anticholinesterase pesticides (metabolism, neurotoxicity, and epidemiology) ||...

3BUTYRYLCHOLINESTERASE: OVERVIEW,STRUCTURE, AND FUNCTION

OKSANA LOCKRIDGE, ELLEN G. DUYSEN, AND PATRICK MASSON

University of Nebraska Medical Center, Eppley Institute, Omaha, Nebraska

3.1 Introduction 25

3.2 Name 26

3.3 History 26

3.4 Why was Butyrylcholinesterase Rather thanAcetylcholinesterase Chosen as a Therapeutic? 27

3.5 Function of Butyrylcholinesterase Deduced fromStudies In People 283.5.1 Detoxify Succinylcholine and Mivacurium 283.5.2 Proposed But Not Yet Proven: Detoxify

Organophosphorus Agents, Carbamates,and Cocaine 28

3.5.3 Proposed But Not Yet Proven:Fat Metabolism 28

3.6 Function of Butyrylcholinesterase Deduced from theKnockout Mouse 283.6.1 Fat Mouse 283.6.2 Detoxify Succinylcholine and Mivacurium 283.6.3 Detoxify Cocaine 293.6.4 Hydrolysis of Acetylcholine 29

3.7 Function of Butyrylcholinesterase Deduced fromthe G117H Transgenic Mouse 29

3.8 Function of Butyrylcholinesterase Deduced fromthe AChE Knockout Mouse 30

3.8.1 Hydrolysis of Acetylcholine 303.8.2 Regulation of Acetylcholine Release 303.8.3 Controversy 30

3.9 Function of Butyrylcholinesterase Deduced fromTissue-Specific Deletions of Acetylcholinesteraseand Anchor Proteins 313.9.1 Neurotransmission and Thermoregulation 31

3.10 Function of Butyrylcholinesterase Deduced fromProtection Experiments with PurifiedButyrylcholinesterase 323.10.1 Detoxify Organophosphorus Agents 323.10.2 Reverse Succinylcholine and

Mivacurium Apnea 323.10.3 Aryl Acylamidase Activity of

Butyrylcholinesterase 33

3.11 Butyrylcholinesterase in Human Tissues 33

3.12 Summary: Functions of Butyrylcholinesterase 33

3.13 Structure of Human Butyrylcholinesterase 34

3.14 Conclusions and Future Directions 34

Acknowledgments 36

References 36

3.1 INTRODUCTION

Human butyrylcholinesterase is a soluble sugar-coatedglobular molecule that is synthesized in the liver and secretedinto the blood. Almost all tissues contain butyrylcholinester-ase, but the enzyme in plasma is the most studied. Butyryl-cholinesterase first gained the attention of researchers

and clinicians when it was discovered that the abnormalresponse to the muscle relaxant drug succinyldicholine wasexplained by natural genetic variants of plasma butyrylcho-linesterase (Kalow and Staron, 1957). Butyrylcholinesterasewas one of the first examples in the new field of pharmaco-genetics. The study of natural genetic variants of butyrylcho-linesterase led to the realization that some people have no

Anticholinesterase Pesticides: Metabolism, Neurotoxicity, and Epidemiology. Edited by Tetsuo Satoh and Ramesh C. GuptaCopyright # 2010 John Wiley & Sons, Inc.

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butyrylcholinesterase activity (Liddell et al., 1962). Despitethe complete absence of butyrylcholinesterase activity,people with silent butyrylcholinesterase are healthy (Hodgkinet al., 1965; Manoharan et al., 2007). On this basis it was con-cluded that butyrylcholinesterase has no essential functionthat cannot be compensated by other enzymes. The only situ-ation in which a function for butyrylcholinesterase was appar-ent was in response to drugs administered intravenously. Theesterase activity of butyrylcholinesterase was responsible fordestroying a large percentage of the drug before it reached itssite of activity. People with silent or atypical butyrylcholin-esterase were unable to hydrolyze the drug and thereforegot an overdose.

The butyrylcholinesterase knockout mouse and the acetyl-cholinesterase knockout mouse were made to try to uncover aphysiological function for butyrylcholinesterase (Li et al.,2008a; Xie et al., 2000). Testing in our laboratory led to theconclusion that butyrylcholinesterase has a supporting rolein neurotransmission, in that butyrylcholinesterase hydro-lyzes acetylcholine that has diffused out of the nerve synapse.This role becomes evident only when acetylcholinesterase isinhibited. Drugs currently approved for slowing the progres-sion of Alzheimer’s disease are inhibitors of acetylcholin-esterase. Toxic side effects from donepezil and huperzine Aare expected in people with the silent butyrylcholinesterasegenotype (Duysen et al., 2007).

Butyrylcholinesterase is a serine esterase. Its activity isinhibited by organophosphorus esters that make a covalentbond with the active site serine located in the consensussequence GlyXSerXGly (Lockridge, 1988). Butyrylcholin-esterase is exquisitely sensitive to inhibition by organo-phosphorus pesticides and nerve agents. The ability ofbutyrylcholinesterase to scavenge organophosphorus poisonshas made butyrylcholinesterase the new therapeutic for pro-tection against nerve agent toxicity (Doctor and Saxena,2005; Lenz et al., 2007). The U.S. Department of Defensehas invested millions of dollars to produce huge quantitiesof pure human butyrylcholinesterase to be used for protectionagainst nerve agents.

3.2 NAME

Human butyrylcholinesterase (EC 3.1.1.8) accession numberP06276 in Swiss Protein Database, accession number gi116353 in NCBI protein database, accession numberM16541 in NCBI nucleotide database, code number 1p0min the Protein Data Bank. In the older literature butyrylcholin-esterase is called pseudocholinesterase and serum cholin-esterase. In 1989 the Human Gene Mapping NomenclatureCommittee assigned the abbreviation BCHE for the geneand BChE for the butyrylcholinesterase protein. Frenchscientists and their associates use the abbreviation BuChEbecause buche is the word for a section of a tree or log in

French. The French enjoy the fact that the abbreviation foracetylcholinesterase, AChE, is the word ax in the Frenchlanguage, which nicely complements the BuChE log.

3.3 HISTORY

Butyrylcholinesterase has historically been regarded as anuninteresting second cousin of the important acetylcholin-esterase (EC 3.1.1.7). The few laboratories that engaged inbutyrylcholinesterase research were interested in human gen-etic variants, based on the fact that people with mutations intheir butyrylcholinesterase enzyme were unable to breathe forseveral hours after receiving a dose of muscle relaxantintended to paralyze for 3 min (Kalow and Staron, 1957;Lockridge, 1990; McGuire et al., 1989). Clinicians wereinterested in assays for plasma butyrylcholinesterase activitybecause low activity is associated with poisoning by organo-phosphorus pesticides and nerve agents (Ohbu et al., 1997;Yeary et al., 1993).

In 1989 Clarence Broomfield had the idea that pretreat-ment with butyrylcholinesterase could prevent nerve agenttoxicity. He tested this idea in monkeys and found that themonkeys were completely protected from two LD50 dosesof soman (Broomfield et al., 1991). The monkeys had nosalivation, lacrimation, tremor, no respiratory difficulties orseizures, and no cognitive impairment. This success was fol-lowed by many reports over the next decades confirmingthe protective effect of pretreatment with butyrylcholinester-ase in mice, rats, guinea pigs, and monkeys (Allon et al.,1998; Ashani et al., 1991; Brandeis et al., 1993; Doctoret al., 1991; Genovese and Doctor, 1995; Lenz et al., 2005,2007; Raveh et al., 1993, 1997; Saxena et al., 2006; Wolfeet al., 1992).

Doctor convinced the Pentagon generals that humanbutyrylcholinesterase was an effective therapeutic for protec-tion against nerve agent toxicity. The U.S. Department ofDefense invested in two methods for production of gramquantities of human butyrylcholinesterase. Native humanbutyrylcholinesterase was purified by Baxter Pharmaceuti-cals from outdated human plasma, starting with Cohn fractionIV-4, using a method developed by Luo in the laboratory ofSaxena and Doctor (Saxena et al., 2006). Recombinanthuman butyrylcholinesterase was expressed in the milk oftransgenic goats by Nexia Biotechnologies Inc., Canada(now PharmAthene Inc., Canada and Maryland) and purifiedin gram quantities (Huang et al., 2007, 2008).

In the meantime, Pharmavene Inc. (Rockville, Maryland)decided to develop butyrylcholinesterase for treatment ofcocaine toxicity. It was known that wild-type butyrylcholin-esterase was the esterase responsible for converting cocaineto inactive products. However, wild-type butyrylcholin-esterase destroyed cocaine slowly (Lynch et al., 1997;Stewart et al., 1977). Recombinant DNA methods allowed

26 BUTYRYLCHOLINESTERASE: OVERVIEW, STRUCTURE, AND FUNCTION

scientists to make mutants with improved cocaine hydrolaseactivity (Duysen et al., 2002a; Gao and Brimijoin, 2004;Sun et al., 2002; Xie et al., 1999; Zheng et al., 2008). SteveBrimijoin now has a quadruple mutant that hydrolyzescocaine 1000-fold faster than it is hydrolyzed by wild-typebutyrylcholinesterase. This mutant protects rats from cocainetoxicity and blocks drug seeking in rats that had previouslyself-administered cocaine (Brimijoin et al., 2008).

3.4 WHY WAS BUTYRYLCHOLINESTERASERATHER THAN ACETYLCHOLINESTERASECHOSEN AS A THERAPEUTIC?

Acetylcholinesterase has the important physiologic functionof hydrolyzing the neurotransmitter acetylcholine in nervesynapses. The lethality of organophosphorus agents isexplained by inhibition of acetylcholinesterase, followed byoverstimulation of acetylcholine receptors, influx of calcium,and a cascade of imbalances that lead to respiratory arrest(McDonough and Shih, 1997). Acetylcholinesterase reactsrapidly with the most toxic stereoisomers of nerve agentsand organophosphorus agents, and has been shown to protectanimals from toxicity (Ashani et al., 1991; Doctor et al.,1991; Maxwell et al., 1992, 1993; Raveh et al., 1989;Wolfe et al., 1992). Acetylcholinesterase would seem to bea good candidate for use as a therapeutic. However, to dateacetylcholinesterase has not been selected for mass pro-duction and clinical trials.

The requirements for an enzyme to be an effective scaven-ger for organophosphorus toxicants were defined as follows(Doctor et al., 1991). (1) The enzyme must react rapidlywith organophosphorus toxicants. (2) It should be availablein sufficient quantities. (3) It should be stable on prolongedstorage. (4) The enzyme should have a long half-life in vivo.(5) It should not be immunoreactive. (6) The stoichiometrybetween the scavenger enzyme and the toxic agent shouldapproach 1 :1. (7) Large quantities of the enzyme shouldcause no side effects or performance decrements.

