Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides

Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides

Biosensors & Bioelectronics Vol. 13. No. 2, pp. 157-164, 1998 © 1998 Elsevier Science S.A. All rights reserved Printed in Great Britain PII: S0956-56...

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Biosensors & Bioelectronics Vol. 13. No. 2, pp. 157-164, 1998 © 1998 Elsevier Science S.A. All rights reserved Printed in Great Britain

PII: S0956-5663(97)00108-5

ELSEVIER

0956-5663/98/$19.00

Engineering sensitive acetylcholinesterase for detection of organophosphate and carbamate insecticides Frangois Villatte,* Veronique Marcel, Sandino Estrada-Mondaca & Didier Fournier Laboratoire d'entomologie appliqufe, Universit6 Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France (Received 5 June 1997; revised form received 25 September 1997; accepted 25 September 1997)

Abstract: High quantitites of various acetylcholinesterases can now be produced

following in vitro expression and it is possible to use them as biosensors to detect organophosphates and carbamates insecticides. In order to check the potentialities of acetylcholinesterase from various sources, we have studied enzyme from bovine erythrocyte, Electrophorus electricus, Drosophila melanogaster, Torpedo californica and Caenorhabditis elegans. It appears that insect acetylcholinesterase is more susceptible to a broad range of organophosphates and carbamates insecticides than the other tested enzymes. D. melanogaster is 8-fold more sensitive than E. electricus enzyme and this sensitivity has been increased to 12-fold by introducing a mutation at position 408. © 1998 Elsevier Science S.A. All rights reserved Keywords: biosensor, acetylcholinesterase, organophosphate, carbamate, insecticide

INTRODUCTION Intensive use of organophosphates and carbamates pesticides has led to many ecotoxicological problems. Their primary toxicity comes from an irreversible inhibition of a nervous system key enzyme, the acetylcholinesterase (ACHE, EC 3.1.1.7). Phosphorylation or carbamoylation of the serine of the active site blocks metabolization of the neurotransmitter acethylcholine, the post synaptic membrane remains depolarised and then

*To whom correspondence should be addressed. Fax: 33.05.61.55.69.10. E-maih [email protected]

the synaptic transmission does not take place. These compounds are used to control various agricultural pests: insects, acari and nematodes, but, as cholinergic transmission is well conserved, they are potentially toxic for all animals. Organophosphorus and carbamate compounds can be chemically detected by gaz chromatography-mass spectroscopy but this technique is expensive and time consuming. An alternative for their detection is to use AChE which is efficiently inhibited by these compounds. This strategy has been used to detect low levels of these contaminants in crop, water or soil samples and biosensors based on the use of immobilised enzyme have been described. Several entrapment or cross157

F. Villatte et al.

linking methods and signal detection techniques have been reported (Battacharya et al., 1981; Durand & Thomas, 1984; Razumas et al., 1981; Morelis & Coulet, 1990; Salamoun & Remien, 1993; Marty et al., 1992). Electrophorus electricus AChE is the most often used enzyme in biosensors, because this enzyme can be obtained in large amounts from the electric organ and is commercially available. However, some differences in insecticide sensitivity of AChE from various sources have been reported (Simeon & Reiner, 1973; Loewenstein et al., 1993; Rodriguez et al., 1997) suggesting that electrophorus AChE is not the best enzyme to be used in biosensors. However, other AChEs were not available in large enough quantity. This limitation has recently been overpassed by cloning the genes for coding for AChE from several species and by their in vitro expression in eucaryote systems. Here we compare three in vitro expressed AChEs to two commercially available enzymes for insecticide sensitivity. Furthermore, as in vitro expression allows mutated enzymes to be produced, modified in their primary sequence and consequently catalytic properties, we modified AChE to further increase its sensitivity.

