Lockridge O


Full name : Lockridge Oksana

First name : Oksana

Mail : University of Nebraska Medical Center, Eppley Institute, 985950 Nebraska Medical Center, Omaha, NE 68198-5950

Zip Code : 68198-6805

City : Omaha

Country : USA

Email : olockrid@unmc.edu

Phone : 402 559-6032

Fax : 402 559-4651

Website : \/\/www.unmc.edu\/eppley\/about\/faculty\/lockridge.html

Directory :

References (294)

Title : Review: Organophosphorus toxicants, in addition to inhibiting acetylcholinesterase activity, make covalent adducts on multiple proteins and promote protein crosslinking into high molecular weight aggregates - Lockridge_2023_Chem.Biol.Interact_14ChEPon_110460
Author(s) : Lockridge O , Schopfer LM
Ref : Chemico-Biological Interactions , :110460 , 2023
Abstract : The acute effects of exposure to organophosphorus toxicants are explained by inhibition of acetylcholinesterase activity. However, the mechanisms that explain long term illness associated with organophosphorus exposure are still under investigation. We find that organophosphorus nerve agents and organophosphorus pesticides make covalent adducts not only on the serine from acetylcholinesterase, but also on tyrosine, lysine, glutamate, serine and threonine from a variety of proteins. Almost any protein can be modified by a high dose of organophosphorus toxicant. A low dose of 10 microM chlorpyrifos oxon added to the serum-free culture medium of human neuroblastoma SH-SY5Y cells resulted in tyrosine adducts on 48 proteins immunopurified from the cell lysate. We identified the adducted proteins by mass spectrometry after immunopurifying modified proteins with a rabbit anti-diethoxyphospho-tyrosine monoclonal antibody which biased this study for tyrosine adducts. In cultured cells, the primary organophosphate targets are abundant proteins. Organophosphate-modified proteins may disrupt physiological processes. In separate experiments we identified organophosphate adducts on lysine. Organophosphylation activates the lysine for protein crosslinking. The activated lysine reacts with glutamic acid or aspartic acid protein side chains to form an isopeptide bond between proteins, resulting in high molecular weight crosslinked proteins. Crosslinked proteins form insoluble aggregates that may lead to neurogenerative disease.
ESTHER : Lockridge_2023_Chem.Biol.Interact_14ChEPon_110460
PubMedSearch : Lockridge_2023_Chem.Biol.Interact_14ChEPon_110460
PubMedID: 36963650

Title : Overview of Adductomics in Toxicology - Lockridge_2023_Curr.Protoc_3_e672
Author(s) : Lockridge O
Ref : Curr Protoc , 3 :e672 , 2023
Abstract : Adductomics is epidemiology at the molecular level. Untargeted adductomics compares levels of chemical adducts on albumin, hemoglobin, and DNA between healthy and exposed individuals. The goal is to determine a cause-and-effect relationship between chemical exposure and illness. Chemical exposures are not necessarily due to synthetic chemicals but are often due to oxidation products of naturally occurring lipids, for example, 4-hydroxynonenal and acrolein produced by lipid peroxidation of arachidonic and linoleic acids. The preferred method used in adductomics is ultra-high pressure liquid chromatography coupled to with nanoelectrospray tandem mass spectrometry. The mass of the adduct indicates its structure and identifies the chemical. The advantages of molecular epidemiology include information about the many toxicants to which a person is exposed over a period of weeks or months and the relative exposure levels. The disadvantage is the absence of information about the mechanism of toxicity. Untargeted adductomics examines albumin and hemoglobin adducts, which serve as biomarkers of exposure but do not identify the proteins and genes responsible for the toxicity. Targeted adductomics is used when the origin of the toxicity is known. This can be either an adducted protein, such as the butyrylcholinesterase protein modified by nerve agents, or a toxicant, such as acetaminophen. Untargeted adductomics methods have identified potential protein adduct biomarkers of breast cancer, colorectal cancer, childhood leukemia, and lung cancer. Adductomics is a new research area that offers structural insights into chemical exposures and a platform for the discovery of disease biomarkers. 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.
ESTHER : Lockridge_2023_Curr.Protoc_3_e672
PubMedSearch : Lockridge_2023_Curr.Protoc_3_e672
PubMedID: 36799690

Title : Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin - Schopfer_2023_PLoS.One_18_e0280380
Author(s) : Schopfer LM , David E , Hinrichs SH , Lockridge O
Ref : PLoS ONE , 18 :e0280380 , 2023
Abstract : Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.
ESTHER : Schopfer_2023_PLoS.One_18_e0280380
PubMedSearch : Schopfer_2023_PLoS.One_18_e0280380
PubMedID: 36638134

Title : Drug and pro-drug substrates and pseudo-substrates of human butyrylcholinesterase - Masson_2023_Biochem.Pharmacol_218_115910
Author(s) : Masson P , Shaihutdinova Z , Lockridge O
Ref : Biochemical Pharmacology , 218 :115910 , 2023
Abstract : Butyrylcholinesterase (BChE) is present in plasma and numerous cells and organs. Its physiological function(s) is(are) still unclear. However, this enzyme is of pharmacological and toxicological importance. It displays a broad specificity and is capable of hydrolyzing a wide range of substrates with turnovers differing by several orders of magnitude. Nowaday, these substrates include more than two dozen carboxyl-ester drugs, numerous acetylated prodrugs, and transition state analogues of acetylcholine. In addition, BChE displays a promiscuous hydrolytic activity toward amide bonds of arylacylamides, and slowly hydrolyzes carbamyl- and phosphoryl-esters. Certain pseudo-substrates like carbamates and organophosphates are major drugs of potential medical interest. The existence of a large genetic poly-allelism, affecting the catalytic properties of BChE is at the origin of clinical complications in the use of certain drugs catabolized by BChE. The number of drugs and prodrugs hydrolyzed by BChE is expected to increase in the future. However, very few quantitative data (K(m), k(cat)) are available for most marketed drugs, and except for myorelaxants like succinylcholine and mivacurium, the impact of BChE genetic mutations on catalytic parameters has not been evaluated for most of these drugs.
ESTHER : Masson_2023_Biochem.Pharmacol_218_115910
PubMedSearch : Masson_2023_Biochem.Pharmacol_218_115910
PubMedID: 37972875

Title : Organophosphorus Pesticides Promote Protein Cross-Linking - Schopfer_2022_Chem.Res.Toxicol__
Author(s) : Schopfer LM , Onder S , Lockridge O
Ref : Chemical Research in Toxicology , : , 2022
Abstract : Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 microg of MAP-rich tubulin with 100 microM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.
ESTHER : Schopfer_2022_Chem.Res.Toxicol__
PubMedSearch : Schopfer_2022_Chem.Res.Toxicol__
PubMedID: 36048166

Title : Butyrylcholinesterase in SH-SY5Y human neuroblastoma cells - Onder_2022_Neurotoxicol__
Author(s) : Onder S , Schopfer LM , Jiang W , Tacal O , Lockridge O
Ref : Neurotoxicology , : , 2022
Abstract : Cultured SH-SY5Y human neuroblastoma cells are used in neurotoxicity assays. These cells express markers of the cholinergic and dopaminergic systems. Acetylcholinesterase (AChE) activity has been reported in these cells. Neurotoxic organophosphate compounds that inhibit AChE, also inhibit butyrylcholinesterase (BChE). We confirmed the presence of AChE in the cell lysate by activity assays, Western blot, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of immunopurified AChE. A nondenaturing gel stained for AChE activity identified the catalytically active AChE in SH-SY5Y cells as the unstable monomer. We also identified immature BChE in the cell lysate. The concentration of active BChE protein was similar to that of active AChE protein. The rate of substrate hydrolysis by AChE was 10-fold higher than substrate hydrolysis by BChE. The higher rate was due to the 10-fold higher specific activity of AChE over BChE (5000 units/mg for AChE; 500 units/mg for BChE). Neither cholinesterase was secreted. Tryptic peptides of immunopurified AChE and BChE were identified by LC-MS/MS on an Orbitrap Lumos Fusion mass spectrometer. The unfolded protein chaperone, binding immunoglobulin protein BiP/GRP78, was identified in the mass spectral data from all cholinesterase samples, suggesting that BiP was co-extracted with cholinesterase. This suggests that the cytoplasmic cholinesterases are immature forms of AChE and BChE that bind to BiP. It was concluded that SH-SY5Y cells express active AChE and active BChE, but the proteins do not mature to glycosylated tetramers.
ESTHER : Onder_2022_Neurotoxicol__
PubMedSearch : Onder_2022_Neurotoxicol__
PubMedID: 35189179

Title : Evaluation of mass spectrometry MS\/MS spectra for the presence of isopeptide crosslinked peptides - Schopfer_2021_PLoS.One_16_e0254450
Author(s) : Schopfer LM , Onder S , Lockridge O
Ref : PLoS ONE , 16 :e0254450 , 2021
Abstract : Isopeptide crosslinked proteins can be the product of transglutaminase or of exposure to organophosphorus toxicants (OP). Transglutaminase links glutamine to lysine with loss of ammonia. OP toxicants induce a link between glutamic acid and lysine with loss of water. Our goal was to establish criteria to distinguish real from false isopeptide crosslinks reported by software searches of mass spectrometry data. We used fragmentation spectra of tryptic peptides from MAP-rich tubulin Sus scrofa as a test system for detection of naturally-occurring isopeptide crosslinks. Data were analyzed with Protein Prospector. Criteria for the assignments included the presence of at least 1 crosslink specific product ion, fragment ions from both peptides, Protein Prospector scores <=20, and best fit of the MS/MS data to the crosslinked peptide as opposed to a linear peptide. Out of 301,364 spectra, 15 potential transglutaminase-type crosslinked peptide candidates were identified. Manual evaluation of these MS/MS spectra reduced the number to 1 valid crosslink between Q112 of NFH and K368 of Tau. Immunopurification with anti-isopeptide 81D1C2 confirmed that MAP-rich tubulin contained only one isopeptide. Support for this isopeptide bond was obtained by showing that transglutaminase was capable of incorporating dansyl-aminohexyl -QQIV into K368. A model of the KIETHK-QLEAHNR isopeptide was synthesized with the aid of transglutaminase. MS/MS spectra of the model validated our interpretation of the native isopeptide. An OP-induced isopeptide bond between K163 of tubulin alpha-1A and E158 of tubulin beta-4B was induced by treating MAP-rich tubulin with 100 microM chlorpyrifos oxon. This crosslink was supported by the criteria described above and by the presence of diethoxyphospho-lysine 163 in the tubulin alpha-1A peptide. The information obtained in this work is valuable for future studies that aim to understand why exposure to OP is associated with increased risk of neurodegenerative disease.
ESTHER : Schopfer_2021_PLoS.One_16_e0254450
PubMedSearch : Schopfer_2021_PLoS.One_16_e0254450
PubMedID: 34242352

Title : Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry - Onder_2021_J.Proteome.Res__
Author(s) : Onder S , van Grol M , Fidder A , Xiao G , Noort D , Yerramalla U , Tacal O , Schopfer LM , Lockridge O
Ref : J Proteome Res , : , 2021
Abstract : Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (K(d) 3.2 x 10(-8) M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 microM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.
ESTHER : Onder_2021_J.Proteome.Res__
PubMedSearch : Onder_2021_J.Proteome.Res__
PubMedID: 34469172

Title : Proline 285 is integral for the reactivation of organophosphate-inhibited human butyrylcholinesterase by 2-PAM - diTargiani_2020_Chem.Biol.Interact__109092
Author(s) : diTargiani RC , Belinskaya T , Tipparaju P , Lockridge O , Saxena A
Ref : Chemico-Biological Interactions , :109092 , 2020
Abstract : Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger that protects from the toxicity of nerve agents. Non-human primates are suitable models for toxicity studies that cannot be performed in humans. We evaluated the biochemical properties of native macaque (MaBChE) tetramers, compared to recombinant MaBChE monomers, PEGylated recombinant MaBChE tetramers and monomers, and native HuBChE tetramers. Km and kcat values for butyrylthiocholine were independent of subunit assembly status. The Km for all forms of MaBChE was about 70muM, compared to 13muM for HuBChE. The kcat was about 100,000 min(-1) for MaBChE and 30,000 min(-1) for HuBChE. The reversible inhibitor ethopropazine had similar Ki values of 0.05muM for all MaBChE forms and HuBChE. The bimolecular rate constant, ki, for inhibition by diisopropylfluorophosphate (DFP), an analog of sarin, was 2.2 to 2.5x10(7)M(-1)min(-1) for all MaBChE forms and for HuBChE. A major difference between MaBChE and HuBChE was the rate of reactivation by 2-PAM. The second order rate constant for reactivation of DFP-inhibited MaBChE by 2-PAM was 1.4M(-1)min(-1), but was 380 fold faster for DFP-inhibited HuBChE (kr 531M(-1)min(-1)). The acyl pocket of MaBChE has Leu285 in place of Pro285 in HuBChE. The reactivation rate of DFP-inhibited HuBChE mutant P285L by 2-PAM was reduced 5.8% (kr 92M(-1)min(-1)) indicating that P285 determines whether 2-PAM binds in an orientation that favors release of diisopropylphosphate. DFP-inhibited MaBChE treated with 0.2M 2-PAM recovered 10% of its original activity, whereas DFP-inhibited HuBChE recovered 80% activity. It was concluded that the biochemical properties of MaBChE are similar to those of HuBChE except for the reactivation of DFP-inhibited BChE.
ESTHER : diTargiani_2020_Chem.Biol.Interact__109092
PubMedSearch : diTargiani_2020_Chem.Biol.Interact__109092
PubMedID: 32278739

Title : Characteristic fragment ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine - Biberoglu_2020_Anal.Biochem__113718
Author(s) : Biberoglu K , Schopfer LM , Tacal O , Lockridge O
Ref : Analytical Biochemistry , :113718 , 2020
Abstract : Glutamine residues susceptible to transglutaminase-catalyzed crosslinking can be identified by incorporation of dansyl cadaverine or biotin cadaverine. Bacterial transglutaminase and human transglutaminase 2 were used to modify residues in beta-casein with dansyl cadaverine. Bacterial transglutaminase was used to modify residues in human butyrylcholinesterase with biotin cadaverine. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector searches of mass spectrometry data. The MS/MS spectra from modified casein included intense peaks at 336.2, 402.2, and 447.2 for fragments of dansyl cadaverine adducts on glutamine. The MS/MS spectra from modified butyrylcholinesterase included intense peaks at 329.2, 395.2, and 440.2 for fragments of biotin cadaverine adducts on glutamine. No evidence for transglutaminase-catalyzed adducts on glutamic acid, aspartic acid, or asparagine was found. Consistent with expectation, it was concluded that bacterial transglutaminase and human transglutaminase 2 specifically modify glutamine. The characteristic ions associated with dansyl cadaverine and biotin cadaverine adducts on glutamine are useful markers for modified peptides.
ESTHER : Biberoglu_2020_Anal.Biochem__113718
PubMedSearch : Biberoglu_2020_Anal.Biochem__113718
PubMedID: 32335065

Title : Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers - Toker_2020_Chem.Biol.Interact__109007
Author(s) : Toker L , Silman I , Zeev-Ben-Mordehai T , Sussman JL , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , :109007 , 2020
Abstract : Acetylcholinesterase (AChE) terminates cholinergic neurotransmission by hydrolyzing acetylcholine. The collagen-tailed AChE tetramer is a product of 2 genes, ACHE and ColQ. The AChE tetramer consists of 4 identical AChE subunits and one polyproline-rich peptide, whose function is to hold the 4 AChE subunits together. Our goal was to determine the amino acid sequence of the polyproline-rich peptide(s) in Torpedo californica AChE (TcAChE) tetramers to aid in the analysis of images that will be acquired by cryo-EM. Collagen-tailed AChE was solubilized from Torpedo californica electric organ, converted to 300kDa tetramers by digestion with trypsin, and purified by affinity chromatography. Polyproline-rich peptides were released by denaturing the TcAChE tetramers in a boiling water bath, and reducing disulfide bonds with dithiothreitol. Carbamidomethylated peptides were separated from TcAChE protein on a spin filter before they were analyzed by liquid chromatography tandem mass spectrometry on a high resolution Orbitrap Fusion Lumos mass spectrometer. Of the 64 identified collagen-tail (ColQ) peptides, 60 were from the polyproline-rich region near the N-terminus of ColQ. The most abundant proline-rich peptides were SVNKCCLLTPPPPPMFPPPFFTETNILQE, at 40% of total mass-spectral signal intensity, and SVNKCCLLTPPPPPMFPPPFFTETNILQEVDLNNLPLEIKPTEPSCK, at 27% of total intensity. The high abundance of these 2 peptides makes them candidates for the principal form of the polyproline-rich peptide in the trypsin-treated TcAChE tetramers.
ESTHER : Toker_2020_Chem.Biol.Interact__109007
PubMedSearch : Toker_2020_Chem.Biol.Interact__109007
PubMedID: 32087110

Title : Signature Ions in MS\/MS Spectra for Dansyl-Aminohexyl-QQIV Adducts on Lysine - Schopfer_2020_Molecules_25_
Author(s) : Schopfer LM , Lockridge O
Ref : Molecules , 25 : , 2020
Abstract : Bacterial transglutaminase was used to label human plasma proteins with fluorescent tags. Protein lysines were modified with dansyl-epsilon-aminohexyl-Gln-Gln-Ile-Val-OH (dansylQQIV), while protein glutamines were modified with dansyl cadaverine. Labeled proteins included human butyrylcholinesterase, apolipoprotein A-1, haptoglobin, haptoglobin-related protein, immunoglobulin heavy chain, and hemopexin. Tryptic peptides were analyzed by LC-MS/MS on an Orbitrap Fusion Lumos mass spectrometer. Modified residues were identified in Protein Prospector and Proteome Discoverer searches of mass spectrometry data. The MS/MS fragmentation spectra from dansylQQIV-modified peptides gave intense peaks at 475.2015, 364.1691, 347.1426, 234.0585, and 170.0965 m/z. These signature ions are useful markers for identifying modified peptides. Human butyrylcholinesterase retained full activity following modification by dansylQQIV or dansyl cadaverine.
ESTHER : Schopfer_2020_Molecules_25_
PubMedSearch : Schopfer_2020_Molecules_25_
PubMedID: 32521655

Title : Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase - Biberoglu_2020_Molecules_25_
Author(s) : Biberoglu K , Tacal O , Schopfer LM , Lockridge O
Ref : Molecules , 25 : , 2020
Abstract : A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 A, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.
ESTHER : Biberoglu_2020_Molecules_25_
PubMedSearch : Biberoglu_2020_Molecules_25_
PubMedID: 31991818

Title : Half-life of chlorpyrifos oxon and other organophosphorus esters in aqueous solution - Lockridge_2019_Chem.Biol.Interact__108788
Author(s) : Lockridge O , Verdier L , Schopfer LM
Ref : Chemico-Biological Interactions , :108788 , 2019
Abstract : Aqueous solutions of chlorpyrifos oxon are used to study the ability of chlorpyrifos oxon to catalyze protein crosslinking. Assays for protein crosslinking can avoid artifacts by using information on the stability of chlorpyrifos oxon in solution. We undertook to determine the half-life of chlorpyrifos oxon in aqueous solution because literature values do not exist. The rate of conversion of chlorpyrifos oxon to 3,5,6-trichloro-2-pyridinol was measured at 23 degrees C in 20mM TrisCl pH 8 and pH 9 by recording loss of absorbance at 290nm for chlorpyrifos oxon and increase in absorbance at 320nm for 3,5,6-trichloro-2-pyridinol. The half-life of chlorpyrifos oxon was 20.9 days at pH 8 and 6.7 days at pH 9. Literature reports for the stability of other organophosphorus toxicants were summarized because our current studies suggest that other organophosphorus toxicants are also crosslinking agents.
ESTHER : Lockridge_2019_Chem.Biol.Interact__108788
PubMedSearch : Lockridge_2019_Chem.Biol.Interact__108788
PubMedID: 31401088

Title : Mass Spectrometry Identifies Isopeptide Cross-Links Promoted by Diethylphosphorylated Lysine in Proteins Treated with Chlorpyrifos Oxon - Schopfer_2019_Chem.Res.Toxicol_32_762
Author(s) : Schopfer LM , Lockridge O
Ref : Chemical Research in Toxicology , 32 :762 , 2019
Abstract : Exposure to chlorpyrifos at doses that do not inhibit acetylcholinesterase can be followed by chronic illness in adults and developmental deficits in children. A mechanism to explain these effects is not available. Using mass spectrometry, we have found that chlorpyrifos oxon is a cross-linking agent. Pure proteins incubated with 1.5 mM chlorpyrifos oxon were diethylphosphorylated on lysine and tyrosine. The diethylphospho-lysine reacted with the carboxyl side-chain of aspartic and glutamic acid to form an isopeptide cross-link, releasing diethylphosphate in the process. Of the 14 proteins tested, 9 had cross-links between distinct proteins or between monomers of the same protein, whereas 8 had a cyclic structure created by joining side-chains of nearby residues through an isopeptide bond. The precursor lysine in the isopeptide bond was diethylphosphorylated on the epsilon-amino group. Tubulin was more susceptible to chlorpyrifos-oxon-induced cross-linking than the other proteins (10 cross-links in tubulin, 2 in human albumin). The role of diethylphospho-tyrosine was not examined. We hypothesize that the protein misfolding and protein cross-linking induced by exposure to chlorpyrifos oxon, via metabolism of chlorpyrifos, could disrupt function, particularly of tubulin, thus leading to chronic illness. Our proposed mechanism is hypothetical until the many questions it raises have been addressed.
ESTHER : Schopfer_2019_Chem.Res.Toxicol_32_762
PubMedSearch : Schopfer_2019_Chem.Res.Toxicol_32_762
PubMedID: 30844252

Title : Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography - Schopfer_2019_PLoS.One_14_e0209795
Author(s) : Schopfer LM , Lockridge O , David E , Hinrichs SH
Ref : PLoS ONE , 14 :e0209795 , 2019
Abstract : Human butyrylcholinesterase (HuBChE) is being developed as a therapeutic for protection from the toxicity of nerve agents. An enriched source of HuBChE is Cohn fraction IV-4 from pooled human plasma. For the past 40 years, purification of HuBChE has included affinity chromatography on procainamide-Sepharose. The present report supports a new affinity sorbent, Hupresin, for purification of HuBChE from Cohn fraction IV-4. Nine batches of 70-80 kg frozen Cohn fraction were extracted with water, filtered, and chromatographed on 30 L of Q-Ceramic ion exchange sorbent at pH 4.5. The 4% pure Q-eluent was pumped onto 4.2 L Hupresin, where contaminants were washed off with 0.3 M NaCl in 20 mM sodium phosphate pH 8.0, before 99% pure HuBChE was eluted with 0.1 M tetramethylammonium bromide. The average yield was 1.5 g of HuBChE from 80 kg Cohn paste. Recovery of HuBChE was reduced by 90% when the paste was stored at -20 degrees C for 1 year, and reduced 100% when stored at 4 degrees C for 24h. No reduction in HuBChE recovery occurred when paste was stored at -80 degrees C for 3 months or 3 years. Hupresin and procainamide-Sepharose were equally effective at purifying HuBChE from Cohn fraction. HuBChE in Cohn fraction required 1000-fold purification to attain 99% purity, but 15,000-fold purification when the starting material was plasma. HuBChE (P06276) purified from Cohn fraction was a 340 kDa tetramer of 4 identical N-glycated subunits, stable for years in solution or as a lyophilized product.
ESTHER : Schopfer_2019_PLoS.One_14_e0209795
PubMedSearch : Schopfer_2019_PLoS.One_14_e0209795
PubMedID: 30625168

Title : Computer-designed active human butyrylcholinesterase double mutant with a new catalytic triad - Grigorenko_2019_Chem.Biol.Interact_13ChEPon_306_138
Author(s) : Grigorenko BL , Novichkova DA , Lushchekina SV , Zueva IV , Schopfer LM , Nemukhin AV , Varfolomeev SD , Lockridge O , Masson P
Ref : Chemico-Biological Interactions , 306 :138 , 2019
Abstract : A computer-designed mutant of human butyrylcholinesterase (BChE), N322E/E325G, with a novel catalytic triad was made. The catalytic triad of the wild-type enzyme (S198.H438.E325) was replaced by S198.H438.N322E in silico. Molecular dynamics for 1.5 mus and Markov state model analysis showed that the new catalytic triad should be operative in the mutant enzyme, suggesting functionality. QM/MM modeling performed for the reaction of wild-type BChE and double mutant with echothiophate showed high reactivity of the mutant towards the organophosphate. A truncated monomeric (L530 stop) double mutant was expressed in Expi293cells. Non-purified transfected cell culture medium was analyzed. Polyacrylamide gel electrophoresis under native conditions followed by activity staining with BTC as the substrate provided evidence that the monomeric BChE mutant was active. Inhibition of the double mutant by echothiophate followed by polyacrylamide gel electrophoresis and activity staining showed that this enzyme slowly self-reactivated. However, because Expi293cells secrete an endogenous BChE tetramer and several organophosphate-reacting enzymes, catalytic parameters and self-reactivation constants after phosphorylation of the new mutant were not determined in the crude cell culture medium. The study shows that the computer-designed double mutant (N322E/E325G) with a new catalytic triad (S198.H438.N322E) is a suitable template for design of novel active human BChE mutants that display an organophosphate hydrolase activity.
ESTHER : Grigorenko_2019_Chem.Biol.Interact_13ChEPon_306_138
PubMedSearch : Grigorenko_2019_Chem.Biol.Interact_13ChEPon_306_138
PubMedID: 31009643

Title : Delipidation of Plasma Has Minimal Effects on Human Butyrylcholinesterase - Onder_2018_Front.Pharmacol_9_117
Author(s) : Onder S , Tacal O , Lockridge O
Ref : Front Pharmacol , 9 :117 , 2018
Abstract : Human butyrylcholinesterase (BChE) is purified in large quantities from Cohn fraction IV-4 to use for protection against the toxicity of chemical warfare agents. Small scale preliminary experiments use outdated plasma from the American Red Cross as the starting material for purifying BChE (P06276). Many of the volunteer donor plasma samples are turbid with fat, the donor having eaten fatty food before the blood draw. The turbid fat interferes with enzyme assays performed in the spectrophotometer and with column chromatography. Our goal was to find a method to remove fat from plasma without loss of BChE activity. Satisfactory delipidation was achieved by adding a solution of 10% dextran sulfate and calcium chloride to fatty plasma, followed by centrifugation, and filtration through a 0.8 mum filter. Treatment with Aerosil also delipidated fatty plasma, but was accompanied by loss of 50% of the plasma volume. BChE activity and the BChE isozyme pattern on nondenaturing gel electrophoresis were unaffected by delipidation. BChE in delipidated plasma was efficiently captured by immobilized monoclonal antibodies B2 18-5 and mAb2. The immunopurified BChE was released from antibody binding with acid and visualized as a highly enriched, denatured BChE preparation by SDS gel electrophoresis. In conclusion, delipidation with dextran sulfate/CaCl2 preserves BChE activity and the tetramer structure of BChE.
ESTHER : Onder_2018_Front.Pharmacol_9_117
PubMedSearch : Onder_2018_Front.Pharmacol_9_117
PubMedID: 29497381

Title : Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis - Napon_2018_Comp.Med_68_367
Author(s) : Napon G , Dafferner AJ , Saxena A , Lockridge O
Ref : Comp Med , 68 :367 , 2018
Abstract : Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with alpha- or beta-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.
ESTHER : Napon_2018_Comp.Med_68_367
PubMedSearch : Napon_2018_Comp.Med_68_367
PubMedID: 30278860

Title : Tetramer organizing polyproline-rich peptides identified by mass spectrometry after release of the peptides from Hupresin-purified butyrylcholinesterase tetramers isolated from milk of domestic pig (Sus scrofa) - Saxena_2018_Data.Brief_20_1607
Author(s) : Saxena A , Belinskaya T , Schopfer LM , Lockridge O
Ref : Data Brief , 20 :1607 , 2018
Abstract : Milk of the domestic pig has 10 times more butyrylcholinesterase (BChE) per mL than porcine serum. We purified BChE from porcine milk by affinity chromatography on Hupresin-Sepharose. The pure porcine BChE (PoBChE) was a tetramer with a molecular weight of 340,000, similar to that of human BChE tetramers. The C-terminal 40 residues of PoBChE constitute the tetramerization domain. The glue that holds the 4 BChE subunits together is a polyproline-rich peptide. Mass spectrometry analysis of trypsin-digested PoBChE identified a variety of polyproline-rich peptides originating from 12 different proteins. The donor proteins exist in the nucleus or cytoplasm of cells and contribute their polyproline-rich peptides after a cell is degraded. The secreted PoBChE scavenges the polyproline-rich peptides and incorporates one polyproline peptide per PoBChE tetramer, where the polyproline peptide is bound noncovalently but very tightly with an estimated dissociation constant of 10(-12) M. The most abundant polyproline-rich peptides were derived from acrosin, homeobox protein HoxB4, lysine-specific demethylase 6B, proline-rich protein 12, and proline-rich membrane anchor 1 (PRiMA). The research article associated with the data in this report can be found in Saxena et al. (2018). The Data in Brief report lists all the polyproline-rich peptides identified in PoBChE tetramers.
ESTHER : Saxena_2018_Data.Brief_20_1607
PubMedSearch : Saxena_2018_Data.Brief_20_1607
PubMedID: 30263913

Title : Purification of recombinant human butyrylcholinesterase on Hupresin(R) - Lockridge_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1102-1103_109
Author(s) : Lockridge O , David E , Schopfer LM , Masson P , Brazzolotto X , Nachon F
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 1102-1103 :109 , 2018
Abstract : Affinity chromatography on procainamide-Sepharose has been an important step in the purification of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) since its introduction in 1978. The procainamide affinity gel has limitations. In the present report a new affinity gel called Hupresin(R) was evaluated for its ability to purify truncated, recombinant human butyrylcholinesterase (rHuBChE) expressed in a stably transfected Chinese Hamster Ovary cell line. We present a detailed example of the purification of rHuBChE secreted into 3940mL of serum-free culture medium. The starting material contained 13,163units of BChE activity (20.9mg). rHuBChE was purified to homogeneity in a single step by passage over 82mL of Hupresin(R) eluted with 0.1M tetramethylammonium bromide in 20mM TrisCl pH7.5. The fraction with the highest specific activity of 630units/mg contained 11mg of BChE. Hupresin(R) is superior to procainamide-Sepharose for purification of BChE, but is not suitable for purifying native AChE because Hupresin(R) binds AChE so tightly that AChE is not released with buffers, but is desorbed with denaturing solvents such as 50% acetonitrile or 1% trifluoroacetic acid. Procainamide-Sepharose will continue to be useful for purification of AChE.
ESTHER : Lockridge_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1102-1103_109
PubMedSearch : Lockridge_2018_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_1102-1103_109
PubMedID: 30384187

Title : Cryo-EM structure of the native butyrylcholinesterase tetramer reveals a dimer of dimers stabilized by a superhelical assembly - Leung_2018_Proc.Natl.Acad.Sci.U.S.A_115_13270
Author(s) : Leung MR , van Bezouwen LS , Schopfer LM , Sussman JL , Silman I , Lockridge O , Zeev-Ben-Mordehai T
Ref : Proc Natl Acad Sci U S A , 115 :13270 , 2018
Abstract : The quaternary structures of the cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are essential for their localization and function. Of practical importance, BChE is a promising therapeutic candidate for intoxication by organophosphate nerve agents and insecticides, and for detoxification of addictive substances. Efficacy of the recombinant enzyme hinges on its having a long circulatory half-life; this, in turn, depends strongly on its ability to tetramerize. Here, we used cryoelectron microscopy (cryo-EM) to determine the structure of the highly glycosylated native BChE tetramer purified from human plasma at 5.7 A. Our structure reveals that the BChE tetramer is organized as a staggered dimer of dimers. Tetramerization is mediated by assembly of the C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit as a superhelical assembly around a central lamellipodin-derived oligopeptide with a proline-rich attachment domain (PRAD) sequence that adopts a polyproline II helical conformation and runs antiparallel. The catalytic domains within a dimer are asymmetrically linked to the WAT/PRAD. In the resulting arrangement, the tetramerization domain is largely shielded by the catalytic domains, which may contribute to the stability of the human BChE (HuBChE) tetramer. Our cryo-EM structure reveals the basis for assembly of the native tetramers and has implications for the therapeutic applications of HuBChE. This mode of tetramerization is seen only in the cholinesterases but may provide a promising template for designing other proteins with improved circulatory residence times.
ESTHER : Leung_2018_Proc.Natl.Acad.Sci.U.S.A_115_13270
PubMedSearch : Leung_2018_Proc.Natl.Acad.Sci.U.S.A_115_13270
PubMedID: 30538207
Gene_locus related to this paper: human-BCHE

Title : Optimization of Cholinesterase-Based Catalytic Bioscavengers Against Organophosphorus Agents - Lushchekina_2018_Front.Pharmacol_9_211
Author(s) : Lushchekina SV , Schopfer LM , Grigorenko BL , Nemukhin AV , Varfolomeev SD , Lockridge O , Masson P
Ref : Front Pharmacol , 9 :211 , 2018
Abstract : Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km > 10(6) M(-1)min(-1)) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.
ESTHER : Lushchekina_2018_Front.Pharmacol_9_211
PubMedSearch : Lushchekina_2018_Front.Pharmacol_9_211
PubMedID: 29593539

Title : Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity - Schopfer_2018_J.Biol.Chem_293_13566
Author(s) : Schopfer LM , Lockridge O
Ref : Journal of Biological Chemistry , 293 :13566 , 2018
Abstract : Exposure to organophosphorus toxicants (OP) can have chronic adverse effects that are not explained by inhibition of acetylcholinesterase, the cause of acute OP toxicity. We therefore hypothesized that OP-induced chronic illness is initiated by the formation of organophosphorus adducts on lysine residues in proteins, followed by protein cross-linking and aggregation. Here, Western blots revealed that exposure to the OP chlorpyrifos oxon converted porcine tubulin from its original 55-kDa mass to high-molecular-weight aggregates. Liquid chromatography-tandem MS analysis of trypsin-digested samples identified several diethoxyphospho-lysine residues in the OP-treated tubulin. Using a search approach based on the Batch Tag program, we identified cross-linked peptides and found that these chemically activated lysines reacted with acidic amino acid residues creating gamma-glutamyl--lysine or aspartyl--lysine isopeptide bonds between beta- and alpha-tubulin. Of note, these cross-linked tubulin molecules accounted for the high-molecular-weight aggregates. To the best of our knowledge, this is the first report indicating that chlorpyrifos oxon-exposed tubulin protein forms intermolecular cross-links with other tubulin molecules, resulting in high-molecular-weight protein aggregates. It is tempting to speculate that chronic illness from OP exposure may be explained by a mechanism that starts with OP adduct formation on protein lysines followed by protein cross-linking. We further speculate that OP-modified or cross-linked tubulin can impair axonal transport, reduce neuron connections, and result in neurotoxicity.
ESTHER : Schopfer_2018_J.Biol.Chem_293_13566
PubMedSearch : Schopfer_2018_J.Biol.Chem_293_13566
PubMedID: 30006344

Title : Use of Hupresin To Capture Red Blood Cell Acetylcholinesterase for Detection of Soman Exposure - Onder_2018_Anal.Chem_90_974
Author(s) : Onder S , Schopfer LM , Cashman JR , Tacal O , Johnson RC , Blake TA , Lockridge O
Ref : Analytical Chemistry , 90 :974 , 2018
Abstract : Toxicity from acute exposure to nerve agents and organophosphorus toxicants is due to irreversible inhibition of acetylcholinesterase (AChE) in the nervous system. AChE in red blood cells is a surrogate for AChE in the nervous system. Previously we developed an immunopurification method to enrich red blood cell AChE (RBC AChE) as a biomarker of exposure. The goal of the present work was to provide an alternative RBC AChE enrichment strategy, by binding RBC AChE to Hupresin affinity gel. AChE was solubilized from frozen RBC by addition of 1% Triton X-100. Insoluble debris was removed by centrifugation. The red, but not viscous, RBC AChE solution was loaded on a Hupresin affinity column. Hemoglobin and other proteins were washed off with 3 M NaCl, while retaining AChE bound to Hupresin. Denatured AChE was eluted with 1% trifluoroacetic acid. The same protocol was used for 20 mL of RBC AChE inhibited with a soman model compound. The acid denatured protein was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. A targeted method identified the aged soman adduct on serine 203 in peptide FGESAGAAS. It was concluded that Hupresin can be used to enrich soman-inhibited AChE solubilized from 8 mL of frozen human erythrocytes, yielding a quantity sufficient for detecting soman exposure.
ESTHER : Onder_2018_Anal.Chem_90_974
PubMedSearch : Onder_2018_Anal.Chem_90_974
PubMedID: 29172437

Title : Characterization of butyrylcholinesterase from porcine milk - Saxena_2018_Arch.Biochem.Biophys_652_38
Author(s) : Saxena A , Belinskaya T , Schopfer LM , Lockridge O
Ref : Archives of Biochemistry & Biophysics , 652 :38 , 2018
Abstract : Human butyrylcholinesterase (HuBChE) is under development for use as a pretreatment antidote against nerve agent toxicity. Animals are used to evaluate the efficacy of HuBChE for protection against organophosphorus nerve agents. Pharmacokinetic studies of HuBChE in minipigs showed a mean residence time of 267h, similar to the half-life of HuBChE in humans, suggesting a high degree of similarity between BChE from 2 sources. Our aim was to compare the biochemical properties of PoBChE purified from porcine milk to HuBChE purified from human plasma. PoBChE hydrolyzed acetylthiocholine slightly faster than butyrylthiocholine, but was sensitive to BChE-specific inhibitors. PoBChE was 50-fold less sensitive to inhibition by DFP than HuBChE and 5-fold slower to reactivate in the presence of 2-PAM. The amino acid sequence of PoBChE determined by liquid chromatography tandem mass spectrometry was 91% identical to HuBChE. Monoclonal antibodies 11D8, mAb2, and 3E8 (HAH 002) recognized both PoBChE and HuBChE. Assembly of 4 identical subunits into tetramers occurred by noncovalent interaction with polyproline-rich peptides in PoBChE as well as in HuBChE, though the set of polyproline-rich peptides in milk-derived PoBChE was different from the set in plasma-derived HuBChE tetramers. It was concluded that the esterase isolated from porcine milk is PoBChE.
ESTHER : Saxena_2018_Arch.Biochem.Biophys_652_38
PubMedSearch : Saxena_2018_Arch.Biochem.Biophys_652_38
PubMedID: 29908755
Gene_locus related to this paper: pig-BCHE

Title : Immunopurification of Acetylcholinesterase from Red Blood Cells for Detection of Nerve Agent Exposure - Dafferner_2017_Chem.Res.Toxicol_30_1897
Author(s) : Dafferner AJ , Schopfer LM , Xiao G , Cashman JR , Yerramalla U , Johnson RC , Blake TA , Lockridge O
Ref : Chemical Research in Toxicology , 30 :1897 , 2017
Abstract : Nerve agents and organophosphorus pesticides make a covalent bond with the active site serine of acetylcholinesterase (AChE), resulting in inhibition of AChE activity and toxic symptoms. AChE in red blood cells (RBCs) serves as a surrogate for AChE in the nervous system. Mass spectrometry analysis of adducts on RBC AChE could provide evidence of exposure. Our goal was to develop a method of immunopurifying human RBC AChE in quantities adequate for detecting exposure by mass spectrometry. For this purpose, we immobilized 3 commercially available anti-human acetylcholinesterase monoclonal antibodies (AE-1, AE-2, and HR2) plus 3 new monoclonal antibodies. The monoclonal antibodies were characterized for binding affinity, epitope mapping by pairing analysis, and nucleotide and amino acid sequences. AChE was solubilized from frozen RBCs with 1% (v/v) Triton X-100. A 16 mL sample containing 5.8 mug of RBC AChE was treated with a quantity of soman model compound that inhibited 50% of the AChE activity. Native and soman-inhibited RBC AChE samples were immunopurified on antibody-Sepharose beads. The immunopurified RBC AChE was digested with pepsin and analyzed by liquid chromatography tandem mass spectrometry on a 6600 Triple-TOF mass spectrometer. The aged soman-modified PheGlyGluSerAlaGlyAlaAlaSer (FGESAGAAS) peptide was detected using a targeted analysis method. It was concluded that all 6 monoclonal antibodies could be used to immunopurify RBC AChE and that exposure to nerve agents could be detected as adducts on the active site serine of RBC AChE.
ESTHER : Dafferner_2017_Chem.Res.Toxicol_30_1897
PubMedSearch : Dafferner_2017_Chem.Res.Toxicol_30_1897
PubMedID: 28892361

Title : Hupresin Retains Binding Capacity for Butyrylcholinesterase and Acetylcholinesterase after Sanitation with Sodium Hydroxide - Onder_2017_Front.Pharmacol_8_713
Author(s) : Onder S , David E , Tacal O , Schopfer LM , Lockridge O
Ref : Front Pharmacol , 8 :713 , 2017
Abstract : Hupresin is a new affinity resin that binds butyrylcholinesterase (BChE) in human plasma and acetylcholinesterase (AChE) solubilized from red blood cells (RBC). Hupresin is available from the CHEMFORASE company. BChE in human plasma binds to Hupresin and is released with 0.1 M trimethylammonium bromide (TMA) with full activity and 10-15% purity. BChE immunopurified from plasma by binding to immobilized monoclonal beads has fewer contaminating proteins than the one-step Hupresin-purified BChE. However, when affinity chromatography on Hupresin follows ion exchange chromatography at pH 4.5, BChE is 99% pure. The membrane bound AChE, solubilized from human RBC with 0.6% Triton X-100, binds to Hupresin and remains bound during washing with sodium chloride. Human AChE is not released in significant quantities with non-denaturing solvents, but is recovered in 1% trifluoroacetic acid. The denatured, partially purified AChE is useful for detecting exposure to nerve agents by mass spectrometry. Our goal was to determine whether Hupresin retains binding capacity for BChE and AChE after Hupresin is washed with 0.1 M NaOH. A 2 mL column of Hupresin equilibrated in 20 mM TrisCl pH 7.5 was used in seven consecutive trials to measure binding and recovery of BChE from 100 mL human plasma. Between each trial the Hupresin was washed with 10 column volumes of 0.1 M sodium hydroxide. A similar trial was conducted with red blood cell AChE in 0.6% Triton X-100. It was found that the binding capacity for BChE and AChE was unaffected by washing Hupresin with 0.1 M sodium hydroxide. Hupresin could be washed with sodium hydroxide at least seven times without losing binding capacity.
ESTHER : Onder_2017_Front.Pharmacol_8_713
PubMedSearch : Onder_2017_Front.Pharmacol_8_713
PubMedID: 29066970

