Fetal Bovine Serum Acetylcholinesterase Research Articles

Low levels of endogenous cholinesterases support the choice of cows, sheep and goats for the transgenic expression of human butyrylcholinesterase in milk

Butyrylcholinesterase purified from human plasma (Hu BChE) as well as recombinant (r) Hu BChE are candidate enzymes that can protect humans from toxicity of organophosphorus compounds (OPs). Domestic animals such as cows, pigs, sheep, and goats have been used for the transgenic expression of a variety of valuable therapeutic proteins. Indeed, rHu BChE was successfully expressed in the milk of transgenic goats, but the presence of any endogenous cholinesterases (ChE) in milk would interfere with the isolation of expressed rHu BChE. The aim of this study was to determine the presence of endogenous ChEs in bovine, ovine, caprine, and porcine milk to determine the suitability of these species for the production of rHu BChE. Using acetyl- and butyryl- thiocholine as substrates, ChE activity (2–4 U/mL) was detected in pig milk only. ChE activities in milk from other animals were <0.01 U/mL and could only be detected following enrichment on procainamide-Sepharose gel. Two different methods based on measuring activity in the presence of acetylcholinesterase (AChE)- or BChE- specific inhibitors were used to estimate the proportions of AChE and BChE activities in enriched milk. Monoclonal antibodies (MAbs), against fetal bovine serum AChE that recognize AChEs from ruminants only, were also used to confirm the identity of AChEs. While bovine and ovine milk contain both AChE and BChE activities, caprine and porcine milk contain predominantly BChE activity. The presence of very low ChE activity supports the choice of cows, sheep, and goats for the transgenic expression of rHu BChE in milk.

Monoclonal antibodies to fetal bovine serum acetylcholinesterase distinguish between acetylcholinesterases from ruminant and non-ruminant species

Two types of cholinesterases (ChEs) are present in mammalian blood and tissues: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). While AChE regulates neurotransmission by hydrolyzing acetylcholine at the postsynaptic membranes and neuromuscular junctions, BChE in plasma has been suggested to be involved in detoxifying toxic compounds. This study was undertaken to establish the identity of circulating ChE activity in plasmas from domestic animals (bovine, ovine, caprine, porcine and equine) by assessing sensitivity to AChE-specific inhibitors (BW284c51 and edrophonium) and BChE-specific inhibitors (dibucaine, ethopropazine and Iso-OMPA) as well as binding to anti-FBS AChE monoclonal antibodies (MAbs). Based on the inhibition of ChE activity by ChE-specific inhibitors, it was determined that bovine, ovine and caprine plasma predominantly contain AChE, while porcine and equine plasma contain BChE. Three of the anti-FBS AChE MAbs, 4E5, 5E8 and 6H9, inhibited 85–98% of enzyme activity in bovine, ovine and caprine plasma, confirming that the esterase in these plasmas was AChE. These MAbs did not bind to purified recombinant human or mouse AChE, demonstrating that these MAbs were specific for AChEs from ruminant species. These MAbs did not inhibit the activity of purified human BChE, or ChE activity in porcine and equine plasma, confirming that the ChE in these plasmas was BChE. Taken together, these results demonstrate that anti-FBS AChE MAbs can serve as useful tools for distinguishing between AChEs from ruminant and non-ruminant species and BChEs.

Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase

Acetylcholinesterase (AChE) in the serum of fetal cow is a tetramer. The related enzyme, butyrylcholinesterase (BChE), in the sera of humans and horse requires polyproline peptides for assembly into tetramers. Our goal was to determine whether soluble tetrameric AChE includes tetramer organizing peptides in its structure. Fetal bovine serum AChE was denatured by boiling to release non-covalently bound peptides. Bulk protein was separated from peptides by filtration and by high performance liquid chromatography. Peptide mass and amino acid sequence of the released peptides were determined by MALDI–TOF–TOF and LTQ-Orbitrap mass spectrometry. Twenty polyproline peptides, divided into 5 families, were identified. The longest peptide contained 25 consecutive prolines and no other amino acid. Other polyproline peptides included one non-proline amino acid, for example serine at the C-terminus of 20 prolines. A search of the mammalian proteome database suggested that this assortment of polyproline peptides originated from at least 5 different precursor proteins, none of which were the ColQ or PRiMA of membrane-anchored AChE. To date, AChE and BChE are the only proteins known that include polyproline tetramer organizing peptides in their tetrameric structure.

