Shafferman A

General

Full name : Shafferman Avigdor

First name : Avigdor

Mail : Israel Institute for Biological Research\; POB 19\; Ness Ziona 74100

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Country : Israel

Email : avigdors@iibr.gov.il

Phone : +97289381595

Fax : 972-8-401404

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References (102)

Title : Progress and novel strategies in vaccine development and treatment of anthrax - Chitlaru_2011_Immunol.Rev_239_221
Author(s) : Chitlaru T , Altboum Z , Reuveny S , Shafferman A
Ref : Immunol Rev , 239 :221 , 2011
Abstract : The lethal anthrax disease is caused by spores of the gram-positive Bacillus anthracis, a member of the cereus group of bacilli. Although the disease is very rare in the Western world, development of anthrax countermeasures gains increasing attention due to the potential use of B. anthracis spores as a bio-terror weapon. Protective antigen (PA), the non-toxic subunit of the bacterial secreted exotoxin, fulfills the role of recognizing a specific receptor and mediating the entry of the toxin into the host target cells. PA elicits a protective immune response and represents the basis for all current anthrax vaccines. Anti-PA neutralizing antibodies are useful correlates for protection and for vaccine efficacy evaluation. Post exposure anti-toxemic and anti-bacteremic prophylactic treatment of anthrax requires prolonged antibiotic administration. Shorter efficient postexposure treatments may require active or passive immunization, in addition to antibiotics. Although anthrax is acknowledged as a toxinogenic disease, additional factors, other than the bacterial toxin, may be involved in the virulence of B. anthracis and may be needed for the long-lasting protection conferred by PA immunization. The search for such novel factors is the focus of several high throughput genomic and proteomic studies that are already leading to identification of novel targets for therapeutics, for vaccine candidates, as well as biomarkers for detection and diagnosis.
ESTHER : Chitlaru_2011_Immunol.Rev_239_221
PubMedSearch : Chitlaru_2011_Immunol.Rev_239_221
PubMedID: 21198675

Title : Next generation OP-bioscavengers: a circulatory long-lived 4-PEG hypolysine mutant of F338A-HuAChE with optimal pharmacokinetics and pseudo-catalytic characteristics - Kronman_2010_Chem.Biol.Interact_187_253
Author(s) : Kronman C , Cohen O , Mazor O , Ordentlich A , Raveh L , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 187 :253 , 2010
Abstract : We have shown previously that conjugation of polyethylene glycol (PEG) chains to recombinant human acetylcholinesterase (rHuAChE) results in the extension of its residence time in the circulation of mice and monkeys [1,2]. By profiling the pharmacokinetic behavior of an array of well-defined hypolysine human mutant AChE molecules following PEGylation, we now determine that the duration of these enzyme forms in the circulation of rhesus macaques correlates with their number of appended PEG moieties, and is influenced by the actual location of the PEG chains at the molecule surface, as well. These findings, which concur with those we have previously established in mice, indicate that a common set of rules dictates the circulatory fate of PEGylated HuAChEs in rodents and non-human primates. In addition to its effect on circulatory residence, PEGylation reduces the ability of the rHuAChE bioscavenger to elicit an immune response in the heterologous mouse animal system. Thus, an inverse relationship between anti-AChE antibody production and PEG loading was observed following repeated administration of the different PEGylated hypolysine human AChEs to mice. We note however, that in rhesus macaques, the essentially homologous (human) AChE does not induce specific anti-AChE antibodies after repeated administration of high doses of the enzyme in its PEGylated form, and even in its non-PEGylated form. Taken together, these findings indicate that PEG acts by veiling enzyme-related epitopes, which would otherwise interact with host circulatory elimination pathways and immune system. The barring of such interactions by obstructive PEGs, confers the enzyme molecule with both extended circulatory residence and mitigated immunogenic properties. Further modulation by incorporation of the F338A mutation into the PEGylated hypolysine rHuAChE enzyme mold, resulted in the generation of an OP-bioscavenger that displayed reduced aging rates and could effectively protect mice against repeated exposure to CW agents. This selected 4-PEG F338A-AChE can serve as a paradigm for new generation OP-bioscavengers, specifically tailored for prophylactic treatment against toxic OP-agents.
ESTHER : Kronman_2010_Chem.Biol.Interact_187_253
PubMedSearch : Kronman_2010_Chem.Biol.Interact_187_253
PubMedID: 20005217

Title : Postexposure immunization with modified vaccinia virus Ankara or conventional Lister vaccine provides solid protection in a murine model of human smallpox - Paran_2009_J.Infect.Dis_199_39
Author(s) : Paran N , Suezer Y , Lustig S , Israely T , Schwantes A , Melamed S , Katz L , Preuss T , Hanschmann KM , Kalinke U , Erez N , Levin R , Velan B , Lower J , Shafferman A , Sutter G
Ref : J Infect Dis , 199 :39 , 2009
Abstract : BACKGROUND: Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain.
METHODS: We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV).
RESULTS: In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses.
CONCLUSIONS: ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.
ESTHER : Paran_2009_J.Infect.Dis_199_39
PubMedSearch : Paran_2009_J.Infect.Dis_199_39
PubMedID: 19012492

Title : Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response - Lustig_2009_Vaccine_27_1691
Author(s) : Lustig S , Maik-Rachline G , Paran N , Melamed S , Israely T , Erez N , Orr N , Reuveny S , Ordentlich A , Laub O , Shafferman A , Velan B
Ref : Vaccine , 27 :1691 , 2009
Abstract : The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.
ESTHER : Lustig_2009_Vaccine_27_1691
PubMedSearch : Lustig_2009_Vaccine_27_1691
PubMedID: 19195492

Title : Accommodation of physostigmine and its analogues by acetylcholinesterase is dominated by hydrophobic interactions - Barak_2009_Biochem.J_417_213
Author(s) : Barak D , Ordentlich A , Stein D , Yu QS , Greig NH , Shafferman A
Ref : Biochemical Journal , 417 :213 , 2009
Abstract : The role of the functional architecture of the HuAChE (human acetylcholinesterase) in reactivity toward the carbamates pyridostigmine, rivastigmine and several analogues of physostigmine, that are currently used or considered for use as drugs for Alzheimer's disease, was analysed using over 20 mutants of residues that constitute the interaction subsites in the active centre. Both steps of the HuAChE carbamylation reaction, formation of the Michaelis complex as well as the nucleophilic process, are sensitive to accommodation of the ligand by the enzyme. For certain carbamate/HuAChE combinations, the mode of inhibition shifted from a covalent to a noncovalent type, according to the balance between dissociation and covalent reaction rates. Whereas the charged moieties of pyridostigmine and rivastigmine contribute significantly to the stability of the corresponding HuAChE complexes, no such effect was observed for physostigmine and its analogues, phenserine and cymserine. Moreover, physostigmine-like ligands carrying oxygen instead of nitrogen at position -1 of the tricyclic moiety (physovenine and tetrahydrofurobenzofuran analogues) displayed comparable structure-function characteristics toward the various HuAChE enzymes. The essential role of the HuAChE hydrophobic pocket, comprising mostly residues Trp(86) and Tyr(337), in accommodating (-)-physostigmine and in conferring approximately 300-fold stereoselectivity toward physostigmines, was elucidated through examination of the reactivity of selected HuAChE mutations toward enantiomeric pairs of different physostigmine analogues. The present study demonstrates that certain charged and uncharged ligands, like analogues of physostigmine and physovenine, seem to be accommodated by the enzyme mostly through hydrophobic interactions.
ESTHER : Barak_2009_Biochem.J_417_213
PubMedSearch : Barak_2009_Biochem.J_417_213
PubMedID: 18729824

Title : Flexibility versus rigidity of the functional architecture of AChE active center - Shafferman_2008_Chem.Biol.Interact_175_166
Author(s) : Shafferman A , Barak D , Stein D , Kronman C , Velan B , Greig NH , Ordentlich A
Ref : Chemico-Biological Interactions , 175 :166 , 2008
Abstract : Functional architecture of the AChE active center appears to be characterized by both structural "rigidity", necessary to stabilize the catalytic triad as well as by flexibility in accommodating the different, high affinity AChE ligands. These seemingly conflicting structural properties of the active center are demonstrated through combination of structural methods with kinetic studies of the enzyme and its mutant derivatives with plethora of structurally diverse ligands and in particular with series of stereoselective covalent and noncovalent AChE ligands. Thus, steric perturbation of the acyl pocket precipitates in a pronounced stereoselectivity toward methylphosphonates by disrupting the stabilizing environment of the catalytic histidine rather than through steric exclusion demonstrating the functional importance of the "rigid" environment of the catalytic machinery. The acyl pocket, the cation-binding subsite (Trp86) and the peripheral anionic subsite were also found to be directly involved in HuAChE stereoselectivity toward charged chiral phosphonates, operating through differential positioning of the ligand cationic moiety within the active center. Residue Trp86 is also a part of the "hydrophobic patch" which seems flexible enough to accommodate the structurally diverse ligands like tacrine, galanthamine and the two diastereomers of huperzine A. Also, we have recently discovered further aspects of the role of both the unique structure and the flexibility of the "hydrophobic patch" in determining the reactivity and stereoselectivity of HuAChE toward certain carbamates including analogs of physostigmine. In these cases the ligands are accommodated mostly through hydrophobic interactions and their stereoselectivity delineates precisely the steric limits of the pocket. Hence, the HuAChE stereoselectivity provides a sensitive tool in the in depth exploration of the functional architecture of the active center. These studies suggest that the combination of "rigidity" and flexibility within the HuAChE gorge are an essential element of its molecular design.
ESTHER : Shafferman_2008_Chem.Biol.Interact_175_166
PubMedSearch : Shafferman_2008_Chem.Biol.Interact_175_166
PubMedID: 18471807

Title : Aging-resistant organophosphate bioscavenger based on polyethylene glycol-conjugated F338A human acetylcholinesterase - Mazor_2008_Mol.Pharmacol_74_755
Author(s) : Mazor O , Cohen O , Kronman C , Raveh L , Stein D , Ordentlich A , Shafferman A
Ref : Molecular Pharmacology , 74 :755 , 2008
Abstract : The high reactivity of cholinesterases (ChEs) toward organophosphorus (OP) compounds has led to propose recombinant ChEs as bioscavengers of nerve agents. The bioscavenging potential of recombinant ChEs can be enhanced by conjugation of polyethylene glycol (PEG) moieties, to extend their circulatory residence. However, the ability of exogenously administered ChEs to confer long-term protection against repeated exposures to nerve agents is still limited due to the aging process, whereby organophosphate-ChE adducts undergo irreversible dealkylation, which precludes oxime-mediated reactivation of the enzyme. To generate an optimal acetylcholinesterase (AChE)-based OP bioscavenger, the F338A mutation, known to decelerate the rate of aging of AChE-OP conjugates, was incorporated into polyethylene glycol-conjugated (PEGylated) human AChE. The PEGylated F338A-AChE displayed unaltered rates of hydrolysis, inhibition, phosphylation, and reactivation and could effectively protect mice against exposure to soman (pinacolylmethyl phosphonofluoridate), sarin (O-isopropyl methylphosphonofluoridate), or O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothioate (VX). Unlike PEGylated wild-type (WT)-AChE, the PEGylated F338A-AChE exhibits significantly reduced aging rates after soman inhibition and can be efficiently reactivated by the 1-[[[4(aminocarbonyl)-pyridinio]methoxy]methyl]-2(hydroxyimino)methyl]pyridinium dichloride (HI-6) oxime, both in vitro and in vivo. Accordingly, oxime administration to PEG-F338A-AChE-pretreated mice enabled them to withstand repeated soman exposure (5.4 and 4 LD(50)/dose), whereas same regime treatment of non-PEGylated F338A-AChE- or PEGylated WT-AChE-pretreated mice failed to protect against the second challenge, due to rapid clearance or irreversible aging of the latter enzymes. Thus, judicious incorporation of selected mutations into the AChE mold in conjunction with its chemical modification provides means to engineer an optimal ChE-based OP bioscavenger in terms of circulatory longevity, resistance to aging, and reduced doses required for protection, even against repeated exposures to nerve agents, such as soman.
ESTHER : Mazor_2008_Mol.Pharmacol_74_755
PubMedSearch : Mazor_2008_Mol.Pharmacol_74_755
PubMedID: 18523134