Title : The C5 Variant of the Butyrylcholinesterase Tetramer Includes a Noncovalently Bound 60 kDa Lamellipodin Fragment - Schopfer_2017_Molecules_22_
Author(s) : Schopfer LM , Delacour H , Masson P , Leroy J , Krejci E , Lockridge O
Ref : Molecules , 22 : , 2017
Abstract : Humans with the C5 genetic variant of butyrylcholinesterase (BChE) have 30-200% higher plasma BChE activity, low body weight, and shorter duration of action of the muscle relaxant succinylcholine. The C5 variant has an extra, slow-moving band of BChE activity on native polyacrylamide gel electrophoresis. This band is about 60 kDa larger than wild-type BChE. Umbilical cord BChE in 100% of newborn babies has a C5-like band. Our goal was to identify the unknown, 60 kDa protein in C5. Both wild-type and C5 BChE are under the genetic control of two independent loci, the BCHE gene on Chr 3q26.1 and the RAPH1 (lamellipodin) gene on Chr 2q33. Wild-type BChE tetramers are assembled around a 3 kDa polyproline peptide from lamellipodin. Western blot of boiled C5 and cord BChE showed a positive response with an antibody to the C-terminus of lamellipodin. The C-terminal exon of lamellipodin is about 60 kDa including an N-terminal polyproline. We propose that the unknown protein in C5 and cord BChE is encoded by the last exon of the RAPH1 gene. In 90% of the population, the 60 kDa fragment is shortened to 3 kDa during maturation to adulthood, leaving only 10% of adults with C5 BChE.
ESTHER : Schopfer_2017_Molecules_22_
PubMedSearch : Schopfer_2017_Molecules_22_
PubMedID: 28661448
Gene_locus related to this paper: human-BCHE

Title : Monoclonal Antibody That Recognizes Diethoxyphosphotyrosine-Modified Proteins and Peptides Independent of Surrounding Amino Acids - Onder_2017_Chem.Res.Toxicol_30_2218
Author(s) : Onder S , Dafferner AJ , Schopfer LM , Xiao G , Yerramalla U , Tacal O , Blake TA , Johnson RC , Lockridge O
Ref : Chemical Research in Toxicology , 30 :2218 , 2017
Abstract : Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are irreversibly inhibited by organophosphorus pesticides through formation of a covalent bond with the active site serine. Proteins that have no active site serine, for example albumin, are covalently modified on tyrosine and lysine. Chronic illness from pesticide exposure is not explained by inhibition of AChE and BChE. Our goal was to produce a monoclonal antibody that recognizes proteins diethoxyphosphorylated on tyrosine. Diethoxyphosphate-tyrosine adducts for 13 peptides were synthesized. The diethoxyphosphorylated (OP) peptides cross-linked to four different carrier proteins were used to immunize, boost, and screen mice. Monoclonal antibodies were produced with hybridoma technology. Monoclonal antibody depY was purified and characterized by ELISA, western blotting, Biacore, and Octet technology to determine binding affinity and binding specificity. DepY recognized diethoxyphosphotyrosine independent of the amino acid sequence around the modified tyrosine and independent of the identity of the carrier protein or peptide. It had an IC50 of 3 x 10(-9) M in a competition assay with OP tubulin. Kd values measured by Biacore and OctetRED96 were 10(-8) M for OP-peptides and 1 x 10(-12) M for OP-proteins. The limit of detection measured on western blots hybridized with 0.14 mug/mL of depY was 0.025 mug of human albumin conjugated to YGGFL-OP. DepY was specific for diethoxyphosphotyrosine (chlorpyrifos oxon adduct) as it failed to recognize diethoxyphospholysine, phosphoserine, phosphotyrosine, phosphothreonine, dimethoxyphosphotyrosine (dichlorvos adduct), dimethoxyphosphoserine, monomethoxyphosphotyrosine (aged dichlorvos adduct), and cresylphosphoserine. In conclusion, a monoclonal antibody that specifically recognizes diethoxyphosphotyrosine adducts has been developed. The depY monoclonal antibody could be useful for identifying new biomarkers of OP exposure.
ESTHER : Onder_2017_Chem.Res.Toxicol_30_2218
PubMedSearch : Onder_2017_Chem.Res.Toxicol_30_2218
PubMedID: 29137457

Title : Characterization of butyrylcholinesterase in bovine serum - Dafferner_2017_Chem.Biol.Interact_266_17
Author(s) : Dafferner AJ , Lushchekina SV , Masson P , Xiao G , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 266 :17 , 2017
Abstract : Human butyrylcholinesterase (HuBChE) protects from nerve agent toxicity. Our goal was to determine whether bovine serum could be used as a source of BChE. Bovine BChE (BoBChE) was immunopurified from 100 mL fetal bovine serum (FBS) or 380 mL adult bovine serum by binding to immobilized monoclonal mAb2. Bound proteins were digested with trypsin and analyzed by liquid chromatography-tandem mass spectrometry. The results proved that FBS and adult bovine serum contain BoBChE. The concentration of BoBChE was estimated to be 0.04 mug/mL in FBS, and 0.03 mug/mL in adult bovine serum, values lower than the 4 mug/mL BChE in human serum. Nondenaturing gel electrophoresis showed that monoclonal mAb2 bound BoBChE but not bovine acetylcholinesterase (BoAChE) and confirmed that FBS contains BoBChE and BoAChE. Recombinant bovine BChE (rBoBChE) expressed in serum-free culture medium spontaneously reactivated from inhibition by chlorpyrifos oxon at a rate of 0.0023 min-1 (t1/2 = 301 min-1) and aged at a rate of 0.0138 min-1 (t1/2 = 50 min-1). Both BoBChE and HuBChE have 574 amino acids per subunit and 90% sequence identity. However, the apparent size of serum BoBChE and rBoBChE tetramers was much greater than the 340,000 Da of HuBChE tetramers. Whereas HuBChE tetramers include short polyproline rich peptides derived from lamellipodin, no polyproline peptides have been identified in BoBChE. We hypothesize that BoBChE tetramers use a large polyproline-rich protein to organize subunits into a tetramer and that the low concentration of BoBChE in serum is explained by limited quantities of an unidentified polyproline-rich protein.
ESTHER : Dafferner_2017_Chem.Biol.Interact_266_17
PubMedSearch : Dafferner_2017_Chem.Biol.Interact_266_17
PubMedID: 28189703
Gene_locus related to this paper: bovin-BCHE

Title : Tetramer-organizing polyproline-rich peptides differ in CHO cell-expressed and plasma-derived human butyrylcholinesterase tetramers - Schopfer_2016_Biochim.Biophys.Acta_1864_706
Author(s) : Schopfer LM , Lockridge O
Ref : Biochimica & Biophysica Acta , 1864 :706 , 2016
Abstract : Tetrameric butyrylcholinesterase (BChE) in human plasma is the product of multiple genes, namely one BCHE gene on chromosome 3q26.1 and multiple genes that encode polyproline-rich peptides. The function of the polyproline-rich peptides is to assemble BChE into tetramers. CHO cells transfected with human BChE cDNA express BChE monomers and dimers, but only low quantities of tetramers. Our goal was to identify the polyproline-rich peptides in CHO-cell derived human BChE tetramers. CHO cell-produced human BChE tetramers were purified from serum-free culture medium. Peptides embedded in the tetramerization domain were released from BChE tetramers by boiling and identified by liquid chromatography-tandem mass spectrometry. A total of 270 proline-rich peptides were sequenced, ranging in size from 6-41 residues. The peptides originated from 60 different proteins that reside in multiple cell compartments including the nucleus, cytoplasm, and endoplasmic reticulum. No single protein was the source of the polyproline-rich peptides in CHO cell-expressed human BChE tetramers. In contrast, 70% of the tetramer-organizing peptides in plasma-derived BChE tetramers originate from lamellipodin. No protein source was identified for polyproline peptides containing up to 41 consecutive proline residues. In conclusion, the use of polyproline-rich peptides as a tetramerization motif is documented only for the cholinesterases, but is expected to serve other tetrameric proteins as well. The CHO cell data suggest that the BChE tetramer-organizing peptide can arise from a variety of proteins.
ESTHER : Schopfer_2016_Biochim.Biophys.Acta_1864_706
PubMedSearch : Schopfer_2016_Biochim.Biophys.Acta_1864_706
PubMedID: 26947244

Title : Monoclonal antibodies to human butyrylcholinesterase reactive with butyrylcholinesterase in animal plasma - Peng_2016_Chem.Biol.Interact_243_82
Author(s) : Peng H , Brimijoin S , Hrabovska A , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 243 :82 , 2016
Abstract : Five mouse anti-human butyrylcholinesterase (BChE) monoclonal antibodies bind tightly to native human BChE with nanomolar dissociation constants. Pairing analysis in the Octet system identified the monoclonal antibodies that bind to overlapping and independent epitopes on human BChE. The nucleotide and amino acid sequences of 4 monoclonal antibodies are deposited in GenBank. Our goal was to determine which of the 5 monoclonal antibodies recognize BChE in the plasma of animals. Binding of monoclonal antibodies 11D8, B2 18-5, B2 12-1, mAb2 and 3E8 to BChE in animal plasma was measured using antibody immobilized on Pansorbin cells and on Dynabeads Protein G. A third method visualized binding by the shift of BChE activity bands on nondenaturing gels stained for BChE activity. Gels were counterstained for carboxylesterase activity. The three methods agreed that B2 18-5 and mAb2 have broad species specificity, but the other monoclonal antibodies interacted only with human BChE, the exception being 3E8, which also bound chicken BChE. B2 18-5 and mAb2 recognized BChE in human, rhesus monkey, horse, cat, and tiger plasma. A weak response was found with rabbit BChE. Monoclonal mAb2, but not B2 18-5, bound pig and bovine BChE. Gels stained for carboxylesterase activity confirmed that plasma from humans, monkey, pig, chicken, and cow does not contain carboxylesterase, but plasma from horse, cat, tiger, rabbit, guinea pig, mouse, and rat has carboxylesterase. Rabbit plasma carboxylesterase hydrolyzes butyrylthiocholine. In conclusion monoclonal antibodies B2 18-5 and mAb2 can be used to immuno extract BChE from the plasma of humans, monkey and other animals.
ESTHER : Peng_2016_Chem.Biol.Interact_243_82
PubMedSearch : Peng_2016_Chem.Biol.Interact_243_82
PubMedID: 26585590

Title : Origin of polyproline-rich peptides in human butyrylcholinesterase tetramers - Peng_2016_Chem.Biol.Interact_259_63
Author(s) : Peng H , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 259 :63 , 2016
Abstract : The human butyrylcholinesterase (HuBChE) tetramer is composed of 4 identical subunits and a noncovalently bound polyproline-rich peptide. In a previous report we identified lamellipodin as the source of the polyproline-rich peptides in HuBChE tetramers purified from plasma. Our current goal was to identify proteins in addition to lamellipodin that donate polyproline-rich peptides to plasma HuBChE tetramers. Peptides were released from 1 mg of pure plasma-derived HuBChE tetramers by boiling. Mass spectrometry identified 74 polyproline-rich peptides. MALDI-TOF mass spectra and spectral counting of the LC-MS/MS data supported the conclusion that lamellipodin accounted for 70% of the polyproline-rich peptides. Additional precursor proteins were matched through BLASTp searches, suggesting but not proving, that 20 proteins including UDP-N-acetyl glucosamine transferase ALG13 homolog, leiomodin 2, and zinc finger homeobox protein 2 are sources of polyproline-rich peptides found in HuBChE tetramers. Eighteen polyproline-rich peptides had no match in the human protein database. In conclusion, HuBChE assembles into tetramers through interaction of its C-terminal domain with polyproline peptides derived from a variety of proteins.
ESTHER : Peng_2016_Chem.Biol.Interact_259_63
PubMedSearch : Peng_2016_Chem.Biol.Interact_259_63
PubMedID: 26876904

Title : Naturally Occurring Genetic Variants of Human Acetylcholinesterase and Butyrylcholinesterase and Their Potential Impact on the Risk of Toxicity from Cholinesterase Inhibitors - Lockridge_2016_Chem.Res.Toxicol_29_1381
Author(s) : Lockridge O , Norgren RB, Jr. , Johnson RC , Blake TA
Ref : Chemical Research in Toxicology , 29 :1381 , 2016
Abstract : Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides.
ESTHER : Lockridge_2016_Chem.Res.Toxicol_29_1381
PubMedSearch : Lockridge_2016_Chem.Res.Toxicol_29_1381
PubMedID: 27551784

Title : Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay - Brawner_2016_Chem.Biol.Interact_249_19
Author(s) : Brawner A , Hinrichs SH , Larson MA , Lockridge O
Ref : Chemico-Biological Interactions , 249 :19 , 2016
Abstract : The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min-1 and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 degrees C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.
ESTHER : Brawner_2016_Chem.Biol.Interact_249_19
PubMedSearch : Brawner_2016_Chem.Biol.Interact_249_19
PubMedID: 26915974

Title : Monoclonal Antibodies That Recognize Various Folding States of Pure Human Butyrylcholinesterase Can Immunopurify Butyrylcholinesterase from Human Plasma Stored at Elevated Temperatures - Peng_2016_ACS.Omega_1_1182
Author(s) : Peng H , Blake TA , Johnson RC , Dafferner AJ , Brimijoin S , Lockridge O
Ref : ACS Omega , 1 :1182 , 2016
Abstract : Human plasma to be analyzed for exposure to cholinesterase inhibitors is stored at 4 degrees C or lower to prevent denaturation of human butyrylcholinesterase (HuBChE), the biomarker of exposure. Currently published protocols immunopurify HuBChE using antibodies that bind native HuBChE before analysis by mass spectrometry. It is anticipated that the plasma collected from human casualties may be stored nonideally at elevated temperatures of up to 45 degrees C for days or maybe weeks. At 45 degrees C, the plasma loses 50% of its HuBChE activity in 8 days and 95% in 40 days. Our goal was to identify a set of monoclonal antibodies that could be used to immunopurify HuBChE from plasma stored at 45 degrees C. The folding states of pure human HuBChE stored at 4 and 45 degrees C and boiled at 100 degrees C were visualized on nondenaturing gels stained with Coomassie blue. Fully active pure HuBChE tetramers had a single band, but pure HuBChE stored at 45 degrees C had four bands, representing native, partly unfolded, aggregated, and completely denatured, boiled tetramers. The previously described monoclonal B2 18-5 captured native, partly unfolded, and aggregated HuBChE tetramers, whereas a new monoclonal, C191 developed in our laboratory, was found to selectively capture completely denatured, boiled HuBChE. The highest quantity of HuBChE protein was extracted from 45 degrees C heat-denatured human plasma when HuBChE was immunopurified with a combination of monoclonals B2 18-5 and C191. Using a mixture of these two antibodies in future emergency response assays may increase the capability to confirm exposure to cholinesterase inhibitors.
ESTHER : Peng_2016_ACS.Omega_1_1182
PubMedSearch : Peng_2016_ACS.Omega_1_1182
PubMedID: 28058292

Title : Human butyrylcholinesterase polymorphism: Molecular modeling - Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S80
Author(s) : Lushchekina SV , Delacour H , Lockridge O , Masson P
Ref : Int J Risk Saf Med , 27 Suppl 1 :S80 , 2015
Abstract : BACKGROUND: Prolonged apnoea following injection of ester-containing myoralaxants was first described in 1953. Because a large part of administered succinylcholine is shortly hydrolyzed by plasma butyrylcholinesterase (BChE) under normal conditions, prolonged apnoea was attributed to deficiency in BChE. It was found that BChE deficiency was due to genetic variations. Human BChE gene shows a large polyallelism. About 75 natural mutations of the BCHE gene have been documented so far [1]. Most of them cause alteration in BChE activity through point mutation effect on catalytic activity. Frame shifts and stop codons may also affect expression, or cause truncations in the sequence. OBJECTIVE: Recently, two novel BChE "silent" variants, Val204Asp [2] and Ala34Val [3], causing prolonged neuromuscular block after administration of mivacurium, were discovered. Mutations were genetically and kinetically characterized. The aim of the current study was to understand how these mutations determine "silent" phenotype.
METHODS: Molecular dynamics studies were carried out with NAMD 2.9 software at the Lomonosov supercomputer. Charmm 36 force field was used, periodical boundary conditions, 1 atm pressure, 298 K. 100 ns molecular dynamics runs were performed for the wild-type BChE and its mutants Val204Asp and Ala34Val.
RESULTS: Unlike wild-type BChE, which retained its operative catalytic triad through the whole MD simulation, the catalytic triad of mutants was disrupted, making chemical step impossible. Val204Asp mutation leads to reorganization of hydrogen bonding network around the catalytic triad, which in turn increases the distance between catalytic residue main chains. Mutation Ala34Val, located on the protein surface, leads to increased fluctuations in the Omega-loop and subsequent disruption of the gorge structure, including disruption of the catalytic triad and formation of new hydrogen bonds involving catalytic center residues.
CONCLUSIONS: Comparative study of the "silent" Ala328Asp mutant and the catalytically active mutant Ala328Cys shows that MD approach can discriminate between the differential effects of point mutations at a same position.
ESTHER : Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S80
PubMedSearch : Lushchekina_2015_Int.J.Risk.Saf.Med_27 Suppl 1_S80
PubMedID: 26639724

Title : Comparison of 5 monoclonal antibodies for immunopurification of human butyrylcholinesterase on Dynabeads: K values, binding pairs, and amino acid sequences - Peng_2015_Chem.Biol.Interact_240_336
Author(s) : Peng H , Brimijoin S , Hrabovska A , Targosova K , Krejci E , Blake TA , Johnson RC , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 240 :336 , 2015
Abstract : Human butyrylcholinesterase (HuBChE) is a stoichiometric bioscavenger of nerve agents and organophosphorus pesticides. Mass spectrometry methods detect stable nerve agent adducts on the active site serine of HuBChE. The first step in sample preparation is immunopurification of HuBChE from plasma. Our goal was to identify monoclonal antibodies that could be used to immunopurify HuBChE on Dynabeads Protein G. Mouse anti-HuBChE monoclonal antibodies were obtained in the form of ascites fluid, dead hybridoma cells stored frozen at -80 degrees C for 30 years, or recently frozen hybridoma cells. RNA from 4 hybridoma cell lines was amplified by PCR for determination of their nucleotide and amino acid sequences. Full-length light and heavy chains were expressed, and the antibodies purified from culture medium. A fifth monoclonal was purchased. The 5 monoclonal antibodies were compared for ability to capture HuBChE from human plasma on Dynabeads Protein G. In addition, they were evaluated for binding affinity by Biacore and ELISA. Epitope mapping by pairing analysis was performed on the Octet Red96 instrument. The 5 monoclonal antibodies, B2 12-1, B2 18-5, 3E8, mAb2, and 11D8, had similar KD values of 10-9 M for HuBChE. Monoclonal B2 18-5 outperformed the others in the Dynabeads Protein G assay where it captured 97% of the HuBChE in 0.5 ml plasma. Pairing analysis showed that 3E8 and B2 12-1 share the same epitope, 11D8 and B2 18-5 share the same epitope, but mAb2 and B2 12-1 or mAb2 and 3E8 bind to different epitopes on HuBChE. B2 18-5 was selected for establishment of a stable CHO cell line for production of mouse anti-HuBChE monoclonal.
ESTHER : Peng_2015_Chem.Biol.Interact_240_336
PubMedSearch : Peng_2015_Chem.Biol.Interact_240_336
PubMedID: 26343001

Title : Review of human butyrylcholinesterase structure, function, genetic variants, history of use in the clinic, and potential therapeutic uses - Lockridge_2015_Pharmacol.Ther_148C_34
Author(s) : Lockridge O
Ref : Pharmacol Ther , 148C :34 , 2015
Abstract : 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.
ESTHER : Lockridge_2015_Pharmacol.Ther_148C_34
PubMedSearch : Lockridge_2015_Pharmacol.Ther_148C_34
PubMedID: 25448037

Title : Butyrylcholinesterase genotype and enzyme activity in relation to Gulf War illness: preliminary evidence of gene-exposure interaction from a case-control study of 1991 Gulf War veterans - Steele_2015_Environ.Health_14_4
Author(s) : Steele L , Lockridge O , Gerkovich MM , Cook MR , Sastre A
Ref : Environ Health , 14 :4 , 2015
Abstract : BACKGROUND: Epidemiologic studies have implicated wartime exposures to acetylcholinesterase (AChE)-inhibiting chemicals as etiologic factors in Gulf War illness (GWI), the multisymptom condition linked to military service in the 1991 Gulf War. It is unclear, however, why some veterans developed GWI while others with similar exposures did not. Genetic variants of the enzyme butyrylcholinesterase (BChE) differ in their capacity for metabolizing AChE-inhibiting chemicals, and may confer differences in biological responses to these compounds. The current study assessed BChE enzyme activity and BChE genotype in 1991 Gulf War veterans to evaluate possible association of this enzyme with GWI.
METHODS: This case-control study evaluated a population-based sample of 304 Gulf War veterans (144 GWI cases, meeting Kansas GWI criteria, and 160 controls). BChE enzyme activity levels and genotype were compared, overall, in GWI cases and controls. Potential differences in risk associated with cholinergic-related exposures in theater were explored using stratified analyses to compare associations between GWI and exposures in BChE genetic and enzyme activity subgroups.
RESULTS: Overall, GWI cases and controls did not differ by mean BChE enzyme activity level or by BChE genotype. However, for the subgroup of Gulf War veterans with less common, generally less active, BChE genotypes (K/K, U/AK, U/A, A/F, AK/F), the association of wartime use of pyridostigmine bromide (PB) with GWI (OR = 40.00, p = 0.0005) was significantly greater than for veterans with the more common U/U and U/K genotypes (OR = 2.68, p = 0.0001).
CONCLUSIONS: Study results provide preliminary evidence that military personnel with certain BChE genotypes who used PB during the 1991 Gulf War may have been at particularly high risk for developing GWI. Genetic differences in response to wartime exposures are potentially important factors in GWI etiology and should be further evaluated in conjunction with exposure effects.
ESTHER : Steele_2015_Environ.Health_14_4
PubMedSearch : Steele_2015_Environ.Health_14_4
PubMedID: 25575675

Title : Pure human butyrylcholinesterase hydrolyzes octanoyl ghrelin to desacyl ghrelin - Schopfer_2015_Gen.Comp.Endocrinol_224_61
Author(s) : Schopfer LM , Lockridge O , Brimijoin S
Ref : General & Comparative Endocrinology , 224 :61 , 2015
Abstract : The ghrelin hormone is a 28 amino acid peptide esterified on serine 3 with octanoic acid. Ghrelin is inactivated by hydrolysis of the ester bond. Previous studies have relied on inhibitors to identify human butyrylcholinesterase (BChE) as the hydrolase in human plasma that converts ghrelin to desacyl ghrelin. The reaction of BChE with ghrelin is unusual because the rate of hydrolysis is very slow and the substrate is ten times larger than standard BChE substrates. These unusual features prompted us to re-examine the reaction, using human BChE preparations that were more than 98% pure. Conversion of ghrelin to desacyl ghrelin was monitored by MALDI TOF mass spectrometry. It was found that 5 different preparations of pure human BChE all hydrolyzed ghrelin, including BChE purified from human plasma, from Cohn fraction IV-4, BChE immunopurified by binding to monoclonals mAb2 and B2 18-5, and recombinant human BChE purified from culture medium. We reasoned that it was unlikely that a common contaminant that could be responsible for ghrelin hydrolysis would appear in all of these preparations. km was <1muM, and kcat was approximately 1.4min(-1). A Michaelis-Menten analysis employing these kinetic values together with serum concentrations of ghrelin and BChE demonstrated that BChE could hydrolyze all of the ghrelin in serum in approximately 1h. It was concluded that BChE is physiologically relevant for the hydrolysis of ghrelin.
ESTHER : Schopfer_2015_Gen.Comp.Endocrinol_224_61
PubMedSearch : Schopfer_2015_Gen.Comp.Endocrinol_224_61
PubMedID: 26073531

Title : Detection of cresyl phosphate-modified butyrylcholinesterase in human plasma for chemical exposure associated with aerotoxic syndrome - Schopfer_2014_Anal.Biochem_461C_17
Author(s) : Schopfer LM , Masson P , Lamourette P , Simon S , Lockridge O
Ref : Analytical Biochemistry , 461C :17 , 2014
Abstract : Flight crews complain of illness following a fume event in aircraft. A chemical in jet engine oil, the neurotoxicant tri-o-cresyl phosphate, after metabolic activation to cresyl saligenin phosphate makes a covalent adduct on butyrylcholinesterase (BChE). We developed a mass spectrometry method for detection of the cresyl phosphate adduct on human BChE as an indicator of exposure. Monoclonal mAb2, whose amino acid sequence is provided, was crosslinked to cyanogen bromide-activated Sepharose 4B and used to immunopurify plasma BChE treated with cresyl saligenin phosphate. BChE was released with acetic acid, digested with pepsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MSMS) on the Triple TOF 5600 mass spectrometer. Peptide FGES198AGAAS with an added mass of 170Da from cresyl phosphate on serine 198 (Ser198) was detected as parent ion 966.4Da. When characteristic daughter ions were monitored in the MSMS spectrum, the limit of detection was 0.1% cresyl saligenin phosphate inhibited plasma BChE. This corresponds to 2x10-9g in 0.5ml or 23x10-15moles of inhibited BChE in 0.5ml of plasma. In conclusion, a sensitive assay for exposure to tri-o-cresyl phosphate was developed. Laboratories that plan to use this method are cautioned that a positive result gives no proof that tri-o-cresyl phosphate is toxic at low levels.
ESTHER : Schopfer_2014_Anal.Biochem_461C_17
PubMedSearch : Schopfer_2014_Anal.Biochem_461C_17
PubMedID: 24892986

Title : Polyproline promotes tetramerization of recombinant human butyrylcholinesterase - Larson_2014_Biochem.J_462_329
Author(s) : Larson MA , Lockridge O , Hinrichs SH
Ref : Biochemical Journal , 462 :329 , 2014
Abstract : Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65 muM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 muM) for 1.5 h at 25 degrees C. However, rBChE tetramerization was inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellular machinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.
ESTHER : Larson_2014_Biochem.J_462_329
PubMedSearch : Larson_2014_Biochem.J_462_329
PubMedID: 24916051

Title : Characterization of a Novel BCHE Silent Allele: Point Mutation (p.Val204Asp) Causes Loss of Activity and Prolonged Apnea with Suxamethonium - Delacour_2014_PLoS.One_9_e101552
Author(s) : Delacour H , Lushchekina SV , Mabboux I , Bousquet A , Ceppa F , Schopfer LM , Lockridge O , Masson P
Ref : PLoS ONE , 9 :e101552 , 2014
Abstract : Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivacurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of suxamethonium leading to the discovery of a novel BCHE variant (c.695T>A, p.Val204Asp). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation disrupts the catalytic triad and determines a "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with heterozygous atypical silent genotype. Electrophoretic analysis of plasma BChE of the proband and his mother showed that patient has a reduced amount of tetrameric enzyme in plasma and that minor fast-moving BChE components: monomer, dimer, and monomer-albumin conjugate are missing. Kinetic analysis showed that the p.Val204Asp/p.Asp70Gly-p.Ala539Thr BChE displays a pure Michaelian behavior with BTC as the substrate. Both catalytic parameters Km = 265 microM for BTC, two times higher than that of the atypical enzyme, and a low Vmax are consistent with the absence of activity against suxamethonium. Molecular dynamic (MD) simulations showed that the overall effect of the mutation p.Val204Asp is disruption of hydrogen bonding between Gln223 and Glu441, leading Ser198 and His438 to move away from each other with subsequent disruption of the catalytic triad functionality regardless of the type of substrate. MD also showed that the enzyme volume is increased, suggesting a pre-denaturation state. This fits with the reduced concentration of p.Ala204Asp/p.Asp70Gly-p.Ala539Thr tetrameric enzyme in the plasma and non-detectable fast moving-bands on electrophoresis gels.
ESTHER : Delacour_2014_PLoS.One_9_e101552
PubMedSearch : Delacour_2014_PLoS.One_9_e101552
PubMedID: 25054547

Title : Characterization of a novel butyrylcholinesterase point mutation (p.Ala34Val), silent with mivacurium - Delacour_2014_Biochem.Pharmacol_92_476
Author(s) : Delacour H , Lushchekina SV , Mabboux I , Ceppa F , Masson P , Schopfer LM , Lockridge O
Ref : Biochemical Pharmacology , 92 :476 , 2014
Abstract : Butyrylcholinesterase deficiency is characterized by prolonged apnea after the use of muscle relaxants (suxamethonium or mivarcurium) in patients who have mutations in the BCHE gene. Here, we report a case of prolonged neuromuscular block after administration of mivacurium leading to the discovery of a novel BCHE variant (c.185C>T, p.Ala34Val). Inhibition studies, kinetic analysis and molecular dynamics were undertaken to understand how this mutation remote from the active center determines the "silent" phenotype. Low activity of patient plasma butyrylcholinesterase with butyrylthiocholine (BTC) and benzoylcholine, and values of dibucaine and fluoride numbers fit with a heterozygous enzyme of type atypical/silent. Kinetic analysis with succinyldithiocholine (SCdTC) as the substrate showed that Ala34Val BChE was inactive against this substrate. However, with BTC, the mutant enzyme was active, displaying an unexpected activation by excess substrate. Competitive inhibition of BTC by mivacurium gave a Ki=1.35mM consistent with the lack of activity with the related substrate SCdTC, and with the clinical data. Molecular dynamic simulations revealed the mechanism by which mutation Ala34Val determines the silent phenotype: a chain of intramolecular events leads to disruption of the catalytic triad, so that His438 no longer interacts with Ser198, but instead forms hydrogen bonds either with residues Glu197 and Trp82, or peripheral site residue Tyr332. However, at high BTC concentration, initial binding of substrate to the peripheral site triggers restoration of a functional catalytic triad, and activity with BTC.
ESTHER : Delacour_2014_Biochem.Pharmacol_92_476
PubMedSearch : Delacour_2014_Biochem.Pharmacol_92_476
PubMedID: 25264279

Title : Effects of viscosity and osmotic stress on the reaction of human butyrylcholinesterase with cresyl saligenin phosphate, a toxicant related to aerotoxic syndrome: kinetic and molecular dynamics studies - Masson_2013_Biochem.J_454_387
Author(s) : Masson P , Lushchekina SV , Schopfer LM , Lockridge O
Ref : Biochemical Journal , 454 :387 , 2013
Abstract : CSP (cresyl saligenin phosphate) is an irreversible inhibitor of human BChE (butyrylcholinesterase) that has been involved in the aerotoxic syndrome. Inhibition under pseudo-first-order conditions is biphasic, reflecting a slow equilibrium between two enzyme states E and E'. The elementary constants for CSP inhibition of wild-type BChE and D70G mutant were determined by studying the dependence of inhibition kinetics on viscosity and osmotic pressure. Glycerol and sucrose were used as viscosogens. Phosphorylation by CSP is sensitive to viscosity and is thus strongly diffusion-controlled (kon approximately 108 M-1.min-1). Bimolecular rate constants (ki) are about equal to kon values, making CSP one of the fastest inhibitors of BChE. Sucrose caused osmotic stress because it is excluded from the active-site gorge. This depleted the active-site gorge of water. Osmotic activation volumes, determined from the dependence of ki on osmotic pressure, showed that water in the gorge of the D70G mutant is more easily depleted than that in wild-type BChE. This demonstrates the importance of the peripheral site residue Asp70 in controlling the active-site gorge hydration. MD simulations provided new evidence for differences in the motion of water within the gorge of wild-type and D70G enzymes. The effect of viscosogens/osmolytes provided information on the slow equilibrium Eright harpoon over left harpoonE', indicating that alteration in hydration of a key catalytic residue shifts the equilibrium towards E'. MD simulations showed that glycerol molecules that substitute for water molecules in the enzyme active-site gorge induce a conformational change in the catalytic triad residue His438, leading to the less reactive form E'.
ESTHER : Masson_2013_Biochem.J_454_387
PubMedSearch : Masson_2013_Biochem.J_454_387
PubMedID: 23782236

Title : PHOS-Select Iron Affinity Beads Enrich Peptides for the Detection of Organophosphorus Adducts on Albumin - Jiang_2013_Chem.Res.Toxicol_26_1917
Author(s) : Jiang W , Dubrovskii YA , Podolskaya EP , Murashko EA , Babakov VN , Nachon F , Masson P , Schopfer LM , Lockridge O
Ref : Chemical Research in Toxicology , 26 :1917 , 2013
Abstract : Albumin is covalently modified by organophosphorus toxicants (OP) on tyrosine 411, but less than 1% of albumin is modified in humans by lethal OP doses that inhibit 95% of plasma butyrylcholinesterase. A method that enriches OP-modified albumin peptides could aid analysis of low dose exposures. Soman or chlorpyrifos oxon treated human plasma was digested with pepsin. Albumin peptides were enriched by binding to Fe(3+) beads at pH 11 and eluted with pH 2.6 buffer. Similarly, mouse and guinea pig albumin modified by chlorpyrifos oxon were digested with pepsin and enriched by binding to Fe(3+) beads. Peptides were identified by MALDI-TOF/TOF mass spectrometry. PHOS-select iron affinity beads specifically enriched albumin peptides VRY411TKKVPQVST and LVRY411TKKVPQVST in a pepsin digest of human plasma. The unmodified as well as OP-modified peptides bound to the beads. The binding capacity of 500 muL of beads was the pepsin digest of 2.1 muL of human plasma. The limit of detection was 0.2% of OP-modified albumin peptide in 0.43 muL of plasma. Enrichment of OP-modified albumin peptides by binding to Fe(3+) beads is a method with potential application to diagnosis of OP pesticide and nerve agent exposure in humans, mice, and guinea pigs.
ESTHER : Jiang_2013_Chem.Res.Toxicol_26_1917
PubMedSearch : Jiang_2013_Chem.Res.Toxicol_26_1917
PubMedID: 24187955

Title : Detectable organophosphorus pesticide exposure in the blood of Nebraska and Iowa residents measured by mass spectrometry of butyrylcholinesterase adducts - Jiang_2013_Chem.Biol.Interact_203_91
Author(s) : Jiang W , Lockridge O
Ref : Chemico-Biological Interactions , 203 :91 , 2013
Abstract : The Centers for Disease Control and Prevention detected organophosphorus pesticide (OP) metabolites in the urine of 96% of Americans, for urine collected before the ban on nonagricultural use of OP in December 2001. It was not known whether exposure was to OP degradation products or to live OP. Our goal was to determine whether exposure was to live OP in the years 2001, 2003, and 2005. Our test for exposure was the presence of OP adducts on plasma butyrylcholinesterase (BChE) detected by mass spectrometry. We purified three lots of BChE from the pooled plasma of 600-800 individuals each, in the years 2001, 2003, and 2005. Blood donors were healthy adults living in Nebraska and Iowa, two agricultural states that grow corn and soybeans. The purified BChE was tested for the presence of OP adducts on serine 198 using MALDI-TOF/TOF mass spectrometry. Low levels of methoxyphosphate-labeled BChE were found. The amount of adducted BChE was highest (1%) in blood collected in the year 2001 and lowest (0.2%) in blood collected in the year 2005. A negative control sample of BChE purified from cord blood collected in the year 2012 had no detectable adducts. It was concluded that Nebraska and Iowa residents were exposed to very low levels of live, intact organophosphorus pesticides, and that exposure levels in the pooled samples declined after the year 2001.
ESTHER : Jiang_2013_Chem.Biol.Interact_203_91
PubMedSearch : Jiang_2013_Chem.Biol.Interact_203_91
PubMedID: 22989774

Title : Mass spectrometry method to identify aging pathways of Sp- and Rp-tabun adducts on human butyrylcholinesterase based on the acid labile P-N bond - Jiang_2013_Toxicol.Sci_132_390
Author(s) : Jiang W , Cashman JR , Nachon F , Masson P , Schopfer LM , Lockridge O
Ref : Toxicol Sci , 132 :390 , 2013
Abstract : The phosphoramidate nerve agent tabun inhibits butyrylcholinesterase (BChE) and acetylcholinesterase by making a covalent bond on the active site serine. The adduct loses an alkyl group in a process called aging. The mechanism of aging of the tabun adduct is controversial. Some studies claim that aging proceeds through deamination, whereas crystal structure studies show aging by O-dealkylation. Our goal was to develop a method that clearly distinguishes between deamination and O-dealkylation. We began by studying the tetraisopropyl pyrophosphoramide adduct of BChE because this adduct has two P-N bonds. Mass spectra showed that the P-N bonds were stable during trypsin digestion at pH 8 but were cleaved during pepsin digestion at pH 2. The P-N bond in tabun was also acid labile, whereas the P-O bond was stable. A scheme to distinguish aging by deamination from aging by O-dealkylation was based on the acid labile P-N bond. BChE was inhibited with Sp- and Rp-tabun thiocholine nerve agent model compounds to make adducts identical to those of tabun with known stereochemistry. After aging and digestion with pepsin at pH 2, peptide FGES198AGAAS from Sp-tabun thiocholine had a mass of 902.2 m/z in negative mode, indicating that it had aged by deamination, whereas peptide FGES198AGAAS from Rp-tabun thiocholine had a mass of 874.2 m/z in negative mode, indicating that it had aged by O-dealkylation. BChE inhibited by authentic, racemic tabun yielded both 902.2 and 874.2 m/z peptides, indicating that both stereoisomers reacted with BChE and aged either by deamination or dealkylation.
ESTHER : Jiang_2013_Toxicol.Sci_132_390
PubMedSearch : Jiang_2013_Toxicol.Sci_132_390
PubMedID: 23345579

Title : Application of chromosomal DNA and protein targeting for the identification of Yersinia pestis - Larson_2013_Proteomics.Clin.Appl_7_416
Author(s) : Larson MA , Ding SJ , Slater SR , Hanway A , Bartling AM , Fey PD , Lockridge O , Francesconi SC , Hinrichs SH
Ref : Proteomics Clin Appl , 7 :416 , 2013
Abstract : PURPOSE: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis. EXPERIMENTAL DESIGN: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates.
RESULTS: The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts. CONCLUSIONS AND CLINICAL RELEVANCE: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.
ESTHER : Larson_2013_Proteomics.Clin.Appl_7_416
PubMedSearch : Larson_2013_Proteomics.Clin.Appl_7_416
PubMedID: 23436733

Title : Human serum albumin-based design of a diflunisal prodrug - Yang_2013_Eur.J.Pharm.Biopharm_84_549
Author(s) : Yang F , Ma ZY , Zhang Y , Li GQ , Li M , Qin JK , Lockridge O , Liang H
Ref : Eur J Pharm Biopharm , 84 :549 , 2013
Abstract : The cyclooxygenase-2 inhibitor, diflunisal, is used in the clinic for its anti-inflammatory activity. About 99% of a dose of diflunisal is unavailable for reaction with the target enzyme, because diflunisal strongly binds to human serum albumin (HSA). To reduce the binding affinity of diflunisal to albumin, we designed and synthesized the prodrug acetyldiflunisal. The crystal structure of HSA complexed with fatty acid and acetyldiflunisal revealed that acetyldiflunisal binds to the IIA subdomain and that upon binding, it acetylates lysine 199. Mass spectrometry confirmed that acetyldiflunisal acetylates Lys199. The acetylated albumin had twofold weaker binding affinity for diflunisal as demonstrated by fluorescence quenching. Reduced binding affinity means that diflunisal is more easily released from acetylated albumin into the circulation. Therefore, lower doses of acetyldiflunisal compared to diflunisal will be required. Taken together, our results not only provide a template for design of HSA-based prodrugs, but also pave the way toward more effective use of diflunisal in the clinic.
ESTHER : Yang_2013_Eur.J.Pharm.Biopharm_84_549
PubMedSearch : Yang_2013_Eur.J.Pharm.Biopharm_84_549
PubMedID: 23416065

Title : Newly observed spontaneous activation of ethephon as a butyrylcholinesterase inhibitor - Liyasova_2013_Chem.Res.Toxicol_26_422
Author(s) : Liyasova MS , Schopfer LM , Kodani S , Lantz SR , Casida JE , Lockridge O
Ref : Chemical Research in Toxicology , 26 :422 , 2013
Abstract : The plant growth regulator ethephon (2-chloroethylphosphonic acid) inhibits human butyrylcholinesterase (BChE) by making a covalent adduct on the active site serine 198. Our goal was to extend earlier studies on ethephon inhibition. Addition of freshly prepared ethephon to BChE in buffered medium, at pH 7.0 and 22 degrees C, resulted in no inhibition initially. However, inhibition developed progressively over 60 min of incubation. Preincubation of ethephon in pH 7-9 buffers increased its initial inhibitory potency. These observations indicated that ethephon itself was not the inhibitor. About 3% of the initial ethephon could be trapped as a BChE adduct. Mass spectral analysis of the active site peptide from inhibited BChE showed that the inhibitor added a mass of 108 Da to the active site serine on peptide FGES198AGAAS. This result rules out a previous hypothesis that ethephon adds HPO3 to BChE (added mass of 80 Da). To accommodate these observations, we propose that in aqueous media at neutral to slightly alkaline pH about 3% of the ethephon is converted (t1/2 = 9.9 h at pH 7.0) into a cyclic oxaphosphetane which is the actual BChE inhibitor forming the 2-hydroxyethylphosphonate adduct on BChE at Ser198 while about 97% of the ethephon breaks down to ethylene (t1/2 = 11-48 h at pH 7.0) which is responsible for plant growth regulation.
ESTHER : Liyasova_2013_Chem.Res.Toxicol_26_422
PubMedSearch : Liyasova_2013_Chem.Res.Toxicol_26_422
PubMedID: 23410221

Title : Polyclonal antibody to soman-tyrosine - Li_2013_Chem.Res.Toxicol_26_584
Author(s) : Li B , Duysen EG , Froment MT , Masson P , Nachon F , Jiang W , Schopfer LM , Thiele GM , Klassen LW , Cashman JR , Williams GR , Lockridge O
Ref : Chemical Research in Toxicology , 26 :584 , 2013
Abstract : Soman forms a stable, covalent bond with tyrosine 411 of human albumin, with tyrosines 257 and 593 in human transferrin, and with tyrosine in many other proteins. The pinacolyl group of soman is retained, suggesting that pinacolyl methylphosphonate bound to tyrosine could generate specific antibodies. Tyrosine in the pentapeptide RYGRK was covalently modified with soman simply by adding soman to the peptide. The phosphonylated-peptide was linked to keyhole limpet hemocyanin, and the conjugate was injected into rabbits. The polyclonal antiserum recognized soman-labeled human albumin, soman-mouse albumin, and soman human transferrin but not nonphosphonylated control proteins. The soman-labeled tyrosines in these proteins are surrounded by different amino acid sequences, suggesting that the polyclonal recognizes soman-tyrosine independent of the amino acid sequence. Antiserum obtained after 4 antigen injections over a period of 18 weeks was tested in a competition ELISA where it had an IC50 of 10(-11) M. The limit of detection on Western blots was 0.01 mug (15 picomoles) of soman-labeled albumin. In conclusion, a high-affinity, polyclonal antibody that specifically recognizes soman adducts on tyrosine in a variety of proteins has been produced. Such an antibody could be useful for identifying secondary targets of soman toxicity.
ESTHER : Li_2013_Chem.Res.Toxicol_26_584
PubMedSearch : Li_2013_Chem.Res.Toxicol_26_584
PubMedID: 23469927