Cholinesterases in the Milk of Domestic Animals

Human serum butyrylcholinesterase (Hu BChE) is currently under development as a prophylaxis that can protect soldiers from organophosphate (OP) nerve agents. Due to the high cost of production of this enzyme, recombinant Hu BChE is being produced in the milk of transgenic goats. The objective of this study was to determine the presence of endogenous cholinesterases (ChEs) in the milk of cow, goat, sheep, and pig to determine the suitability of goats for the production of recombinant Hu BChE. ChE activity was measured using acetyl-, butyryl- and propionylthiocholine as substrates using the Ellman assay. Detectable levels of ChEs (2-4 U/ml) were present in pig milk only. ChEs in milk from other animals were < 0.1 U/ml and could be detected following centrifugation and enrichment on a procainamide column only. ChE activity in milk varied with the number of pregnancy. ChE-specific inhibitors were used to calculate the proportions of acetylcholinesterase (AChE) and BChE activities. Milk from all four species contained predominantly BChE activity, varying from 100 % in pig to 55 % in sheep. Monoclonal antibodies (MAbs), produced against fetal bovine serum AChE that inhibit AChEs from ruminants only, were also used to confirm the identity of AChEs. Studies with MAbs showed that BChE activity in milk varied from 100 % in pig to 40 % in sheep. Taken together, these results support the choice of goat as the transgenic animal for the expression of recombinant Hu BChE.

Bioscavengers for the protection of humans against organophosphate toxicity

Current antidotes for organophosphorus compounds (OP) poisoning consist of a combination of pretreatment with carbamates (pyridostigmine bromide), to protect acetylcholinesterase (AChE) from irreversible inhibition by OP compounds, and post-exposure therapy with anti-cholinergic drugs (atropine sulfate) to counteract the effects of excess acetylcholine and oximes (e.g., 2-PAM chloride) to reactivate OP-inhibited AChE. These antidotes are effective in preventing lethality from OP poisoning, but they do not prevent post-exposure incapacitation, convulsions, seizures, performance decrements, or in many cases permanent brain damage. These symptoms are commonly observed in experimental animals and are likely to occur in humans. The problems intrinsic to these antidotes stimulated attempts to develop a single protective drug, itself devoid of pharmacological effects, which would provide protection against the lethality of OP compounds and prevent post-exposure incapacitation. One approach is the use of enzymes such as cholinesterases (ChEs), β-esterases in general, as single pretreatment drugs to sequester highly toxic OP anti-ChEs before they reach their physiological targets. This approach turns the irreversible nature of the OP: ChE interaction from disadvantage to an advantage; instead of focusing on OP as an anti-ChE, one can use ChE as an anti-OP. Using this approach, it was shown that administration of fetal bovine serum AChE (FBSAChE) or equine serum butyrylcholinesterase (EqBChE) or human serum BChE (HuBChE) protected the animals from multiple LD 50s of a variety of highly toxic OPs without any toxic effects or performance decrements. The bioscavengers that have been explored to date for the detoxification of OPs fall into three categories: (A) those that can catalytically hydrolyze OPs and thus render them non-toxic, such as OP hydrolase and OP anhydrase; (B) those that stoichiometrically bind to OPs, that is, 1 mol of enzyme neutralizes one or 2 mol of OP inactivating both, such as ChEs and related enzymes; and (C) and those generally termed as “pseudo catalytic”, e.g., a combination of ChE and an oxime pre-treatment such that the catalytic activity of OP-inhibited ChE can rapidly and continuously be restored in the presence of an oxime. Since the biochemical mechanism underlying prophylaxis by exogenous esterases such as ChEs is established and tested in several animal species, including non-human primates, this concept should allow a reliable extrapolation of results from animal experiments to human application. Having being extensively investigated by several groups, plasma derived HuBChE is judged to be the most suitable bioscavenger for its advancement for human use. The program is being developed at the present time for conducting a safety clinical trial in human volunteers. Several other candidate bioscavengers will follow; e.g., recombinant HuBChE expressed in the milk of transgenic goats, pseudo catalytic scavenger(s), e.g., a combination of ChE and oxime, and possibly PON 1 as a catalytic scavenger in the future.

Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases.

E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.

A theoretical expression for the protection associated with stoichiometric and catalytic scavengers in a single compartment model of organophosphorus poisoning

The ability of certain organophosphorus (OP) compounds to inhibit acetylcholinesterase (AChE) has made them useful for industrial (insecticides) and military (nerve agents) purposes. We have previously published a single compartment mathematical model of the interactions between OP nerve agents and the enzymes affected by these agents. That model, which could be used to predict the LD50 of seven nerve agents in rats, has been extended to include the protective actions of stoichiometric and catalytic OP–scavenger enzymes (delivered as pretreatments) so that protective ratios attributable to the scavengers may be predicted. Prediction of expected human protection from in vitro rate constant and initial enzyme level measurements is the ultimate goal for this work. The enhanced model predicts the LD50 from rate constants of the OP agent’s binding reactions with AChE, carboxylesterase (CaE) and a stoichiometric scavenger (S); a first-order OP elimination rate (including a contribution due to a catalytic scavenger); and whole body estimates of AChE, CaE and S. The ratio of the scavenger-treated LD50 estimate to the scavenger-free LD50 estimate provided a theoretical expression describing the scavenger’s contributions to the protective ratio. Published in vivo protective ratios for two stoichiometric scavengers (fetal bovine serum AChE and human utyrylcholinesterase) against challenge by several OP agents in mice were compared with ratios predicted by the model. A linear regression analysis of in vivo protective ratios in mice versus the ratios predicted by the model from the in vitro measurements resulted in an R 2 value of 0.902. The catalytic scavenger portion of the theory could not be validated due to a lack of published data. We conclude that the one-compartment model can be used to make reasonable estimates of the protective ratio attributable to stoichiometric scavengers, but can make no conclusions regarding the ability of the model to predict catalytic scavenger protection ratios.

Nerve Agent Bioscavengers: Protection With Reduced Behavioral Effects

Although treatments for intoxication by organophosphorus nerve agents exist, the treatment regimens suffer from undesirable side effects. To overcome these disadvantages, the use of bioscavengers has emerged as a new approach to reduce the in vivo toxicity of chemical warfare nerve agents. Bioscavengers fall into two broad categories: stoichiometric (i.e., proteins that bind a poison in some fixed ratio) and catalytic (i.e., proteins that can cause the breakdown of a molecule of a poison, regenerate, and then repeat the process until all of the poison molecules have been destroyed). To be an improvement of current treatments, a biological scavenger should have no or minimal behavioral or physiological side effects, should provide protection against one or more nerve agents up to 5 times the median lethal dose (5 LD50), and should reduce or eliminate any behavioral or physiological side effects normally associated with the currently fielded therapy. Studies with equine or human butyryl-cholinesterase or fetal bovine serum acetylcholinesterase show that none of these scavengers exhibit behavioral side effects when administered to rats or monkeys. These three scavengers as well as carboxylesterase are each capable of providing protection against 2 to 16 LD50s of GD, GB, or VX depending on the scavenger and the test species (rat, mouse, rabbit, guinea pig, or rhesus monkey). When behavioral testing was performed on animals pretreated with a bioscavenger and then administered up to 5 LD50s of GD or VX, either no, or only very minor, transient deficits were reported. These results are in stark contrast to the prolonged (1 to 2 week) behavioral in-capacitation experienced by animals pretreated with pyridostigmine and then exposed to the same dose of nerve agent followed by the standard atropine, oxime therapy with or without diazepam. Although several challenges still remain before bioscavengers could augment or replace the current therapeutic regimes for nerve agent intoxication, the results to date offer impressive evidence for the value of this approach as the next generation of pharmaceuticals to afford protection against nerve agent poisoning with a virtual absence of behavioral side effects.