Title : Controlled concealment of exposed clearance and immunogenic domains by site-specific polyethylene glycol attachment to acetylcholinesterase hypolysine mutants - Cohen_2007_J.Biol.Chem_282_35491
Author(s) : Cohen O , Kronman C , Lazar A , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 282 :35491 , 2007
Abstract : Cholinesterases are efficient scavengers of organophosphates and are currently being developed as drugs for treatment against poisoning by such compounds. Recombinant ChE bioscavengers have very short circular longevity, a limitation that can be overcome by complex post-translation manipulations or by chemical modification such as polyethylene glycol conjugation. Series of multiple Lys-Ala mutants of human acetylcholinesterase were prepared allowing the generation of homogenous and well defined polyethylene-glycol conjugated AChEs with either one, two, three, four, or five appended polyethylene glycol (PEG) moieties/molecule. The rank order of circulatory longevity of these molecules was dependent on the number of PEG appendages up to a certain threshold: 5 = 4 > 3 > 2 > 1 > 0. Hypolysine acetylcholinesterases (AChEs) carrying the same number of PEGs, and therefore with identical masses, allowed us to demonstrate that circulatory longevity correlates with the predicted extent of concealment of the AChE surface. Furthermore, circulatory profiles of high number and low number PEG-AChEs differing in their sialic acid contents demonstrate a direct relationship between PEG loading and the effective seclusion of AChE from the hepatic asialoglycoprotein receptor clearance system. Finally, an inverse relationship is found between the extent of PEG loading and the ability of the human acetylcholinesterase to elicit specific anti-HuAChE antibodies. In conclusion, these findings suggest that for the extension of circulatory longevity, protein surface domain concealment exerted by polyethylene glycol attachment is at least as important as its effect on size enlargement and highlights the role of PEG attachment in masking interactions between biomolecules and their cognate receptors.
ESTHER : Cohen_2007_J.Biol.Chem_282_35491
PubMedSearch : Cohen_2007_J.Biol.Chem_282_35491
PubMedID: 17932038

Title : Polyethylene-glycol conjugated recombinant human acetylcholinesterase serves as an efficacious bioscavenger against soman intoxication - Kronman_2007_Toxicology_233_40
Author(s) : Kronman C , Cohen O , Raveh L , Mazor O , Ordentlich A , Shafferman A
Ref : Toxicology , 233 :40 , 2007
Abstract : Extensive pharmacokinetic studies in both mice and rhesus macaques, with biochemically well defined forms of native and recombinant AChEs from bovine, rhesus and human origin, allowed us to determine an hierarchical pattern by which post-translation-related factors and specific amino-acid epitopes govern the pharmacokinetic performance of the enzyme molecule. In parallel, we demonstrated that controlled conjugation of polyethylene-glycol (PEG) side-chains to lysine residues of rHuAChE also results in the generation of active enzyme with improved pharmacokinetic performance. Here, we show that equally efficient extension of circulatory residence can be achieved by specific conditions of PEGylation, regardless of the post-translation-modification state of the enzyme. The masking effect of PEGylation, which is responsible for extending circulatory lifetime, also contributes to the elimination of immunological responses following repeated administration of AChE. Finally, in vivo protection studies in mice allowed us to determine that the PEGylated AChE protects the animal from a high lethal dose (2.5 LD(50)) of soman. On a mole basis, both the recombinant AChE and its PEGylated form provide higher levels of protection against soman poisoning than the native serum-derived HuBChE. The findings that circulatory long-lived PEGylated AChE can confer superior protection to mice against OP-compound poisoning while exhibiting reduced immunogenicity, suggest that this chemically modified version of rHuAChE may serve as a highly effective bioscavenger for prophylactic treatment against OP-poisoning.
ESTHER : Kronman_2007_Toxicology_233_40
PubMedSearch : Kronman_2007_Toxicology_233_40
PubMedID: 17045722

Title : Inhibition of human acetyl- and butyrylcholinesterase by novel carbamates of (-)- and (+)-tetrahydrofurobenzofuran and methanobenzodioxepine - Luo_2006_J.Med.Chem_49_2174
Author(s) : Luo W , Yu QS , Kulkarni SS , Parrish DA , Holloway HW , Tweedie D , Shafferman A , Lahiri DK , Brossi A , Greig NH
Ref : Journal of Medicinal Chemistry , 49 :2174 , 2006
Abstract : A new enantiomeric synthesis utilizing classical resolution provided two novel series of optically active inhibitors of cholinesterase: (-)- and (+)-O-carbamoyl phenols of tetrahydrofurobenzofuran and methanobenzodioxepine. An additional two series of (-)- and (+)-O-carbamoyl phenols of pyrroloindole and furoindole were obtained by known procedures, and their anticholinesterase actions were similarly quantified against freshly prepared human acetyl- (AChE) and butyrylcholinesterase (BChE). Both enantiomeric forms of each series demonstrated potent cholinesterase inhibitory activity (with IC(50) values as low as 10 nM for AChE and 3 nM for BChE), with the exception of the (+)-O-carbamoyl phenols of pyrroloindole, which lacked activity (IC(50) values >1 microM). Based on the biological data of these four series, a structure-activity relationship (SAR) analysis was provided by molecular volume calculations. In addition, a probable transition-state model was established according to the known X-ray structure of a transition-state complex of Torpedo californica AChE-m-(N,N,N-trimethylammonio)-2,2,2-trifluoroacetophenone (TcAChE-TMTFA). This model proved valuable in explaining the enantioselectivity and enzyme subtype selectivity of each series. These carbamates are more potent than, or similarly potent to, anticholinesterases in current clinical use, providing not only inhibitors of potential clinical relevance but also pharmacological tools to define drug-enzyme binding interactions within an enzyme crucial in the maintenance of cognition and numerous systemic physiological functions in health, aging, and disease.
ESTHER : Luo_2006_J.Med.Chem_49_2174
PubMedSearch : Luo_2006_J.Med.Chem_49_2174
PubMedID: 16570913

Title : Comparison of polyethylene glycol-conjugated recombinant human acetylcholinesterase and serum human butyrylcholinesterase as bioscavengers of organophosphate compounds - Cohen_2006_Mol.Pharmacol_70_1121
Author(s) : Cohen O , Kronman C , Raveh L , Mazor O , Ordentlich A , Shafferman A
Ref : Molecular Pharmacology , 70 :1121 , 2006
Abstract : Comparative protection studies in mice demonstrate that on a molar basis, recombinant human acetylcholinesterase (rHuAChE) confers higher levels of protection than native human butyrylcholinesterase (HuBChE) against organophosphate (OP) compound intoxication. For example, mice challenged with 2.5 LD50 of O-isopropyl methylphosphonofluoridate (sarin), pinacolylmethyl phosphonofluoridate (soman), and O-ethyl-S-(2-isopropylaminoethyl) methylphosphonothiolate (VX) after treatment with equimolar amounts of the two cholinesterases displayed 80, 100, and 100% survival, respectively, when pre-treatment was carried out with rHuAChE and 0, 20, and 60% survival, respectively, when pretreatment was carried out with HuBChE. Kinetic studies and active site titration analyses of the tested OP compounds with acetylcholinesterases (AChEs) and butyrylcholinesterases (BChEs) from different mammalian species demonstrate that the superior in vivo efficacy of acetyl-cholinesterases is in accordance with the higher stereoselectivity of AChE versus BChE toward the toxic enantiomers comprising the racemic mixtures of the various OP agents. In addition, we show that polyethylene glycol-conjugated (PEGy-lated) rHuAChE, which is characterized by a significantly extended circulatory residence both in mice and monkeys ( Biochem J 357: 795-802, 2001 ; Biochem J 378: 117-128, 2004 ), retains full reactivity toward OP compounds both in vitro and in vivo and provides a higher level of protection to mice against OP poisoning, compared with native serum-derived HuBChE. Indeed, PEGylated rHuAChE also confers superior prophylactic protection when administered intravenously or intramuscularly over 20 h before exposure of mice to a lethal dose of VX (1.3-1.5 LD50). These findings together with the observations that the PEGylated rHuAChE exhibits unaltered biodistribution and high bioavailability present a case for using PEGylated rHuAChE as a very efficacious bioscavenger of OP agents.
ESTHER : Cohen_2006_Mol.Pharmacol_70_1121
PubMedSearch : Cohen_2006_Mol.Pharmacol_70_1121
PubMedID: 16801396

Title : The role of AChE active site gorge in determining stereoselectivity of charged and noncharged VX enantiomers - Ordentlich_2005_Chem.Biol.Interact_157-158_191
Author(s) : Ordentlich A , Barak D , Sod-Moriah G , Kaplan D , Mizrahi D , Segall Y , Kronman C , Karton Y , Lazar A , Marcus D , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 157-158 :191 , 2005
Abstract : The reactivity of human acetylcholinesterase (HuAChE) toward the chemical warfare agent VX [O-ethyl S-[2-(diisopropylamino)ethyl] methyl-phosphonothioate] and its stereoselectivity toward the P(S)-enantiomer were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere nc-VX [O-ethyl S-(3-isopropyl-4-methyl-pentyl) methylphosphonothioate]. Stereoselectivity of the wild-type HuAChE toward VX(S) is manifested by a 115-fold higher bimolecular rate constant (1.4 x 10(8) min(-1) M(-1)) as compared to that of VX(R). HuAChE was also 12,500-fold more reactive toward VX(S) than toward nc-VX(S), demonstrating the significance of the polar interactions of the ammonium substituent to their overall affinity toward VX. Indeed, substitution of the cation-binding subsite residue Trp86 by alanine resulted in a decrease of three orders of magnitude in HuAChE reactivity toward both VX enantiomers, with only a marginal effect on the reactivity toward the enantiomers of nc-VX. These results demonstrate that accommodation of the charged moieties of both VX enantiomers depends predominantly on interactions with the aromatic moiety of Trp86. Yet, these interactions seem to limit the stereoselectivity toward the P(S)-enantiomer, which for charged methylphosphonates is much lower than for the noncharged analogs, like sarin or soman. Marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 by asparagine (D74N) practically abolished stereoselectivity toward VX(S) (a 130-fold decrease), while substitution which retained the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers originates predominantly from a different orientation of the charged leaving group with respect to residue Asp74. Such different orientations of the charged leaving group in the HuAChE adducts of the VX enantiomers seem to be a consequence of intramolecular interactions with the bulky phosphorus alkoxy group.
ESTHER : Ordentlich_2005_Chem.Biol.Interact_157-158_191
PubMedSearch : Ordentlich_2005_Chem.Biol.Interact_157-158_191
PubMedID: 16289014

