RESEARCH ARTICLE

Acetylcholinesterases from the Disease

Vectors Aedes aegypti and Anopheles
gambiae: Functional Characterization and
Comparisons with Vertebrate Orthologues
Cecilia Engdahl1, Sofie Knutsson1, Sten-Åke Fredriksson2, Anna Linusson1,
Göran Bucht2*, Fredrik Ekström2*
1 Department of Chemistry, Umeå University, Umeå, Sweden, 2 Swedish Defence Research Agency,
CBRN Defence and Security, Umeå, Sweden

* [email protected] (GB); [email protected] (FE)

Abstract
OPEN ACCESS
Mosquitoes of the Anopheles (An.) and Aedes (Ae.) genus are principal vectors of human
diseases including malaria, dengue and yellow fever. Insecticide-based vector control is an
Citation: Engdahl C, Knutsson S, Fredriksson SÅ,
Linusson A, Bucht G, Ekström F (2015) established and important way of preventing transmission of such infections. Currently
Acetylcholinesterases from the Disease Vectors used insecticides can efficiently control mosquito populations, but there are growing con-
Aedes aegypti and Anopheles gambiae: Functional cerns about emerging resistance, off-target toxicity and their ability to alter ecosystems. A
Characterization and Comparisons with Vertebrate
potential target for the development of insecticides with reduced off-target toxicity is the cho-
Orthologues. PLoS ONE 10(10): e0138598.
doi:10.1371/journal.pone.0138598 linergic enzyme acetylcholinesterase (AChE). Herein, we report cloning, baculoviral expres-
sion and functional characterization of the wild-type AChE genes (ace-1) from An. gambiae
Editor: Israel Silman, Weizmann Institute of Science,
ISRAEL and Ae. aegypti, including a naturally occurring insecticide-resistant (G119S) mutant of An.
gambiae. Using enzymatic digestion and liquid chromatography-tandem mass spectrome-
Received: July 1, 2015
try we found that the secreted proteins were post-translationally modified. The Michaelis-
Accepted: September 1, 2015
Menten constants and turnover numbers of the mosquito enzymes were lower than those of
Published: October 8, 2015 the orthologous AChEs from Mus musculus and Homo sapiens. We also found that the
Copyright: © 2015 Engdahl et al. This is an open G119S substitution reduced the turnover rate of substrates and the potency of selected
access article distributed under the terms of the covalent inhibitors. Furthermore, non-covalent inhibitors were less sensitive to the G119S
Creative Commons Attribution License, which permits
substitution and differentiate the mosquito enzymes from corresponding vertebrate
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are enzymes. Our findings indicate that it may be possible to develop selective non-covalent
credited. inhibitors that effectively target both the wild-type and insecticide resistant mutants of mos-
Data Availability Statement: All relevant data are quito AChE.
within the paper and its Supporting Information files.

Funding: This work was supported by Centrum för

Miljövetenskaplig Forskning (CMF), http://www8.umu.
se/cmf/index_eng.htm, AL; and Vetenskapsrådet
(VR), http://www.vr.se/inenglish.4. Introduction
12fff4451215cbd83e4800015152.html, AL. The
funders had no role in study design, data collection
Mosquitoes are estimated to transmit infectious diseases to more than 500 million people in
and analysis, decision to publish, or preparation of Africa, Americas and Asia, with millions of deaths annually. Climate change, globalization and
the manuscript. other factors indicate that there is a growing risk that these infectious diseases may also become

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 1 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