Human acetylcholinesterase fulfills all the criteria except(2) and (4). There is no good source of soluble, nativehuman acetylcholinesterase. Human red blood cells haveacetylcholinesterase bound to their outer surface via a glyco-lipid anchor (Roberts et al., 1988). The acetylcholinesterase isa disulfide linked dimer containing three carbohydrate chainsper monomer (Rosenberry and Scoggin, 1984; Velan et al.,1993). A liter of whole blood contains about 0.5 mg acetyl-cholinesterase, a quantity that is 20% of the amount ofbutyrylcholinesterase in whole blood. Release of acetylcho-linesterase from red blood cells is not as simple as extractionof the soluble butyrylcholinesterase from plasma (Lockridgeet al., 2005; Rosenberry et al., 1981). Monomeric and dimericacetylcholinesterase are cleared from the circulation of ani-mals within a few minutes (Chitlaru et al., 2001; Saxena

et al., 1998). Another disadvanatage is that purified acetyl-cholinesterase sticks to plastic and glass resulting in largelosses unless the protein is highly concentrated or albuminis added.

Human butyrylcholinesterase fulfills all the criteria for aneffective scavenger.

1. It reacts rapidly with organophosphorus toxicants(Raveh et al., 1993).

2. Outdated human plasma contains soluble butyryl-cholinesterase at a concentration of 5 mg/L.

3. The sugarcoated surface of the butyrylcholinesterasetetramer protects it from proteases. The purifiedenzyme is stable for years (Grunwald et al., 1997;Lockridge et al., 2005; Saxena et al., 2006).

4. Injected purified human butyrylcholinesterase tetra-mers have a half-life in the human circulation of 8 to12 days (Jenkins et al., 1967; Ostergaard et al., 1988;Stovner and Stadskleiv, 1976).

5. Human butyrylcholinesterase injected into humans isnot expected to cause an immune reaction. The phaseI clinical trials currently underway are testing this.Preliminaryevidence from injection of monkey butyryl-cholinesterase into monkeys has shown no immunereaction (Rosenberg et al., 2002). Multiple transfusionsof blood containing human butyrylcholinesterase havecaused no adverse effects in humans despite the exist-ence of naturally occurring mutations in butyrylcholin-esterase (Souza et al., 2005).

6. Human butyrylcholinesterase covalently binds onemolecule of toxicant per molecule of enzyme (Ravehet al., 1993, 1997).

7. No adverse side effects or performance decrementshave been observed following administration of quan-tities of butyrylcholinesterase up to 800 times higherthan the endogenous blood levels in animals (Brandeiset al., 1993; Genovese and Doctor, 1995; Lynch et al.,1997; Matzke et al., 1999; Raveh et al., 1993).

Another advantage of butyrylcholinesterase is its widesubstrate specificity. It hydrolyzes many more esters thanare hydrolyzed by acetylcholinesterase. It can be used notonly for protection from the toxicity of nerve agents and pes-ticides, but also for protection from succinylcholine apnea,mivacurium apnea, and cocaine toxicity (Ashani, 2000).

The scientists who chose to develop butyrylcholinesteraseas a therapeutic also looked into the future when catalyticbioscavengers would replace native butyrylcholinesterase.The limitation of native butyrylcholinesterase is that it inacti-vates a stoichiometric equivalent of nerve agent so that a hugedose (200 to 300 mg) of costly enzyme is needed to protectone human against several LD50 doses of nerve agent. Anideal therapeutic would inactivate hundreds of molecules of

3.4 WHY WAS BUTYRYLCHOLINESTERASE RATHER THAN ACETYLCHOLINESTERASE CHOSEN AS A THERAPEUTIC? 27

nerve agent per molecule of enzyme. Butyrylcholinesterasehas been successfully mutated to a catalytic bioscavenger inthe glycine 117 histidine mutant (Millard et al., 1995). Therate of hydrolysis is not yet fast enough to justify large-scale production of the G117H mutant, but other mutationsmay be found that increase its organophosphate hydrolaseactivity. To date mutagenesis of acetylcholinesterase hasyielded no mutant capable of hydrolyzing organophosphoruspesticides and nerve agents at a better rate than the G117Hmutant of butyrylcholinesterase (Poyot et al., 2006).

A limitation of butyrylcholinesterase is that it preferen-tially scavenges the less toxic stereoisomers of nerve agents.

3.5 FUNCTION OF BUTYRYLCHOLINESTERASEDEDUCED FROM STUDIES IN PEOPLE

3.5.1 Detoxify Succinylcholine and Mivacurium

People with silent butyrylcholinesterase are healthy, fertile,and live to old age (Manoharan et al., 2007). An inheritedmutation in their butyrylcholinesterase gene results in com-plete absence of butyrylcholinesterase activity (Manoharanet al., 2006). The observation that people with the silentbutyrylcholinesterase genotype have no health problemsuntil they are challenged with drugs has led to the conclusionthat the normal physiological function of butyrylcholin-esterase, if any, can be compensated by other systems.

The drugs for which there is documented evidence thatbutyrylcholinesterase functions in detoxication are themuscle relaxants succinylcholine and mivacurium. Peoplewith silent and atypical (Asp 70 Gly) butyrylcholinesteraserequire mechanical ventilation for up to 2 hours after an intra-venous dose that paralyzes most people for 3 min (Gatkeet al., 2001; Kalow and Staron, 1957; Liddell et al., 1962).

3.5.2 Proposed But Not Yet Proven: DetoxifyOrganophosphorus Agents, Carbamates, and Cocaine

A role for butyrylcholinesterase in detoxication of poisonousesters such as organophosphorus pesticides, nerve agents,carbamates, and natural toxic esters like cocaine has been pro-posed but not proven (Hoffman et al., 1992; Jbilo et al., 1994;Kalow, 2004). In vitro and animal studies support the notionthat butyrylcholinesterase has a role in detoxication of thesepoisons but human data are not available. It is not knownwhether people with silent butyrylcholinesterase are moresusceptible to the toxicity of these agents.

3.5.3 Proposed But Not Yet Proven: Fat Metabolism

Butyrylcholinesterase inactivates octanoyl ghrelin, a28-amino-acid hormone that stimulates feeding and promotesweight gain through its metabolic actions, decreasing energyexpenditure and fat catabolism (De Vriese et al., 2004; Ueno

et al., 2005). It was expected that butyrylcholinesterasedeficiency would be associated with obesity. However, thesilent butyrylcholinesterase subjects had normal weight(Manoharan et al., 2007), suggesting that alternative routesof hydrolysis of octanoyl ghrelin compensate for the absenceof butyrylcholinesterase. Mice deficient in butyrylcholin-esterase did become obese, but only when fed a high-fatdiet (Li et al., 2008b). Wild-type mice fed the same high-fat diet did not become obese.

3.6 FUNCTION OF BUTYRYLCHOLINESTERASEDEDUCED FROM THE KNOCKOUT MOUSE

3.6.1 Fat Mouse

The butyrylcholinesterase knockout mouse has no butyryl-cholinesterase in any organ or tissue (Li et al., 2008a). Ithas no obvious phenotype when fed a standard 5% fat mousediet. However, when it is fed an 11% fat diet it becomesobese, weighing 30% more than wild-type adult mice (Liet al., 2008b). Butyrylcholinesterase is one of the esterasesthat inactivates octanoyl-ghrelin by hydrolyzing the esterbond (De Vriese et al., 2004). A possible mechanism toexplain the obesity might involve inadequate deesterificationof octanoyl-ghrelin, a peptide hormone that stimulates feed-ing and is involved in energy utilization (Tschop et al.,2000). An ELISA method using antibodies to acylated andunacylated ghrelin did not support this hypothesis becauseacylated ghrelin levels were lower, rather than higher, inplasma of the knockout mice compared to wild-type mice.Measurement of food intake and motor activity showed thatobesity of the knockout mice was not explained by increasedfood intake or by lack of exercise. It was concluded thatbutyrylcholinesterase deficiency resulted in decreased fatmetabolism. A role for butyrylcholinesterase in fat utilizationhas previously been suggested by others (Kutty et al., 1981).

3.6.2 Detoxify Succinylcholine and Mivacurium

Succinylcholine and mivacurium muscle relaxants are admi-nistered to humans intravenously. The drugs relax the throatmuscles allowing tracheal intubation of a plastic tube throughwhich oxygen and anesthesia gases are delivered to the lungs.About 90% of the succinylcholine is destroyed by butyryl-cholinesterase in the plasma, so that only 10% reaches thenerve synapses. People with silent or atypical genetic variantsof butyrylcholinesterase destroy none of the injected dose.The huge overdose accumulates in the neuromuscular junc-tions and paralyzes the muscles for 2 hours until the succinyl-choline diffuses away (Lockridge, 1990).

Butyrylcholinesterase knockout mice died when they weretreated with a dose of succinylcholine that was safe forwild-type mice (Li et al., 2008a). This shows that the miceare suitable models for succinylcholine apnea of humans.


3.6.3 Detoxify Cocaine

In man, butyrylcholinesterase hydrolyzes cocaine to the non-toxic products benzoic acid and ecgonine methyl ester(Stewart et al., 1977). An alternative metabolic route oxidizescocaine through the action of cytochrome P450 enzymes tonorcocaine nitroxide, a reactive metabolite that binds to pro-teins and causes hepatotoxicity (Ndikum-Moffor et al., 1998;Ndikum-Moffor and Roberts, 2003).

Butyrylcholinesterase knockout mice treated with cocainedaily for 7 days had abnormal liver histology and cardiacfibrosis (Duysen et al., 2008). Wild-type mice treated withthe same schedule of cocaine had less hepatotoxicity andno cardiac fibrosis. It was concluded that absence of butyryl-cholinesterase drove cocaine metabolism through the cyto-chrome P450 path to produce high levels of the toxiccocaine metabolite. This could explain the hepatotoxicity ofcocaine to butyrylcholinesterase knockout mice. The impli-cation for humans is that people with silent butyrylcholin-esterase are likely to suffer hepatotoxicity from doses ofcocaine that most people can tolerate.

3.6.4 Hydrolysis of Acetylcholine

When butyrylcholinesterase knockout mice with 0% ofnormal butyrylcholinesterase activity, heterozygotes with50%, and wild-type mice with 100% of normal butyrylcholin-esterase activity were treated with the specific acetylcholin-esterase inhibitors huperzine A and donepezil, all showedsigns of toxicity. The most severe signs were in mice comple-tely deficient in butyrylcholinesterase; these mice died within3 h of subcutaneous treatment with 10 mg/kg donepezil, andwithin 10 min of subcutaneous treatment with 1.5 mg/kghuperzine A (Duysen et al., 2007). The heterozygote micehad intermediate signs of toxicity but were moribund by24 h. The wild-type mice completely recovered by 24 h.Plasma acetylcholinesterase activity was inhibited 80% at30 min post dosing with huperzine A but had returned tonearly normal by 18 h. Such a dose of huperzine A did notinhibit plasma butyrylcholinesterase. The specificity ofhuperzine A and donepezil as acetylcholinesterase inhibitorswas confirmed by the finding that acetylcholinesterase knock-out mice were completely resistant to the effects of thesedrugs (Boudinot et al., 2005; Duysen et al., 2007).

