Camp. Biochem. Phpiol. Printed in Great Britain
Vol. 103C, No. 1, pp. 129-l 34, 1992
0306~4492/92 $5.00 + 0.00 :o 1992 Pergamon Press Ltd
CHARACTERIZATION OF CHOLINESTERASES FROM THE PARASITIC NEMATODE TRICHINELLA SPIRALIS THEO DEVOS
Department
of Zoology,
University
and
TERRY
A. DICK*
of Manitoba, Winnipeg, Mb. Canada, Fax: 204-269-7431
R3T 2N2. Tel.: 204-474-9896;
(Received 24 January 1992; accepted 6 March 1992) Abstract-l. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors. 2. The 5.3 and 13s forms identified in Trichinella extracts are also found in C. e/egans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident. 3. The level of cholinesterases in excretory/secretory products is low. 4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.
S. dentatus C. eleguns,
had a similar size distribution to that of including the 7s form (Kolson and Russell, 1985). Western blot analysis also revealed multiple molecular forms of CHE in somatic extracts of N. americanus (Pritchard et al., 1991) while the antigenic CHE of the digenean parasite Schistosoma mansoni was a 7.5s molecule (Tarrab-Hazdai et al., 1984). From the above it is evident that comparative information on nematode CHE activity and characterization of the antigenic forms of these enzymes from parasites is limited. To our knowledge the CHE of Trichinellu, indeed for any aphasmidian nematode, have not been characterized. Consequently, this study partially characterized the cholinesterases of the porcine isolate of T. spiralis by examining the size distribution, inhibitor profiles and kinetics of the different molecular forms. We have also determined that anti-CHE antibodies were not present in mice infected with
INTRODUCTION
Cholinesterase (CHE) enzymes from nematodes occur in multiple molecular forms (Ogilvie et al., 1973; Johnson and Russell, 1983), vary in kinetic properties and interaction with inhibitors (Johnson and Russell, 1983), are secreted at high levels from some species (Ogilvie et al., 1973; Rhoads, 1981) and are antigenic (Ogilvie et al., 1973; Rhoads, 1981; Rathuar et al., 1987; Pritchard et al., 1991). Despite the potential antigenicity of CHE and its role in the nematode neuromuscular system, CHE has been characterized in few nematode species. Characterization of the CHEs of parasitic nematodes has focused on secretory forms (Ogilvie et al., 1973; Rhoads, 1981; Rathuar et al., 1987; Pritchard et al., 1991) and an association between the level of secretory CHE and an anti-CHE antibody response was established (Ogilvie et al., 1973; Pritchard et al., 1991). However, secretory CHE has been extensively characterized only in Stephanurus dentatus (Rhoads, 1981), and partially characterized in Brugia maluyi (Rathuar et al., 1987) and Necator americanus (Pritchard et al., 1991), all of which elicit anti-CHE antibodies following infection. Secretory CHE in S. dentatus occurred in a single form (MW 100 Kd) while in B. malayi and N. americanus multiple forms were detected ranging from 100-200 kDa in the former and 32-220 kDa in the latter. Nonsecreted CHE has been studied in the free living nematode C. eZegans in which there are several molecular forms. Three major classes (5.3, 7 and 13s) were characterized based on size, Km, inhibition by carbamates and activity with the substrate butyrylthiocholine (BTCI) and three separate genetic loci were identified (Johnson and Russell, 1983; Johnson et al., 1988). Multiple molecular CHE forms which differed in size (7S, 8S, 12S, and 13s) and sensitivity to carbamates were also described from two species of the plant parasitic nematode Meloidogyne (Chang and
Opperman,
1991). The nonsecreted
*Author to whom correspondence
should
CHEs
Trichinella. MATERIALS AND METHODS
All reagents were obtained from Sigma (St. Louis, MO). The Pl isolate of Trichinella was passaged in outbred Swiss
Webster mice (CRL:COBS CFW) as described previously (Dick et al., 1988). Larvae were isolated by sieving following a 2 hr 1% HCL Pepsin digest at 37°C. Larvae were washed 5 x with saline, 3 x with ddH,O and stored at -70°C. Larvae cultured for excretory/secretory products (E/S) were washed 5 x by settling thrbugh ste& PBS and cultured 24 hr at 37°C in sterile PBS at 4000 larvae/ml. E/S samples were concentrated by ultrafiltration using a YM-IO membrane (Amicon). The N2 strain of C. elegans was cultured using standard conditions (Sulston and Hodgkin, 1988) and extracted using the methods described below. Extraction Larvae were suspended I:3 (larvae: buffer) in 0.1 M phosphate buffer, pH 7.5 and homogenized by grinding on ice for 15 min in a ground glass homogenizer with a motor driven teflon pestle. The homogenate was centrifuged at 31,000 RPM for 1hr in a Ti50 rotor with an L8-55 ultracentrifuge (Beckman). The supernatant (Sl) was kept on ice, and the pellet was rehomogenized in the same buffer with a
of
be addressed. 129
THEO DEVOS and TERRY A. DICK
130
Kinematica tissue blender. The homogenate was centrifuged as above, the supernatant stored on ice (S2), and the pellet rehomogenized as above in an equal volume of the same buffer + 1% Triton X-100. The homogenate was centrifuged as above, the supernatant (S3) stored and the pellet rehomogenized as above in an equal volume of the Triton buffer. This final homogenate was centrifuged, the supernatant (S4) kept, and the pellet discarded. Sucrose
density centrifugation
Sucrose gradients were prepared in 0.1 M phosphate buffer, pH 7.5, including 1% Triton X-100 for those samples containing the detergent. 500~1 samples were loaded on 8 ml 5-20% linear gradients, and centrifuged for 18 hr at 4°C in an SW 41Ti Rotor on an L8-55 ultracentrifuge. B-galactosidase (16S), Catalase (1I .3S) and alkaline phosphatase (6.1 S) were included as molecular weight standards. Sedimentation coefficients were calculated by comparison with the molecular weight markers as described by Martin and Ames (1961). Enzyme
assays
Protein concentrations were determined by the BioRad protein microassay, using bovine serum albumin (BSA) as a protein standard. Cholinesterase activity was determined by a modification of the method of Ellman et al. (1961). In the standard assay, 900 ~1 assay solution (0.3 mM DTNB, 0.1 mM acetvlcholine iodide (ATCI). 0. I M ohosahate. DH 7.5) was added to IO ~1 of sample, and the changein optical density (O.D.) over time at 412 nm was assayed using a Milton Roy Spec 601. In the microassay, 200 ~1 of assay solution was added to 10 ~1 of sample in a 96 well microtitre plate, and the activity was calculated by comparing the O.D. at 410nm before and after incubation at 37°C using a BioTek EL308 Microplate Reader. BTCI was substituted for ATCI in the same assay solution to determine the rate of BTCI hydrolysis. B-galactosidase activity was measured by the change in O.D. at 410 nm in microtitre plates in which 200~1 assay solution (0.1 M phosphate, pH 7.5, 20mM o-nitrophenyl B-D-galactopyranoside, 10 mM NaCI, 1mM MgCI, and 0. I M B-mercaptoethanol) was added to 10 ~1 sample. Alkaline phosphatase activity was measured by the change in O.D. at 410 nm in microtitre plates in which 200 ~1 assay solution (0.1 M Tris/HCl, pH 8.5, 1 mM sodium-pnitrophenyl phosphate) was added to 10~1 of sample. Catalase activity was determined by measuring the change in O.D. over time at 240 nm in a Milton Roy Spec 601, following the addition of 1 ml assay solution (0.05 M phosphate buffer, pH 7.0, 0.02% H,Or) to 10~1 sample. Inhibition Samples were incubated with the specific inhibitors Eserine, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3one (BW284c51), and tetraisopropyl phosphoramide (Iso Ompa) at various concentrations. Inhibition was calculated as a percentage of the activity of positive controls, as measured in the microassay. Concanavalin