Allosteric control of acetylcholinesterase activity by monoclonal antibodies

Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.

Characterization of O,O-diethylphosphoryl oximes as inhibitors of cholinesterases and substrates of phosphotriesterases

Reactivators of organophosphate (OP)-inhibited cholinesterases (ChEs) are believed to give rise to phosphorylated oximes (POX) that reinhibit the enzyme. Diethylphosphoryl oximes (DEP-OX) that were generated in situ were demonstrated in the past to be unstable, yet were more potent inhibitors of acetylcholinesterase (AChE) than the parent OPs. In view of the inconsistencies among reported results, and the potential toxicity of POXs, it seemed important to characterize authentic DEP-OXs, and to evaluate their interference with reactivation of diethylphosphoryl-ChE (DEP-ChE) conjugates. To this end, the diethylphosphoric acid esters of 1-methyl-2-pyridinium carboxaldehyde oxime (DEP-2PAM) and 1-methyl-4 pyridinium carboxaldehyde oxime (DEP-4PAM) were synthesized and chemically defined. The half-lives of DEP-2PAM and DEP-4PAM in 10 mM Tris buffer, pH 7.8, at 29° were found to be 10 and 980 sec, respectively. The two DEP-OXs inhibited ChEs with the following ranking order: for DEP-2PAM, human butyrylcholinesterase (HuBChE, k i = 2.03 × 10 9 M −1 min −1) > mouse AChE (MoAChE) ≅ fetal bovine serum AChE (FBS-AChE) ≅ equine BChE (EqBChE); for DEP-4PAM, HuBChE ( k i = 0.71 × 10 9 M −1 min −1) > EqBChE > MoAChE > FBS-AChE. A dialkylarylphosphate hydrolase (phosphotriesterase; PTE) from Pseudomonas sp. catalyzed the hydrolysis of DEP-4PAM with k cat/ K m = 3.56 × 10 7 M −1 min −1 and K m = 0.78 mM. Reactivation of DEP-ChEs was enhanced by PTE when 4-PAM-based oximes were used as reactivators, whereas reactivation with 2-PAM-based oximes was not affected by PTE. This observation is attributed primarily to the short half-life of DEP-OXs derived from the latter oximes. Relatively low doses of PTE can detoxify large quantities of DEP-OXs rapidly, and thereby augment the efficacy of antidotes that contain the oxime function in position 4 of the pyridine ring.

Inhibition of Acetylcholinesterase and Butyrylcholinesterase by Chlorpyrifos-oxon

Phosphorothionate insecticides such as parathion ( O, O-diethyl O- p-nitrophenyl phosphorothioate) and chlorpyrifos (CPS; O, O-diethyl O-3,5,6-trichloro-2-pyridyl phosphorothioate; Dursban) are metabolically converted by oxidative desulfuration into paraoxon and chlorpyrifos-oxon (CPO). The insecticidal action of chlorpyrifos stems from inhibition of acetylcholinesterase (AChE) by CPO, resulting in severe cholinergic toxicity. Sensory peripheral neuropathy was observed in people exposed environmentally to chlorpyrifos sprayed in confined areas. We have examined the kinetics of inhibition of AChE and butyrylcholinesterase (BChE) by paraoxon and CPO. The bimolecular rate constants ( k i ) for inhibition by paraoxon of recombinant human (rH) AChE, recombinant mouse (rM) AChE, and fetal bovine serum (FBS) AChE were 7.0, 4.0, and 3.2 × 10 5 M −1 min −1. The k i values for the inhibition by CPO of rH AChE, fetal bovine serum AChE, human RBC AChE, Torpedo AChE, and recombinant mouse (rM) AChE were 9.3, 2.2, 3.8, 8.0, and 5.1 × 10 6 M −1 min −1, respectively. Inhibition of human serum BChE, rH BChE, and rM BChE by CPO yielded k i values of 1.65, 1.67, and 0.78 × 10 9 M −1 min −1, respectively. The k i values obtained for BChE from various species were 160- to 750-fold larger than those of AChE from parallel sources. Inhibition of the single-site mutant A 328Y of rH BChE by CPO displayed a 21-fold lower rate than that of wild-type rH BChE ( k i , 7.9 × 10 7 vs 1.67 × 10 9 M −1 min −1). The double mutant of acyl pocket residues of rH AChE, F 295L/F 297V, was inhibited by CPO with a 150-fold larger k i than wild type (1.5 × 10 9 vs 1.0 × 10 7 M −1 min −1). The increased rate obtained with the double mutant displaying characteristics of the BChE active center provides a rationale for higher efficacy of CPO scavenging by BChE, compared with AChE.