Title : Matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry of titanium oxide-enriched peptides for detection of aged organophosphorus adducts on human butyrylcholinesterase - Jiang_2013_Anal.Biochem_439_132
Author(s) : Jiang W , Murashko EA , Dubrovskii YA , Podolskaya EP , Babakov VN , Mikler J , Nachon F , Masson P , Schopfer LM , Lockridge O
Ref : Analytical Biochemistry , 439 :132 , 2013
Abstract : Exposure to nerve agents or organophosphorus (OP) pesticides can have life-threatening effects. Human plasma butyrylcholinesterase (BChE) inactivates these poisons by binding them to Ser198. After hours or days, these OP adducts acquire a negative charge by dealkylation in a process called aging. Our goal was to develop a method for enriching the aged adduct to facilitate detection of exposure. Human BChE inhibited by OP toxicants was incubated for 4days to 6years. Peptides produced by digestion with pepsin were enriched by binding to titanium oxide (TiO2) and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. It was found that with two exceptions, all aged OP adducts in peptide FGES198AGAAS were enriched by binding to Titansphere tips. Cresyl saligenin phosphate yielded two types of aged adduct, cresylphosphate and phosphate, but only the phosphate adduct bound to Titansphere. The nerve agent VR yielded no aged adduct, supporting crystal structure findings that the VR adduct on BChE does not age. The irreversible nature of aged OP adducts was demonstrated by the finding that after 6years at room temperature in sterile pH 7.0 buffer, the adducts were still detectable. It was concluded that TiO2 microcolumns can be used to enrich aged OP-modified BChE peptide.
ESTHER : Jiang_2013_Anal.Biochem_439_132
PubMedSearch : Jiang_2013_Anal.Biochem_439_132
PubMedID: 23624322

Title : Polyproline tetramer organizing peptides in fetal bovine serum acetylcholinesterase - Biberoglu_2013_Biochim.Biophys.Acta_1834_745
Author(s) : Biberoglu K , Schopfer LM , Saxena A , Tacal O , Lockridge O
Ref : Biochimica & Biophysica Acta , 1834 :745 , 2013
Abstract : 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.
ESTHER : Biberoglu_2013_Biochim.Biophys.Acta_1834_745
PubMedSearch : Biberoglu_2013_Biochim.Biophys.Acta_1834_745
PubMedID: 23352838

Title : Inhibition pathways of the potent organophosphate CBDP with cholinesterases revealed by X-ray crystallographic snapshots and mass spectrometry - Carletti_2013_Chem.Res.Toxicol_26_280
Author(s) : Carletti E , Colletier JP , Schopfer LM , Santoni G , Masson P , Lockridge O , Nachon F , Weik M
Ref : Chemical Research in Toxicology , 26 :280 , 2013
Abstract : Tri-o-cresyl-phosphate (TOCP) is a common additive in jet engine lubricants and hydraulic fluids suspected to have a role in aerotoxic syndrome in humans. TOCP is metabolized to cresyl saligenin phosphate (CBDP), a potent irreversible inhibitor of butyrylcholinesterase (BChE), a natural bioscavenger present in the bloodstream, and acetylcholinesterase (AChE), the off-switch at cholinergic synapses. Mechanistic details of cholinesterase (ChE) inhibition have, however, remained elusive. Also, the inhibition of AChE by CBDP is unexpected, from a structural standpoint, i.e., considering the narrowness of AChE active site and the bulkiness of CBDP. In the following, we report on kinetic X-ray crystallography experiments that provided 2.7-3.3 A snapshots of the reaction of CBDP with mouse AChE and human BChE. The series of crystallographic snapshots reveals that AChE and BChE react with the opposite enantiomers and that an induced-fit rearrangement of Phe297 enlarges the active site of AChE upon CBDP binding. Mass spectrometry analysis of aging in either H(2)(16)O or H(2)(18)O furthermore allowed us to identify the inhibition steps, in which water molecules are involved, thus providing insights into the mechanistic details of inhibition. X-ray crystallography and mass spectrometry show the formation of an aged end product formed in both AChE and BChE that cannot be reactivated by current oxime-based therapeutics. Our study thus shows that only prophylactic and symptomatic treatments are viable to counter the inhibition of AChE and BChE by CBDP.
ESTHER : Carletti_2013_Chem.Res.Toxicol_26_280
PubMedSearch : Carletti_2013_Chem.Res.Toxicol_26_280
PubMedID: 23339663
Gene_locus related to this paper: human-BCHE , mouse-ACHE

Title : Resistance of human butyrylcholinesterase to methylene blue-catalyzed photoinactivation\; mass spectrometry analysis of oxidation products - Tacal_2013_Photochem.Photobiol_89_336
Author(s) : Tacal O , Li B , Lockridge O , Schopfer LM
Ref : Photochem Photobiol , 89 :336 , 2013
Abstract : Methylene blue, 3, 7-bis(dimethylamino)-phenothiazin-5-ium chloride, is a reversible inhibitor of human butyrylcholinesterase (BChE) in the absence of light. In the presence of light and oxygen, methylene blue promotes irreversible inhibition of human BChE as a function of time, requiring 3 h irradiation to inhibit 95% activity. Inactivation was accompanied by a progressive loss of Coomassie-stained protein bands on native and denaturing polyacrylamide gels, suggesting backbone fragmentation. Aggregation was not detected. MALDI-TOF/TOF mass spectrometry identified oxidized tryptophan (W52, 56, 231, 376, 412, 490, 522), oxidized methionine (M81, 144, 302, 532, 554, 555), oxidized histidine (H214), oxidized proline (P230), oxidized cysteine (C519) and oxidized serine (S215). A 20 min irradiation in the presence of methylene blue resulted in 17% loss of BChE activity, suggesting that BChE is relatively resistant to methylene blue-catalyzed photoinactivation and that therefore this process could be used to sterilize BChE preparations.
ESTHER : Tacal_2013_Photochem.Photobiol_89_336
PubMedSearch : Tacal_2013_Photochem.Photobiol_89_336
PubMedID: 23136924

Title : Cresyl saligenin phosphate makes multiple adducts on free histidine, but does not form an adduct on histidine 438 of human butyrylcholinesterase - Liyasova_2013_Chem.Biol.Interact_203_103
Author(s) : Liyasova MS , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 203 :103 , 2013
Abstract : Cresyl saligenin phosphate (CBDP) is a suspected causative agent of "aerotoxic syndrome", affecting pilots, crew members and passengers. CBDP is produced in vivo from ortho-containing isomers of tricresyl phosphate (TCP), a component of jet engine lubricants and hydraulic fluids. CBDP irreversibly inhibits butyrylcholinesterase (BChE) in human plasma by forming adducts on the active site serine (Ser-198). Inhibited BChE undergoes aging to release saligenin and o-cresol. The active site histidine (His-438) was hypothesized to abstract o-hydroxybenzyl moiety from the initial adduct on Ser-198. Our goal was to test this hypothesis. Mass spectral analysis of CBDP-inhibited BChE digested with Glu-C showed an o-hydroxybenzyl adduct (+106amu) on lysine 499, a residue far from the active site, but not on His-438. Nevertheless, the nitrogen of the imidazole ring of free l-histidine formed a variety of adducts upon reaction with CBDP, including the o-hydroxybenzyl adduct, suggesting that histidine-CBDP adducts may form on other proteins.
ESTHER : Liyasova_2013_Chem.Biol.Interact_203_103
PubMedSearch : Liyasova_2013_Chem.Biol.Interact_203_103
PubMedID: 22898212

Title : Loss of Dnmt3b function upregulates the tumor modifier Ment and accelerates mouse lymphomagenesis - Hlady_2012_J.Clin.Invest_122_163
Author(s) : Hlady RA , Novakova S , Opavska J , Klinkebiel D , Peters SL , Bies J , Hannah J , Iqbal J , Anderson KM , Siebler HM , Smith LM , Greiner TC , Bastola D , Joshi S , Lockridge O , Simpson MA , Felsher DW , Wagner KU , Chan WC , Christman JK , Opavsky R
Ref : J Clinical Investigation , 122 :163 , 2012
Abstract : DNA methyltransferase 3B (Dnmt3b) belongs to a family of enzymes responsible for methylation of cytosine residues in mammals. DNA methylation contributes to the epigenetic control of gene transcription and is deregulated in virtually all human tumors. To better understand the generation of cancer-specific methylation patterns, we genetically inactivated Dnmt3b in a mouse model of MYC-induced lymphomagenesis. Ablation of Dnmt3b function using a conditional knockout in T cells accelerated lymphomagenesis by increasing cellular proliferation, which suggests that Dnmt3b functions as a tumor suppressor. Global methylation profiling revealed numerous gene promoters as potential targets of Dnmt3b activity, the majority of which were demethylated in Dnmt3b-/- lymphomas, but not in Dnmt3b-/- pretumor thymocytes, implicating Dnmt3b in maintenance of cytosine methylation in cancer. Functional analysis identified the gene Gm128 (which we termed herein methylated in normal thymocytes [Ment]) as a target of Dnmt3b activity. We found that Ment was gradually demethylated and overexpressed during tumor progression in Dnmt3b-/- lymphomas. Similarly, MENT was overexpressed in 67% of human lymphomas, and its transcription inversely correlated with methylation and levels of DNMT3B. Importantly, knockdown of Ment inhibited growth of mouse and human cells, whereas overexpression of Ment provided Dnmt3b+/+ cells with a proliferative advantage. Our findings identify Ment as an enhancer of lymphomagenesis that contributes to the tumor suppressor function of Dnmt3b and suggest it could be a potential target for anticancer therapies.
ESTHER : Hlady_2012_J.Clin.Invest_122_163
PubMedSearch : Hlady_2012_J.Clin.Invest_122_163
PubMedID: 22133874

Title : The proline-rich tetramerization peptides in equine serum butyrylcholinesterase - Biberoglu_2012_Febs.J_279_3844
Author(s) : Biberoglu K , Schopfer LM , Tacal O , Lockridge O
Ref : Febs J , 279 :3844 , 2012
Abstract : Soluble, tetrameric, plasma butyrylcholinesterase from horse has previously been shown to include a non-covalently attached polyproline peptide in its structure. The polyproline peptide matched the polyproline-rich region of human lamellipodin. Our goal was to examine the tetramer-organizing peptides of horse butyrylcholinesterase in more detail. Horse butyrylcholinesterase was denatured by boiling, thus releasing a set of polyproline peptides ranging in mass from 1173 to 2098 Da. The peptide sequences were determined by fragmentation in MALDI-TOF-TOF and linear ion trap quadrupole Orbitrap mass spectrometers. Twenty-seven polyproline peptides grouped into 13 families were identified. Peptides contained a minimum of 11 consecutive proline residues and as many as 21. Many of the peptides had a non-proline amino acid at the N-terminus. A search of the protein databanks matched peptides to nine proteins, although not all peptides matched a known protein. It is concluded that polyproline peptides of various lengths and sequences are included in the tetramer structure of horse butyrylcholinesterase. The function of these polyproline peptides is to serve as tetramer-organizing peptides.
ESTHER : Biberoglu_2012_Febs.J_279_3844
PubMedSearch : Biberoglu_2012_Febs.J_279_3844
PubMedID: 22889087

Title : Analytical approaches for monitoring exposure to organophosphorus and carbamate agents through analysis of protein adducts - Schopfer_2012_Drug.Test.Anal_4_246
Author(s) : Schopfer LM , Lockridge O
Ref : Drug Test Anal , 4 :246 , 2012
Abstract : Appropriate treatment of a poisoned patient requires knowing the identity of the poison. This review summarizes the methods for identifying poisoning by organophosphorus and carbamate poisons. Mass spectrometry methods identify the poison from the adducts they form with proteins in blood. The most sensitive method uses potassium fluoride to release the organophosphorus agent from its covalent binding to serine 198 of human butyrylcholinesterase. The released poison is identified by gas chromatography-mass spectrometry. The drawback of this method is that it does not detect exposure to agents such as soman, because butyrylcholinesterase adducts with these agents age to a non-reactivatable form. A method that detects both aged and non-aged organophosphylated adducts as well as carbamate adducts is one that digests butyrylcholinesterase with a protease and analyzes the modified peptide by mass spectrometry. This method does not distinguish between poisons that have the same mass after reaction with butyrylcholinesterase--for example, between exposure to chlorpyrifos oxon and paraoxon. Albumin forms a stable, covalent bond with organophosphates on tyrosine 411. The rate of reaction with albumin is much slower than with butyrylcholinesterase, but albumin adducts persist for a longer time in the circulation; they do not age; and they do not release the organophosphate when a patient is treated with an oxime.
ESTHER : Schopfer_2012_Drug.Test.Anal_4_246
PubMedSearch : Schopfer_2012_Drug.Test.Anal_4_246
PubMedID: 22359362

Title : Human butyrylcholinesterase produced in insect cells: huprine-based affinity purification and crystal structure - Brazzolotto_2012_Febs.J_279_2905
Author(s) : Brazzolotto X , Wandhammer M , Ronco C , Trovaslet M , Jean L , Lockridge O , Renard PY , Nachon F
Ref : Febs J , 279 :2905 , 2012
Abstract : Butyrylcholinesterase is a serine hydrolase present in all mammalian tissues. It can accommodate larger substrates or inhibitors than acetylcholinesterase, the enzyme responsible for hydrolysis of the neurotransmitter acetylcholine in the central nervous system and neuromuscular junctions. AChE is the specific target of organophosphorous pesticides and warfare nerve agents, while BChE is their stoichiometric bioscavenger. Conversion of BChE into a catalytic bioscavenger by rational design or designing reactivators specific to BChE required structural data obtained with a recombinant low-glycosylated human BChE expressed in Chinese hamster ovary cells. This expression system yields about 1 mg of pure enzyme per liter of cell culture. Here, we report an improved expression system with 4-fold higher yield for truncated human BChE with all glycosylation sites present using insect cells. We developed a fast purification protocol of the recombinant protein using a huprine-based affinity chromatography superior to the classical procainamide-based affinity. The purified BChE crystallized in different conditions and space group than those for the recombinant low-glycosylated protein produced in Chinese hamster ovary cells. The crystals diffracted to 2.5 A. The overall monomer structure is similar to the low-glycosylated structure but for the presence of the additional glycans. Remarkably, the carboxylic acid molecule systematically bound to the catalytic serine in the low-glycosylated structure is also present in this new structure, despite the different expression system, purification protocol and crystallization conditions.
ESTHER : Brazzolotto_2012_Febs.J_279_2905
PubMedSearch : Brazzolotto_2012_Febs.J_279_2905
PubMedID: 22726956
Gene_locus related to this paper: human-BCHE

Title : Cresyl saligenin phosphate, an organophosphorus toxicant, makes covalent adducts with histidine, lysine, and tyrosine residues of human serum albumin - Liyasova_2012_Chem.Res.Toxicol_25_1752
Author(s) : Liyasova MS , Schopfer LM , Lockridge O
Ref : Chemical Research in Toxicology , 25 :1752 , 2012
Abstract : CBDP [2-(2-cresyl)-4H-1-3-2-benzodioxaphosphorin-2-oxide] is a toxic organophosphorus compound. It is generated in vivo from tri-ortho-cresyl phosphate (TOCP), a component of jet engine oil and hydraulic fluids. Exposure to TOCP was proven to occur on board aircraft by finding CBDP-derived phospho-butyrylcholinesterase in the blood of passengers. Adducts on BChE, however, do not explain the toxicity of CBDP. Critical target proteins of CBDP are yet to be identified. Our goal was to facilitate the search for the critical targets of CBDP by determining the range of amino acid residues capable of reacting with CBDP and characterizing the types of adducts formed. We used human albumin as a model protein. Mass spectral analysis of the tryptic digest of CBDP-treated human albumin revealed adducts on His-67, His-146, His-242, His-247, His-338, Tyr-138, Tyr-140, Lys-199, Lys-351, Lys-414, Lys-432, and Lys-525. Adducts formed on tyrosine residues were different from those formed on histidines and lysines. Tyrosines were organophosphorylated by CBDP, while histidine and lysine residues were alkylated. This is the first report of an organophosphorus compound with both phosphorylating and alkylating properties. The o-hydroxybenzyl adduct on histidine is novel. The ability of CBDP to form stable adducts on histidine, tyrosine, and lysine allows one to consider new mechanisms of toxicity from TOCP exposure.
ESTHER : Liyasova_2012_Chem.Res.Toxicol_25_1752
PubMedSearch : Liyasova_2012_Chem.Res.Toxicol_25_1752
PubMedID: 22793878

Title : Mice treated with a nontoxic dose of chlorpyrifos oxon have diethoxyphosphotyrosine labeled proteins in blood up to 4 days post exposure, detected by mass spectrometry - Jiang_2012_Toxicology_295_15
Author(s) : Jiang W , Duysen EG , Lockridge O
Ref : Toxicology , 295 :15 , 2012
Abstract : Inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity is an established biomarker of exposure to organophosphorus poisons (OP). Inhibition of activity is due to covalent binding of the OP to the active site serine. Mass spectrometry has made it possible to monitor OP exposure by analyzing OP adducts on tyrosine in proteins that have no active site serine. Our goal was to test the hypothesis that OP-tyrosine may serve as a biomarker of OP exposure in mice. A MALDI-TOF mass spectrometry strategy to analyze diethoxyphosphate-tyrosine of m/z 318 was developed. The adduct was synthesized by incubating l-tyrosine with chlorpyrifos oxon at pH 8.1. The adduct eluted from a reverse phase HPLC column with 22-23% acetonitrile. The fragmentation spectrum of the m/z 318 precursor ion confirmed its identity as diethoxyphosphate-tyrosine. Diethoxyphosphate-tyrosine was isolated from chlorpyrifos oxon treated mouse albumin after digesting the protein with pronase. Mice (n=3 per group) were treated with a nontoxic dose of chlorpyrifos oxon (3 mg/kg) and a toxic dose (10 mg/kg transdermally). The pronase digested plasma yielded diethoxyphosphate-tyrosine up to 120 h after treatment with 3 mg/kg chlorpyrifos oxon and up to 144 h after 10 mg/kg. In contrast plasma AChE activity returned to normal after 24-72 h. In conclusion MALDI-TOF mass spectrometry can be used to diagnose exposure to chlorpyrifos oxon days after AChE inhibition assays are uninformative.
ESTHER : Jiang_2012_Toxicology_295_15
PubMedSearch : Jiang_2012_Toxicology_295_15
PubMedID: 22406659

Title : Differential sensitivity of plasma carboxylesterase-null mice to parathion, chlorpyrifos and chlorpyrifos oxon, but not to diazinon, dichlorvos, diisopropylfluorophosphate, cresyl saligenin phosphate, cyclosarin thiocholine, tabun thiocholine, and carbofuran - Duysen_2012_Chem.Biol.Interact_195_189
Author(s) : Duysen EG , Cashman JR , Schopfer LM , Nachon F , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 195 :189 , 2012
Abstract : Mouse blood contains four esterases that detoxify organophosphorus compounds: carboxylesterase, butyrylcholinesterase, acetylcholinesterase, and paraoxonase-1. In contrast human blood contains the latter three enzymes but not carboxylesterase. Organophosphorus compound toxicity is due to inhibition of acetylcholinesterase. Symptoms of intoxication appear after approximately 50% of the acetylcholinesterase is inhibited. However, complete inhibition of carboxylesterase and butyrylcholinesterase has no known effect on an animal's well being. Paraoxonase hydrolyzes organophosphorus compounds and is not inhibited by them. Our goal was to determine the effect of plasma carboxylesterase deficiency on response to sublethal doses of 10 organophosphorus toxicants and one carbamate pesticide. Homozygous plasma carboxylesterase deficient ES1(-/-) mice and wild-type littermates were observed for toxic signs and changes in body temperature after treatment with a single sublethal dose of toxicant. Inhibition of plasma acetylcholinesterase, butyrylcholinesterase, and plasma carboxylesterase was measured. It was found that wild-type mice were protected from the toxicity of 12.5mg/kg parathion applied subcutaneously. However, both genotypes responded similarly to paraoxon, cresyl saligenin phosphate, diisopropylfluorophosphate, diazinon, dichlorvos, cyclosarin thiocholine, tabun thiocholine, and carbofuran. An unexpected result was the finding that transdermal application of chlorpyrifos at 100mg/kg and chlorpyrifos oxon at 14mg/kg was lethal to wild-type but not to ES1(-/-) mice, showing that with this organochlorine, the presence of carboxylesterase was harmful rather than protective. It was concluded that carboxylesterase in mouse plasma protects from high toxicity agents, but the amount of carboxylesterase in plasma is too low to protect from low toxicity compounds that require high doses to inhibit acetylcholinesterase.
ESTHER : Duysen_2012_Chem.Biol.Interact_195_189
PubMedSearch : Duysen_2012_Chem.Biol.Interact_195_189
PubMedID: 22209767

Title : Production of ES1 plasma carboxylesterase knockout mice for toxicity studies - Duysen_2011_Chem.Res.Toxicol_24_1891
Author(s) : Duysen EG , Koentgen F , Williams GR , Timperley CM , Schopfer LM , Cerasoli DM , Lockridge O
Ref : Chemical Research in Toxicology , 24 :1891 , 2011
Abstract : The LD(50) for soman is 10-20-fold higher for a mouse than a human. The difference in susceptibility is attributed to the presence of carboxylesterase in mouse but not in human plasma. Our goal was to make a mouse lacking plasma carboxylesterase. We used homologous recombination to inactivate the carboxylesterase ES1 gene on mouse chromosome 8 by deleting exon 5 and by introducing a frame shift for amino acids translated from exons 6 to 13. ES1-/- mice have no detectable carboxylesterase activity in plasma but have normal carboxylesterase activity in tissues. Homozygous ES1-/- mice and wild-type littermates were tested for response to a nerve agent model compound (soman coumarin) at 3 mg/kg sc. This dose intoxicated both genotypes but was lethal only to ES1-/- mice. This demonstrated that plasma carboxylesterase protects against a relatively high toxicity organophosphorus compound. The ES1-/- mouse should be an appropriate model for testing highly toxic nerve agents and for evaluating protection strategies against the toxicity of nerve agents.
ESTHER : Duysen_2011_Chem.Res.Toxicol_24_1891
PubMedSearch : Duysen_2011_Chem.Res.Toxicol_24_1891
PubMedID: 21875074
Gene_locus related to this paper: mouse-Ces1c

Title : Gene-Delivered Butyrylcholinesterase Is Prophylactic against the Toxicity of Chemical Warfare Nerve Agents and Organophosphorus Compounds - Parikh_2011_J.Pharmacol.Exp.Ther_337_92
Author(s) : Parikh K , Duysen EG , Snow B , Jensen NS , Manne V , Lockridge O , Chilukuri N
Ref : Journal of Pharmacology & Experimental Therapeutics , 337 :92 , 2011
Abstract : Gene delivery using an adenoviral system has been effective in introducing therapeutic proteins in vitro and in vivo. This study tested the feasibility of using adenovirus to deliver clinically relevant amounts of butyrylcholinesterase (BChE), a proven bioscavenger of nerve agents. The adenovirus construct expressed full-length mouse BChE. Mice were injected with a single dose of adenovirus (1.5 10(10) infectious units) in the tail vein; plasma was collected through day 11 and assayed for BChE activity. Maximum activity, representing a 300- to 3400-fold increase over baseline, was found on day 4. Expression levels returned to baseline by day 10. Nondenaturing gel electrophoresis showed the recombinant BChE was a dimer that could be converted to tetramers by addition of polyproline. The toxic compounds chosen for protection studies were positively charged organophosphorus agents, echothiophate, and O-ethyl-S-2-N,N-diisopropylaminoethyl methylphosphonothiolate (VX). Mice containing elevated blood levels of BChE (300- to 3,000-fold over the control mice) were challenged with incremental doses of echothiophate or VX. Mice showed no signs of toxicity and were protected from up to 30 LD(50) dose of echothiophate and 5 LD(50) dose of VX. A good correlation was observed between tolerated echothiophate dose and plasma BChE levels at time of challenge. The absolute increases in levels of circulating BChE and the sustained nature of the response resulted in a very high enzyme concentration, deemed critical in acute toxicity (5 LD(50) or more) scenarios. These results suggest that gene-delivered BChE is a prophylactic and affords protection equivalent to that of a multimilligram injection of the same.
ESTHER : Parikh_2011_J.Pharmacol.Exp.Ther_337_92
PubMedSearch : Parikh_2011_J.Pharmacol.Exp.Ther_337_92
PubMedID: 21205915

Title : Transglutaminase-mediated remodeling of the human erythrocyte membrane skeleton: relevance for erythrocyte diseases with shortened cell lifespan -
Author(s) : Lorand L , Murthy SN , Khan AA , Xue W , Lockridge O , Chishti AH
Ref : Adv Enzymol Relat Areas Mol Biol , 78 :385 , 2011
PubMedID: 22220479

Title : The kinetic study of the inhibition of human cholinesterases by demeton-S-methyl shows that cholinesterase-based titration methods are not suitable for this organophosphate - Bazire_2011_Toxicol.In.Vitro_25_754
Author(s) : Bazire A , Gillon E , Lockridge O , Vallet V , Nachon F
Ref : Toxicol In Vitro , 25 :754 , 2011
Abstract : The organophosphorus insecticide, demeton-S-methyl (DSM), is considered as a good surrogate of the highly toxic nerve agent VX for skin absorption studies due to similar physico-chemical properties and in vitro percutaneous penetration profile. But, when skin distribution was estimated by measuring inhibition of cholinesterase activity, the results were poorly reproducible. The various grades of commercial DSM solutions were suspected to be the origin of the discrepancies. This hypothesis was tested by measuring inhibition of human acetyl- and butyrylcholinesterase by two commercial DSM solutions. The inhibition rate was independent on the enzyme concentration confirming pseudo-first order conditions. But complete inhibition of butyrylcholinesterase activity was achieved only when the DSM concentration was at least 1500-fold higher than the enzyme concentration. Besides, complete inhibition of acetylcholinesterase was never achieved. Mass spectrometry analysis of the inhibited butyrylcholinesterase adducts identified monomethoxyphosphorylated-serine, the aged product of inhibition by DSM or a derivative with a modified leaving group. Neither spontaneous reactivation nor aging of the dimethoxyphosphorylated-serine could account for the inhibition kinetics observed, suggesting an overly complicated kinetic scheme not compatible with the requirement of a titration experiment. In conclusion, cholinesterase-based analytical methods should be avoided for DSM titration in skin penetration studies.
ESTHER : Bazire_2011_Toxicol.In.Vitro_25_754
PubMedSearch : Bazire_2011_Toxicol.In.Vitro_25_754
PubMedID: 21238577

Title : Adenovirus-mediated human paraoxonase1 gene transfer to provide protection against the toxicity of the organophosphorus pesticide toxicant diazoxon - Duysen_2011_Gene.Ther_18_250
Author(s) : Duysen EG , Parikh K , Aleti V , Manne V , Lockridge O , Chilukuri N
Ref : Gene Therapy , 18 :250 , 2011
Abstract : Human paraoxonase1 (hPON1) is a potential therapeutic against the toxicity of organophosphorus (OP) pesticides and chemical warfare nerve agents. We tested whether PON1 gene transfer using adenovirus provides protection against the toxicity of the OP diazoxon. Using an adenovirus construct containing hPON1 gene, we showed elevated levels of recombinant hPON1 in vitro in 293A cells and in vivo in mice. The recombinant enzyme was secreted by 293A cells into culture medium and into the systemic circulation of mice. Western blotting revealed that the virally expressed hPON1 had the expected molecular weight of 45 kDa. Recombinant hPON1 in mice was in complex with mouse high-density lipoprotein (HDL) and migrated more slowly than endogenous hPON1 in the human HDL complex. Mice injected with adenovirus expressed PON1 at 600-3480 U ml(-1) on day 5 post-treatment, which is 8-50-fold above endogenous. Six mice expressing hPON1 survived 2LD(50) doses of diazoxon. Four of the six mice survived a second dose of diazoxon (for a total of 4LD(50)) administered 24 h later. In contrast, none of the three mice in the control group survived one 2LD(50) dose. These results show that hPON1 in mice functions as a prophylactic and offers significant protection against lethal doses of diazoxon.
ESTHER : Duysen_2011_Gene.Ther_18_250
PubMedSearch : Duysen_2011_Gene.Ther_18_250
PubMedID: 20981111

Title : Biomarkers of organophosphorus (OP) exposures in humans - Marsillach_2011_Neurotoxicol_32_656
Author(s) : Marsillach J , Richter RJ , Kim JH , Stevens RC , Maccoss MJ , Tomazela D , Suzuki SM , Schopfer LM , Lockridge O , Furlong CE
Ref : Neurotoxicology , 32 :656 , 2011
Abstract : There are ongoing events where aircraft engine lubricant containing tricresyl phosphates (TCPs) contaminates aircraft cabins. Some individuals have experienced tremors or other neurological symptoms that may last for many months following exposures. Mass spectrometric (MS) protocols are being developed to determine the percentage of "biomarker proteins" that are modified by such exposures, specifically on active site serines. Both plasma butyrylcholinesterase (BChE) and red cell acylpeptide hydrolase (APH) are readily inhibited by 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP) or phenyl saligenin cyclic phosphate (PSP) and have the potential to provide information about the level of exposure of an individual. We have developed immunomagnetic bead-based single-step purification protocols for both BChE and APH and have characterized the active site serine adducts of BChE by MS.
ESTHER : Marsillach_2011_Neurotoxicol_32_656
PubMedSearch : Marsillach_2011_Neurotoxicol_32_656
PubMedID: 21767566

Title : Role of acetylcholinesterase on the structure and function of cholinergic synapses: insights gained from studies on knockout mice - Adler_2011_Cell.Mol.Neurobiol_31_909
Author(s) : Adler M , Sweeney RE , Hamilton TA , Lockridge O , Duysen EG , Purcell AL , Deshpande SS
Ref : Cellular Molecular Neurobiology , 31 :909 , 2011
Abstract : Electrophysiological and ultrastructural studies were performed on phrenic nerve-hemidiaphragm preparations isolated from wild-type and acetylcholinesterase (AChE) knockout (KO) mice to determine the compensatory mechanisms manifested by the neuromuscular junction to excess acetylcholine (ACh). The diaphragm was selected since it is the primary muscle of respiration, and it must adapt to allow for survival of the organism in the absence of AChE. Nerve-elicited muscle contractions, miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) were recorded by conventional electrophysiological techniques from phrenic nerve-hemidiaphragm preparations isolated from 1.5- to 2-month-old wild-type (AChE(+/+)) or AChE KO (AChE(-/-)) mice. These recordings were chosen to provide a comprehensive assessment of functional alterations of the diaphragm muscle resulting from the absence of AChE. Tension measurements from AChE(-/-) mice revealed that the amplitude of twitch tensions was potentiated, but tetanic tensions underwent a use-dependent decline at frequencies below 70 Hz and above 100 Hz. MEPPs recorded from hemidiaphragms of AChE(-/-) mice showed a reduction in frequency and a prolongation in decay (37%) but no change in amplitude compared to values observed in age-matched wild-type littermates. In contrast, MEPPs recorded from hemidiaphragms of wild-type mice that were exposed for 30 min to the selective AChE inhibitor 5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW284C51) exhibited a pronounced increase in amplitude (42%) and a more marked prolongation in decay (76%). The difference between MEPP amplitudes and decays in AChE(-/-) hemidiaphragms and in wild-type hemidiaphragms treated with BW284C51 represents effective adaptation by the former to a high ACh environment. Electron microscopic examination revealed that diaphragm muscles of AChE(-/-) mice had smaller nerve terminals and diminished pre- and post-synaptic surface contacts relative to neuromuscular junctions of AChE(+/+) mice. The morphological changes are suggested to account, in part, for the ability of muscle from AChE(-/-) mice to function in the complete absence of AChE.
ESTHER : Adler_2011_Cell.Mol.Neurobiol_31_909
PubMedSearch : Adler_2011_Cell.Mol.Neurobiol_31_909
PubMedID: 21538119

Title : Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons - Duysen_2011_Toxicol.Appl.Pharmacol_255_214
Author(s) : Duysen EG , Lockridge O
Ref : Toxicol Appl Pharmacol , 255 :214 , 2011
Abstract : The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase.
ESTHER : Duysen_2011_Toxicol.Appl.Pharmacol_255_214
PubMedSearch : Duysen_2011_Toxicol.Appl.Pharmacol_255_214
PubMedID: 21767560

Title : X-ray crystallographic snapshots of reaction intermediates in the G117H mutant of human butyrylcholinesterase, a nerve agent target engineered into a catalytic bioscavenger - Nachon_2011_Biochem.J_434_73
Author(s) : Nachon F , Carletti E , Wandhammer M , Nicolet Y , Schopfer LM , Masson P , Lockridge O
Ref : Biochemical Journal , 434 :73 , 2011
Abstract : OPs (organophosphylates) exert their acute toxicity through inhibition of acetylcholinesterase, by phosphylation of the catalytic serine residue. Engineering of human butyrylcholinesterase, by substitution of a histidine residue for the glycine residue at position 117, led to the creation of OP hydrolase activity. However, the lack of structural information and poor understanding of the hydrolytic mechanism of the G117H mutant has hampered further improvements in the catalytic activity. We have solved the crystallographic structure of the G117H mutant with a variety of ligands in its active site. A sulfate anion bound to the active site suggested the positioning for an OP prior to phosphylation. A fluoride anion was found in the active site when NaF was added to the crystallization buffer. In the fluoride complex, the imidazole ring from the His117 residue was substantially shifted, adopting a relaxed conformation probably close to that of the unliganded mutant enzyme. Additional X-ray structures were obtained from the transient covalent adducts formed upon reaction of the G117H mutant with the OPs echothiophate and VX [ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl](methyl)phosphinate]. The position of the His117 residue shifted in response to the introduction of these adducts, overlaying the phosphylserine residue. These structural data suggest that the dephosphylation mechanism involves either a substantial conformational change of the His117 residue or an adjacent nucleophilic substitution by water.
ESTHER : Nachon_2011_Biochem.J_434_73
PubMedSearch : Nachon_2011_Biochem.J_434_73
PubMedID: 21091433
Gene_locus related to this paper: human-BCHE

Title : Reaction of cresyl saligenin phosphate, the organophosphorus agent implicated in aerotoxic syndrome, with human cholinesterases: mechanistic studies employing kinetics, mass spectrometry, and X-ray structure analysis - Carletti_2011_Chem.Res.Toxicol_24_797
Author(s) : Carletti E , Schopfer LM , Colletier JP , Froment MT , Nachon F , Weik M , Lockridge O , Masson P
Ref : Chemical Research in Toxicology , 24 :797 , 2011
Abstract : Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate (TCP), an antiwear additive in jet engine lubricants and hydraulic fluid. CBDP (2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one) is the toxic metabolite of triortho-cresylphosphate, a component of TCP. Human butyrylcholinesterase (BChE; EC and human acetylcholinesterase (AChE; EC are irreversibly inhibited by CBDP. The bimolecular rate constants of inhibition (k(i)), determined under pseudo-first-order conditions, displayed a biphasic time course of inhibition with k(i) of 1.6 x 10(8) M(-1) min(-1) and 2.7 x 10(7) M(-1) min(-1) for E and E' forms of BChE. The inhibition constants for AChE were 1 to 2 orders of magnitude slower than those for BChE. CBDP-phosphorylated cholinesterases are nonreactivatable due to ultra fast aging. Mass spectrometry analysis showed an initial BChE adduct with an added mass of 170 Da from cresylphosphate, followed by dealkylation to a structure with an added mass of 80 Da. Mass spectrometry in (18)O-water showed that (18)O was incorporated only during the final aging step to form phospho-serine as the final aged BChE adduct. The crystal structure of CBDP-inhibited BChE confirmed that the phosphate adduct is the ultimate aging product. CBDP is the first organophosphorus agent that leads to a fully dealkylated phospho-serine BChE adduct.
ESTHER : Carletti_2011_Chem.Res.Toxicol_24_797
PubMedSearch : Carletti_2011_Chem.Res.Toxicol_24_797
PubMedID: 21438623
Gene_locus related to this paper: human-BCHE

Title : Cocrystallization studies of full-length recombinant butyrylcholinesterase (BChE) with cocaine - Asojo_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_434
Author(s) : Asojo OA , Ngamelue MN , Homma K , Lockridge O
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 67 :434 , 2011
Abstract : Human butyrylcholinesterase (BChE; EC is a 340kDa tetrameric glycoprotein that is present in human serum at about 5mgl(-1) and has well documented therapeutic effects on cocaine toxicity. BChE holds promise as a therapeutic that reduces and finally eliminates the rewarding effects of cocaine, thus weaning an addict from the drug. There have been extensive computational studies of cocaine hydrolysis by BChE. Since there are no reported structures of BChE with cocaine or any of the hydrolysis products, full-length monomeric recombinant wild-type BChE was cocrystallized with cocaine. The refined 3 A resolution structure appears to retain the hydrolysis product benzoic acid in sufficient proximity to form a hydrogen bond to the active-site Ser198.
ESTHER : Asojo_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_434
PubMedSearch : Asojo_2011_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_67_434
PubMedID: 21505234
Gene_locus related to this paper: human-BCHE

Title : Exposure to tri-o-cresyl phosphate detected in jet airplane passengers - Liyasova_2011_Toxicol.Appl.Pharmacol_256_337
Author(s) : Liyasova M , Li B , Schopfer LM , Nachon F , Masson P , Furlong CE , Lockridge O
Ref : Toxicol Appl Pharmacol , 256 :337 , 2011
Abstract : The aircraft cabin and flight deck ventilation are supplied from partially compressed unfiltered bleed air directly from the engine. Worn or defective engine seals can result in the release of engine oil into the cabin air supply. Aircrew and passengers have complained of illness following such "fume events". Adverse health effects are hypothesized to result from exposure to tricresyl phosphate mixed esters, a chemical added to jet engine oil and hydraulic fluid for its anti-wear properties. Our goal was to develop a laboratory test for exposure to tricresyl phosphate. The assay was based on the fact that the active-site serine of butyrylcholinesterase reacts with the active metabolite of tri-o-cresyl phosphate, cresyl saligenin phosphate, to make a stable phosphorylated adduct with an added mass of 80 Da. No other organophosphorus agent makes this adduct in vivo on butyrylcholinesterase. Blood samples from jet airplane passengers were obtained 24-48 h after completing a flight. Butyrylcholinesterase was partially purified from 25 ml serum or plasma, digested with pepsin, enriched for phosphorylated peptides by binding to titanium oxide, and analyzed by mass spectrometry. Of 12 jet airplane passengers tested, 6 were positive for exposure to tri-o-cresyl phosphate that is, they had detectable amounts of the phosphorylated peptide FGEpSAGAAS. The level of exposure was very low. No more than 0.05 to 3% of plasma butyrylcholinesterase was modified. None of the subjects had toxic symptoms. Four of the positive subjects were retested 3 to 7 months following their last airplane trip and were found to be negative for phosphorylated butyrylcholinesterase. In conclusion, this is the first report of an assay that detects exposure to tri-o-cresyl phosphate in jet airplane travelers.
ESTHER : Liyasova_2011_Toxicol.Appl.Pharmacol_256_337
PubMedSearch : Liyasova_2011_Toxicol.Appl.Pharmacol_256_337
PubMedID: 21723309

Title : Prolonged toxic effects after cocaine challenge in butyrylcholinesterase\/plasma carboxylesterase double knockout mice: a model for butyrylcholinesterase-deficient humans - Duysen_2011_Drug.Metab.Dispos_39_1321
Author(s) : Duysen EG , Lockridge O
Ref : Drug Metabolism & Disposition: The Biological Fate of Chemicals , 39 :1321 , 2011
Abstract : Death and toxicity after cocaine use do not correlate with cocaine blood levels. One explanation for this observation is that cocaine abusers may posses one or more of the 58 possible known mutations in the butyrylcholinesterase gene (BCHE). Butyrylcholinesterase (BChE) serves as the primary cocaine hydrolase producing a nontoxic product ecgonine methyl ester. A reduction in endogenous levels of BChE may result in increased metabolism by hepatic carboxylesterase to produce norcocaine, a toxic product. Humans have carboxylesterase in tissues but not in plasma, whereas wild-type mice have significant amounts of carboxylesterase in tissues and plasma. Knockout mice with no plasma carboxylesterase were created to eliminate the contribution of plasma carboxylesterase in cocaine hydrolysis, thereby simulating human enzyme levels. This study tested the hypothesis that reductions in BChE such as those in humans with BChE mutations contribute to increased toxicity after cocaine use. Carboxylesterase and BChE double knockout mice, models for humans with BChE deficiency, were challenged with a nonlethal dose of 100 mg/kg (-)-cocaine. Carboxylesterase/BChE double knockout mice demonstrated toxic signs significantly longer than did wild-type and carboxylesterase knockout mice. The carboxylesterase/BChE-deficient mice took approximately 2.5 times as long to recover from cocaine toxicities, including the following: hypothermia, hyperactivity, stereotypical behavior, ocular effects, and dorsiflexion of the tail. The carboxylesterase/BChE double knockout mouse model demonstrates the importance of endogenous BChE for protection against cocaine toxicity and provides an in vivo system for studying drug sensitivity of humans who carry a BChE mutation.
ESTHER : Duysen_2011_Drug.Metab.Dispos_39_1321
PubMedSearch : Duysen_2011_Drug.Metab.Dispos_39_1321
PubMedID: 21540357

Title : Drastic decrease in dopamine receptor levels in the striatum of acetylcholinesterase knock-out mouse - Hrabovska_2010_Chem.Biol.Interact_183_194
Author(s) : Hrabovska A , Farar V , Bernard V , Duysen EG , Brabec J , Lockridge O , Myslivecek J
Ref : Chemico-Biological Interactions , 183 :194 , 2010
Abstract : BACKGROUND: The acetylcholinesterase knock-out mouse lives to adulthood despite 60-fold elevated acetylcholine concentrations in the brain that are lethal to wild-type animals. Part of its mechanism of survival is a 50% decrease in muscarinic and nicotinic receptors and a 50% decrease in adrenoceptor levels. HYPOTHESIS: The hypothesis was tested that the dopaminergic neuronal system had also adapted. METHODS: Radioligand binding assays measured dopamine receptor level and binding affinity in the striatum. Immunohistochemistry of brain sections with specific antibodies visualized dopamine transporter. Effects on the intracellular compartment were measured as cAMP content, PI-phospholipase C activity. RESULTS: Dopamine receptor levels were decreased 28-fold for the D(1)-like, and more than 37-fold for the D(2)-like receptors, though binding affinity was normal. Despite these huge changes in receptor levels, dopamine transporter levels were not affected. The intracellular compartment had normal levels of cAMP and PI-phospholipase C activity. CONCLUSION: Survival of the acetylcholinesterase knock-out mouse could be linked to adaptation of many neuronal systems during development including the cholinergic, adrenergic and dopaminergic. These adaptations balance the overstimulation of cholinergic receptors caused by high acetylcholine concentrations and thus maintain homeostasis inside the cell, allowing the animal to live.
ESTHER : Hrabovska_2010_Chem.Biol.Interact_183_194
PubMedSearch : Hrabovska_2010_Chem.Biol.Interact_183_194
PubMedID: 19818744