Title : Host-regulated disposition of mammalian AChEs - Kronman_2005_Chem.Biol.Interact_157-158_51
Author(s) : Kronman C , Cohen O , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 157-158 :51 , 2005
Abstract : Primates are characterized by a paucity of soluble acetylcholinesterase (AChE) in the circulation at the adult stage, where the predominant circulating cholinesterase is butyrylcholinesterase. In recent years, we subjected recombinant human and bovine acetylcholinesterase to extensive pharmacokinetic studies in mice, an animal system which also displays very low levels of circulating AChE. In this system, a post-translation-related hierarchical pattern governing circulatory residence through AChE sialylation, subunit tetramerization and glycan loading was elucidated. Based on these studies, coordinated modulation of the sialic acid contents, state of subunit assembly and number of glycans allowed us to generate human or bovine AChE forms which reside in the circulation of mice for long periods of time, mimicking the pharmacokinetic behavior of native serum-derived cholinesterases. However, extension of the pharmacokinetic studies to primates, revealed an additional element, which affects circulatory residence of AChEs in this animal system. Unlike in the case of bovine AChE, optimization of subunit assembly and glycan loading of the primate versions of AChE (human or rhesus) did not increase their circulatory lifetime in rhesus macaques. This differential pharmacokinetic behavior of bovine and primate AChEs in macaques appears to be related to the 35 diverging bovine/primate AChE amino acids which are clustered within three defined domains at the enzyme surface, and thereby may facilitate the specific removal of "self" or "self-like" cholinesterases from the circulation of monkeys and thus provide an explanation for the absence of soluble AChE in the circulation of primates.
ESTHER : Kronman_2005_Chem.Biol.Interact_157-158_51
PubMedSearch : Kronman_2005_Chem.Biol.Interact_157-158_51
PubMedID: 16289063

Title : Functional requirements for the optimal catalytic configuration of the AChE active center - Shafferman_2005_Chem.Biol.Interact_157-158_123
Author(s) : Shafferman A , Barak D , Kaplan D , Ordentlich A , Kronman C , Velan B
Ref : Chemico-Biological Interactions , 157-158 :123 , 2005
Abstract : Functional analysis of the HuAChE active center architecture revealed that accommodation of structurally diverse substrates and other ligands is achieved through interactions with specific subsites such as the acyl pocket, cation binding site, hydrophobic site or the oxyanion hole. Recent studies have begun to unravel the role of this active center architecture in maintaining the optimal catalytic facility of the enzyme through inducing proper alignment of the catalytic triad. The exact positioning of the catalytic glutamate (Glu334) seems to be determined by a hydrogen bond network including several polar residues and water molecules. Disruption of this network by replacement of Ser229 by alanine is thought to remove the Glu334 carboxylate from the vicinity of His447 abolishing catalytic activity. The proper orientation of the catalytic histidine side chain is maintained by these polar interactions as well as through "aromatic trapping" by residues lining the HuAChE active center gorge. Thus, replacement of aromatic residues in the vicinity of His447, as in the F295A/F338A or in the Y72N/Y124Q/W286A/F295L/F297V/Y337A (hexamutant which mimicks the aromatic lining of HuBChE) enzymes, resulted in a dramatic decrease in catalytic activity, which was proposed to originate from catalytically nonproductive mobility of His447. Yet, HuBChE is catalytically efficient indicating that "aromatic trapping" is not the only way to conformationally stabilize the His447 side chain. A possible restriction of this mobility in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Both simulations and reactivities of the actual F295X/F338A enzymes, carrying various aliphatic residues at position 295, indicate that of the bulky amino acids, like leucine or isoleucine, only methionine was capable of maintaining the catalytically viable conformation of His447. The F295M/F338A HuAChE was only two-fold less reactive than the F338A enzyme toward acetylthiocholine, and exhibited wild type-like reactivity toward covalent modifiers of the catalytic Ser203. The findings are consistent with the notion that different combinations of steric interference and specific polar interactions serve to maintain the position of His447 and thereby the high efficiency of the catalytic machinery. The two seemingly conflicting demands on the architecture of the active center-flexible accommodation of substrate and optimal juxtaposition of residues of the catalytic triad, demonstrate the truly amazing molecular design of the AChE active center.
ESTHER : Shafferman_2005_Chem.Biol.Interact_157-158_123
PubMedSearch : Shafferman_2005_Chem.Biol.Interact_157-158_123
PubMedID: 16256968

Title : Effects of spontaneous deamidation on the cytotoxic activity of the Bacillus anthracis protective antigen - Zomber_2005_J.Biol.Chem_280_39897
Author(s) : Zomber G , Reuveny S , Garti N , Shafferman A , Elhanany E
Ref : Journal of Biological Chemistry , 280 :39897 , 2005
Abstract : Protective antigen (PA) is a central virulence factor of Bacillus anthracis and a key component in anthrax vaccines. PA binds to target cell receptors, is cleaved by the furin protease, self-aggregates to heptamers, and finally internalizes as a complex with either lethal or edema factors. Under mild room temperature storage conditions, PA cytotoxicity decreased (t(1/2) approximately 7 days) concomitant with the generation of new acidic isoforms, probably through deamidation of Asn residues. Ranking all 68 Asn residues in PA based on their predicted deamidation rates revealed five residues with half-lives of <60 days, and these residues were further analyzed: Asn10 in the 20-kDa region, Asn162 at P6 vicinal to the furin cleavage site, Asn306 in the pro-pore translocation loop, and both Asn713 and Asn719 in the receptor-binding domain. We found that PA underwent spontaneous deamidation at Asn162 upon storage concomitant with decreased susceptibility to furin. A panel of model synthetic furin substrates was used to demonstrate that Asn162 deamidation led to a 20-fold decrease in the bimolecular rate constant (k(cat)/Km) of proteolysis due to the new negatively charged residue at P6 in the furin recognition sequence. Furthermore, reduced PA cytotoxicity correlated with a decrease in PA cell binding and also with deamidation of Asn713 and Asn719. On the other hand, neither deamidation of Asn10 or Asn306 nor impairment of heptamerization could be observed upon prolonged PA storage. We suggest that PA inactivation during storage is associated with susceptible deamidation sites, which are intimately involved in both mechanisms of PA cleavage by furin and PA-receptor binding.
ESTHER : Zomber_2005_J.Biol.Chem_280_39897
PubMedSearch : Zomber_2005_J.Biol.Chem_280_39897
PubMedID: 16188881

Title : Lessons from functional analysis of AChE covalent and noncovalent inhibitors for design of AD therapeutic agents - Barak_2005_Chem.Biol.Interact_157-158_219
Author(s) : Barak D , Ordentlich A , Kaplan D , Kronman C , Velan B , Shafferman A
Ref : Chemico-Biological Interactions , 157-158 :219 , 2005
Abstract : Determination of the 3D-structure of acetylcholinesterase (AChE) of Torpedo californica over a decade ago, and more recently that of human enzyme together with extensive targeted mutagenesis of the mammalian AChEs led to a fine mapping of the multiple functional domains within the active center of the enzyme. Many of the contributions of this active center architecture to accommodation of noncovalent ligands could be deduced from the X-ray structures of the corresponding HuAChE complexes. Yet, Michaelis complexes leading to transient covalent adducts are not amenable to structural analysis. Since the rates of formation of the covalent adducts depend predominantly on the stabilities of the corresponding Michaelis complexes, it is essential to characterize the specific interactions contributing to stabilization of these complexes. Functional analysis of interactions with HuAChE enzymes allows for such characterization for carbamates, like pyridostigmine or rivastigmine, much in the same way as that for the noncovalent therapeutic ligands nivalin or aricept. In fact, the observed differences between the affinities toward carbamates and the noncovalent ligands seem to result from specific structural characteristics of the inhibitors rather than from the decomposition path of the particular complex. Replacements at the cation binding site (Trp86), hydrogen bond network (Glu202, Tyr133, Glu450), and hydrophobic pocket result in similar effects for the covalent as well as for the noncovalent inhibitors. Also, while the effects of perturbing the aromatic trapping of the catalytic His447 for pyridostigmine and nivalin were analogous to those for the substrate, the corresponding effects for rivastigmine and aricept were quite different. Thus, elucidation of the functional architecture of the HuAChE active center is bound to be of considerable utility in the current effort to design novel covalent AChE inhibitors as therapeutics for Alzheimer's disease (AD).
ESTHER : Barak_2005_Chem.Biol.Interact_157-158_219
PubMedSearch : Barak_2005_Chem.Biol.Interact_157-158_219
PubMedID: 16289124

Title : Poster (35) The hierarchy of post-translation modifications determining circulatory longevity of acetylcholinesterase. -
Author(s) : Velan B , Kronman C , Chitlaru T , Cohen O , Ordentlich A , Shafferman A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :339 , 2004
PubMedID:

Title : Surprising findings from the functional analysis of human acetylcholinesterase adducts of Alzheimer?s disease drugs. -
Author(s) : Ordentlich A , Barak D , Ariel N , Kronman C , Kaplan D , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :177 , 2004
PubMedID:

Title : Some basic rules governing oligosaccharide-dependent circulatory residence of glycoproteins are revealed by MALDI-TOF mapping of the multiple N-glycans associated with recombinant bovine acetylcholinesterase. -
Author(s) : Kronman C , Chitlaru T , Seliger N , Lazar S , Lazar A , Zilberstein L , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :613 , 2004
PubMedID:

Title : Effect of post-translation modifications of human acetylcholinesterase on its circulatory residence. -
Author(s) : Chitlaru T , Kronman C , Lazar S , Seliger N , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :511 , 2004
PubMedID:

Title : Generation of pharmacokinetically improved recombinant human acetylcholinesterase by polyethylene glycol modification. -
Author(s) : Cohen O , Kronman C , Chitlaru T , Ordentlich A , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :519 , 2004
PubMedID:

Title : Attempts to engineer an enzyme mimic of butyrylcholinesterase by substitution of the six divergent aromatic amino acids in the active center of acetylcholinesterase. -
Author(s) : Kaplan D , Ordentlich A , Barak D , Ariel N , Kronman C , Baruch V , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :601 , 2004
PubMedID:

Title : A complex array of post-translation modifications determines the circulatory longevity of acetylcholinesterase in a hierarchical manner. -
Author(s) : Shafferman A , Chitlaru T , Ordentlich A , Velan B , Kronman C
Ref : Cholinergic Mechanisms, CRC Press :245 , 2004
PubMedID:

Title : MALDI-TOF\/MS analysis of tabun-acetylcholinesterase conjugate. -
Author(s) : Elhanany E , Ordentlich A , Dgany OR , Kaplan D , Segall Y , Barak R , Velan B , Shafferman A
Ref : Cholinergic Mechanisms, CRC Press :563 , 2004
PubMedID:

Title : Poster (14) The aromatic trapping of histidine 447 in catalysis of acetylcholinesterases -
Author(s) : Shafferman A , Barak D , Kaplan D , Ordentlich A , Ariel N , Velan B
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :327 , 2004
PubMedID:

Title : The aromatic trapping of histidine 447 in catalysis of acetylcholinesterases -
Author(s) : Shafferman A , Barak D , Kaplan D , Ordentlich A , Ariel N , Velan B
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :181 , 2004
PubMedID:

Title : Stereoselectivity toward VX is determined by interactions with residues of the acyl pocket as well as of the peripheral anionic site of AChE - Ordentlich_2004_Biochemistry_43_11255
Author(s) : Ordentlich A , Barak D , Sod-Moriah G , Kaplan D , Mizrahi D , Segall Y , Kronman C , Karton Y , Lazar A , Marcus D , Velan B , Shafferman A
Ref : Biochemistry , 43 :11255 , 2004
Abstract : The origins of human acetylcholinesterase (HuAChE) reactivity toward the lethal chemical warfare agent O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (VX) and its stereoselectivity toward the P(S)-VX enantiomer (VX(S)) were investigated by examining the reactivity of HuAChE and its mutant derivatives toward purified enantiomers of VX and its noncharged isostere O-ethyl S-(3-isopropyl-4-methylpentyl) methylphosphonothioate (nc-VX) as well as echothiophate and its noncharged analogue. Reactivity of wild-type HuAChE toward VX(S) was 115-fold higher than that toward VX(R), with bimolecular rate constants of 1.4 x 10(8) and 1.2 x 10(6) min(-1) M(-1). HuAChE was also 12500-fold more reactive toward VX(S) than toward nc-VX(S). Substitution of the cation binding subsite residue Trp86 with alanine resulted in a 3 order of magnitude decrease in HuAChE reactivity toward both VX enantiomers, while this replacement had an only marginal effect on the reactivity toward the enantiomers of nc-VX and the noncharged echothiophate. These results attest to the critical role played by Trp86 in accommodating the charged moieties of both VX enantiomers. A marked decrease in stereoselectivity toward VX(S) was observed following replacements of Phe295 at the acyl pocket (F295A and F295A/F297A). Replacement of the peripheral anionic site (PAS) residue Asp74 with asparagine (D74N) practically abolished stereoselectivity toward VX(S) (130-fold decrease), while a substitution which retains the negative charge at position 74 (D74E) had no effect. The results from kinetic studies and molecular simulations suggest that the differential reactivity toward the VX enantiomers is mainly a result of a different interaction of the charged leaving group with Asp74.
ESTHER : Ordentlich_2004_Biochemistry_43_11255
PubMedSearch : Ordentlich_2004_Biochemistry_43_11255
PubMedID: 15366935

Title : Poster (21) Analysis of acetylcholinesterase adducts of alzheimer's drugs -
Author(s) : Ordentlich A , Kronman C , Barak D , Ariel N , Kaplan D , Velan B , Shafferman A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :332 , 2004
PubMedID:

Title : Poster (23) Phosphorylation and aging of AChE - insights from mutagenesis, mass spectrometry and structural studies -
Author(s) : Barak D , Ordentlich A , Kaplan D , Elhanani E , Segall Y , Barak R , Velan B , Shafferman A
Ref : In: Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects , (Inestrosa NC, Campos EO) P. Universidad Catolica de Chile-FONDAP Biomedicina :332 , 2004
PubMedID:

Title : Amino acid domains control the circulatory residence time of primate acetylcholinesterases in rhesus macaques (Macaca mulatta) - Cohen_2004_Biochem.J_378_117
Author(s) : Cohen O , Kronman C , Velan B , Shafferman A
Ref : Biochemical Journal , 378 :117 , 2004
Abstract : An array of 13 biochemically well defined molecular forms of bovine, human and newly cloned rhesus macaque (Macaca mulatta) AChEs (acetylcholinesterases) differing in glycosylation and subunit assembly status were subjected to comparative pharmacokinetic studies in mice and rhesus macaques. The circulatory lifetimes of recombinant bovine, macaque and human AChEs in mice were governed by previously determined hierarchical rules; the longest circulatory residence time was obtained when AChE was fully sialylated and tetramerized [Kronman, Chitlaru, Elhanany, Velan and Shafferman (2000) J. Biol. Chem. 275, 29488-29502; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625]. In rhesus macaques, bovine molecular forms still obeyed the same hierarchical rules, whereas primate AChEs showed significant deviation from this behaviour. Residence times of human and rhesus AChEs were effectively extended by extensive sialylation, but subunit tetramerization and N-glycan addition had a marginal effect on their circulatory longevity in macaques. It appears that the major factor responsible for the differential pharmacokinetics of bovine and primate AChEs in macaques is related to differences in primary structure, suggesting the existence of a specific mechanism for the circulatory clearance of primate AChEs in rhesus macaques. The 35 amino acids that differ between bovine and primate AChEs are clustered within three defined domains, all located at the enzyme surface, and may therefore mediate the facilitated removal of primate cholinesterases specifically from the circulation of monkeys. These surface domains can be effectively masked by poly(ethylene glycol) appendage, resulting in the generation of chemically modified human and macaque AChEs that reside in the circulation for extraordinarily long periods of time (mean residence time of 10000 min). This extended residence time is similar to that displayed by native macaque butyrylcholinesterase (9950 min), which is the prevalent cholinesterase form in the circulation of adult macaques.
ESTHER : Cohen_2004_Biochem.J_378_117
PubMedSearch : Cohen_2004_Biochem.J_378_117
PubMedID: 14575524
Gene_locus related to this paper: macmu-ACHE

Title : Is aromaticity essential for trapping the catalytic histidine 447 in human acetylcholinesterase? - Kaplan_2004_Biochemistry_43_3129
Author(s) : Kaplan D , Barak D , Ordentlich A , Kronman C , Velan B , Shafferman A
Ref : Biochemistry , 43 :3129 , 2004
Abstract : Replacement of both the acyl pocket residue Phe295 as well as residue Phe338, adjacent to the catalytic His447 in human acetylcholinesterase (HuAChE), resulted in a 680-fold decline in catalytic activity due to conformational destabilization of the histidine side chain [Barak et al. (2002) Biochemistry 41, 8245]. A possible restriction of this catalytically nonproductive mobility of His447 in a series of F295X/F338A HuAChEs was examined in silico followed by site-directed mutagenesis. Simulations suggested that of the 12 aliphatic residues substituted at position 295, including hydrophobic and polar amino acids, only methionine was capable of maintaining the catalytically viable conformation of His447. Examination of the reactivities of the actual F295X/F338A HuAChEs showed that indeed the F295M/F338A enzyme was only 2-fold less reactive than the F338A mutant toward acetylthiocholine, while enzymes substituted by the similarly bulky residues leucine and isoleucine were catalytically impaired. Furthermore, only the F295M/F338A enzyme exhibited wild-type-like reactivity toward covalent modifiers of the catalytic Ser203 including the methylphosphonate soman and transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), as well as a facile dealkylation of the F295M/F338A-soman adduct. A different behavior was observed for bulkier ligands which introduce a deformation in the acyl pocket, and therefore their activity seems only marginally affected by the positioning of His447. The findings emphasize the importance of the precise positioning of His447 for catalysis and indicate that, in the absence of aromatic "trapping", restriction of the histidine mobility in F295X/F338A HuAChEs requires a combination of steric interference and a specific polar interaction. The results also underscore the role of the acyl pocket subsite of cholinesterases in maintaining the catalytically viable conformation of the catalytic histidine.
ESTHER : Kaplan_2004_Biochemistry_43_3129
PubMedSearch : Kaplan_2004_Biochemistry_43_3129
PubMedID: 15023064

Title : Overloading and removal of N-glycosylation targets on human acetylcholinesterase: effects on glycan composition and circulatory residence time - Chitlaru_2002_Biochem.J_363_619
Author(s) : Chitlaru T , Kronman C , Velan B , Shafferman A
Ref : Biochemical Journal , 363 :619 , 2002
Abstract : Optimization of post-translational modifications was shown to affect the ability of recombinant human acetylcholinesterase (rHuAChE) produced in HEK-293 cells to be retained in the circulation for prolonged periods of time [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959-967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647-658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613-625]. To evaluate the possible contribution of the number of appended N-glycans in determining the pharmacokinetic behaviour of AChE, a series of sixteen recombinant human AChE glycoforms, differing in their number of appended N-glycans (2, 3, 4 or 5 glycans), state of assembly (dimeric or tetrameric) and terminal glycan sialylation (partially or fully sialylated) were generated. Extensive structural analysis of N-glycans demonstrated that the various glycan types associated with all the different rHuAChE glycoforms are essentially similar both in structure and abundance, and that production of the various glycoforms in the sialyltransferase-overexpressing 293ST-2D6 cell line resulted in the generation of enzyme species that carry glycans sialylated to the same extent. Pharmacokinetic profiling of the rHuAChE glycoforms in their fully tetramerized and sialylated state clearly demonstrated that circulatory longevity correlated directly with the number of attached N-glycans (mean residence times for rHuAChE glycoforms harbouring 2, 3, and 4 glycans=200, 740, and 1055 min respectively). This study provides evidence that glycan loading, together with N-glycan terminal processing and enzyme subunit oligomerization, operate in a hierarchical and concerted manner in determining the pharmacokinetic characteristics of AChE.
ESTHER : Chitlaru_2002_Biochem.J_363_619
PubMedSearch : Chitlaru_2002_Biochem.J_363_619
PubMedID: 11964163

Title : Pressure and heat inactivation of recombinant human acetylcholinesterase. Importance of residue E202 for enzyme stability - Clery-Barraud_2002_Eur.J.Biochem_269_4297
Author(s) : Clery-Barraud C , Ordentlich A , Grosfeld H , Shafferman A , Masson P
Ref : European Journal of Biochemistry , 269 :4297 , 2002
Abstract : The effects of pressure on structure and activity of recombinant human acetylcholinesterase (rHuAChE) were investigated up to a pressure of 300 MPa using gel electrophoresis under elevated hydrostatic pressure, fluorescence of bound 8-anilinonaphthalene-1-sulfonate (ANS) and activity measurements following exposure to high pressure. Study of wild-type enzyme and three single mutants (D74N, E202Q, E450A) and one sextuple mutant (E84Q/E292A/D349N/E358Q/E389Q/D390N) showed that pressure exerts a differential action on wild-type rHuAChE and its mutants, allowing estimation of the contribution of carboxylic amino acid side-chains to enzyme stability. Mutation of negatively charged residues D74 and E202 by polar side-chains strengthened heat or pressure stability. The mutation E450A and the sextuple mutation caused destabilization of the enzyme to pressure. Thermal inactivation data on mutants showed that all of them were stabilized against temperature. In conclusion, pressure and thermal stability of mutants provided evidence that the residue E202 is a determinant of structural and functional stability of HuAChE.
ESTHER : Clery-Barraud_2002_Eur.J.Biochem_269_4297
PubMedSearch : Clery-Barraud_2002_Eur.J.Biochem_269_4297
PubMedID: 12199708

Title : The aromatic trapping of the catalytic histidine is essential for efficient catalysis in acetylcholinesterase - Barak_2002_Biochemistry_41_8245
Author(s) : Barak D , Kaplan D , Ordentlich A , Ariel N , Velan B , Shafferman A
Ref : Biochemistry , 41 :8245 , 2002
Abstract : While substitution of the aromatic residues (Phe295, Phe338), located in the vicinity of the catalytic His447 in human acetylcholinesterase (HuAChE) had little effect on catalytic activity, simultaneous replacement of both residues by aliphatic amino acids resulted in a 680-fold decrease in catalytic activity. Molecular simulations suggested that the activity decline is related to conformational destabilization of His447, similar to that observed for the hexamutant HuAChE which mimics the active center of butyrylcholinesterase. On the basis of model structures of other cholinesterases (ChEs), we predicted that catalytically nonproductive mobility of His447 could be restricted by introduction of aromatic residue in a different location adjacent to this histidine (Val407). Indeed, the F295A/F338A/V407F enzyme is 170-fold more reactive than the corresponding double mutant and only 3-fold less reactive than the wild-type HuAChE. However, analogous substitution of Val407 in the hexamutant HuAChE (generating the heptamutant Y72N/Y124Q/W286A/F295L/F297V/Y337A/V407F) did not enhance catalytic activity. Reactivity of these double, triple, hexa, and hepta mutant HuAChEs was monitored toward covalent ligands such as organophosphates and the transition state analogue TMFTA, which probe, respectively, the facility of the enzymes to accommodate Michaelis complexes and to undergo the acylation process. The findings suggest that in the F295A/F338A mutant the two His447 conformational states, which are essential for the different stages of the catalytic process, seem to be destabilized. On the other hand, in the F295A/F338A/V407F mutant only the state involved in acylation is impaired. Such differential effects on the His447 conformational properties demonstrate the general role of aromatic residues in cholinesterases, and probably in other serine hydrolases, in "trapping" of the catalytic histidine and thereby in optimization of catalytic activity.
ESTHER : Barak_2002_Biochemistry_41_8245
PubMedSearch : Barak_2002_Biochemistry_41_8245
PubMedID: 12081473

