Oksana Lockridge

Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse

Abstract: We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X‐100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15‐fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X‐100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/‐ mice had 50% of normal AChE activity and AChE ‐/‐ mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X‐100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl‐binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X‐100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild‐type mice as well.

Butyrylcholinesterase for protection from organophosphorus poisons; catalytic complexities and hysteretic behavior

Butyrylcholinesterase is a promiscuous enzyme that displays complex kinetic behavior. It is toxicologically important because it detoxifies organophosphorus poisons (OP) by making a covalent bond with the OP. The OP and the butyrylcholinesterase are both inactivated in the process. Inactivation of butyrylcholinesterase has no adverse effects. However, inactivation of acetylcholinesterase in nerve synapses can be lethal. OP-inhibited butyrylcholinesterase and acetylcholinesterase can be reactivated with oximes provided the OP has not aged. Strategies for preventing the toxicity of OP include (a) treatment with an OP scavenger, (b) reaction of non-aged enzyme with oximes, (c) reactivation of aged enzyme, (d) slowing down aging with peripheral site ligands, and (e) design of mutants that rapidly hydrolyze OP. Option (a) has progressed through phase I clinical trials with human butyrylcholinesterase. Option (b) is in routine clinical use. The others are at the basic research level. Butyrylcholinesterase displays complex kinetic behavior including activation by positively charged esters, ability to hydrolyze amides, and a lag time (hysteresis) preceding hydrolysis of benzoylcholine and N-methylindoxyl acetate. Mass spectrometry has identified new OP binding motifs on tyrosine and lysine in proteins that have no active site serine. It is proposed, but not yet proven, that low dose exposure involves OP modification of proteins that have no active site serine.

Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet; phenotype of the adult acetylcholinesterase deficient mouse

Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE -/- mice were born alive and survived an average of 14 days. The emaciated appearance of AChE -/- mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (+/-) were fed 11% high fat chow supplemented with liquid Ensure. AChE -/- pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7-8 vs. 4), and estrous (week 15-16 vs. 6-7). Significant physical findings in adult AChE -/- mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function.

Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses

Phase I clinical trials have shown that pure human butyrylcholinesterase (BChE) is safe when administered to humans. A potential therapeutic use of BChE is for prevention of nerve agent toxicity. A recombinant mutant of BChE that rapidly inactivates cocaine is being developed as a treatment to help recovering cocaine addicts avoid relapse into drug taking. These clinical applications rely on knowledge of the structure, stability, and properties of BChE, information that is reviewed here. Gene therapy with a vector that sustains expression for a year from a single injection is a promising method for delivering therapeutic quantities of BChE.

Carbamates with Differential Mechanism of Inhibition Toward Acetylcholinesterase and Butyrylcholinesterase

Most carbamates are pseudoirreversible inhibitors of cholinesterases. Phenothiazine carbamates exhibit this inhibition of acetylcholinesterase but produce reversible inhibition of butyrylcholinesterase, suggesting that they do not form a covalent bond with the catalytic serine. This atypical inhibition is attributable to pi-pi interaction of the phenothiazine moiety with F329 and Y332 in butyrylcholinesterase. These residues are in a helical segment, referred to here as the E-helix because it contains E325 of the catalytic triad. The involvement of the E-helix in phenothiazine carbamate reversible inhibition of butyrylcholinesterase is confirmed using mutants of this enzyme at A328, F329, or Y332 that show typical pseudoirreversible inhibition. Thus, in addition to various domains of the butyrylcholinesterase active site gorge, such as the peripheral anionic site and the pi-cationic site of the Omega-loop, the E-helix represents a domain that could be exploited for development of specific inhibitors to treat dementias.

Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity

Central cholinergic systems are involved in a plethora of brain functions and are severely and selectively damaged in neurodegenerative diseases such as Alzheimer’s disease and dementia with Lewy bodies. Cholinergic dysfunction is treated with inhibitors of acetylcholinesterase (AChE) while the role of butyrylcholinesterase (BChE) for brain cholinergic function is unclear. We have used in vivo microdialysis to investigate the regulation of hippocampal acetylcholine (ACh) levels in mice that are devoid of AChE (AChE‐/‐ mice). Extracellular ACh levels in the hippocampus were 60‐fold elevated in AChE‐/‐ mice compared with wild‐type (AChE+/+) animals. In AChE‐/‐ mice, calcium‐free conditions reduced hippocampal ACh levels by 50%, and infusion of tetrodotoxin by more than 90%, indicating continuous ACh release. Infusion of a selective AChE inhibitor (BW284c51) caused a dose‐dependent, up to 16‐fold increase of extracellular ACh levels in AChE+/+ mice but did not change ACh levels in AChE‐/‐ mice. In contrast, infusion of a selective inhibitor of BChE (bambuterol) caused up to fivefold elevation of ACh levels in AChE‐/‐ mice, but was without effect in AChE+/+ animals. These results were corroborated with two other specific inhibitors of AChE and BChE, tolserine and bis‐norcymserine, respectively. We conclude that lack of AChE causes dramatically increased levels of extracellular ACh in the brain. Importantly, in the absence of AChE, the levels of extracellular ACh in the brain are controlled by the activity of BChE. These results point to a potential usefulness of BChE inhibitors in the treatment of central cholinergic dysfunction in which brain AChE activity is typically reduced.

