Lawrence M. Schopfer

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.

Binding and Hydrolysis of Soman by Human Serum Albumin

Human plasma and fatty acid free human albumin were incubated with soman at pH 8.0 and 25 degrees C. Four methods were used to monitor the reaction of albumin with soman: progressive inhibition of the aryl acylamidase activity of albumin, the release of fluoride ion from soman, 31P NMR, and mass spectrometry. Inhibition (phosphonylation) was slow with a bimolecular rate constant of 15 +/- 3 M(-1) min (-1). MALDI-TOF and tandem mass spectrometry of the soman-albumin adduct showed that albumin was phosphonylated on tyrosine 411. No secondary dealkylation of the adduct (aging) occurred. Covalent docking simulations and 31P NMR experiments showed that albumin has no enantiomeric preference for the four stereoisomers of soman. Spontaneous reactivation at pH 8.0 and 25 degrees C, measured as regaining of aryl acylamidase activity and decrease of covalent adduct (pinacolyl methylphosphonylated albumin) by NMR, occurred at a rate of 0.0044 h (-1), indicating that the adduct is quite stable ( t1/2 = 6.5 days). At pH 7.4 and 22 degrees C, the covalent soman-albumin adduct, measured by MALDI-TOF mass spectrometry, was more stable ( t1/2 = 20 days). Though the concentration of albumin in plasma is very high (about 0.6 mM), its reactivity with soman (phosphonylation and phosphotriesterase activity) is too slow to play a major role in detoxification of the highly toxic organophosphorus compound soman. Increasing the bimolecular rate constant of albumin for organophosphates is a protein engineering challenge that could lead to a new class of bioscavengers to be used against poisoning by nerve agents. Soman-albumin adducts detected by mass spectrometry could be useful for the diagnosis of soman exposure.

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.

Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines

Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5–6 h there was partial acetylation of 16–17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with β-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 °C was 61 ± 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.

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.

Mice Treated with Chlorpyrifos or Chlorpyrifos Oxon Have Organophosphorylated Tubulin in the Brain and Disrupted Microtubule Structures, Suggesting a Role for Tubulin in Neurotoxicity Associated with Exposure to Organophosphorus Agents

Exposure to organophosphorus (OP) agents can lead to learning and memory deficits. Disruption of axonal transport has been proposed as a possible explanation. Microtubules are an essential component of axonal transport. In vitro studies have demonstrated that OP agents react with tubulin and disrupt the structure of microtubules. Our goal was to determine whether in vivo exposure affects microtubule structure. One group of mice was treated daily for 14 days with a dose of chlorpyrifos that did not significantly inhibit acetylcholinesterase. Beta-tubulin from the brains of these mice was diethoxyphosphorylated on tyrosine 281 in peptide GSQQY(281)RALTVPELTQQMFDSK. A second group of mice was treated with a single sublethal dose of chlorpyrifos oxon (CPO). Microtubules and cosedimenting proteins from the brains of these mice were visualized by atomic force microscopy nanoimaging and by Coomassie blue staining of polyacrylamide gel electrophoresis bands. Proteins in gel slices were identified by mass spectrometry. Nanoimaging showed that microtubules from control mice were decorated with many proteins, whereas microtubules from CPO-treated mice had fewer associated proteins, a result confirmed by mass spectrometry of proteins extracted from gel slices. The dimensions of microtubules from CPO-treated mice (height 8.7 +/- 3.1 nm and width 36.5 +/- 15.5 nm) were about 60% of those from control mice (height 13.6 +/- 3.6 nm and width 64.8 +/- 15.9 nm). A third group of mice was treated with six sublethal doses of CPO over 50.15 h. Mass spectrometry identified diethoxyphosphorylated serine 338 in peptide NS(338)NFVEWIPNNVK of beta-tubulin. In conclusion, microtubules from mice exposed to chlorpyrifos or to CPO have covalently modified amino acids and abnormal structure, suggesting disruption of microtubule function. Covalent binding of CPO to tubulin and to tubulin-associated proteins is a potential mechanism of neurotoxicity.

A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters.