Competing Interests: The authors have declared a problem in Europe [1]. Malaria and dengue are the most common mosquito-borne infectious
that no competing interests exist. diseases, each responsible for millions of cases per year. The uncertainty in the number of
reported cases is large with approximately 200 million cases of malaria in 2013, along with
more than half a million deaths due to the disease [2]. In addition there were almost 100 mil-
lion cases of dengue infections in 2010 [3]. The responsible agents of these diseases are trans-
mitted through the bites of infected mosquitoes. Species of the Anopheles genus transmit the
malaria parasite, with Anopheles (An.) gambiae as the main vector [4]. Similarly, Aedes (Ae.)
mosquitoes such as Ae. aegypti and Ae. albopictus are the principal vectors of dengue as well as
yellow fever and chikungunya viruses [5, 6].
Reducing the numbers of disease-transmitting mosquitoes is a proven strategy for control-
ling disease transmission. The four main classes of insecticides used for mosquito vector con-
trol are chlorinated hydrocarbons, organophosphates, carbamates and pyrethroids [7].
Although their molecular targets differ, these insecticides have similar effects on their target
organisms, leading to paralysis and death. Chlorinated hydrocarbons, such as dichlorodiphe-
nyltrichloroethane (DDT), and pyrethroids target the voltage-gated ion channels of neurons,
while organophosphates and carbamates inhibit the activity of acetylcholinesterase (AChE, EC
3.1.1.7), an essential enzyme for the function of the nervous system. The large-scale production
and frequent use of insecticides has caused their accumulation in ecosystems, resulting in envi-
ronmental contamination and toxicity to many different species including humans [7]. The
spread of insecticide resistance also threatens the effectiveness of currently used insecticides
[8–10]. In particular, there have been alarming reports from 27 countries in sub-Saharan Africa
of pyrethroid resistance among Anopheles vectors [11]. New vector control strategies such as
the use of microorganisms, viruses, biological toxins or natural products, collectively called bio-
pesticides, are under development [12–14]. However, to maximize the effectiveness of vector
control and combat the spread of mosquito-borne diseases, it may be useful to adopt combina-
tions of different approaches [15]
AChE is not only a target for insecticides but also for chemically related warfare agents (i.e.
nerve agents) and naturally occurring toxins (e.g. snake venoms). It is responsible for terminat-
ing nerve signals at the synaptic cleft by hydrolyzing the neurotransmitter acetylcholine (ACh
(1), Fig 1). Disruption of this mechanism leads to accumulation of ACh causing overstimula-
tion and eventual blockage of neurotransmission. Currently used anticholinesterase insecti-
cides for vector control are inhibitors that form a covalent bond with a conserved serine at the
active site of the enzyme (reviewed in [16]). This active site serine (Ser203, human AChE num-
bering) is located at the base of a deep and narrow gorge, where it forms one part of the cata-
lytic triad together with His447 and Glu334 [17, 18]. The gorge is lined with aromatic residues
and penetrates halfway through the enzyme.
Mammals have one ace gene encoding for AChE and this is also the case for Drosophila (D.)
melanogaster [19]. However, it is now established that most investigated insects carry multiple
AChE genes. Mosquitoes have two AChE genes due to an old duplication; the paralogous ace-1
gene and the orthologous ace-2 gene which is homologous to the gene in Drosophila [20–22].
D. melanogaster, and other flies, possess only one gene, probably due to a secondary loss during
the evolution of the Diptera [23]. It is AChE1, encoded by the ace-1 gene, which is responsible
for catalytic acetylcholinesterase activity and AChE-mediated insecticide resistance in mosqui-
toes [21, 24]. A naturally occurring mutation that confers resistance to organophosphates and
carbamates in mosquitoes is a glycine to serine mutation at position 119 (G119S) in AChE1
[24]. This mutation has been identified in populations of An. gambiae, as well as in the West
Nile and Japanese encephalitis vector Culex pipiens, found in different geographical locations
[24]. However, this mutation has not been found in Ae. aegypti ace-1, probably because this

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 2 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

Fig 1. Chemical structures of the substrates and inhibitors investigated in this study. Substrates: acetylcholine (1), acetylthiocholine (2),
propionylthiocholine (3), butyrylthiocholine (4). Covalent inhibitors: propoxur (5), eserine (6). Non-covalent inhibitors: C7653 (7), donepezil (8), C5681R (9),
C5681S (10), ethopropazine (11).
doi:10.1371/journal.pone.0138598.g001

gene uses a different codon for glycine at this position, necessitating two point mutations for
the conversion of glycine to serine [25].
The crystal structure of AChE from D. melanogaster has been determined [26], but no struc-
ture of mosquito AChE is currently available. AChE from D. melanogaster shows an amino
acid sequence identity of 39 and 37% to the corresponding enzymes from An. gambiae and Ae.
aegypti, respectively. This is comparable to the sequence identity of 38% between ace-1 and
ace-2 within An. gambiae. Notably, the mosquito AChE1 and the human enzyme exhibit a
slightly higher sequence identity (48–49%). Most studies on AChEs have focused on AChE
from Homo sapiens, Mus musculus, D. melanogaster and the electric rays Torpedo californica
or marmorata. Recently, AChE1 from An. gambiae (AgAChE1), and to some extent, Ae.

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 3 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

aegypti (AaAChE1), has been expressed and biochemically characterized [27–29]. In addition,
unique residues in the active site of AChE1 have been identified and suggested as targets for
insecticides [30, 31]. It has also been shown that some covalent inhibitors targeting AgAChE1
show selectivity over human AChE [32].
Here we report the cloning, expression and profiling of catalytically active wild-type
enzymes encoded by the ace-1 genes of An. gambiae and Ae. aegypti as well as the G119S
mutant of An. gambiae. Post-translational modifications of these enzymes were investigated
using LC-MS/MS and their basic catalytic parameters and substrate preferences were deter-
mined. Furthermore, ligand-binding properties were investigated using a set of inhibitors (5–
11, Fig 1) and compared to those of vertebrate AChEs from Homo sapiens (hAChE) and Mus
musculus (mAChE).