These results showed that butyrylcholinesterase protectedmice from the toxic effects of acetylcholinesterase inhibitors.A mechanism to explain protection by butyrylcholinesterasewas needed. A role for butyrylcholinesterase as a scavengerwas ruled out because huperzine A did not inhibit butyryl-cholinesterase in vivo, and was a poor inhibitor of purifiedenzyme (Ashani et al., 1992). The protection could beexplained by hydrolysis of acetylcholine by butyrylcholin-esterase. Wild-type mice whose acetylcholinesterase had beeninhibited still had butyrylcholinesterase available for hydro-lyzing acetylcholine. However, the butyrylcholinesterase

knockout mice had no backup acetylcholine hydrolaseactivity to hydrolyze acetylcholine; they died after tonic con-vulsions, a symptom characteristic of excess acetylcholine inthe brain. It was concluded that butyrylcholinesterase has aphysiological function in hydrolysis of acetylcholine in vivo.

Although acetylcholine is hydrolyzed by butyrylcholin-esterase fourfold more slowly than by acetylcholinesterase,butyrylcholinesterase may compensate in a crisis situationfollowing exposure to inhibitors and make the differencebetween life and death. Humans can have unusually lowbutyrylcholinesterase activity due to genetic variation, liverdisease, malnutrition, or iatrogenic causes (Whittaker,1980). On the basis of the results with butyrylcholinesterasedeficient mice it can be predicted that people with reducedbutyrylcholinesterase activity may have toxic side effectsfollowing treatment with the specific acetylcholinesteraseinhibitors donepezil HCl (Ariceptw), currently licensed forAlzheimer’s disease, and (2)-huperzine A, a drug in clinicaltrials for Alzheimer’s disease (clinical trial NCT00083590)(Little et al., 2008).

3.7 FUNCTION OF BUTYRYLCHOLINESTERASEDEDUCED FROM THE G117H TRANSGENICMOUSE

The Gly117 His mutant of human butyrylcholinesterasehydrolyzes organophosphorus agents (Lockridge et al., 1997;Millard et al., 1995, 1998). It also hydrolyzes acetylcho-line and butyrylcholine. Transgenic mice that express humanG117H butyrylcholinesterase in all tissues were created bygenetic engineering (Wang et al., 2004). The level of G117Hactivity was no more than 25% of the endogenous butyrylcho-linesterase activity. In addition to G117H, the transgenic micehad normal endogenous acetylcholinesterase and butyryl-cholinesterase activities. When G117H transgenic and wild-type mice were treated with echothiophate, the G117Htransgenic mice survived but the wild-type mice died. Thepossibility was considered that the survival of the transgenicmice was due to hydrolysis of echothiophate by G117H. Ifturnover of echothiophate was a factor then the acetylcholin-esterase and butyrylcholinesterase activities in the transgenicmice should have been protected. However, it was found thatthe endogenous acetylcholinesterase and butyrylcholin-esterase activities were inhibited to a similar extent in bothgroups. The G117H butyrylcholinesterase was not inhibitedbecause G117H butyrylcholinesterase is resistant to inhibitionby organophosphorus agents. The survival of the G117Htransgenic mice can be explained by the fact that these micehad G117H butyrylcholinesterase available to hydrolyzeacetylcholine. The G117H butyrylcholinesterasewas assumedto be outside the nerve synapse. It suggests that butyryl-cholinesterase located outside the synaptic cleft hydrolyzesacetylcholine that diffuses out of the synapse and that thishydrolysis is sufficient to keep the mouse alive.

3.7 FUNCTION OF BUTYRYLCHOLINESTERASE DEDUCED FROM THE G117H TRANSGENIC MOUSE 29

3.8 FUNCTION OF BUTYRYLCHOLINESTERASEDEDUCED FROM THE AChE KNOCKOUTMOUSE

3.8.1 Hydrolysis of Acetylcholine

The acetylcholinesterase knockout mouse has no acetylcho-linesterase in any tissue (Li et al., 2000; Xie et al., 2000).AChE2/2 mice live to adulthood but only with special care(Duysen et al., 2002b). They are fed a liquid diet in a dishon the floor of the cage. Their muscles are so weak thatthey cannot lift their head to eat food pellets placed on thecage lid. They are smaller than their normal litter mates atall stages of life. As adults they weigh about 60% ofnormal. They do not climb. They have no grip strength.They are sexually dysfunctional as they do not mate and donot reproduce. They are blind (Bytyqi et al., 2004).Acetylcholine levels in the hippocampus are 60-fold higherthan normal (Hartmann et al., 2007). They have adapted tothe high acetylcholine levels by reducing muscarinic receptorlevels in the brain (Bernard et al., 2003; Li et al., 2003;Volpicelli-Daley et al., 2003), and nicotinic receptor levelsin the diaphragm muscle to 50% of normal (Adler et al.,2004). Adrenoceptors that oppose the action of muscarinicreceptors have also been downregulated, thus maintaining aproper balance of receptors and accounting for lung functionin these animals (Myslivecek et al., 2007). Soleus muscleshave reduced numbers of slow muscle fibers, reducedmuscle weight and cross-sectional area, and reduced absolutemaximal isometric force (Vignaud et al., 2008).

The diaphragm muscle endplates of AChE2/2 miceexhibit marked abnormalities, including fragmented nerveterminals, irregular junctional folds, regions of postjunctionalmembrane lacking innervation, and extension of Schwanncell processes into the synaptic cleft (Adler et al., 2004;Girard et al., 2005). It was suggested that the cumulativeeffect of synaptic remodeling may be to reduce the presyn-aptic and postsynaptic contacts and to open additional path-ways for diffusion of acetylcholine.

Acetylcholinesterase knockout mice are alive because theyhave butyrylcholinesterase to hydrolyze the acetylcholinethat diffuses out of the synaptic cleft. Their requirement forbutyrylcholinesterase activity is demonstrated by studies inwhich butyrylcholinesterase activity is inhibited with bam-buterol, a selective inhibitor of butyrylcholinesterase. Abambuterol dose that inhibits mouse plasma butyrylcho-linesterase 94% causes respiratory failure and death in acetyl-cholinesterase knockout mice but has no effect on wild-typemice (Chatonnet et al., 2003). It was concluded that butyryl-cholinesterase compensates for the absent acetylcholin-esterase by hydrolyzing acetylcholine at the neuromuscularjunction (Adler et al., 2004; McArdle et al., 2005; Mesulamet al., 2002). Butyrylcholinesterase is not a good substitute,however, since the AChE2/2 mouse is greatly impaired.

Acetylcholinesterase knockout mice displayed the samecholinergic signs of poisoning as wild-type mice followingtreatment with the nerve agent VX (Duysen et al., 2001).The cholinergic signs of toxicity—lacrimation, salivation,mucus in the eyes, tremor, loss of motor activity, flattenedposture, peripheral vasodilation, hypothermia, gasping, uri-nation, and tonic convulsions—are attributed to overstimu-lation of acetylcholine receptors by excess acetylcholine.Since acetylcholinesterase knockout mice have no acetyl-cholinesterase, the excess acetylcholine resulting from VXtreatment can only have come from inhibition of butyryl-cholinesterase. The butyrylcholinesterase was inhibited50% in these mice. It was concluded that butyrylcholin-esterase hydrolyzes acetylcholine in acetylcholinesteraseknockout mice.

A similar conclusion was reached in microdialysisstudies of acetylcholine levels in acetylcholinesterase knock-out brain where inhibition of butyrylcholinesterase activitywith bambuterol or bis-norcymserine elevated the acetyl-choline level fivefold (Hartmann et al., 2007).

3.8.2 Regulation of Acetylcholine Release

Electrophysiological recordings of end plate potential inhemidiaphragm preparations from wild-type and acetyl-cholinesterase knockout mice were compared (Minic et al.,2003). It was concluded that inhibition of butyrylcholin-esterase by iso-OMPA or bambuterol reduces the numberof quanta of acetylcholine released from presynaptic nervetermini. A similar conclusion was reached in studies of respir-ation (Chatonnet et al., 2003). When acetylcholinesteraseknockout mice were treated with the butyrylcholinesteraseinhibitor bambuterol, they had weaker respiratory musclecontraction, measured as reduced tidal volume of the lungs.In contrast, inhibition of acetylcholinesterase in wild-typemice resulted in stronger muscle contractions, measured asincreased tidal volume. These results were taken to meanthat there is too little acetylcholine after butyrylcholin-esterase is inhibited. It was concluded that butyrylcholin-esterase has no function in hydrolysis of acetylcholine,and instead it was proposed that butyrylcholinesterase func-tions to modulate release of acetylcholine quanta from ves-icles in the presynaptic nerve termini. It was hypothesizedthat the lethal effect of butyrylcholinesterase inhibition inacetylcholinesterase knockout mice is due to decreasedrelease of transmitter, so that the postsynaptic membraneis inadequately stimulated.

3.8.3 Controversy

The interpretation that butyrylcholinesterase inhibitionresults in too little acetylcholine rather than too much is atodds with our conclusion. It does not fit our observationthat the acetylcholinesterase knockout mouse after treatment


with a butyrylcholinesterase inhibitor has the classic cholin-ergic signs of toxicity. Lacrimation, salivation, and tremorare attributed to excess acetylcholine, rather than to insuffi-cient acetylcholine. Direct measurement of acetylcholinelevels in the brain of the acetylcholinesterase knockoutmouse showed that acetylcholine levels increased fivefoldafter butyrylcholinesterase inhibition (Hartmann et al.,2007). It could be argued that peripheral and central effectsof butyrylcholinesterase inhibition are different, so that theexcess acetylcholine found in the brain does not representwhat happens in the muscle. But how does inadequate acetyl-choline account for cholinergic signs of toxicity in the periph-ery? Insufficient acetylcholine, as in poisoning by botulinumtoxin, causes dry mouth, not salivation. This controversycould be resolved by directly measuring acetylcholinelevels in muscle of acetylcholinesterase knockout mice.

3.9 FUNCTION OF BUTYRYLCHOLINESTERASEDEDUCED FROM TISSUE-SPECIFIC DELETIONSOF ACETYLCHOLINESTERASE AND ANCHORPROTEINS

3.9.1 Neurotransmission and Thermoregulation

Acetylcholinesterase is encoded by a single gene on humanchromosome 7q22 and mouse chromosome 5 (Getmanet al., 1992). Acetylcholinesterase has soluble andmembrane-bound forms created by alternative splicing andby binding to anchor proteins (Noureddine et al., 2008).Butyrylcholinesterase is encoded by a single gene onhuman chromosome 3q26 and mouse chromosome 3, butthere are no alternatively spliced variants (Allderdice et al.,1991). Butyrylcholinesterase is anchored to membranes viathe same anchor proteins, PRiMA and COLQ, used by

acetylcholinesterase. The soluble butyrylcholinesterase tetra-mer has lamellipodin-derived polyproline-rich peptideswhose function is to organize the four subunits into a tetramer(Li et al., 2008c). Similar lamellipodin-derived polyproline-rich peptides may be present in soluble acetylcholinesterasetetramers, but this has not yet been reported.

Deletion of a small section of intron 1 of the ACHE gene(Table 3.1) produces a mouse that has no acetylcholinesterasein muscle, but has normal expression in the central nervoussystem (Camp et al., 2008). Inhibition of butyrylcholinester-ase with bambuterol did not affect breathing frequency, butdid lower tidal volume and body temperature (Boudinotet al., 2009) in the intron 1 deletion mutant but not in wild-type mice. It was concluded that butyrylcholinesterase has arole in thermoregulation.