Predicted Michaelis-Menten Complexes of Cocaine-Butyrylcholinesterase ENGINEERING EFFECTIVE BUTYRYLCHOLINESTERASE MUTANTS FOR COCAINE DETOXICATION

Butyrylcholinesterase (BChE) is important in cocaine metabolism, but it hydrolyzes (−)-cocaine only one-two thousandth as fast as the unnatural (+)-stereoisomer. A starting point in engineering BChE mutants that rapidly clear cocaine from the bloodstream, for overdose treatment, is to elucidate structural factors underlying the stereochemical difference in catalysis. Here, we report two three-dimensional Michaelis-Menten complexes of BChE liganded with natural and unnatural cocaine molecules, respectively, that were derived from molecular modeling and supported by experimental studies. Such complexes revealed that the benzoic ester group of both cocaine stereoisomers must rotate toward the catalytic Ser198 for hydrolysis. Rotation of (−)-cocaine appears to be hindered by interactions of its phenyl ring with Phe329 and Trp430. These interactions do not occur with (+)-cocaine. Because the rate of (−)-cocaine hydrolysis is predicted to be determined mainly by the re-orientation step, it should not be greatly influenced by pH. In fact, measured rates of this reaction were nearly constant over the pH range from 5.5 to 8.5, despite large rate changes in hydrolysis of (+)-cocaine. Our models can explain why BChE hydrolyzes (+)-cocaine faster than (−)-cocaine, and they suggest that mutations of certain residues in the catalytic site could greatly improve catalytic efficiency and the potential for detoxication.

Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice

Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic neurotransmission. Overstimulation of cholinergic receptors by excess acetylcholine is known to be lethal. However, AChE knockout mice live to adulthood, although they have weak muscles, do not eat solid food, and die early from seizures. We wanted to know what compensatory factors allowed these mice to survive. We had previously shown that their butyrylcholinesterase activity was normal and had not increased. In this report, we tested the hypothesis that AChE-/- mice adapted to the absence of AChE by downregulating cholinergic receptors. Receptor downregulation is expected to reduce sensitivity to agonists and to increase sensitivity to antagonists. Physiological response to the muscarinic agonists, oxotremorine (OXO) and pilocarpine, showed that AChE-/- mice were resistant to OXO-induced hypothermia, tremor, salivation, and analgesia, and to pilocarpine-induced seizures. AChE+/- mice had an intermediate response. The muscarinic receptor binding sites measured with [3H]quinuclinyl benzilate, as well as the protein levels of M1, M2, and M4 receptors measured with specific antibodies on Western blots, were reduced to be approximately 50% in AChE-/- brain. However, mRNA levels for muscarinic receptors were unchanged. These results indicate that one adaptation to the absence of AChE is downregulation of muscarinic receptors, thus reducing response to cholinergic stimulation.

Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers.

BChE (butyrylcholinesterase) protects the cholinergic nervous system from organophosphorus nerve agents by scavenging these toxins. Recombinant human BChE produced from transgenic goat to treat nerve agent intoxication is currently under development. The therapeutic potential of BChE relies on its ability to stay in the circulation for a prolonged period, which in turn depends on maintaining tetrameric quaternary configuration. Native human plasma BChE consists of 98% tetramers and has a half-life (t((1/2))) of 11-14 days. BChE in the neuromuscular junctions and the central nervous system is anchored to membranes through interactions with ColQ (AChE-associated collagen tail protein) and PRiMA (proline-rich membrane anchor) proteins containing proline-rich domains. BChE prepared in cell culture is primarily monomeric, unless expressed in the presence of proline-rich peptides. We hypothesized that a poly-proline peptide is an intrinsic component of soluble plasma BChE tetramers, just as it is for membrane-bound BChE. We found that a series of proline-rich peptides was released from denatured human and horse plasma BChE. Eight peptides, with masses from 2072 to 2878 Da, were purified by HPLC and sequenced by electrospray ionization tandem MS and Edman degradation. All peptides derived from the same proline-rich core sequence PSPPLPPPPPPPPPPPPPPPPPPPPLP (mass 2663 Da) but varied in length at their N- and C-termini. The source of these peptides was identified through database searching as RAPH1 [Ras-associated and PH domains (pleckstrin homology domains)-containing protein 1; lamellipodin, gi:82581557]. A proline-rich peptide of 17 amino acids derived from lamellipodin drove the assembly of human BChE secreted from CHO (Chinese-hamster ovary) cells into tetramers. We propose that the proline-rich peptides organize the 4 subunits of BChE into a 340 kDa tetramer, by interacting with the C-terminal BChE tetramerization domain.

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetylcholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.