Title : Effect of human acetylcholinesterase subunit assembly on its circulatory residence - Chitlaru_2001_Biochem.J_354_613
Author(s) : Chitlaru T , Kronman C , Velan B , Shafferman A
Ref : Biochemical Journal , 354 :613 , 2001
Abstract : Sialylated recombinant human acetylcholinesterase (rHuAChE), produced by stably transfected cells, is composed of a mixed population of monomers, dimers and tetramers and manifests a time-dependent circulatory enrichment of the higher-order oligomeric forms. To investigate this phenomenon further, homogeneous preparations of rHuAChE differing in their oligomerization statuses were generated: (1) monomers, represented by the oligomerization-impaired C580A-rHuAChE mutant, (2) wild-type (WT) dimers and (3) tetramers of WT-rHuAChE generated in vitro by complexation with a synthetic ColQ-derived proline-rich attachment domain ('PRAD') peptide. Three different series of each of these three oligoform preparations were produced: (1) partly sialylated, derived from HEK-293 cells; (2) fully sialylated, derived from engineered HEK-293 cells expressing high levels of sialyltransferase; and (3) desialylated, after treatment with sialidase to remove sialic acid termini quantitatively. The oligosaccharides associated with each of the various preparations were extensively analysed by matrix-assisted laser desorption ionization-time-of-flight MS. With the enzyme preparations comprising the fully sialylated series, a clear linear relationship between oligomerization and circulatory mean residence time (MRT) was observed. Thus monomers, dimers and tetramers exhibited MRTs of 110, 195 and 740 min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5 min). These observations suggest that multiple removal systems contribute to the elimination of AChE from the circulation. Here we also demonstrate that by the combined modulation of sialylation and tetramerization it is possible to generate a rHuAChE displaying a circulatory residence exceeding that of all other known forms of native or recombinant human AChE.
ESTHER : Chitlaru_2001_Biochem.J_354_613
PubMedSearch : Chitlaru_2001_Biochem.J_354_613
PubMedID: 11237866

Title : Effect of chemical modification of recombinant human acetylcholinesterase by polyethylene glycol on its circulatory longevity - Cohen_2001_Biochem.J_357_795
Author(s) : Cohen O , Kronman C , Chitlaru T , Ordentlich A , Velan B , Shafferman A
Ref : Biochemical Journal , 357 :795 , 2001
Abstract : Post-translational modifications were recently shown to be responsible for the short circulatory mean residence time (MRT) of recombinant human acetylcholinesterase (rHuAChE) [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959--967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647--658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613--625], which is one of the major obstacles to the fulfilment of its therapeutic potential as a bioscavenger. In the present study we demonstrate that the MRT of rHuAChE can be significantly increased by the controlled attachment of polyethylene glycol (PEG) side chains to lysine residues. Attachment of as many as four PEG molecules to monomeric rHuAChE had minimal effects, if any, on either the catalytic activity (K(m)=0.09 mM and k(cat)=3.9 x 10(5) min(-1)) or the reactivity of the modified enzyme towards active-centre inhibitors, such as edrophonium and di-isopropyl fluorophosphate, or to peripheral-site ligands, such as propidium, BW284C51 and even the bulky snake-venom toxin fasciculin-II. The increase in MRT of the PEG-modified monomeric enzyme is linearly dependent, in the tested range, on the number of attached PEG molecules, as well as on their size. It appears that even low level PEG-conjugation can overcome the deleterious effect of under-sialylation on the pharmacokinetic performance of rHuAChE. At the highest tested ratio of attached PEG-20000/rHuAChE (4:1), an MRT of over 2100 min was attained, a value unmatched by any other known form of recombinant or native serum-derived AChE reported to date.
ESTHER : Cohen_2001_Biochem.J_357_795
PubMedSearch : Cohen_2001_Biochem.J_357_795
PubMedID: 11463350

Title : Does butyrylization of acetylcholinesterase through substitution of the six divergent aromatic amino acids in the active center gorge generate an enzyme mimic of butyrylcholinesterase? - Kaplan_2001_Biochemistry_40_7433
Author(s) : Kaplan D , Ordentlich A , Barak D , Ariel N , Kronman C , Velan B , Shafferman A
Ref : Biochemistry , 40 :7433 , 2001
Abstract : The active center gorge of human acetylcholinesterase (HuAChE) is lined by 14 aromatic residues, whereas in the closely related human butyrylcholinesterase (HuBChE) 3 of the aromatic active center residues (Phe295, Phe297, Tyr337) as well as 3 of the residues at the gorge entrance (Tyr72, Tyr124, Trp286) are replaced by aliphatic amino acids. To investigate whether this structural variability can account for the reactivity differences between the two enzymes, gradual replacement of up to all of the 6 aromatic residues in HuAChE by the corresponding residues in HuBChE was carried out. The affinities of the hexamutant (Y72N/Y124Q/W286A/F295L/F297V/Y337A) toward tacrine, decamethonium, edrophonium, huperzine A, or BW284C51 differed by about 5-, 80-, 170-, 25000-, and 17000-fold, respectively, from those of the wild-type HuAChE. For most of these prototypical noncovalent active center and peripheral site ligands, the hexamutant HuAChE displayed a reactivity phenotype closely resembling that of HuBChE. These results support the accepted view that the active center architectures of AChE and BChE differ mainly by the presence of a larger void space in BChE. Nevertheless, reactivity of the hexamutant HuAChE toward the substrates acetylthiocholine and butyrylthiocholine, or covalent ligands such as phosphonates and the transition state analogue m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA), is about 45-170-fold lower than that of HuBChE. Most of this reduction in reactivity can be related to the combined replacements of the three aromatic residues at the active center, Phe295, Phe297, and Tyr337. We propose that the hexamutant HuAChE, unlike BChE, is impaired in its capacity to accommodate certain tetrahedral species in the active center. This impairment may be related to the enhanced mobility of the catalytic histidine His447, which is observed in molecular dynamics simulations of the hexamutant and the F295L/F297V/Y337A HuAChE enzymes but not in the wild-type HuAChE.
ESTHER : Kaplan_2001_Biochemistry_40_7433
PubMedSearch : Kaplan_2001_Biochemistry_40_7433
PubMedID: 11412096

Title : Resolving pathways of interaction of covalent inhibitors with the active site of acetylcholinesterases: maldi-tof\/ms analysis of various nerve agent phosphyl adducts - Elhanany_2001_Chem.Res.Toxicol_14_912
Author(s) : Elhanany E , Ordentlich A , Dgany O , Kaplan D , Segall Y , Barak R , Velan B , Shafferman A
Ref : Chemical Research in Toxicology , 14 :912 , 2001
Abstract : Understanding reaction pathways of phosphylation, reactivation, and "aging" of AChE with toxic organophosphate compounds is both a biochemical and a pharmacological challenge. Here we describe experiments which allowed to resolve some of the less well understood reaction pathways of phosphylation and "aging" of acetylcholinesterase (AChE) involving phosphoroamidates (P-N agents) such as tabun or the widely used pesticide methamidophos. Tryptic digests of phosphylated AChEs (from human and Torpedo californica), ZipTip peptide fractionation and matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF/MS) enabled reproducible signal enrichment of the isotopically resolved peaks of organophosphoroamidate conjugates of the AChE active site Ser peptides. For tabun and its hexadeuterio analogue, we find, as expected, that the two phosphoramidate adducts of the active site peptide differ by 6.05 mass units but following aging we find that the two corresponding phospho-peptides have identical molecular weights. We further show that the aging product of paraoxon-AChE adduct is identical to the aging product of the tabun-AChE conjugate. These results unequivocally demonstrate that the pathway of aging of tabun adducts of the human or the Torpedo californica AChEs proceeds through P-N bond scission. For methamidophos, we show that phosphylation of AChE involves elimination of the thiomethyl moiety and that the spontaneous reactivation of the resulting organophosphate adduct generates the phosphorus free AChE active site Ser-peptide.
ESTHER : Elhanany_2001_Chem.Res.Toxicol_14_912
PubMedSearch : Elhanany_2001_Chem.Res.Toxicol_14_912
PubMedID: 11453739

Title : Hierarchy of post-translational modifications involved in the circulatory longevity of glycoproteins. Demonstration of concerted contributions of glycan sialylation and subunit assembly to the pharmacokinetic behavior of bovine acetylcholinesterase - Kronman_2000_J.Biol.Chem_275_29488
Author(s) : Kronman C , Chitlaru T , Elhanany E , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 275 :29488 , 2000
Abstract : The tetrameric form of native serum-derived bovine acetylcholinesterase is retained in the circulation for much longer periods (mean residence time, MRT = 1390 min) than recombinant bovine acetylcholinesterase (rBoAChE) produced in the HEK-293 cell system (MRT = 57 min). Extensive matrix-assisted laser desorption ionization-time of flight analyses established that the basic structures of the N-glycans associated with the native and recombinant enzymes are similar (the major species (50-60%) are of the biantennary fucosylated type and 20-30% are of the triantennary type), yet the glycan termini of the native enzyme are mostly capped with sialic acid (82%) and alpha-galactose (12%), whereas glycans of the recombinant enzyme exhibit a high level of exposed beta-galactose residues (50%) and a lack of alpha-galactose. Glycan termini of both fetal bovine serum and rBoAChE were altered in vitro using exoglycosidases and sialyltransferase or in vivo by a HEK-293 cell line developed specifically to allow efficient sialic acid capping of beta-galactose-exposed termini. In addition, the dimeric and monomeric forms of rBoAChE were quantitatively converted to tetramers by complexation with a synthetic peptide representing the human ColQ-derived proline-rich attachment domain. Thus by controlling both the level and nature of N-glycan capping and subunit assembly, we generated and characterized 9 distinct bovine AChE glycoforms displaying a 400-fold difference in their circulatory lifetimes (MRT = 3.5-1390 min). This revealed some general rules and a hierarchy of post-translation factors determining the circulatory profile of glycoproteins. Accordingly, an rBoAChE was generated that displayed a circulatory profile indistinguishable from the native form.
ESTHER : Kronman_2000_J.Biol.Chem_275_29488
PubMedSearch : Kronman_2000_J.Biol.Chem_275_29488
PubMedID: 10867010

