows strong similarities, e.g. acid and alkaline phosphatases, esterase, lipase, aminopeptidases, a range of glycosidases and a 25 kDa cysteine proteinase (major allergen Der p 1) [5, 13, 21]. Whole-body extracts of the four species of mites from three phylogenetic orders and with diering dietary habits showed considerable uniformity in their range of phosphatase, esterase and aminopeptidase enzymes. These types of enzymes are present in extracts from the tick Boophilus microplus [21], where they are associated with digestion within the midgut. Acid phosphatase activity was particularly strong in all four species and has previously been localised associated with periphery of membranes surrounding the mesenteron wall and phagolysosomes within the digestive tract of Oribatid mites [22]. Non-speci®c esterases have also previously been demonstrated in mite extracts [23]. Lipase activity was absent from extracts of P. cuniculi [14] but was present, albeit at low levels, in extracts of each of the species tested in the present study. There is strong genotypic evidence to suggest
23
Fig. 2. Inhibition of haemoglobin hydrolysis by Triton-X 100 soluble fraction of mite extract. Mite extract was pre-incubated with 1 mM E-64, 5 mM EDTA or 1 nM pepstatin A prior to the addition of haemoglobin solution and further incubation. The soluble polypeptides were then precipitated using trichloroacetic acid and removed by centrifugation before the absorbance of the supernatant was measured at 280 nm. Percentage activity was calculated using the hydrolysis of a comparative control sample containing carrier solvent (methanol) at a concentration of 1% in the case of pepstatin A. Data shown are the means (2S.E.) of three replicates in three repetitions of the experiment (n = 9). Each repetition of the experiment used dierent extracts of each mite species. *Value signi®cantly dierent from 100% activity observed in controls (P < 0.05).
that P. cuniculi and P. ovis are conspeci®c [24] but phenotypically adapted to rabbit or sheep hosts, respectively. The absence of detectable lipase activity in the former may be a re¯ection of the diet utilised on the rabbit. Whole-blood is consumed on the rabbit compared with a more sebaceous diet of epidermis and serous exudate on the sheep [25]. Similarly, P. cuniculi extracts digest haemoglobin more eectively than P. ovis extracts [15]. The two parasitic species from dierent orders, D. gallinae and P. ovis, had very similar pro®les of glycosidases and contained low activities of this enzyme class, whereas the polyphagous species A. siro contained a wide range of glycosidase activities. The phytophagous species, T. urticae, only displayed high activities of b-galactosidase and b-glucuronidase. bGalactosidase activity has previously been described in T. urticae [26] and also in phytophagous Oribatid mites [22]. The natural substrates for aryl b-galactosi-
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A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
Table 2 Rates of hydrolysis of aspartic proteinase substrate by mite extracts and its inhibition by pepstatin A Species
Hydrolysis rate (mOD unit min-1)
IC50 (pM)
D. gallinae P. ovis A. siro T. urticae
0.4112 0.004a 0.2142 0.014b 0.4392 0.020a 0.0582 0.003c
80.522.4 51.425.5 82.521.6 ±
Values shown are means of three replicates in a single repetition of the experiment (2S.E.). The experiment was repeated using dierent extracts of each mite species with similar results.Values annotated with the same letter are not signi®cantly dierent (P < 0.05)
dases in phytophagous insects are thought to be galactolipids which are particularly abundant in photosynthetic tissues [27], and this may explain the high activity of this enzyme class in T. urticae. Contrary to a previous study [26], b-glucosidase activity was demonstrated in T. urticae whereas a-galactosidase and a-glucosidase were absent. This may result from rearing a polyphagous species of mite on a dierent host plant or it may re¯ect the presence of dierent gut
Fig. 3. Inhibition of hydrolysis of H-Pro-Thr-Glu-Phe-Phe(NO2)Arg-Leu-OH by mite extracts using pepstatin A. Ten microlitres of soluble mite extract were combined with 0.9 ml of 0.1 M sodium formate buer (pH 3) containing a range of concentrations of pepstatin A for 30 min at 308C. This was followed by the addition of substrate and incubation at 308C for a further 2 h prior to the measurement of reduction in absorbance at 300 nm. Values shown are means of three replicates in a single experiment (2S.E.). The experiment was repeated using dierent extracts of each mite species with similar results.