Exon 5 is the alternatively spliced exon used for glycolipidanchored acetylcholinesterase found in red blood cells. Exon6 is the alternatively spliced exon found in soluble G4 tetra-mers as well as in acetylcholinesterase anchored to the col-lagen tail and anchored to PRiMA. Deletion of exons 5 and6 produces a mouse with very low acetylcholinesteraseactivity in the brain and neuromuscular junction, butincreased monomeric acetylcholinesterase in plasma (Campet al., 2008). Inhibition of butyrylcholinesterase with bam-buterol caused a decrease in tidal volume and body tempera-ture in the mutant mice, but not in wild-type mice (Boudinotet al., 2009). It was concluded that butyrylcholinesterase hasa role in thermoregulation.

The PRiMA gene codes for a protein that anchors acetyl-cholinesterase and butyrylcholinesterase to cell membranesin the brain and muscle (Perrier et al., 2002). Deletion ofthe PRiMA gene results in a mouse that has no acetylcholin-esterase or butyrylcholinesterase in brain. The phenotype ofthe PRiMA mutant was indistinguishable from that of the

TABLE 3.1 Gene Deletions that Affect Expression of Acetylcholinesterase and Butyrylcholinesterase

Gene Deletion Tissue Affected Phenotype References

Intron 1 of ACHE gene No AChE in muscle; normal AChEin brain and spinal cord

Inhibition of BChE reduces temperature,reduces tidal volume

Boudinot et al. (2009);Camp et al. (2008)

Exons 5 and 6 ofACHE gene

Very low AChE in brain andmuscle; high AChE in serum

Inhibition of BChE reduces temperature,reduces tidal volume

Boudinot et al. (2009);Camp et al. (2008)

PRiMA gene No AChE or BChE in brain Normal respiration, normal body weight,normal temperature; inhibition of BChEhas no effect

Boudinot et al. (2009)

COLQ gene No AChE or BChE atneuromuscular junction

Weak muscles Feng et al. (1999)

ACHE gene No AChE in any tissue Weak muscles, seizures, low body weight,hyperventilates, poor temperaturecontrol; inhibition of BChE is lethal

Duysen et al. (2002b);Boudinot et al. (2009)

BCHE gene No BChE in any tissue Succinylcholine apnea; hepatotoxicity fromcocaine; sensitive to huperzine A anddonepezil; inhibition of AChE is lethal

Duysen et al. (2007,2008); Li et al. (2008a)

3.9 BUTYRYLCHOLINESTERASE DEDUCED FROM TISSUE-SPECIFIC DELETIONS OF ACETYLCHOLINESTERASE 31

wild-type mouse. It was concluded that acetylcholinesteraseand butyrylcholinesterase in the central nervous systemappear dispensable in the control of respiration (Boudinotet al., 2009).

The COLQ gene codes for the collagen tail that anchorsacetylcholinesterase and butyrylcholinesterase in the neuro-muscular junction (Krejci et al., 1997). Inhibition of butyryl-cholinesterase affects muscle contraction in mice that lackacetylcholinesterase at the neuromuscular junction, but isnot lethal when the mice have acetylcholinestrase and butyr-ylcholinesterase in other locations. Survival may be explainedby the presence of acetylcholinesterase and butyrylcholin-esterase in the circulation of these animals. It was suggestedthat nerve-released acetylcholine can diffuse from the neuro-muscular junction into the blood where it is hydrolyzed byacetylcholinesterase and butyrylcholinesterase. This givesbutyrylcholinesterase a role in neurotransmission.

3.10 FUNCTION OF BUTYRYLCHOLINESTERASEDEDUCED FROM PROTECTION EXPERIMENTSWITH PURIFIED BUTYRYLCHOLINESTERASE

3.10.1 Detoxify Organophosphorus Agents

Pure human butyrylcholinesterase injected into monkeys,rats, mice, and guinea pigs protected animals from the lethal-ity of sarin, soman, tabun, and VX (Allon et al., 1998; Ashaniet al., 1991; Brandeis et al., 1993; Doctor et al., 1991; Doctorand Saxena, 2005; Genovese and Doctor, 1995; Lenz et al.,2005, 2007; Raveh et al., 1993, 1997; Saxena et al., 2006;Wolfe et al., 1992). Stoichiometric quantities of butyrylcholi-nesterase were required to scavenge 2 to 8 LD50 doses ofnerve agent. Protection worked best when nerve agent wasgiven in small repeated doses rather than in a single dose(Doctor and Saxena, 2005; Lenz et al., 2007; Raveh et al.,1997). Administration of a single large dose allowed someof the nerve agent to escape inactivation by butyrylcholin-esterase. The nerve agent entered compartments not accessibleto the 340 kDa butyrylcholinesterase.

There are no reports in animals that pure butyrylcholin-esterase protects from the toxicity of organophosphorus pesti-cides, though protection from pesticides is expected. Thereare three case reports of the clinical use of partially purifiedhuman butyrylcholinesterase to treat poisoning by organo-phosphorus pesticides. Patients were treated with 5% purebutyrylcholinesterase purified from human plasma, a productsold by Behringwerke (Marburg, Germany).

1. The patient poisoned with 15 g parathion was still onartificial respiration three weeks later (Goedde andAltland, 1971). He was treated with three doses ofbutyrylcholinesterase over a period of 7 days. Afterthe third dose, his plasma butyrylcholinesterase

activity went from zero to 40% of normal and there-after continued to rise. The patient recovered. The455 mg of partially pure enzyme he received calculatesto 23 mg of pure butyrylcholinesterase.

2. A 26-year-old man injected himself intramuscularlywith 3.75 g of demeton methyl in a suicide attempt(Klose and Gutensohn, 1976). He lost consciousnessand ceased breathing. Standard therapy with atropineand obidoxime did not improve his condition. He wastreated with 540 mg of partially purified butyrylcholin-esterase (containing 25 mg of pure butyrylcholinester-ase) on days 3 and 4. He continued to receiveatropine for an additional 8 days. The patient recovered.

3. A patient who attempted suicide with parathion deve-loped coma, respiratory arrest, muscle fasciculations,miosis, and incontinence. He was treated with atropine,pralidoxime, and purified butyrylcholinesterase. Thebutyrylcholinesterase was administered on days 2 to14 for a total of 1272 mg crude enzyme containing64 mg butyrylcholinesterase. Improvement in thepatient’s condition correlated with increased plasmabutyrylcholinesterase activity.

No adverse effects from treatment with butyrylcholinester-ase were found in these cases. It is not clear whether recoverywas due to treatment with butyrylcholinesterase or to treat-ment with standard therapeutics.

3.10.2 Reverse Succinylcholineand Mivacurium Apnea

Succinylcholine is a fast acting muscle relaxant used in con-junction with anesthetics prior to surgery. It is also known assuxamethonium, Anectine, and Scoline. Most patientsresume spontaneous breathing within 3 to 5 min of a standarddose. However, people with genetic variants of butyrylcholin-esterase are paralyzed for up to 2 h and require assistedventilation. Succinylcholine blocks neurotransmission bybinding to nicotinic acetylcholine receptors in the muscles.

Mivacurium (Mivacronw, Abbott Labs) is a short-actingmuscle relaxant used to facilitate intubation as an adjunct togeneral anesthesia. Its manufacture has been discontinuedin the United States for business reasons.

The literature reports 155 humans treated with puri-fied butyrylcholinesterase to reverse succinylcholine- ormivacurium-induced apnea (Table 3.2). The injected butyryl-cholinesterase was effective in restoring spontaneous breath-ing, but only if the butyrylcholinesterase was given duringcomplete neuromuscular block. For a person with the atypicalgenetic variant (Asp 70 Gly) complete neuromuscularblock lasted for 30 to 50 min after succinylcholine. If thebutyrylcholinesterase was injected 90 min after succinyl-choline, it had no effect (Viby-Mogensen, 1981).


3.10.3 Aryl Acylamidase Activityof Butyrylcholinesterase

Butyrylcholinesterase was found to hydrolyze aryl acyla-mides. It was hypothesized that this activity may be relatedto possible noncholinergic functions of this enzyme(Balasubramanian and Bhanumathy, 1993; Darvesh et al.,2006). However, poor catalytic properties of purified humanbutyrylcholinesterase with different aryl acylamide substratesdo not support this hypothesis (Masson et al., 2007, 2008).

3.11 BUTYRYLCHOLINESTERASEIN HUMAN TISSUES

The butyrylcholinesterase enzyme is best known for itspresence in human plasma where its concentration is about5 mg/L. However, tissues other than blood are also richin butyrylcholinesterase. Butyrylcholinesterase mRNA ispresent in human tissues, in the following relative quantities:liver . lung . brain . heart . skeletal muscle . pancreas(Jbilo et al., 1994). No butyrylcholinesterase mRNA wasdetected in placenta. A study of post-mortem tissuesshowed butyrylcholinesterase activity in human liver .

lung . spleen . stomach . kidney . small intestine . cere-bellum . plasma . medulla oblongata . cerebral cortex .

heart . leg muscle . skin . thyroid (Manoharan et al.,2007). The presence of butyrylcholinesterase activity inskin has been confirmed (Schallreuter et al., 2007). Humansemen shows butyrylcholinesterase activity (Pedron, 1983).

Butyrylcholinesterase in cerebrospinal fluid originatesfrom serum and the central nervous system (Kluge et al.,2001). Sweat, urine, lacrimal fluid, and saliva have about1/1500 of the butyrylcholinesterase activity of humanplasma (Ryhanen, 1983).

Elevated butyrylcholinesterase levels occur in humanglioblastoma (Razon et al., 1984), and in amyloid plaquesin brains of Alzheimer disease patients (Mesulam andGeula, 1994).

3.12 SUMMARY: FUNCTIONS OFBUTYRYLCHOLINESTERASE

In summary, endogenous butyrylcholinesterase in humanplasma, liver, lung, intestinal tract, and skin serves to detoxifypoisons. By scavenging organophosphorus pesticides andnerve agents butyrylcholinesterase protects the neurotrans-mitter function of acetylcholinesterase, preventing respiratoryfailure and death. By inactivating cocaine, butyrylcholin-esterase protects overstimulation by dopamine, thus pre-venting seizures, heart failure, and death. By inactivatingsuccinylcholine and mivacurium, butyrylcholinesterase pro-tects acetylcholine receptors, where blocking of neurotrans-mission paralyzes the breathing muscles.

Butyrylcholinesterase acts as a backup for acetylcholin-esterase by hydrolyzing acetylcholine that has diffused outof nerve synapses.

Butyrylcholinesterase has a role in thermoregulation, andin fat utilization.