Methods
Construction of synthetic genes and cloning into the baculovirus
chromosome
Full-length sequences of AChE1 (ace-1) from An. gambiae (XM_321792) [33] and Ae. aegypti
(EF209048) [28] were downloaded from GenBank and codon-optimized for expression in Spo-
1
doptera frugiperda-9 (Sf 9) cells (ATCC CRL-1711™) according to the codon database [34].
Synthetic genes covering the complete open-reading frames were produced (Eurofins MWG
Operon, Germany) and cloned in frame to a C-terminal 6xHis-tag in the baculovirus donor
vector pFastBac/CT-TOPO (Invitrogen, Waltham, MA, USA). Correct sequences of individual
clones were verified by sequencing plasmid DNA from One Shot Mach1-T1R E. coli. The AgA-
ChE1-G119S mutant was constructed using the Quick-Change II XL Site Directed Mutagenesis
kit (Agilent Technologies, Santa Clara, CA, USA) and the mutation was confirmed by
sequencing.
The donor plasmids carrying the ace-1 genes were then transformed into MAX Efficiency
DH10Bac competent E. coli carrying the bacmid chromosome along with a helper plasmid that
enables recombination. The genes of interest were recombined into the baculovirus chromosome
according to the Bac-to-Bac TOPO Expression system manual (Invitrogen) and bacterial colo-
nies carrying the ace-1 genes from An. gambiae or Ae. aegypti were identified by blue/white
screening before sequencing. Bacmid DNA with the expected sequence was gently isolated from
1
the bacteria prior to transfection using Fugene HD (Roche Applied Science, Penzberg, Ger-
many) into Sf9 cells grown in Sf900 serum-free insect cell growth medium (Invitrogen).

Expression
The baculovirus infections induced as described above were verified 48–72 h post-infection by
GFP fluorescence in Sf9 Easy Titer (Sf9-ET) cells maintained in HyClone SFX Insect medium
supplemented with geniticin (100 μg/ml) and FBS (5%) [35] and by measuring the AChE1
activity of supernatants of baculovirus-infected Sf 9 cells (see the section on “determination of
enzymatic activity”). Viral titers in expanded viral stocks were determined at day five by end-
point dilution assays in the Sf 9-ET cell line by scoring GFP-positive wells [36]. The expression
of ace-1 gene products was optimized by analysing AChE activity at multiple time-points after
infection. Briefly, adherent Sf9 cells at approximately 70-80% confluence were infected with a
multiplicity of infection (MOI), i.e. ratio of virus to insect cells, of 0.1, 1.0 and 10. Infected cells
were thereafter maintained for 48 h at 28°C before the media was replaced and the cells were
incubated for an additional 3–7 days at 20°C or 28°C. Cells were harvested by scraping and sep-
arated from the media by centrifugation for 10 min at 500 rcf. The enzymatic activity was

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 4 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

determined as described below in samples of infected cells and in corresponding cell culture
media using a PerkinElmer Lambda 650 UV/VIS spectrometer at 412 nm. Once an optimal
MOI, temperature and duration of infection had been determined, the production of AChE1
was scaled up. AChE1 proteins were expressed from full-length genes and not truncated prior
to cloning, these recombinant proteins were used for all experiments. The cloning and expres-
sion of hAChE and mAChE have been described previously [37, 38].

Purification
The expressed wild-type proteins were purified by affinity chromatography using the active
site ligand procainamide coupled to epoxy-activated sepharose (GE healthcare LifeScience, Lit-
tle Chalfont, UK) [38]. Cell culture supernatants were mixed with procainamide sepharose and
the slurry was incubated for 16 h at 4°C under constant rotation. It was then loaded on Eco col-
umns (Bio-Rad Laboratories Inc., Hercules, CA, USA), washed with 2 mM MES (pH 6.5) con-
taining 250 mM NaCl and finally eluted with the same buffer supplemented with 50 mM
procainamide. Attempts to use the C-terminal His-tag for Ni-NTA agarose purification were
not successful. The purified samples were analysed by SDS-PAGE and subsequently used for
the analysis of post-translational modifications and for kcat calculations. Non-purified secreted
enzymes were used for all other experiments.

Glycosylation analysis
The glycosylation of the expressed recombinant AChE1s was investigated using enzymatic
digestion and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The purified
protein was digested in a molecular weight cut-off centrifugal filter (Microcon 10,000 MWCO,
Millipore, Billerica, MA, USA) [39]. A 100 μl aliquot was concentrated on the filter at 12,000 x
g, after which the filter was washed twice with 200 μl ammonium bicarbonate (100 mM). The
protein was subsequently digested with 1 μg of proteinase K in 100 μl of 100 mM ammonium
bicarbonate for 4h at 40°C and the peptides were recovered by centrifugation and washing of
the filter with 100 μl of 50% acetonitrile. The digests were stored at -20°C before LC-MS
analysis.
The digested proteins were analysed by LC-MS and MS/MS on a Waters Nano-Acquity
UPLC system connected to a Waters Qtof Ultima mass spectrometer equipped with a nano-
electrospray ion source (Waters Inc). The peptides were separated on a 100 mm, 75 μm i.d.
C18 UPLC column (Waters Inc.) using a water:acetonitrile gradient containing 0.1% formic
acid from 3–40% acetonitrile over 25 min at a flow rate of 400 nl/min. The samples were ana-
lysed by LC-MS using alternate scanning at low and elevated collision energies with argon as
the collision target. Carbohydrate marker ions at m/z 204.1 and 366.2 formed by collision-
induced dissociation (CID) at 40 eV were used to detect glycopeptides. The corresponding low
energy mass spectrum was examined and the product ion spectra of selected precursor ions
were acquired in a separate LC-MS/MS run.