A precipitation
Con A was added at a 1 mg: I mg ratio to I mg/ml Trichinella extract and incubated 14 hr at 4°C. The Con A suspension was tested for activity in the standard cholinesterase assay, then centrifuged in a microfuge. The supernatant and pellet were tested for activity to determine interaction with Con A. Similar assays were performed in the presence of 0.5 M methyl-alphaID-mannopyranoside, which inhibits specific Con A binding by interaction with its binding site. Antigenicity Antibodies to electric eel cholinesterase (EECHE) (Sigma Type III) were raised in mice by intraperitonal injection. Mice were first injected with antigen (50 pg in 100 ~1
phosphate buffered saline) at a I: 1 ratio with complete Freund’s adjuvant, and were given a similar dose in incomplete Freund’s adjuvant 14 days later. Serum samples were obtained by tail bleeding mice. Antibody levels were assessed by an enzyme linked immunosorbent assay. Microtiter plates were coated with antigen at 1 rg/well for Trichinella or 0.25 pg/well for cholinesterase by incubation overnight at 4°C in carbonate buffer (pH 9.6). Plates were washed with PBS + 0.1% Tween 20 (PBST), and blocked with 3% BSA in PBS, for 2 hr at RT. Plates were washed as above, incubated for 1hr at RT with test serum, then washed, and incubated for 1 hr with the second antibody (antimouse IgG or IgA horse radish peroxidase conjugate, 1: 1000 dilution in PBS + 3% BSA) at RT, then washed with PBST and incubated for l/2 hr at RT with substrate (0.04% o-phenylenediamine, 0.012% H,O,, in 0.2M phosphate, 0.1 M citrate buffer, pH 5.0). The reaction was stopped by adding 2.5 N HCl, and the activity determined by measuring the O.D. at 490nm on a Biotek EL-308 microplate reader. Specific antibodies to CHE were also measured in a capture ELISA. Plates were coated with 0.5 pg/well sheep antimouse IgG or IgA in carbonate buffer O/N. Plates were washed with PBST, blocked for 2 hr with 0.5% gelatin in PBST, washed with PBST, and dilutions of serum and bile samples were added. Plates were incubated for 2 hr, washed with PBST, and dilutions of EECHE or TrichineNa antigen were added. Following overnight incubation plates were washed with PBST, CHE substrate was added and the reaction monitored as described above. Double diffusion experiments were carried out as described by Ouchterlony (1968). Precipitin reaction were detected by staining with Amido Schwarz and CHE activity in precipitates was determined by specific staining (Karnovsky and Roots, 1964). Heat denaturation studies were carried out as described by Michaeli et al. (1969). Briefly, Trichinella antigen and EECHE were incubated with serum or mucosal samples for 1hr then heated to 60°C for 30 min and CHE activity was determined in the microassay described above. Protection was expressed as a percentage of control CHE activity.
RESULTS Approximately 86% of the Trichinellu cholinesterase activity detected in crude homogenates was solubilized with a combination of phosphate and detergent (Triton X-100) buffers. The protein concentration and enzyme activity for a typical extraction are summarized in Table 1. Activity with the substrate BTCI was 60% that determined with ATCI. Extractions under high salt (1 M NaCl) conditions released no further activity. Low levels of ES CHE activity were also detected, as listed in Table 1. The phosphate and detergent extracts were fractionated by sucrose density gradient centrifugation (Fig. 1). Separation of the phosphate extract produced peaks of activity at 13s and 5.3S, of approximately equal magnitude, and a small peak at 7s. The majority of the CHE activity in the detergent extract was 13S, with a minor 5.3s peak, and no 7S activity (Fig. 1B). We were surprised at the absence of the 7S peak, since it has been reported from other nematodes (Johnson and Russell, 1983; Kolson et al., 1985). To confirm that the absence of this peak was not an artifact of our extraction procedure, C. eleguns homogenates were prepared and the CHE size distribution determined as described above. The profiles were similar to that described previously (Johnson and Russell, 1983) including the presence of the 7s peak.
Trichineliacholinesterases Table
I. Tvoical
extraction
of cholinesterase Protein
from
Specific activity*
Trichinella
Table
%
units
Rec.t
(mg)
Homogenate
82
1.0 x 10-Z
0.82
SI: 0.1 M PO,
60
5.0 x lo-’
0.294
9.9
8.0 x IO-’
0.080
9.7
20.7
1.2 x 10-f
0.248
30.2
S4: 0.1 M PO:
1% TX-100
8.7
I.0 x 10-S
0.087
-10.6 86.3
Excretory/secretory *Specific
1.0
activity:
1
unit = I pmole
3.5 x 10-3 ATCI
of cholinesterase
in extracts
BGAL
CC.T
+
z,+
0
tMolar
0.045
s
02
of activity
in
Phosphate extract
+
1 20
BGAL
0.4
0.6
ALP
CAT
0.6
1.0
Detergent extract
0 96 0.72 / 0.46
i /
024-
0.00 *._e-•i 0.0
( 0.2
i
l \. \
,.-D-0-.. l. %. C.-m l-W..-c._‘,
0.4 Gradient
0.6
0.6
1.0
L%)
Fig. 1. Sucrose density gradient profiles for phosphate and detergent extracts of Trichinella. Cholinesterase activity was measured with ATCI and is presented as the change in optical density at 410 nm for 10 ~1 aliquots of 250 ~1 fractions from 5520% sucrose gradients. Sedimentation coefficients were determined by comparison with standards: B-galactosidase 16s (BGAL), Catalase 1IS (CAT) and Alkaline phosphatase 6.1s (ALP), as indicated on the profiles. 010
r
0.06
-
0.06
-
004
-
r.
c z
ki ::kii
I
purified
iso-Ompa
I
I.5 x 10-7
5.5 x 10-4
I.5 x 10-u
4.8 x IO -’
iodide.
Km determined
from
plots. of
inhibitor
to untreated
producing
50%
inhibition
as
controls.
Centrifugation of the detergent extracts in the absence of detergent produced similar profiles (data not shown). In profiles produced for both the phosphate and detergent extracts using BTCI as substrate, the 5.3s peak was reduced in magnitude to a greater extent than the 13s peak. Activity of the 5.3s and 13s peaks were 30% and 60% respectively of the activity with ATCI as substrate. The kinetic constants of the 5.3s and 13s forms and interactions with specific cholinesterase inhibitors were examined. Both forms showed inhibition at high substrate concentrations (Fig. 2), typical of cholinesterases (Silver, 1974). The two forms differed slightly in Km (Table 2) and differences were also observed in the interaction with specific inhibitors (Table 2). The 5.3s form was more sensitive to Iso Ompa than was the 13s form, while the 13s form was more sensitive to both Eserine and BW284c51 than was the 5.3s form. The glycoprotein nature of the Trichinelfa cholinesterases was assessed by precipitation with Concanavalin A (Con A). Following overnight precipitation with 1 mg/ml Con A, and centrifugation, the supernatant activity of both the phosphate and detergent extracts was reduced by 88-89% and the activity was concentrated in the Con A precipitate. The precipitate of cholinesterase activity was inhibited by the inclusion of 0.5 M methyl-alpha-d-mannopyranoside in the reaction solution. Crude Trichinella extracts and partially purified cholinesterase positive samples were highly antigenic as revealed by ELISA with both serum and secretory antigens (Table 3). However, CHE specific antibodies were not detected by capture ELISA in serum and mucosal samples of infected mice. Furthermore, incubation of Trichinella extracts with anti-Trichinella serum did not precipitate the cholinesterase activity and did not protect CHE from heat denaturation. With both the phosphate and detergent extracts, as well as partially purified samples, the protection from heat denaturation was less than lo%, as compared to Table
O~O_o
with acetylcholine
concentration
compared
,*‘\
00
z
partially
BW284c5
1.5 x 10-g
determined
Eadie-Hofstee
0.4 F
of
Trichinda larvae
bydrolysed/min/mg
as a percentage
ALP
from
5.5 x 10-8
80pM
13s *Activity
crude home-genate.