Title : Attenuated nontoxinogenic and nonencapsulated recombinant Bacillus anthracis spore vaccines protect against anthrax - Cohen_2000_Infect.Immun_68_4549
Author(s) : Cohen S , Mendelson I , Altboum Z , Kobiler D , Elhanany E , Bino T , Leitner M , Inbar I , Rosenberg H , Gozes Y , Barak R , Fisher M , Kronman C , Velan B , Shafferman A
Ref : Infect Immun , 68 :4549 , 2000
Abstract : Several highly attenuated spore-forming nontoxinogenic and nonencapsulated Bacillus anthracis vaccines differing in levels of expression of recombinant protective antigen (rPA) were constructed. Biochemical analyses (including electrospray mass spectroscopy and N terminus amino acid sequencing) as well as biological and immunological tests demonstrated that the rPA retains the characteristics of native PA. A single immunization of guinea pigs with 5 x 10(7) spores of one of these recombinant strains, MASC-10, expressing high levels of rPA (>/=100 microgram/ml) from a constitutive heterologous promoter induced high titers of neutralizing anti-PA antibodies. This immune response was long lasting (at least 12 months) and provided protection against a lethal challenge of virulent (Vollum) anthrax spores. The recombinant B. anthracis spore vaccine appears to be more efficacious than the vegetative cell vaccine. Furthermore, while results clearly suggest a direct correlation between the level of expression of PA and the potency of the vaccine, they also suggest that some B. anthracis spore-associated antigen(s) may contribute in a significant manner to protective immunity.
ESTHER : Cohen_2000_Infect.Immun_68_4549
PubMedSearch : Cohen_2000_Infect.Immun_68_4549
PubMedID: 10899854

Title : Structures of recombinant native and E202Q mutant human acetylcholinesterase complexed with the snake-venom toxin fasciculin-II - Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
Author(s) : Kryger G , Harel M , Giles K , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
Ref : Acta Crystallographica D Biol Crystallogr , 56 :1385 , 2000
Abstract : Structures of recombinant wild-type human acetylcholinesterase and of its E202Q mutant as complexes with fasciculin-II, a 'three-finger' polypeptide toxin purified from the venom of the eastern green mamba (Dendroaspis angusticeps), are reported. The structure of the complex of the wild-type enzyme was solved to 2.8 A resolution by molecular replacement starting from the structure of the complex of Torpedo californica acetylcholinesterase with fasciculin-II and verified by starting from a similar complex with mouse acetylcholinesterase. The overall structure is surprisingly similar to that of the T. californica enzyme with fasciculin-II and, as expected, to that of the mouse acetylcholinesterase complex. The structure of the E202Q mutant complex was refined starting from the corresponding wild-type human acetylcholinesterase structure, using the 2.7 A resolution data set collected. Comparison of the two structures shows that removal of the charged group from the protein core and its substitution by a neutral isosteric moiety does not disrupt the functional architecture of the active centre. One of the elements of this architecture is thought to be a hydrogen-bond network including residues Glu202, Glu450, Tyr133 and two bridging molecules of water, which is conserved in other vertebrate acetylcholinesterases as well as in the human enzyme. The present findings are consistent with the notion that the main role of this network is the proper positioning of the Glu202 carboxylate relative to the catalytic triad, thus defining its functional role in the interaction of acetylcholinesterase with substrates and inhibitors.
ESTHER : Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
PubMedSearch : Kryger_2000_Acta.Crystallogr.D.Biol.Crystallogr_56_1385
PubMedID: 11053835
Gene_locus related to this paper: human-ACHE

Title : Evidence for P-N bond scission in phosphoroamidate nerve agent adducts of human acetylcholinesterase - Barak_2000_Biochemistry_39_1156
Author(s) : Barak D , Ordentlich A , Kaplan D , Barak R , Mizrahi D , Kronman C , Segall Y , Velan B , Shafferman A
Ref : Biochemistry , 39 :1156 , 2000
Abstract : Acetylcholinesterases (AChEs) form conjugates with certain highly toxic organophosphorus (OP) agents that become gradually resistant to reactivation. This phenomenon termed "aging" is a major factor limiting the effectiveness of therapy in certain cases of OP poisoning. While AChE adducts with phosphonates and phosphates are known to age through scission of the alkoxy C-O bond, the aging path for adducts with phosphoroamidates (P-N agents) like the nerve agent N,N-dimethylphosphonocyanoamidate (tabun) is not clear. Here we report that conjugates of tabun and of its butyl analogue (butyl-tabun) with the E202Q and F338A human AChEs (HuAChEs) age at similar rates to that of the wild-type enzyme. This is in marked contrast to the large effect of these substitutions on the aging of corresponding adducts with phosphates and phosphonates, suggesting that a different aging mechanism may be involved. Both tabun and butyl-tabun appear to be similarly accommodated in the active center, as suggested by molecular modeling and by kinetic studies of phosphylation and aging with a series of HuAChE mutants (E202Q, F338A, F295A, F297A, and F295L/F297V). Mass spectrometric analysis shows that HuAChE adduct formation with tabun and butyl-tabun occurs through loss of cyanide and that during the aging process both of these adducts show a mass decrease of 28 +/- 4 Da. Due to the nature of the alkoxy substituent, such mass decrease can be unequivocally assigned to loss of the dimethylamino group, at least for the butyl-tabun conjugate. This is the first demonstration that AChE adducts with toxic P-N agents can undergo aging through scission of the P-N bond.
ESTHER : Barak_2000_Biochemistry_39_1156
PubMedSearch : Barak_2000_Biochemistry_39_1156
PubMedID: 10653663

Title : Crystal structures of aged phosphonylated acetylcholinesterase: nerve agent reaction products at the atomic level - Millard_1999_Biochemistry_38_7032
Author(s) : Millard CB , Kryger G , Ordentlich A , Greenblatt HM , Harel M , Raves ML , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
Ref : Biochemistry , 38 :7032 , 1999
Abstract : Organophosphorus acid anhydride (OP) nerve agents are potent inhibitors which rapidly phosphonylate acetylcholinesterase (AChE) and then may undergo an internal dealkylation reaction (called "aging") to produce an OP-enzyme conjugate that cannot be reactivated. To understand the basis for irreversible inhibition, we solved the structures of aged conjugates obtained by reaction of Torpedo californica AChE (TcAChE) with diisopropylphosphorofluoridate (DFP), O-isopropylmethylphosponofluoridate (sarin), or O-pinacolylmethylphosphonofluoridate (soman) by X-ray crystallography to 2.3, 2.6, or 2.2 A resolution, respectively. The highest positive difference density peak corresponded to the OP phosphorus and was located within covalent bonding distance of the active-site serine (S200) in each structure. The OP-oxygen atoms were within hydrogen-bonding distance of four potential donors from catalytic subsites of the enzyme, suggesting that electrostatic forces significantly stabilize the aged enzyme. The active sites of aged sarin- and soman-TcAChE were essentially identical and provided structural models for the negatively charged, tetrahedral intermediate that occurs during deacylation with the natural substrate, acetylcholine. Phosphorylation with DFP caused an unexpected movement in the main chain of a loop that includes residues F288 and F290 of the TcAChE acyl pocket. This is the first major conformational change reported in the active site of any AChE-ligand complex, and it offers a structural explanation for the substrate selectivity of AChE.
ESTHER : Millard_1999_Biochemistry_38_7032
PubMedSearch : Millard_1999_Biochemistry_38_7032
PubMedID: 10353814
Gene_locus related to this paper: torca-ACHE

Title : Reaction Products of Acetylcholinesterase and Vx Reveal a Mobile Histidine in the Catalytic Triad - Millard_1999_J.Am.Chem.Soc_121_9883
Author(s) : Millard CB , Koellner G , Ordentlich A , Shafferman A , Silman I , Sussman JL
Ref : Journal of the American Chemical Society , 121 :9883 , 1999
Abstract : The presence of a precisely aligned active-site triad (Ser-His-Asp/Glu) in the three-dimensional structures of widely different hydrolytic enzymes has generated intense interest in the chemical modus operandi of this catalytic motif. 1 One hypothesis, which has not received wide acceptance, proposes that the imidazole of the catalytic His is mobile during enzyme function. 2 We solved the structures of the phosphonylation and dealkylation ("aging") reaction products of acetylcholinesterase (AChE; EC 3.1.1.7) and an organophosphorus (OP) inhibitor, O-ethyl-S-[2-[bis(1-meth-ylethyl) amino]ethyl] methylphosphonothioate (VX) by X-ray crystallography. The structures clearly demonstrate reversible movement of the catalytic His. Moreover, the conformational change apparently involves a hydrogen (H-) bond with a glutamate (E199) which had been implicated previously in OP and substrate reactions.
ESTHER : Millard_1999_J.Am.Chem.Soc_121_9883
PubMedSearch : Millard_1999_J.Am.Chem.Soc_121_9883
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : A preliminary comparison of structural models for catalytic intermediates of acetylcholinesterase - Silman_1999_Chem.Biol.Interact_119-120_43
Author(s) : Silman I , Millard CB , Ordentlich A , Greenblatt HM , Harel M , Barak D , Shafferman A , Sussman JL
Ref : Chemico-Biological Interactions , 119-120 :43 , 1999
Abstract : Determination of the three dimensional structure of Torpedo Californica acetylcholinesterase (TcAChE) provided an experimental tool for directly visualizing interaction of AChE with cholinesterase inhibitors of fundamental, pharmacological and toxicological interest. The structure revealed that the active site is located near the bottom of a deep and narrow gorge lined with 14 conserved aromatic amino acids. The structure of a complex of TcAChE with the powerful 'transition state analog' inhibitor, TMTFA, suggested that its orientation in the experimentally determined structure was very similar to that proposed for the natural substrate, acetylcholine, by manual docking. The array of enzyme-ligand interactions visualized in the TMFTA complex also are expected to envelope the unstable TI that forms with acetylcholine during acylation, and to sequester it from solvent. In our most recent studies, the crystal structures of several 'aged' conjugates of TcAChE obtained with OP nerve agents have been solved and compared with that of the native enzyme. The methylphosphonylated-enzyme obtained by reaction with soman provides a useful structural analog for the TI that forms during deacylation after the reaction of TcAChE with acetylcholine. By comparing these structures, we conclude that the same 'oxyanion hole' residues, as well as the aromatic side chains constituting the 'acyl pocket', participate in acylation (TMTFA-AChE) and deacylation (OP-AChE), and that AChE can accommodate both TIs at the bottom of the gorge without major conformational movements.
ESTHER : Silman_1999_Chem.Biol.Interact_119-120_43
PubMedSearch : Silman_1999_Chem.Biol.Interact_119-120_43
PubMedID: 10421437
Gene_locus related to this paper: torca-ACHE

Title : Exploring the active center of human acetylcholinesterase with stereomers of an organophosphorus inhibitor with two chiral centers - Ordentlich_1999_Biochemistry_38_3055
Author(s) : Ordentlich A , Barak D , Kronman C , Benschop HP , De Jong LP , Ariel N , Barak R , Segall Y , Velan B , Shafferman A
Ref : Biochemistry , 38 :3055 , 1999
Abstract : The stereoselectivity of the phosphonylation reaction and the effects of adduct configuration on the aging process were examined for human acetylcholinesterase (HuAChE) and its selected active center mutants, using the four stereomers of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman). The reactivity of wild type HuAChE toward the PS-soman diastereomers was 4.0-7.5 x 10(4)-fold higher than that toward the PR-diastereomers. Aging of the PSCS-somanyl-HuAChE conjugate was also >1.6 x 10(4)-fold faster than that of the corresponding PRCS-somanyl adduct, as shown by both reactivation and electrospray mass spectrometry (ESI/MS) experiments. On the other hand, both processes exhibited very limited sensitivity to the chirality of the alkoxy group Calpha of either PS- or PR-diastereomers. These stereoselectivities presumably reflect the relative participation of the enzyme in stabilization of the Michaelis complexes and in dealkylation of the respective covalent conjugates, and therefore could be utilized for further probing of the HuAChE active center functional architecture. Reactivities of HuAChE enzymes carrying replacements at the acyl pocket (F295A, F297A, and F295L/F297V) indicate that stereoselectivity with respect to the soman phosphorus chirality depends on the structure of this binding subsite, but this stereoselectivity cannot be explained only by limitation in the capacity to accommodate the PR-diastereomers. In addition, these acyl pocket enzyme mutants display some (5-10-fold) preference for the PRCR-soman over the PRCS-stereomer, while reactivity of the hydrophobic pocket mutant enzyme W86F toward the PRCS-soman resembles that of the wild type HuAChE. Residue substitutions in the H-bond network (E202Q, E450A, Y133F, and Y133A) and the hydrophobic pocket (F338A, W86A, W86F, and Y337A) result in a limited stereoselectivity for the PSCS- over the PSCR-stereomer. Aging of the PS-somanyl conjugates with all the HuAChE mutant enzymes tested practically lacked stereoselectivity with respect to the Calpha of the alkoxy moiety. Thus, the inherent asymmetry of the active center does not seem to affect the rate-determining step of the dealkylation process, possibly because both the PSCS- and the PSCR-somanyl moieties yield the same carbocationic intermediate.
ESTHER : Ordentlich_1999_Biochemistry_38_3055
PubMedSearch : Ordentlich_1999_Biochemistry_38_3055
PubMedID: 10074358