Activity assay and determination of enzymatic activity

The enzymatic activity was investigated using the Ellman assay [40]. The assay was performed
using the substrate acetylthiocholine iodide (ATChI, 2) at a concentration of 1 mM in 0.1 M
sodium phosphate buffer (pH 7.4) at 30°C. The reagent 5,5'-dithiobis-(2-nitrobenzoic acid)
(DTNB) was present at a final concentration of 0.2 mM. Enzyme-catalysed hydrolysis of the
substrate was quantified by monitoring a time-dependent change in absorbance at 412 nm due
to the colour change caused by the reaction between DTNB and the hydrolysis product. Enzy-
matic activities were evaluated by plotting absorption per minute (dA/min) versus substrate

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 5 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

concentration ([S]). The substrates’ rates of autohydrolysis were subtracted from the measured
enzyme-catalyzed rates. All experiments were performed at least in duplicate and values are
given with a 95% confidence interval (CI).

Michaelis-Menten kinetics
Michaelis-Menten constants (Km) and maximum velocity (Vmax) values for AgAChE1, AgA-
ChE1-G119S, AaAChE1, mAChE and hAChE were determined by measuring their initial rates
(V0) at different substrate concentrations. Substrate concentrations where substrate inhibition
was recognizable were avoided and the Km and Vmax values were obtained from non-linear
regression curve fit using the Michaelis-Menten equation in GraphPad Prism version 6.04 for
Windows (GraphPad Software, La Jolla, CA, USA, www.graphpad.com). The substrate prefer-
ences of AgAChE1, AgAChE1-G119S and AaAChE1 were investigated using ATChI, propio-
nylthiocholine iodide (PTChI) and butyrylthiocholine iodide (BTChI) (2–4, Fig 1). Each
enzyme’s turnover number (kcat) was determined from the relationship between Vmax and pro-
tein concentration. The protein concentration as the total number of active sites in solution at
a given volume was determined experimentally by active site titrations using the covalent
inhibitor O-ethyl S-[2-(diisopropylamino) ethyl] methylphosphonothioate (VX). The wild-
type mosquito enzymes were incubated with different concentrations of VX for 120 min to
achieve complete inactivation of the enzymes by both enantiomers of VX. Enzymatic activity
was then measured and a titration curve was constructed by plotting the relative activity against
the VX concentration. The number of active sites was determined from the titration and used
together with Vmax for the calculation of kcat.

Inhibition kinetics
Inhibition profiles of different AChEs were determined by studying a number of previously
described AChE inhibitors (5–11, Fig 1) [41–43]. The non-covalent inhibitors (7–11) were
characterized by determination of their half-maximal inhibitory concentration (IC50) values
and covalently binding inhibitors (5–7) were investigated by determination of their inhibition
constants (ki). Stock solutions of the inhibitors were prepared in dimethyl sulfoxide (DMSO) at
a concentration of 100 mM and working dilutions thereof were prepared in 0.1 M sodium
phosphate buffer pH 7.4. For IC50 determinations, the enzymatic activity was determined
immediately following addition of inhibitor solutions of different concentrations up to a maxi-
mum of 1 mM. IC50-values were calculated using non-linear regression (curve fitting) in
GraphPad Prism and the log (inhibitor) vs. response variable slope equation with four parame-
ters. For ki-determinations, samples were pre-incubated in the presence of the relevant inhibi-
tor at the specified concentration and activity was measured at several time-points (1, 3, 5, 10,
20, 30, 40, 50 and 60 min) until no further decrease in activity could be seen. kobs values were
obtained using non-linear regression (curve fitting) in GraphPad Prism software with the one
phase decay equation. These values were plotted as a function of the inhibitor concentration
and the value of ki was obtained from the resulting slope.

Cluster analysis based on kinetic data

The relationships between different AChEs (AaAChE1, AgAChE1, AgAChE1-G119S, hAChE
and mAChE) with respect to their kinetic parameters were visualized by cluster analysis. The
analysis was based on our experimental kinetic measurements together with previously pub-
lished data, and its results were presented in the form of un-rooted trees.
Three cluster analyses of the AChEs were performed based on all of the parameters consid-
ered in this work and two different sub-sets of these parameters. Sub-set A contains parameters

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 6 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

related to basal enzymatic function that are dependent on a reaction with the catalytic serine
residue (Vmax, KM, kcat and ki) while sub-set B contains parameters related to affinity for non-
covalent inhibitors alone (IC50). Prior to the cluster analysis, the parameter values were scaled
to unit variance and the Vmax values of the investigated substrates of each enzyme were nor-
malized against that enzyme’s Vmax for ATChI. Euclidean distances between the enzymes were
calculated for each of the three datasets. The enzymes were clustered according to these Euclid-
ean distances and the Neighbor-Joining method [44] with a randomized order (seed 5) of the
enzymes using PHYLIP [45]. The clustering is visualized as un-rooted trees, with the angle of
the arc set to 360 degrees, where the branch lengths correspond to the Euclidean distances
between the enzymes, reflecting the similarities and dissimilarities in the underlying data.