0.5
parameters
fractions
Eserine
14OpM
5.3s
protein. tRecovery
inhibitory
lnhibitort
35.8
1% TX-100
Total
and
Km*
Fraction
S3: 0.1 M PO:
S2: 0.1 M PO,
Kinetic
cholinesterase
Total
Extraction
2.
131
3. Antibody
response
to
Trichinella (PI)
eel acetvlcholinesterase
-2
-Log ATCI Fig. 2. Specific activity 0(moles/min/mg) of the 5.3 and 13s CHE fractions determined for a range of substrate (ATCI) concentrations. Each point represents the mean specific activity _+standard deviation from triplicated assays.
extracts
and electric
IEECHE)
anti-PI
an&PI
Sample
IgG”
IgAb
IgG’
Sld
2.900
0.170
0.080
S3’
0.730
anti-EECHE
2.590
0.120 0.684’
0.010
5.3s 13s
2.490
1.119’
0.050
E/Ss
2.300
0.135
0.010
EECHE
0.010
0.040
‘l/l00
Dilution intestinal serum of
of lumen
from
intestinal
secretory
serum samples
EECHE lumen
antigen.
from from
infected
0.040
1.160 mice.
infected
bl/10
Dilution
of
mice. ‘l/100
Dilution
of
injected
mice. “See
samples
from
Table
infected
I. ‘l/2
mice.
Dilution
BExcretory/
132
THEO DEVOS and TERRY A. DICK
up to 100% protection of electric eel acetylcholinesterase with anti-eel cholinesterase antibodies. Similar results were observed with the immunoglobulins sIgA and IgG from the gall bladder and intestinal lumen of Trichinella infected mice. Cholinesterase staining of Trichinellu anti-Trichinella immunodiffusions revealed no positive bands. Immunological cross reactivity between Trichinella extracts and anti-EECHE antibodies was examined by ELISA (Table III) using serum recovered from mice injected with EECHE. While the ELISA titre was slightly greater than that of normal serum, the anti-eel serum did not produce cholinesterase positive bands in immunodiffusion with Trichinellu extracts, and did not protect Trichinella CHE from heat denaturation, indicating that Trichinellu CHE and EECHE are not immunologically cross reactive. DISCUSSION
The CHE of T. spiralis occurred in multiple molecular forms differing in size, activity with BTCI, kinetics and interaction with inhibitors. These characteristics were compared with values from other nematodes summarized in Table 4. The size distribution (5.3s and 13s) and predominance of the 13s form in the detergent extract was similar to CHE from C. elegans (Johnson and Russell, 1983). However, the 7s form which occurred in detergent extracts of C. elegans (Johnson and Russell, 1983) and verified in this study did not occur in Trichinella. This also differs from the distribution of CHE in Meloidogyne (Chang and Opperman, 1991) and S. dentatus in which the 7s form predominates in detergent extracts (Kolson et al., 1985) as well as other Nippostrongylus brasiliensis and Trichostrongylus colubriformes in which a single molecular form of 65-70 kd (5.3s like) was reported (Hogarth-Scott et al., 1973). This also differs from the CHE of the digenean S. mansoni from which a single antigenic 7.4s form was reported (Tarrab-Hazdai et al., 1984). Differences in the rate of reaction with ATCI and BTCI substrates are used to differentiate vertebrate CHE’s. For acetylcholinesterase in vertebrates hydrolysis of BTCI occurs at l/60 the rate of ATCI Table 4. Characteristics Species
Source”
Size 100 + 200 kDa
c. elegans
ES ext. ext.
Meloidogyne
ext.
M. apri N. americanus
ext.
B. malayi
S. denmrus T. spiralis
ES ext. ES ext.
ext.
13SA’ 7s B/C 5.3s A/B 7s B/C’ 8s A l2SA l3SA 30-220 kDa 30-220 kDa 5.8s 7s 13s 5.3s
hydrolysis (Johnson and Russell, 1983). The reported rates of nematode BTCI hydrolysis expressed as a percentage of activity with ATCI varied, at 6-30% for several gastrointestinal nematodes (Ogilvie et al., 1973), 12.5% for S. dentatus secretory CHE (Rhoads, 1981) 25-33% for B. maluyi secretory CHE (Rathuar et al., 1987) and 3% and 24% for secretory and somatic CHE respectively from N. americanus (Pritchard et al., 1991) (Table 4). The rates for T. spiralis were 30% and 60% for the 5.3s and 13s forms respectively. The 5.3s activity of Trichinellu was intermediate between C. elegans class A and class B, while the 13s activity was similar to the 13s class A CHE in C. elegans (Johnson and Russell, 1983). Despite the variability in activity described above, it is evident that hydrolysis of BTCI occurs at a higher relative rate for nematode CHEs compared with vertebrate CHEs. A characteristic of true CHE activity is the inhibition of enzyme activity at high substrate concentration (Silver, 1974). Inhibitory substrate concentrations reported previously were 10 mM for N. brasiliensis (Sanderson et al., 1967) 10mM for Metastrongylus upri, (Reiner et al., 1978) and 20 mM for S. dentatus (Rhoads, 1981). Both the 5.3 and 13s forms in Trichinella showed the typical bell shaped activity curve (Silver, 1974) and were inhibited at substrate concentrations beyond 0.5-I mM ATCI. The Km values for the 5.3s (140pM) and 13s (80 PM) CHE’s from Trichinellu were similar to that reported for M. upri (100 NM) (Reiner et al., 1978), but less than that reported for the secretroy CHE from S. dentatus (560 PM) (Rhoads, 1981). The Km values for C. elegans class A and class B cholinesterases were 10 and 80pM respectively (Johnson and Russell, 1983) and ranged from 24 to 100 PM in Meloidogyne but differences in the substrate used limit direct comparisons with C. elegans and Meloidogyne. The vertebrate acetyl and butyryl CHE’s can be distinguished by differences in the concentration of specific inhibitors required for a 50% reduction in enzyme activity (Silver, 1974). Differences were also observed in specific inhibition of the CHE classes from C. elegans, though the nematode CHE’s do not fit the vertebrate acetyl/butyryl CHE model (Johnson of nematode
Km (PM)
I2 671 I 6’ I S/80 32/8d 35 24 100 100
560 16’ 80 140
cholinesterases BTClb
Reference
33% 25% 70% 25%
Rathuar er nl., 1987 Rathuar er al., 1987 Johnson and Russell, 1983 Johnson and Russell, 1983 + Kolson and Russell, 1985 Johnson and Russell, 1983 Chang and Opperman, 1991 Chang and Opperman, 1991 Chang and Opperman, 1991 Chang and Opperman, 1991 Reiner er al., 1978 Pritchard ef crl., 1991 Pritchard er al., 1991 Rhoads, I98 I Kolson and Russell, 1985 This study This studv
64118%
6.1% 2.1% 24% 12.5% 60% 30%
“Source of sample: ES = excretory secretory product, ext. = worm extract bActivity with BTCI as a percentage of ATCI activity. ‘Class designations for C. &guns and Meloidogyne. dKm in nM for Class C cholinesterases.