Title : Theoretical and experimental investigations of electrostatic effects on acetylcholinesterase catalysis and inhibition - Malany_1999_Chem.Biol.Interact_119-120_99
Author(s) : Malany S , Baker NA , Verweyst M , Medhekar R , Quinn DM , Velan B , Kronman C , Shafferman A
Ref : Chemico-Biological Interactions , 119-120 :99 , 1999
Abstract : The role of electrostatics in the function of acetylcholinesterase (AChE) has been investigated by both theoretical and experimental approaches. Second-order rate constants (kE = k(cat)/Km) for acetylthiocholine (ATCh) turnover have been measured as a function of ionic strength of the reaction medium for wild-type and mutant AChEs. Also, binding and dissociation rate constants have been measured as a function of ionic strength for the respective charged and neutral transition state analog inhibitors m-(N,N,N-trimethylammonio)trifluoroacetophenone (TMTFA) and m-(t-butyl)trifluoroacetophenone (TBTFA). Linear free-energy correlations between catalytic rate constants and inhibition constants indicate that kE for ATCh turnover is rate limited by terminal binding events. Comparison of binding rate constants for TMTFA and TBTFA attests to the sizable electrostatic discrimination of AChE. Free energy profiles for cationic ligand release from the active sites of wild-type and mutant AChEs have been calculated via a model that utilizes the structure of T. californica AChE, a spherical ligand, and energy terms that account for electrostatic and van der Waals interactions and chemical potential. These calculations indicate that EA and EI complexes are not bound with respect to electrostatic interactions, which obviates the need for a 'back door' for cationic ligand release. Moreover, the computed energy barriers for ligand release give linear free-energy correlations with log(kE) for substrate turnover, which supports the general correctness of the computational model.
ESTHER : Malany_1999_Chem.Biol.Interact_119-120_99
PubMedSearch : Malany_1999_Chem.Biol.Interact_119-120_99
PubMedID: 10421443

Title : Contribution of Primary Sequence and Post-Translation Modification to the Pharmacokinetics of Human and Bovine Acetylcholinesterases -
Author(s) : Velan B , Kronman C , Chitlaru T , Mendelson I , Ordentlich A , Shafferman A
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. :291 , 1998
PubMedID:

Title : 3D Structure at 2.7 Resolution of Native and E202Q Mutant Human Acetylcholinesterase Complexed with Fasciculin-II -
Author(s) : Kryger G , Giles K , Harel M , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
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. :323 , 1998
PubMedID:

Title : 3D Structure of a Complex of Human Acetylcholinesterase with Fasciculin-II at 2.7 Resolution -
Author(s) : Kryger G , Giles K , Harel M , Toker L , Velan B , Lazar A , Kronman C , Barak D , Ariel N , Shafferman A , Silman I , Sussman JL
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. :370 , 1998
PubMedID:

Title : Compatibility of Structures Inferred from Mutagenesis and from X-Ray Crystallography for Various AChE Complexes -
Author(s) : Ariel N , Ordentlich A , Barak D , Bino T , Velan B , Shafferman A
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. :375 , 1998
PubMedID:

Title : The 'aromatic patch' of three proximal residues in the human acetylcholinesterase active centre allows for versatile interaction modes with inhibitors - Ariel_1998_Biochem.J_335_95
Author(s) : Ariel N , Ordentlich A , Barak D , Bino T , Velan B , Shafferman A
Ref : Biochemical Journal , 335 :95 , 1998
Abstract : The role of the functional architecture of the human acetylcholinesterase (HuAChE) active centre in accommodating the non-covalent inhibitors tacrine and huperzine A, or the carbamates pyridostigmine and physostigmine, was analysed using 16 mutants of residues lining the active-centre gorge. Despite the structural diversity of the ligands, certain common properties of the complexes could be observed: (a) replacement of aromatic residues Tyr133, Tyr337 and especially Trp86, resulted in pronounced changes in stability of all the complexes examined; (b) effects due to replacements of the five other aromatic residues along the active-centre gorge, such as the acyl pocket (Phe295, Phe297) or at the peripheral anionic site (Tyr124, Trp286, Tyr341) were relatively small; (c) effects due to substitution of the carboxylic residues in the gorge (Glu202, Glu450) were moderate. These results and molecular modelling indicate that the aromatic side chains of residues Trp86, Tyr133 and Tyr337 form together a continuous 'aromatic patch' lining the wall of the active-centre gorge, allowing for the accommodation of the different ligands via multiple modes of interaction. Studies with HuAChE mutants carrying replacements at positions 86, 133 and 337 indicate that the orientations of huperzine A and tacrine in the HuAChE complexes in solution are significantly different from those observed in X-ray structures of the corresponding complexes with Torpedo californica AChE (TcAChE). These discrepancies may be explained in terms of structural differences between the complexes of HuAChE and TcAChE or, more likely, by the enhanced flexibility of the AChE active-centre gorge in solution as compared with the crystalline state.
ESTHER : Ariel_1998_Biochem.J_335_95
PubMedSearch : Ariel_1998_Biochem.J_335_95
PubMedID: 9742217

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
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. :425 , 1998
PubMedID:

Title : Crystal structure of Aged phosphorylated and phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
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. :425 , 1998
PubMedID:
Gene_locus related to this paper: torca-ACHE

Title : Assembly of Acetylcholinesterase Subunits in vitro -
Author(s) : Giles K , Ben-Yohanan R , Velan B , Shafferman A , Sussman JL , Silman I
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. :442 , 1998
PubMedID:

Title : Modulation of circulatory residence of recombinant acetylcholinesterase through biochemical or genetic manipulation of sialylation levels - Chitlaru_1998_Biochem.J_336_647
Author(s) : Chitlaru T , Kronman C , Zeevi M , Kam M , Harel A , Ordentlich A , Velan B , Shafferman A
Ref : Biochemical Journal , 336 :647 , 1998
Abstract : Sialylation of N-glycans associated with recombinant human acetylcholinesterase (rHuAChE) has a central role in determining its circulatory clearance rate. Human embryonal kidney 293 (HEK-293) cells, which are widely used for the expression of recombinant proteins, seem to be limited in their ability to sialylate overexpressed rHuAChE. High-resolution N-glycan structural analysis, by gel permeation, HPLC anion-exchange chromatography and high-pH anion-exchange chromatography (HPAEC), revealed that the N-glycans associated with rHuAChE produced in HEK-293 cells belong mainly to the complex-biantennary class and are only partly sialylated, with approx. 60% of the glycans being monosialylated. This partial sialylation characterizes rHuAChE produced by cells selected for high-level expression of the recombinant protein. In low-level producer lines, the enzyme exhibits a higher sialic acid content, suggesting that undersialylation of rHuAChE in high-level producer lines stems from a limited endogenous glycosyltransferase activity. To improve sialylation in HEK-293 cells, rat liver beta-galactoside alpha-2,6-sialyltransferase cDNA was stably transfected into cells expressing high levels of rHuAChE. rHuAChE produced by the modified cells displayed a significantly higher proportion of fully sialylated glycans as shown by sialic acid incorporation assays, direct measurement of sialic acid, and HPAEC glycan profiling. Genetically modified sialylated rHuAChE exhibited increased circulatory retention (the slow-phase half-life, t12beta, was 130 min, compared with 80 min for the undersialylated enzyme). Interestingly, the same increase in circulatory residence was observed when rHuAChE was subjected to extensive sialylation in vitro. The engineered HEK-293 cells in which the glycosylation machinery was modified might represent a valuable tool for the high level of expression of recombinant glycoproteins whose sialic acid content is important for their function or for pharmacokinetic behaviour.
ESTHER : Chitlaru_1998_Biochem.J_336_647
PubMedSearch : Chitlaru_1998_Biochem.J_336_647
PubMedID: 9841877

Title : Bovine Acetylcholinestrase Cloning, Expression and Characterization of the Recombinant Enzyme -
Author(s) : Mendelson I , Kronman C , Zeliger N , Seri T , Ordentlich A , Shafferman A , Velan B
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. :148 , 1998
PubMedID:

Title : Structural Modifications of the Omega Loop in Human Acetylcholinesterase -
Author(s) : Ariel N , Velan B , Barak D , Leitner M , Bino T , Ordentlich A , Shafferman A
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. :453 , 1998
PubMedID:

Title : Crystal Structures of Aged Phosphorylated and Phosphonylated Torpedo Californica Acetylcholinesterase -
Author(s) : Millard CB , Kryger G , Ordentlich A , Harel M , Raves ML , Greenblatt HM , Segall Y , Barak D , Shafferman A , Silman I , Sussman JL
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. :454 , 1998
PubMedID:

Title : Functional characteristics of the oxyanion hole in human acetylcholinesterase - Ordentlich_1998_J.Biol.Chem_273_19509
Author(s) : Ordentlich A , Barak D , Kronman C , Ariel N , Segall Y , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 273 :19509 , 1998
Abstract : The contribution of the oxyanion hole to the functional architecture and to the hydrolytic efficiency of human acetylcholinesterase (HuAChE) was investigated through single replacements of its elements, residues Gly-121, Gly-122 and the adjacent residue Gly-120, by alanine. All three substitutions resulted in about 100-fold decrease of the bimolecular rate constants for hydrolysis of acetylthiocholine; however, whereas replacements of Gly-120 and Gly-121 affected only the turnover number, mutation of residue Gly-122 had an effect also on the Michaelis constant. The differential behavior of the G121A and G122A enzymes was manifested also toward the transition state analog m-(N,N, N-trimethylammonio)trifluoroacetophenone (TMTFA), organophosphorous inhibitors, carbamates, and toward selected noncovalent active center ligands. Reactivity of both mutants toward TMTFA was 2000-11, 000-fold lower than that of the wild type HuAChE; however, the G121A enzyme exhibited a rapid inhibition pattern, as opposed to the slow binding kinetics shown by the G122A enzyme. For both phosphates (diethyl phosphorofluoridate, diisopropyl phosphorofluoridate, and paraoxon) and phosphonates (sarin and soman), the decrease in inhibitory activity toward the G121A enzyme was very substantial (2000-6700-fold), irrespective of size of the alkoxy substituents on the phosphorus atom. On the other hand, for the G122A HuAChE the relative decline in reactivity toward phosphonates (500-460-fold) differed from that toward the phosphates (12-95-fold). Although formation of Michaelis complexes with substrates does not seem to involve significant interaction with the oxyanion hole, interactions with this motif are a major stabilizing element in accommodation of covalent inhibitors like organophosphates or carbamates. These observations and molecular modeling suggest that replacements of residues Gly-120 or Gly-121 by alanine alter the structure of the oxyanion hole motif, abolishing the H-bonding capacity of residue at position 121. These mutations weaken the interaction between HuAChE and the various ligands by 2.7-5.0 kcal/mol. In contrast, variations in reactivity due to replacement of residue Gly 122 seem to result from steric hindrance at the active center acyl pocket
ESTHER : Ordentlich_1998_J.Biol.Chem_273_19509
PubMedSearch : Ordentlich_1998_J.Biol.Chem_273_19509
PubMedID: 9677373