Wild-type human butyrylcholinesterase (BuChE) has proven to be an efficient bioscavenger for protection against nerve agent toxicity. Human acetylcholinesterase (AChE) has a similar potential. A limitation to their usefulness is that both cholinesterases (ChEs) react stoichiometrically with organophosphosphorus (OP) esters. Because OPs can be regarded as pseudo-substrates for which the dephosphylation rate constant is almost zero, several strategies have been attempted to promote the dephosphylation reaction. Oxime-mediated reactivation of phosphylated ChEs generates a turnover, but it is too slow to make pseudo-catalytic scavengers of pharmacological interest. Alternatively, it was hypothesized that ChEs could be converted into OP hydrolases by using rational site-directed mutagenesis based upon the crystal structure of ChEs. The idea was to introduce a nucleophile into the oxyanion hole, at an appropriate position to promote hydrolysis of the phospho-serine bond via a base catalysis mechanism. Such mutants, if they showed the desired catalytic and pharmacokinetic properties, could be used as catalytic scavengers. The first mutant of human BuChE that was capable of hydrolyzing OPs was G117H. It had a slow rate. Crystallographic study of the G117H mutant showed that hydrolysis likely occurs by activation of a water molecule rather than direct nucleophilic attack by H117. Numerous BuChE mutants were made later, but none of them was better than the G117H mutant at hydrolyzing OPs, with the exception of soman. Soman aged too rapidly to be hydrolyzed by G117H. Hydrolysis was however accomplished with the double mutant G117H/E197Q, which did not age after phosphonylation with soman. Multiple mutations in the active center of human and Bungarus AChE led to enzymes displaying low catalytic activity towards OPs and unwanted kinetic complexities. A new generation of human AChE mutants has been designed with the assistance of molecular modelling and computational methods. According to the putative water-activation mechanism of G117H BChE, a new histidine/aspartate dyad was introduced into the active center of human AChE at the optimum location for hydrolysis of the OP adduct. Additional mutations were made for optimizing activity of the new dyad. It is anticipated that these new mutants will have OP hydrolase activity.

Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin : A potential mechanism of long term toxicity by organophosphorus agents

Chronic low dose exposure to organophosphorus poisons (OP) results in cognitive impairment. Studies in rats have shown that OP interfere with microtubule polymerization. Since microtubules are required for transport of nutrients from the nerve cell body to the nerve synapse, it has been suggested that disruption of microtubule function could explain the learning and memory deficits associated with OP exposure. Tubulin is a major constituent of microtubules. We tested the hypothesis that OP bind to tubulin by treating purified bovine tubulin with sarin, soman, chlorpyrifos oxon, diisopropylfluorophosphate, and 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin). Tryptic peptides were isolated and analyzed by mass spectrometry. It was found that OP bound to tyrosine 83 of alpha tubulin in peptide TGTYR, tyrosine 59 in beta tubulin peptide YVPR, tyrosine 281 in beta tubulin peptide GSQQYR, and tyrosine 159 in beta tubulin peptide EEYPDR. The OP reactive tyrosines are located either near the GTP binding site or within loops that interact laterally with protofilaments. It is concluded that OP bind covalently to tubulin, and that this binding could explain cognitive impairment associated with OP exposure.

Reaction of human albumin with aspirin in vitro: mass spectrometric identification of acetylated lysines 199, 402, 519, and 545

The aspirin esterase activity of human plasma is due to butyrylcholinesterase and albumin. Our goal was to identify the amino acid residues involved in the aspirin esterase activity of albumin. Fatty acid-free human albumin and human plasma were treated with aspirin for 5 min-24 h. Acetylated residues were identified by LC/MS/MS and MALDI-TOF/TOF mass spectrometry of tryptic peptides. Treatment with 0.3 mM aspirin resulted in acetylation of Lys-199, Lys-402, Lys-519, and Lys-545. Treatment with 20 mM aspirin resulted in acetylation of 26 lysines. There was no acetylation of Tyr-411, under any conditions. Acetylated lysine was stable for at least 21 days at pH 7.4, 37 degrees C. Albumin acetylated by aspirin had reduced esterase activity with beta-naphthyl acetate as shown on gels stained for esterase activity. It was concluded that the aspirin esterase activity of albumin is a pseudo-esterase activity in which aspirin stably acetylates lysines and releases salicylate.

Five Tyrosines and Two Serines in Human Albumin Are Labeled by the Organophosphorus Agent FP-Biotin

Tyrosine 411 of human albumin is an established site for covalent attachment of 10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide (FP-biotin), diisopropylfluorophosphate, chlorpyrifos oxon, soman, sarin, and dichlorvos. This work investigated the hypothesis that other residues in albumin could be modified by organophosphorus agents (OP). Human plasma was aggressively treated with FP-biotin; plasma proteins were separated into high and low abundant portions using a proteome partitioning antibody kit, and the proteins were digested with trypsin. The FP-biotinylated tryptic peptides were isolated by binding to monomeric avidin beads. The major sites of covalent attachment identified by mass spectrometry were Y138, Y148, Y401, Y411, Y452, S232, and S287 of human albumin. Prolonged treatment of pure human albumin with chlorpyrifos oxon labeled Y138, Y150, Y161, Y401, Y411, and Y452. To identify the most reactive residue, albumin was treated for 2 h with DFP, FP-biotin, chlorpyrifos oxon, or soman, digested with trypsin or pepsin, and analyzed by mass spectrometry. The most reactive residue was always Tyr 411. Diethoxyphosphate-labeled Tyr 411 was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to detect OP exposure and to engineer albumin for use as an OP scavenger.