TABLE 3.2 Clinical Use of Purified Human Butyrylcholinesterase

No. of PatientsReason for Using

Butyrylcholinesterase Source References

4 Succinylcholine apnea Cholase from Cohn fraction IV-6 Evans et al. (1953)23 Succinylcholine Cholase Borders et al. (1955)6 Succinylcholine Behring, Marburg Goedde and Altland (1971)1 Succinylcholine Behringwerke Stovner and Stadskleiv (1976)1 Succinylcholine Behringwerke Ploier (1975)15 Succinylcholine Behringwerke Scholler et al. (1977)1 Succinylcholine Behringwerke, Marburg Schuh (1977)13 Succinylcholine Cholase Viby-Mogensen (1981)1 Succinylcholine Behringwerke Benzer et al. (1992)2 Mivacurium Behringwerke Ostergaard et al. (1995)40 Mivacurium Behringwerke Naguib et al. (1995a)1 Mivacurium Behringwerke Naguib et al. (1995b)20 Mivacurium Behringwerke Naguib et al. (1996a)16 Mivacurium Behringwerke Naguib et al. (1996b)11 Mivacurium Centeon Pharma, Marburg Ostergaard et al. (2005)Total 155

Note: The butyrylcholinesterase preparation contained 90 mg protein recovered from 1000 mL serum. Since human serum contains 4 to 5 mg of butyrylcholin-esterase in 1000 mL, the preparation is estimated to be 5% pure.

3.12 SUMMARY: FUNCTIONS OF BUTYRYLCHOLINESTERASE 33

3.13 STRUCTURE OF HUMANBUTYRYLCHOLINESTERASE

Human butyrylcholinesterase in serum is a soluble, globulartetramer of four identical subunits. Each subunit has 574amino acids, nine asparagine-linked carbohydrate chains,and three internal disulfide bonds (Lockridge et al., 1987b).The internal disulfide bonds are between Cys65-Cys92,Cys252-Cys263, and Cys400-Cys519. A disulfide bond atCys571 joins two subunits (Lockridge et al., 1987a). Thisinterchain disulfide bond is located four amino acids fromthe carboxy terminus. The tetramer is a dimer of dimers.Reduction of the interchain disulfide bond has no effect ontetramer assembly. The butyrylcholinesterase remains a tetra-mer because the four subunits are linked through the tetramer-ization domain at the carboxy terminus (Altamirano andLockridge, 1999; Blong et al., 1997). The 40 amino acidsat the C-terminus form an alpha helix and the four alphahelices interact through a proline-rich peptide which threadsthrough the center of the four-helix bundle (Li et al.,2008c). The tetramerization domain extends out of the tetra-mer globule, so that it is outside the protective shield of thesugarcoated surface of the tetramer. Its location outside theglobule makes the tetramerization domain susceptible to pro-teolysis. Tetramers can be dissociated to monomers by treat-ment with proteases (Lockridge and La Du, 1982), thoughthis is an inefficient process because once the tetramershave dissociated to monomers they are no longer enclosedby a protease-resistant sugar coating. The monomers arerapidly degraded. Purified truncated monomers that lack thetetramerization domain have full catalytic activity.

The molecular weight of the tetramer is 340 kDa.Carbohydrates contribute 23.9% of the weight (Haupt et al.,1966). The nine asparagine-linked glycans per subunit areincompletely sialylated (Kolarich et al., 2008). Each subunitcontains one cysteine (Cys 66) that is not in a disulfide bondand is not available for reaction with sulfhydryl agents. It ishypothesized that Cys 66 is oxidized.

The crystal structure of a truncated monomer ofhuman butyrylcholinesterase has been solved (Nicoletet al., 2003). The structure of butyrylcholinesterase is verysimilar to that of acetylcholinesterase. A detailed review ofthe crystal structure and a comparison of the structures ofbutyrylcholinesterase and acetylcholinesterase are presentedby Nachon et al. (2003).

The diameter of globular butyrylcholinesterase is about 50to 60 Angstroms. The active site is located about half-waydown the middle of the globule, 20 Angstroms from the sur-face, at the bottom of an opening called the “active sitegorge.” The active site consists of the catalytic triad aminoacids: serine 198, histidine 438, and glutamic acid 325. Thepositively charged end of a choline ester is oriented againstthe indole ring of tryptophan 80. Before the crystal structurewas known, the choline binding site was speculated to be

negatively charged and was therefore called the “anionic”site. The anionic site has now been renamed the p-cationsite. The neutral end of the choline ester fits into the acylpocket, which is defined by the side chains of Leu286,Val288, and Trp231. The residues in the oxyanion hole,Gly116, Gly117, and Ala199, stabilize the tetrahedral inter-mediate that forms when serine makes a covalent bond withthe substrate.

The important residues near the top of the active site gorgeare Asp 70 and Tyr 332 (Masson et al., 1997). Their sidechains are within hydrogen bonding distance. These two resi-dues constitute the peripheral anionic site of butyrylcholin-esterase (Masson et al., 1996; Nachon et al., 1998). Thenegatively charged Asp 70 attracts positively charged sub-strates to enter the gorge. When Asp 70 is mutated to glycine,the binding affinity for positively charged compounds isseverely reduced. The naturally occurring Asp 70 Glymutation in humans, called the atypical variant, is responsiblefor prolonged apnea from succinylcholine because thepositively charged succinylcholine has a poor affinity forthe Asp 70 Gly mutant and is therefore not bound and nothydrolyzed (McGuire et al., 1989).

The crystal structure of butyrylcholinesterase revealed amechanism to explain why inhibition by organophosphorusagents is irreversible (Carletti et al., 2008; Nachon et al.,2005). The covalently bound agent dealkylates in a processrequiring the assistance of His 438. The dealkylation leadsto a new negative charge on one of the oxygens of thephosphyl group. A salt bridge forms between His 438 andthe oxyanion of the dealkylated organophosphorus agent.This salt bridge captures a proton on His 438. For reactivationthat proton must be released from His 438. The inability torelease the proton on His 438 makes it impossible for His438 to activate a water molecule. There is no activatedwater molecule to displace the organophosphorus agentfrom serine. The consequence is irreversible inhibition ofenzyme activity.

In conclusion, knowledge of the crystal structure of humanbutyrylcholinesterase is useful for the design of mutants thathydrolyze organophosphorus agents and cocaine. It is alsouseful for understanding the basis of succinylcholine apneain people, and for understanding why nerve agents irreversi-bly inhibit butyrylcholinesterase.

3.14 CONCLUSIONS AND FUTUREDIRECTIONS

Butyrylcholinesterase functions in neurotransmission tohydrolyze acetylcholine. This function is apparent onlywhen acetylcholinesterase is inhibited. A second functionfor butyrylcholinesterase is to detoxify poisons such ascocaine and organophosphorus agents. Butyrylcholinesterasehas a role in fat utilization, though this role is not understood.


Butyrylcholinesterase is being mass produced foruse as an antidote against chemical warfare nerve agents. Abutyrylcholinesterase variant, genetically engineered toincrease its cocaine hydrolase activity, has the potential tobecome a treatment for cocaine addiction and cocainetoxicity.

In the year 2010, the following questions were still openfor investigation.

1. Are humans with genetic variants of butyrylcholin-esterase unusually susceptible to the toxicity of nerveagents, organophosphorus pesticides, carbamates,cocaine, and Alzheimer drugs? To begin to addressthis question a mouse model of human butyrylcholin-esterase deficiency is being made. The new mousemodel will express neither butyrylcholinesterase norcarboxylesterase. The presently available butyryl-cholinesterase knockout mouse is an inadequatemodel of human butyrylcholinesterase deficiencybecause mice have 30- to 100-fold more carboxyl-esterase than butyrylcholinesterase in serum. In con-trast humans have no carboxylesterase in serum. Themouse carboxylesterase reacts with many of the samecompounds that react with butyrylcholinesterase.Its high abundance obscures the contribution ofbutyrylcholinesterase to detoxication.

2. The function of butyrylcholinesterase in acetylcholinehydrolysis is controversial. One group favors a role forbutyrylcholinesterase as a backup for acetylcholin-esterase in hydrolysis of acetylcholine. Anothergroup concludes that butyrylcholinesterase has norole in terminating neurotransmission. Instead theypropose that butyrylcholinesterase modulates therelease of acetylcholine vesicles from presynapticnerve terminals, so that inhibition of butyrylcholin-esterase activity results in less acetylcholine in theneuromuscular junction. They predict that butyryl-cholinesterase knockout mice and humans with buty-rylcholinesterase deficiency fatigue more quickly.

3. What is the structure of the tetramerization domain ofbutyrylcholinesterase and how do the four subunitsinteract to form a tetramer? The crystal structure ofthe butyrylcholinesterase tetramer is not yet available.The crystal structure would reveal whether a family ofproline-rich peptides occupies the four helix bundle ofthe tetramerization domain. The released peptideshave various lengths. Are these the natural lengthsor do the peptides break apart when they are released?

4. How is lamellipodin processed to yield thepolyproline-rich peptides in the butyrylcholinesterasetetramerization domain? The short polyproline-richregion of lamellipodin is in the middle of the 1303-amino-acid protein. How does it get cleaved out of

lamellipodin and transported for assembly into thefour-helix bundle of butyrylcholinesterase?

5. Do soluble acetylcholinesterase tetramers contain thesame polyproline-rich peptides that are present inbutyrylcholinesterase? A good start for this questionwould be to purify acetylcholinesterase from fetalbovine serum, and look for the peptides that arereleased when the purified protein is denatured.

6. What mutations will improve the organophosphorushydrolase activity of butyrylcholinesterase? A 100-to 1000-fold improvement in the rate of catalysis ofnerve agents by G117H butyrylcholinesterase isneeded. Several laboratories are working on thisproblem but so far without success. Random DNAshuffling has the best chance of success.

7. How is butyrylcholinesterase expression regulated?Why do some people have two- to threefold higherlevels of butyrylcholinesterase? These people areresistant to muscle relaxants.

8. Why does the C5 variant of butyrylcholinesterasehave increased butyrylcholinesterase activity? It isassumed that the C5 variant is a complex between abutyrylcholinesterase tetramer and another protein.What is the other protein? Could the other proteinbe partially processed lamellipodin or another proteinencoded by a gene on chromosome 2?

9. Why does butyrylcholinesterase accumulate inAlzheimer plaques? Is there a benefit to inhibitingbutyrylcholinesterase activity in Alzheimer patients?

10. Why do obese people have high levels of plasmabutyrylcholinesterase activity? What is the role ofbutyrylcholinesterase in fat metabolism?

11. The chemical state of cysteine 66 is unknown. Thiscysteine does not react with sulfhydryl reagents andis assumed to be oxidized.

12. Clinical trials are needed to test the ability of exo-genous butyrylcholinesterase to reverse cocainetoxicity and cocaine addiction in people.

13. A significant fraction of covalent dimer (10% to 15%)is not reducible by thiol agents; what are the natureand significance of additional cross-links betweensubunits?

14. Large-scale production of butyrylcholinesterase fromhuman plasma or recombinant enzyme from goat milkis expensive. Expression in bacteria and yeast hasfailed to yield functional butyrylcholinesterasedespite 20 years of effort by several groups. Newexpression systems are needed that will lower thecost of producing human butyrylcholinesterase.

15. Human butyrylcholinesterase expressed in bacteriais an inactive aggregate found in inclusionbodies. An understanding of the process by which

3.14 CONCLUSIONS AND FUTURE DIRECTIONS 35

butyrylcholinesterase is folded into active enzymemay aid in converting the inactive aggregated proteininto active enzyme.

16. Butyrylcholinesterase mutants with high efficiencyagainst organophosphorus pesticides and nerveagents would be ideal catalytic bioscavengers for pro-tection against poisoning. Design of efficient catalystsrequires knowledge of dephosphorylation transition-state structures. Quantum mechanics and molecularmechanics simulations will help to make suchevolved mutants.