Trichinella cholinesterases
and Russell, 1983). For example, inhibition of vertebrate butyrylcholinesterase with iso-Ompa occurs at a 100-1000 fold lower concentration of inhibitor than inhibition of the CHEs from C. eregans (Johnson and Russell, 1983). The inhibition of CHE activity by eserine, BW284c51 and iso-Ompa in Trichinella was generally similar to that observed for class A and B cholinesterases from C. elegans, with some minor differences. Among other nematodes, the 5’. dentatus secretory CHE was more resistant to both eserine and BW284c51 inhibition (Rhoads, 1981) than our results for T. spiralis CHEs, while CHEs from the plant parasitic nematodes Meloidogyne arenaria and M. incognita were more sensitive to eserine inhibition (Opperman and Chang, 1990). The precipitation of the Trichinella cholinesterases with Con A demonstrates the high mannose nature of the carbohydrates of this glycoprotein. This is similar to the cholinesterase in B. malayi which was fractionated by Con A chromatography (Rathuar et al., 1987). Similarly, vertebrate cholinesterase enzymes are also high mannose glycoproteins, as demonstrated by precipitation with lectins, of which Con A was most efficient (Canovas-Munoz and Vidal, 1989). Cross reactivity with antibodies raised against EECHE has been demonstrated in B. malayi (Rathuar et al., 1987), as well as in Schistosoma mansoni (Tarrab-Hazdai et al., 1984). In the latter study, this cross reactivity mediated a cytotoxic effect on schistosomulae. In contrast, no cross reactivity was detected between the secretory cholinesterases of several closely related nematode species (Rothwell et al., 1973). In this study, we did not detect cross reactivity between electric eel cholinesterase and crude or partially purified extracts of Trichinella, by ELISA or heat denaturation. Antigenicity of CHEs in infected hosts has been demonstrated for a number of nematodes (Ogilvie et al., 1973; Rhoads, 1981; Rathuar et al., 1987, Pritchard, 1991). In Trichinella we have found no evidence for antigenicity of CHE in infected mice by ELISA, precipitation, heat denaturation, or staining of double diffusion gels. Comparison of several gastrointestinal nematodes indicated a correlation between the level of cholinesterase secretion and antibody production (Ogilvie et al., 1973). While it is difficult to compare the secretory products because of the way in which activity was expressed, the Trichinefla secretory activity is comparatively low, which may account for the lack of antigenicity. The lack of antigenicity may also relate to differences in Trichinelfa CHEs, and in particular the lack of the 7s form. When comparing CHE characteristics among nematodes it became apparent that despite a number of publications, detailed information was available for few species (e.g. C. elegans and S. dentatus). Our understanding of the evolution of CHE, its role in nematode physiology, as well as the potential to exploit CHEs as antigens or drug targets would benefit from further comparative studies. To summarize, CHEs from Trichinefla have a number of characteristics in common with CHEs from other nematodes. They occur in multiple molecular forms (5.3s and 13s) which are differentially extracted in the presence of detergent. The relative hydrolysis of BTCI is similar to C. elegans CHEs, enzyme activity
133
is inhibited at high substrate concentrations, and the effects of specific inhibitors occur at similar concentrations. However, there are differences which include the absence of the 7S form from detergent extracts, differences in Km values, small differences in concentrations of inhibitors, variation in the level of CHE in excretory/secretory products (Ogilvie et al., 1973; Rhoads et al., 1981), and lack of antigenicity of CHE in mice infected with Trichinefla. The importance of multiple CHE forms is unclear since C. elegans mutants lacking different CHEs are phenotypically normal (Johnson et al., 1988). Also, CHE is coded for by a single gene in Drosophila melanogaster (Fournier et al., 1989) and limited forms occur in other insects (Belzunces et al., 1988). It is worth noting that Trichinella is placed in the class Aphasmida which is distant from the other nematodes discussed in this report. Perhaps the absence of the 7S form in Trichinella reflects selection against CHE redundancy in this class, similar to that described in insects. To determine whether this is a common feature of the aphasmids will require analysis of CHEs from other species in this group. Furthermore, if the degree of species variability among nematodes in the kinetics, secretion, genetics and antigenicity of the CHEs is an indicator for other enzymes, data from “model” nematode systems such as C. elegans should be interpreted with caution. Finally, from the limited data available it is evident that there are similarities among nematodes, insects and vertebrates, but cholinesterases from nematodes do not fit the vertebrate acetyl/butyryl cholinesterase model (Taylor, 1991). Acknowledgements-The
authors acknowledge a University of Manitoba Alumni Graduate Fellowship to T. deVos, and a Natural Sciences and Engineering Research Council of Canada operating grant to T. Dick.
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Canovas-Munoz M. D. and Vidal C. J. (I 989) Acetylcholinesterase from muscle microsomes: interaction with lectins. Trans. Biochem. Sot. 17, 677-679.
Chang S. and Opperman C. H. (1991) Characterization of acetylcholinesterase molecular forms of the root-knot nematode, Meloidogyne. Mol. Biochem. Parasitol. 49, 205-214.
Dick T. A., Dougherty D. A. and Wassom D. L. (1988) TrichineNa spiralis infections of inbred mice: Genetics of the host response following infection with different Trichineila isolates. J. Parasitol. 74, 665-669. Ellman G. L., Courtney K. D., Andres V. and Featherstone R. (1961) A new and rapid calorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7, 88-95.
Fournier D., Karch F., Bride J., Hall L. M. C., Berge J. and Spierer P. (1989) Drosophila melanogaster acetylcholinesterase gene. Structure, evolution and mutations. J. mol. Biol. 210, 15-22.
Hogath-Scott R. S.. Watt B. J., Ogilvie B. M. and Rothwell T. L. W. (1973) The molecular size of nematode acetylchoIine$terases and their separation from nematode allereens. my Int. J. Parasitol. 3. 735-741.
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THEODEVOSand TERRYA.
Johnson C. D. and Russell R. L. (1983) Multiple molecular forms of acetylcholinesterase in the nematode Caenorhabditis elegans. J. Neurochem. 41, 30-46. Johnson C. D., Rand J. B., Herman R. K., Stern B. D. and Russell R. L. (1988) The acetylcholinesterase genes of C. elegans. Identification of a third gene (ace-3) and mosaic mapping of a synthetic lethal phenotype. Neuron 1, 165-173. Karnovsky M. and Roots L. (1964) A “direct-coloring” thiocholine method for cholinesterases. J. Hisrochem. Cyfochem. 12, 219-221.
Kolson D. L. and Russell R. L. (1985) A novel class of acetylcholinesterases, revealed by mutations, in the nematode Caenorhabditis elegans. J. Neurogen. 2, 93-110.
Martin R. G. and Ames B. N. (1961) A method for determining the sedimentation bkhaviour of enzymes: application to protein mixtures. J. biol. Chem. 236, 1372-1379. Michaeli D., Pinto J., Beniamini E. and deBmen F. P. (1969) Immunoenzymology of acetylcholinesterase-I. Substrate specificity and heat stability of acetylcholinesterase and of acetylcholinesterase-antibody complexes. Immunochemisrry 6, 101-109.