Title : Reaction of human albumin with aspirin in vitro: mass spectrometric identification of acetylated lysines 199, 402, 519, and 545 - Liyasova_2010_Biochem.Pharmacol_79_784
Author(s) : Liyasova MS , Schopfer LM , Lockridge O
Ref : Biochemical Pharmacology , 79 :784 , 2010
Abstract : 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.
ESTHER : Liyasova_2010_Biochem.Pharmacol_79_784
PubMedSearch : Liyasova_2010_Biochem.Pharmacol_79_784
PubMedID: 19836360

Title : 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 - Jiang_2010_Toxicol.Sci_115_183
Author(s) : Jiang W , Duysen EG , Hansen H , Shlyakhtenko L , Schopfer LM , Lockridge O
Ref : Toxicol Sci , 115 :183 , 2010
Abstract : 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.
ESTHER : Jiang_2010_Toxicol.Sci_115_183
PubMedSearch : Jiang_2010_Toxicol.Sci_115_183
PubMedID: 20142434

Title : Review of tyrosine and lysine as new motifs for organophosphate binding to proteins that have no active site serine - Lockridge_2010_Chem.Biol.Interact_187_344
Author(s) : Lockridge O , Schopfer LM
Ref : Chemico-Biological Interactions , 187 :344 , 2010
Abstract : The accepted target for organophosphorus agent (OP) binding to enzymes is the active site serine in the consensus sequence Gly X Ser X Gly. New motifs have been identified by using mass spectrometry to fragment OP-labeled peptides. It has been found that OP can make covalent bonds with tyrosine and lysine in proteins that have no active site serine. The OP-tyrosine bond is stable, and does not undergo the decay seen with OP-serine. Information on OP binding to tyrosine has been applied to diagnosis of OP exposure, through the use of mass spectrometry to detect OP-labeled albumin in human and animal plasma. It is expected that the new OP binding motif will aid in the search for a mechanism of low dose OP toxicity. It is hypothesized that proteins involved in axonal transport, especially proteins whose function depends on reversible phosphorylation, are prime candidates for a role in OP-induced neurodegeneration. Treatment of neurodegenerative disorders could be developed by identifying methods to reverse OP binding to tyrosine.
ESTHER : Lockridge_2010_Chem.Biol.Interact_187_344
PubMedSearch : Lockridge_2010_Chem.Biol.Interact_187_344
PubMedID: 20211158

Title : Visualization of exogenous delivery of nanoformulated butyrylcholinesterase to the central nervous system - Gaydess_2010_Chem.Biol.Interact_187_295
Author(s) : Gaydess A , Duysen E , Li Y , Gilman V , Kabanov A , Lockridge O , Bronich T
Ref : Chemico-Biological Interactions , 187 :295 , 2010
Abstract : Butyrylcholinesterase (BChE) is an efficient bioscavenger of highly toxic organophosphorus poisons and nerve agents. However, BChE administered into the periphery does not provide significant protection of the central nervous system (CNS) due to rejection by the blood-brain barrier. In this study, we evaluated the feasibility of delivering BChE to the CNS by packing it into a block ionomer complex of nanoscale size with a cationic poly(l-lysine)-graft-poly(ethylene oxide) (PLL-g-PEO) copolymer. The multimolecular structure of BChE/PLL-g-PEO complexes was further reinforced by formation of cross-links between the polymer chains. The resulting cross-linked complexes were stable against dilution without significant loss of BChE enzymatic activity. In some cases the BChE was labeled with fluorescent IRDye 800CW before it was incorporated into nanoparticles. BChE/PLL-g-PEO complexes were injected into mice intramuscularly and intravenously. In vivo imaging showed incorporation of the fluorescently labeled BChE in brain. Activity assays showed that BChE remained active in the brain at 72-h post-injection. It was concluded that nanocomplexes can deliver the 340 kDa BChE tetramer to the brain.
ESTHER : Gaydess_2010_Chem.Biol.Interact_187_295
PubMedSearch : Gaydess_2010_Chem.Biol.Interact_187_295
PubMedID: 20060815

Title : Mass spectral characterization of organophosphate-labeled, tyrosine-containing peptides: characteristic mass fragments and a new binding motif for organophosphates - Schopfer_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1297
Author(s) : Schopfer LM , Grigoryan H , Li B , Nachon F , Masson P , Lockridge O
Ref : Journal of Chromatography B Analyt Technol Biomed Life Sciences , 878 :1297 , 2010
Abstract : We have identified organophosphorus agent (OP)-tyrosine adducts on 12 different proteins labeled with six different OP. Labeling was achieved by treating pure proteins with up to 40-fold molar excess of OP at pH 8-8.6. OP-treated proteins were digested with trypsin, and peptides were separated by HPLC. Fragmentation patterns for 100 OP-peptides labeled on tyrosine were determined in the mass spectrometer. The goals of the present work were (1) to determine the common features of the OP-reactive tyrosines, and (2) to describe non-sequence MSMS fragments characteristic of OP-tyrosine peptides. Characteristic ions at 272 and 244 amu for tyrosine-OP immonium ions were nearly always present in the MSMS spectrum of peptides labeled on tyrosine by chlorpyrifos-oxon. Characteristic fragments also appeared from the parent ions that had been labeled with diisopropylfluorophosphate (216 amu), sarin (214 amu), soman (214 amu) or FP-biotin (227, 312, 329, 691 and 708 amu). In contrast to OP-reactive serines, which lie in the consensus sequence GXSXG, the OP-reactive tyrosines have no consensus sequence. Their common feature is the presence of nearby positively charged residues that activate the phenolic hydroxyl group. The significance of these findings is the recognition of a new binding motif for OP to proteins that have no active site serine. Modified peptides are difficult to find when the OP bears no radiolabel and no tag. The characteristic MSMS fragment ions are valuable because they are identifiers for OP-tyrosine, independent of the peptide.
ESTHER : Schopfer_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1297
PubMedSearch : Schopfer_2010_J.Chromatogr.B.Analyt.Technol.Biomed.Life.Sci_878_1297
PubMedID: 19762289

Title : Methamidophos, dichlorvos, O-methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry - Tacal_2010_J.Appl.Toxicol_30_469
Author(s) : Tacal O , Lockridge O
Ref : J Appl Toxicol , 30 :469 , 2010
Abstract : Organophosphorus pesticides used most commonly in Turkey include methamidophos, dichlorvos, O-methoate and diazinon. These toxic chemicals or their metabolites make a covalent bond with the active site serine of butyrylcholinesterase. Our goal was to identify the adducts that result from the reaction of human butyrylcholinesterase with these pesticides. Highly purified human butyrylcholinesterase was treated with a 20-fold molar excess of pesticide. The protein was denatured by boiling and digested with trypsin. MS and MSMS spectra of HPLC-purified peptides were acquired on a MALDI-TOF-TOF 4800 mass spectrometer. It was found that methamidophos added a mass of +93, consistent with addition of methoxy aminophosphate. A minor amount of adduct with an added mass of +109 was also found. Dichlorvos and O-methoate both made dimethoxyphosphate (+108) and monomethoxyphosphate adducts (+94). Diazinon gave a novel adduct with an added mass of +152 consistent with diethoxythiophosphate. Inhibition of enzyme activity in the presence of diazinon developed slowly (15 h), concomitant with isomerization of diazinon via a thiono-thiolo rearrangement. The isomer of diazinon yielded diethoxyphosphate and monoethoxyphosphate adducts with added masses of +136 and +108. MSMS spectra confirmed that each of the pesticides studied made a covalent bond with serine 198 of butyrylcholinesterase. These results can be used to identify the class of pesticides to which a patient was exposed.
ESTHER : Tacal_2010_J.Appl.Toxicol_30_469
PubMedSearch : Tacal_2010_J.Appl.Toxicol_30_469
PubMedID: 20229498

Title : Contributions of selective knockout studies to understanding cholinesterase disposition and function - Camp_2010_Chem.Biol.Interact_187_72
Author(s) : Camp S , Zhang L , Krejci E , Dobbertin A , Bernard V , Girard E , Duysen EG , Lockridge O , De Jaco A , Taylor P
Ref : Chemico-Biological Interactions , 187 :72 , 2010
Abstract : The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.
ESTHER : Camp_2010_Chem.Biol.Interact_187_72
PubMedSearch : Camp_2010_Chem.Biol.Interact_187_72
PubMedID: 20153304

Title : Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose - Li_2010_J.Appl.Toxicol_30_559
Author(s) : Li B , Ricordel I , Schopfer LM , Baud F , Megarbane B , Masson P , Lockridge O
Ref : J Appl Toxicol , 30 :559 , 2010
Abstract : The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60-84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase.
ESTHER : Li_2010_J.Appl.Toxicol_30_559
PubMedSearch : Li_2010_J.Appl.Toxicol_30_559
PubMedID: 20809544

Title : Detection of adduct on tyrosine 411 of albumin in humans poisoned by dichlorvos - Li_2010_Toxicol.Sci_116_23
Author(s) : Li B , Ricordel I , Schopfer LM , Baud F , Megarbane B , Nachon F , Masson P , Lockridge O
Ref : Toxicol Sci , 116 :23 , 2010
Abstract : Studies in mice and guinea pigs have shown that albumin is a new biomarker of organophosphorus toxicant (OP) and nerve agent exposure. Our goal was to determine whether OP-labeled albumin could be detected in the blood of humans exposed to OP. Blood from four OP-exposed patients was prepared for mass spectrometry analysis by digesting 0.010 ml of serum with pepsin and purifying the labeled albumin peptide by offline high performance liquid chromatography. Dimethoxyphosphate-labeled tyrosine 411 was identified in albumin peptides VRY(411)TKKVPQVSTPTL and LVRY(411)TKKVPQVSTPTL from two patients who had attempted suicide with dichlorvos. The butyrylcholinesterase activity in these serum samples was inhibited 80%. A third patient whose serum BChE activity was inhibited 8% by accidental inhalation of dichlorvos had undetectable levels of adduct on albumin. A fourth patient whose BChE activity was inhibited 60% by exposure to chlorpyrifos had no detectable adduct on albumin. This is the first report to demonstrate the presence of OP-labeled albumin in human patients. It is concluded that tyrosine 411 of human albumin is covalently modified in the serum of humans poisoned by dichlorvos and that the modification is detectable by mass spectrometry. The special reactivity of tyrosine 411 with OP suggests that other proteins may also be modified on tyrosine. Identification of other OP-modified proteins may lead to an understanding of neurotoxic symptoms that appear long after the initial OP exposure.
ESTHER : Li_2010_Toxicol.Sci_116_23
PubMedSearch : Li_2010_Toxicol.Sci_116_23
PubMedID: 20395308

Title : Structural approach to the aging of phosphylated cholinesterases - Masson_2010_Chem.Biol.Interact_187_157
Author(s) : Masson P , Nachon F , Lockridge O
Ref : Chemico-Biological Interactions , 187 :157 , 2010
Abstract : Phosphylated cholinesterases (ChE) can undergo a side reaction that progressively decreases their reactivatability. This process, termed "aging", results from dealkylation of the adduct and depends on the structure of the organophosphyl moiety. Aged ChEs are resistant to reactivation by oximes. Owing to the toxicological importance of OPs, the molecular mechanism of aging has been the subject of research for decades. It was not clear whether aging involves the same bond breakage regardless the type of OP or is a scission of P-O-C bonds (P-O or O-C) in phosphates/phosphonates, P-N-C bonds in phosphoramidates, and P-S-C bonds in phosphonothionates. It was assumed that the resulting negatively charged atom on phosphorus of the aged adduct prevented nucleophilic attack by oximates, but studies on negatively charged model molecules do not support this hypothesis. Decrease in conformational flexibility of aged enzymes may contribute to their non-reactivatability by preventing proper adjustment of reactivators in the active site gorge. MALDI-TOF mass spectrometry of phosphylated human butyrylcholinesterase (hBChE) in water and in (18)O-water provided evidence that aging results from O-C breakage, i.e. O-dealkylation. In contrast, the isomalathion-BChE conjugate ages mostly through P-S bond cleavage, but a minor product results from O-C and/or S-C breakage. The crystal structures of hBChE and hAChE inhibited by tabun showed that aging of tabun-ChE conjugates results from O-dealkylation. However, depending on the nature of O-alkyl and N-alkyl chains, aging of BChE inhibited by other phosphoramidates results either from O-C breakage or deamination, i.e. P-N breakage. It was found that dealkylation of branched alkoxy involves a transient carbocation. Dealkylation of OP-ChE conjugates is accompanied by enzyme conformational changes. Urea, organic solvent, heat and pressure denaturation of human BChE showed that the conformational stability of aged OP-BChE conjugates is dramatically increased compared to native enzyme. Determination of the three-dimensional structure of BChE and AChE conjugated to different OPs showed that aged adducts form a salt bridge with the protonated catalytic histidine. Structure alteration of aged enzymes is accompanied by exit of water molecules from the enzyme's active site gorge. In addition, neutron scattering studies provided evidence that the structural dynamics of aged BChE is dramatically altered compared to native enzyme. Knowledge of the molecular basis of aging will help to design reactivators of aged ChEs, molecules capable of slowing the aging process, and pseudocatalytic ChE-based bioscavengers.
ESTHER : Masson_2010_Chem.Biol.Interact_187_157
PubMedSearch : Masson_2010_Chem.Biol.Interact_187_157
PubMedID: 20338153

Title : Development of diagnostics in the search for an explanation of aerotoxic syndrome - Schopfer_2010_Anal.Biochem_404_64
Author(s) : Schopfer LM , Furlong CE , Lockridge O
Ref : Analytical Biochemistry , 404 :64 , 2010
Abstract : Aerotoxic syndrome is assumed to be caused by exposure to tricresyl phosphate, an additive in engine lubricants and hydraulic fluids that is activated to the toxic 2-(ortho-cresyl)-4H-1,3,2-benzodioxaphosphoran-2-one (CBDP). Currently, there is no laboratory evidence to support intoxication of airline crew members by CBDP. Our goal was to develop methods for testing in vivo exposure by identifying and characterizing biomarkers. Mass spectrometry was used to study the reaction of CBDP with human albumin, free tyrosine, and human butyrylcholinesterase. Human albumin made a covalent bond with CBDP, adding a mass of 170amu to Tyr411 to yield the o-cresyl phosphotyrosine derivative. Human butyrylcholinesterase made a covalent bond with CBDP on Ser198 to yield five adducts with added masses of 80, 108, 156, 170, and 186amu. The most abundant adduct had an added mass of 80amu from phosphate (HPO(3)), a surprising result given that no pesticide or nerve agent is known to yield phosphorylated serine with an added mass of 80amu. The next most abundant adduct had an added mass of 170amu to form o-cresyl phosphoserine. It is concluded that toxic gases or oil mists in cabin air may form adducts on plasma butyrylcholinesterase and albumin, detectable by mass spectrometry.
ESTHER : Schopfer_2010_Anal.Biochem_404_64
PubMedSearch : Schopfer_2010_Anal.Biochem_404_64
PubMedID: 20447373

Title : Butyrylcholinesterase for protection from organophosphorus poisons: catalytic complexities and hysteretic behavior - Masson_2010_Arch.Biochem.Biophys_494_107
Author(s) : Masson P , Lockridge O
Ref : Archives of Biochemistry & Biophysics , 494 :107 , 2010
Abstract : 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.
ESTHER : Masson_2010_Arch.Biochem.Biophys_494_107
PubMedSearch : Masson_2010_Arch.Biochem.Biophys_494_107
PubMedID: 20004171

Title : Mass spectral characterization of organophosphate-labeled lysine in peptides. - Grigoryan_2009_Anal.Biochem_394_92
Author(s) : Grigoryan H , Li B , Xue W , Grigoryan M , Schopfer LM , Lockridge O
Ref : Analytical Biochemistry , 394 :92 , 2009
Abstract : Organophosphate (OP) esters bind covalently to the active site serine of enzymes in the serine hydrolase family. Recently, mass spectrometry identified covalent binding of OPs to tyrosine in a wide variety of proteins when purified proteins were incubated with OPs. In the current work, manual inspection of tandem mass spectrometry (MS/MS) data led to the realization that lysines also make a covalent bond with OPs. OP-labeled lysine residues were found in seven proteins that had been treated with either chlorpyrifos oxon (CPO) or diisopropylfluorophosphate (DFP): human serum albumin (K212, K414, K199, and K351), human keratin 1 (K211 and K355), human keratin 10 (K163), bovine tubulin alpha (K60, K336, K163, K394, and K401), bovine tubulin beta (K58), bovine actin (K113, K291, K326, K315, and K328), and mouse transferrin (K296 and K626). These results suggest that OP binding to lysine is a general phenomenon. Characteristic fragments specific for CPO-labeled lysine appeared at 237.1, 220.0, 192.0, 163.9, 128.9, and 83.9amu. Characteristic fragments specific for DFP-labeled lysine appeared at 164.0, 181.2, and 83.8amu. This new OP-binding motif to lysine suggests new directions to search for mechanisms of long-term effects of OP exposure and in the search for biomarkers of OP exposure.
ESTHER : Grigoryan_2009_Anal.Biochem_394_92
PubMedSearch : Grigoryan_2009_Anal.Biochem_394_92
PubMedID: 19596251

Title : Mass spectrometry identifies multiple organophosphorylated sites on tubulin - Grigoryan_2009_Toxicol.Appl.Pharmacol_240_149
Author(s) : Grigoryan H , Schopfer LM , Peeples ES , Duysen EG , Grigoryan M , Thompson CM , Lockridge O
Ref : Toxicol Appl Pharmacol , 240 :149 , 2009
Abstract : Acute toxicity of organophosphorus poisons (OP) is explained by inhibition of acetylcholinesterase in nerve synapses. Low-dose effects are hypothesized to result from modification of other proteins, whose identity is not yet established. The goal of the present work was to obtain information that would make it possible to identify tubulin as a target of OP exposure. Tubulin was selected for study because live mice injected with a nontoxic dose of a biotinylated organophosphorus agent appeared to have OP-labeled tubulin in brain as determined by binding to avidin beads and mass spectrometry. The experiments with live mice were not conclusive because binding to avidin beads could be nonspecific. To be convincing, it is necessary to find and characterize the OP-labeled tubulin peptide. The search for OP-labeled tubulin peptides was begun by identifying residues capable of making a covalent bond with OP. Pure bovine tubulin (0.012 mM) was treated with 0.01-0.5 mM chlorpyrifos oxon for 24 h at 37 degrees C in pH 8.3 buffer. The identity of labeled amino acids and percent labeling was determined by mass spectrometry. Chlorpyrifos oxon bound covalently to tyrosines 83, 103, 108, 161, 224, 262, 272, 357, and 399 in bovine alpha tubulin, and to tyrosines 50, 51, 59, 106, 159, 281, 310, and 340 in bovine beta tubulin. The most reactive were tyrosine 83 in alpha and tyrosine 281 in beta tubulin. In the presence of 1 mM GTP, percent labeling increased 2-fold. Based on the crystal structure of the tubulin heterodimer (PDB 1jff) tyrosines 83 and 281 are well exposed to solvent. In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals.
ESTHER : Grigoryan_2009_Toxicol.Appl.Pharmacol_240_149
PubMedSearch : Grigoryan_2009_Toxicol.Appl.Pharmacol_240_149
PubMedID: 19632257

Title : Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine - Grigoryan_2009_Chem.Biol.Interact_180_492
Author(s) : Grigoryan H , Li B , Anderson EK , Xue W , Nachon F , Lockridge O , Schopfer LM
Ref : Chemico-Biological Interactions , 180 :492 , 2009
Abstract : Organophosphorus (OP) esters are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-biotin. The proteins were digested with trypsin and the labeled peptides enriched by binding to monomeric avidin. Peptides were purified by HPLC and fragmented by collision induced dissociation in a tandem ion trap mass spectrometer. Eight proteins were labeled and six were not. Tyrosine was labeled in human alpha-2-glycoprotein 1 zinc-binding protein (Tyr 138, Tyr 174 and Tyr 181), human kinesin 3C motor domain (Tyr 145), human keratin 1 (Tyr 230), bovine actin (Tyr 55 and Tyr 200), murine ATP synthase beta (Tyr 431), murine adenine nucleotide translocase 1 (Tyr 81), bovine chymotrypsinogen (Tyr 201) and porcine pepsin (Tyr 310). Only 1-3 tyrosines per protein were modified, suggesting that the reactive tyrosine was activated by nearby residues that facilitated ionization of the hydroxyl group of tyrosine. These results suggest that OP binding to tyrosine is a general phenomenon. It is concluded that organophosphorus-reactive proteins include not only enzymes in the serine hydrolase family, but also proteins that have no active site serine. The recognition of a new OP-binding motif to tyrosine suggests new directions to search for mechanisms of long-term effects of OP exposure. Another application is in the search for biomarkers of organophosphorus agent exposure. Previous searches have been limited to serine hydrolases. Now proteins such as albumin and keratin can be considered.
ESTHER : Grigoryan_2009_Chem.Biol.Interact_180_492
PubMedSearch : Grigoryan_2009_Chem.Biol.Interact_180_492
PubMedID: 19539807

Title : Tyrosines of human and mouse transferrin covalently labeled by organophosphorus agents: a new motif for binding to proteins that have no active site serine - Li_2009_Toxicol.Sci_107_144
Author(s) : Li B , Schopfer LM , Grigoryan H , Thompson CM , Hinrichs SH , Masson P , Lockridge O
Ref : Toxicol Sci , 107 :144 , 2009
Abstract : The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation.
ESTHER : Li_2009_Toxicol.Sci_107_144
PubMedSearch : Li_2009_Toxicol.Sci_107_144
PubMedID: 18930948

Title : Nerve agent analogues that produce authentic soman, sarin, tabun, and cyclohexyl methylphosphonate-modified human butyrylcholinesterase - Gilley_2009_Chem.Res.Toxicol_22_1680
Author(s) : Gilley C , MacDonald M , Nachon F , Schopfer LM , Zhang J , Cashman JR , Lockridge O
Ref : Chemical Research in Toxicology , 22 :1680 , 2009
Abstract : The goal was to test 14 nerve agent model compounds of soman, sarin, tabun, and cyclohexyl methylphosphonofluoridate (GF) for their suitability as substitutes for true nerve agents. We wanted to know whether the model compounds would form the identical covalent adduct with human butyrylcholinesterase that is produced by reaction with true nerve agents. Nerve agent model compounds containing thiocholine or thiomethyl in place of fluorine or cyanide were synthesized as Sp and Rp stereoisomers. Purified human butyrylcholinesterase was treated with a 45-fold molar excess of nerve agent analogue at pH 7.4 for 17 h at 21 degrees C. The protein was denatured by boiling and was digested with trypsin. Aged and nonaged active site peptide adducts were quantified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry of the tryptic digest mixture. The active site peptides were isolated by HPLC and analyzed by MALDI-TOF-TOF mass spectrometry. Serine 198 of butyrylcholinesterase was covalently modified by all 14 compounds. Thiocholine was the leaving group in all compounds that had thiocholine in place of fluorine or cyanide. Thiomethyl was the leaving group in the GF thiomethyl compounds. However, sarin thiomethyl compounds released either thiomethyl or isopropyl, while soman thiomethyl compounds released either thiomethyl or pinacolyl. Thiocholine compounds reacted more rapidly with butyrylcholinesterase than thiomethyl compounds. Labeling with the model compounds resulted in aged adducts that had lost the O-alkyl group (O-ethyl for tabun, O-cyclohexyl for GF, isopropyl for sarin, and pinacolyl for soman) in addition to the thiocholine or thiomethyl group. The nerve agent model compounds containing thiocholine and the GF thiomethyl analogue were found to be suitable substitutes for true soman, sarin, tabun, and GF in terms of the adduct that they produced with human butyrylcholinesterase. However, the soman and sarin thiomethyl compounds yielded two types of adducts, one of which was thiomethyl phosphonate, a modification not found after treatment with authentic soman and sarin.
ESTHER : Gilley_2009_Chem.Res.Toxicol_22_1680
PubMedSearch : Gilley_2009_Chem.Res.Toxicol_22_1680
PubMedID: 19715348

Title : Intrathecal delivery of fluorescent labeled butyrylcholinesterase to the brains of butyrylcholinesterase knock-out mice: visualization and quantification of enzyme distribution in the brain - Johnson_2009_Neurotoxicol_30_386
Author(s) : Johnson ND , Duysen EG , Lockridge O
Ref : Neurotoxicology , 30 :386 , 2009
Abstract : Exogenously delivered butyrylcholinesterase (BChE) has proven to be an efficient bioscavenger against highly toxic organophosphorus poisons and nerve agents. The scavenger properties of BChE when delivered via intramuscular, intravenous, subcutaneous, or intraperitoneal routes are limited to the body's peripheral sites because the 340 kDa enzyme does not cross the blood-brain barrier (BBB). Overcoming the BBB is an important step toward evaluating the neuroprotective properties of BChE within the central nervous system (CNS). This study examines the feasibility of delivering BChE to the brain and spinal cord by intrathecal (IT) injection. Mice completely devoid of BChE were injected intrathecally with either BChE (80 units) that was labeled with near-infrared fluorescent dye (BChE/IRDye) or a molar equivalent amount of carboxylate dye. The BChE/IRDye and carboxylate dye were tracked using an in vivo imaging system demonstrating the real-time distribution of BChE in the brain and the residence time in the brain and spinal cord through 25 h post-dosing. BChE/IRdye levels in the brain peaked at 6h post-dosing. BChE enzyme activity was quantified in plasma and brain sections by BChE activity assays of plasma and of perfused tissues. Average BChE activity levels were 0.6 units/g in the brains of mice treated with BChE/IRDye at 4h post-dosing. Intense fluorescent signal in the cortex, dentate gyrus and ventricles of the brain at 25 h post-dosing was visualized by confocal microscopy and the presence of BChE was confirmed with activity assays of frozen sections. This procedure proved to be an efficient, safe and rapid method to deliver BChE to the CNS of mice, providing a research tool for determining neural protection by BChE following OP exposure.
ESTHER : Johnson_2009_Neurotoxicol_30_386
PubMedSearch : Johnson_2009_Neurotoxicol_30_386
PubMedID: 19442823

Title : Effects of rivastigmine and donepezil on brain acetylcholine levels in acetylcholinesterase-deficient mice - Naik_2009_J.Pharm.Pharm.Sci_12_79
Author(s) : Naik RS , Hartmann J , Kiewert C , Duysen EG , Lockridge O , Klein J
Ref : J Pharm Pharm Sci , 12 :79 , 2009
Abstract : PURPOSE: Alzheimer s disease is characterized by a dysfunction of central cholinergic systems and is treated by inhibitors of acetylcholinesterase (AChE). This study tests the effect of two AChE inhibitors in therapeutic use, rivastigmine and donepezil, in mice that are devoid of AChE (AChE-/- mice). Rivastigmine is an inhibitor of both AChE and butyrylcholinesterase (BChE) whereas donepezil is a selective inhibitor of AChE. METHODS: We have used in vivo microdialysis to investigate the effects of the two drugs on the extracellular concentration of acetylcholine (ACh) in the hippocampus of AChE-/- mice. RESULTS: Extracellular ACh levels in the hippocampus were 30-fold elevated in AChE-/- mice compared to wild-type (AChE+/+) animals. Infusion of rivastigmine (1 and 10 microM) caused a further doubling of ACh levels in AChE-/- mice within 90-120 min. In contrast, infusion of donepezil (1 microM) did not affect hippocampal ACh levels in AChE-/- mice although it increased ACh levels more than twofold in wild-type mice. CONCLUSIONS: In the absence of AChE, rivastigmine enhances the levels of extracellular ACh by inhibiting BChE. This finding may be of therapeutic relevance because BChE activity is preserved, but AChE activity is strongly decreased, in late-stage Alzheimer s disease.
ESTHER : Naik_2009_J.Pharm.Pharm.Sci_12_79
PubMedSearch : Naik_2009_J.Pharm.Pharm.Sci_12_79
PubMedID: 19470293

Title : Nanoimages show disruption of tubulin polymerization by chlorpyrifos oxon: implications for neurotoxicity - Grigoryan_2009_Toxicol.Appl.Pharmacol_240_143
Author(s) : Grigoryan H , Lockridge O
Ref : Toxicol Appl Pharmacol , 240 :143 , 2009
Abstract : Organophosphorus agents cause cognitive deficits and depression in some people. We hypothesize that the mechanism by which organophosphorus agents cause these disorders is by modification of proteins in the brain. One such protein could be tubulin. Tubulin polymerizes to make the microtubules that transport cell components to nerve axons. The goal of the present work was to measure the effect of the organophosphorus agent chlorpyrifos oxon on tubulin polymerization. An additional goal was to identify the amino acids covalently modified by chlorpyrifos oxon in microtubule polymers and to compare them to the amino acids modified in unpolymerized tubulin dimers. Purified bovine tubulin (0.1 mM) was treated with 0.005-0.1 mM chlorpyrifos oxon for 30 min at room temperature and then polymerized by addition of 1 mM GTP to generate microtubules. Microtubules were visualized by atomic force microscopy. Chlorpyrifos oxon-modified residues were identified by tandem ion trap electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry of tryptic peptides. Nanoimaging showed that low concentrations (0.005 and 0.01 mM) of chlorpyrifos oxon yielded short, thin microtubules. A concentration of 0.025 mM stimulated polymerization, while high concentrations (0.05 and 0.1 mM) caused aggregation. Of the 17 tyrosines covalently modified by chlorpyrifos oxon in unpolymerized tubulin dimers, only 2 tyrosines were labeled in polymerized microtubules. The two labeled tyrosines in polymerized tubulin were Tyr 103 in EDAANNYR of alpha tubulin, and Tyr 281 in GSQQYR of beta tubulin. In conclusion, chlorpyrifos oxon binding to tubulin disrupts tubulin polymerization. These results may lead to an understanding of the neurotoxicity of organophosphorus agents.
ESTHER : Grigoryan_2009_Toxicol.Appl.Pharmacol_240_143
PubMedSearch : Grigoryan_2009_Toxicol.Appl.Pharmacol_240_143
PubMedID: 19631231

Title : The butyrylcholinesterase knockout mouse a research tool in the study of drug sensitivity, bio-distribution, obesity and Alzheimer's disease - Duysen_2009_Expert.Opin.Drug.Metab.Toxicol_5_523
Author(s) : Duysen EG , Li B , Lockridge O
Ref : Expert Opin Drug Metab Toxicol , 5 :523 , 2009
Abstract : Butyrylcholinesterase (BChE) mutations common in the human population may result in complete or partial BChE deficiency, making the BChE knockout (KO) mouse a model for human deficiencies. The BChE KO mouse cannot tolerate standard doses of the muscle relaxant succinylcholine or the Alzheimer's disease drugs huperzine A and donepezil. It is resistant to the asthma drug bambuterol. The importance of BChE in detoxication of cocaine has been demonstrated by hepatotoxicity and cardiotoxicity in cocaine-challenged BChE KO mice. The BChE KO mouse becomes obese on a high-fat diet, suggesting a role for BChE in fat metabolism. BChE serves as a backup for acetylcholinesterase by hydrolyzing the neurotransmitter acetylcholine in acetylcholinesterase knockout mice. Imaging studies show that BChE injected intrathecally crosses the blood-brain barrier. Mice, but not humans, have carboxylesterase in their blood. Carboxylesterase obscures the role of BChE in detoxication of organophosphorus pesticides. Future studies will make a double knockout that has neither BChE nor carboxylesterase. The double knockout is expected to be unusually sensitive to the toxicity of organophosphorus pesticides. Knowledge of drug sensitivities in the mouse model of human BChE deficiency will aid in understanding adverse drug effects in humans.
ESTHER : Duysen_2009_Expert.Opin.Drug.Metab.Toxicol_5_523
PubMedSearch : Duysen_2009_Expert.Opin.Drug.Metab.Toxicol_5_523
PubMedID: 19416087

Title : Adenovirus-transduced human butyrylcholinesterase in mouse blood functions as a bioscavenger of chemical warfare nerve agents - Chilukuri_2009_Mol.Pharmacol_76_612
Author(s) : Chilukuri N , Duysen EG , Parikh K , diTargiani RC , Doctor BP , Lockridge O , Saxena A
Ref : Molecular Pharmacology , 76 :612 , 2009
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents. We have showed previously that recombinant (r) Hu BChE can be expressed at very high levels, 400 to 600 U/ml in mouse blood, by delivering the Hu BChE gene using adenovirus (Ad). Here, we report the biochemical properties of the Ad-expressed full-length and truncated rHu BChE in mouse blood. The molecular sizes of the full-length rHu BChE subunit and its oligomers were similar to those of native Hu BChE, although only a small portion of the full-length rHu BChE subunit underwent assembly into dimers and tetramers. As expected, Ad containing the truncated Hu BChE gene transduced the expression of monomeric rHu BChE only. Compared with 415 U of rHu BChE per milliliter in blood, tissues including liver, lung, heart, brain, kidney, muscle, intestine, diaphragm, salivary gland, and fat expressed <10 U/g of rHu BChE activity. Ad-expressed rHu BChE in mouse blood neutralized soman and O-ethyl S-2-N,N-diisopropylaminoethyl methylphosphonothiolate at rates similar to those of native Hu BChE and rHu BChE expressed in vitro. Because the expression of rHu BChE rapidly decreased 6 days after virus administration, sera were assayed for the presence of anti-Hu BChE antibodies. Anti-Hu BChE antibodies were detected on day 7 and in increased amounts thereafter, which coincided with the loss of Hu BChE expression in sera. In conclusion, the delivery of Hu BChE gene using Ad can be a promising strategy that can provide protection against multiple lethal doses of chemical warfare nerve agents in vivo.
ESTHER : Chilukuri_2009_Mol.Pharmacol_76_612
PubMedSearch : Chilukuri_2009_Mol.Pharmacol_76_612
PubMedID: 19542320

Title : Carbofuran poisoning detected by mass spectrometry of butyrylcholinesterase adduct in human serum - Li_2009_J.Appl.Toxicol_29_149
Author(s) : Li H , Ricordel I , Tong L , Schopfer LM , Baud F , Megarbane B , Maury E , Masson P , Lockridge O
Ref : J Appl Toxicol , 29 :149 , 2009
Abstract : Carbofuran is a pesticide whose acute toxicity is due to inhibition of acetylcholinesterase. Butyrylcholinesterase (BChE) in plasma is inhibited by carbofuran and serves as a biomarker of poisoning by carbofuran. The goal was to develop a method to positively identify poisoning by carbofuran. Sera from an attempted murder and an attempted suicide were analyzed for the presence of carbofuran adducts on BChE. The BChE from 1 ml of serum was rapidly purified on a 0.2 ml procainamide-Sepharose column. Speed was essential because the carbofuran-BChE adduct decarbamylates with a half-life of about 2 h. The partially purified BChE was boiled to denature the protein, thus stopping decarbamylation and making the protein vulnerable to digestion with trypsin. The labeled peptide was partially purified by HPLC before analysis by LC/MS/MS in the multiple reaction monitoring mode on the QTRAP 2000 mass spectrometer. Carbofuran was found to be covalently bound to Ser 198 of human BChE in serum samples from two poisoning cases. Multiple reaction monitoring triggered MS/MS spectra positively identified the carbofuran-BChE adduct. In conclusion a mass spectrometry method to identify carbofuran poisoning in humans has been developed. The method uses 1 ml of serum and detects low-level exposure associated with as little as 20% inhibition of plasma butyrylcholinesterase.
ESTHER : Li_2009_J.Appl.Toxicol_29_149
PubMedSearch : Li_2009_J.Appl.Toxicol_29_149
PubMedID: 18937214

Title : A collaborative endeavor to design cholinesterase-based catalytic scavengers against toxic organophosphorus esters - Masson_2008_Chem.Biol.Interact_175_273
Author(s) : Masson P , Nachon F , Broomfield CA , Lenz DE , Verdier L , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 175 :273 , 2008
Abstract : 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.
ESTHER : Masson_2008_Chem.Biol.Interact_175_273
PubMedSearch : Masson_2008_Chem.Biol.Interact_175_273
PubMedID: 18508040

Title : Adenovirus-mediated gene transfer of human butyrylcholinesterase results in persistent high-level transgene expression in vivo - Chilukuri_2008_Chem.Biol.Interact_175_327
Author(s) : Chilukuri N , Duysen EG , Parikh K , Sun W , Doctor BP , Lockridge O , Saxena A
Ref : Chemico-Biological Interactions , 175 :327 , 2008
Abstract : Human serum butyrylcholinesterase (Hu BChE) is a promising therapeutic against the toxicity of chemical warfare nerve agents, pesticide intoxication, and cocaine overdose. However, its widespread application is hampered by difficulties in large-scale production of the native protein from human plasma and/or availability as a recombinant protein suitable for use in vivo. This limitation may be resolved by in vivo delivery and expression of the Hu BChE gene. In this study, recombinant (r) adenoviruses (Ads) encoding full-length and truncated rHu BChEs were tested for in vivo expression in mice. Mice injected with these rAds intraperitoneally failed to express rHu BChE. However, a single tail vein injection of both rAds resulted in persistent high serum levels of rHu BChE in BChE knockout mice, which peaked on days 4/5 at 377+/-162U/ml for full-length rHu BChE and 574+/-143U/ml for truncated rHu BChE. These activity levels are orders of magnitude higher than 1.9U/ml of mouse BChE present in wild-type mouse serum. Thereafter, rHu BChE levels dropped rapidly and very little or no activity was detected in the serum 10 days post-virus administration. In conclusion, the present study demonstrates the potential of rAd-mediated Hu BChE gene therapy to counteract multiple lethal doses of chemical warfare nerve agent toxicity.
ESTHER : Chilukuri_2008_Chem.Biol.Interact_175_327
PubMedSearch : Chilukuri_2008_Chem.Biol.Interact_175_327
PubMedID: 18499092

Title : Binding and hydrolysis of soman by human serum albumin - Li_2008_Chem.Res.Toxicol_21_421
Author(s) : Li B , Nachon F , Froment MT , Verdier L , Debouzy JC , Brasme B , Gillon E , Schopfer LM , Lockridge O , Masson P
Ref : Chemical Research in Toxicology , 21 :421 , 2008
Abstract : 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.
ESTHER : Li_2008_Chem.Res.Toxicol_21_421
PubMedSearch : Li_2008_Chem.Res.Toxicol_21_421
PubMedID: 18163544

Title : Carbamates with differential mechanism of inhibition toward acetylcholinesterase and butyrylcholinesterase - Darvesh_2008_J.Med.Chem_51_4200
Author(s) : Darvesh S , Darvesh KV , McDonald RS , Mataija D , Walsh R , Mothana S , Lockridge O , Martin E
Ref : Journal of Medicinal Chemistry , 51 :4200 , 2008
Abstract : 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.
ESTHER : Darvesh_2008_J.Med.Chem_51_4200
PubMedSearch : Darvesh_2008_J.Med.Chem_51_4200
PubMedID: 18570368

Title : Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines - Lockridge_2008_J.Biol.Chem_283_22582
Author(s) : Lockridge O , Xue W , Gaydess A , Grigoryan H , Ding SJ , Schopfer LM , Hinrichs SH , Masson P
Ref : Journal of Biological Chemistry , 283 :22582 , 2008
Abstract : 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 beta-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 degrees 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.
ESTHER : Lockridge_2008_J.Biol.Chem_283_22582
PubMedSearch : Lockridge_2008_J.Biol.Chem_283_22582
PubMedID: 18577514

Title : Increased hepatotoxicity and cardiac fibrosis in cocaine-treated butyrylcholinesterase knockout mice - Duysen_2008_Basic.Clin.Pharmacol.Toxicol_103_514
Author(s) : Duysen EG , Li B , Carlson M , Li YF , Wieseler S , Hinrichs SH , Lockridge O
Ref : Basic Clin Pharmacol Toxicol , 103 :514 , 2008
Abstract : In mice, cocaine is detoxified to inactive products by butyrylcholinesterase (BChE) and carboxylesterase. In human beings, cocaine detoxification is primarily by BChE. The focus of this investigation was to elucidate the importance of BChE in reducing pathophysiological effects following cocaine exposure. Previous studies examining the effects of cocaine on BChE deficient animals relied on chemical inhibition of BChE with tetraisopropyl pyrophosphoramide (iso-OMPA). The creation of the BChE knockout mouse has provided a model for studying pathological effects of cocaine in mice free of chemical confounders. We hypothesized that mice with low or no BChE activity would have reduced cocaine metabolism, leading to hepatotoxicity and cardiomyopathy. A high-resolution in vivo imaging system recorded cardiac and respiratory function following treatment with a carboxylesterase inhibitor and a high dose of cocaine (100 mg/kg, intraperitoneally). The BChE-/- mice demonstrated depressed respiration through 12 hr after dosing and abnormal respiratory patterns consisting of a pause at full inspiration (apneusis), whereas BChE+/+ mice had recovered normal respiration rates by 30 min. after dosing and exhibited no apneusis. Liver and cardiac histology sections were analysed following a 20 mg/kg intraperitoneally dose of cocaine administered daily for 7 days. BChE-/- mice treated for 7 days with the chronic low dose showed significant hepatotoxicity and cardiac perivascular fibrosis compared to BChE+/+ mice. The observed functional changes following acute high-dose and chronic low-dose cocaine in BChE-/- and +/- mice warrants further investigation into the possibility of increased cocaine toxicity in human beings with BChE deficiency.
ESTHER : Duysen_2008_Basic.Clin.Pharmacol.Toxicol_103_514
PubMedSearch : Duysen_2008_Basic.Clin.Pharmacol.Toxicol_103_514
PubMedID: 19067679

Title : Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin - Ding_2008_Chem.Res.Toxicol_21_1787
Author(s) : Ding SJ , Carr J , Carlson JE , Tong L , Xue W , Li Y , Schopfer LM , Li B , Nachon F , Asojo OA , Thompson CM , Hinrichs SH , Masson P , Lockridge O
Ref : Chemical Research in Toxicology , 21 :1787 , 2008
Abstract : 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.
ESTHER : Ding_2008_Chem.Res.Toxicol_21_1787
PubMedSearch : Ding_2008_Chem.Res.Toxicol_21_1787
PubMedID: 18707141

Title : 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 - Grigoryan_2008_Chem.Biol.Interact_175_180
Author(s) : Grigoryan H , Schopfer LM , Thompson CM , Terry AV , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 175 :180 , 2008
Abstract : 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.
ESTHER : Grigoryan_2008_Chem.Biol.Interact_175_180
PubMedSearch : Grigoryan_2008_Chem.Biol.Interact_175_180
PubMedID: 18502412