symbionts in dierent populations of T. urticae. bGlucosidase activity was found in all of the mite extracts and may be responsible for hemicellulose digestion or the cleavage of carbohydrate moieties from glycoproteins dependent on the diet of each species [27]. N-Acetyl-b-glucosaminidase was also present in each species. This enzyme is involved in the moulting process in invertebrates and, as mixed stages of mites were used for the extractions, may be expected to be present. Trypsin activity was absent from all mite species, but A. siro extracts displayed a low chymotrypsin activity. Chymotrypsin-like enzymes constitute the group 6 allergens characterised from pyroglyphid dust mites [28] with which A. siro has allergenic crossreactivity [29]. Tetranychus urticae extracts are also responsible for occupational allergic disease in agricultural workers, but do not display antigenic crossreactivity with dust-mite extracts [30]. The acidic pH optimum for haemoglobin and the lack of hydrolysis above pH 6 suggests the absence, or very low activity, of detectable serine proteinases in the mite extracts. Only pepstatin A, a speci®c aspartic proteinase inhibitor, signi®cantly reduced haemoglobin hydrolysis by every mite extract, although E-64, a cysteine proteinase inhibitor, reduced the proteolysis by ca. 50% in A. siro and T. urticae extracts. Enhancement of haemoglobin hydrolysis in the presence of EDTA was also suggestive of cysteine proteinase activity in T. urticae. Overall, the dierences in the eects of dierent enzyme inhibitors on the hydrolysis of haemoglobin can be largely attributed to the relative amounts of aspartic and cysteine proteinases present in the extracts. Thus, hydrolysis of haemoglobin by P. ovis extract, which was low in cysteine proteinase activity, was not signi®cantly aected by a cysteine proteinase inhibitor. In contrast, haemoglobin hydrolysis in the remaining species was signi®cantly reduced by the presence of a cysteine proteinase inhibitor, and each of these species had signi®cantly higher cysteine proteinase activity than P. ovis. The cysteine proteinase substrate Z-Phe-Arg-pNA and the inhibitors used (Z-Phe-Ala-DMK and biotinyl-Phe-Ala-FMK) are speci®c to the plant enzyme papain and cathepsins B and L which are lysosomal exo- and endopeptidases, respectively [31]. Cysteine proteinases with sequence homology to cathepsin Blike molecules comprise the group 1 allergens of Dermatophagoides spp. [5], and their presence in faecal pellets suggests a digestive role. Aspartic proteinases were also present in each of the extracts, along with a leucine aminopeptidase. The presence of these enzymes alongside histological evidence from a variety of tick and mite species [32±36] supports their role in lysoso-
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
25
Fig. 4. Detection of mite cysteine proteinase activity using biotinyl-Phe-Ala-FMK. Mite extracts were pre-incubated with 50 mM sodium acetate (pH 5.0) containing 5 mM cysteine and 1 mM EDTA for 20 min at 378C, then split into two fractions. Z-Phe-Ala-DMK was then added to one fraction (+lanes) to a ®nal concentration of 1.5 mM and the same volume of methanol without inhibitor was added to the other fraction (ÿ lanes). After a further incubation, biotinyl-Phe-Ala-FMK in was added to both fractions to a ®nal concentration of 0.075 mM. Following incubation, samples were boiled in reducing loading buer and fractions containing 3.5 mg protein separated on a 12.5% polyacrylamide minigel prior to overnight transfer to nitrocellulose membrane. Subsequent detection of the enzyme:biotinylated inhibitor complexes and biotinylated molecular mass markers on the nitrocellulose membranes was performed as previously described [20]. Molecular mass markers, in kDa, are indicated on each blot.
mal, intracellular digestion of food in each of the species. This involves the internalisation of solid and liquid food items by pinocytosis and phagocytosis by specialised digestive cells in the gut, lysosomal proteol-
ysis by endopeptidases (cathepsins D and L) followed by intra- or extracellular digestion by cytosolic or membrane-bound exopeptidases (cathepsin B or membrane-bound aminopeptidase) [37].
Table 3 Cysteine proteinase activity in mite extracts Treatment
pH 3.5
D. gallinae P. ovis A. siro T. urticae a
4.5
Control
Methanola
E-64
Z-Phe-Ala-DMK
Control
Methanol
E-64
Z-Phe-Ala-DMK
2.0620.05b 0.0620.06 1.2220.04 0.6620.02
2.342 0.06 0 1.242 0.02 0.692 0.01
0.0720.07 0 0.0420.02 0.0420.02
0.0320.02 0 0 0
2.032 0.05 0.312 0.01 1.882 0.05 0.802 0.01
2.20 20.09 0.24 20.04 1.72 20.05 0.91 20.05
0 0 0.0520.01 0
0 0.05 20.04 0.02 20.01 0
Control reaction for Z-Phe-Ala-DMK inhibition containing methanol at a concentration of 4.75%. Figures shown are mean reaction rates (mOD units min ÿ 1) 2S.E.M. (n = 3).
b
26
A.J. Nisbet, P.F. Billingsley / International Journal for Parasitology 30 (2000) 19±27
The cysteine proteinases detected may also be responsible for inducing allergic-type responses; Der p 1, the house-dust-mite cysteine proteinase, possesses several properties related to its enzymatic activities which make it a potent allergen [38, 39]. Determination of the mechanistic class of proteolytic allergens from mite species is important, because desensitisation to the allergen may be achieved by exposure of the host or atopic individual to enzyme:inhibitor complexes [40].
Acknowledgements We are very grateful to Dr M. Taylor and Mr B. Groves, CVL, Surrey, UK; Dr M. Solomon, HRI, Kent, UK; Dr J.A.T. Woodford, SCRI, Dundee, UK; and Mr T. Pennycott, SAC, Auchincruive, UK for provision of mites; Professor J.H. McKerrow, University of California, San Francisco, CA, USA and Dr A.J. Walker, University of Manchester, UK for advice on cysteine proteinase detection; and to the Ministry of Agriculture, Fisheries and Food, UK for funding this research under MAFF Open Contract: CSA 3402.
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