Ogilvie B. M., Rothwell T. L. W., Bremner K. C., Schnitzerling H. J., Nolan J. and Keith R. K. (1973) Acetylcholinesterase secretion by parasitic nematodes-I. Evidence for secretion of the enzyme by a number of species. Int. J. Parasitol. 3, 589-597. Opperman C. H. and Chang S. (1990) Plant-parasitic nematode acetylcholinesterase inhibition by carbamate and organophosphate nematicides. J. Nemarol. 22, 481488.
Ouchterlony 0. (1968) Handbook of Immunod@ision and Immunoelectrophoresis. 215 pp. Ann Arbor, Michigan.
DICK
Pritchard D. I., Legget K. V., Rogan M. T., McKean P. G. and Brown A. (1991) Necafor americanus secretory cholinesterase and its purification from excretory-secretory products by affinity chromatography. Par. Immunol. 13, 187-199.
Rathuar S., Robertson B. D., Selkirk M. E. and Maizels R. M. (1987) Secretory acetylcholinesterase from Brugia malayi adult and microfilarial parasites. Mol. Biochem. Parasitol. 26, 257-265.
Reiner E., Skrinjaric-Spoljar M., Kralg M. and Krvavica S. (1978) Kinetic properties of the chohnesterase in Metastrongylus apri (Nematoda): substrate hydrolysis and reaction with organophosphorus compounds. Camp. Biochem. Physiol. 6OC, 155-l 57. Rhoads M. L. (1981) Cholinesterase in the parasitic nematode. Sieohanurus dentatus. J. biol. Chem. 256. 9316-9323. _ Rothwell T. L. W., Ogilvie B. M. and Love R. J. (1973) Acetylcholinesterase secretion by parasitic nematodesII. Trichostrongylus spp. Im. J. Parasitol. 3, 5999608. Sanderson B. E.- (1969) Acetylcholinesterase activity in Nippostrongylus brasiliensis (Nematoda). Camp. Biochem. Physiol. 29, 1207-1213.
Silver A. (1974) The Biology of Cholinesterases, 596 pp. North-Holland, New York. Sulston J. and Hodgkin J. (1988) Methods. In The Nematode Caenorhabditis elegans. Woods, E. W. ed. Cold Spring Harbor Laboratory Press, pp. 667. Tarrab-Hazdai R., LeviSchaffer F., Smolarsky M. and Arnon R. (1984) Acetylcholinesterase of Schistosoma mansoni: antigenic cross-reactivity with Elecfrophorus electricus and its functional implications. Eur. J. Immunol. 14, 205-209.
Taylor P. (1991) The cholinesterases. J. biol. Chem, 266, 4025-4028.
Vol. 103C, No. 1, pp. 129-l 34, 1992
0306~4492/92 $5.00 + 0.00 :o 1992 Pergamon Press Ltd
CHARACTERIZATION OF CHOLINESTERASES FROM THE PARASITIC NEMATODE TRICHINELLA SPIRALIS THEO DEVOS
Department
of Zoology,
University
and
TERRY
A. DICK*
of Manitoba, Winnipeg, Mb. Canada, Fax: 204-269-7431
R3T 2N2. Tel.: 204-474-9896;
(Received 24 January 1992; accepted 6 March 1992) Abstract-l. Trichinella cholinesterases occur in multiple molecular forms which differ in size, kinetics, activity with butyrylthiocholine, and effects of inhibitors. 2. The 5.3 and 13s forms identified in Trichinella extracts are also found in C. e/egans and other nematodes but the 7S form which occurs in other nematodes was absent from Trichinella detergent extracts. Differences in kinetic and inhibition properties among nematode species were also evident. 3. The level of cholinesterases in excretory/secretory products is low. 4. Trichinella cholinesterases did not elicit a detectable antibody response in mice.
S. dentatus C. eleguns,
had a similar size distribution to that of including the 7s form (Kolson and Russell, 1985). Western blot analysis also revealed multiple molecular forms of CHE in somatic extracts of N. americanus (Pritchard et al., 1991) while the antigenic CHE of the digenean parasite Schistosoma mansoni was a 7.5s molecule (Tarrab-Hazdai et al., 1984). From the above it is evident that comparative information on nematode CHE activity and characterization of the antigenic forms of these enzymes from parasites is limited. To our knowledge the CHE of Trichinellu, indeed for any aphasmidian nematode, have not been characterized. Consequently, this study partially characterized the cholinesterases of the porcine isolate of T. spiralis by examining the size distribution, inhibitor profiles and kinetics of the different molecular forms. We have also determined that anti-CHE antibodies were not present in mice infected with
INTRODUCTION
Cholinesterase (CHE) enzymes from nematodes occur in multiple molecular forms (Ogilvie et al., 1973; Johnson and Russell, 1983), vary in kinetic properties and interaction with inhibitors (Johnson and Russell, 1983), are secreted at high levels from some species (Ogilvie et al., 1973; Rhoads, 1981) and are antigenic (Ogilvie et al., 1973; Rhoads, 1981; Rathuar et al., 1987; Pritchard et al., 1991). Despite the potential antigenicity of CHE and its role in the nematode neuromuscular system, CHE has been characterized in few nematode species. Characterization of the CHEs of parasitic nematodes has focused on secretory forms (Ogilvie et al., 1973; Rhoads, 1981; Rathuar et al., 1987; Pritchard et al., 1991) and an association between the level of secretory CHE and an anti-CHE antibody response was established (Ogilvie et al., 1973; Pritchard et al., 1991). However, secretory CHE has been extensively characterized only in Stephanurus dentatus (Rhoads, 1981), and partially characterized in Brugia maluyi (Rathuar et al., 1987) and Necator americanus (Pritchard et al., 1991), all of which elicit anti-CHE antibodies following infection. Secretory CHE in S. dentatus occurred in a single form (MW 100 Kd) while in B. malayi and N. americanus multiple forms were detected ranging from 100-200 kDa in the former and 32-220 kDa in the latter. Nonsecreted CHE has been studied in the free living nematode C. eZegans in which there are several molecular forms. Three major classes (5.3, 7 and 13s) were characterized based on size, Km, inhibition by carbamates and activity with the substrate butyrylthiocholine (BTCI) and three separate genetic loci were identified (Johnson and Russell, 1983; Johnson et al., 1988). Multiple molecular CHE forms which differed in size (7S, 8S, 12S, and 13s) and sensitivity to carbamates were also described from two species of the plant parasitic nematode Meloidogyne (Chang and
Opperman,
1991). The nonsecreted
*Author to whom correspondence
should
CHEs
Trichinella. MATERIALS AND METHODS
All reagents were obtained from Sigma (St. Louis, MO). The Pl isolate of Trichinella was passaged in outbred Swiss
Webster mice (CRL:COBS CFW) as described previously (Dick et al., 1988). Larvae were isolated by sieving following a 2 hr 1% HCL Pepsin digest at 37°C. Larvae were washed 5 x with saline, 3 x with ddH,O and stored at -70°C. Larvae cultured for excretory/secretory products (E/S) were washed 5 x by settling thrbugh ste& PBS and cultured 24 hr at 37°C in sterile PBS at 4000 larvae/ml. E/S samples were concentrated by ultrafiltration using a YM-IO membrane (Amicon). The N2 strain of C. elegans was cultured using standard conditions (Sulston and Hodgkin, 1988) and extracted using the methods described below. Extraction Larvae were suspended I:3 (larvae: buffer) in 0.1 M phosphate buffer, pH 7.5 and homogenized by grinding on ice for 15 min in a ground glass homogenizer with a motor driven teflon pestle. The homogenate was centrifuged at 31,000 RPM for 1hr in a Ti50 rotor with an L8-55 ultracentrifuge (Beckman). The supernatant (Sl) was kept on ice, and the pellet was rehomogenized in the same buffer with a