Title : Whole body and tissue imaging of the butyrylcholinesterase knockout mouse injected with near infrared dye labeled butyrylcholinesterase - Duysen_2008_Chem.Biol.Interact_175_119
Author(s) : Duysen EG , Lockridge O
Ref : Chemico-Biological Interactions , 175 :119 , 2008
Abstract : Butyrylcholinesterase (BChE) has proven to be an effective bioscavenger against nerve agents and organophosphates. Phase I safety trials of human BChE are currently being conducted and large-scale production of recombinant BChE is underway. Information on the real-time distribution of BChE from the injection site has not been well characterized. This study utilized the BChE nullizygote (BChE-/-) mouse and tetrameric equine BChE labeled with LI-COR fluorescent IRDye 800CW to track, quantify and determine the retention time of BChE in vivo following intramuscular injection. In vivo images were acquired with Xenogen's IVIS 200 imager and the LI-COR Odyssey Imaging System fitted with the MousePOD. Plasma and tissues were tested for BChE activity. The 2 mg of BChE spread from the injection site to heart, liver, intestine, kidneys, lungs, salivary glands, and muscle, but did not enter the brain or the skin. Fluorescence intensity in organs and BChE activity in plasma peaked on day 1. BChE activity in plasma was undetectable by day 16, at a time when there was still significant fluorescent signal and BChE activity in the liver (0.32 units/g), injected quadriceps (0.13 units/g) and in most of the organs analyzed. It is concluded that the tetrameric BChE glycoprotein of 340 kDa diffuses from the muscle injection site to blood and peripheral organs and has a longer residence time in the organs than in blood.
ESTHER : Duysen_2008_Chem.Biol.Interact_175_119
PubMedSearch : Duysen_2008_Chem.Biol.Interact_175_119
PubMedID: 18486120

Title : Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice - Hartmann_2008_Neurochem.Int_52_972
Author(s) : Hartmann J , Kiewert C , Duysen EG , Lockridge O , Klein J
Ref : Neurochem Int , 52 :972 , 2008
Abstract : Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 microM in AChE-deficient mice and 4.36 microM in wild-type mice. Addition of choline (10 microM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 microM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.
ESTHER : Hartmann_2008_Neurochem.Int_52_972
PubMedSearch : Hartmann_2008_Neurochem.Int_52_972
PubMedID: 18023504

Title : Kinetic analysis of effector modulation of butyrylcholinesterase-catalysed hydrolysis of acetanilides and homologous esters - Masson_2008_FEBS.J_275_2617
Author(s) : Masson P , Froment MT , Gillon E , Nachon F , Lockridge O , Schopfer LM
Ref : Febs J , 275 :2617 , 2008
Abstract : The effects of tyramine, serotonin and benzalkonium on the esterase and aryl acylamidase activities of wild-type human butyrylcholinesterase and its peripheral anionic site mutant, D70G, were investigated. The kinetic study was carried out under steady-state conditions with neutral and positively charged aryl acylamides [o-nitrophenylacetanilide, o-nitrotrifluorophenylacetanilide and m-(acetamido) N,N,N-trimethylanilinium] and homologous esters (o-nitrophenyl acetate and acetylthiocholine). Tyramine was an activator of hydrolysis for neutral substrates and an inhibitor of hydrolysis for positively charged substrates. The affinity of D70G for tyramine was lower than that of the wild-type enzyme. Tyramine activation of hydrolysis for neutral substrates by D70G was linear. Tyramine was found to be a pure competitive inhibitor of hydrolysis for positively charged substrates with both wild-type butyrylcholinesterase and D70G. Serotonin inhibited both esterase and aryl acylamidase activities for both positively charged and neutral substrates. Inhibition of wild-type butyrylcholinesterase was hyperbolic (i.e. partial) with neutral substrates and linear with positively charged substrates. Inhibition of D70G was linear with all substrates. A comparison of the effects of tyramine and serotonin on D70G versus the wild-type enzyme indicated that: (a) the peripheral anionic site is involved in the nonlinear activation and inhibition of the wild-type enzyme; and (b) in the presence of charged substrates, the ligand does not bind to the peripheral anionic site, so that ligand effects are linear, reflecting their sole interaction with the active site binding locus. Benzalkonium acted as an activator at low concentrations with neutral substrates. High concentrations of benzalkonium caused parabolic inhibition of the activity with neutral substrates for both wild-type butyrylcholinesterase and D70G, suggesting multiple binding sites. Benzalkonium caused linear, noncompetitive inhibition of the positively charged aryl acetanilide m-(acetamido) N,N,N-trimethylanilinium for D70G, and an unusual mixed-type inhibition/activation (alpha > beta > 1) for wild-type butyrylcholinesterase with this substrate. No fundamental difference was observed between the effects of ligands on the butyrylcholinesterase-catalysed hydrolysis of esters and amides. Thus, butyrylcholinesterase uses the same machinery, i.e. the catalytic triad S198/H448/E325, for the hydrolysis of both types of substrate. The differences in response to ligand binding depend on whether the substrates are neutral or positively charged, i.e. the differences depend on the function of the peripheral site in wild-type butyrylcholinesterase, or the absence of its function in the D70G mutant. The complex inhibition/activation effects of effectors, depending on the integrity of the peripheral anionic site, reflect the allosteric 'cross-talk' between the peripheral anionic site and the catalytic centre.
ESTHER : Masson_2008_FEBS.J_275_2617
PubMedSearch : Masson_2008_FEBS.J_275_2617
PubMedID: 18422653

Title : Fast affinity purification coupled with mass spectrometry for identifying organophosphate labeled plasma butyrylcholinesterase - Li_2008_Chem.Biol.Interact_175_68
Author(s) : Li H , Tong L , Schopfer LM , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 175 :68 , 2008
Abstract : Classical plasma butyrylcholinesterase (BChE) purification involves dialysis and multiple steps of chromatography. We describe a procainamide affinity gel purification scheme that takes 15-30 min to purify BChE from 1 ml plasma. The method uses a microfuge spin column to build a 0.2 ml procainamide affinity column. The eluted BChE contains 3-4 microg of 500-fold purified BChE, free from 99% of contaminating plasma proteins. The BChE was further purified by gel electrophoresis. Tryptic peptides from the BChE containing gel electrophoresis band were prepared by in-gel digestion, separated by reverse phase liquid chromatography and identified by mass spectrometry. The 29 residue active site tryptic peptide labeled with the nerve agents soman or sarin was identified.
ESTHER : Li_2008_Chem.Biol.Interact_175_68
PubMedSearch : Li_2008_Chem.Biol.Interact_175_68
PubMedID: 18586231

Title : Five new naturally occurring mutations of the BCHE gene and frequencies of 12 butyrylcholinesterase alleles in a Brazilian population - Mikami_2008_Pharmacogenet.Genomics_18_213
Author(s) : Mikami LR , Wieseler S , de Souza RLR , Schopfer LM , Nachon F , Lockridge O , Chautard-Freire-Maia EA
Ref : Pharmacogenet Genomics , 18 :213 , 2008
Abstract : Human butyrylcholinesterase (BChE; EC is codified by the BCHE gene (3q26.1-q26.2) in which 65 variants have been identified. BChE is a scavenger of organophosphorus and carbamate compounds and hydrolyzes succinylcholine, mivacurium and cocaine. The present study describes 12 naturally occurring BCHE mutations including five new mutations (K12R, G15G, V294M, G333C and R470W) identified in 366 blood donors from Southern Brazil. Exons 2 and 4 of the BCHE gene were examined by PCR-SSCA and samples with unexpected electrophoretic patterns were sequenced. The respective nucleotide substitution that characterizes each of the four new nonsynonymous mutations was introduced into BCHE cDNA by site directed mutagenesis and transfected into human embryonic kidney 293T cells and/or Chinese hamster ovary cells. The catalyzed hydrolysis of butyrylthiocholine (BTC) by BChE was measured by the Ellman method. Enzyme kinetic parameters obtained after the expression of the respective recombinant BChE evaluated the effects of the four nonsynonymous mutations. Thirty-four out of 366 individuals carried a BChE mutation in exon 2. The K variant mutation, A539T in exon 4, was present in one out of three persons. Gene expression showed that only one of the newly identified mutations (G333C) altered BChE activity, leading to a decrease of about 80% in relation to the wild-type enzyme.
ESTHER : Mikami_2008_Pharmacogenet.Genomics_18_213
PubMedSearch : Mikami_2008_Pharmacogenet.Genomics_18_213
PubMedID: 18300943

Title : The butyrylcholinesterase knockout mouse as a model for human butyrylcholinesterase deficiency - Li_2008_J.Pharmacol.Exp.Ther_324_1146
Author(s) : Li B , Duysen EG , Carlson M , Lockridge O
Ref : Journal of Pharmacology & Experimental Therapeutics , 324 :1146 , 2008
Abstract : Butyrylcholinesterase (BChE) is an important enzyme for metabolism of ester drugs. Many humans have partial or complete BChE deficiency due to genetic variation. Our goal was to create a mouse model of BChE deficiency to allow testing of drug toxicity. For this purpose, we created the BChE knockout mouse by gene-targeted deletion of a portion of the BCHE gene (accession number M99492). The BChE(-/-) mouse had no BChE activity in plasma, but it had low residual butyrylthiocholine hydrolase activity in all other tissues attributed to carboxylesterase ES-10. The BChE(-/-) mouse had a normal phenotype except when challenged with drugs. Nicotinic receptor function as indicated by response to nicotine seemed to be normal in BChE(-/-) mice, but muscarinic receptor function as measured by response to oxotremorine and pilocarpine was altered. Heart rate, blood pressure, and respiration, measured in a Vevo imager, were similar in BChE(+/+) and BChE(-/-) mice. Like BChE(-/-) humans, the BChE(-/-) mouse responded to succinylcholine with prolonged respiratory arrest. Bambuterol was not toxic to BChE(-/-) mice, suggesting it is safe in BChE(-/-) humans. Challenge with 150 mg/kg pilocarpine i.p., a muscarinic agonist, or with 50 mg/kg butyrylcholine i.p., induced tonicclonic convulsions and death in BChE(-/-) mice. This suggests that butyrylcholine, like pilocarpine, binds to muscarinic receptors. In conclusion, the BChE(-/-) mouse is a suitable model for human BChE deficiency.
ESTHER : Li_2008_J.Pharmacol.Exp.Ther_324_1146
PubMedSearch : Li_2008_J.Pharmacol.Exp.Ther_324_1146
PubMedID: 18056867

Title : Lamellipodin proline rich peptides associated with native plasma butyrylcholinesterase tetramers - Li_2008_Biochem.J_411_425
Author(s) : Li H , Schopfer LM , Masson P , Lockridge O
Ref : Biochemical Journal , 411 :425 , 2008
Abstract : 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.
ESTHER : Li_2008_Biochem.J_411_425
PubMedSearch : Li_2008_Biochem.J_411_425
PubMedID: 18076380

Title : The butyrylcholinesterase knockout mouse is obese on a high-fat diet - Li_2008_Chem.Biol.Interact_175_88
Author(s) : Li B , Duysen EG , Lockridge O
Ref : Chemico-Biological Interactions , 175 :88 , 2008
Abstract : Butyrylcholinesterase (BChE) inactivates the appetite stimulating hormone octanoyl-ghrelin. The hypothesis was tested that BChE-/- mice would have abnormally high body weight and high levels of octanoyl-ghrelin. It was found that BChE-/- mice fed a standard 5% fat diet had normal body weight. However, BChE-/- mice fed a diet containing 11% fat became obese. Their obesity was not explained by increased levels of octanoyl-ghrelin, or by increased caloric intake, or by decreased exercise. Instead, a role for BChE in fat utilization was suggested.
ESTHER : Li_2008_Chem.Biol.Interact_175_88
PubMedSearch : Li_2008_Chem.Biol.Interact_175_88
PubMedID: 18452903

Title : Aging of cholinesterases phosphylated by tabun proceeds through O-dealkylation - Carletti_2008_J.Am.Chem.Soc_130_16011
Author(s) : Carletti E , Li H , Li B , Ekstrom F , Nicolet Y , Loiodice M , Gillon E , Froment MT , Lockridge O , Schopfer LM , Masson P , Nachon F
Ref : Journal of the American Chemical Society , 130 :16011 , 2008
Abstract : Human butyrylcholinesterase (hBChE) hydrolyzes or scavenges a wide range of toxic esters, including heroin, cocaine, carbamate pesticides, organophosphorus pesticides, and nerve agents. Organophosphates (OPs) exert their acute toxicity through inhibition of acetylcholinesterase (AChE) by phosphorylation of the catalytic serine. Phosphylated cholinesterase (ChE) can undergo a spontaneous, time-dependent process called "aging", during which the OP-ChE conjugate is dealkylated. This leads to irreversible inhibition of the enzyme. The inhibition of ChEs by tabun and the subsequent aging reaction are of particular interest, because tabun-ChE conjugates display an extraordinary resistance toward most current oxime reactivators. We investigated the structural basis of oxime resistance for phosphoramidated ChE conjugates by determining the crystal structures of the non-aged and aged forms of hBChE inhibited by tabun, and by updating the refinement of non-aged and aged tabun-inhibited mouse AChE (mAChE). Structures for non-aged and aged tabun-hBChE were refined to 2.3 and 2.1 A, respectively. The refined structures of aged ChE conjugates clearly show that the aging reaction proceeds through O-dealkylation of the P(R) enantiomer of tabun. After dealkylation, the negatively charged oxygen forms a strong salt bridge with protonated His438N epsilon2 that prevents reactivation. Mass spectrometric analysis of the aged tabun-inhibited hBChE showed that both the dimethylamine and ethoxy side chains were missing from the phosphorus. Loss of the ethoxy is consistent with the crystallography results. Loss of the dimethylamine is consistent with acid-catalyzed deamidation during the preparation of the aged adduct for mass spectrometry. The reported 3D data will help in the design of new oximes capable of reactivating tabun-ChE conjugates.
ESTHER : Carletti_2008_J.Am.Chem.Soc_130_16011
PubMedSearch : Carletti_2008_J.Am.Chem.Soc_130_16011
PubMedID: 18975951
Gene_locus related to this paper: human-BCHE , mouse-ACHE

Title : Aryl acylamidase activity of human serum albumin with o-nitrotrifluoroacetanilide as the substrate - Masson_2007_J.Enzyme.Inhib.Med.Chem_22_463
Author(s) : Masson P , Froment MT , Darvesh S , Schopfer LM , Lockridge O
Ref : J Enzyme Inhib Med Chem , 22 :463 , 2007
Abstract : Albumin is generally regarded as an inert protein with no enzyme activity. However, albumin has esterase activity as well as aryl acylamidase activity. A new acetanilide substrate, o-nitrotrifluoroacetanilide (o-NTFNAC), which is more reactive than the classical o-nitroacetanilide, made it possible to determine the catalytic parameters for hydrolysis by fatty-acid free human serum albumin. Owing to the low enzymatic activity of albumin, kinetic studies were performed at high albumin concentration (0.075 mM). The albumin behavior with this substrate was Michaelis-Menten like. Kinetic analysis was performed according to the formalism used for catalysis at high enzyme concentration. This approach provided values for the turnover and dissociation constant of the albumin-substrate complex: k(cat) = 0.13 +/- 0.02 min(-1) and Ks = 0.67 +/- 0.04 mM. MALDI-TOF experiments showed that unlike the ester substrate p-nitrophenyl acetate, o-NTFNAC does not form a stable adduct (acetylated enzyme). Kinetic analysis and MALDI-TOF experiments demonstrated that hydrolysis of o-NTFNAC by albumin is fully rate-limited by the acylation step (k(cat) = k2). Though the aryl acylamidase activity of albumin is low (k(cat)/Ks = 195 M(-1)min(-1)), because of its high concentration in human plasma (0.6-1 mM), albumin may participate in hydrolysis of aryl acylamides through second-order kinetics. This suggests that albumin may have a role in the metabolism of endogenous and exogenous aromatic amides, including drugs and xenobiotics.
ESTHER : Masson_2007_J.Enzyme.Inhib.Med.Chem_22_463
PubMedSearch : Masson_2007_J.Enzyme.Inhib.Med.Chem_22_463
PubMedID: 17847714

Title : Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity - Hartmann_2007_J.Neurochem_100_1421
Author(s) : Hartmann J , Kiewert C , Duysen EG , Lockridge O , Greig NH , Klein J
Ref : Journal of Neurochemistry , 100 :1421 , 2007
Abstract : 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.
ESTHER : Hartmann_2007_J.Neurochem_100_1421
PubMedSearch : Hartmann_2007_J.Neurochem_100_1421
PubMedID: 17212694

Title : Matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411 - Li_2007_Anal.Biochem_361_263
Author(s) : Li B , Schopfer LM , Hinrichs SH , Masson P , Lockridge O
Ref : Analytical Biochemistry , 361 :263 , 2007
Abstract : Our goal was to determine whether chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate (DFP), and sarin covalently bind to human albumin. Human albumin or plasma was treated with organophosphorus (OP) agent at alkaline pH, digested with pepsin at pH 2.3, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Two singly charged peaks m/z 1718 and 1831, corresponding to the unlabeled peptide fragments containing the active site Tyr411 residue, were detected in all samples. The sequences of the two peptides were VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. The peptide-OP adducts of these peptides were also found. They had masses of 1854 and 1967 for chlorpyrifos oxon, 1825 and 1938 for dichlorvos, 1881 and 1994 for DFP, and 1838 and 1938 for sarin; these masses fit a mechanism whereby OP bound covalently to Tyr411. The binding of DFP to Tyr411 of human albumin was confirmed by electrospray tandem mass spectrometry and analysis of product ions. None of the OP-albumin adducts lost an alkoxy group, leading to the conclusion that aging did not occur. Our results show that OP pesticides and nerve agents bind covalently to human albumin at Tyr411. The presence of Tyr411 on an exposed surface of albumin suggests that an antibody response could be generated against OP-albumin adducts.
ESTHER : Li_2007_Anal.Biochem_361_263
PubMedSearch : Li_2007_Anal.Biochem_361_263
PubMedID: 17188226

Title : A novel method for purifying recombinant human host defense cathelicidin LL-37 by utilizing its inherent property of aggregation - Li_2007_Protein.Expr.Purif_54_157
Author(s) : Li Y , Li X , Li H , Lockridge O , Wang G
Ref : Protein Expr Purif , 54 :157 , 2007
Abstract : The importance of human LL-37 in host defense and innate immunity is well appreciated as reflected by an exponential increase of relevant literature in Pub-Med. Although several articles reported the expression and purification of this cathelicidin, some protocols suffered from low efficiency in enzyme cleavage of fusion proteins due to aggregation and poor separation of recombinant LL-37 from the carrier protein on reverse-phase HPLC. We present a new method for purifying LL-37 that avoids both problems. In this method, the fusion protein (a tetramer) purified by metal affinity chromatography was readily cleaved at a thrombin site 30-residue upstream of the LL-37 sequence. The released LL-37-containing fragment formed a large soluble aggregate (approximately 95 kDa) at pH approximately 7, allowing a rapid and clean separation from the carrier thioredoxin (approximately 14 kDa) by size-exclusion chromatography. Recombinant LL-37 was released from the isolated aggregate by chemical cleavage in 50% formic acid at 50 degrees C for 32 h. Due to a dramatic difference in retention time, recombinant LL-37 was well resolved from the S-Tag-containing peptide by RP-HPLC. Compared to previous procedures, the new method involves fewer steps and is highly reproducible. It increases peptide yield by 53%. NMR data support the aggregation of LL-37 into a tetramer with increase of pH as well as the feasibility of structural studies of an isotope-labeled antimicrobial peptide in the lipid micelle of dioctanoyl phosphatidylglycerol (D8PG) for the first time.
ESTHER : Li_2007_Protein.Expr.Purif_54_157
PubMedSearch : Li_2007_Protein.Expr.Purif_54_157
PubMedID: 17382559

Title : Sensitivity of butyrylcholinesterase knockout mice to (--)-huperzine A and donepezil suggests humans with butyrylcholinesterase deficiency may not tolerate these Alzheimer's disease drugs and indicates butyrylcholinesterase function in neurotransmission - Duysen_2007_Toxicology_233_60
Author(s) : Duysen EG , Li B , Darvesh S , Lockridge O
Ref : Toxicology , 233 :60 , 2007
Abstract : Butyrylcholinesterase (EC BChE) is present in all human and mouse tissues, and is more abundant than acetylcholinesterase (EC AChE) in all tissues except brain. People who have no BChE activity due to a genetic variation are healthy. This has led to the hypothesis that BChE has no physiological function. We tested this hypothesis by challenging BChE and AChE knockout mice, as well as wild-type mice, with the AChE specific inhibitors, (--)-huperzine A and donepezil, and with serine hydrolase inhibitors, echothiophate and chlorpyrifos oxon. (--)-Huperzine A and donepezil caused mortality and significant toxicity in the BChE-/- animals. The BChE heterozygote (BCHE+/-) mice with approximately one-half the BChE activity of the BChE wild type (BChE+/+) exhibited intermediate toxic symptoms, and survived a longer period. The BChE+/+ animals displayed comparatively minor toxic symptoms and recovered by 24h post-dosing. Plasma AChE activity was inhibited to the same extent in BChE-/-, +/-, and +/+ mice, whereas BChE activity was not inhibited. This indicated that the protective effect of BChE was not due to scavenging (--)-huperzine A. AChE-/- mice were unaffected by (--)-huperzine A and donepezil, demonstrating the specificity of these inhibitors for AChE. AChE-/- mice treated with chlorpyrifos oxon lost all BChE activity, had severe cholinergic symptoms and died of convulsions. This showed that BChE activity was essential for survival of AChE-/- mice. In conclusion, we propose that the protective effect of BChE is explained by hydrolysis of excess acetylcholine in physiologically relevant regions such as diaphragm, cardiac muscle, and brain. Thus, BChE has a function in neurotransmission. People with BChE deficiency are expected to be intolerant of standard doses of the anti-Alzheimer's drugs, (--)-huperzine A and donepezil.
ESTHER : Duysen_2007_Toxicology_233_60
PubMedSearch : Duysen_2007_Toxicology_233_60
PubMedID: 17194517

Title : Hydrolysis of oxo- and thio-esters by human butyrylcholinesterase - Masson_2007_Biochim.Biophys.Acta_1774_16
Author(s) : Masson P , Froment MT , Gillon E , Nachon F , Lockridge O , Schopfer LM
Ref : Biochimica & Biophysica Acta , 1774 :16 , 2007
Abstract : Catalytic parameters of human butyrylcholinesterase (BuChE) for hydrolysis of homologous pairs of oxo-esters and thio-esters were compared. Substrates were positively charged (benzoylcholine versus benzoylthiocholine) and neutral (phenylacetate versus phenylthioacetate). In addition to wild-type BuChE, enzymes containing mutations were used. Single mutants at positions: G117, a key residue in the oxyanion hole, and D70, the main component of the peripheral anionic site were tested. Double mutants containing G117H and mutations on residues of the oxyanion hole (G115, A199), or the pi-cation binding site (W82), or residue E197 that is involved in stabilization of tetrahedral intermediates were also studied. A mathematical analysis was used to compare data for BuChE-catalyzed hydrolysis of various pairs of oxo-esters and thio-esters and to determine the rate-limiting step of catalysis for each substrate. The interest and limitation of this method is discussed. Molecular docking was used to analyze how the mutations could have altered the binding of the oxo-ester or the thio-ester. Results indicate that substitution of the ethereal oxygen for sulfur in substrates may alter the adjustment of substrate in the active site and stabilization of the transition-state for acylation. This affects the k2/k3 ratio and, in turn, controls the rate-limiting step of the hydrolytic reaction. Stabilization of the transition state is modulated both by the alcohol and acyl moieties of substrate. Interaction of these groups with the ethereal hetero-atom can have a neutral, an additive or an antagonistic effect on transition state stabilization, depending on their molecular structure, size and enantiomeric configuration.
ESTHER : Masson_2007_Biochim.Biophys.Acta_1774_16
PubMedSearch : Masson_2007_Biochim.Biophys.Acta_1774_16
PubMedID: 17182295

Title : Neuropathological and immunochemical studies of brain parenchyma in acetylcholinesterase knockout mice: implications in Alzheimer's disease - Rice_2007_J.Alzheimers.Dis_11_481
Author(s) : Rice SG , Nowak L , Duysen EG , Lockridge O , Lahiri DK , Reyes PF
Ref : J Alzheimers Dis , 11 :481 , 2007
Abstract : The 'cholinergic hypothesis', based on the correlation of the reduction of cholinergic activity in Alzheimer's disease (AD) with cognition and memory, is currently the most widely-held view for AD. Drug treatments for AD focus mainly on inhibition of acetylcholinesterase (AChE), and to some extent butyrylcholinesterase (BChE). In addition to changes in AChE in AD, there is a rise in the level of the sister enzyme BChE. However, the role of the two cholinesterases is poorly understood in vivo. We characterized several proteins immunohistochemically in brain sections from AChE nullizygote (AChE-/-) and wild type AChE+/+ mice. Previous studies had shown that AChE-/- mouse tissues are devoid of AChE activity and that the overall cholinesterase activity is significantly decreased in the knockout group [16]. Despite the differences of cholinesterase activity, we found no significant structural alterations between the experimental groups. Immunohistochemical examination revealed no neuronal, dendritic, astrocytic, synaptic, microglial, and endothelial differences between AChE-/- and AChE+/+ mice. Similarly, the histochemical examination showed no morphologic alterations between AChE-/- and AChE+/+ mice. Our studies show that neither the absence of AChE nor the presence exclusively of BChE is associated with neuroglial and vascular pathology.
ESTHER : Rice_2007_J.Alzheimers.Dis_11_481
PubMedSearch : Rice_2007_J.Alzheimers.Dis_11_481
PubMedID: 17656828

Title : Adaptation to excess acetylcholine by downregulation of adrenoceptors and muscarinic receptors in lungs of acetylcholinesterase knockout mice - Myslivecek_2007_Naunyn.Schmiedebergs.Arch.Pharmacol_376_83
Author(s) : Myslivecek J , Duysen EG , Lockridge O
Ref : Naunyn Schmiedebergs Arch Pharmacol , 376 :83 , 2007
Abstract : The acetylcholinesterase knockout mouse has elevated acetylcholine levels due to the complete absence of acetylcholinesterase. Our goal was to determine the adaptive changes in lung receptors that allow these animals to tolerate excess neurotransmitter. The hypothesis was tested that not only muscarinic receptors but also alpha(1)-adrenoceptors and beta-adrenoceptors are downregulated, thus maintaining a proper balance of receptors and accounting for lung function in these animals. The quantity of alpha(1A), alpha(1B), alpha(1D), beta(1), and beta(2)-adrenoceptors and muscarinic receptors was determined by binding of radioligands. G-protein coupling was assessed using pseudo-competition with agonists. Phospholipase C activity was measured by an enzymatic assay. Cyclic AMP (cAMP) content was measured by immunoassay. Muscarinic receptors were decreased to 50%, alpha(1)-adrenoceptors to 23%, and beta-adrenoceptors to about 50% of control. Changes were subtype specific, as alpha(1A), alpha(1B), and beta(2)-adrenoceptors, but not alpha(1D)-adrenoceptor, were decreased. In contrast, receptor signaling into the cell as measured by coupling to G proteins, cAMP content, and PI-phospholipase C activity was the same as in control. This shows that the nearly normal lung function of these animals was explained by maintenance of a correct balance of adrenoceptors and muscarinic receptors. In conclusion, knockout mice have adapted to high concentrations of acetylcholine by downregulating receptors that bind acetylcholine, as well as by downregulating receptors that oppose the action of muscarinic receptors. Tolerance to excess acetylcholine is achieved by reducing the levels of muscarinic receptors and adrenoceptors.
ESTHER : Myslivecek_2007_Naunyn.Schmiedebergs.Arch.Pharmacol_376_83
PubMedSearch : Myslivecek_2007_Naunyn.Schmiedebergs.Arch.Pharmacol_376_83
PubMedID: 17805515

Title : Comparison of cognitive functions between people with silent and wild-type butyrylcholinesterase - Manoharan_2007_J.Neural.Transm.(Vienna)_114_939
Author(s) : Manoharan I , Kuznetsova A , Fisk JD , Boopathy R , Lockridge O , Darvesh S
Ref : J Neural Transm (Vienna) , 114 :939 , 2007
Abstract : In the human brain, butyrylcholinesterase (BuChE) is expressed in neurons and glia. For example, many nuclei in the human thalamus, with projections to the cerebral cortex, contain a large number of neurons with intense BuChE activity. Thalamocortical projections subserve a variety of cognitive functions. Due to genetic mutations, there are individuals who do not have detectable BuChE activity (silent BuChE). While the prevalence of silent BuChE is only 1:100,000 in European and American populations, it is 1:24 in the Vysya community in Coimbatore, India. To examine whether there are differences in cognitive functions between individuals with silent BuChE and those expressing normal BuChE (wild-type), twelve healthy individuals with silent BuChE and thirteen healthy individuals with wild-type BuChE, all from the Vysya community in Coimbatore, were tested for cognitive function using the Automated Neuropsychological Assessment Metrics test battery. The silent BuChE group was slightly faster on simple reaction tasks, but slower on a visual perceptual matching task. Furthermore, discriminant function analyses correctly classified 11/12 silent and 8/13 wild-type BuChE subjects (76% correct classification overall) based on BuChE status. Different profiles of cognitive test performance between individuals with silent and wild-type BuChE were observed. These observations suggest a function for BuChE in cognition.
ESTHER : Manoharan_2007_J.Neural.Transm.(Vienna)_114_939
PubMedSearch : Manoharan_2007_J.Neural.Transm.(Vienna)_114_939
PubMedID: 17318303

Title : A medical health report on individuals with silent butyrylcholinesterase in the Vysya community of India - Manoharan_2007_Clin.Chim.Acta_378_128
Author(s) : Manoharan I , Boopathy R , Darvesh S , Lockridge O
Ref : Clinica Chimica Acta , 378 :128 , 2007
Abstract : BACKGROUND: Butyrylcholinesterase (BChE; gi:116353) deficiency has adverse effects on the response to succinylcholine and mivacurium. A physiological function of BChE is to inactivate octanoyl ghrelin. We determined the health effect of complete absence of BChE in humans.
METHODS: Clinical tests of cardiac, lung, liver, and kidney function, body weight, sperm counts and motility were performed on 5 men, age 20-32 y, in the Vysya community of Coimbatore, India who had silent BChE. Postmortem tissues from 2 cadavers with wild-type BChE were assayed.
RESULTS: Test results were normal, except for lung function, which indicated mild obstruction in silent as well as in wild-type BChE subjects. Creatine kinase-MB levels were high in 2 subjects, but there were no other indications of damage to the heart. Body weight was normal. Family histories revealed no trend in disease susceptibility. The human body contains 10 times more BChE than acetylcholinesterase molecules. CONCLUSION: Individuals completely deficient in BChE have only minor abnormalities in clinical test results. However, they respond abnormally to standard doses of succinylcholine and mivacurium. It is expected, but not proven, that they are unusually susceptible to the toxicity of cocaine and organophosphorus pesticides, and resistant to bambuterol and irinotecan. Their normal body weight suggests alternative routes for deactivation of octanoyl ghrelin.
ESTHER : Manoharan_2007_Clin.Chim.Acta_378_128
PubMedSearch : Manoharan_2007_Clin.Chim.Acta_378_128
PubMedID: 17182021

Title : Expression of three naturally occurring genetic variants (G75R, E90D, I99M) of the BCHE gene of human butyrylcholinesterase - Mikami_2007_Pharmacogenet.Genomics_17_681
Author(s) : Mikami LR , Wieseler S , de Souza RLR , Schopfer LM , Lockridge O , Chautard-Freire-Maia EA
Ref : Pharmacogenet Genomics , 17 :681 , 2007
Abstract : The present paper examined the effects of three non synonymous BCHE mutations (G75R, E90D and /99M) on enzyme kinetic parameters obtained after the expression of the respective recombinant BChEs. The respective nucleotide substitution that characterizes each of the three variants was introduced into BCHE cDNA by site directed mutagenesis and transfected into human embryonic kidney 293 T cells and Chinese hamster ovary cells (for E90D). BChE catalysed hydrolysis of butyrylthiocoline (BTC) was measured by Ellman method. The expression results showed that: (1) the activity of the G75R enzyme represents approximately 45% of the wild-type activity, whereas that of the I99M enzyme does not differ from the wild-type; (2) the E90D enzyme presents a silent phenotype; disruption of the salt bridge between E90 and R42 may cause the enzyme to be rapidly degraded inside the cells. In homozygous form the E90D enzyme may confer increased susceptibility to succinylcholine, but may delay cognitive impairment in aged individuals. BChE genotyping may become important for estimating prognosis, and the knowledge of the genetic variants of BChE in a particular population may be useful for carrying out the genotyping assays.
ESTHER : Mikami_2007_Pharmacogenet.Genomics_17_681
PubMedSearch : Mikami_2007_Pharmacogenet.Genomics_17_681
PubMedID: 17700357

Title : Crystallization and X-ray structure of full-length recombinant human butyrylcholinesterase - Ngamelue_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_723
Author(s) : Ngamelue MN , Homma K , Lockridge O , Asojo OA
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :723 , 2007
Abstract : Human butyrylcholinesterase (BChE) has been shown to function as an endogenous scavenger of diverse poisons. BChE is a 340 kDa tetrameric glycoprotein that is present in human serum at a concentration of 5 mg l(-1). The well documented therapeutic effects of BChE on cocaine toxicity and organophosphorus agent poisoning has increased the need for effective methods of producing recombinant therapeutic BChE. In order to be therapeutically useful, BChE must have a long circulatory residence time or associate as tetramers. Full-length recombinant BChE produced in Chinese hamster ovary (CHO) cells or human embryonic kidney cells has been shown to associate as monomers, with a shorter circulatory residence time than the naturally occurring tetrameric serum protein. Based on the preceding observation as well as the need to develop novel methodologies to facilitate the mass production of therapeutic recombinant BChE, studies have been initiated to determine the structural basis of tetramer formation. Towards these ends, full-length monomeric recombinant BChE has been crystallized for the first time. A 2.8 A X-ray structure was solved in space group P42(1)2, with unit-cell parameters a = b = 156, c = 146 A.
ESTHER : Ngamelue_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_723
PubMedSearch : Ngamelue_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_723
PubMedID: 17768338
Gene_locus related to this paper: human-BCHE

Title : Aging pathways for organophosphate-inhibited human butyrylcholinesterase, including novel pathways for isomalathion, resolved by mass spectrometry - Li_2007_Toxicol.Sci_100_136
Author(s) : Li H , Schopfer LM , Nachon F , Froment MT , Masson P , Lockridge O
Ref : Toxicol Sci , 100 :136 , 2007
Abstract : Some organophosphorus compounds are toxic because they inhibit acetylcholinesterase (AChE) by phosphylation of the active site serine, forming a stable conjugate: Ser-O-P(O)-(Y)-(XR) (where X can be O, N, or S and Y can be methyl, OR, or SR). The inhibited enzyme can undergo an aging process, during which the X-R moiety is dealkylated by breaking either the P-X or the X-R bond depending on the specific compound, leading to a nonreactivatable enzyme. Aging mechanisms have been studied primarily using AChE. However, some recent studies have indicated that organophosphate-inhibited butyrylcholinesterase (BChE) may age through an alternative pathway. Our work utilized matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry to study the aging mechanism of human BChE inhibited by dichlorvos, echothiophate, diisopropylfluorophosphate (DFP), isomalathion, soman, sarin, cyclohexyl sarin, VX, and VR. Inhibited BChE was aged in the presence of H2O18 to allow incorporation of (18)O, if cleavage was at the P-X bond. Tryptic-peptide organophosphate conjugates were identified through peptide mass mapping. Our results showed no aging of VX- and VR-treated BChE at 25 degrees C, pH 7.0. However, BChE inhibited by dichlorvos, echothiophate, DFP, soman, sarin, and cyclohexyl sarin aged exclusively through O-C bond cleavage, i.e., the classical X-R scission pathway. In contrast, isomalathion aged through both X-R and P-X pathways; the main aged product resulted from P-S bond cleavage and a minor product resulted from O-C and/or S-C bond cleavage.
ESTHER : Li_2007_Toxicol.Sci_100_136
PubMedSearch : Li_2007_Toxicol.Sci_100_136
PubMedID: 17698511

Title : Naturally occurring mutation Leu307Pro of human butyrylcholinesterase in the Vysya community of India - Manoharan_2006_Pharmacogenet.Genomics_16_461
Author(s) : Manoharan I , Wieseler S , Layer PG , Lockridge O , Boopathy R
Ref : Pharmacogenet Genomics , 16 :461 , 2006
Abstract : BACKGROUND: People with genetic variants of butyrylcholinesterase (EC, BChE) can have hours of prolonged apnea after a normal dose of succinylcholine or mivacurium.
METHODS: Plasma samples from 226 people in the Vysya community in Coimbatore, India were tested for BChE activity.
RESULTS: Nine unrelated individuals had no detectable activity. DNA sequencing revealed a novel mutation in exon 2 of the BCHE gene, responsible for the silent phenotype of human serum BChE. All silent BChE samples were homozygous for a point mutation at codon 307 (CTT-->CCT), resulting in substitution of leucine 307 by proline. Western blot analysis with a monoclonal antibody showed no BChE protein in plasma. Silent BChE plasma samples had no organophosphate-reactive BChE, as measured with FP-biotin. Expression of recombinant Leu307Pro BChE in cell culture confirmed that this mutant is expressed at very low levels. The proline substitution most likely destabilizes the BChE structure and causes the protein to be misfolded and rapidly degraded.
CONCLUSIONS: This is the first report of a molecularly defined BChE mutation in the Indian population. The frequency of homozygous silent BChE in the Vysya community is 1 in 24, a value 4000-fold higher than the frequency of homozygous silent BChE in European and American populations.
ESTHER : Manoharan_2006_Pharmacogenet.Genomics_16_461
PubMedSearch : Manoharan_2006_Pharmacogenet.Genomics_16_461
PubMedID: 16788378
Gene_locus related to this paper: human-BCHE

Title : Gene transfer of acetylcholinesterase protects the knockout mouse from the toxicity of DFP - Li_2006_J.Mol.Neurosci_30_79
Author(s) : Li B , Duysen EG , Lockridge O
Ref : Journal of Molecular Neuroscience , 30 :79 , 2006
Abstract : Acetylcholinesterase (AChE) has a clear role in nerve impulse transmission. Organophosphorus esters are highly toxic chemicals used as pesticides, fire retardants, plasticizers, and chemical warfare agents. The acute toxicity of organophosphorus poisons is attributed to inhibition of AChE in nerve synapses. This leads to seizures, respiratory arrest, and death. Our goal was to find a new therapeutic for protection against the toxicity of organophosphates (OPs). We investigated the feasibility of using a gene therapy vector to deliver AChE over long time periods and in quantities sufficiently high to provide protection against diisopropylfluorophosphate (DFP) toxicity. We used the AChE-/- mouse for these studies because this mouse has no endogenous AChE activity (Xie et al., 2000). Any AChE activity found in tissues could only come from the viral vector.
ESTHER : Li_2006_J.Mol.Neurosci_30_79
PubMedSearch : Li_2006_J.Mol.Neurosci_30_79
PubMedID: 17192637

Title : Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters - Poyot_2006_Biochim.Biophys.Acta_1764_1470
Author(s) : Poyot T , Nachon F , Froment MT , Loiodice M , Wieseler S , Schopfer LM , Lockridge O , Masson P
Ref : Biochimica & Biophysica Acta , 1764 :1470 , 2006
Abstract : Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.
ESTHER : Poyot_2006_Biochim.Biophys.Acta_1764_1470
PubMedSearch : Poyot_2006_Biochim.Biophys.Acta_1764_1470
PubMedID: 16962835

Title : Production of the butyrylcholinesterase knockout mouse - Li_2006_J.Mol.Neurosci_30_193
Author(s) : Li B , Duysen EG , Saunders TL , Lockridge O
Ref : Journal of Molecular Neuroscience , 30 :193 , 2006
Abstract : The butyrylcholinesterase (BChE [EC]) knockout mouse is a model for BChE deficiency in humans. The existence of genetic variants of human BChE was discovered after a new muscle relaxant, succinylcholine, was introduced into the practice of medicine in the late 1950s. People with the atypical variant were unable to breathe for 2 h after receiving a dose intended to paralyze for 3-5 min (Kalow and Gunn, 1957, 1959). The atypical variant was later found to have a single-amino-acid mutation at Asp-70 (McGuire et al., 1989), which decreased the affinity of BChE for all positively charged compounds. Though the atypical BChE mutant is the one most commonly encountered in cases of succinylcholine apnea, an additional 58 mutations in the BChE coding sequence have been reported. The frequency of BChE mutations in the American population is known (Lockridge, 1990). One person out of 25 carries one atypical allele (D70G), whereas 1 out of 2500 is homozygous for D70G. The most frequent mutation, A539T, is carried by 1 person out of every 4 and is found in homozygous form in 1 person out of 69 (Bartels et al., 1992). The homozygous A539T form is associated with a 33% decrease in plasma BChE activity. Some people have no detectable BChE activity in plasma, owing to a mutation that truncates the protein, or inactivates it. The frequency of silent BChE is 1 out of 160 for carriers, and 1 out of 110,000 for homozygotes. The BChE knockout mice are models for silent BChE in humans. The literature contains no documentation of the health of people with silent BChE, other than to say they are healthy. We know nothing about their life expectancy, fertility, risk of cognitive impairment, risk of heart disease, or susceptibility to toxins. The BChE knockout mouse will allow us to test the hypothesis that the function of BChE is to detoxify poisons and will allow us to test the role of BChE in other physiological functions.
ESTHER : Li_2006_J.Mol.Neurosci_30_193
PubMedSearch : Li_2006_J.Mol.Neurosci_30_193
PubMedID: 17192674

Title : Phenotype comparison of three acetylcholinesterase knockout strains - Duysen_2006_J.Mol.Neurosci_30_91
Author(s) : Duysen EG , Lockridge O
Ref : Journal of Molecular Neuroscience , 30 :91 , 2006
Abstract : The phenotypes of three mouse strains that carry the acetylcholinesterase knockout (AChE KO) mutation have been compared. The AChE KO mouse was developed from embryonic stem (ES) cells, originating from a 129/Sv blastocyst. Animals generated from strain 129/Sv suffer from dysgenesis of the corpus callosum and possibly a number of other neuroanatomical deficiencies. To determine the contribution of background genes to phenotype, 129/Sv AChE heterozygote (AChE+/-) mice were backcrossed 10 generations with wild-type inbred C57/BL6 (C57) mice and with wild-type outbred CD1(R) mice. AChE-/- mice in strains C57 and CD1died during seizures before postnatal day (P) 21, whereas mice in strain 129/Sv lived to adulthood.
ESTHER : Duysen_2006_J.Mol.Neurosci_30_91
PubMedSearch : Duysen_2006_J.Mol.Neurosci_30_91
PubMedID: 17192642

