Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Jiang, Haobo; Liu, Siwei; Zhao, Peng; Pope, Carey

doi:10.1016/j.ibmb.2009.07.002

Cited by 24 publications

(45 citation statements)

References 39 publications

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“…Two AChE inhibitors (tacrine and galantamine), approved for treatment of Alzheimer’s disease, were shown to have poor Ag AChE/ h AChE inhibition selectivity, and a potent peripheral site inhibitor of h AChE (0.4 nM IC 50 , Fasciculin II, 4 kDa peptide) was shown to cause no measurable inhibition of Ag AChE at 100 nM [20]. Purified WT Ag AChE (catalytic domain) suitable for kinetic characterization was first obtained by expression of the catalytic subunit in Sf21 cells [21] K m , specific activity at saturating substrate ( V max , U/mg), and k cat were determined for the standard substrate analog acetylthiocholine (ATCh) and three other analogs. Kinetic inhibition constants were also determined for carbaryl, and the active metabolites (oxons) of malathion and parathion (Table 1) [21].…”

Section: An Gambiae Acetylcholinesterasementioning

confidence: 99%

“…Purified WT Ag AChE (catalytic domain) suitable for kinetic characterization was first obtained by expression of the catalytic subunit in Sf21 cells [21] K m , specific activity at saturating substrate ( V max , U/mg), and k cat were determined for the standard substrate analog acetylthiocholine (ATCh) and three other analogs. Kinetic inhibition constants were also determined for carbaryl, and the active metabolites (oxons) of malathion and parathion (Table 1) [21]. …”

Section: An Gambiae Acetylcholinesterasementioning

confidence: 99%

“…However, the measured k cat (7.4 × 10 3 min −1 ) of the WT Ag AChE enzyme was significantly lower than that obtained by other groups (cf. Table 2) [21,22]. Finally, transcript levels of ace -1 and ace -2 Ag AChE were studied in An.…”

Section: An Gambiae Acetylcholinesterasementioning

confidence: 99%

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Discovery of Species-selective and Resistance-breaking Anticholinesterase Insecticides for the Malaria Mosquito

Carlier

Bloomquist

Totrov

et al. 2017

CMC

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Great reductions in malaria mortality have been accomplished in the last 15 years, in part due to the widespread roll-out of insecticide-treated bednets across sub-Saharan Africa. To date, these nets only employ pyrethroids, insecticides that target the voltage-gated sodium ion channel of the malaria vector, Anopheles gambiae. Due to the growing emergence of An. gambiae strains that are resistant to pyrethroids, there is an urgent need to develop new public health insecticides that engage a different target and possess low mammalian toxicity. In this review, we will describe efforts to develop highly species-specific and resistance-breaking inhibitors of An. gambiae acetylcholinesterase (AgAChE). These efforts have been greatly aided by advances in knowledge of the structure of the enzyme, and two major inhibitor design strategies have been explored. Since AgAChE possesses an unpaired Cys residue not present in mammalian AChE, a logical strategy to achieve selective inhibition involves design of compounds that could ligate that Cys. A second strategy involves the design of new molecules to target the catalytic serine of the enzyme. Here the challenge is not only to achieve high inhibition selectivity vs human AChE, but also to demonstrate toxicity to An. gambiae that carry the G119S resistance mutation of AgAChE. The advances made and challenges remaining will be presented. This review is part of the special issue “Insecticide Mode of Action: From Insect to Mammalian Toxicity.

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Section: An Gambiae Acetylcholinesterasementioning

confidence: 99%

Section: An Gambiae Acetylcholinesterasementioning

confidence: 99%

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Discovery of Species-selective and Resistance-breaking Anticholinesterase Insecticides for the Malaria Mosquito

Carlier

Bloomquist

Totrov

et al. 2017

CMC

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“…Heretofore, the expression systems developed for AChE expression include insect (Shi et al 2001), baculovirus-infected cells (Shang et al 2007;Jiang et al 2009), COS cells (Cousin et al 1996) and Pichia pastoris expression system (Xu et al 2007). The baculovirus-insect cell expression system has proven to be an extremely valuable tool for recombinant insect AChE protein production (Shang et al 2007).…”

Section: Discussionmentioning

confidence: 99%

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Lang

Shang

Chen

et al. 2010

World J Microbiol Biotechnol

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Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.

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“…However, several types of ChE have been amplified from insects such as Aedes aegypti (Anthony et al, 1995), Aphis gossypii (Li and Han, 2002), Plutella xylostella (Ni et al, 2003), Pseudophacopteron canarium (Lin et al, 2007), Culex pipiens , Drosophila melanogaster (Zhang, 2008), and Anopheles gambiae (Jiang et al, 2009). Among these, some have been expressed in strains of bacteria or fungi (Anthony et al, 1995;Zhang, 2008;Jiang et al, 2009). The results of these studies were helpful for designing primers and selecting vectors and hosts in the present study.…”

Section: Introductionmentioning

confidence: 99%

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna

Liu

Yuan

2016

J. Zhejiang Univ. Sci. B

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Abstract:To yield cholinesterase (ChE) from prokaryotic expression, the ChE gene that belongs to Daphnia magna was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using forward primer 5'-CCCYGGNGCSAT GATGTG-3' and reverse primer 5'-GYAAGTTRGCCCAATATCT-3'. To express the gene, one sequence of the amplified DNA, which was able to encode a putative protein containing two conserved carboxylesterase domains, was connected to the prokaryotic expression vector PET-29a(+). The recombinant vector was transformed into Escherichia coil BL21 (DE3). Protein expression was induced by isopropy-D-thiogalactoside. The expressed ChE was used as an immunogen to immunize BALB/c mice. The obtained antibodies were tested for their specificity towards crude enzymes from species such as Alona milleri, Macrobrachium nipponense, Bombyx mori, Chironomus kiiensis, Apis mellifera, Eisenia foetida, Brachydanio rerio, and Xenopus laevis. Results indicated that the antibodies had specificity suitable for detecting ChE in Daphnia magna. A type of indirect and non-competitive enzyme-linked immunosorbent assay (IN-ELISA) was used to test the immunoreactive content of ChE (ChE-IR) in Daphina magna. The detection limit of the IN-ELISA was found to be 14.5 ng/ml at an antiserum dilution of 1:22 000. Results from tests on Daphnia magna exposed to sublethal concentrations of triazophos indicated a maximal induction of 57.2% in terms of ChE-IR on the second day after the animals were exposed to a concentration of 2.10 μg/L triazophos. Testing on animals acclimatized to a temperature of 16 °C indicated that ChE-IR was induced by 16.9% compared with the ChE-IR content detected at 21 °C, and the rate of induction was 25.6% at 10 °C. The IN-ELISA was also used to test the stability of ChE-IR in collected samples. Repeated freezing and thawing had no influence on the outcome of the test. All these results suggest that the polyclonal antibodies developed against the recombinant ChE are as efficient as those developed against the native ChE in detecting ChE content in Daphnia magna.

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Recombinant expression and biochemical characterization of the catalytic domain of acetylcholinesterase-1 from the African malaria mosquito, Anopheles gambiae

Cited by 24 publications

References 39 publications

Discovery of Species-selective and Resistance-breaking Anticholinesterase Insecticides for the Malaria Mosquito

Discovery of Species-selective and Resistance-breaking Anticholinesterase Insecticides for the Malaria Mosquito

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Developing antibodies from cholinesterase derived from prokaryotic expression and testing their feasibility for detecting immunogen content in Daphnia magna

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