MATERIALS AND METHODS

Enzymes Electrophorus electricus AChE (Ee.AChE) and bovine erythrocyte AChE (Be.AChE) were obtained from Sigma Chemical Co. AChE from Torpedo californica (Tc.AChE), from Drosophila melanogaster (Dm.AChE) and Caenorhabditis elegans (Ce.AChE) were produced in baculovirus infected cells as previously described (Gibney & Taylor, 1990; Chaabihi et al., 1994; Arpagaus et al., 1994).

Mutagenesis Mutagenesis was performed according to Kunkel (1985). Briefly, cDNA encoding Dm.AChE was transferred in a vector containing the origin of replication of phage FI allowing to produce single stranded DNA. This single strand was obtained in a u n g - strain (CJ236) replacing some thimidines by uracyls. Following hybridisation with a primer containing the mutation, the second strand 158

Biosensors & Bioelectronics

was in vitro synthesised using T4 DNA polymerase. The resulting plasmid was used to transform ung+ strain (TG1) where the parent DNA strand was not replicated. Colonies bearing the mutated plasmid were screened by PCR-based amplification of specific allele and the presence of the mutation was further verified by sequencing. The plasmid was co-transfected in Sf9 cells with baculovirus previously linearized (Kitts & Possee, 1993), and the virus was purified and titrated after ten days in culture.

Production and purification of recombinant proteins Production of AChEs was done in protein-free medium (ISFM (Sigma)/SF900 (BRL), 50/50) to avoid any copurification of bovine serum ACHE. The cell culture medium, containing typically 2 mg of AChE per liter, was centrifuged and loaded onto an affinity chromatography column using procainamide as ligand. The gel was washed with 25 mM Tris HC1, pH 9, 1 M NaC1, 0.1% CHAPS and then with 25mM Tris HC1 pH 9, 0.1% CHAPS. AChE was eluted with 25 mM Tris HC1 pH 9, M NaC1, 0-1% CHAPS, 10 mM procainamide, allowing to yield 80% of injected enzyme. AChE was then diafiltred onto a 50 kDa membrane (Amicon) with 25 mM phosphate buffer pH 7, lyophilised and stored at 4°C. Following these steps, samples appeared devoid of contaminants. Before experiments, enzyme was solved in 25 mM phosphate buffer pH 7 and stabilised with 0.5 mg/ml of bovine serum albumine. In these conditions, enzyme is stable for more than one week at room temperature.

Kinetics of thiocholine substrate hydrolysis Kinetic analysis were performed at 25°C in 25 mM phosphate buffer pH 7.0 with 10 100 ng of enzyme. Hydrolysis of thiocholine iodide ester was followed at 412nm using the method of Ellman et al. (1961), with substrate concentrations that ranged from 1/~M to 300mM. Substrate concentration profiles were fitted by non-linear regression, considering that there are two binding sites for the substrate: the esterasic site responsible for the metabolisation and the peripheral site responsible for the activation and inhibition (Cauet et al., 1987; Radic et al., 1993; Stojan et al., in preparation). AChE active site titration was performed with 7-(methylethoxyphosphinyloxy)l-

Biosensors&Bioelectronics

Acetylcholinesterasebiosensor

methyl-quinolinium iodide (MEPQ) according to Levy & Ashani (1986). The generally accepted inhibition mechanism of AChE by organophosphates and carbamates compounds has been described by Aldridge (1950):

RESULTS

where ki obs represents the ki calculated for one insecticide, ki Dm represents the ki calculated for the same insecticide with Dm AChE and n represents the number of tested insecticides.

