Purification of transgenic plant-derived recombinant human acetylcholinesterase-R

Geyer, Brian C.; Muralidharan, Mrinalini; Cherni, Irene; Doran, Jeffrey D.; Fletcher, Samuel P.; Evron, Tama; Soreq, Hermona; Mor, Tsafrir S.

doi:10.1016/j.cbi.2005.10.097

Cited by 25 publications

(26 citation statements)

References 10 publications

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“…Finally, to benchmark our methods, we determined the specific activity of the commercial h AChE. The value we obtained (4,100 U/mg) is higher than that quoted by Sigma, but is lower than the 6,000 U/mg value reported in the literature [30], [31], [32].…”

Section: Resultsmentioning

confidence: 61%

Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Wong

Chen

et al. 2012

PLoS ONE

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Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (k cat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.

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Section: Resultsmentioning

confidence: 61%

Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Wong

Chen

et al. 2012

PLoS ONE

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“…The spectrophotometric Ellman assay was used to assess basic BChE activity with BTC (Sigma) as the substrate (1 mM). Assays were run at 30°C in a Spectramax 190 spectrophotometer (Molecular Devices) as previously described [8]. To evaluate cocaine hydrolysis, a previously described radiometric assay was used with 3 H cocaine as substrate over a wide range of concentrations [2].…”

Section: Methodsmentioning

confidence: 99%

Plants as a source of butyrylcholinesterase variants designed for enhanced cocaine hydrolase activity

Larrimore

Barcus

Kannan

et al. 2013

Chemico-Biological Interactions

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Cocaine addiction affects millions of people with disastrous personal and social consequences. Cocaine is one of the most reinforcing of all drugs of abuse, and even those who undergo rehabilitation and experience long periods of abstinence have an over 80% chance of relapse. Yet there is no FDA-approved treatment to decrease the likelihood of relapse in rehabilitated addicts. Recent studies, however, have demonstrated a promising potential treatment option with the help of the serum enzyme butyrylcholinesterase (BChE), which is capable of breaking down naturally occurring (−)-cocaine before the drug can influence the reward centers of the brain or affect other areas of the body. This activity of wild-type (WT) BChE, however, is relatively low. This prompted the design of variants of BChE which exhibit significantly improved catalytic activity against (−)-cocaine. Plants are a promising means to produce large amounts of these cocaine hydrolase variants of BChE, cheaply, safely with no concerns regarding human pathogens and functionally equivalent to enzymes derived from other sources. Here, in expressing cocaine-hydrolyzing mutants of BChE in Nicotiana benthamiana using the MagnICON virus-assisted transient expression system, and in reporting their initial biochemical analysis, we provide proof-of-principle that plants can express engineered BChE proteins with desired properties.

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“…Purification of WT pBChE and pAChE-R from transgenic Nicotiana benthamiana plants was recently described (29,31,32). The G117H/E197Q variant of pBChE was transiently expressed in WT plants using the MagnICON vector system (59) and was purified following a similar protocol used for WT pBChE.…”

Section: Methodsmentioning

confidence: 99%

“…As an alternative to animal-based technologies, we have evaluated the capacity of plant production of recombinant human cholinesterases (29)(30)(31) to meet this need. We report herein that human BChE assembles in planta into tetramers, unlike recombinant BChE from other sources.…”

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confidence: 99%

Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents

Geyer

Kannan

Garnaud

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...

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Purification of transgenic plant-derived recombinant human acetylcholinesterase-R

Cited by 25 publications

References 10 publications

Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Select Small Core Structure Carbamates Exhibit High Contact Toxicity to “Carbamate-Resistant” Strain Malaria Mosquitoes, Anopheles gambiae (Akron)

Plants as a source of butyrylcholinesterase variants designed for enhanced cocaine hydrolase activity

Plant-derived human butyrylcholinesterase, but not an organophosphorous-compound hydrolyzing variant thereof, protects rodents against nerve agents

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