Title : A mutation linked with autism reveals a common mechanism of endoplasmic reticulum retention for the alpha,beta-hydrolase fold protein family - De Jaco_2006_J.Biol.Chem_281_9667
Author(s) : De Jaco A , Comoletti D , Kovarik Z , Gaietta G , Radic Z , Lockridge O , Ellisman MH , Taylor P
Ref : Journal of Biological Chemistry , 281 :9667 , 2006
Abstract : A mutation linked to autistic spectrum disorders encodes an Arg to Cys replacement in the C-terminal portion of the extracellular domain of neuroligin-3. The solvent-exposed Cys causes virtually complete retention of the protein in the endoplasmic reticulum when the protein is expressed in transfected cells. An identical Cys substitution was reported for butyrylcholinesterase through genotyping patients with post-succinylcholine apnea. Neuroligin, butyrylcholinesterase, and acetylcholinesterase are members of the alpha,beta-hydrolase fold family of proteins sharing sequence similarity and common tertiary structures. Although these proteins have distinct oligomeric assemblies and cellular dispositions, homologous Arg residues in neuroligin-3 (Arg-451), in butyrylcholinesterase (Arg-386), and in acetylcholinesterase (Arg-395) are conserved in all studied mammalian species. To examine whether an homologous Arg to Cys mutation affects related proteins similarly despite their differing capacities to oligomerize, we inserted homologous mutations in the acetylcholinesterase and butyrylcholinesterase cDNAs. Using confocal fluorescence microscopy and analysis of oligosaccharide processing, we find that the homologous Arg to Cys mutation also results in endoplasmic reticulum retention of the two cholinesterases. Small quantities of mutated acetylcholinesterase exported from the cell retain activity but show a greater K(m), a much smaller k(cat), and altered substrate inhibition. The nascent proteins associate with chaperones during processing, but the mutation presumably restricts processing through the endoplasmic reticulum and Golgi apparatus, because of local protein misfolding and inability to oligomerize. The mutation may alter the capacity of these proteins to dissociate from their chaperone prior to oligomerization and processing for export.
ESTHER : De Jaco_2006_J.Biol.Chem_281_9667
PubMedSearch : De Jaco_2006_J.Biol.Chem_281_9667
PubMedID: 16434405

Title : Protection from the toxicity of diisopropylfluorophosphate by adeno-associated virus expressing acetylcholinesterase - Li_2006_Toxicol.Appl.Pharmacol_214_152
Author(s) : Li B , Duysen EG , Poluektova LY , Murrin LC , Lockridge O
Ref : Toxicol Appl Pharmacol , 214 :152 , 2006
Abstract : Organophosphorus esters (OP) are highly toxic chemicals used as pesticides and nerve agents. Their acute toxicity is attributed to inhibition of acetylcholinesterase (AChE, EC in nerve synapses. Our goal was to find a new therapeutic for protection against OP toxicity. We used a gene therapy vector, adeno-associated virus serotype 2 (AAV-2), to deliver murine AChE to AChE-/- mice that have no endogenous AChE activity. The vector encoded the most abundant form of AChE: exons 2, 3, 4, and 6. Two-day old animals, with an immature immune system, were injected. AChE delivered intravenously was expressed up to 5 months in plasma, liver, heart, and lung, at 5-15% of the level in untreated wild-type mice. A few mice formed antibodies, but antibodies did not block AChE activity. The plasma AChE was a mixture of dimers and tetramers. AChE delivered intramuscularly had 40-fold higher activity levels than in wild-type muscle. None of the AChE was collagen-tailed. No retrograde transport through the motor neurons to the central nervous system was detected. AChE delivered intrastriatally assembled into tetramers. In brain, the AAV-2 vector transduced neurons, but not astrocytes and microglia. Vector-treated AChE-/- mice lived longer than saline-treated controls. AChE-/- mice were protected from diisopropylfluorophosphate-induced respiratory failure when the vector was delivered intravenously, but not intrastriatally. Since vector-treated animals had no AChE activity in diaphragm muscle, protection from respiratory failure came from AChE in other tissues. We conclude that AChE scavenged OP and in this way protected the activity of butyrylcholinesterase (BChE, EC in motor endplates.
ESTHER : Li_2006_Toxicol.Appl.Pharmacol_214_152
PubMedSearch : Li_2006_Toxicol.Appl.Pharmacol_214_152
PubMedID: 16443250

Title : Markers of organophosphate exposure in human serum - Wieseler_2006_J.Mol.Neurosci_30_93
Author(s) : Wieseler S , Schopfer LM , Lockridge O
Ref : Journal of Molecular Neuroscience , 30 :93 , 2006
Abstract : The goal of this work is to identify novel serum proteins that are labeled with organophosphates (OPs) and to create a protocol for identification using mass spectroscopy. The use of OP-labeled proteins for identification of exposure is useful because such proteins will remain in circulation for weeks (Van Der Schans et al., 2004). Currently, both butyrylcholinesterase (BChE) and albumin have been shown to bind OPs in blood. Peeples et al. (2005) showed that albumin is labeled by OPs, specifically 6-Nbiotinylaminohexyl isopropyl phosphorofluoridate hemihydrate, in living mice. Albumin is the major protein in human serum, and its reaction with OPs tends to overwhelm the identification of other proteins. In vitro studies of human serum require removal of the serum albumin without depleting the less abundant proteins. Following this step, identifying the remaining proteins is simply a matter of labeling the proteins with an OP, separating the labeled from nonlabeled proteins, and using Q-trap mass spectrometry for identification.
ESTHER : Wieseler_2006_J.Mol.Neurosci_30_93
PubMedSearch : Wieseler_2006_J.Mol.Neurosci_30_93
PubMedID: 17192643

Title : Role of water in aging of human butyrylcholinesterase inhibited by echothiophate: the crystal structure suggests two alternative mechanisms of aging - Nachon_2005_Biochemistry_44_1154
Author(s) : Nachon F , Asojo OA , Borgstahl GE , Masson P , Lockridge O
Ref : Biochemistry , 44 :1154 , 2005
Abstract : Organophosphorus poisons (OP) bind covalently to the active-site serine of cholinesterases. The inhibited enzyme can usually be reactivated with powerful nucleophiles such as oximes. However, the covalently bound OP can undergo a suicide reaction (termed aging) yielding nonreactivatable enzyme. In human butyrylcholinesterase (hBChE), aging involves the residues His438 and Glu197 that are proximal to the active-site serine (Ser198). The mechanism of aging is known in detail for the nerve gases soman, sarin, and tabun as well as the pesticide metabolite isomalathion. Aging of soman- and sarin-inhibited acetylcholinesterase occurs by C-O bond cleavage, whereas that of tabun- and isomalathion-inhibited acetylcholinesterase occurs by P-N and P-S bond cleavage, respectively. In this work, the crystal structures of hBChE inhibited by the ophthalmic reagents echothiophate (nonaged and aged) and diisopropylfluorophosphate (aged) were solved and refined to 2.1, 2.25, and 2.2 A resolution, respectively. No appreciable shift in the position of the catalytic triad histidine was observed between the aged and nonaged conjugates of hBChE. This absence of shift contrasts with the aged and nonaged crystal structures of Torpedo californica acetylcholinesterase inhibited by the nerve agent VX. The nonaged hBChE structure shows one water molecule interacting with Glu197 and the catalytic triad histidine (His438). Interestingly, this water molecule is ideally positioned to promote aging by two mechanisms: breaking either a C-O bond or a P-O bond. Pesticides and certain stereoisomers of nerve agents are expected to undergo aging by breaking the P-O bond.
ESTHER : Nachon_2005_Biochemistry_44_1154
PubMedSearch : Nachon_2005_Biochemistry_44_1154
PubMedID: 15667209
Gene_locus related to this paper: human-BCHE

Title : Life without acetylcholinesterase: the implications of cholinesterase inhibitor toxicity in AChE-knockout mice - Lockridge_2005_Environ.Toxicol.Pharmacol_19_463
Author(s) : Lockridge O , Duysen EG , Voelker T , Thompson CM , Schopfer LM
Ref : Environ Toxicol Pharmacol , 19 :463 , 2005
Abstract : The acetylcholinesterase (AChE)-knockout mouse is a new tool for identifying physiologically relevant targets of organophosphorus toxicants (OP). If AChE were the only important target for OP toxicity, then mice with zero AChE would have been expected to be resistant to OP. The opposite was found. AChE-/- mice were more sensitive to the lethality of DFP, chlorpyrifos oxon, iso-OMPA, and the nerve agent VX. A lethal dose of OP caused the same cholinergic signs of toxicity in mice with zero AChE as in mice with normal amounts of AChE. This implied that the mechanism of toxicity of a lethal dose of OP in AChE-/- mice was the same as in mice that had AChE, namely accumulation of excess acetylcholine followed by overstimulation of receptors. OP lethality in AChE-/- mice could be due to inhibition of BChE, or to inhibition of a set of proteins. A search for additional targets used biotinylated-OP as a marker. In vitro experiments found that biotinylated-OP appeared to label as many as 55 proteins in the 100,000xg supernatant of mouse brain. Chlorpyrifos oxon bound a set of proteins (bands 12, 41, 45) that did not completely overlap with the set of proteins bound by diazoxon (bands 9, 12, 41, 47) or dichlorvos (bands 12, 23, 24, 32, 44, 45, 51) or malaoxon (band 9). These results support the idea that a variety of proteins could be interacting with a given OP to give the neurotoxic symptoms characteristic of a particular OP.
ESTHER : Lockridge_2005_Environ.Toxicol.Pharmacol_19_463
PubMedSearch : Lockridge_2005_Environ.Toxicol.Pharmacol_19_463
PubMedID: 21783513

Title : Delivery of human acetylcholinesterase by adeno-associated virus to the acetylcholinesterase knockout mouse - Hrabovska_2005_Chem.Biol.Interact_157-158_71
Author(s) : Hrabovska A , Duysen EG , Sanders JD , Murrin LC , Lockridge O
Ref : Chemico-Biological Interactions , 157-158 :71 , 2005
Abstract : The purpose of this work was to develop a gene delivery system that expressed acetylcholinesterase (AChE) for prolonged periods. An adeno-associated virus (AAV) expressing human AChE was constructed by co-transfecting three plasmids into HEK 293T cells. The purified vector expressed 0.17 microg AChE per 1 million viral particles in culture medium in 23 h, or 0.8 U/ml. The AAV/hAChE was injected into muscle of adult AChE knockout mice and into the brains of 3-6 week old AChE knockout mice. Intramuscular injection yielded plasma AChE levels approaching 50% of the AChE activity of wild-type mouse plasma. The highest AChE activity was found on day 3 post-injection. AChE activity declined thereafter to a constant 7% of normal. The decreased level was accompanied by the appearance of anti-human AChE antibodies, suggesting partial clearance of AChE from plasma by antibodies. Intrastriatal injection resulted in AChE expression in the striatum. No antibodies were detected in animals treated intrastriatally. Motor coordination was improved and the lifespan of intrastriatally-treated AChE knockout mice was prolonged. Human AChE was expressed in mouse brain for up to 7 months after intrastriatal injection of an AAV/hAChE construct. Gene-therapy to supply AChE to the striatum improved motor coordination and prolonged the life of mice genetically deficient in AChE, probably by reducing their susceptibility to spontaneous seizures. This supports the hypothesis that their seizures are induced by excess acetylcholine.
ESTHER : Hrabovska_2005_Chem.Biol.Interact_157-158_71
PubMedSearch : Hrabovska_2005_Chem.Biol.Interact_157-158_71
PubMedID: 16243306

Title : Characteristic mass spectral fragments of the organophosphorus agent FP-biotin and FP-biotinylated peptides from trypsin and bovine albumin (Tyr410) - Schopfer_2005_Anal.Biochem_345_122
Author(s) : Schopfer LM , Champion MM , Tamblyn N , Thompson CM , Lockridge O
Ref : Analytical Biochemistry , 345 :122 , 2005
Abstract : A mass spectrometry-based method was developed for selective detection of FP-biotinylated peptides in complex mixtures. Mixtures of peptides, at the low-picomole level, were analyzed by liquid chromatography and positive ion, nanospray, triple quadrupole, linear ion trap mass spectrometry. Peptides were fragmented by collision-activated dissociation in the mass spectrometer. The free FP-biotin and peptides containing FP-biotinylated serine or FP-biotinylated tyrosine yielded characteristic fragment ions at 227, 312, and 329 m/z. FP-biotinylated serine yielded an additional characteristic fragment ion at 591 m/z. Chromatographic peaks containing FP-biotinylated peptides were indicated by these diagnostic ions. Data illustrating the selectivity of the approach are presented for tryptic digests of FP-biotinylated trypsin and FP-biotinylated serum albumin. A 16-residue peptide from bovine trypsin was biotinylated on the active site serine. A 3-residue peptide from bovine albumin, YTR, was biotinylated on Tyr410. This latter result confirms that the organophosphorus binding site of albumin is a tyrosine. This method can be used to search for new biomarkers of organophosphorus agent exposure.
ESTHER : Schopfer_2005_Anal.Biochem_345_122
PubMedSearch : Schopfer_2005_Anal.Biochem_345_122
PubMedID: 16125664

Title : Reaction kinetics of biotinylated organophosphorus toxicant, FP-biotin, with human acetylcholinesterase and human butyrylcholinesterase - Schopfer_2005_Chem.Res.Toxicol_18_747
Author(s) : Schopfer LM , Voelker T , Bartels CF , Thompson CM , Lockridge O
Ref : Chemical Research in Toxicology , 18 :747 , 2005
Abstract : A biotinylated organophosphate could be useful for identifying proteins that react with organophosphorus toxicants (OP). FP-biotin, 10-(fluoroethoxyphosphinyl)-N-(biotinamidopentyl)decanamide, was synthesized and found to be stable in methanol and chloroform but less stable in water. Because acetylcholinesterase (AChE, EC and butyrylcholinesterase (BChE, EC are known to be sensitive targets of OP, their reactivity with FP-biotin was tested. The rate constant for reaction with human AChE was 1.8 x 10(7) M(-1) min(-1), and for human BChE, it was 1.6 x 10(8) M(-1) min(-1). A phosphorus stereoisomer, constituting about 50% of the FP-biotin preparation, appeared to be the reactive species. The binding affinity was estimated to be >85 nM for AChE and >5.8 nM for BChE. It was concluded that FP-biotin is a potent OP, well-suited for searching for new biomarkers of OP exposure.
ESTHER : Schopfer_2005_Chem.Res.Toxicol_18_747
PubMedSearch : Schopfer_2005_Chem.Res.Toxicol_18_747
PubMedID: 15833035

Title : Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry - Peeples_2005_Toxicol.Sci_83_303
Author(s) : Peeples ES , Schopfer LM , Duysen EG , Spaulding R , Voelker T , Thompson CM , Lockridge O
Ref : Toxicol Sci , 83 :303 , 2005
Abstract : The classical laboratory tests for exposure to organophosphorus toxicants (OP) are inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activity in blood. In a search for new biomarkers of OP exposure, we treated mice with a biotinylated organophosphorus agent, FP-biotin. The biotinylated proteins in muscle were purified by binding to avidin-Sepharose, separated by gel electrophoresis, digested with trypsin, and identified from their fragmentation patterns on a quadrupole time-of-flight mass spectrometer. Albumin and ES1 carboxylesterase (EC were found to be major targets of FP-biotin. These FP-biotinylated proteins were also identified in mouse plasma by comparing band patterns on nondenaturing gels stained for albumin and carboxylesterase activity, with band patterns on blots hybridized with Streptavidin Alexa-680. Two additional FP-biotin targets, AChE (EC and BChE (EC, were identified in mouse plasma by finding that enzyme activity was inhibited 50-80%. Mouse plasma contained eight additional FP-biotinylated bands whose identity has not yet been determined. In vitro experiments with human plasma showed that chlorpyrifos oxon, echothiophate, malaoxon, paraoxon, methyl paraoxon, diazoxon, diisopropylfluorophosphate, and dichlorvos competed with FP-biotin for binding to human albumin. Though experiments with purified albumin have previously shown that albumin covalently binds OP, this is the first report of OP binding to albumin in a living animal. Carboxylesterase is not a biomarker in man because humans have no carboxylesterase in blood. It is concluded that OP bound to albumin could serve as a new biomarker of OP exposure in man.
ESTHER : Peeples_2005_Toxicol.Sci_83_303
PubMedSearch : Peeples_2005_Toxicol.Sci_83_303
PubMedID: 15525694

Title : Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma - Li_2005_Biochem.Pharmacol_70_1673
Author(s) : Li B , Sedlacek M , Manoharan I , Boopathy R , Duysen EG , Masson P , Lockridge O
Ref : Biochemical Pharmacology , 70 :1673 , 2005
Abstract : The goal of this work was to identify the esterases in human plasma and to clarify common misconceptions. The method for identifying esterases was nondenaturing gradient gel electrophoresis stained for esterase activity. We report that human plasma contains four esterases: butyrylcholinesterase (EC, paraoxonase (EC, acetylcholinesterase (EC, and albumin. Butyrylcholinesterase (BChE), paraoxonase (PON1), and albumin are in high enough concentrations to contribute significantly to ester hydrolysis. However, only trace amounts of acetylcholinesterase (AChE) are present. Monomeric AChE is seen in wild-type as well as in silent BChE plasma. Albumin has esterase activity with alpha- and beta-naphthylacetate as well as with p-nitrophenyl acetate. Misconception #1 is that human plasma contains carboxylesterase. We demonstrate that human plasma contains no carboxylesterase (EC, in contrast to mouse, rat, rabbit, horse, cat, and tiger that have high amounts of plasma carboxylesterase. Misconception #2 is that lab animals have BChE but no AChE in their plasma. We demonstrate that mice, unlike humans, have substantial amounts of soluble AChE as well as BChE in their plasma. Plasma from AChE and BChE knockout mice allowed identification of AChE and BChE bands without the use of inhibitors. Human BChE is irreversibly inhibited by diisopropylfluorophosphate, echothiophate, and paraoxon, but mouse BChE spontaneously reactivates. Since human plasma contains no carboxylesterase, only BChE, PON1, and albumin esterases need to be considered when evaluating hydrolysis of an ester drug in human plasma.
ESTHER : Li_2005_Biochem.Pharmacol_70_1673
PubMedSearch : Li_2005_Biochem.Pharmacol_70_1673
PubMedID: 16213467

Title : Structural data on the aging of diethylphosphoryl-butyrylcholinesterase -
Author(s) : Nachon F , Asojo OA , Borgstahl GE , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 157-158 :408 , 2005
PubMedID: 16429549

Title : Identification of organophosphate-reactive proteins by tandem mass spectrometry -
Author(s) : Li H , Schopfer LM , Spaulding R , Thompson CM , Lockridge O
Ref : Chemico-Biological Interactions , 157-158 :383 , 2005
PubMedID: 16429512

Title : Large scale purification of butyrylcholinesterase from human plasma suitable for injection into monkeys\; A potential new therapeutic for protection against cocaine end nerve agent toxicity - Lockridge_2005_J.Med.Chem.Biol.Radiol.Def_3_nihms5095
Author(s) : Lockridge O , Schopfer LM , Winger G , Woods JH
Ref : Journal of Medicinal Chemistryical Biology Radiol Def , 3 :nihms5095 , 2005
Abstract : Pretreatment of animals with butyrylcholinesterase (EC BChE) provides complete protection from the acute effects of organophosphorus nerve agents. Butyrylcholinesterase has also been shown to protect from cocaine toxicity. Large amounts of highly purified butyrylcholinesterase are needed to test the effectiveness of this new therapeutic agent in monkeys. Only a minimum amount of endotoxin can be present in a therapeutic intended for injection into monkeys. Our goal was to develop a large scale purification method for human BChE from human plasma with precautions to minimize endotoxin content. A protocol was developed that processed up to 100 L of human plasma at a time. Dialysis in pH 4.0 buffer, ion exchange chromatography at pH 4, affinity chromatography on procainamide-Sepharose, and HPLC ion exchange at pH 7.4 yielded highly purified human BChE containing a low endotoxin level of about 800 EU/ml. The purified BChE produced by this method had a mean residence time of 56 h in mice and 93 h in monkeys, and caused no toxic effects. The absence of a toxic effect in monkeys demonstrates that the endotoxin level of 800 EU/ml was well tolerated by monkeys.
ESTHER : Lockridge_2005_J.Med.Chem.Biol.Radiol.Def_3_nihms5095
PubMedSearch : Lockridge_2005_J.Med.Chem.Biol.Radiol.Def_3_nihms5095
PubMedID: 16788731

Title : Intraperitoneal administration of 340 kDa human plasma butyrylcholinesterase increases the level of the enzyme in the cerebrospinal fluid of rats - Saez-Valero_2005_Neurosci.Lett_383_93
Author(s) : Saez-Valero J , de Gracia JA , Lockridge O
Ref : Neuroscience Letters , 383 :93 , 2005
Abstract : Human butyrylcholinesterase (BuChE) is being developed as a new therapeutic for protection against the toxicity of organophosphorus agents and cocaine. The purified BuChE consists predominantly of 340 kDa tetramers and contains less than 5% monomers and dimers. Our goal was to determine whether BuChE crosses the blood-cerebrospinal fluid (CSF) barrier. Rats were injected intraperitoneally with 1mg of purified human BuChE. Plasma BuChE activity increased nearly 400-fold, while BuChE activity in the CSF increased three-fold. Sucrose density centrifugation showed that the human BuChE molecule in the rat CSF was a tetramer. Immunoprecipitation confirmed the identity of the CSF BuChE as human BuChE. The lower amount of human BuChE in the CSF (0.04%) than of smaller proteins (0.1-1%), with respect to their levels in plasma, supports the idea that passage through the blood-CSF barrier depends on molecular size. BuChE in the CSF could serve to protect the brain from the neurotoxicity of organophosphorus pesticides and cocaine.
ESTHER : Saez-Valero_2005_Neurosci.Lett_383_93
PubMedSearch : Saez-Valero_2005_Neurosci.Lett_383_93
PubMedID: 15936518

Title : Hysteresis of butyrylcholinesterase in the approach to steady-state kinetics - Masson_2005_Chem.Biol.Interact_157-158_143
Author(s) : Masson P , Schopfer LM , Froment MT , Debouzy JC , Nachon F , Gillon E , Lockridge O , Hrabovska A , Goldstein BN
Ref : Chemico-Biological Interactions , 157-158 :143 , 2005
Abstract : Butyrylcholinesterase (BChE) displays hysteretic behavior with certain neutral and charged substrates in the approach to steady state. Previous studies led us to interpret this phenomenon in terms of slow transitions between two enzyme conformers E and E'. This kinetic peculiarity is observed in human, horse and rat BChE. Oscillations that superimpose on the hysteretic lag are observed when benzoylcholine and N-alkyl derivatives of benzoylcholine are used as substrate. Hysteresis of BChE can be modulated by medium parameters (pH, salts, temperature, and pressure). Though mutant enzymes show different hysteretic behavior, so far attempts to provide a molecular mechanism of BChE hysteresis from mutagenesis studies have been unproductive. However, the substrate dependence of the hysteretic induction times, using wild-type BChE and several mutants, allowed us to build a general, mechanistic model for the hysteresis. In this model, substrate can bind to E, E', or both conformers, and ES and/or E'S can be catalytically active. The exact pathway followed depends on both the nature of the substrate and the structure of the BChE mutant under study. We propose that oscillations develop when substrate exists in different, slowly interconvertible, conformational and/or aggregation forms, of which only the minor form is capable of reacting with BChE. In support of this proposal, NMR studies have provided direct evidence for slow equilibria between monomeric and micellar forms of long-chain, alkyl derivatives of benzoyl-(N-substituted) choline. There is no direct evidence that hysteresis plays a role in BChE function(s). However, the "new view" of protein dynamics proposes that proteins are normally in equilibrium between pre-existing, functional and non-functional conformers; and that binding a ligand to the functional form shifts that equilibrium towards the functional conformation. Therefore, a physiological or toxicological relevance for the hysteresis in BChE cannot be ruled out.
ESTHER : Masson_2005_Chem.Biol.Interact_157-158_143
PubMedSearch : Masson_2005_Chem.Biol.Interact_157-158_143
PubMedID: 16256969

Title : Helicobacter hepaticus infection in acetylcholinesterase knockout mice results in severe intestinal distension -
Author(s) : Duysen EG , Kolar CH , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :301 , 2004

Title : Acetylcholinesterase wild-type and knockout mice show different locomotor activity after scopolamine injection -
Author(s) : Hrabovska A , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :315 , 2004

Title : Resistance to organophosphorus agent toxicity in transgenic mice expressing the G117H mutant of human butyrylcholinesterase - Wang_2004_Toxicol.Appl.Pharmacol_196_356
Author(s) : Wang Y , Boeck AT , Duysen EG , Van Keuren M , Saunders TL , Lockridge O
Ref : Toxicol Appl Pharmacol , 196 :356 , 2004
Abstract : Organophosphorus toxicants (OP) include chemical nerve agents and pesticides. The goal of this work was to find out whether an animal could be made resistant to OP toxicity by genetic engineering. The human butyrylcholinesterase (BChE) mutant G117H was chosen for study because it has the unusual ability to hydrolyze OP as well as acetylcholine, and it is resistant to inhibition by OP. Human G117H BChE, under the control of the ROSA26 promoter, was expressed in all tissues of transgenic mice. A stable transgenic mouse line expressed 0.5 microg/ml of human G117H BChE in plasma as well as 2 microg/ml of wild-type mouse BChE. Intestine, kidneys, stomach, lungs, heart, spleen, liver, brain, and muscle expressed 0.6-0.15 microg/g of G117H BChE. Transgenic mice were normal in behavior and fertility. The LD50 dose of echothiophate for wild-type mice was 0.1 mg/kg sc. This dose caused severe cholinergic signs of toxicity and lethality in wild-type mice, but caused no deaths and only mild toxicity in transgenic animals. The mechanism of protection was investigated by measuring acetylcholinesterase (AChE) and BChE activity. It was found that AChE and endogenous BChE were inhibited to the same extent in echothiophate-treated wild type and transgenic mice. This led to the hypothesis that protection against echothiophate toxicity was not explained by hydrolysis of echothiophate. In conclusion, the transgenic G117H BChE mouse demonstrates the factors required to achieve protection from OP toxicity in a vertebrate animal.
ESTHER : Wang_2004_Toxicol.Appl.Pharmacol_196_356
PubMedSearch : Wang_2004_Toxicol.Appl.Pharmacol_196_356
PubMedID: 15094306

Title : Reduced acetylcholine receptor density, morphological remodeling, and butyrylcholinesterase activity can sustain muscle function in acetylcholinesterase knockout mice - Adler_2004_Muscle.Nerve_30_317
Author(s) : Adler M , Manley HA , Purcell AL , Deshpande SS , Hamilton TA , Kan RK , Oyler G , Lockridge O , Duysen EG , Sheridan RE
Ref : Muscle & Nerve , 30 :317 , 2004
Abstract : Nerve-evoked contractions were studied in vitro in phrenic nerve-hemidiaphragm preparations from strain 129X1 acetylcholinesterase knockout (AChE-/-) mice and their wild-type littermates (AChE+/+). The AChE-/- mice fail to express AChE but have normal levels of butyrylcholinesterase (BChE) and can survive into adulthood. Twitch tensions elicited in diaphragms of AChE-/- mice by single supramaximal stimuli had larger amplitudes and slower rise and decay times than did those in wild-type animals. In AChE-/- preparations, repetitive stimulation at frequencies of 20 and 50 Hz and at 200 and 400 Hz produced decremental muscle tensions; however, stimulation at 70 and 100 Hz resulted in little or no loss of tension during trains. Muscles from AChE+/+ mice maintained tension at all frequencies examined but exhibited tetanic fade after exposure to the selective AChE inhibitor 1,5-bis(4-allyldimethyl-ammoniumphenyl)pentane-3-one (BW 284C51). The ability of diaphragm muscles from AChE-/- mice to maintain tension at 70 and 100 Hz suggests a partial compensation for impairment of acetylcholine (ACh) hydrolysis. Three mechanisms--including a reliance on BChE activity for termination of ACh action, downregulation of nicotinic acetylcholine receptors (nAChRs), and morphological remodeling of the endplate region--were identified. Studies of neuromuscular transmission in this model system provide an excellent opportunity to evaluate the role of AChE without complications arising from use of inhibitors.
ESTHER : Adler_2004_Muscle.Nerve_30_317
PubMedSearch : Adler_2004_Muscle.Nerve_30_317
PubMedID: 15318343

Title : Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants - Wang_2004_Anal.Biochem_329_131
Author(s) : Wang Y , Schopfer LM , Duysen EG , Nachon F , Masson P , Lockridge O
Ref : Analytical Biochemistry , 329 :131 , 2004
Abstract : Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC; BChE) and acetylcholinesterase (EC; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.
ESTHER : Wang_2004_Anal.Biochem_329_131
PubMedSearch : Wang_2004_Anal.Biochem_329_131
PubMedID: 15136175

Title : Poster (49) Crystallographic basis for substrate\/product exchange in cholinesterases. -
Author(s) : Nicolet Y , Lockridge O , Masson P , Fontecilla-Camps JC , Nachon F
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :347 , 2004

Title : Rate-determining step of butyrylcholinesterase-catalyzed hydrolysis of benzoylcholine and benzoylthiocholine. Volumetric study of wild-type and D70G mutant behavior - Masson_2004_Eur.J.Biochem_271_1980
Author(s) : Masson P , Bec N , Froment MT , Nachon F , Balny C , Lockridge O , Schopfer LM
Ref : European Journal of Biochemistry , 271 :1980 , 2004
Abstract : The rate-limiting step for hydrolysis of the positively charged oxoester benzoylcholine (BzCh) by human butyrylcholinesterase (BCHE) is deacylation (k(3)), whereas it is acylation (k(2)) for hydrolysis of the homologous thioester benzoylthiocholine (BzSCh). Steady-state hydrolysis of BzCh and BzSCh by wild-type BCHE and its peripheral anionic site mutant D70G was investigated at different hydrostatic pressures, which allowed determination of volume changes associated with substrate binding, and the activation volumes for the chemical steps. A differential nonlinear pressure-dependence of the catalytic parameters for hydrolysis of both substrates by both enzymes was shown. Nonlinearity of the plots may be explained in terms of compressibility changes or rate-limiting changes. To distinguish between these two possibilities, enzyme phosphorylation by diisopropylfluorophosphate (DFP) in the presence of substrate (BzSCh) under pressure was studied. There was no pressure dependence of volume changes for DFP binding or for phosphorylation of either wild-type or D70G. Analysis of the pressure dependence for steady-state hydrolysis of substrates, and for phosphorylation by DFP provided evidence that no enzyme compressibility changes occurred during the catalyzed reactions. Thus, the nonlinear pressure dependence of substrate hydrolysis reflects changes in the rate-limiting step with pressure. Change in rate-determining step occurred at a pressure of 100 MPa for hydrolysis of BzCh by wild-type and at 75 MPa for D70G. For hydrolysis of BzSCh the change occurred at higher pressures because k(2) << k(3) at atmospheric pressure for this substrate. Elementary volume change contributions upon initial binding, productive binding, acylation and deacylation were calculated from the pressure differentiation of kinetic constants. This analysis shed light on the molecular events taking place along the hydrolysis pathways of BzCh and BzSCh by wild-type BCHE and the D70G mutant. In addition, volume change differences between wild-type and D70G provided new evidence that residue D70 in the peripheral site controls hydration of the active site gorge and the dynamics of the water molecule network during catalysis. Finally, a steady-state kinetic study of the oxyanion hole mutant (G117H) showed that substitution of the ethereal sulfur for oxygen in the substrate alters the final adjustment of substrate in the active site and stabilization of the acylation transition state.
ESTHER : Masson_2004_Eur.J.Biochem_271_1980
PubMedSearch : Masson_2004_Eur.J.Biochem_271_1980
PubMedID: 15128307

Title : Damped oscillatory hysteretic behaviour of butyrylcholinesterase with benzoylcholine as substrate - Masson_2004_Eur.J.Biochem_271_220
Author(s) : Masson P , Goldstein BN , Debouzy JC , Froment MT , Lockridge O , Schopfer LM
Ref : European Journal of Biochemistry , 271 :220 , 2004
Abstract : Steady-state kinetics for the hydrolysis of benzoylcholine (BzCh) and benzoylthiocholine (BzSCh) by wild-type human butyrylcholinesterase (BuChE) and by the peripheral anionic site mutant D70G were compared. kcat/Km for the hydrolysis of BzSCh was 17-fold and 32-fold lower than that for hydrolysis of BzCh by wild-type and D70G, respectively. The rate-limiting step for hydrolysis of BzCh was deacylation, whereas acylation was rate-limiting for hydrolysis of BzSCh. Wild-type enzyme and the D70G mutant were found to reach steady-state velocity slowly with BzCh as the substrate. At pH 6, the approach to steady-state for both enzymes consisted of a mono-exponential acceleration upon which a set of damped oscillations was superimposed. From pH 7 to 8.5, the approach to steady-state consisted of a simple exponential acceleration. The damped oscillations were analyzed by both a numerical approximation and simulation based on a theoretical model. BuChE-catalyzed hydrolysis of the thiocholine analogue of BzCh showed neither lags nor oscillations, under the same conditions. The frequency and amplitude of the damped oscillations decreased as the BzCh concentration increased. The apparent induction time for the exponential portion of the lag was calculated from the envelope of the damped oscillations or from the smooth lag. Wild-type BuChE showed a hyperbolic increase in induction time as the BzCh concentration increased (tau max = 210 s at pH 6.0). However, the induction time for D70G was constant over the whole range of BzCh concentrations (tau max = 60 s at pH 6.0). Thus, the induction time does not conform to a simple hysteretic model in which there is a slow conformational transition of the enzyme from an inactive form E to an active form E'. No pH-dependence of the induction time was found between pH 6.0 and 8.5 in sodium phosphate buffers of various concentrations (from 1 mm to 1 m). However, increasing the pH tended to abolish the oscillations (increase the damping factor). This effect was more pronounced for D70G than for wild-type. Although the lyotropic properties of phosphate change from chaotropic at pH 6.0 to kosmotropic at pH > 8.0, no effect of phosphate concentration on the oscillations was noticed at the different pH values, suggesting that the oscillations are not related to a pH-dependent Hofmeister effect of phosphate ions. Simulation and theoretical analysis of the oscillatory behaviour of the approach to the steady-state for BuChE led us to propose a model for the hysteresis of BuChE with BzCh. In this model, the substrate-free enzyme is present as an equilibrium mixture of two forms, E and E'. Substrate binds to E and E', but only Epsilon'S makes products. It is proposed that oscillations originate from a time-dependent change in the local concentration, solvation and/or conformation of substrate in the bulk solution. 1H-NMR measurements provided evidence for a slow equilibrium between two BzCh conformers. Binding of the conformationally preferred substrate conformer leads to products.
ESTHER : Masson_2004_Eur.J.Biochem_271_220
PubMedSearch : Masson_2004_Eur.J.Biochem_271_220
PubMedID: 14686935

Title : Poster (55) Life without acetylcholinesterase -
Author(s) : Lockridge O , Duysen EG , Li B , Hrabovska A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :350 , 2004

Title : Poster (59) Complete postnatal degeneration of the photoreceptor layer in an AChE knockout mouse -
Author(s) : Bytyqi AH , Duysen EG , Lockridge O , Layer PG
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :352 , 2004

Title : Poster (60) Early distortion of network formation in the inner retina of an AChE knockout mouse -
Author(s) : Bytyqi AH , Paraoanu LE , Duysen EG , Lockridge O , Layer PG
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :353 , 2004

Title : Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptors - Bytyqi_2004_Eur.J.Neurosci_20_2953
Author(s) : Bytyqi AH , Lockridge O , Duysen E , Wang Y , Wolfrum U , Layer PG
Ref : European Journal of Neuroscience , 20 :2953 , 2004
Abstract : Blinding diseases can be assigned predominantly to genetic defects of the photoreceptor/pigmented epithelium complex. As an alternative, we show here for an acetylcholinesterase (AChE) knockout mouse that photoreceptor degeneration follows an impaired development of the inner retina. During the first 15 postnatal days of the AChE-/- retina, three major calretinin sublaminae of the inner plexiform layer (IPL) are disturbed. Thereby, processes of amacrine and ganglion cells diffusely criss-cross throughout the IPL. In contrast, parvalbumin cells present a nonlaminar IPL pattern in the wild-type, but in the AChE-/- mouse their processes become structured within two 'novel' sublaminae. During this early period, photoreceptors become arranged regularly and at a normal rate in the AChE-/- retina. However, during the following 75 days, first their outer segments, and then the entire photoreceptor layer completely degenerate by apoptosis. Eventually, cells of the inner retina also undergo apoptosis. As butyrylcholinesterase (BChE) is present at a normal level in the AChE-/- mouse, the observed effects must be solely due to the missing AChE. These are the first in vivo findings to show a decisive role for AChE in the formation of the inner retinal network, which, when absent, ultimately results in photoreceptor degeneration.
ESTHER : Bytyqi_2004_Eur.J.Neurosci_20_2953
PubMedSearch : Bytyqi_2004_Eur.J.Neurosci_20_2953
PubMedID: 15579149

Title : Poster (78) H. hepaticus infection in acetylcholinesterase knockout mice results in severe intestinal distension. -
Author(s) : Duysen EG , Kolar CH , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :362 , 2004

Title : Phenotype of the adult acetylcholinesterase-deficient mouse. -
Author(s) : Duysen EG , Stribley JA , Fry DL , Hinrichs SH , Lockridge O
Ref : Cholinergic Mechanisms, CRC Press :559 , 2004

Title : Dramatic depletion of cell surface muscarinic receptor due to limited delivery from intracytoplasmic stores in neurons of acetylcholinesterase-deficient mice. -
Author(s) : Bernard V , Brana C , Liste I , Lockridge O , Bloch B
Ref : Cholinergic Mechanisms, CRC Press :477 , 2004

Title : Poster (82) Acetylcholinesterase wild-type and knock-out mice show different locomotor activity after scopolamine injection -
Author(s) : Hrabovska A , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :364 , 2004

Title : X-Ray structures of native and soman-aged human butyrylcholinesterase. -
Author(s) : Nachon F , Nicolet Y , Masson P , Lockridge O , Fontecilla-Camps JC
Ref : Cholinergic Mechanisms, CRC Press :165 , 2004

Title : Poster (90) Acetylcholinesterase knockout mice are resistant oxotremorine-induced hypothermia and pilocarpine-induced seizures -
Author(s) : Li B , Duysen EG , Lockridge O
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :368 , 2004

Title : Poster (93) How do the crystal structures of human butyrylcholinesterase compare to torpedo californica acetylcholinesterases structures? -
Author(s) : Nachon F , Nicolet Y , Masson P , Lockridge O , Fontecilla-Camps JC
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :369 , 2004

Title : Acetylcholinesterase knockout mice have increased sensitivity to scopolamine and atropine. -
Author(s) : Hrabovska A , Duysen EG , Lockridge O
Ref : Cholinergic Mechanisms, CRC Press :593 , 2004

Title : Mutants of human butyrylcholinesterase with organophosphate hydrolase activity\; evidence that His117 is a general base catalyst for hydrolysis of echothiophate - Schopfer_2004_J.Med.Chem.Biol.Radiol.Def_2_1
Author(s) : Schopfer LM , Boeck AT , Broomfield CA , Lockridge O
Ref : Journal of Medicinal Chemistryical Biology Radiol Def , 2 :1 , 2004
Abstract : Human butyrylcholinesterase (BChE, EC is an efficient scavenger of nerve agents and organophosphorus (OP) pesticides; one molecule of BChE inactivates one molecule of OP in a suicide reaction that irreversibly inhibits BChE. By contrast the BChE mutant, G117H, inactivates many molecules of OP. The OP makes a covalent bond with the active site serine and then the serine is dephosphorylated by the action of His117. In an effort to understand the mechanism by which is 117 achieves dephosphorylation, 62 new mutants of human BChE were tested for OP hydrolase activity, using a new screening assay. It was found that not only G117H, but also G117D, G117E, and L286H mutants were OP hydrolases. These results support the hypothesis that a hydrogen-bond acceptor acts as a general base t activate a water molecule which in turn dephosphorylates the active site serine The screening assay provides a convenient means for identifying cholinesterase mutants with OP hydrolase activity.
ESTHER : Schopfer_2004_J.Med.Chem.Biol.Radiol.Def_2_1
PubMedSearch : Schopfer_2004_J.Med.Chem.Biol.Radiol.Def_2_1

Title : Hysteretic behavior of butyrylcholinesterase: Kinetic curiosity or catalytically and physiologicaly significant? -
Author(s) : Masson P , Froment MT , Nachon F , Lockridge O , Schopfer LM
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :191 , 2004

Title : Poster (20) Hysteretic behavior of butyrylcholinesterase : kinetic curiosity or catalytically and physiologically significant? -
Author(s) : Masson P , Froment MT , Nachon F , Lockridge O , Schopfer LM
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :331 , 2004

Title : Muscarinic acetylcholine receptors are down-regulated in acetylcholinesterase knockout mice. -
Author(s) : Li B , Duysen EG , Lockridge O
Ref : Cholinergic Mechanisms, CRC Press :619 , 2004

Title : Regulation of muscarinic acetylcholine receptor function in acetylcholinesterase knockout mice - Li_2003_Pharmacol.Biochem.Behav_74_977
Author(s) : Li B , Duysen EG , Volpicelli-Daley LA , Levey AI , Lockridge O
Ref : Pharmacol Biochem Behav , 74 :977 , 2003
Abstract : 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..
ESTHER : Li_2003_Pharmacol.Biochem.Behav_74_977
PubMedSearch : Li_2003_Pharmacol.Biochem.Behav_74_977
PubMedID: 12667913

Title : Altered hippocampal muscarinic receptors in acetylcholinesterase-deficient mice - Volpicelli-Daley_2003_Ann.Neurol_53_788
Author(s) : Volpicelli-Daley LA , Duysen EG , Lockridge O , Levey AI
Ref : Annals of Neurology , 53 :788 , 2003
Abstract : A primary therapeutic strategy for Alzheimer's disease includes acetylcholinesterase (AChE) inhibitors with the goal of enhancing cholinergic transmission. Stimulation of muscarinic acetylcholine receptors (mAChRs) by elevated levels of ACh plays a role in the effects of AChE inhibitors on cognition and behavior. However, AChE inhibitors only demonstrate modest symptomatic improvements. Chronic treatment with these drugs may cause mAChR downregulation and consequently limit the treatment efficacy. AChE knockout (-/-) mice were utilized in this study as a model for investigating the effects of selective, complete, and chronic diminished AChE activity on mAChR expression and function. In AChE -/- mice, the M(1), M(2), and M(4) mAChRs showed strikingly 50 to 80% decreased expression in brain regions associated with memory. In addition, mAChRs showed decreased presynaptic, cell surface, and dendritic distributions and increased localization to intracellular puncta. Furthermore, mAChR agonist-induced activation of extracellular signal-regulated kinase, a signaling pathway associated with synaptic plasticity and amyloidogenesis, is diminished in the hippocampus and cortex of AChE -/- mice. Therefore, chronic diminished ACh metabolism produces profound effects on mAChR expression and function. The alterations of mAChRs in AChE -/- mice suggest that mAChR downregulation may contribute to the limited efficacy of AChE inhibitors in Alzheimer's disease treatment.
ESTHER : Volpicelli-Daley_2003_Ann.Neurol_53_788
PubMedSearch : Volpicelli-Daley_2003_Ann.Neurol_53_788
PubMedID: 12783426