Fig. 1 (A and B) shows the activity (kobs) versus substrate concentration for each enzyme. The maximal kob~ are varied: 3560 s- ~at 400/xM for Ee.AChE, 650 s i at 1 mM for Tc.AChE, 950 sat 1.5 mM for Dm.AChE, 750s -~ at 4 m M for Ce.AChE and 1100 s ~ at 900 ~M for Be.AChE. So it appears that, among the enzymes that were tested, Ee.AChE is the most active. The different AChEs presented various sensitivities to organophosphate and carbamate insecticides (Fig. 2): bimolecular reaction constants, kis, show that Dm.AChE is more sensitive than the other AChEs to 14 out of 19 insecticides, Tc.AChE is more sensitive to 2 compounds, Ee.AChE is more sensitive to 2 insecticides, BeAChE is more sensitive to 1 insecticide, and Ce.AChE is never the most sensitive to all tested molecules (Table 1). The average sensitivity of each enzyme is shown in Fig. 3. It appears that the Y408F mutant is more susceptible than the wild type to 11 compounds, and is more sensitive to 15 out of 19 insecticides, compared to other tested AChEs (Table 1), and its global sensitivity is higher than all other AChEs. The maximum activity of this mutant (kobs = 718 s -~ at 850/xM) is equal to 70% of the maximum activity of the wild type (Fig. 1B), reflecting that hydrolysis of ASCh is affected by the Y-F replacement. Modelisation of paraoxon ethyl located in the Tc.AChE active site shows that the phenyl ring of the insecticide (Fig. 2) may interact with the ring of the F330, which is the counterpart of Y408 in Dm.AChE, by aromatic interactions (Fig. 4). Indeed, the phenyl ring of the insecticide is located between F330 and W84, which is the main part of the socalled "anionic site" and implicated in substrate recognition. Replacement of this tyrosine in Dm.AChE by a phenylalanine may modify these interactions, leading to a higher stability of the insecticide molecule in the active site of the mutant Y408F than in the wild type active site.

Active site modelling

DISCUSSION

kl

k2

E+CX

=

ECX

k3 "*

EC+X

"

E+C

k-1

with e = enzyme, cX = carbamate or organophosphate, X = leaving group, Kd = k_~/k~, k 2 = carbamoylation or phosphorylation rate constant, k3 = decarbamylation or dephosphorylation rate constant, and ki = bimolecular rate constant ( = k2/Kd). For ki determination, insecticides were previously dissolved in acetone and then diluted in water. The maximum acetone concentration was 1%. Enzymes were incubated for various times with different insecticide concentrations, such as [I] always at least ten-fold superior to [E], in 25 mM phosphate buffer pH 7 at 25°C. Variation of the concentration of the free enzyme [E] with time was estimated by the Ellman method. This variation follows a pseudoorder, Ln[E]/[Eo] = -ki[I]t where t represents the time of incubation and I the inhibitor. All graphs obtained for each inhibitor were linear, whatever the variable, [I] or t, suggesting that reactivation (k3) remained negligible during the time that lasted the experiment (20 min). The average sensitivity to insecticides of each AChE have been calculated following the formula:

(~, {kiohs~) Llntkl~omOm} e

n

Which acetylcholinesterase for a biosensor? Active site modelling was performed on a Biosym Discover computer (Silicon graphic) using the Insight II software and atomic coordinates of Tc.AChE (Sussman 1991).

et al.,

The best suited enzyme to be used in a biosensor should be the most sensitive to insecticides to place the threshold of detection as low as poss159

F. Villatte et al.

Biosensors & Bioelectronics

I~1 4000

b 4000 *

300°

Ee.AChE

:

3000

I *DIn.AChE • Y408F 2000

1

0

0," ~ ' ' -

0

, 10

0

, 100

~

1000

, , 1000 10000 [ASCh (~M)]

i , , 0 100000 1000000 1

10

100

1000 [ASCh (~tM)]

10000

100000 1000000

Fig. 1. Concentration dependencies for acetylthiocholine hydrolysis by AChE from various sources (A) and by wild type and Y408F mutant Dm.AChE (B). TABLE 1

Insecticides Organophosphates Paraoxon ethyl Paraoxon methyl Malaoxon Heptenophos Dichlorvos Azametiphos Demethon S methyl Demethon S ethyl Profenfos Carbamates Aldicarbe Triazamate Carbaryl Pirimicarb Carbofuran Oxamyl Methomyl Propoxur Butocarboxim Thiofanox