17. The hysteretic behavior of butyrylcholinesterase withcertain substrates suggests that the enzyme exists inseveral conformational states (inactive and active) inslow equilibrium. The molecular mechanism of hys-teresis and its possible physiological relevance areunknown.

18. Large populations of butyrylcholinesterase-positiveneurons are present in the human brain, particularlyin the amygdala, hippocampal formation, and thala-mus. This suggests a role for butyrylcholinesterasein regulating the activity of the neurotransmitteracetylcholine. It is also possible, but unproven, thatbutyrylcholinesterase acts on a novel neurotransmitterwhose identity is not yet known.

ACKNOWLEDGMENTS

This work was supported by U.S. Army Medical Researchand Materiel Command W81XWH-07-2-0034 (to OL), NIHCounterACT grant U01 NS058056 (to OL), Eppley Cancer Centergrant P30CA36727 and DGA grant 03co010-05/PEA 01 08 7(to PM).

REFERENCES

Adler M, Manley HA, Purcell AL, Deshpande SS, Hamilton TA,Kan RK, Oyler G, Lockridge O, Duysen EG and Sheridan RE(2004). Reduced acetylcholine receptor density, morphologicalremodeling, and butyrylcholinesterase activity can sustainmuscle function in acetylcholinesterase knockout mice. MuscleNerve 30:317–327.

Allderdice PW, Gardner HA, Galutira D, Lockridge O, LaDu BNand McAlpine PJ (1991). The cloned butyrylcholinesterase(BCHE) gene maps to a single chromosome site, 3q26.Genomics 11:452–454.

Allon N, Raveh L, Gilat E, Cohen E, Grunwald J and Ashani Y(1998). Prophylaxis against soman inhalation toxicity in guineapigs by pretreatment alone with human serum butyrylcholines-terase. Toxicol Sci 43:121–128.

Altamirano CV and Lockridge O (1999). Conserved aromaticresidues of the C-terminus of human butyrylcholinesterase

mediate the association of tetramers. Biochemistry38:13414–13422.

Ashani Y (2000). Prospective of human butyrylcholinesterase as adetoxifying antidote and potential regulator of controlled-releasedrugs. Drug Develop Res 50:298–308.

Ashani Y, Shapira S, Levy D, Wolfe AD, Doctor BP and Raveh L(1991). Butyrylcholinesterase and acetylcholinesterase prophy-laxis against soman poisoning in mice. Biochem Pharmacol41:37–41.

Ashani Y, Peggins JO, III and Doctor BP (1992). Mechanism ofinhibition of cholinesterases by huperzine A. Biochem BiophysRes Commun 184:719–726.

Balasubramanian AS and Bhanumathy CD (1993). Noncholinergicfunctions of cholinesterases. Faseb J 7:1354–1358.

Benzer A, Luz G, Oswald E, Schmoigl C and Menardi G (1992).Succinylcholine-induced prolonged apnea in a 3-week-oldnewborn: treatment with human plasma cholinesterase. AnesthAnalg 74:137–138.

Bernard V, Brana C, Liste I, Lockridge O and Bloch B (2003).Dramatic depletion of cell surface m2 muscarinic receptordue to limited delivery from intracytoplasmic stores in neuronsof acetylcholinesterase-deficient mice. Mol Cell Neurosci23:121–133.

Blong RM, Bedows E and Lockridge O (1997). Tetramerizationdomain of human butyrylcholinesterase is at the C-terminus.Biochem J 327 (Pt 3):747–757.

Borders RW, Stephen CR, Nowill WK and Martin R (1955). Theinterrelationship of succinylcholine and the blood cholinester-ases during anesthesia. Anesthesiology 16:401–422.

Boudinot E, Taysse L, Daulon S, Chatonnet A, Champagnat J andFoutz AS (2005). Effects of acetylcholinesterase and butyryl-cholinesterase inhibition on breathing in mice adapted or notto reduced acetylcholinesterase. Pharmacol Biochem Behav80:53–61.

Boudinot E, Bernard V, Camp S, Taylor P, Champagnat J, Krejci Eand Foutz AS (2009). Influence of differential expression ofacetylcholinesterase in brain and muscle on respiration. RespirPhysiol Neurobiol 165:40–48.

Brandeis R, Raveh L, Grunwald J, Cohen E and Ashani Y (1993).Prevention of soman-induced cognitive deficits by pretreatmentwith human butyrylcholinesterase in rats. Pharmacol BiochemBehav 46:889–896.

Brimijoin S, Gao Y, Anker JJ, Gliddon LA, Lafleur D, Shah R,Zhao Q, Singh M and Carroll ME (2008). A cocaine hydrolaseengineered from human butyrylcholinesterase selectivelyblocks cocaine toxicity and reinstatement of drug seeking inrats. Neuropsychopharmacology 33:2715–2725.

Broomfield CA, Maxwell DM, Solana RP, Castro CA, Finger AVand Lenz DE (1991). Protection by butyrylcholinesterase againstorganophosphorus poisoning in nonhuman primates.J Pharmacol Exp Ther 259:633–638.

Bytyqi AH, Lockridge O, Duysen E, Wang Y, Wolfrum U and LayerPG (2004). Impaired formation of the inner retina in an AChEknockout mouse results in degeneration of all photoreceptors.Eur J Neurosci 20:2953–2962.


Camp S, De Jaco A, Zhang L, Marquez M, De la Torre B andTaylor P (2008). Acetylcholinesterase expression in muscle isspecifically controlled by a promoter-selective enhancesomein the first intron. J Neurosci 28:2459–2470.

Carletti E, Li H, Li B, Ekstrom F, Nicolet Y, Loiodice M,Gillon E, Froment MT, Lockridge O, Schopfer LM, Masson Pand Nachon F (2008). Aging of cholinesterases phosphylatedby tabun proceeds through o-dealkylation. J Am Chem Soc.130:16011–16020.

Chatonnet F, Boudinot E, Chatonnet A, Taysse L, Daulon S,Champagnat J and Foutz AS (2003). Respiratory survival mech-anisms in acetylcholinesterase knockout mouse. Eur J Neurosci18:1419–1427.

Chitlaru T, Kronman C, Velan B and Shafferman A (2001). Effect ofhuman acetylcholinesterase subunit assembly on its circulatoryresidence. Biochem J 354:613–625.

Darvesh S, McDonald RS, Darvesh KV, Mataija D, Mothana S,Cook H, Carneiro KM, Richard N, Walsh R and Martin E(2006). On the active site for hydrolysis of aryl amides andcholine esters by human cholinesterases. Bioorg Med Chem14:4586–4599.

De Vriese C, Gregoire F, Lema-Kisoka R, Waelbroeck M,Robberecht P and Delporte C (2004). Ghrelin degradation byserum and tissue homogenates: identification of the cleavagesites. Endocrinology 145:4997–5005.

Doctor BP and Saxena A (2005). Bioscavengers for the protection ofhumans against organophosphate toxicity. Chem Biol Interact157–158:167–171.

Doctor BP, Raveh L, Wolfe AD, Maxwell DM and Ashani Y (1991).Enzymes as pretreatment drugs for organophosphate toxicity.Neurosci Biobehav Rev 15:123–128.

Duysen EG, Li B, Xie W, Schopfer LM, Anderson RS, BroomfieldCA and Lockridge O (2001). Evidence for nonacetylcholines-terase targets of organophosphorus nerve agent: supersensitivityof acetylcholinesterase knockout mouse to VX lethality.J Pharmacol Exp Ther 299:528–535.

Duysen EG, Bartels CF and Lockridge O (2002a). Wild-type andA328W mutant human butyrylcholinesterase tetramersexpressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity.J Pharmacol Exp Ther 302:751–758.

Duysen EG, Stribley JA, Fry DL, Hinrichs SH and Lockridge O(2002b). Rescue of the acetylcholinesterase knockout mouseby feeding a liquid diet; phenotype of the adult acetylcholinester-ase deficient mouse. Brain Res Dev Brain Res 137:43–54.

Duysen EG, Li B, Darvesh S and Lockridge O (2007). Sensitivity ofbutyrylcholinesterase knockout mice to (2)-huperzine A anddonepezil suggests humans with butyrylcholinesterase defi-ciency may not tolerate these Alzheimer’s disease drugs andindicates butyrylcholinesterase function in neurotransmission.Toxicology 233:60–69.

Duysen EG, Li B, Carlson M, Li Y, Wieseler S, Hinrichs SHand Lockridge O (2008). Increased hepatotoxicity and cardiacfibrosis in cocaine-treated butyrylcholinesterase knockout mice.Basic Clin Pharmacol Toxicol 103:514–521.

Evans FT, Gray PW, Lehmann H and Silk E (1953). Effect ofpseudo-cholinesterase level on action of succinylcholine inman. Br Med J 1:136–138.

Feng G, Krejci E, Molgo J, Cunningham JM, Massoulie J andSanes JR (1999). Genetic analysis of collagen Q: roles in acetyl-cholinesterase and butyrylcholinesterase assembly and in syn-aptic structure and function. J Cell Biol 144:1349–1360.

Gao Y and Brimijoin S (2004). An engineered cocaine hydrolaseblunts and reverses cardiovascular responses to cocaine in rats.J Pharmacol Exp Ther 310:1046–1052.

Gatke MR, Ostergaard D, Bundgaard JR, Varin F and Viby-Mogensen J (2001). Response to mivacurium in a patient com-pound heterozygous for a novel and a known silent mutation inthe butyrylcholinesterase gene: genotyping by sequencing.Anesthesiology 95:600–606.

Genovese RF and Doctor BP (1995). Behavioral and pharmacolo-gical assessment of butyrylcholinesterase in rats. PharmacolBiochem Behav 51:647–654.

Getman DK, Eubanks JH, Camp S, Evans GA and Taylor P(1992). The human gene encoding acetylcholinesterase is locatedon the long arm of chromosome 7. Am J Hum Genet51:170–177.

Girard E, Barbier J, Chatonnet A, Krejci E and Molgo J (2005).Synaptic remodeling at the skeletal neuromuscular junctionof acetylcholinesterase knockout mice and its physiologicalrelevance. Chem Biol Interact 157–158:87–96.

Goedde HW and Altland K (1971). Suxamethonium sensitivity. AnnNY Acad Sci 179:695–703.

Grunwald J, Marcus D, Papier Y, Raveh L, Pittel Z and Ashani Y(1997). Large-scale purification and long-term stability ofhuman butyrylcholinesterase: a potential bioscavenger drug.J Biochem Biophys Methods 34:123–135.

Hartmann J, Kiewert C, Duysen EG, Lockridge O, Greig NH andKlein J (2007). Excessive hippocampal acetylcholine levels inacetylcholinesterase-deficient mice are moderated by butyryl-cholinesterase activity. J Neurochem 100:1421–1429.

Haupt H, Heide K, Zwisler O and Schwick HG (1966). [Isolationand physico-chemical characterization of cholinesterase inhuman serum]. Blut 14:65–75.

Hodgkin W, Giblett ER, Levine H, Bauer W and Motulsky AG(1965). Complete pseudocholinesterase deficiency: genetic andimmunologic characterization. J Clin Invest 44:486–493.