Title : Altered striatal function and muscarinic cholinergic receptors in acetylcholinesterase knockout mice - Volpicelli-Daley_2003_Mol.Pharmacol_64_1309
Author(s) : Volpicelli-Daley LA , Hrabovska A , Duysen EG , Ferguson SM , Blakely RD , Lockridge O , Levey AI
Ref : Molecular Pharmacology , 64 :1309 , 2003
Abstract : Cholinesterase inhibitors are commonly used to improve cognition and treat psychosis and other behavioral symptoms in Alzheimer's disease, Parkinson's disease, and other neuropsychiatric conditions. However, mechanisms may exist that down-regulate the synaptic response to altered cholinergic transmission, thus limiting the efficacy of cholinomimetics in treating disease. Acetylcholinesterase knockout (AChE-/-) mice were used to investigate the neuronal adaptations to diminished synaptic acetylcholine (ACh) metabolism. The striatum of AChE-/- mice showed no changes in choline acetyltransferase activity or levels of the vesicular ACh transporter but showed striking 60% increases in the levels of the highaffinity choline transporter. This transporter takes choline from the synapse into the neuron for resynthesis of ACh. In addition, the striata of AChE-/- mice showed dramatic reductions in levels of the M1, M2, and M4 muscarinic ACh receptors (mAChRs), but no alterations in dopamine receptors or the beta2 subunit of nicotinic receptors. M1, M2, and M4 also showed decreased dendritic and cell surface distributions and enhanced intracellular localizations in striatal neurons of AChE-/- mice. mAChR antagonist treatment reversed the shifts in mAChR distribution, indicating that internalized receptors in AChE-/- mice can recover to basal distributions. Finally, AChE-/- mice showed increased sensitivity to mAChR antagonist-induced increases in locomotor activity, demonstrating functional mAChR down-regulation. mAChR downregulation in AChE-/- mice has important implications for the long-term use of cholinesterase inhibitors and other cholinomimetics in treating disorders characterized by perturbed cholinergic function.
ESTHER : Volpicelli-Daley_2003_Mol.Pharmacol_64_1309
PubMedSearch : Volpicelli-Daley_2003_Mol.Pharmacol_64_1309
PubMedID: 14645660

Title : Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases - Saxena_2003_Eur.J.Biochem_270_4447
Author(s) : Saxena A , Fedorko JM , Vinayaka CR , Medhekar R , Radic Z , Taylor P , Lockridge O , Doctor BP
Ref : European Journal of Biochemistry , 270 :4447 , 2003
Abstract : 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.
ESTHER : Saxena_2003_Eur.J.Biochem_270_4447
PubMedSearch : Saxena_2003_Eur.J.Biochem_270_4447
PubMedID: 14622273

Title : Crystal structure of human butyrylcholinesterase and of its complexes with substrate and products - Nicolet_2003_J.Biol.Chem_278_41141
Author(s) : Nicolet Y , Lockridge O , Masson P , Fontecilla-Camps JC , Nachon F
Ref : Journal of Biological Chemistry , 278 :41141 , 2003
Abstract : Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser(198) has been modeled as bound butyrate.
ESTHER : Nicolet_2003_J.Biol.Chem_278_41141
PubMedSearch : Nicolet_2003_J.Biol.Chem_278_41141
PubMedID: 12869558
Gene_locus related to this paper: human-BCHE

Title : Application of directed evolution technology to optimize the cocaine hydrolase activity of human butyrylcholinesterase -
Author(s) : Pancook JD , Pecht G , Ader M , Mosko M , Lockridge O , Watkins JD
Ref : FASEB Journal , 17 :A565 , 2003

Title : Dramatic depletion of cell surface m2 muscarinic receptor due to limited delivery from intracytoplasmic stores in neurons of acetylcholinesterase-deficient mice - Bernard_2003_Mol.Cell.Neurosci_23_121
Author(s) : Bernard V , Brana C , Liste I , Lockridge O , Bloch B
Ref : Molecular & Cellular Neurosciences , 23 :121 , 2003
Abstract : We have studied the consequences of the constitutive acetylcholinesterase (AChE) deficiency in knockout mice for the AChE gene on the subcellular localization of the m2 receptor (m2R) and the regulation of its intraneuronal compartmentalization by the cholinergic environment, using immunohistochemistry at light and electron microscopic levels. (1) In AChE +/+ mice in vivo, m2R is mainly located at the neuronal membrane in striatum, hippocampus, and cortex. In AChE -/- mice, m2R is almost absent at the membrane but is accumulated in the endoplasmic reticulum and Golgi complex. (2) In vivo and in vitro (organotypic culture) dynamic studies demonstrate that the balance between membrane and intracytoplasmic m2R can be regulated by the cholinergic influence: In AChE -/- mice, m2R is translocated from the cytoplasm to the cell surface after (1) blockade of muscarinic receptors by atropine, (2) supplementation of AChE -/- neurons with AChE in vitro, and (3) disruption of the cortical and hippocampal cholinergic afferents in vitro. Our results suggest that the neurochemical environment may contribute to the control of the abundance and availability of cell surface receptors, and consequently to the control of neuronal sensitivity to neurotransmitters or drugs, by regulating their delivery from the endoplasmic reticulum and Golgi complex.
ESTHER : Bernard_2003_Mol.Cell.Neurosci_23_121
PubMedSearch : Bernard_2003_Mol.Cell.Neurosci_23_121
PubMedID: 12799142

Title : High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine - Masson_2003_Eur.J.Biochem_270_315
Author(s) : Masson P , Nachon F , Bartels CF , Froment MT , Ribes F , Matthews C , Lockridge O
Ref : European Journal of Biochemistry , 270 :315 , 2003
Abstract : Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH
ESTHER : Masson_2003_Eur.J.Biochem_270_315
PubMedSearch : Masson_2003_Eur.J.Biochem_270_315
PubMedID: 12605682

Title : Substrate activation in acetylcholinesterase induced by low pH or mutation in the pi-cation subsite - Masson_2002_Biochim.Biophys.Acta_1594_313
Author(s) : Masson P , Schopfer LM , Bartels CF , Froment MT , Ribes F , Nachon F , Lockridge O
Ref : Biochimica & Biophysica Acta , 1594 :313 , 2002
Abstract : Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.
ESTHER : Masson_2002_Biochim.Biophys.Acta_1594_313
PubMedSearch : Masson_2002_Biochim.Biophys.Acta_1594_313
PubMedID: 11904227

Title : Engineering of a monomeric and low-glycosylated form of human butyrylcholinesterase: expression, purification, characterization and crystallization - Nachon_2002_Eur.J.Biochem_269_630
Author(s) : Nachon F , Nicolet Y , Viguie N , Masson P , Fontecilla-Camps JC , Lockridge O
Ref : European Journal of Biochemistry , 269 :630 , 2002
Abstract : Human butyrylcholinesterase (BChE; EC is of particular interest because it hydrolyzes or scavenges a wide range of toxic compounds including cocaine, organophosphorus pesticides and nerve agents. The relative contribution of each N-linked glycan for the solubility, the stability and the secretion of the enzyme was investigated. A recombinant monomeric BChE lacking four out of nine N-glycosylation sites and the C-terminal oligomerization domain was stably expressed as a monomer in CHO cells. The purified recombinant BChE showed catalytic properties similar to those of the native enzyme. Tetragonal crystals suitable for X-ray crystallography studies were obtained; they were improved by recrystallization and found to diffract to 2.0 A resolution using synchrotron radiation. The crystals belong to the tetragonal space group I422 with unit cell dimensions a = b = 154.7 A, c = 124.9 A, giving a Vm of 2.73 A3 per Da (estimated 60% solvent) for a single molecule of recombinant BChE in the asymmetric unit. The crystal structure of butyrylcholinesterase will help elucidate unsolved issues concerning cholinesterase mechanisms in general.
ESTHER : Nachon_2002_Eur.J.Biochem_269_630
PubMedSearch : Nachon_2002_Eur.J.Biochem_269_630
PubMedID: 11856322

Title : DNA sequence of butyrylcholinesterase from the rat: expression of the protein and characterization of the properties of rat butyrylcholinesterase - Boeck_2002_Biochem.Pharmacol_63_2101
Author(s) : Boeck AT , Schopfer LM , Lockridge O
Ref : Biochemical Pharmacology , 63 :2101 , 2002
Abstract : The rat is the model animal for toxicity studies. Butyrylcholinesterase (BChE), being sensitive to inhibition by some organophosphorus and carbamate pesticides, is a biomarker of toxic exposure. The goal of this work was to characterize the purified rat BChE enzyme. The cDNA sequence showed eight amino acid differences between the active site gorge of rat and human BChE, six clustered around the acyl binding pocket and two below the active site serine. A prominent difference in rat was the substitution of arginine for leucine at position 286 in the acyl pocket. Wild-type rat BChE, the mutant R286L, wild-type human BChE, and the mutant L286R were expressed in CHO cells and purified. Arg286 was found responsible for the resistance of rat BChE to inhibition by Triton X-100. Replacement of Arg286 with leucine caused the affinity for Triton X-100 to increase 20-fold, making it as sensitive as human BChE to inhibition by Triton X-100. Wild-type rat BChE had an 8- to 9-fold higher K(m) for the positively charged substrates butyrylthiocholine, acetylthiocholine, propionylthiocholine, benzoylcholine, and cocaine compared with wild-type human BChE. Wild-type rat BChE catalyzed turnover 2- to 7-fold more rapidly than human BChE, showing the highest turnover with propionylthiocholine (201,000 min(-1)). Human BChE does not reactivate spontaneously after inhibition by echothiophate, but rat BChE reactivates with a half-life of 4.3hr. Human serum contains 5mg/L of BChE and 0.01mg/L of AChE. Male rat serum contains 0.2mg/L of BChE and approximately 0.2mg/L of AChE.
ESTHER : Boeck_2002_Biochem.Pharmacol_63_2101
PubMedSearch : Boeck_2002_Biochem.Pharmacol_63_2101
PubMedID: 12110369
Gene_locus related to this paper: ratno-BCHE

Title : Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet\; phenotype of the adult acetylcholinesterase deficient mouse - Duysen_2002_Brain.Res.Dev.Brain.Res_137_43
Author(s) : Duysen EG , Stribley JA , Fry DL , Hinrichs SH , Lockridge O
Ref : Brain Research Developmental Brain Research , 137 :43 , 2002
Abstract : 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.
ESTHER : Duysen_2002_Brain.Res.Dev.Brain.Res_137_43
PubMedSearch : Duysen_2002_Brain.Res.Dev.Brain.Res_137_43
PubMedID: 12128253

Title : Early weaning and culling eradicated Helicobacter hepaticus from an acetylcholinesterase knockout 129S6\/SvEvTac mouse colony - Duysen_2002_Comp.Med_52_461
Author(s) : Duysen EG , Fry DL , Lockridge O
Ref : Comp Med , 52 :461 , 2002
Abstract : The finding of Helicobacter hepaticus infection in our acetylcholinesterase (AChE) knockout mouse colony led to a search for a treatment. One-hundred percent of AChE +/+, 100% of AChE +/-, and 35% of AChE -/- mice tested positive. The lower infection rate in AChE -/- mice, who are routinely weaned on day 15, suggested that early weaning might be an effective eradication strategy. The AChE +/+ and +/- mice were weaned on days 13, 14, 15, or 16. Litters were placed in sterile, heated, isolator cages. Animals were fed liquid Ensure Fiber and 11% fat pelleted diet. Feces were tested for the presence of H. hepaticus by use of DNA amplification. Litters weaned on days 14, 15, or 16 had a high rate (68, 63, and 100%, respectively), whereas litters weaned on day 13 had a lower (8%) rate of infection. Uninfected animals have remained free of H. hepaticus through day 120. Pups weaned on day 13 lost body weight, beginning on day 14, but recovered by day 16. It is concluded that the non-coprophagic behavior of AChE -/- mice accounts for a low infection rate and that the combination of early weaning, routine testing, and culling provide an effective method for eradication of H. hepaticus.
ESTHER : Duysen_2002_Comp.Med_52_461
PubMedSearch : Duysen_2002_Comp.Med_52_461
PubMedID: 12405641

Title : Acetylcholinesterase knockouts establish central cholinergic pathways and can use butyrylcholinesterase to hydrolyze acetylcholine - Mesulam_2002_Neurosci_110_627
Author(s) : Mesulam MM , Guillozet A , Shaw P , Levey AI , Duysen EG , Lockridge O , Levey A
Ref : Neuroscience , 110 :627 , 2002
Abstract : Acetylcholinesterase is one of the most prominent constituents of central cholinergic pathways. It terminates the synaptic action of acetylcholine through hydrolysis and yields the choline moiety that is necessary for transmitter recycling. Despite these pivotal relationships, mice nullizygous for acetylcholinesterase established all principal anatomical components of central cholinergic pathways. No compensatory increase in the distribution of butyrylcholinesterase was detected. However, both the wild-type and nullizygous mice showed that butyrylcholinesterase enzyme activity extended to all parts of the brain receiving cholinergic innervation and that it could hydrolyze the acetylcholine surrogate acetylthiocholine. As opposed to acetylcholinesterase which was mostly of neuronal origin, butyrylcholinesterase appeared to be mostly of glial origin. These experiments lead to the unexpected conclusion that acetylcholinesterase is not necessary for the establishment of cholinergic pathways. They also show that butyrylcholinesterase can potentially substitute for acetylcholinesterase and that this enzyme is likely to play a constitutive (rather than just back-up) role in the hydrolysis of acetylcholine in the normal brain. The inhibition of butyrylcholinesterase may therefore provide a desirable feature of cholinergic therapies, including those aimed at treating Alzheimer's disease.
ESTHER : Mesulam_2002_Neurosci_110_627
PubMedSearch : Mesulam_2002_Neurosci_110_627
PubMedID: 11934471

Title : Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity - Duysen_2002_J.Pharmacol.Exp.Ther_302_751
Author(s) : Duysen EG , Bartels CF , Lockridge O
Ref : Journal of Pharmacology & Experimental Therapeutics , 302 :751 , 2002
Abstract : Human butyrylcholinesterase (BChE) hydrolyzes cocaine to inactive metabolites. A mutant of human BChE, A328W, hydrolyzed cocaine 15-fold faster compared with wild-type BChE. Although the catalytic properties of human BChE secreted by Chinese hamster ovary (CHO) cells are identical to those of native BChE, a major difference became evident when the recombinant BChE was injected into rats and mice. Recombinant BChE disappeared from the circulation within minutes, whereas native BChE stayed in the blood for a week. Nondenaturing gel electrophoresis showed that the recombinant BChE consisted mainly of monomers and dimers. In contrast, native BChE is a tetramer. The problem of the short residence time was solved by finding a method to assemble the recombinant BChE into tetramers. Coexpression in CHO cells of BChE and 45 residues from the N terminus of the COLQ protein yielded 70% tetrameric BChE. The resulting purified recombinant BChE tetramers had a half-life of 16 h in the circulation of rats and mice. The 16-h half-life was achieved without modifying the carbohydrate content of recombinant BChE. The protective effect of recombinant wild-type and A328W mutant BChE against cocaine toxicity was tested by measuring locomotor activity in mice. Pretreatment with wild-type BChE or A328W tetramers at a dose of 2.8 units/g i.p. reduced cocaine-induced locomotor activity by 50 and 80%. These results indicate that recombinant human BChE could be useful for treating cocaine toxicity in humans.
ESTHER : Duysen_2002_J.Pharmacol.Exp.Ther_302_751
PubMedSearch : Duysen_2002_J.Pharmacol.Exp.Ther_302_751
PubMedID: 12130740

Title : Specificity of ethephon as a butyrylcholinesterase inhibitor and phosphorylating agent - Haux_2002_Chem.Res.Toxicol_15_1527
Author(s) : Haux JE , Lockridge O , Casida JE
Ref : Chemical Research in Toxicology , 15 :1527 , 2002
Abstract : Butyrylcholinesterase (BChE) is inhibited by the plant growth regulator (2-chloroethyl)phosphonic acid (ethephon) as observed 25 years ago both in vitro and in vivo in rats and mice and more recently in subchronic studies at low doses with human subjects. The proposed mechanism is phosphorylation of the BChE active site at S198 by ethephon dianion. The present study tests this hypothesis directly using [(33)P]ethephon and recombinant BChE (rBChE) with single amino acid substitutions and further evaluates if BChE is the most sensitive esterase target in vitro and with mice in vivo. [(33)P]Ethephon labels purified rBChE but not enzymatically inactive diethylphosphoryl-rBChE (derivatized at S198 by preincubation with chlorpyrifos oxon) or several other esterases and proteins. Amino acid substitutions that greatly reduce rBChE sensitivity to ethephon are G117H and G117K in the oxyanion hole (which may interfere with hydrogen bonding between glycine-N-H and ethephon dianion) and A328F, A328W, and A328Y (perhaps by impeding access to the active site gorge). Other substitutions that do not affect sensitivity are D70N, D70K, D70G, and E197Q which are not directly involved in the catalytic triad. The effect of pH and buffer composition on inhibition supports the hypothesis that ethephon dianion is the actual phosphorylating agent without activation by divalent cations. Human plasma BChE in vitro and mouse plasma BChE in vitro and in vivo are more sensitive to ethephon than any other esterases detected by butyrylthiocholine or 1-naphthyl acetate hydrolysis in native-PAGE. All mouse liver esterases observed are less sensitive than plasma BChE to ethephon in vitro and in vivo. More than a dozen other esterases examined are 10-100-fold less sensitive than BChE to ethephon. Thus, BChE inhibition continues to be the most sensitive marker of ethephon exposure.
ESTHER : Haux_2002_Chem.Res.Toxicol_15_1527
PubMedSearch : Haux_2002_Chem.Res.Toxicol_15_1527
PubMedID: 12482234

Title : Naturally occurring mutation, Asp70his, in human butyrylcholinesterase - Boeck_2002_Ann.Clin.Biochem_39_154
Author(s) : Boeck AT , Fry DL , Sastre A , Lockridge O
Ref : Annals of Clinical Biochemistry , 39 :154 , 2002
Abstract : BACKGROUND: People with genetic variants of butyrylcholinesterase can have hours of prolonged apnoea after a normal dose of succinylcholine or mivacurium.
METHODS: Serum samples from 308 persons living in mid-USA were phenotyped to identify the atypical and fluoride variants. 308 samples were analysed for the K variant by DNA amplification, digestion with Mae III and gel electrophoresis. Amplified DNA from 16 samples was sequenced to identify the D70G, T243M and D70H mutations. Values for kcat and Km were determined for the D70H mutant BChE expressed in 293T cells.
RESULTS: A new mutation, Asp70His, was identified. This mutation is located in the peripheral anionic site of butyrylcholinesterase, where it causes a 10-fold decrease in binding affinity for positively charged substrates. CONCLUSION: People homozygous for the Asp70His mutation are expected to have prolonged apnoea in response to succinylcholine or mivacurium, similar to people with the Asp70Gly mutation.
ESTHER : Boeck_2002_Ann.Clin.Biochem_39_154
PubMedSearch : Boeck_2002_Ann.Clin.Biochem_39_154
PubMedID: 11928765

Title : Cocaine metabolism accelerated by a re-engineered human butyrylcholinesterase - Sun_2002_J.Pharmacol.Exp.Ther_302_710
Author(s) : Sun H , Shen ML , Pang YP , Lockridge O , Brimijoin S
Ref : Journal of Pharmacology & Experimental Therapeutics , 302 :710 , 2002
Abstract : Plasma butyrylcholinesterase (BChE) is important in the metabolism of cocaine, but natural human BChE has limited therapeutic potential for detoxication because of low catalytic efficiency with cocaine. Here we report pharmacokinetics of cocaine in rats treated with A328W/Y332A BChE, an excellent cocaine hydrolase designed with the aid of molecular modeling. Compared with wild-type BChE, this enzyme hydrolyzes cocaine with 40-fold improved k(cat) (154 min(-1) versus 4.1 min(-1)) and only slightly increased K(M) (18 microM versus 4.5 microM). In rats given this hydrolase (3 mg/kg i.v.) 10 min before cocaine challenge (6.8 mg/kg i.v.), cocaine half-life was reduced from 52 min to 18 min. Mirroring the reductions of plasma cocaine were large increases in benzoic acid, a product of BChE-mediated cocaine hydrolysis. All other pharmacokinetic parameters confirmed a large, dose-dependent acceleration of cocaine removal by the injected cocaine hydrolase. These results show that A328W/Y332A, an efficient cocaine hydrolase in vivo as well as in vitro, might promote cocaine detoxication in a clinical setting.
ESTHER : Sun_2002_J.Pharmacol.Exp.Ther_302_710
PubMedSearch : Sun_2002_J.Pharmacol.Exp.Ther_302_710
PubMedID: 12130735
Gene_locus related to this paper: human-BCHE

Title : Re-engineering butyrylcholinesterase as a cocaine hydrolase - Sun_2002_Mol.Pharmacol_62_220
Author(s) : Sun H , Pang YP , Lockridge O , Brimijoin S
Ref : Molecular Pharmacology , 62 :220 , 2002
Abstract : To address the problem of acute cocaine overdose, we undertook molecular engineering of butyrylcholinesterase (BChE) as a cocaine hydrolase so that modest doses could be used to accelerate metabolic clearance of this drug. Molecular modeling of BChE complexed with cocaine suggested that the inefficient hydrolysis (k(cat) = 4 min(-1)) involves a rotation toward the catalytic triad, hindered by Tyr332. To eliminate rotational hindrance and retain substrate affinity, we introduced two amino acid substitutions (Ala328Trp/Tyr332Ala). The resulting mutant BChE reduced cocaine burden in tissues, accelerated plasma clearance by 20-fold, and prevented cocaine-induced hyperactivity in mice. The enzyme's kinetic properties (k(cat) = 154 min(-1), K(M) = 18 microM) satisfy criteria suggested previously for treating cocaine overdose (k(cat) >120 min(-1), K(M) < 30 microM). This success demonstrates that computationally guided mutagenesis can generate functionally novel enzymes with clinical potential.
ESTHER : Sun_2002_Mol.Pharmacol_62_220
PubMedSearch : Sun_2002_Mol.Pharmacol_62_220
PubMedID: 12130672
Gene_locus related to this paper: human-BCHE

Title : Predicted Michaelis-Menten complexes of cocaine-butyrylcholinesterase. Engineering effective butyrylcholinesterase mutants for cocaine detoxication. - Sun_2001_J.Biol.Chem_276_9330
Author(s) : Sun H , El Yazal J , Lockridge O , Schopfer LM , Brimijoin S , Pang YP
Ref : Journal of Biological Chemistry , 276 :9330 , 2001
Abstract : 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 Ser(198) for hydrolysis. Rotation of (-)-cocaine appears to be hindered by interactions of its phenyl ring with Phe(329) and Trp(430). 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.
ESTHER : Sun_2001_J.Biol.Chem_276_9330
PubMedSearch : Sun_2001_J.Biol.Chem_276_9330
PubMedID: 11104759

Title : Evidence for nonacetylcholinesterase targets of organophosphorus nerve agent: supersensitivity of acetylcholinesterase knockout mouse to vx lethality - Duysen_2001_J.Pharmacol.Exp.Ther_299_528
Author(s) : Duysen EG , Li B , Xie W , Schopfer LM , Anderson RS , Broomfield CA , Lockridge O
Ref : Journal of Pharmacology & Experimental Therapeutics , 299 :528 , 2001
Abstract : The possibility that organophosphate toxicity is due to inhibition of targets other than acetylcholinesterase (AChE, EC was examined in AChE knockout mice. Mice (34-55 days old) were grouped for this study, after it was determined that AChE, butyrylcholinesterase (BChE), and carboxylesterase activities had reached stable values by this age. Mice with 0, 50, or 100% AChE activity were treated subcutaneously with the nerve agent VX. The LD50 for VX was 10 to 12 microg/kg in AChE-/-, 17 microg/kg in AChE+/-, and 24 microg/kg in AChE+/+ mice. The same cholinergic signs of toxicity were present in AChE-/- mice as in wild-type mice, even though AChE-/- mice have no AChE whose inhibition could lead to cholinergic signs. Wild-type mice, but not AChE-/- mice, were protected by pretreatment with atropine. Tissues were extracted from VX-treated and untreated animals and tested for AChE, BChE, and acylpeptide hydrolase activity. VX treatment inhibited 50% of the AChE activity in brain and muscle of AChE+/+ and +/- mice, 50% of the BChE activity in all three AChE genotypes, but did not significantly inhibit acylpeptide hydrolase activity. It was concluded that the toxicity of VX must be attributed to inhibition of nonacetylcholinesterase targets in the AChE-/- mouse. Organophosphorus ester toxicity in wild-type mice is probably due to inhibition or binding to several proteins, only one of which is AChE.
ESTHER : Duysen_2001_J.Pharmacol.Exp.Ther_299_528
PubMedSearch : Duysen_2001_J.Pharmacol.Exp.Ther_299_528
PubMedID: 11602663

Title : Effects of mutations of active site residues and amino acids interacting with the Omega loop on substrate activation of butyrylcholinesterase - Masson_2001_Biochim.Biophys.Acta_1544_166
Author(s) : Masson P , Xie W , Froment MT , Lockridge O
Ref : Biochimica & Biophysica Acta , 1544 :166 , 2001
Abstract : The peripheral anionic site (PAS) of human butyrylcholinesterase is involved in the mechanism of substrate activation by positively charged substrates and ligands. Two substrate binding loci, D70 in the PAS and W82 in the active site, are connected by the Omega loop. To determine whether the Omega loop plays a role in the signal transduction between the PAS and the active site, residues involved in stabilization of the loop, N83, K339 and W430, were mutated. Mutations N83A and N83Q caused loss of substrate activation, suggesting that N83 which interacts with the D70 backbone may be an element of the transducing system. The K339M and W430A mutant enzymes retained substrate activation. Residues W82, E197, and A328 in the active site gorge have been reported to be involved in substrate activation. At butyrylthiocholine concentrations greater then 2 mM, W82A showed apparent substrate activation. Mutations E197Q and E197G strongly reduced substrate activation, while mutation E197D caused a moderate effect, suggesting that the carboxylate of residue E197 is involved in substrate activation. Mutations A328F and A328Y showed no substrate activation, whereas A328G retained substrate activation. Substrate activation can result from an allosteric effect due to binding of the second substrate molecule on the PAS. Mutation W430A was of special interest because this residue hydrogen bonds to W82 and Y332. W430A had strongly reduced affinity for tetramethylammonium. The bimolecular rate constant for reaction with diisopropyl fluorophosphate was reduced 10000-fold, indicating severe alteration in the binding area in W430A. The kcat values for butyrylthiocholine, o-nitrophenyl butyrate, and succinyldithiocholine were lower. This suggested that the mutation had caused misfolding of the active site gorge without altering the Omega loop conformation/dynamics. W430 as well as W231 and W82 appear to form the wall of the active site gorge. Mutation of any of these tryptophans disrupts the architecture of the active site.
ESTHER : Masson_2001_Biochim.Biophys.Acta_1544_166
PubMedSearch : Masson_2001_Biochim.Biophys.Acta_1544_166
PubMedID: 11341926
Gene_locus related to this paper: human-BCHE

Title : The active site of human paraoxonase (PON1) - Josse_2001_J.Appl.Toxicol_21 Suppl 1_S7
Author(s) : Josse D , Lockridge O , Xie W , Bartels CF , Schopfer LM , Masson P
Ref : J Appl Toxicol , 21 Suppl 1 :S7 , 2001
Abstract : Ideally we would like to treat people exposed to nerve agents with an enzyme that rapidly destroys nerve agents. The enzymes considered for such a role include human butyrylcholinesterase (BChE), acetylcholinesterase (AChE), carboxylesterase and paraoxonase (PON1). Success has been achieved in endowing BChE with the ability to hydrolyze organophosphates. The G117H mutant of BCHE hydrolyzes sarin and VX, whereas the double mutant G117H/E197Q hydrolyzes soman (Millard et al. Biochemistry 1995; 34: 15925-15933; 1998; 37: 237-247). However, the rates of organophosphate hydrolysis are slow and a faster organophosphate hydrolase is being sought. Native PON1 hydrolyzes paraoxon with a catalytic efficiency, of 2.4 x 10(6) M(-1) x min(-1), and our goal is to improve the organophosphate hydrolase activity of PON1. To achieve this we need to identify the amino acids in the active site of PON1. Using site-directed mutagenesis and expression in human 293T cells, we have identified the following eight amino acids as being essential to PON1 activity: W280, H114, H133, H154, H242, H284, E52 and D53. Fluorescence of PON1 complexed to terbium ion shows that at least one tryptophan is close to the calcium binding site.
ESTHER : Josse_2001_J.Appl.Toxicol_21 Suppl 1_S7
PubMedSearch : Josse_2001_J.Appl.Toxicol_21 Suppl 1_S7
PubMedID: 11920913

Title : Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse - Li_2000_J.Neurochem_75_1320
Author(s) : Li B , Stribley JA , Ticu A , Xie W , Schopfer LM , Hammond P , Brimijoin S , Hinrichs SH , Lockridge O
Ref : Journal of Neurochemistry , 75 :1320 , 2000
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.
ESTHER : Li_2000_J.Neurochem_75_1320
PubMedSearch : Li_2000_J.Neurochem_75_1320
PubMedID: 10936216

Title : The butyrylcholinesterase K-variant shows similar cellular protein turnover and quaternary interaction to the wild-type enzyme - Altamirano_2000_J.Neurochem_74_869
Author(s) : Altamirano CV , Bartels CF , Lockridge O
Ref : Journal of Neurochemistry , 74 :869 , 2000
Abstract : A recent study has linked the butyrylcholinesterase (BChE) K-variant and the apolipoprotein epsilon4 isoform to late-onset Alzheimer's disease. These findings have been controversial and have led us to examine the differences between wild-type and K-variant BChE in enzyme activity, protein stability, and quaternary structure. J-variant BChE (E497V/A539T) was also studied because it is associated with the K-variant mutation. The K-variant mutation (A539T) is located in the C-terminal tetramerization domain. Wild-type, K-variant, and J-variant BChE were expressed in Chinese hamster ovary cells and purified. The purified enzymes had similar binding affinity (Km) values and catalytic rates for butyrylthiocholine and benzoylcholine. In pulse-chase studies the K-variant, J-variant, and wildtype BChE were degraded rapidly within the cell, with a half-time of approximately 1.5 h. Less than 5% of the intracellular BChE was exported. The C-terminal peptide containing the K-variant mutation interacted with itself as strongly as did the wild-type peptide in the yeast two-hybrid system. Both K-variant and wild-type BChE assembled into tetramers in the presence of poly-L-proline or the proline-rich attachment domain of the collagen tail. The native K-variant BChE in serum showed the same proportion of tetramers as the native serum wild-type BChE. We conclude that the K-variant BChE is similar to wild-type BChE in enzyme activity, protein turnover, and tetramer formation.
ESTHER : Altamirano_2000_J.Neurochem_74_869
PubMedSearch : Altamirano_2000_J.Neurochem_74_869
PubMedID: 10646540

Title : Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma - Bartels_2000_Biochem.Pharmacol_60_479
Author(s) : Bartels CF , Xie W , Miller-Lindholm AK , Schopfer LM , Lockridge O
Ref : Biochemical Pharmacology , 60 :479 , 2000
Abstract : Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC; this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.
ESTHER : Bartels_2000_Biochem.Pharmacol_60_479
PubMedSearch : Bartels_2000_Biochem.Pharmacol_60_479
PubMedID: 10874122
Gene_locus related to this paper: felca-ACHE , felca-BCHE , tiger-BCHE

Title : Pesticides and susceptible populations: people with butyrylcholinesterase genetic variants may be at risk - Lockridge_2000_Neurotoxicol_21_113
Author(s) : Lockridge O , Masson P
Ref : Neurotoxicology , 21 :113 , 2000
Abstract : Butyrylcholinesterase (BChE) scavenges low doses of organophosphorus (for example, paraoxon) and carbamate pesticides (for example, carbaryl) and in this way protects people from the toxic effects of these poisons. The protective role of BChE is demonstrated by the finding that pesticide applicators can have reduced BChE activity with no clinical signs of poisoning. The question has arisen whether people with genetic variants of BChE are less protected. Seventy-six percent of the population is homozygous for wild-type BChE, while 24% carry at least one genetic variant allele. Most genetic variants of BChE have reduced activity. The clinically most important variant is atypical (D70G) BChE because people with this variant have 2 hours of apnea after receiving a dose of succinylcholine that is intended to paralyze muscles for 3-5 minutes. In test tube experiments the atypical variant reacts more slowly with all positively charged compounds (for example physostigmine, echothiophate). This leaves more toxin available for reaction with acetylcholinesterase in nerve synapses and predicts that people with atypical BChE will be less protected. Variants with low activity, such as silent BChE, are predicted to be at increased risk from organophosphorus pesticides based on experiments in monkeys and rodents where injection of purified BChE protected animals from the toxic effects of nerve agents. More studies are needed to strengthen the hypothesis that people with genetic variants of BChE are at higher risk of intoxication from pesticides.
ESTHER : Lockridge_2000_Neurotoxicol_21_113
PubMedSearch : Lockridge_2000_Neurotoxicol_21_113
PubMedID: 10794391

Title : Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase - Xie_2000_J.Pharmacol.Exp.Ther_293_896
Author(s) : Xie W , Stribley JA , Chatonnet A , Wilder PJ , Rizzino A , McComb RD , Taylor P , Hinrichs SH , Lockridge O
Ref : Journal of Pharmacology & Experimental Therapeutics , 293 :896 , 2000
Abstract : Acetylcholinesterase (AChE; EC is the primary terminator of nerve impulse transmission at cholinergic synapses and is believed to play an important role in neural development. Targeted deletion of four exons of the ACHE gene reduced AChE activity by half in heterozygous mutant mice and totally eliminated AChE activity in nullizygous animals. Butyrylcholinesterase (EC activity was normal in AChE -/- mice. Although nullizygous mice were born alive and lived up to 21 days, physical development was delayed. The neuromuscular junction of 12-day-old nullizygous animals appeared normal in structure. Nullizygous mice were highly sensitive to the toxic effects of the organophosphate diisopropylfluorophosphate and to the butyrylcholinesterase-specific inhibitor bambuterol. These findings indicate that butyrylcholinesterase and possibly other enzymes are capable of compensating for some functions of AChE and that the inhibition of targets other than AChE by organophosphorus agents results in death.
ESTHER : Xie_2000_J.Pharmacol.Exp.Ther_293_896
PubMedSearch : Xie_2000_J.Pharmacol.Exp.Ther_293_896
PubMedID: 10869390
Gene_locus related to this paper: mouse-ACHE

Title : Identification of residues essential for human paraoxonase (PON1) arylesterase\/organophosphatase activities - Josse_1999_Biochemistry_38_2816
Author(s) : Josse D , Xie W , Renault F , Rochu D , Schopfer LM , Masson P , Lockridge O
Ref : Biochemistry , 38 :2816 , 1999
Abstract : Human serum paraoxonase (PON1) is a calcium-dependent organophosphatase. To identify residues essential for PON1 activity, we adopted complementary approaches based on chemical modification and site-directed mutagenesis. To detect 45Ca2+ binding to native and chemically modified PON1, we performed nondenaturating gel electrophoresis. The environment of calcium-binding sites was probed using the Ca2+ analogue, terbium. Tb3+ binds to calcium-binding sites as shown by displacement of 45Ca2+ by Tb3+. Binding of Tb3+ is accompanied by a complete loss of enzyme activity. PON1 chemical modification with the Trp-selective reagent, N-bromosuccinimide, and the Asp/Glu-selective, dicyclohexylcarbodiimide, established that Trp and Asp/Glu residues are components of the PON1 active center and calcium-binding sites. Additional evidence for the presence of a Trp residue in the PON1 calcium-binding sites was a characteristic fluorescence emission at 545 nm from the PON1-Tb3+ complex and abolishment of that fluorescence upon modification by N-bromosuccinimide. The importance of aromatic/hydrophobic character of the residue 280 was demonstrated by site-directed mutagenesis: the W280F mutant was fully active while the W280A and W280L mutants had markedly reduced activity. Twelve amino acids among conserved His and Asp/Glu residues were found essential for PON1 arylesterase and organophosphatase activities: H114, H133, H154, H242, H284, D53, D168, D182, D268, D278, E52, and E194. Finally, the cysteines constituting the PON1 disulfide bond (C41 and C352) were essential, but the glycan chains linked to Asn 252 and 323 were not essential for PON1 secretion and activity.
ESTHER : Josse_1999_Biochemistry_38_2816
PubMedSearch : Josse_1999_Biochemistry_38_2816
PubMedID: 10052953

Title : Knockout of one acetylcholinesterase allele in the mouse - Xie_1999_Chem.Biol.Interact_119-120_289
Author(s) : Xie W , Wilder PJ , Stribley J , Chatonnet A , Rizzino A , Taylor P , Hinrichs SH , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :289 , 1999
Abstract : One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.
ESTHER : Xie_1999_Chem.Biol.Interact_119-120_289
PubMedSearch : Xie_1999_Chem.Biol.Interact_119-120_289
PubMedID: 10421464

Title : An improved cocaine hydrolase: the A328Y mutant of human butyrylcholinesterase is 4-fold more efficient - Xie_1999_Mol.Pharmacol_55_83
Author(s) : Xie W , Altamirano CV , Bartels CF , Speirs RJ , Cashman JR , Lockridge O
Ref : Molecular Pharmacology , 55 :83 , 1999
Abstract : Butyrylcholinesterase (BChE) has a major role in cocaine detoxication. The rate at which human BChE hydrolyzes cocaine is slow, with a kcat of 3.9 min(-1) and Km of 14 microM. Our goal was to improve cocaine hydrolase activity by mutating residues near the active site. The mutant A328Y had a kcat of 10.2 min(-1) and Km of 9 microM for a 4-fold improvement in catalytic efficiency (kcat/Km). Since benzoylcholine (kcat 15,000 min(-1)) and cocaine form the same acyl-enzyme intermediate but are hydrolyzed at 4000-fold different rates, it was concluded that a step leading to formation of the acyl-enzyme intermediate was rate-limiting. BChE purified from plasma of cat, horse, and chicken was tested for cocaine hydrolase activity. Compared with human BChE, horse BChE had a 2-fold higher kcat but a lower binding affinity, cat BChE was similar to human, and chicken BChE had only 10% of the catalytic efficiency. Naturally occurring genetic variants of human BChE were tested for cocaine hydrolase activity. The J and K variants (E497V and A539T) had k(cat) and Km values similar to wild-type, but because these variants are reduced to 66 and 33% of normal levels in human blood, respectively, people with these variants may be at risk for cocaine toxicity. The atypical variant (D70G) had a 10-fold lower binding affinity for cocaine, suggesting that persons with the atypical variant of BChE may experience severe or fatal cocaine intoxication when administered a dose of cocaine that is not harmful to others.
ESTHER : Xie_1999_Mol.Pharmacol_55_83
PubMedSearch : Xie_1999_Mol.Pharmacol_55_83
PubMedID: 9882701

Title : Differences in active-site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase - Saxena_1999_Chem.Biol.Interact_119-120_61
Author(s) : Saxena A , Redman AM , Jiang X , Lockridge O , Doctor BP
Ref : Chemico-Biological Interactions , 119-120 :61 , 1999
Abstract : We examined the role of A328(F330) in the binding of various inhibitors to cholinesterases (ChEs) using human butyrylcholinesterase (BChE) mutants to determine if the conclusions drawn from studies with acetylcholinesterase (AChE) mutants could be extended to BChE. For huperzine A and edrophonium, the results obtained with AChE mutants could be directly correlated with those obtained with native ChEs and site-specific mutants of human BChE. Inhibition studies of ethopropazine with BChE mutants, where A328 was modified to either F or Y, suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active-site gorge showed that the poor inhibitory activity of ethopropazine towards AChE was due to the smaller dimension of the active-site gorge. The volume of the BChE active-site gorge is approximately 200 A3 larger than that of the AChE gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations. These results suggest that, although the overall scaffolding of the two enzymes may be highly similar, the dimensions and the micro-environment of the gorge play a significant role in determining the selectivity of substrate and inhibitors for ChEs.
ESTHER : Saxena_1999_Chem.Biol.Interact_119-120_61
PubMedSearch : Saxena_1999_Chem.Biol.Interact_119-120_61
PubMedID: 10421439

Title : Human serum paraoxonase (PON1): identification of essential amino acid residues by group-selective labelling and site-directed mutagenesis - Josse_1999_Chem.Biol.Interact_119-120_71
Author(s) : Josse D , Xie W , Masson P , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :71 , 1999
Abstract : Human serum paraoxonase/arylesterase (PON1, EC is a calcium-dependent enzyme which hydrolyzes a wide variety of organophosphates, including paraoxon, DFP, sarin and soman. Although the 3-D structure of PON has not yet been determined and its sequence shows no similarity with any other crystallized proteins, we undertook to identify some of its essential amino acid residues by two complementary approaches: group-specific labelling and site-directed mutagenesis. Group-specific labelling studies, performed on the purified native enzyme, indicated that one or more Trp, His and Asp/Glu are potentially important residues for PON activity. Based on these results, we identified some of these residues, conserved in the sequenced mammalian PON1, by site-directed mutagenesis. PON1 mutants were transiently expressed in 293T cells. The catalytic constants k(cat) and Km (relative to k(cat) and Km of the wild-type) determined with four different substrates (phenylacetate, paraoxon, diazoxon, chlorpyrifos oxon), were not significantly changed for the following mutants: W193A, W201A, W253A, H160N, H245N, H250N, H347N, E32A, E48A, D88A, D107A, D121A, D273A. By contrast, k(cat) was less than 1% for eight mutants: W280A, H114N, H133N, H154N, H242N, H284N, E52A and D53A. The essential amino acid residues identified in this work could be part of the PON1 active site, acting either as calcium ligands (E52 and D53?) or as substrate binding (W280?) or nucleophilic (His residues?) sites. However, we cannot rule out that the effects of mutations on catalytic properties resulted from a remote conformational change and/or misfolding of mutant proteins.
ESTHER : Josse_1999_Chem.Biol.Interact_119-120_71
PubMedSearch : Josse_1999_Chem.Biol.Interact_119-120_71
PubMedID: 10421440