Bimolecular rate constants (M-~.min ') of various AChEs to insecticides

Dm.AChE

1.4 4.2 2.6 1.3 5.0

10+6 10+5 10+6 10 +5 10 +5

Tc.AChE

2.6 10+4 4.0 10+4 2.7 10+4

5.8 2.1 3.0 3.1 1.9 3.5 1.4 3.1 3.3

5.5 10+3

7.6 10 +3

1.3 10 +7

3.3 5.3 1.9 2.3 4.9

3.3 10 +7

1.7 2.4 5.1 8.8 9.0 8.3 4.6

10+5 10+5 10+6 10+5 10+4 10+5 10+2

8.8 10+4

10+4 10 ÷5 10 +5 10+4 10 +3 10+5 10+3 10 +2 10 +3

Be.AChE

Ee.AChE

Dm.Y408F

2.7 10 +4

2.8 10+5 2.2 10+5 2.9 10+6

2.9 10 ÷5

3.2 10+6 1.5 10+6

1.1 10+5

8.7 10 +6

10+2 10 +4 10 +5 10 +2 10 +3

4.9 10+4

2.0 10 +3

< 10+3 2.2 10+5 1.0 10+3

< 10 +3 2.9 10+5 1.0 10+3 < 1 0 +2

6.6 10+5 3.5 10+5

1.9 10 +3

1.3 10+4

< 1 0 +3

9.0 10 +3

2.4 10 +7

9.7 10+3 1.6 10 +4

2.6 3.6 1.2 8.0 1.2

3.2 10 +3

7.5 10 +4

10+6 10+4 10+4 10+6 ]0 +5

1.4 10+3 4.4 10+4 9.1 10 +3

5.3 10+3 1.8 10+5 3.8 10+3

1.3 10+4 3.2 10 +5 6.6 10+4

4.3 10 +3

8.7 10 +3

2.2 10 +3 2.9 10 +6

7.2 10 +4

2.9 10+4

6.4 10 +4

6.0 10+4 1.6 10+2 8.4 10+3

1.5 10+3 3.6 10+5

ible. It a p p e a r s h e r e t h a t t h e A C h E w i t h t h e h i g h e s t s e n s i t i v i t y to i n s e c t i c i d e s is t h e i n s e c t enzyme: bimolecular reaction constants of D m . A C h E are h i g h e r f o r 15 o u t o f t h e 19 t e s t e d i n s e c t i c i d e s . S o m e o f t h e m , s u c h as p a r a o x o n e t h y l o r m e t h y l are l a r g e l y u s e d i n F r a n c e . T h e a v e r a g e s e n s i t i v i t y i n c r e a s e f r o m E e . A C h E to D m . A C h E is 8 - f o l d a n d this d i f f e r e n c e in s e n s i tivity reaches several thousand-fold for some 160

Ce.AChE

6.3 10+5 8.9 10+4

3.3 1.2 7.2 5.9 3.5

10+5 10 +5 10+4 10+4 10+2

7.7 10 +4

4.2 2.1 1.5 7.6

10 +5 10 +5 10 +5 10+2

2.8 10 +5

2.5 10 +7

1.7 10+4 2.4 10 +4

7.1 10+3 2.0 2.4 8.7 1.3 8.2 1.1

10 +6 10 +5 10+6 10+6 10+4 10+6

1.1 10 +3 8.5 10+4

c o m p o u n d s s u c h as d i c h l o r v o s . T h i s p h e n o m e n o n could be explained by the fact that tested comp o u n d s h a v e b e e n c h o s e n to b e a c t i v e a g a i n s t insects and not against vertebrates. If the toxicological specificity against insects originates from s e v e r a l f a c t o r s s u c h as p e n e t r a t i o n , m e t a b o l i z a t i o n o r a c t i v a t i o n o f t h e i n s e c t i c i d e , it a l s o d e p e n d s on target protein sensitivity. E e A C h E is t h e m o r e e f f i c i e n t o f all s t u d i e d