Acceleration of oxime-induced reactivation of organophosphate-inhibited fetal bovine serum acetylcholinesterase by monoquaternary and bisquaternary ligands.

Reactivation of organophosphate (OP)-inhibited acetylcholinesterase (AChE) by oximes is the primary reason for their effectiveness in the treatment of OP poisoning. Reactivation is reported to accelerate by quaternary ligands such as decamethonium, which is devoid of nucleophilicity. The mechanism of this enhancement is not known. To better understand the acceleration phenomenon, we examined ligand modulations of oxime-induced reactivation of methylphosphonylated AChE using 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide and fetal bovine serum AChE. Edrophonium, decamethonium, and propidium, three quaternary AChE ligands of different types, were tested as potential accelerators. Experiments were carried out with both soluble enzyme preparation and AChE conjugated to polyurethane. Kinetic measurements with oximes 2-[hydroxyiminomethyl]-1-methylpyridinium chloride, 1,1'-trimethylene bis-(4-hydroxyimino methyl)-pyridinium dibromide, and 1, 1'-[oxybis-methylene)bis[4-(hydroxyimino)methyl]pyridiniu um dichloride showed that in the presence of 50 microM edrophonium, the reactivation rate constants increased 3.3-12.0-fold; 200 microM decamethonium produced a 1.6-3.0-fold enhancement of reactivation rate constants by the same oximes. Reactivation of the inhibited enzyme by 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride, 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(3-carboxy-aminopyridinium )-d imethyl ether hydrochloride, and 1-[[[4-(aminocarbonyl)pyridino]methoxy]methyl]-2, 4, -bis(hydroxyimino)methyl pyridinium dichloride was not affected by either ligand. Propidium slowed the reactivation of 7-(methylethoxyphosphinyloxy)-1- methylquinolinium iodide-inhibited AChE by all oximes. Results suggest that the accelerator site may reside inside the catalytic gorge rather than at its entrance and acceleration may be due to the prevention of reinhibition of the regenerated enzyme by the putative product, the phosphonylated oxime. In addition to the nucleophilic property of the oximate anion, some of the reactivators may carry an accelerating determinant, as characterized with respect to edrophonium and decamethonium. Results offer possible explanations for the superiority of 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxy-aminopyridinium )-d imethyl ether hydrochloride over other oximes in the reactivation of specific AChE-OP conjugates.

Role of oligosaccharides in the pharmacokinetics of tissue-derived and genetically engineered cholinesterases.