Results and Discussion

Design and cloning of expression constructs
To facilitate post-translational modifications such as translocation and cleavage of a putative
secretion signal, an Sf 9 based baculovirus expression system was evaluated. Codon-optimized
full-length ace-1 genes of An. gambiae and Ae. aegypti were produced and cloned into donor
plasmids. All plasmids containing the ace-1 genes were verified by sequencing before being
recombined into bacmid chromosomes. Recombinant baculovirus chromosomes were identi-
fied by blue/white screening and the flanking sequences of the inserts were reconfirmed by
sequencing. Bacmid chromosomes were finally isolated and used for transfection into Sf 9 cell
lines. Cells with typical morphological changes and granular appearance were noticed within
one week of infection in Sf 9 cells, and a clear GFP fluorescence signal was subsequently
observed in Sf 9-ET cells. Infected primary cultures (P1) were harvested and used for reinfec-
tion of Sf 9 cells before viral titers were determined in amplified viral stocks. The median tissue
culture infective doses (TCID50) of the P2 stocks were calculated to be 2 x 108 and 4 x 108 / ml
for An. gambiae and Ae. aegypti, respectively. The G119S mutant was made by site-directed
mutagenesis of the wild type An. gambiae ace-1 gene in the baculovirus donor vector before
being recombined onto a baculovirus chromosome and expressed in Sf 9 cells like the wild type
enzymes.

Optimization of protein expression

The production and secretion of AChE1 was optimized by exploring the influence of incuba-
tion temperature, MOI and time of cultivation. The amount of expressed and secreted AChE1
was determined in the culture media and the highest secretion levels were obtained when
adherent cells were infected at an approximate cell density of 70–80% confluence with a MOI
of 0.1. The amount of secreted AChE1 at 5–7 days post-infection was estimated to be around
10 mg/L at 28°C, and slightly higher levels of expression were detected in suspension cultures.
Of the total measured enzyme activity, about thirty percent was detected in cell culture media
and the remaining enzyme activity was detected in samples of lysed cells. Under comparable
expression conditions, media containing the AgAChE1-G119S mutant enzyme displayed a sig-
nificantly lower enzymatic activity than media collected from cells expressing the correspond-
ing wild type enzyme.

Characterization of AChE1 from mosquitoes

SDS-PAGE gel electrophoresis of the purified mature proteins showed that the secreted pro-
teins migrated with an apparent molecular weight between 60–70 kDa (S1 Fig). Assuming
that the mature proteins start at the amino acids D162NDP in the case of AgAChE1 [27] and

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 Characterization of AChEs from Disease Transmitting Mosquitoes

Table 1. AChE1 glycopeptides detected by LC-MS.

Amino acid sequence and Corresponding glycan Glycopeptide m/ Glycopeptide Glycan Peptide
position z mass mass mass
Aedes aegypti
GLNTT 507–511 -GlcNAc (Fuc) GlcNAc Man (Man) Man 772.272+ 1542.52 1038.29 504.23
GLNTT 507–511 -GlcNAc (Fuc) GlcNAc Man (Man) Man 873.792+ 1745.57 1241.34 504.23
GalNAc
GLNTT 507–511 -GlcNAc (Fuc) GlcNAc Man (Man GalNAc) 975.322+ 1948.63 1444.40 504.23
Man GalNAc
Anopheles gambiae
GLNTS 507–511 -GlcNAc (Fuc) GlcNAc Man (Man) Man 765.262+ 1528.51 1038.27 490.24
GLNTS 507–511 -GlcNAc (Fuc) GlcNAc Man (Man) Man 866.802+ 1731.58 n. d. n. d.
GalNAc
GLNTS 507–511 -GlcNAc (Fuc) GlcNAc Man (Man GalNAc) 968.332+ 1934.65 n. d. n. d.
Man GalNAc

n.d. = not determined

doi:10.1371/journal.pone.0138598.t001

D129NDP in that of AaAChE1 [28], their theoretical weight is 64 kDa. To assess the glycosyla-
tion of the recombinant enzymes, purified mature proteins were studied after digestion with
Proteinase K followed by LC-MS/MS analysis. Carbohydrate marker ions in the high energy
spectrum indicated the presence of glycopeptides (S2 Fig) [46]. Further analysis (see also sup-
porting information section S1 Text) revealed that the mass spectral data corresponded to a N-
glycosylated core structure -GlcNAc(Fuc)-GlcNAc-Man3 (Table 1). In addition to the core
structure, two additional glycoforms of the peptide containing terminal galactosamine were
identified (Table 1). The structures were common to both species and consistent with the bian-
tennary complex-type structures reported previously in fetal bovine serum AChE and equine
serum BuChE [47]. To identify the specific glycosylation site, the peptide mass was matched to
the amino acid sequence of the respective protein and we found that the glycan structures were
attached to Asn509 in both species. Jiang et al have previously shown an increase in migration
on SDS-PAGE for N-glycosidase-treated AgAChE1 compared to non-treated samples, indicat-
ing the presence of N-glycosylated amino acids in An. gambiae [27].