of
be addressed. 129
THEO DEVOS and TERRY A. DICK
130
Kinematica tissue blender. The homogenate was centrifuged as above, the supernatant stored on ice (S2), and the pellet rehomogenized as above in an equal volume of the same buffer + 1% Triton X-100. The homogenate was centrifuged as above, the supernatant (S3) stored and the pellet rehomogenized as above in an equal volume of the Triton buffer. This final homogenate was centrifuged, the supernatant (S4) kept, and the pellet discarded. Sucrose
density centrifugation
Sucrose gradients were prepared in 0.1 M phosphate buffer, pH 7.5, including 1% Triton X-100 for those samples containing the detergent. 500~1 samples were loaded on 8 ml 5-20% linear gradients, and centrifuged for 18 hr at 4°C in an SW 41Ti Rotor on an L8-55 ultracentrifuge. B-galactosidase (16S), Catalase (1I .3S) and alkaline phosphatase (6.1 S) were included as molecular weight standards. Sedimentation coefficients were calculated by comparison with the molecular weight markers as described by Martin and Ames (1961). Enzyme
assays
Protein concentrations were determined by the BioRad protein microassay, using bovine serum albumin (BSA) as a protein standard. Cholinesterase activity was determined by a modification of the method of Ellman et al. (1961). In the standard assay, 900 ~1 assay solution (0.3 mM DTNB, 0.1 mM acetvlcholine iodide (ATCI). 0. I M ohosahate. DH 7.5) was added to IO ~1 of sample, and the changein optical density (O.D.) over time at 412 nm was assayed using a Milton Roy Spec 601. In the microassay, 200 ~1 of assay solution was added to 10 ~1 of sample in a 96 well microtitre plate, and the activity was calculated by comparing the O.D. at 410nm before and after incubation at 37°C using a BioTek EL308 Microplate Reader. BTCI was substituted for ATCI in the same assay solution to determine the rate of BTCI hydrolysis. B-galactosidase activity was measured by the change in O.D. at 410 nm in microtitre plates in which 200~1 assay solution (0.1 M phosphate, pH 7.5, 20mM o-nitrophenyl B-D-galactopyranoside, 10 mM NaCI, 1mM MgCI, and 0. I M B-mercaptoethanol) was added to 10 ~1 sample. Alkaline phosphatase activity was measured by the change in O.D. at 410 nm in microtitre plates in which 200 ~1 assay solution (0.1 M Tris/HCl, pH 8.5, 1 mM sodium-pnitrophenyl phosphate) was added to 10~1 of sample. Catalase activity was determined by measuring the change in O.D. over time at 240 nm in a Milton Roy Spec 601, following the addition of 1 ml assay solution (0.05 M phosphate buffer, pH 7.0, 0.02% H,Or) to 10~1 sample. Inhibition Samples were incubated with the specific inhibitors Eserine, 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3one (BW284c51), and tetraisopropyl phosphoramide (Iso Ompa) at various concentrations. Inhibition was calculated as a percentage of the activity of positive controls, as measured in the microassay. Concanavalin
A precipitation
Con A was added at a 1 mg: I mg ratio to I mg/ml Trichinella extract and incubated 14 hr at 4°C. The Con A suspension was tested for activity in the standard cholinesterase assay, then centrifuged in a microfuge. The supernatant and pellet were tested for activity to determine interaction with Con A. Similar assays were performed in the presence of 0.5 M methyl-alphaID-mannopyranoside, which inhibits specific Con A binding by interaction with its binding site. Antigenicity Antibodies to electric eel cholinesterase (EECHE) (Sigma Type III) were raised in mice by intraperitonal injection. Mice were first injected with antigen (50 pg in 100 ~1
phosphate buffered saline) at a I: 1 ratio with complete Freund’s adjuvant, and were given a similar dose in incomplete Freund’s adjuvant 14 days later. Serum samples were obtained by tail bleeding mice. Antibody levels were assessed by an enzyme linked immunosorbent assay. Microtiter plates were coated with antigen at 1 rg/well for Trichinella or 0.25 pg/well for cholinesterase by incubation overnight at 4°C in carbonate buffer (pH 9.6). Plates were washed with PBS + 0.1% Tween 20 (PBST), and blocked with 3% BSA in PBS, for 2 hr at RT. Plates were washed as above, incubated for 1hr at RT with test serum, then washed, and incubated for 1 hr with the second antibody (antimouse IgG or IgA horse radish peroxidase conjugate, 1: 1000 dilution in PBS + 3% BSA) at RT, then washed with PBST and incubated for l/2 hr at RT with substrate (0.04% o-phenylenediamine, 0.012% H,O,, in 0.2M phosphate, 0.1 M citrate buffer, pH 5.0). The reaction was stopped by adding 2.5 N HCl, and the activity determined by measuring the O.D. at 490nm on a Biotek EL-308 microplate reader. Specific antibodies to CHE were also measured in a capture ELISA. Plates were coated with 0.5 pg/well sheep antimouse IgG or IgA in carbonate buffer O/N. Plates were washed with PBST, blocked for 2 hr with 0.5% gelatin in PBST, washed with PBST, and dilutions of serum and bile samples were added. Plates were incubated for 2 hr, washed with PBST, and dilutions of EECHE or TrichineNa antigen were added. Following overnight incubation plates were washed with PBST, CHE substrate was added and the reaction monitored as described above. Double diffusion experiments were carried out as described by Ouchterlony (1968). Precipitin reaction were detected by staining with Amido Schwarz and CHE activity in precipitates was determined by specific staining (Karnovsky and Roots, 1964). Heat denaturation studies were carried out as described by Michaeli et al. (1969). Briefly, Trichinella antigen and EECHE were incubated with serum or mucosal samples for 1hr then heated to 60°C for 30 min and CHE activity was determined in the microassay described above. Protection was expressed as a percentage of control CHE activity.
RESULTS Approximately 86% of the Trichinellu cholinesterase activity detected in crude homogenates was solubilized with a combination of phosphate and detergent (Triton X-100) buffers. The protein concentration and enzyme activity for a typical extraction are summarized in Table 1. Activity with the substrate BTCI was 60% that determined with ATCI. Extractions under high salt (1 M NaCl) conditions released no further activity. Low levels of ES CHE activity were also detected, as listed in Table 1. The phosphate and detergent extracts were fractionated by sucrose density gradient centrifugation (Fig. 1). Separation of the phosphate extract produced peaks of activity at 13s and 5.3S, of approximately equal magnitude, and a small peak at 7s. The majority of the CHE activity in the detergent extract was 13S, with a minor 5.3s peak, and no 7S activity (Fig. 1B). We were surprised at the absence of the 7S peak, since it has been reported from other nematodes (Johnson and Russell, 1983; Kolson et al., 1985). To confirm that the absence of this peak was not an artifact of our extraction procedure, C. eleguns homogenates were prepared and the CHE size distribution determined as described above. The profiles were similar to that described previously (Johnson and Russell, 1983) including the presence of the 7s peak.