Hoffman RS, Henry GC, Howland MA, Weisman RS, Weil L andGoldfrank LR (1992). Association between life-threateningcocaine toxicity and plasma cholinesterase activity. Ann EmergMed 21:247–253.

Huang YJ, Huang Y, Baldassarre H, Wang B, Lazaris A, Leduc M,Bilodeau AS, Bellemare A, Cote M, Herskovits P, Touati M,Turcotte C, Valeanu L, Lemee N, Wilgus H, Begin I, Bhatia B,Rao K, Neveu N, Brochu E, Pierson J, Hockley DK, CerasoliDM, Lenz DE, Karatzas CN and Langermann S (2007).Recombinant human butyrylcholinesterase from milk of trans-genic animals to protect against organophosphate poisoning.Proc Natl Acad Sci USA 104:13603–13608.

Huang YJ, Lundy PM, Lazaris A, Huang Y, Baldassarre H, Wang B,Turcotte C, Cote M, Bellemare A, Bilodeau AS, Brouillard S,

REFERENCES 37

Touati M, Herskovits P, Begin I, Neveu N, Brochu E, Pierson J,Hockley DK, Cerasoli DM, Lenz DE, Wilgus H, Karatzas CNand Langermann S (2008). Substantially improved pharmaco-kinetics of recombinant human butyrylcholinesterase by fusionto human serum albumin. BMC Biotechnol 8:50.

Jbilo O, Bartels CF, Chatonnet A, Toutant JP and Lockridge O(1994). Tissue distribution of human acetylcholinesteraseand butyrylcholinesterase messenger RNA. Toxicon 32:1445–1457.

Jenkins T, Balinsky D and Patient DW (1967). Cholinesterase inplasma: first reported absence in the Bantu; half-life determi-nation. Science 156:1748–1750.

Kalow W (2004). Atypical plasma cholinesterase. A personaldiscovery story: a tale of three cities. Can J Anaesth 51:206–211.

Kalow W and Staron N (1957). On distribution and inheritance ofatypical forms of human serum cholinesterase, as indicated bydibucaine numbers. Can J Med Sci 35:1305–1320.

Klose R and Gutensohn G (1976). [Treatment of alkyl phosphatepoisoning with purified serum cholinesterase (author’s transl)].Prakt Anaesth 11:1–7.

Kluge WH, Kluge HH, Hochstetter A, Vollandt R, Bauer HI andVenbrocks R (2001). Butyrylcholinesterase in lumbar and ventri-cular cerebrospinal fluid. Acta Neurol Scand 104:17–23.

Kolarich D, Weber A, Pabst M, Stadlmann J, Teschner W,Ehrlich H, Schwarz HP and Altmann F (2008). Glycoproteomiccharacterization of butyrylcholinesterase from human plasma.Proteomics 8:254–263.

Krejci E, Thomine S, Boschetti N, Legay C, Sketelj J and MassoulieJ (1997). The mammalian gene of acetylcholinesterase-associated collagen. J Biol Chem 272:22840–22847.

Kutty KM, Huang SN and Kean KT (1981). Pseudocholinesterase inobesity: hypercaloric diet induced changes in experimental obesemice. Experientia 37:1141–1142.

Lenz DE, Maxwell DM, Koplovitz I, Clark CR, Capacio BR,Cerasoli DM, Federko JM, Luo C, Saxena A, Doctor BP andOlson C (2005). Protection against soman or VX poisoning byhuman butyrylcholinesterase in guinea pigs and cynomolgusmonkeys. Chem Biol Interact 157–158:205–210.

Lenz DE, Yeung D, Smith JR, Sweeney RE, Lumley LA andCerasoli DM (2007). Stoichiometric and catalytic scavengersas protection against nerve agent toxicity: a mini review.Toxicology 233:31–39.

Li B, Stribley JA, Ticu A, Xie W, Schopfer LM, Hammond P,Brimijoin S, Hinrichs SH and Lockridge O (2000). Abundanttissue butyrylcholinesterase and its possible function in theacetylcholinesterase knockout mouse. J Neurochem75:1320–1331.

Li B, Duysen EG, Volpicelli-Daley LA, Levey AI and Lockridge O(2003). Regulation of muscarinic acetylcholine receptor functionin acetylcholinesterase knockout mice. Pharmacol BiochemBehav 74:977–986.

Li B, Duysen EG, Carlson M and Lockridge O (2008a). Thebutyrylcholinesterase knockout mouse as a model for humanbutyrylcholinesterase deficiency. J Pharmacol Exp Ther324:1146–1154.

Li B, Duysen EG and Lockridge O (2008b). The butyrylcholinester-ase knockout mouse is obese on a high-fat diet. Chem BiolInteract 175:88–91.

Li H, Schopfer LM, Masson P and Lockridge O (2008c).Lamellipodin proline rich peptides associated with native plasmabutyrylcholinesterase tetramers. Biochem J 411:425–432.

Liddell J, Lehmann H and Silk E (1962). A “silent” pseudo-cholinesterase gene. Nature 193:561–562.

Little JT, Walsh S and Aisen PS (2008). An update on huperzine Aas a treatment for Alzheimer’s disease. Expert Opin InvestigDrugs 17:209–215.

Lockridge O (1988). Structure of human serum cholinesterase.Bioessays 9:125–128.

Lockridge O (1990). Genetic variants of human serum cholinester-ase influence metabolism of the muscle relaxant succinylcholine.Pharmacol Ther 47:35–60.

Lockridge O and La Du BN (1982). Loss of the interchain disulfidepeptide and dissociation of the tetramer following limited pro-teolysis of native human serum cholinesterase. J Biol Chem257:12012–12018.

Lockridge O, Adkins S and La Du BN. (1987a). Location of disul-fide bonds within the sequence of human serum cholinesterase.J Biol Chem 262:12945–12952.

Lockridge O, Bartels CF, Vaughan TA, Wong CK, Norton SE andJohnson LL (1987b). Complete amino acid sequence of humanserum cholinesterase. J Biol Chem 262:549–557.

Lockridge O, Blong RM, Masson P, Froment MT, Millard CB andBroomfield CA (1997). A single amino acid substitution,Gly117 His, confers phosphotriesterase (organophosphorusacid anhydride hydrolase) activity on human butyrylcholinester-ase. Biochemistry 36:786–795.

Lockridge O, Schopfer LM, Winger G and Woods JH (2005). Largescale purification of butyrylcholinesterase from human plasmasuitable for injection into monkeys: a potential new therapeuticfor protection against cocaine and nerve agent toxicity. J MedCBR Def 3 [online publication].

Lynch TJ, Mattes CE, Singh A, Bradley RM, Brady ROand Dretchen KL (1997). Cocaine detoxification by humanplasma butyrylcholinesterase. Toxicol Appl Pharmacol145:363–371.

Manoharan I, Wieseler S, Layer PG, Lockridge O and Boopathy R(2006). Naturally occurring mutation Leu307Pro of humanbutyrylcholinesterase in the Vysya community of India.Pharmacogenet Genomics 16:461–468.

Manoharan I, Boopathy R, Darvesh S and Lockridge O (2007). Amedical health report on individuals with silent butyrylcholines-terase in the Vysya community of India. Clin Chim Acta378:128–135.

Masson P, Froment MT, Bartels CF and Lockridge O (1996). Asp7Oin the peripheral anionic site of human butyrylcholinesterase.Eur J Biochem 235:36–48.

Masson P, Legrand P, Bartels CF, Froment MT, Schopfer LM andLockridge O (1997). Role of aspartate 70 and tryptophan 82 inbinding of succinyldithiocholine to human butyrylcholinester-ase. Biochemistry 36:2266–2277.


Masson P, Froment MT, Gillon E, Nachon F, Darvesh S andSchopfer LM (2007). Kinetic analysis of butyrylcholinesterase-catalyzed hydrolysis of acetanilides. Biochim Biophys Acta1774:1139–1147.

Masson P, Froment MT, Gillon E, Nachon F, Lockridge O andSchopfer LM (2008). Kinetic analysis of effector modulationof butyrylcholinesterase-catalysed hydrolysis of acetanilidesand homologous esters. Febs J 275:2617–2631.

Matzke SM, Oubre JL, Caranto GR, Gentry MK and Galbicka G(1999). Behavioral and immunological effects of exogenousbutyrylcholinesterase in rhesus monkeys. Pharmacol BiochemBehav 62:523–530.

Maxwell DM, Castro CA, De La Hoz DM, Gentry MK, Gold MB,Solana RP, Wolfe AD and Doctor BP (1992). Protection ofrhesus monkeys against soman and prevention of performancedecrement by pretreatment with acetylcholinesterase. ToxicolAppl Pharmacol 115:44–49.

Maxwell DM, Brecht KM, Doctor BP and Wolfe AD (1993).Comparison of antidote protection against soman by pyridostig-mine, HI-6 and acetylcholinesterase. J Pharmacol Exp Ther264:1085–1089.

McArdle JJ, Sellin LC, Coakley KM, Potian JG, Quinones-LopezMC, Rosenfeld CA, Sultatos LG and Hognason K (2005).Mefloquine inhibits cholinesterases at the mouse neuromuscularjunction. Neuropharmacology 49:1132–1139.

McDonough JH, Jr. and Shih TM (1997). Neuropharmacologicalmechanisms of nerve agent-induced seizure and neuropathology.Neurosci Biobehav Rev 21:559–579.

McGuire MC, Nogueira CP, Bartels CF, Lightstone H, Hajra A, Vander Spek AF, Lockridge O and La Du BN (1989). Identificationof the structural mutation responsible for the dibucaine-resistant(atypical) variant form of human serum cholinesterase. Proc NatlAcad Sci USA 86:953–957.

Mesulam MM and Geula C (1994). Butyrylcholinesterase reactivitydifferentiates the amyloid plaques of aging from those of demen-tia. Ann Neurol 36:722–727.

Mesulam MM, Guillozet A, Shaw P, Levey A, Duysen EG andLockridge O (2002). Acetylcholinesterase knockouts establishcentral cholinergic pathways and can use butyrylcholinesteraseto hydrolyze acetylcholine. Neuroscience 110:627–639.

Millard CB, Lockridge O and Broomfield CA (1995). Designand expression of organophosphorus acid anhydride hydrolaseactivity in human butyrylcholinesterase. Biochemistry 34:15925–15933.

Millard CB, Lockridge O and Broomfield CA (1998).Organophosphorus acid anhydride hydrolase activity in humanbutyrylcholinesterase: synergy results in a somanase.Biochemistry 37:237–247.

Minic J, Chatonnet A, Krejci E and Molgo J (2003).Butyrylcholinesterase and acetylcholinesterase activity andquantal transmitter release at normal and acetylcholinesteraseknockout mouse neuromuscular junctions. Br J Pharmacol138:177–187.

Myslivecek J, Duysen EG and Lockridge O (2007). Adaptation toexcess acetylcholine by downregulation of adrenoceptors and

muscarinic receptors in lungs of acetylcholinesterase knockoutmice. Naunyn Schmiedebergs Arch Pharmacol 376:83–92.