Substrate preferences and substrate inhibition

To investigate the substrate preferences of AgAChE1, AgAChE1-G119S and AaAChE1, their
Michaelis-Menten constants (KM) and maximum reaction rates (Vmax) were determined using
the potential substrates ATChI (2), PTChI (3) and BTChI (4) (Figs 1 and 2A). For the wild-
type enzymes, the KM values were similar for all tested substrates, ranging from 24–36 μM for
AgAChE1 and 20–45 μM for AaAChE1. Conversely, the AgAChE1-G119S mutant exhibited a
significant decrease in substrate affinity with increasing substrate size (MWATChI < MWPTChI
< MWBTChI); its KM values were 58, 315 and 431 μM for ATChI, PTChI and BTChI respec-
tively. For all enzymes, the Vmax values decreased with increasing substrate size
(ATChI < PTChI < BTChI) and were markedly lower for the hydrolysis of the largest sub-
strate, BTChI (see Table 2). The relative catalytic efficiencies given by the Vmax/KM-ratios for
the substrates ATChI, PTChI and BTChI were 19.7, 16.6 and 1.6 for AgAChE1; 16.2, 13.2 and
0.8 for AaAChE1; and 5.5, 0.27 and 0.08 for the G119S mutant, respectively. The AChE1
enzymes hydrolysed ATChI more efficiently than PTChI and BTChI, which is consistent with
the behavior of mAChE and hAChE (Table 2) [48–50] and also with results previously
obtained for various invertebrate AChE enzymes [27, 51, 52]. A common feature of vertebrate

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 8 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

Fig 2. Enzyme kinetics. (A) Substrate preference for AaAChE1 illustrated by Michaelis-Menten curves. The same trend was observed for AgAChE1 and
AgAChE1-G119S. (B) Substrate inhibition of AaAChE1, AgAChE1 and AgAChE1-G119S using ATChI as substrate. (C) Typical graph for ki-calculations of
AgAChE1 with eserine as inhibitor. (D) Typical dose-response curve of AaAChE1 with inhibitor, used for determination of IC50-values for various compounds.
doi:10.1371/journal.pone.0138598.g002

AChEs is that they show substrate inhibition at elevated substrate concentrations. Our analysis
revealed that AgAChE1, AgAChE1-G119S and AaAChE1 are also subject to such inhibition
(Fig 2B), in agreement with a study on the biochemical profile of AgAChE1 [27].

Michaelis-Menten kinetics
The kinetics was further investigated using the substrate analogue ATChI. The initial enzyme
activities were monitored at different substrate concentrations and the KM and Vmax values
were determined and compared to the corresponding constants for the vertebrate AChEs (Fig
2A and Table 2). The mosquito enzymes displayed a higher affinity for ATChI than the verte-
brate AChEs. The KM values for AgAChE1, AgAChE1-G119S and AaAChE1 were 27, 58 and
25 μM, respectively, compared to 84 μM and 146 μM for mAChE and hAChE, respectively.
The kinetics of inhibition and the relationship between the protein concentration (i.e. the
total number of active sites in solution) and enzymatic activity were investigated by means of a
time-course experiment using the covalent inhibitor VX. The biphasic shape of the resulting
curves indicates that both enantiomers of VX reacted (S3A Fig). For AgAChE1 and AaAChE1,
the covalent modification progressed slowly and reached a plateau after approximately 120

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 9 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

Table 2. Kinetic parameters of different AChEs.

Substrate Vmax (mA/min) Km (μM) Vmax/KM Kcat (s-1)

Aedes aegypti AChE1
ATChI 406 (389–422) 25 (20–30) 16.2 140
PTChI 266 (258–275) 20 (17–23) 13.3 92
BTChI 38 (36–40) 45 (36–54) 0.8 13
Anopheles gambiae AChE1
ATChI 528 (505–552) 27 (21–33) 19.6 124
PTChI 407 (393–421) 25 (20–29) 16.3 95
BTChI 58 (55–61) 36 (27–45) 1.6 14
Anopheles gambiae AChE1-G119S
ATChI 318 (310–326) 58 (52–65) 5.5 n. d.
PTChI 105 (87–123) 303 (172–435) 0.27 n. d.
BTChI 34 (24–45) 431 (136–727) 0.08 n. d.
Mus musculus AChE
ATChI 725 (694–756) 84 (72–95) 8.6 n. d.
PTChI 367 (354–380) 59 (51–66) 6.2 n. d.
Homo sapiens AChE
ATChI 821 (782–860) 146 (128–165) 5.6 n. d.
PTChI 429 (415–442) 150 (137–162) 2.9 n. d.

n.d. = not determined, parentheses = 95% CI

doi:10.1371/journal.pone.0138598.t002

minutes, allowing the construction of a titration plot (S3B Fig). From this titration, the relation
between Vmax and the total number of active sites in solution was established, allowing deter-
mination of the turnover number (kcat). The kcat values were determined to be 124 s-1 and 140
s-1 for AgAChE1 and AaAChE1, respectively. These values are approximately twenty to forty
times lower than those reported for vertebrate AChE [48, 53]. The kcat values presented here
are also lower than the previously reported values for AgAChE1 (650 s-1 [27] and 3000 s-1
[54]); to our knowledge, no kcat has been previously reported for AaAChE1. The large differ-
ences between the reported values for AgAChE1 may be due to different experimental meth-
ods. For example, the constants reported herein were determined using full length secreted
enzyme and the concentration was determined using active site titrations. In contrast, previous
reports use a colorimetric assay to determine the concentration of a purified, truncated con-
struct. Because AgAChE1-G119S was resistant towards the organophosphate VX (its activity
decreased by less than 5% over 120 min of incubation) we were unable to perform similar titra-
tions with this enzyme or to determine its kcat value.