To understand the role of glycosylation in the circulation of cholinesterases, we compared the mean residence time of five tissue-derived and two recombinant cholinesterases (injected intravenously in mice) with their oligosaccharide profiles. Monosaccharide composition analysis revealed differences in the total carbohydrate, galactose, and sialic acid contents. The molar ratio of sialic acid to galactose residues on tetrameric human serum butyrylcholinesterase, recombinant human butyrylcholinesterase, and recombinant mouse acetylcholinesterase was found to be approximately 1.0. For Torpedo californica acetylcholinesterase, monomeric and tetrameric fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase, this ratio was approximately 0.5. However, the circulatory stability of cholinesterases could not be correlated with the sialic acid-to-galactose ratio. Fractionation of the total pool of oligosaccharides obtained after neuraminidase digestion revealed one major oligosaccharide for human serum butyrylcholinesterase and three or four major oligosaccharides in other cholinesterases. The glycans of tetrameric forms of plasma cholinesterases (human serum butyrylcholinesterase, fetal bovine serum acetylcholinesterase, and equine serum butyrylcholinesterase) clearly demonstrated a reduced heterogeneity and higher maturity compared with glycans of monomeric fetal bovine serum acetylcholinesterase, dimeric tissue-derived T. californica acetylcholinesterase, and recombinant cholinesterases. T. californica acetylcholinesterase, recombinant cholinesterases, and monomeric fetal bovine serum acetylcholinesterase showed a distinctive shorter mean residence time (44-304 min) compared with tetrameric forms of plasma cholinesterases (1902-3206 min). Differences in the pharmacokinetic parameters of cholinesterases seem to be due to the combined effect of the molecular weight and charge- and size-based heterogeneity in glycans.

Allosteric control of acetylcholinesterase activity by monoclonal antibodies.

Previous studies showed that monoclonal antibodies raised against phosphorylated fetal bovine serum acetylcholinesterase appeared to modulate the catalytic activity of the enzyme by binding to a conformational epitope located at or near the region of the peripheral anionic site. The mechanism of inhibition of acetylcholinesterase by these monoclonal antibodies was further investigated by determining their effect on (i) substrate inhibition due to the binding of excess substrate to the peripheral anionic site and (ii) binding of peripheral anionic site ligands, such as propidium and fasciculin. Results of these experiments demonstrate that the accessibility of substrate to the peripheral anionic site in these complexes was restricted but not completely blocked, as none of the monoclonal antibodies eliminated the phenomenon of excess substrate inhibition. The results also show that propidium clearly slowed the inhibition of fetal bovine serum acetylcholinesterase by all six inhibitory monoclonal antibodies but to different levels. Complexation of fetal bovine serum acetylcholinesterase with monoclonal antibodies 25B1, 4E5, 6H9, and 5E8 interfered with the binding of fasciculin to the complexed enzyme, suggesting that part of their epitope overlapped with the fasciculin binding site. These monoclonal antibodies bind, in part, at the peripheral anionic site, since polyclonal anti-idiotypic antibodies generated against two monoclonal antibodies, 25B1 and 6H9, bound stoichiometric amounts of propidium. Like fasciculin, binding of these monoclonal antibodies in the vicinity of the peripheral anionic site at the rim of the active site gorge allosterically affects the orientation of W86 located at the base of the gorge, resulting in inhibition of enzyme activity.

Unique push-pull mechanism of dealkylation in soman-inhibited cholinesterases.

The pH-dependence and solvent isotope effects of dealkylation in diastereomeric adducts of Electric eel (Ee) and fetal bovine serum (FBS) acetylcholinesterase (AChE) inactivated with P(-)C(+) and P(-)C(-) 2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) were studied at 4.0 +/- 0.2 degrees C. The rate constant versus pH profiles were fit to a bell-shaped curve for all adducts. Best fit parameters are pK1 4.4-4.6 and pK2 6.3-6.5 for Ee AChE and pK1 4.8-5. 0 and pK2 5.8 for FBS AChE. The pKs are consistent with catalytic participation of the Glu199 anion and HisH+440. Maximal rate constants (kmax) are 13-16 x 10(-3) s-1 for Ee AChE and 8 x 10(-3) s-1 for FBS AChE. The solvent isotope effects at the pH maxima are 1.1-1.3, indicating unlikely proton transfer at the enzymic transition states for the dealkylation reaction. Slopes of log rate constant versus pH plots are near 1 at 25.0 +/- 0.2 degrees C between pH 7.0 and 10.0. In stark contrast, the corresponding adducts of trypsin are very stable even at 37.0 +/- 0.2 degrees C. The rate constants for diastereomers of soman-inhibited trypsin at 37.0 +/- 0.2 degrees C are pH independent and approximately 10(4) times smaller than kmax for analogous adducts with AChE. Dealkylation in soman-inhibited AChEs is estimated to occur at >10(10) times faster than a plausible nonenzymic reaction. Up to 40% of the catalytic acceleration can be attributed to an electrostatic push, and an electrostatic pull provides much of the balance. The results of this work together with results of a product analysis by Michel et al. (1969) can be explained by an initial and rate-determining methyl migration from Cbeta to Calpha. This is driven by the high electron density of residues (Glu199 and Trp84) at a crowded active site and may be concerted with C-O bond breaking. The positive charge at the rate-determining transition state is distributed between Cbeta and His440. A tertiary carbocation may have a fleeting existence before it is trapped by water or neighboring electrons which is likely to be promoted by Glu199 as the proton acceptor.