I

Acetylcholinesterase biosensor

Biosensors & Bioelectronics 0

NO2 ~OI~(OCI-L2CH3) 2

0

o

II

OCNHCH3

II

CI-13CHzS CH2CH2SP(OCH2CI-L3)2 demethon S ethyl

paraoxon ethyl

o

o

NO2--~--olJ(oc%) 2

Br ~ ' ~ O P I I

OCH2CH3 SCH2CH2CHa

CI p a r a o ~ n methyl

~°%.,c% ~ c~ ca rbofuran o II

(CI-13)2 NC OCI=NOCNHCH 3

profenofos

SCH3 oxam~t

o o II II CH CH OCCH CHS--P(OCH3)2 3 2 2/OC H CH II

2

o

3

o II

aldicarb

rna laoxon o II oP(oc%) 2

~

c%

CH3S CCH=NOCNHCH 3 CH~

cI

o II

N~N.-CN(CI-~ (CH3)3C--¢k J

N~sAoccH2CH3 O

he ptenophos

triazamate o II OCNHCH3

O II Cl2C=CHOP (OC1%)2

O

SCH.,. " "C--NOCNHCH3

rnethomyl o II OCNHCH3

~

OCH(CH3)2

propoxur

c% CH~SCHCCH~

NOCNHCH~ dichlor~os ci.

o

>=°

o

carba~l

I

CH~SP(OC%)~ O azarnelJphos o II CH3CH2SCH2CH2SP(OCl-~j)2

butocarboxim

o

CH

N.__~C H3 (CH3)2NIJ~'ON/"OCN(CH3)2 O pin n-icarb

II

OCNHCH 3 I N II

CH3SC1-12CC(CH3)3 thiofanox

demethon S methyl Fig. 2. Structure of insecticides.

enzymes and its maximal kobs is 3.7-fold higher than the maximal kobs of Dm.AChE. Thus, use of Dm.AChE in biosensors would imply a 4-fold higher amount of enzyme than Ee.AChE.

Is it possible to increase this sensitivity? The Y408F Dm.AChE mutant is more sensitive than the wild type enzyme to a broad range of 161

F. Villatte et al.

Biosensors & Bioelectronics

1.6 1.4 1.2 °~

1.0 .2

0.8 0.6

¢13

0.4 0.2 I

0.0

Ce

Ee

Be

Tc

~

-

Dm Y408F

Fig. 3. Average sensitivity to insecticides of each ACHE.

3

3

Fig. 4. Stereoview of paraoxon ethyl located in the active site of the Tc.AChE showing 7r-Tr interactions between the phenyl ring o f the insecticide with F330 and W84. F330 is the counterpart of the drosophila Y408, and W84 is a major component o f the "anionic site".

carbamates and organophosphates. When compared to Ee.AChE, the improve in sensitivity increases to 12. This mutant presents a 1.3-fold decrease of efficiency of ASCh hydrolysis, compared to the wild type. This weak decrease of ko~s can be counterbalanced by a greater amount of enzyme: it is possible to produce in vitro 162

several mg of enzyme per litre of medium (Chaabihi et al., 1994). Consequently, Y408F Dm.AChE mutant is more adapted to insecticide traces detection than other AChEs, but the active site should be rendered even more sensitive. Indeed, active site modelisation suggests that modifying aromatic

Biosensors & Bioelectronics interactions by suppression of hydroxyl group in the mutant may play a role in the higher sensitivity to some insecticides and one may suggest to replace the Y408 by a tryptophane, in order to increase these interactions and thus generate a more sensitive mutant.