Trichineliacholinesterases Table
I. Tvoical
extraction
of cholinesterase Protein
from
Specific activity*
Trichinella
Table
%
units
Rec.t
(mg)
Homogenate
82
1.0 x 10-Z
0.82
SI: 0.1 M PO,
60
5.0 x lo-’
0.294
9.9
8.0 x IO-’
0.080
9.7
20.7
1.2 x 10-f
0.248
30.2
S4: 0.1 M PO:
1% TX-100
8.7
I.0 x 10-S
0.087
-10.6 86.3
Excretory/secretory *Specific
1.0
activity:
1
unit = I pmole
3.5 x 10-3 ATCI
of cholinesterase
in extracts
BGAL
CC.T
+
z,+
0
tMolar
0.045
s
02
of activity
in
Phosphate extract
+
1 20
BGAL
0.4
0.6
ALP
CAT
0.6
1.0
Detergent extract
0 96 0.72 / 0.46
i /
024-
0.00 *._e-•i 0.0
( 0.2
i
l \. \
,.-D-0-.. l. %. C.-m l-W..-c._‘,
0.4 Gradient
0.6
0.6
1.0
L%)
Fig. 1. Sucrose density gradient profiles for phosphate and detergent extracts of Trichinella. Cholinesterase activity was measured with ATCI and is presented as the change in optical density at 410 nm for 10 ~1 aliquots of 250 ~1 fractions from 5520% sucrose gradients. Sedimentation coefficients were determined by comparison with standards: B-galactosidase 16s (BGAL), Catalase 1IS (CAT) and Alkaline phosphatase 6.1s (ALP), as indicated on the profiles. 010
r
0.06
-
0.06
-
004
-
r.
c z
ki ::kii
I
purified
iso-Ompa
I
I.5 x 10-7
5.5 x 10-4
I.5 x 10-u
4.8 x IO -’
iodide.
Km determined
from
plots. of
inhibitor
to untreated
producing
50%
inhibition
as
controls.
Centrifugation of the detergent extracts in the absence of detergent produced similar profiles (data not shown). In profiles produced for both the phosphate and detergent extracts using BTCI as substrate, the 5.3s peak was reduced in magnitude to a greater extent than the 13s peak. Activity of the 5.3s and 13s peaks were 30% and 60% respectively of the activity with ATCI as substrate. The kinetic constants of the 5.3s and 13s forms and interactions with specific cholinesterase inhibitors were examined. Both forms showed inhibition at high substrate concentrations (Fig. 2), typical of cholinesterases (Silver, 1974). The two forms differed slightly in Km (Table 2) and differences were also observed in the interaction with specific inhibitors (Table 2). The 5.3s form was more sensitive to Iso Ompa than was the 13s form, while the 13s form was more sensitive to both Eserine and BW284c51 than was the 5.3s form. The glycoprotein nature of the Trichinelfa cholinesterases was assessed by precipitation with Concanavalin A (Con A). Following overnight precipitation with 1 mg/ml Con A, and centrifugation, the supernatant activity of both the phosphate and detergent extracts was reduced by 88-89% and the activity was concentrated in the Con A precipitate. The precipitate of cholinesterase activity was inhibited by the inclusion of 0.5 M methyl-alpha-d-mannopyranoside in the reaction solution. Crude Trichinella extracts and partially purified cholinesterase positive samples were highly antigenic as revealed by ELISA with both serum and secretory antigens (Table 3). However, CHE specific antibodies were not detected by capture ELISA in serum and mucosal samples of infected mice. Furthermore, incubation of Trichinella extracts with anti-Trichinella serum did not precipitate the cholinesterase activity and did not protect CHE from heat denaturation. With both the phosphate and detergent extracts, as well as partially purified samples, the protection from heat denaturation was less than lo%, as compared to Table
O~O_o
with acetylcholine
concentration
compared
,*‘\
00
z
partially
BW284c5
1.5 x 10-g
determined
Eadie-Hofstee
0.4 F
of
Trichinda larvae
bydrolysed/min/mg
as a percentage
ALP
from
5.5 x 10-8
80pM
13s *Activity
crude home-genate.
0.5
parameters
fractions
Eserine
14OpM
5.3s
protein. tRecovery
inhibitory
lnhibitort
35.8
1% TX-100
Total
and
Km*
Fraction
S3: 0.1 M PO:
S2: 0.1 M PO,
Kinetic
cholinesterase
Total
Extraction
2.
131
3. Antibody
response
to
Trichinella (PI)
eel acetvlcholinesterase
-2
-Log ATCI Fig. 2. Specific activity 0(moles/min/mg) of the 5.3 and 13s CHE fractions determined for a range of substrate (ATCI) concentrations. Each point represents the mean specific activity _+standard deviation from triplicated assays.
extracts
and electric
IEECHE)
anti-PI
an&PI
Sample
IgG”
IgAb
IgG’
Sld
2.900
0.170
0.080
S3’
0.730
anti-EECHE
2.590
0.120 0.684’
0.010
5.3s 13s
2.490
1.119’
0.050
E/Ss
2.300
0.135
0.010
EECHE
0.010
0.040
‘l/l00
Dilution intestinal serum of
of lumen
from
intestinal
secretory
serum samples
EECHE lumen
antigen.
from from
infected
0.040
1.160 mice.
infected
bl/10
Dilution
of
mice. ‘l/100
Dilution
of
injected
mice. “See
samples
from
Table
infected
I. ‘l/2
mice.
Dilution
BExcretory/
132
THEO DEVOS and TERRY A. DICK
up to 100% protection of electric eel acetylcholinesterase with anti-eel cholinesterase antibodies. Similar results were observed with the immunoglobulins sIgA and IgG from the gall bladder and intestinal lumen of Trichinella infected mice. Cholinesterase staining of Trichinellu anti-Trichinella immunodiffusions revealed no positive bands. Immunological cross reactivity between Trichinella extracts and anti-EECHE antibodies was examined by ELISA (Table III) using serum recovered from mice injected with EECHE. While the ELISA titre was slightly greater than that of normal serum, the anti-eel serum did not produce cholinesterase positive bands in immunodiffusion with Trichinellu extracts, and did not protect Trichinella CHE from heat denaturation, indicating that Trichinellu CHE and EECHE are not immunologically cross reactive. DISCUSSION
The CHE of T. spiralis occurred in multiple molecular forms differing in size, activity with BTCI, kinetics and interaction with inhibitors. These characteristics were compared with values from other nematodes summarized in Table 4. The size distribution (5.3s and 13s) and predominance of the 13s form in the detergent extract was similar to CHE from C. elegans (Johnson and Russell, 1983). However, the 7s form which occurred in detergent extracts of C. elegans (Johnson and Russell, 1983) and verified in this study did not occur in Trichinella. This also differs from the distribution of CHE in Meloidogyne (Chang and Opperman, 1991) and S. dentatus in which the 7s form predominates in detergent extracts (Kolson et al., 1985) as well as other Nippostrongylus brasiliensis and Trichostrongylus colubriformes in which a single molecular form of 65-70 kd (5.3s like) was reported (Hogarth-Scott et al., 1973). This also differs from the CHE of the digenean S. mansoni from which a single antigenic 7.4s form was reported (Tarrab-Hazdai et al., 1984). Differences in the rate of reaction with ATCI and BTCI substrates are used to differentiate vertebrate CHE’s. For acetylcholinesterase in vertebrates hydrolysis of BTCI occurs at l/60 the rate of ATCI Table 4. Characteristics Species
Source”
Size 100 + 200 kDa
c. elegans
ES ext. ext.
Meloidogyne
ext.
M. apri N. americanus
ext.
B. malayi
S. denmrus T. spiralis
ES ext. ES ext.
ext.