Probing the active site gorge with inhibitors

The ligand binding properties of the AChEs were investigated by determining the potency of a
set of known inhibitors including the covalent inhibitors propoxur (5) and eserine (6), and
non-covalent inhibitors C7653 (7), donepezil (8), C5685R (9), C5685S (10) and ethopropazine
(11) (Fig 1). Propoxur is used against insect pests for both agricultural and public health pur-
poses [55], eserine and donepezil are used clinically in the treatment of neurological disorders
[43], while ethopropazine has mainly been used as a biochemical tool to distinguish between
cholinesterases and also for determining BuChE-specific tissues [50, 56]. The non-covalent
inhibitory activity of C7653, C5685R and C5685S has been studied against both hAChE and
mAChE [41, 42].

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 10 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

Table 3. Characterization of inhibitors.

IC50-values of reversible inhibitors, μM (95% CI) ki-values of covalent inhibitors, μM-1

min-1 (95% CI)

Enzyme Donepezil C7653 C5685R C5685S Ethopropazine Propoxur Eserine

AgAChE1 0.24 (0.19–0.30) 0.44 (0.29–0.67) >30 >30 8.3 (5.8–11.8) 1.11 (1.02–1.21) 27.3 (23.8–30.8)
AaAChE1 0.28 (0.18–0.43) 0.36 (0.26–0.49) >30 >30 4.6 (3.98–5.3) 1.20 (1.05–1.35) 24.8 (18.1–31.5)
hAChE 0.008 (0.0071–0.0085) 0.36 1.3** 1.4** ~1 mM 0.081 (0.071–0.092) 2.66 (2.28–3.05)
mAChE 0.007 (0.0063–0.008) 0.2* 0.7** 0.7** ~1 mM 0.092 (0.068–0.117) 1.36 (1.15–1.56)
AgAChE1-G119S 0.31 (0.25–0.38) 1.3 (0.8–2) >30 >30 ~1 mM n. d. 1.18 (0.63–1.72)

*[41]
**[42]

doi:10.1371/journal.pone.0138598.t003

The covalent inhibitors propoxur and eserine were approximately 10-fold more potent
inhibitors of AChE1 than of vertebrate AChEs, as shown by their inhibition constants (ki, Fig
2C and Table 3). The ki-values for eserine were 24.8 and 27.3 μM -1 min-1 for the wild-type
mosquito AChE1s and 1.4 and 2.7 μM -1 min-1 for mAChE- and hAChE respectively. Interest-
ingly, with a ki of 1.18 μM -1 min-1, the sensitivity of AgAChE1-G119S was similar to that of
mAChE and hAChE. The selectivity of propoxur followed the same general trend as eserine,
but displayed 15–30 fold lower constants. Notably, the G119S mutant was very resistant to
inhibition by propoxur and only marginal inhibition was observed (data not shown).
The enzymes were further characterized using a set of non-covalent inhibitors of AChE.
The interactions between donepezil, C7653, C5685R, C5685S and vertebrate AChE have been
investigated using X-ray crystallography [41, 42, 57], but no crystal structure of AChE in com-
plex with ethopropazine has yet been published. Donepezil and C7653 spans the entire active
site gorge, with the benzylic moiety (donepezil) or the piperidine ring (C7653) forming a paral-
lel key interaction with the indole ring of Trp86 at the base of the active site gorge [41, 57]. The
enantiomeric pair C5685R and C5685S interact with the entrance of the gorge via the substi-
tuted phenyl ring, while the N-ethyl pyrrolidine moiety extends towards the catalytic site. Even
though the enantiomers are within contact distance of Trp86, they do not form close interac-
tions with the indole ring similar to those observed for donepezil and C7653.
We found that C7653 was a potent inhibitor of AgAChE1 and AaAChE1, with IC50-values
of 440 and 360 nM respectively, which are similar to those determined for hAChE and mAChE
(Fig 2D and Table 3). Compared to the wild-type enzymes, the G119S mutant slightly reduces
the potency of C7653 (IC50 of 1.3 μM). Interestingly, donepezil, C5685R and C5685S all dis-
played selectivity, favoring the vertebrate form of AChE. For these compounds, the IC50 of
AgAChE1-G119S is comparable to the constants determined for AgAChE1 and AaAChE1.
This finding indicates that the G119S mutation has only minor implications for the binding of
this set of non-covalent ligands. The only exception is ethopropazine, which is not an inhibitor
of mAChE, hAChE or the G119S mutant (IC50 > 1 mM) but does inhibit the wild-type mos-
quito enzymes with IC50 values of 4.6 and 8.3 μM.

Cluster analysis of functional descriptors

A cluster analysis based on the experimentally determined parameters kcat, KM, Vmax, ki and
the calculated IC50 values (S1 Table) visualized as an un-rooted tree revealed three distinct clus-
ters consisting of the wild-type mosquito enzymes, the AgAChE1-G119S enzyme and finally
the vertebrate mAChE and hAChE (Fig 3A). The data were further divided into two sub-sets.