Nachon F, Ehret-Sabatier L, Loew D, Colas C, van Dorsselaer Aand Goeldner M (1998). Trp82 and Tyr332 are involved in twoquaternary ammonium binding domains of human butyrylcho-linesterase as revealed by photoaffinity labeling with [3H]DDF.Biochemistry 37:10507–10513.

Nachon F, Masson P, Nicolet Y, Lockridge O and Fontecilla-CampsJC (2003). Comparison of the structures of butyrylcholinesteraseand acetylcholinesterase. Martin Dunitz, London.

Nachon F, Asojo OA, Borgstahl GE, Masson P and Lockridge O(2005). Role of water in aging of human butyrylcholinesteraseinhibited by echothiophate: the crystal structure suggests twoalternative mechanisms of aging. Biochemistry 44:1154–1162.

Naguib M, Daoud W, el-Gammal M, Ammar A, Turkistani A,Selim M, Altamimi W and Sohaibani MO (1995a). Enzymaticantagonism of mivacurium-induced neuromuscular blockadeby human plasma cholinesterase. Anesthesiology 83:694–701.

Naguib M, el-Gammal M, Daoud W, Ammar A, Moukhtar H andTurkistani A (1995b). Human plasma cholinesterase forantagonism of prolonged mivacurium-induced neuromuscularblockade. Anesthesiology 82:1288–1292.

Naguib M, Samarkandi AH, Bakhamees HS, Turkistani A andAlharby SW (1996a). Edrophonium and human plasma cho-linesterase combination for antagonism of mivacurium-induced neuromuscular block. Br J Anaesth 77:424–426.

Naguib M, Selim M, Bakhamees HS, Samarkandi AH andTurkistani A (1996b). Enzymatic versus pharmacologic antagon-ism of profound mivacurium- induced neuromuscular blockade.Anesthesiology 84:1051–1059.

Ndikum-Moffor FM and Roberts SM (2003). Cocaine-proteintargets in mouse liver. Biochem Pharmacol 66:105–113.

Ndikum-Moffor FM, Schoeb TR and Roberts SM (1998). Livertoxicity from norcocaine nitroxide, an N-oxidative metaboliteof cocaine. J Pharmacol Exp Ther 284:413–419.

Nicolet Y, Lockridge O, Masson P, Fontecilla-Camps JC andNachon F (2003). Crystal structure of human butyrylcholinester-ase and of its complexes with substrate and products. J Biol Chem278:41141–41147.

Noureddine H, Carvalho S, Schmitt C, Massoulie J and Bon S(2008). Acetylcholinesterase associates differently with itsanchoring proteins ColQ and PRiMA. J Biol Chem 283:20722–20732.

Ohbu S, Yamashina A, Takasu N, Yamaguchi T, Murai T,Nakano K, Matsui Y, Mikami R, Sakurai K and Hinohara S(1997). Sarin poisoning on Tokyo subway. South Med J 90:587–593.

Ostergaard D, Viby-Mogensen J, Hanel HK and Skovgaard LT(1988). Half-life of plasma cholinesterase. Acta AnaesthesiolScand 32:266–269.

Ostergaard D, Jensen FS and Viby-Mogensen J (1995). Reversal ofintense mivacurium block with human plasma cholinesterase inpatients with atypical plasma cholinesterase. Anesthesiology82:1295–1298.

Ostergaard D, Viby-Mogensen J, Rasmussen SN, Gatke MR andVarin F (2005). Pharmacokinetics and pharmacodynamics of

REFERENCES 39

mivacurium in patients phenotypically homozygous for the aty-pical plasma cholinesterase variant: effect of injection of humancholinesterase. Anesthesiology 102:1124–1132.

Pedron N (1983). Cholinesterase activity of seminal plasma andhuman spermatozoa in normal and infertile subjects. ArchAndrol 10:249–251.

Perrier AL, Massoulie J and Krejci E (2002). PRiMA: the mem-brane anchor of acetylcholinesterase in the brain. Neuron33:275–285.

Ploier R. (1975). [A kin with a “silent” pseudocholinesterase gene(author’s transl)]. Z Kinderheilkd 119:31–36.

Poyot T, Nachon F, Froment MT, Loiodice M, Wieseler S,Schopfer LM, Lockridge O and Masson P (2006). Mutant ofBungarus fasciatus acetylcholinesterase with low affinity andlow hydrolase activity toward organophosphorus esters.Biochim Biophys Acta 1764:1470–1478.

Raveh L, Ashani Y, Levy D, De La Hoz D, Wolfe AD and DoctorBP (1989). Acetylcholinesterase prophylaxis against organopho-sphate poisoning. Quantitative correlation between protectionand blood-enzyme level in mice. Biochem Pharmacol38:529–534.

Raveh L, Grunwald J, Marcus D, Papier Y, Cohen E andAshani Y (1993). Human butyrylcholinesterase as a generalprophylactic antidote for nerve agent toxicity. In vitro andin vivo quantitative characterization. Biochem Pharmacol45:2465–2474.

Raveh L, Grauer E, Grunwald J, Cohen E and Ashani Y (1997). Thestoichiometry of protection against soman and VX toxicity inmonkeys pretreated with human butyrylcholinesterase. ToxicolAppl Pharmacol 145:43–53.

Razon N, Soreq H, Roth E, Bartal A and Silman I (1984).Characterization of activities and forms of cholinesterases inhuman primary brain tumors. Exp Neurol 84:681–695.

Roberts WL, Santikarn S, Reinhold VN and Rosenberry TL (1988).Structural characterization of the glycoinositol phospholipidmembrane anchor of human erythrocyte acetylcholinesteraseby fast atom bombardment mass spectrometry. J Biol Chem263:18776–18784.

Rosenberg Y, Luo C, Ashani Y, Doctor BP, Fischer R, Wolfe G andSaxena A (2002). Pharmacokinetics and immunologic conse-quences of exposing macaques to purified homologous butyryl-cholinesterase. Life Sci 72:125–134.

Rosenberry TL and Scoggin DM (1984). Structure of human eryth-rocyte acetylcholinesterase. Characterization of intersubunitdisulfide bonding and detergent interaction. J Biol Chem259:5643–5652.

Rosenberry TL, Chen JF, Lee MM, Moulton TA and Onigman P(1981). Large scale isolation of human erythrocyte membranesby high volume molecular filtration. J Biochem BiophysMethods 4:39–48.

Ryhanen RJ (1983). Pseudocholinesterase activity in some humanbody fluids. Gen Pharmacol 14:459–460.

Saxena A, Ashani Y, Raveh L, Stevenson D, Patel T and Doctor BP(1998). Role of oligosaccharides in the pharmacokineticsof tissue-derived and genetically engineered cholinesterases.Mol Pharmacol 53:112–122.

Saxena A, Sun W, Luo C, Myers TM, Koplovitz I, Lenz DEand Doctor BP (2006). Bioscavenger for protection fromtoxicity of organophosphorus compounds. J Mol Neurosci30:145–148.

Schallreuter KU, Gibbons NC, Elwary SM, Parkin SM and WoodJM (2007). Calcium-activated butyrylcholinesterase in humanskin protects acetylcholinesterase against suicide inhibition byneurotoxic organophosphates. Biochem Biophys Res Commun355:1069–1074.

Scholler KL, Goedde HW and Benkmann HG (1977). The use ofserum cholinesterase in succinylcholine apnoea. Can AnaesthSoc J 24:396–400.

Schuh FT (1977). Serum cholinesterase: effect on the action ofsuxamethonium following administration to a patient withcholinesterase deficiency. Br J Anaesth 49:269–272.

Souza RL, Mikami LR, Maegawa RO and Chautard-Freire-Maia EA(2005). Four new mutations in the BCHE gene of humanbutyrylcholinesterase in a Brazilian blood donor sample. MolGenet Metab 84:349–353.

Stewart DJ, Inaba T, Tang BK and Kalow W (1977). Hydrolysis ofcocaine in human plasma by cholinesterase. Life Sci20:1557–1563.

Stovner J and Stadskleiv K (1976). Suxamethonium apnoea termi-nated with commercial serum cholinesterase. Acta AnaesthesiolScand 20:211–215.

Sun H, Shen ML, Pang YP, Lockridge O and Brimijoin S (2002).Cocaine metabolism accelerated by a re-engineered humanbutyrylcholinesterase. J Pharmacol Exp Ther 302:710–716.

Tschop M, Smiley DL and Heiman ML (2000). Ghrelin inducesadiposity in rodents. Nature 407:908–913.

Ueno H, Yamaguchi H, Kangawa K and Nakazato M (2005).Ghrelin: a gastric peptide that regulates food intake and energyhomeostasis. Regul Pept 126:11–19.

Velan B, Kronman C, Ordentlich A, Flashner Y, Leitner M, Cohen Sand Shafferman A (1993). N-glycosylation of human acetylcho-linesterase: effects on activity, stability and biosynthesis.Biochem J 296 (Pt 3):649–656.

Viby-Mogensen J (1981). Succinylcholine neuromuscular blockadein subjects homozygous for atypical plasma cholinesterase.Anesthesiology 55:429–434.

Vignaud A, Fougerousse F, Mouisel E, Guerchet N, Hourde C,Bacou F, Butler-Browne GS, Chatonnet A and Ferry A (2008).Genetic inactivation of acetylcholinesterase causes functionaland structural impairment of mouse soleus muscles. CellTissue Res 333:289–296.

Volpicelli-Daley LA, Duysen EG, Lockridge O and Levey AI(2003). Altered hippocampal muscarinic receptors in acetylcho-linesterase-deficient mice. Ann Neurol 53:788–796.

Wang Y, Boeck AT, Duysen EG, Van Keuren M, Saunders TL andLockridge O (2004). Resistance to organophosphorus agenttoxicity in transgenic mice expressing the G117H mutant ofhuman butyrylcholinesterase. Toxicol Appl Pharmacol196:356–366.

Whittaker M (1980). Plasma cholinesterase variants and theanaesthetist. Anaesthesia 35:174–197.


Wolfe AD, Blick DW, Murphy MR, Miller SA, Gentry MK,Hartgraves SL and Doctor BP (1992). Use of cholinesterasesas pretreatment drugs for the protection of rhesus monkeysagainst soman toxicity. Toxicol Appl Pharmacol 117:189–193.

Xie W, Altamirano CV, Bartels CF, Speirs RJ, Cashman JR andLockridge O (1999). An improved cocaine hydrolase: theA328Y mutant of human butyrylcholinesterase is 4-fold moreefficient. Mol Pharmacol 55:83–91.

Xie W, Stribley JA, Chatonnet A, Wilder PJ, Rizzino A,McComb RD, Taylor P, Hinrichs SH and Lockridge O (2000).

Postnatal developmental delay and supersensitivity to organo-phosphate in gene-targeted mice lacking acetylcholinesterase.J Pharmacol Exp Ther 293:896–902.

Yeary RA, Eaton J, Gilmore E, North B and Singell J (1993). Amultiyear study of blood cholinesterase activity in urbanpesticide applicators. J Toxicol Environ Health 39:11–25.

Zheng F, Yang W, Ko MC, Liu J, Cho H, Gao D, Tong M, Tai HH,Woods JH and Zhan CG (2008). Most efficient cocaine hydro-lase designed by virtual screening of transition states. J AmChem Soc 130:12148–12155.

REFERENCES 41

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