Title : Tryptophan residue(s) as major components of the human serum paraoxonase active site - Josse_1999_Chem.Biol.Interact_119-120_79
Author(s) : Josse D , Xie W , Masson P , Schopfer LM , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :79 , 1999
Abstract : Serum paraoxonase (PON1, EC is a high density lipid- (HDL)-associated, calcium-dependent enzyme whose 3D structure, active site residues and physiological substrates are not known. The kinetic parameters k(cat) and Km (relative to k(cat) and Km of the wild-type), determined with four substrates (phenylacetate, paraoxon, diazoxon and chlorpyrifosoxon) were less than 1, and more than 100% for the W280A and W280F mutant enzymes, respectively. These results indicated that the aromatic/hydrophobic character of the amino acid in position 280 is essential for PON1 activity. In this study, we investigated whether this aromatic residue is in the PON1 active site. Group-specific labelling studies with N-bromosuccinimide, an oxidative agent of tryptophan, strongly suggested that one or several Trp could be in the active site of PON1 but we could not conclude either on the specificity of the labelling reaction or on the number of oxidized Trp. However, although PON activity was not altered by the hydrophilic tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl chloride (NBC), it was significantly reduced by the p-nitrophenylacetate analog 2-acetoxy-5-nitrobenzyl chloride (ANBC), whose hydrolysis by PON1 generated NBC in the active site. Moreover, since at least one calcium ion is present in the PON catalytic site, we attempted to probe the metal local environment using the calcium analog terbium. The luminescence spectrum of the PON terbium complex exhibited an emission peak at 545 nm characteristic of an aromatic residue (Trp and/or Tyr)-terbium interaction. In conclusion, both the results obtained with the mechanism-based inhibitor of PON1 (ANBC) and the calcium-binding site luminescent probe terbium support the hypothesis of the presence of at least one Trp residue in the PON1 active site. Trp residue(s) may be involved in the binding of aromatic substrates.
ESTHER : Josse_1999_Chem.Biol.Interact_119-120_79
PubMedSearch : Josse_1999_Chem.Biol.Interact_119-120_79
PubMedID: 10421441

Title : Association of tetramers of human butyrylcholinesterase is mediated by conserved aromatic residues of the carboxy terminus - Altamirano_1999_Chem.Biol.Interact_119-120_53
Author(s) : Altamirano CV , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :53 , 1999
Abstract : Human butyrylcholinesterase (BChE) is composed predominantly of tetramers. Our laboratory has shown that up to 40 carboxy terminal residues of each subunit contribute to the stabilization of tetramers (R.M. Blong, E. Bedows, O. Lockridge, The tetramerization domain of butyrylcholinesterase is at the carboxy-terminus, Biochem. J. 327 (1997) 747-757). To better define the residues which participate in tetramer stabilization, the in vivo interaction of the BChE C-terminus 46 residue peptide was quantitated for wild type and mutant BChE using the yeast two-hybrid system. The wild type C-terminal peptides interacted with one another in this system. The K-variant (A539T) and C571A peptides showed interaction similar to that of the wild type. However, only 11.7% of the interaction seen with the wild type peptide was observed with the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) had been altered to alanines (aromatics off mutant). When these seven mutations were incorporated into the complete BChE molecule and expressed in 293T cells, only monomers and dimers were observed. The addition of poly-L-proline to the medium of 293T cells expressing wild type BChE resulted in the increase of the tetrameric form, similar to that observed by Bon et al. (S. Bon, F. Coussen, J. Massoulie, Quaternary associations of acetylcholinesterase II. The polyproline attachment domain of the collagen tail, J. Biol. Chem. 272 (1997) 3016-3021) for acetylcholinesterase expressed in COS cells. However, no increase in tetramers was observed with poly-L-proline addition to the medium of 293T cells expressing the aromatics off BChE mutant. These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues, Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564, and that the poly-L-proline induced increase in tetrameric BChE is mediated through these seven aromatic residues.
ESTHER : Altamirano_1999_Chem.Biol.Interact_119-120_53
PubMedSearch : Altamirano_1999_Chem.Biol.Interact_119-120_53
PubMedID: 10421438

Title : Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers - Altamirano_1999_Biochemistry_38_13414
Author(s) : Altamirano CV , Lockridge O
Ref : Biochemistry , 38 :13414 , 1999
Abstract : Human butyrylcholinesterase (BChE) in serum is composed predominantly of tetramers. The tetramerization domain of each subunit is contained within 40 C-terminal residues. To identify key residues within this domain participating in tetramer stabilization, the interaction between C-terminal 46 residue peptides was quantitated in the yeast two-hybrid system. The wild-type peptide interacted strongly with another wild-type peptide in the yeast two-hybrid system. The C571A mutant peptides interacted to a similar degree as the wild-type. However, the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C571 were altered to alanines showed only 12% of the interaction seen with the wild-type peptide. The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and expressed in 293T and CHO-K1 cells. Expression of wild-type BChE in these cell lines yielded 10% tetramers. The aromatics-off mutant formed dimers and monomers but no tetramers. The aromatics-off/C571A mutant yielded only monomers. Addition of poly-L-proline to culture medium, or coexpression with the N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRAD), increased the amount of tetrameric wild-type BChE from 10 to 70%, but had no effect on the G534stop (lacking 41 C-terminal residues) and the aromatics-off mutants. Recombinant BChE produced by coexpression with Q(N)PRAD was purified by column chromatography. The purified tetramers contained the FLAG-tagged Q(N)PRAD peptide. These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues and that poly-L-proline and PRAD act through these aromatic residues to induce tetramerization.
ESTHER : Altamirano_1999_Biochemistry_38_13414
PubMedSearch : Altamirano_1999_Biochemistry_38_13414
PubMedID: 10529218

Title : Protein engineering of a human enzyme that hydrolyzes V and G nerve agents: design, construction and characterization - Broomfield_1999_Chem.Biol.Interact_119-120_413
Author(s) : Broomfield CA , Lockridge O , Millard CB
Ref : Chemico-Biological Interactions , 119-120 :413 , 1999
Abstract : Because of deficiencies in the present treatments for organophosphorus anticholinesterase poisoning, we are attempting to develop a catalytic scavenger that can be administered as prophylactic protection. Currently known enzymes are inadequate for this purpose because they have weak binding and slow turnover, so we are trying to make an appropriate enzyme by protein engineering techniques. One butyrylcholinesterase mutant, G117H, has the desired type of activity but reacts much too slowly. This communication describes an attempt to determine the reason for the slow reaction so that a more efficient enzyme might be designed. The results indicate that the mutation at residue 117 has resulted in a distortion of the transition state of the reaction of organophosphorus compounds with the active site serine. This information will be used to develop other mutants that avoid transition state stabilization sites.
ESTHER : Broomfield_1999_Chem.Biol.Interact_119-120_413
PubMedSearch : Broomfield_1999_Chem.Biol.Interact_119-120_413
PubMedID: 10421478

Title : Structural and hydration changes in the active site gorge of phosporhylated butyrylcholinesterase accompanying the aging process - Masson_1999_Chem.Biol.Interact_119-120_17
Author(s) : Masson P , Fortier PL , Albaret C , Clery C , Guerra P , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :17 , 1999
Abstract : Wild-type (wt) butyrylcholinesterase (BuChE) and the E197D and D70G mutants were inhibited by diisopropylfluorophosphate (DFP) or soman under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process of DFP-phosphorylated enzymes (diisopropylphosphoryl-BuChE (DIP-BuChE)) was investigated. Hydrostatic pressure strongly increased the rate of aging of wt enzyme. The activation volumes (deltaV*) for the dealkylation reaction was -150 ml/mol for DIP-wtBuChE. On the other hand, pressure had little effect on the aging of the DIP-E197D mutant and no effect on the DIP-D70G mutant, indicating that the transition state of the aging reaction (dealkylation of an isoproxy chain) was associated with an extended conformation/hydration change in wtBuChE but not in mutants. The rate of aging decreased with osmotic pressure, supporting the idea that water is important for stabilizing the transition state. Molecular dynamics simulations were performed on the wtDIP adduct to relate the kinetic data to hydration changes in the enzyme active site gorge. The pH dependence of the melting temperature (Tm) of native and soman-wtBuChE, as determined by differential scanning calorimetry (DSC), indicated that the stabilization energy of aged BuChE is mainly due to the salt bridge between protonated H438 and PO-, with pK(H438) = 8.3. Electrophoresis under high pressure up to 2.5 kbar showed that aged wtBuChE did not undergo pressure-induced molten globule transition unlike the native enzyme. This transition was not seen for the mutant enzymes, indicating that mutants are resistant to the penetration of water into their structure. Our results support the conclusion that D70 and E197 are major residues for the water/H-bond network dynamics in the active site gorge of BuChE, both residues acting like valves. In mutant enzymes, mutated residues function like check valves: forced penetration of water in the gorge is difficult, release of water is facilitated.
ESTHER : Masson_1999_Chem.Biol.Interact_119-120_17
PubMedSearch : Masson_1999_Chem.Biol.Interact_119-120_17
PubMedID: 10421435

Title : Polyol-induced activation by excess substrate of the D70G butyrylcholinesterase mutant - Levitsky_1999_Biochim.Biophys.Acta_1429_422
Author(s) : Levitsky V , Xie W , Froment MT , Lockridge O , Masson P
Ref : Biochimica & Biophysica Acta , 1429 :422 , 1999
Abstract : Wild-type human butyrylcholinesterase (BCHE) has a non-Michaelian behaviour showing substrate activation with butyrylthiocholine (BTC) as the substrate. The D70G mutant has a catalytic constant identical to that of the wild-type enzyme, but a 10-fold lower affinity for BTC compared to wild-type enzyme, and it does not exhibit activation by excess BTC under conventional conditions. In the present work it was found that addition of polyols or sugars changed the kinetic behaviour of the D70G mutant with BTC. In the presence of 40% sucrose, the D70G mutant enzyme displayed marked activation by excess substrate. Because D70 is hydrogen bonded to Y332, mutants of Y332 were studied. Mutant Y332F had a behaviour similar to that of wild-type BCHE, whereas mutants Y332A, Y332A/D70G and D70G had negligible substrate activation. The behavior of wild-type, Y332F, Y332A and Y332A/D70G did not change in the presence of high concentrations of sugar. Substrate activation has been explained by binding of a second substrate molecule in the peripheral site at D70. The D70G mutant should be incapable of substrate activation, if D70 were the only residue involved in substrate activation. The ability of the D70G mutant to display substrate activation by medium engineering suggests that other residues are involved in initial substrate binding and activation by excess substrate. Osmolyte-induced change in conformation and/or hydration status of Y332 and other solvent-exposed residues may account for the non-Michaelian behaviour of the D70G mutant.
ESTHER : Levitsky_1999_Biochim.Biophys.Acta_1429_422
PubMedSearch : Levitsky_1999_Biochim.Biophys.Acta_1429_422
PubMedID: 9989227

Title : Interaction between the peripheral site residues of human butyrylcholinesterase, D70 and Y332, in binding and hydrolysis of substrates - Masson_1999_Biochim.Biophys.Acta_1433_281
Author(s) : Masson P , Xie W , Froment MT , Levitsky V , Fortier PL , Albaret C , Lockridge O
Ref : Biochimica & Biophysica Acta , 1433 :281 , 1999
Abstract : Human butyrylcholinesterase displays substrate activation with positively charged butyrylthiocholine (BTC) as the substrate. Peripheral anionic site (PAS) residues D70 and Y332 appear to be involved in the initial binding of charged substrates and in activation control. To determine the contribution of PAS residues to binding and hydrolysis of quaternary substrates and activation control, the single mutants D70G/Y and Y332F/A/D and the double mutants Y332A/D70G and Y332D/D70Y were studied. Steady-state hydrolysis of the charged substrates, BTC and succinyldithiocholine, and the neutral ester o-nitrophenyl butyrate was measured. In addition, inhibition of wild-type and mutant enzymes by tetramethylammonium was investigated, at low concentrations of BTC. Single and double mutants of D70 and Y332 showed little or no substrate activation, suggesting that both residues were important for activation control. The effects of double mutations on D70 and Y332 were complex. Double-mutant cycle analysis provided evidence for interaction between these residues. The category of interaction (either synergistic, additive, partially additive or antagonistic) was found to depend on the nature of the substrate and on measured binding or kinetic parameters. This complexity reflects both the cross-talk between residues involved in the sequential formation of productive Michaelian complexes and the effect of peripheral site residues on catalysis. It is concluded that double mutations on the PAS induce a conformational change in the active site gorge of butyrylcholinesterase that can alter both substrate binding and enzyme acylation.
ESTHER : Masson_1999_Biochim.Biophys.Acta_1433_281
PubMedSearch : Masson_1999_Biochim.Biophys.Acta_1433_281
PubMedID: 10446378

Title : Bert La Du ASPET Plenary Lecture: Bert N. La Du, a Summary of His Career to 1998 -
Author(s) : Lockridge O
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :1 , 1998

Title : ACHE Knockout Mouse\; Cat AChE and Cat BChE Sequences\; Tetramers of BChE -
Author(s) : Lockridge O , Xie W , Chatonnet A , Taylor P , Bartels CF
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :41 , 1998

Title : Enzymes hydrolyzing organophosphates as potential catalytic scavengers against organophosphate poisoning - Masson_1998_J.Physiol.Paris_92_357
Author(s) : Masson P , Josse D , Lockridge O , Viguie N , Taupin C , Buhler C
Ref : Journal de Physiologie (Paris) , 92 :357 , 1998
Abstract : Enzymes hydrolyzing organophosphates could be used as catalytic scavengers for treatment of organophosphate poisoning and for decontamination. Two organophosphorus hydrolases (OPH) were selected: the Flavobacterium sp/Pseudomonas diminuta phosphotriesterase (PTE) and human paraoxonase (HuPON). Genes encoding these enzymes were cloned and functional recombinant enzymes expressed. PTE was expressed in E. coli. Natural HuPON was purified from human plasma; recombinant HuPON was expressed in human embryonic kidney 293 T cells. Although HuPON displays interesting catalytic properties, a site-directed mutagenesis program was undertaken to improve its catalytic efficiency. PTE has high efficiency in hydrolysis of organophosphates, including nerve agents. PTE injected in rat has a half-life of 100 min. However, to overcome pharmacokinetic problems of injected OPH and/or immunological incompatibility, the model enzyme (recombinant PTE) was immobilized onto a hollow-fiber reactor. This reactor designed for extracorporeal blood circulation is under experimentation for post-exposure detoxification.
ESTHER : Masson_1998_J.Physiol.Paris_92_357
PubMedSearch : Masson_1998_J.Physiol.Paris_92_357
PubMedID: 9789837

Title : Prolonged paralysis after a test dose of mivacurium in a patient with atypical serum cholinesterase -
Author(s) : Vanlinthout LE , Bartels CF , Lockridge O , Callens K , Booij LH
Ref : Anesthesia & Analgesia , 87 :1199 , 1998
PubMedID: 9806709

Title : Structural Changes in the Active Site Gorge of Phosphylated Cholinesterase Accompanying the Aging Process -
Author(s) : Masson P , Clery C , Guerra P , Fortier PL , Albaret C , Lockridge O
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :419 , 1998

Title : Association of Tetramers of Human Butyrylcholinesterase is Mediated by Conserved Aromatic Residues of the Carboxy Terminus -
Author(s) : Altamirano CV , Lockridge O
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :440 , 1998

Title : Acetylcholinesterase and Butyrylcholinesterase of Cat -
Author(s) : Bartels CF , Xie W , Miller-Lindholm AK , Lockridge O
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :147 , 1998

Title : Expression of Human Butyrylcholinesterase in Trichoplusia ni Insect Larvae -
Author(s) : Platteborze PL , Lockridge O , Broomfield CA
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :150 , 1998

Title : Reaction of Human Butyrylcholinesterase (BChE) H117 Enzymes with Carbamates -
Author(s) : Broomfield CA , Mills KV , Meier BM , Lockridge O , Millard CB
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :223 , 1998

Title : The pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants: Further Evidence for a Push-Pull Mechanism - Saxena_1998_Biochemistry_37_15086
Author(s) : Saxena A , Viragh C , Frazier DS , Kovach IM , Maxwell DM , Lockridge O , Doctor BP
Ref : Biochemistry , 37 :15086 , 1998
Abstract : Bimolecular rate constants for the inactivation of recombinant (r) human (Hu) butyrylcholinesterase (BChE) with P(S)C(S)- and P(S)C(R)-2-(3,3-dimethylbutyl) methylphosphonofluoridate (soman) are (92 +/- 7) x 10(6) M-1 min-1 and (13.7 +/- 0.8) x 10(6) M-1 min-1 at pH 7.4, mu = 0.1 M and 25 degreesC. Mutations of E197(199) to D or Q and W82(84) to A result in reductions in the rate constants for inactivation with P(S)C(S)-soman 4.3-, 11.8-, and 263-fold and with P(S)C(R)-soman by 6.5-, 47.3-, and 685-fold, respectively. The pH dependence of dealkylation (aging) in r mouse (Mo) acetylcholinesterase (AChE) and rHu BChE and their mutants inactivated with P(S)C(S)- and P(S)C(R)-soman was compared. Best-fit parameters for the asymmetric bell curves for the adducts of wild-type Mo AChE are pK1 = pK2 = 4.0-4.9 and pK3 = 5.2-6.6. These pKs are consistent with the involvement of two carboxylic acids, possibly E202(199) and either E334(327) or E450(443), and H447(440)H+ in the dealkylation of AChE. E202Q MoAChE inactivated with the soman diastereomers yielded pK3 = 5.5-5.8. Nearly symmetric pH curves for soman-inhibited wild-type and E197D Hu BChE gave pK2 = 3.7-4.6 and pK3 = 7.3-8.0, but much lower, pK3 approximately 5, for the corresponding adduct of the E197Q mutant. Dealkylation in soman-inhibited BChE is consistent with the participation of one carboxylic acid side chain and H438(440)H+. Maximal rate constants for dealkylation (kmax) are 1-6 min-1 for AChE and 2 min-1 for BChE at 25 degreesC. The W82 to A mutation in BChE results in the largest reduction, 2500-6000-fold, in the rate constant for dealkylation. The reduction in the rate constants for dealkylation in the E197 mutants is highly pH dependent. The solvent isotope effects at the pH maxima are 1.3-1.4, indicating unlikely preprotonation or proton in "flight" at the enzymic transition states. The new results support the push-pull mechanism of dealkylation in soman-inhibited cholinesterases proposed previously.
ESTHER : Saxena_1998_Biochemistry_37_15086
PubMedSearch : Saxena_1998_Biochemistry_37_15086
PubMedID: 9790671

Title : A Comparison of Blood Cholinesterase Activities, Pyridostigmine Inhibition of Red Cell Acetylcholinesterase, and Butyrylcholinesterase Phenotypes in Gulf War Veterans and Normal Controls -
Author(s) : Gentry MK , Powell S , Bitsko N , Bartels CF , Bartko J , Chung R , Lockridge O , Ribas J , Roy M , Malone J , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :300 , 1998

Title : Resistance of butyrylcholinesterase to inactivation by ultrasound: effects of ultrasound on catalytic activity and subunit association - Froment_1998_Biochim.Biophys.Acta_1387_53
Author(s) : Froment MT , Lockridge O , Masson P
Ref : Biochimica & Biophysica Acta , 1387 :53 , 1998
Abstract : The effects of 20 kHz ultrasound on catalytic activity and structure of the tetramer of wild-type human butyrylcholinesterase (BChE) from plasma and recombinant D70G mutant enzyme were studied at constant temperature. Effects on catalytic properties of both enzymes were investigated by kinetic analysis under ultrasound irradiation using a neutral substrate (o-nitrophenylbutyrate), a positively charged substrate (butyrylthiocholine), and a negatively charged substrate (aspirin). Effects on structure of highly purified wild-type BChE were followed by gel electrophoresis and activity measurements at Vmax after ultrasound treatment. Unlike hydrostatic pressure, mild ultrasound had moderate effects on catalytic parameters of BChE-catalyzed hydrolysis of substrates. For both wild-type and D70G, Km increased slightly with butyrylthiocholine and o-nitrophenylbutyrate under ultrasound irradiation, suggesting that these effects of ultrasound were not due to the periodic variation of pressure but rather to shear forces that took off substrate from the peripheral site and altered diffusion to the active site. By contrast, affinity of the D70G mutant for aspirin slightly increased with ultrasound power, suggesting that ultrasound-induced microstreaming unmasked peripheral residues involved in recognition and initial binding of the negatively charged substrate. Results support the contention that Km is a composite affinity constant, including dissociation constant of the first encounter enzyme-substrate complex on the peripheral site. Small changes in catalytic activity may have resulted from ultrasound-induced subtle conformational changes altering the active site reactivity. Short ultrasound irradiation induced a faint transient enzyme activation, but prolonged irradiation caused partial dissociation of the tetrameric enzyme and irreversible inactivation. Partial dissociation was related to enzyme microheterogeneity, i.e., nicked (C-terminal segment depleted) tetramers were less stable than native tetramers. The resistance of the native tetramer to ultrasound-induced dissociation was ascribed to the existence of an aromatic amino acid array on the apolar side of the C-terminal helical segment of subunits, the four subunits being held together in a four-helix bundle containing the aromatic zipper motifs. Aromatic/aromatic interactions between the four helical segments are thought to be enhanced by ultrasound-generated pressure.
ESTHER : Froment_1998_Biochim.Biophys.Acta_1387_53
PubMedSearch : Froment_1998_Biochim.Biophys.Acta_1387_53
PubMedID: 9748500

Title : Butyrylcholinesterase-catalysed hydrolysis of aspirin, a negatively charged ester, and aspirin-related neutral esters - Masson_1998_Biochim.Biophys.Acta_1387_41
Author(s) : Masson P , Froment MT , Fortier PL , Visicchio JE , Bartels CF , Lockridge O
Ref : Biochimica & Biophysica Acta , 1387 :41 , 1998
Abstract : Although aspirin (acetylsalicylic acid) is negatively charged, it is hydrolysed by butyrylcholinesterase (BCHE). Catalytic parameters were determined in 100 mM Tris buffer, pH 7.4, in the presence and absence of metal cations. The presence of Ca2+ or Mg2+ (<100 mM) in buffer did not change the Km, but accelerated the rate of hydrolysis of aspirin by wild-type or D70G mutant BCHE by 5-fold. Turnover numbers were of the order of 5000-12000 min-1 for the wild-type enzyme and the D70G and D70K enzymes in 100 mM Tris, pH 7.4, containing 50 mM CaCl2 at 25 degreesC; Km values were 6 mM for wild-type, 16 mM for D70G and 38 mM for D70K. People with 'atypical' BCHE have the D70G mutation. The apparent inhibition seen at high aspirin concentration was not due to inhibition by excess substrate but to spontaneous hydrolysis of aspirin, causing inhibition by salicylate. The wild-type and D70G enzymes were competitively inhibited by salicylic acid; the D70K enzyme showed a complex parabolic inhibition, suggesting multiple binding. The effect of salicylate was substrate-dependent, the D70K mutant being activated by salicylate with butyrylthiocholine as substrate. Km value for wild-type enzyme was lower than for D70 mutants, suggesting that residue 70 located at the rim of the active site gorge was not the major site for the initial encounter aspirin-BCHE complex. On the other hand, the virtual absence of affinity of the W82A mutant for aspirin indicated that W82 was the major residue involved in formation of the Michaelis complex. Molecular modelling of aspirin binding to BCHE indicated perpendicular interactions between the aromatic rings of W82 and aspirin. Kinetic study of BCHE-catalysed hydrolysis of different acetyl esters showed that the rate limiting step was acetylation. The bimolecular rate constants for hydrolysis of aspirin by wild-type, D70G and D70K enzymes were found to be close to 1x106 M-1 min-1. These results support the contention that the electrostatic steering due to the negative electrostatic field of the enzyme plays a role in substrate binding, but plays no role in the catalytic steps, i.e. in the enzyme acetylation.
ESTHER : Masson_1998_Biochim.Biophys.Acta_1387_41
PubMedSearch : Masson_1998_Biochim.Biophys.Acta_1387_41
PubMedID: 9748494

Title : pH Dependence of Dealkylation in Soman-Inhibited Cholinesterases and Their Mutants -
Author(s) : Viragh C , Saxena A , Frazier DS , Kovach IM , Lockridge O , Doctor BP
Ref : In: Structure and Function of Cholinesterases and Related Proteins - Proceedings of Sixth International Meeting on Cholinesterases , (Doctor, B.P., Taylor, P., Quinn, D.M., Rotundo, R.L., Gentry, M.K. Eds) Plenum Publishing Corp. :247 , 1998

Title : Organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase: synergy results in a somanase - Millard_1998_Biochemistry_37_237
Author(s) : Millard CB , Lockridge O , Broomfield CA
Ref : Biochemistry , 37 :237 , 1998
Abstract : Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.
ESTHER : Millard_1998_Biochemistry_37_237
PubMedSearch : Millard_1998_Biochemistry_37_237
PubMedID: 9425044
Gene_locus related to this paper: human-BCHE

Title : Differences in active site gorge dimensions of cholinesterases revealed by binding of inhibitors to human butyrylcholinesterase - Saxena_1997_Biochemistry_36_14642
Author(s) : Saxena A , Redman AM , Jiang X , Lockridge O , Doctor BP
Ref : Biochemistry , 36 :14642 , 1997
Abstract : Amino acid sequence alignments of cholinesterases revealed that 6 of 14 aromatic amino acid residues lining the active center gorge of acetylcholinesterase are replaced by aliphatic amino acid residues in butyrylcholinesterase. The Y337 (F330) in mammalian acetylcholinesterase, which is replaced by A328 in human butyrylcholinesterase, is implicated in the binding of ligands such as huperzine A, edrophonium, and acridines and one end of bisquaternary compounds such as BW284C51 and decamethonium. Y337 may sterically hinder the binding of phenothiazines such as ethopropazine, which contains a bulky exocyclic substitution. Inhibition studies of (-)-huperzine A with human butyrylcholinesterase mutants, where A328 (KI = 194.6 microM) was modified to either F (KI = 0.6 microM, as in Torpedo acetylcholinesterase) or Y (KI = 0.032 microM, as in mammalian acetylcholinesterase), confirmed previous observations made with acetylcholinesterase mutants that this residue is important for binding huperzine A. Inhibition studies of ethopropazine with butyrylcholinesterase mutants, where A328 (KI = 0.18 microM) was modified to either F (KI = 0.82 microM) or Y (KI = 0.28 microM), suggested that A328 was not solely responsible for the selectivity of ethopropazine. Volume calculations for the active site gorge showed that the poor inhibitory activity of ethopropazine toward acetylcholinesterase was due to the smaller dimension of the active site gorge which was unable to accommodate the bulky inhibitor molecule. The volume of the butyrylcholinesterase active site gorge is approximately 200 A3 larger than that of the acetylcholinesterase gorge, which allows the accommodation of ethopropazine in two different orientations as demonstrated by rigid-body refinement and molecular dynamics calculations.
ESTHER : Saxena_1997_Biochemistry_36_14642
PubMedSearch : Saxena_1997_Biochemistry_36_14642
PubMedID: 9398183

Title : Tetramerization domain of human butyrylcholinesterase is at the C- terminus - Blong_1997_Biochem.J_327_747
Author(s) : Blong RM , Bedows E , Lockridge O
Ref : Biochemical Journal , 327 :747 , 1997
Abstract : Butyrylcholinesterase (BChE) in human serum consists predominantly of tetramers. Recombinant BChE, however, expressed in Chinese hamster ovary (CHO) cells, consists of approx. 55% dimers, 10-30% tetramers and 15-40% monomers. To determine the origin of the monomer species we added the FLAG epitope (epitope tag, amino acid sequence DYKDDDDK) to the C-terminus of the enzyme, and expressed BChE-FLAG in CHO cells. We found that secreted, active monomers had lost their FLAG epitope, suggesting that the monomers were made by proteolysis of dimers or tetramers at the C-terminus. To estimate the number of amino acids that could be deleted from the C-terminus without losing BChE activity, we expressed deletion mutants. We found that deletion of up to 50 amino acids from the C-terminus yielded active monomers, but that deletion of 51 amino acids destroyed BChE activity and caused the inactive protein to remain within the cell. Deletion of eight or more amino acids from the N-terminus also resulted in inactive protein that remained inside the cell. Monomeric BChE had wild-type Km and kcat values (8 microM and 24000 min-1 for butyrylthiocholine) and showed substrate activation. The Cys-571-->Ala mutant, though incapable of forming the interchain disulphide bond, had nearly the same amount of tetrameric BChE as recombinant wild-type BChE. These results support the conclusion that the tetramerization domain of BChE is at the C-terminus, within the terminal 50 amino acids, and that the interchain disulphide bond is not essential for tetramerization. Molecular modelling suggested that the tetramerization domain was a four-helix bundle, stabilized by interactions of seven conserved aromatic amino acids.
ESTHER : Blong_1997_Biochem.J_327_747
PubMedSearch : Blong_1997_Biochem.J_327_747
PubMedID: 9581552

Title : A single amino acid substitution, Gly117His, confers phosphotriesterase (organophosphorus acid anhydride hydrolase) activity on human butyrylcholinesterase - Lockridge_1997_Biochemistry_36_786
Author(s) : Lockridge O , Blong RM , Masson P , Froment MT , Millard CB , Broomfield CA
Ref : Biochemistry , 36 :786 , 1997
Abstract : The G117H mutant of human butyrylcholinesterase (EC was expressed in Chinese hamster ovary cells. Substitution of Gly 117 with His to make the G117H mutant endowed butyrylcholinesterase with the ability to catalyze the hydrolysis of organophosphate esters. G117H was still able to hydrolyze butyrylthiocholine, benzoylcholine, and o-nitrophenyl butyrate, but in addition it had acquired the ability to hydrolyze the antiglaucoma drug echothiophate and the pesticide paraoxon. Wild-type butyrylcholinesterase was irreversibly inhibited by echothiophate and paraoxon, but G117H regained 100% activity within 2-3 min following reaction with these compounds. On a polyacrylamide gel, the same bands that stained for activity with butyrylthiocholine also stained for activity with echothiophate. G117H is the only enzyme known that hydrolyzes echothiophate. Diethoxyphosphorylated G117H aged with a half-time of 5.5 h, a rate 600 times slower than the rate of hydrolysis. Echothiophate and paraoxon were hydrolyzed with the same kcat of 0.75 min-1. This calculates to a rate acceleration of 100,000-fold for hydrolysis of echothiophate and paraoxon by the G117H mutant compared to the nonenzymatic rate.
ESTHER : Lockridge_1997_Biochemistry_36_786
PubMedSearch : Lockridge_1997_Biochemistry_36_786
PubMedID: 9020776

Title : Importance of aspartate-70 in organophosphate inhibition, oxime re-activation and aging of human butyrylcholinesterase - Masson_1997_Biochem.J_325_53
Author(s) : Masson P , Froment MT , Bartels CF , Lockridge O
Ref : Biochemical Journal , 325 :53 , 1997
Abstract : Asp-70 is the defining amino acid in the peripheral anionic site of human butyrylcholinesterase (BCHE), whereas acetylcholinesterase has several additional amino acids, the most important one being Trp-277 (Trp-279 in Torpedo AChE). We studied mutants D70G, D70K and A277W to evaluate the role of Asp-70 and Trp-277 in reactions with organophosphates. We found that Asp-70 was important for binding positively charged echothiophate, but not neutral paraoxon and iso-OMPA. Asp-70 was also important for binding of positively charged pralidoxime (2-PAM) and for activation of re-activation by excess 2-PAM. Excess 2-PAM had an effect similar to substrate activation, suggesting the binding of 2 mol of 2-PAM to wild-type but not to the D70G mutant. A surprising result was that Asp-70 was important for irreversible aging, the D70G mutant having a 3- and 8-fold lower rate of aging for paraoxon-inhibited and di-isopropyl fluorophosphate-inhibited BCHE. Mutants of Asp-70 had the same rate constants for phosphorylation and re-activation by 2-PAM as wild-type. The A277W mutant behaved like wild-type in all assays. Our results predict that people with the atypical (D70G) variant of BCHE will be more sensitive to the toxic effects of echothiophate, but will be equally sensitive to paraoxon and di-isopropyl fluorophosphate. People with the D70G mutation will be resistant to re-activation of their inhibited BCHE by 2-PAM, but this will be offset by the lower rate of irreversible aging of inhibited BCHE, allowing some regeneration by spontaneous hydrolysis.
ESTHER : Masson_1997_Biochem.J_325_53
PubMedSearch : Masson_1997_Biochem.J_325_53
PubMedID: 9224629

Title : Role of aspartate 70 and tryptophan 82 in binding of succinyldithiocholine to human butyrylcholinesterase - Masson_1997_Biochemistry_36_2266
Author(s) : Masson P , Legrand P , Bartels CF , Froment MT , Schopfer LM , Lockridge O
Ref : Biochemistry , 36 :2266 , 1997
Abstract : The atypical variant of human butyrylcholinesterase has Gly in place of Asp 70. Patients with this D70G mutation respond abnormally to the muscle relaxant succinyldicholine, experiencing hours of apnea rather than the intended 3 min. Asp 70 is at the rim of the active site gorge 12 A from the active site Ser 198. An unanswered question in the literature is why the atypical variant has a 10-fold increase in Km for compounds with a single positive charge but a 100-fold increase in Km for compounds with two positive charges. We mutated residues Asp 70, Trp 82, Trp 231, Glu 197, and Tyr 332 and expressed mutant enzymes in mammalian cells. Steady-state kinetic parameters for hydrolysis of butyrylthiocholine, benzoylcholine, succinyldithiocholine, and o-nitrophenyl butyrate were determined. The wild type and the D70G mutant had identical k(cat) values for all substrates. Molecular modeling and molecular dynamics suggested that succinyldicholine could bind in two consecutive orientations in the active site gorge; formation of one complex caused a conformational change in the omega loop involving Asp 70 and Trp 82. We propose the formation of three enzyme-substrate intermediates preceding the acyl-enzyme intermediate; kinetic data support this contention. Substrates with a single positive charge interact with Asp 70 just once, whereas substrates with two positive charges, for example succinyldithiocholine, interact with Asp 70 in two complexes, thus explaining the 10- and 100-fold increases in Km in the D70G mutant.
ESTHER : Masson_1997_Biochemistry_36_2266
PubMedSearch : Masson_1997_Biochemistry_36_2266
PubMedID: 9047329

Title : Aging of di-isopropyl-phosphorylated human butyrylcholinesterase - Masson_1997_Biochem.J_327_601
Author(s) : Masson P , Fortier PL , Albaret C , Froment MT , Bartels CF , Lockridge O
Ref : Biochemical Journal , 327 ( Pt 2) :601 , 1997
Abstract : Organophosphate-inhibited cholinesterases can be reactivated by nucleophilic compounds. Sometimes phosphylated (phosphorylated or phosphonylated) cholinesterases become progressively refractory to reactivation; this can result from different reactions. The most frequent process, termed 'aging', involves the dealkylation of an alkoxy group on the phosphyl moiety through a carbocation mechanism. In attempting to determine the amino acid residues involved in the aging of butyrylcholinesterase (BuChE), the human BuChE gene was mutated at several positions corresponding to residues located at the rim of the active site gorge and in the vicinity of the active site. Mutant enzymes were expressed in Chinese hamster ovary cells. Wild-type BuChE and mutants were inhibited by di-isopropylfluorophosphate at pH 8.0 and 25 degrees C. Di-isopropyl-phosphorylated enzymes were incubated with the nucleophilic oxime 2-pyridine aldoxime methiodide and their reactivatability was determined. Reactivatability was expressed by the first-order rate constant of aging and/or the half-life of aging (t12). The t12 was found to be of the order of 60 min for wild-type BuChE. Mutations on Glu-197 increased t12 60-fold. Mutation W82A increased t12 13-fold. Mutation D70G increased t12 8-fold. Mutations in the vicinity of the active site serine residue had either moderate or no effect on aging; t12 was doubled for F329C and F329A, increased only 4-fold for the double mutant A328G+F329S, and no change was observed for the A328G mutant, indicating that the isopropoxy chain to be dealkylated does not directly interact with Ala-328 and Phe-329. These results were interpreted by molecular modelling of di-isopropylphosphorylated wild-type and mutant enzymes. Molecular dynamics simulations indicated that the isopropyl chain that is lost interacted with Trp-82, suggesting that Trp-82 has a role in stabilizing the carbonium ion that is released in the dealkylation step. This study emphasized the important role of the Glu-197 carboxylate in stabilizing the developing carbocation, and the allosteric control of the dealkylation reaction by Asp-70. Indeed, although Asp-70 does not interact with the phosphoryl moiety, mutation D70G affects the rate of aging. This indirect control was interpreted in terms of change in the conformational state of Trp-82 owing to internal motions of the Omega loop (Cys-65-Cys-92) in the mutant enzyme.
ESTHER : Masson_1997_Biochem.J_327_601
PubMedSearch : Masson_1997_Biochem.J_327_601
PubMedID: 9359435

Title : Asp7O in the peripheral anionic site of human butyrylcholinesterase - Masson_1996_Eur.J.Biochem_235_36
Author(s) : Masson P , Froment MT , Bartels CF , Lockridge O
Ref : European Journal of Biochemistry , 235 :36 , 1996
Abstract : The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though kcat was the same for wild-type and D70G mutant, being 24000 min(-1) at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.
ESTHER : Masson_1996_Eur.J.Biochem_235_36
PubMedSearch : Masson_1996_Eur.J.Biochem_235_36
PubMedID: 8631355

Title : Butyrylcholinesterase Transcription Start Site and Promoter -
Author(s) : Jbilo O , Toutant JP , Chatonnet A , Lockridge O
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :23 , 1995

Title : Acetylcholinesterase Gene Sequence and Copy Number are Normal in Alzheimer's Disease Patients Treated with the Organophosphate Metrifonate -
Author(s) : Bartels CF , Moriearty PL , Becker RE , Mountjoy CP , Lockridge O
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :479 , 1995

Title : Subunit Association and Stabilization of Butyrylcholinesterase (BChE) -
Author(s) : Blong RM , Masson P , Lockridge O
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :129 , 1995

Title : Reconsideration of the catalytic center and mechanism of mammalian paraoxonase\/arylesterase - Sorenson_1995_Proc.Natl.Acad.Sci.U.S.A_92_7187
Author(s) : Sorenson RC , Primo-Parmo SL , Kuo CL , Adkins S , Lockridge O , La Du BN
Ref : Proc Natl Acad Sci U S A , 92 :7187 , 1995
Abstract : For three decades, mammalian paraoxonase (A-esterase, aromatic esterase, arylesterase; PON, EC has been thought to be a cysteine esterase demonstrating structural and mechanistic homologies with the serine esterases (cholinesterases and carboxyesterases). Human, mouse, and rabbit PONs each contain only three cysteine residues, and their positions within PON have been conserved. In purified human PON, residues Cys-41 and Cys-352 form an intramolecular disulfide bond and neither could function as an active-center cysteine. Highly purified, enzymatically active PON contains a single titratable sulfhydryl group. Thus, Cys-283 is the only probable candidate for an active-center cysteine. Through site-directed mutagenesis of the human cDNA, Cys-283 was replaced with either serine (C283S) or alanine (C283A). The expressed C283 (wild-type) enzyme was inactivated by para-hydroxymercuribenzoate, but the C283S and C283A mutant enzymes were not inactivated. C283A and C283S mutant enzymes retained both paraoxonase and arylesterase activities, and the Km values for paraoxon and phenyl acetate were similar to those of the wild type. Clearly, residue Cys-283 is free in active PON, but a free sulfhydryl group is not required for either paraoxonase or arylesterase activities. Consequently, it is necessary to examine other models for the active-site structure and catalytic mechanism of PON.
ESTHER : Sorenson_1995_Proc.Natl.Acad.Sci.U.S.A_92_7187
PubMedSearch : Sorenson_1995_Proc.Natl.Acad.Sci.U.S.A_92_7187
PubMedID: 7638166

Title : Design and expression of organophosphorus acid anhydride hydrolase activity in human butyrylcholinesterase - Millard_1995_Biochemistry_34_15925
Author(s) : Millard CB , Lockridge O , Broomfield CA
Ref : Biochemistry , 34 :15925 , 1995
Abstract : Serine esterases and proteases are rapidly and irreversibly inhibited by organophosphorus (OP) nerve agents. To overcome this limitation, we selected several residues that were predicted to be within 3-10 A of both the active site Ser O gamma and the oxyanion hole of human butyrylcholinesterase for mutation to His (G115H, G117H, Q119H, and G121H). In remarkable contrast with wild-type (WT) and all other His mutants tested, G117H underwent spontaneous reactivation following OP inhibition to regain 100% of original esterase activity with maximum k3 values of approximately 6.8 x 10(-5) and 16 x 10(-5) s-1 for GB (sarin) and VX, respectively, in 0.1 M Bis-Tris, 25 degrees C. The free energy of activation for k3 was 19 kcal mol-1, and measurement of pH dependence suggested that reactivation resulted from an acidic group with pKa 6.2. To evaluate further the importance of His in achieving this result, we changed the same Gly to Lys (G117K) and compared its substrate and inhibitor kinetics with those of G117H. Both mutants retained esterase activity with Km values similar to those of WT for neutral ester hydrolysis, but G117K did not reactivate. Complete reactivation proves that G117H is not irreversibly inhibited but instead functions as a catalyst for OP hydrolysis. Dephosphonylation is the rate-limiting step, and G117H effects overall rate constant enhancements of approximately 100- and 2000-fold above the uncatalyzed hydrolysis of GB and VX, respectively, at pH 6.0, 25.0 degrees C. We conclude that an appropriately positioned imidazolium ion in the oxyanion hole catalyzes dephosphonylation and, thereby, confers a novel organophosphorus acid anhydride hydrolase activity upon butyrylcholinesterase.
ESTHER : Millard_1995_Biochemistry_34_15925
PubMedSearch : Millard_1995_Biochemistry_34_15925
PubMedID: 8519749

Title : Mutation of Human Butyrylcholinesterase Glycine 117 to Histidine Preserves Activity but Confers Resistance to Organophosphorus Inhibitors -
Author(s) : Broomfield CA , Millard CB , Lockridge O , Caviston TL
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :169 , 1995

Title : Peripheral Anionic Site of Wild-Type and Mutant Human Butyrylcholinesterase -
Author(s) : Masson P , Froment MT , Bartels CF , Lockridge O
Ref : In Enzyme of the Cholinesterase Family - Proceedings of Fifth International Meeting on Cholinesterases , (Quinn, D.M., Balasubramanian, A.S., Doctor, B.P., Taylor, P., Eds) Plenum Publishing Corp. :230 , 1995

Title : Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA - Jbilo_1994_Toxicon_32_1445
Author(s) : Jbilo O , Bartels CF , Chatonnet A , Toutant JP , Lockridge O
Ref : Toxicon , 32 :1445 , 1994
Abstract : 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.
ESTHER : Jbilo_1994_Toxicon_32_1445
PubMedSearch : Jbilo_1994_Toxicon_32_1445
PubMedID: 7886701

Title : Bacterial expression and in vitro folding of the beta-subunit of human chorionic gonadotropin (hCG beta) and functional assembly of recombinant hCG beta with hCG alpha - Huth_1994_Endocrinology_135_911
Author(s) : Huth JR , Norton SE , Lockridge O , Shikone T , Hsueh AJ , Ruddon RW
Ref : Endocrinology , 135 :911 , 1994
Abstract : A bacterial expression system for the beta-subunit of hCG (hCG beta) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG beta in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG beta complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG beta (rhCG beta) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG beta were recovered from 1 liter culture, rhCG beta was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCG alpha and