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 11 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

Fig 3. Cluster analysis of functional descriptors. (A) Un-rooted tree based on all experimentally determined parameters (main dataset: kcat, KM, Vmax, ki
and IC50–values). (B) Un-rooted tree based on parameters related to basal enzymatic function and variables dependent on both affinity and reaction rate
(sub-set A: kcat, KM, Vmax, ki-values). (C) Un-rooted tree based on parameters related to affinity (sub-set B: IC50–values).
doi:10.1371/journal.pone.0138598.g003

Sub-set A, containing constants that describe the affinity and/or reactivity of compounds that
form a covalent bond to the catalytic serine residue, yielded a tree where the two mosquito
wild-type enzymes still clustered close together. Interestingly, however, this subset analysis
shifted the AgAChE1-G119S branch towards the vertebrate enzymes, indicating a greater
degree of similarity in the ligand binding properties (Fig 3B). The cluster analysis based on
sub-set B, which contained data for the non-covalent inhibitors (i.e. compounds that do not
form a covalent bond to the catalytic serine residue) yielded a tree where the mosquito wild-
type AChE1s and vertebrate proteins again formed two distinct clusters. In contrast to sub-set
A, the node of the AgAChE1-G119S branch was shifted from the vertebrate cluster towards the
wild-type AChE1 cluster (Fig 3C).

Summary and conclusions

A critical question for the development of insecticides with improved selectivity is whether
AChEs from different species have sufficiently diverse ligand binding properties to allow devel-
opment of synthetic compounds that are selective towards mosquitoes without affecting the
cholinergic transmission in off-target species. Sequence comparisons, crystallographic studies
of AChE from various species, and homology modelling of AgAChE1 all suggest that the three
dimensional structures of vertebrate and mosquito enzymes are similar [26, 58]. Nevertheless,
a free cysteine located in a loop at the entrance of the active gorge, corresponding to Phe295 in
hAChE, structurally diverge from the vertebrate enzymes and is thus a potential target for
selective covalent inhibitors [30, 59, 60]. Likewise, recent work targeting the catalytic serine res-
idue with covalent inhibitors containing a carbamate functionality has shown that considerable
selectivity ratios can be achieved for mosquito over vertebrate enzymes [31, 32, 54, 61]. While
the potential of covalent inhibitors has been investigated in a number of studies, inhibitors
with a different mode of action, such as non-covalent inhibitors, are less well explored [62].
Another major concern in insecticide development is to overcome the increasing resistance to
AChE inhibitors conferred by e.g. the G119S mutation. Novel small-core carbamates have been
designed [54], and it has also been shown that it is possible to target resistant mutant enzymes
with non-covalent inhibitors [62].
In this study, we cloned, expressed and characterized the wild type AChE1 protein from two
important disease-vectors and compared these enzymes to orthologous vertebrate AChEs. We
found that the mosquito AChE1 enzymes share many functional and structural characteristics
with the vertebrate AChE enzymes. For example, analysis of post-translational modifications
show that the mosquito enzymes have N-linked glycosylation sites (Table 1). A substrate

PLOS ONE | DOI:10.1371/journal.pone.0138598 October 8, 2015 12 / 17

 Characterization of AChEs from Disease Transmitting Mosquitoes

preference analysis showed that ATChI is the preferred substrate from a set of substrate ana-
logues (Table 2), and the mosquito AChE1 also exhibited a typical substrate inhibition behav-
ior at high substrate concentrations (Fig 2B). To extend the analysis, we generated the
insecticide-resistant G119S mutant of AgAChE1 and probed the efficacy and ligand binding
properties of the different enzymes with a selection of known substrates and inhibitors. Even
though the chemical diversity of the compounds considered in this study is limited, a cluster
analysis allowed visualization of functional trends (Fig 3A). As the analysis is based on bio-
chemical constants, the trends are related to the ligand binding properties of the different
enzymes. For substrates and covalent inhibitors (i.e. compounds that react with the catalytic
serine residue), we found that the G119S substitution induced significant changes of AgAChE1
and made the ligand binding properties of the mutant protein more similar to the properties of
mAChE and hAChE (Fig 3B). In contrast, visualizing a tree based on the constants determined
for the non-covalent inhibitors (i.e. compounds that do not react with the catalytic serine resi-
due), the G119S substitution instead made the ligand binding properties more similar to the
AgAChE1/AaAChE1 wild type proteins (Fig 3C). This finding indicates that within this group
of ligands, the non-covalent inhibitors are less sensitive to the G119S resistance mutation. It
also indicates that it may be useful to explore compounds that inhibit AChE via a different
mode of action than the currently used covalent inhibitors.

Supporting Information
S1 Fig. SDS-PAGE gel showing the molecular weight of purified mature AgAChE1 and
AaAChE1 proteins.
(DOCX)
S2 Fig. Post-translational modifications.
(DOCX)
S3 Fig. VX-titration curves.
(TIF)
S1 Table. Kinetic constants included in the cluster analysis of functional descriptors.
(DOCX)
S1 Text. Additional results and discussion regarding the analysis of post-translational
modifications in AChE1 from mosquitoes.
(DOCX)

Author Contributions
Conceived and designed the experiments: AL GB FE. Performed the experiments: CE SK S-ÅF.
Analyzed the data: CE SK S-ÅF AL GB FE. Wrote the paper: CE SK S-ÅF AL GB FE.

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