CONCLUSION It appears thus that insect AChE is more adapted to the use as biosensor than vertebrate AChE and mutagenesis provides the possibility to obtain hypersensitive variants. These data, combined with high level of yield production of enzyme in vitro demonstrate the efficiency of insect AChE use in biosensors. One may suggest in addition the use of different enzymes, associated with a neuronal network, to elucidate the identity of the insecticide present in samples.

ACKNOWLEDGEMENTS We thank Dr P. Taylor (UCSD) for providing the recombinant baculovirus to express Torpedo californica enzyme and Fr6d6ric Magn6 for production of enzymes. This study was supported by ACCSV no3 "G6n6tique et Environnement" DRET (94-084) and IFREMER.

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Acetylcholinesterase biosensor aminoethyltrimethylammonium. Biochemical and Cellular Biology 65, 529-535. Chaabihi, H., Fournier, D., Fedon, Y., Bossy, J. P., Ravallec, M., Devauchelle, G. and C6rutti, M. (1994) Biochemical characterization of Drosophila melanogaster acetylcholinesterase expressed by recombinant baculovirus. Biochemical and Biophysical Research Communications 203. 734742. Durand, P. & Thomas, D. (1984). Use of immobilized enzyme coupled with an electrochemical sensor for the determination of organophosphorous and carbamates pesticides. JEPTO, 5-4/5, 51-57. Ellman, G. L., Courtney, K. D., Andres, V. and Featherstone, R. M. (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology 7, 88-95. Gibney, G. and Taylor, P. (1990) Biosynthesis of Torpedo acetylcholinesterase in mammalian cells. Journal of Biological Chemistr3" 265, 1257612583. Kitts, P. A. and Possee, R. D. (1993) A method for producing recombinant baculovirus expression vectors at high frequency. Biotechniques 14, 810-817. Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proceedings of National Academy of Science USA 82, 448-452. Levy, D. and Ashani, Y. (1986) Synthesis and in vivo properties of a powerful quaternary methylphosphonate inhibitor of acetylcholinesterase. Biochemical Pharmacology 35, 1079-1085. Loewenstein, Y., Denarie, M., Zakut, H. and Soreq, H. (1993) Molecular dissection of cholinesterase domains responsible for carbamate toxicity. Chemical-Biological Interactions 87, 209-216. Marty, J. L., Sode, K. and Karube, I. (1992) Biosensor for detection of organophosphate and carbamate insecticides. Electroanalysis 4, 249-252. Morelis, R. M. and Coulet, P. R. (1990) Sensitive biosensor for choline and acetylcholine involving fast immobilization of a bienzyme system on a disposable membrane. Analytical Chimica Acta. 231, 27-32. Radic, Z., Pickering, N. A., Vellom, D. C., Camp, S. and Taylor, P. (1993) Three distinct domains in the cholinesterase molecule confer selectivity for acetyl- and butyrylcholinesterase inhibitors. Biochemistry. 32, 12074-12084. Razumas, V. J., Kulis, J. J. and Malinauskas, A. A. (1981) Highsensitivity bioamperometric determination of organophosphate insecticides. Environmental Science and Technology 15, 360-361. Rodriguez, O. P., Muth, G. W., Berkman, C. E., Kim, K. and Thompson, C. M. (1997) Inhibition of various cholinesterases with the enantiomers of 163

F. Villatte et al. malaoxon. Bulletin of Environnemental Contamination Toxicology 58, 171-176. Salamoun, J. and Remien, J. (1993) Indirect detection of anti-cholinesterase compounds in microcolumn liquid chromatography using packed bed reactor with immobilized human red blood cell acetylcholinesterase and choline oxidase. Journal of Pharmacological and Biomedical Analysis 10, 931-936. Simeon, V. and Reiner, E. (1973) Comparison between

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Biosensors & Bioelectronics inhibition of acetylcholinesterase and cholinesterase by some N-methyl- and N-dimethyl-carbamates. Archiv Za Higijena Radae i Toknikologiju 24, 199-206. Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein. Science 253, 872-877.