13SA’ 7s B/C 5.3s A/B 7s B/C’ 8s A l2SA l3SA 30-220 kDa 30-220 kDa 5.8s 7s 13s 5.3s
hydrolysis (Johnson and Russell, 1983). The reported rates of nematode BTCI hydrolysis expressed as a percentage of activity with ATCI varied, at 6-30% for several gastrointestinal nematodes (Ogilvie et al., 1973), 12.5% for S. dentatus secretory CHE (Rhoads, 1981) 25-33% for B. maluyi secretory CHE (Rathuar et al., 1987) and 3% and 24% for secretory and somatic CHE respectively from N. americanus (Pritchard et al., 1991) (Table 4). The rates for T. spiralis were 30% and 60% for the 5.3s and 13s forms respectively. The 5.3s activity of Trichinellu was intermediate between C. elegans class A and class B, while the 13s activity was similar to the 13s class A CHE in C. elegans (Johnson and Russell, 1983). Despite the variability in activity described above, it is evident that hydrolysis of BTCI occurs at a higher relative rate for nematode CHEs compared with vertebrate CHEs. A characteristic of true CHE activity is the inhibition of enzyme activity at high substrate concentration (Silver, 1974). Inhibitory substrate concentrations reported previously were 10 mM for N. brasiliensis (Sanderson et al., 1967) 10mM for Metastrongylus upri, (Reiner et al., 1978) and 20 mM for S. dentatus (Rhoads, 1981). Both the 5.3 and 13s forms in Trichinella showed the typical bell shaped activity curve (Silver, 1974) and were inhibited at substrate concentrations beyond 0.5-I mM ATCI. The Km values for the 5.3s (140pM) and 13s (80 PM) CHE’s from Trichinellu were similar to that reported for M. upri (100 NM) (Reiner et al., 1978), but less than that reported for the secretroy CHE from S. dentatus (560 PM) (Rhoads, 1981). The Km values for C. elegans class A and class B cholinesterases were 10 and 80pM respectively (Johnson and Russell, 1983) and ranged from 24 to 100 PM in Meloidogyne but differences in the substrate used limit direct comparisons with C. elegans and Meloidogyne. The vertebrate acetyl and butyryl CHE’s can be distinguished by differences in the concentration of specific inhibitors required for a 50% reduction in enzyme activity (Silver, 1974). Differences were also observed in specific inhibition of the CHE classes from C. elegans, though the nematode CHE’s do not fit the vertebrate acetyl/butyryl CHE model (Johnson of nematode
Km (PM)
I2 671 I 6’ I S/80 32/8d 35 24 100 100
560 16’ 80 140
cholinesterases BTClb
Reference
33% 25% 70% 25%
Rathuar er nl., 1987 Rathuar er al., 1987 Johnson and Russell, 1983 Johnson and Russell, 1983 + Kolson and Russell, 1985 Johnson and Russell, 1983 Chang and Opperman, 1991 Chang and Opperman, 1991 Chang and Opperman, 1991 Chang and Opperman, 1991 Reiner er al., 1978 Pritchard ef crl., 1991 Pritchard er al., 1991 Rhoads, I98 I Kolson and Russell, 1985 This study This studv
64118%
6.1% 2.1% 24% 12.5% 60% 30%
“Source of sample: ES = excretory secretory product, ext. = worm extract bActivity with BTCI as a percentage of ATCI activity. ‘Class designations for C. &guns and Meloidogyne. dKm in nM for Class C cholinesterases.
Trichinella cholinesterases
and Russell, 1983). For example, inhibition of vertebrate butyrylcholinesterase with iso-Ompa occurs at a 100-1000 fold lower concentration of inhibitor than inhibition of the CHEs from C. eregans (Johnson and Russell, 1983). The inhibition of CHE activity by eserine, BW284c51 and iso-Ompa in Trichinella was generally similar to that observed for class A and B cholinesterases from C. elegans, with some minor differences. Among other nematodes, the 5’. dentatus secretory CHE was more resistant to both eserine and BW284c51 inhibition (Rhoads, 1981) than our results for T. spiralis CHEs, while CHEs from the plant parasitic nematodes Meloidogyne arenaria and M. incognita were more sensitive to eserine inhibition (Opperman and Chang, 1990). The precipitation of the Trichinella cholinesterases with Con A demonstrates the high mannose nature of the carbohydrates of this glycoprotein. This is similar to the cholinesterase in B. malayi which was fractionated by Con A chromatography (Rathuar et al., 1987). Similarly, vertebrate cholinesterase enzymes are also high mannose glycoproteins, as demonstrated by precipitation with lectins, of which Con A was most efficient (Canovas-Munoz and Vidal, 1989). Cross reactivity with antibodies raised against EECHE has been demonstrated in B. malayi (Rathuar et al., 1987), as well as in Schistosoma mansoni (Tarrab-Hazdai et al., 1984). In the latter study, this cross reactivity mediated a cytotoxic effect on schistosomulae. In contrast, no cross reactivity was detected between the secretory cholinesterases of several closely related nematode species (Rothwell et al., 1973). In this study, we did not detect cross reactivity between electric eel cholinesterase and crude or partially purified extracts of Trichinella, by ELISA or heat denaturation. Antigenicity of CHEs in infected hosts has been demonstrated for a number of nematodes (Ogilvie et al., 1973; Rhoads, 1981; Rathuar et al., 1987, Pritchard, 1991). In Trichinella we have found no evidence for antigenicity of CHE in infected mice by ELISA, precipitation, heat denaturation, or staining of double diffusion gels. Comparison of several gastrointestinal nematodes indicated a correlation between the level of cholinesterase secretion and antibody production (Ogilvie et al., 1973). While it is difficult to compare the secretory products because of the way in which activity was expressed, the Trichinefla secretory activity is comparatively low, which may account for the lack of antigenicity. The lack of antigenicity may also relate to differences in Trichinelfa CHEs, and in particular the lack of the 7s form. When comparing CHE characteristics among nematodes it became apparent that despite a number of publications, detailed information was available for few species (e.g. C. elegans and S. dentatus). Our understanding of the evolution of CHE, its role in nematode physiology, as well as the potential to exploit CHEs as antigens or drug targets would benefit from further comparative studies. To summarize, CHEs from Trichinefla have a number of characteristics in common with CHEs from other nematodes. They occur in multiple molecular forms (5.3s and 13s) which are differentially extracted in the presence of detergent. The relative hydrolysis of BTCI is similar to C. elegans CHEs, enzyme activity
133
is inhibited at high substrate concentrations, and the effects of specific inhibitors occur at similar concentrations. However, there are differences which include the absence of the 7S form from detergent extracts, differences in Km values, small differences in concentrations of inhibitors, variation in the level of CHE in excretory/secretory products (Ogilvie et al., 1973; Rhoads et al., 1981), and lack of antigenicity of CHE in mice infected with Trichinefla. The importance of multiple CHE forms is unclear since C. elegans mutants lacking different CHEs are phenotypically normal (Johnson et al., 1988). Also, CHE is coded for by a single gene in Drosophila melanogaster (Fournier et al., 1989) and limited forms occur in other insects (Belzunces et al., 1988). It is worth noting that Trichinella is placed in the class Aphasmida which is distant from the other nematodes discussed in this report. Perhaps the absence of the 7S form in Trichinella reflects selection against CHE redundancy in this class, similar to that described in insects. To determine whether this is a common feature of the aphasmids will require analysis of CHEs from other species in this group. Furthermore, if the degree of species variability among nematodes in the kinetics, secretion, genetics and antigenicity of the CHEs is an indicator for other enzymes, data from “model” nematode systems such as C. elegans should be interpreted with caution. Finally, from the limited data available it is evident that there are similarities among nematodes, insects and vertebrates, but cholinesterases from nematodes do not fit the vertebrate acetyl/butyryl cholinesterase model (Taylor, 1991). Acknowledgements-The
authors acknowledge a University of Manitoba Alumni Graduate Fellowship to T. deVos, and a Natural Sciences and Engineering Research Council of Canada operating grant to T. Dick.
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