Structure of glycan moieties responsible for the extended circulatory life time of fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase.

Cholinesterases are serine hydrolases that can potentially be used as pretreatment drugs for organophosphate toxicity, as drugs to alleviate succinylcholine-induced apnea, and as detoxification agents for environmental toxins such as heroin and cocaine. The successful application of serum-derived cholinesterases as bioscavengers stems from their relatively long residence time in the circulation. To better understand the relationship between carbohydrate structure and the stability of cholinesterases in circulation, we determined the monosaccharide composition, the distribution of various oligosaccharides, and the structure of the major asparagine-linked oligosaccharides units present in fetal bovine serum acetylcholinesterase and equine serum butyrylcholinesterase. Our findings indicate that 70-80% of the oligosaccharides in both enzymes are negatively charged. This finding together with the molar ratio of galactose to sialic acid clearly suggests that the beta-galactose residues are only partially capped with sialic acid, yet they displayed a long duration in circulation. The structures of the two major oligosaccharides from fetal bovine serum acetylcholinesterase and one major oligosaccharide from equine serum butyrylcholinesterase were determined. The three carbohydrate structures were of the biantennary complex type, but only the ones from fetal bovine serum acetylcholinesterase were fucosylated on the innermost N-acetylglucosamine residue of the core. Pharmacokinetic studies with native, desialylated, and deglycosylated forms of both enzymes indicate that the microheterogeneity in carbohydrate structure may be responsible, in part, for the multiphasic clearance of cholinesterases from the circulation of mice.

A Monoclonal Antibody against Acetylcholinesterase Inhibits the Formation of Amyloid Fibrils Induced by the Enzyme

A monoclonal antibody (mAb) 25B1 directed against fetal bovine serum acetylcholinesterase (FBS AChE) was used to examine the ability of the cholinergic enzyme to promote the assembly of amyloid-β peptides (Aβ) into Alzheimerś fibrils. This mAb binds to the peripheral anionic site of the enzyme and allosterically inhibits catalytic activity of FBS AChE. Several techniques, including thioflavine-T fluorescence, turbidity, and negative-staining at the electron microscopy level, were used to assess amyloid formation. Inhibition of amyloid formation was dependent on the molar ratio AChE:mAb 25B1, and at least 50% of the inhibition of the AChE promoting effect occurs at a molar ratio similar to that required for inhibition of the esterase activity. Our results suggest that mAb 25B1 inhibits the promotion of the amyloid fibril formation triggered by AChE by affecting the lag period of the Aβ aggregation process.

Synthesis of (±)-10,10-dimethylhuperzine A — a huperzine analogue possessing a slower enzyme off-rate

The synthesis of (±)-10,10-dimethylhuperzine A is reported together with its kinetic (k on and k off) and dissociation constants for the inhibition of fetal bovine serum acetylcholinesterase. While somewhat more potent than (±)-hyperzine A, the dimethyl analogue shows a slower off-rate from AChE, a result that may be of potential therapeutic value.

Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance

Both propidium and monoclonal antibody (mAb) 25B 1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.

Characterization of monoclonal antibodies that inhibit the catalytic activity of acetylcholinesterases.

Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to > 98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross-reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at > 98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.