Title : Contribution of the Active Center Functional Architecture to AChE Reactivity Toward Substrates and Inhibitors -
Author(s) : Shafferman A , Ordentlich A , Barak D , Kronman C , Ariel N , Velan B
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. :203 , 1998
PubMedID:

Title : Correlation of Isotope and Viscosity Effects -
Author(s) : Malany S , Taylor P , Quinn DM , Sawai M , Shafferman A , Velan B , Kronman C
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. :232 , 1998
PubMedID:

Title : Does Electrostatic Attraction or Steering by Charged Residues within the Gorge Contribute to the Reactivity of AChE? -
Author(s) : Ordentlich A , Barak D , Stein D , Berman D , Kronman C , Ariel N , Velan B , Shafferman A
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. :234 , 1998
PubMedID:

Title : Bovine acetylcholinesterase: cloning, expression and characterization - Mendelson_1998_Biochem.J_334_251
Author(s) : Mendelson I , Kronman C , Ariel N , Shafferman A , Velan B
Ref : Biochemical Journal , 334 :251 , 1998
Abstract : The bovine acetylcholinesterase (BoAChE) gene was cloned from genomic DNA and its structure was determined. Five exons coding for the AChE T-subunit and the alternative H-subunit were identified and their organization suggests high conservation of structure in mammalian AChE genes. The deduced amino acid sequence of the bovine T-subunit is highly similar to the human sequence, showing differences at 34 positions only. However, the cloned BoAChE sequence differs from the published amino acid sequence of AChE isolated from fetal bovine serum (FBS) by: (1) 13 amino acids, 12 of which are conserved between BoAChE and human AChE, and (2) the presence of four rather than five potential N-glycosylation sites. The full coding sequence of the mature BoAChE T-subunit was expressed in human embryonal kidney 293 cells (HEK-293). The catalytic properties of recombinant BoAChE and its reactivity towards various inhibitors were similar to those of the native bovine enzyme. Soluble recombinant BoAChE is composed of monomers, dimers and tetramers, yet in contrast to FBS-AChE, tetramer formation is not efficient. Comparative SDS/PAGE analysis reveals that all four potential N-glycosylation sites identified by DNA sequencing appear to be utilized, and that recombinant BoAChE comigrates with FBS-AChE. A major difference between the recombinant enzyme and the native enzyme was observed when clearance from circulation was examined. The HEK-293-derived enzyme was cleared from the circulation at a much faster rate than FBS-AChE. This difference in behaviour, together with previous studies on the effect of post-translation modification on human AChE clearance [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959-967] suggests that cell-dependent glycosylation plays a key role in AChE circulatory residence.
ESTHER : Mendelson_1998_Biochem.J_334_251
PubMedSearch : Mendelson_1998_Biochem.J_334_251
PubMedID: 9693127
Gene_locus related to this paper: bovin-ACHE

Title : The Aromatic Moiety at Position-86 of HuAChE Accelerate the Aging of Phosphonyl-AChE Conjugates through Cation- Interactions -
Author(s) : Barak D , Ordentlich A , Stein D , Segall Y , Velan B , Benschop HP , De Jong LP , Shafferman A
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. :246 , 1998
PubMedID:

Title : ESMS as a Unique Tool for the Molecular Monitoring of Reactions between HuAChE and Various OP-Agents -
Author(s) : Ordentlich A , Barak R , Barak D , Fischer M , Benschop HP , De Jong LP , Segall Y , Velan B , Shafferman A
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. :249 , 1998
PubMedID:

Title : Aging of somanyl-acetylcholinesterase adducts: facts and models [letter] -
Author(s) : Shafferman A , Ordentlich A , Barak D , Stein D , Ariel N , Velan B
Ref : Biochemical Journal , 324 :996 , 1997
PubMedID: 9235881

Title : Direct determination of the chemical composition of acetylcholinesterase phosphonylation products utilizing electrospray- ionization mass spectrometry - Barak_1997_FEBS.Lett_407_347
Author(s) : Barak R , Ordentlich A , Barak D , Fischer M , Benschop HP , De Jong LP , Segall Y , Velan B , Shafferman A
Ref : FEBS Letters , 407 :347 , 1997
Abstract : While non-reactivability of cholinesterases from their phosphyl conjugates (aging) is attributed to an unimolecular process involving loss of alkyl group from the phosphyl moiety, no conclusive evidence is available that this is the only reaction path and involvement of other post-inhibitory processes cannot be ruled out. To address this issue, molecular masses of the bacterially expressed recombinant human acetylcholinesterase and of its conjugates with a homologous series of alkyl methylphosphonofluoridates, were measured by electrospray-ionization mass spectrometry (ESI-MS). The measured mass of the free enzyme was 64,700 Da (calculated 64,695 Da) and those of the methylphosphono-HuAChE adducts, bearing isopropyl, isobutyl, 1,2-dimethylpropyl and 1,2,2-trimethylpropyl substituents, were 64,820, 64,840, 64,852 and 64,860 Da, respectively. These values reflect both the addition of the phosphonyl moiety and the gradual mass increase due to branching of the alkoxy substituent. The composition of these adducts change with time to yield a common product with molecular mass of 64,780 Da which is consistent with dealkylation of the phosphonyl moieties. Furthermore, in the case of 1,2-dimethylpropyl methylphosphono-HuAChE, the change in the molecular mass and the kinetics of non-reactivability appear to occur in parallel indicating that dealkylation is indeed the predominant molecular transformation leading to 'aging' of phosphonyl-AChE adducts.
ESTHER : Barak_1997_FEBS.Lett_407_347
PubMedSearch : Barak_1997_FEBS.Lett_407_347
PubMedID: 9175882

Title : Structural modifications of the omega loop in human acetylcholinesterase - Velan_1996_FEBS.Lett_395_22
Author(s) : Velan B , Barak D , Ariel N , Leitner M , Bino T , Ordentlich A , Shafferman A
Ref : FEBS Letters , 395 :22 , 1996
Abstract : Conformational mobility of the surface omega loop (Cys-69-Cys-96) in human acetylcholinesterase (HuAChE) was recently implicated in substrate accessibility to the active center and in the mechanism of allosteric modulation of enzymatic activity. We therefore generated and kinetically evaluated the following modifications or replacements in HuAChE: (a) residues at the loop ends, (b) residues involved in putative hydrogen-bond interactions within the loop and between the loop and the protein core, (c) ChEs conserved proline residues within the loop and (d) a deletion of a conserved segment of 5 residues. All the residue replacements, including those of the prolines, had either limited or no effect on enzyme reactivity. These results suggest that unlike the case of lipase, the omega loop in the HuAChE is not involved in large lid-like displacements. In cases where modifications of the loop sequence had some effect on reactivity, the effects could be attributed to an altered position of residue Trp-86 supporting the proposed coupling between the structure of the omega loop and the positioning of the Trp-86 indole moiety, in catalytic activity and in allosterism.
ESTHER : Velan_1996_FEBS.Lett_395_22
PubMedSearch : Velan_1996_FEBS.Lett_395_22
PubMedID: 8849682

Title : Aging of phosphylated human acetylcholinesterase: catalytic processes mediated by aromatic and polar residues of the active centre - Shafferman_1996_Biochem.J_318_833
Author(s) : Shafferman A , Ordentlich A , Barak D , Stein D , Ariel N , Velan B
Ref : Biochemical Journal , 318 :833 , 1996
Abstract : We have examined the effects of 11 substitutions of active centre gorge residues of human acetylcholinesterase (HuAChE) on the rates of phosphonylation by 1,2,2-trimethylpropyl methyl-phosphonofluoridate (soman) and the aging of the resulting conjugates. The rates of phosphonylation were reduced to as little as one-seventieth, mainly in mutants of the hydrogen-bond network (Glu-202, Glu-450, Tyr-133). These recombinant enzymes as well as the F338A, W86A, W86F and D74N mutant HuAChEs varied in their resistance to aging (15-3300-fold relative to the wild type). The most dramatic resistance to aging was observed for the phosphonyl conjugate of the mutant W86A enzyme (1850-3300-fold relative to the wild type). It is proposed that Trp-86 contributes to the aging process by stabilizing the evolving carbonium ion on the 1,2,2-trimethylpropyl moiety, via charge-pi interaction. The rate-enhancing effect of Trp-86 provides a rationale for the unique facility of aging in soman-inhibited cholinesterases, compared with the corresponding conjugates in other serine hydrolases. Replacements of Glu-202 by aspartic acid, glutamine or alanine residues resulted in a similar (1/130-1/300) decrease of the rates of aging. A comparable decrease was also observed for the conjugate of the F338A mutant. These results, and the similar pH dependence of aging rates for the wild-type and E202Q and F338A mutant HuAChEs, indicate that Glu-202 is not involved in proton transfer to the phosphonyl moiety. On the basis of these findings and of molecular modelling we suggest that Glu-202 and Phe-338 contribute to the aging process by stabilizing the imidazolium of the catalytic triad His-447 via charge-charge and charge-pi interactions respectively, thereby facilitating an oxonium formation on the phosphonyl moiety.
ESTHER : Shafferman_1996_Biochem.J_318_833
PubMedSearch : Shafferman_1996_Biochem.J_318_833
PubMedID: 8836126

Title : Interactions of oxime reactivators with diethylphosphoryl adducts of human acetylcholinesterase and its mutant derivatives - Grosfeld_1996_Mol.Pharmacol_50_639
Author(s) : Grosfeld H , Barak D , Ordentlich A , Velan B , Shafferman A
Ref : Molecular Pharmacology , 50 :639 , 1996
Abstract : Diethylphosphoryl conjugates of human acetylcholinesterase (AChE) and selected mutants, carrying amino acid replacements at the active center and at the peripheral anionic site, were subjected to reactivation with the monopyridinium oxime 2-hydroxy-iminomethyl-1-methylpyridinium chloride and the bispyridinium oximes 1,3-bis(4'-hydroxyiminomethyl-1'-pyridinium),propane dibromide (TMB-4(Trimedoxime)) and 1-(2'-hydroxyiminomethyl-1'-pyridinium)-3-(4"-carbamoyl-1"-pyridinium)-2 - oxapropane dichloride (HI-6). The kinetic profiles for all of the reactivation reactions indicate single populations of reactivatable species. Replacement of Trp86, the anionic subsite in the active center, lowered the affinity of the free enzyme toward all three reactivators, but in the corresponding diethylphosphoryl conjugate, only affinity toward TMB-4(Trimedoxime) was affected. Replacement of other constituents of the hydrophobic subsite (Tyr337, Phe338) had no major effect on either affinity to the free enzymes or rates of reactivation. Substitution of residues of the acyl pocket (Phe295, Phe297) lowered the affinities toward reactivators except for the 20-fold increase in affinity of F295A toward HI-6. Replacement of the acidic residues in the active center (Glu202, Glu450) affected mainly the rates of nucleophilic displacement of the phosphoryl moiety. The effect of substituting residues constituting the peripheral anionic site at the rim of the active site gorge (Tyr72, Asp74, Trp286) was particularly puzzling because for 2-hydroxy-iminomethyl-1-methylpyridinium chloride and HI-6, mainly the nucleophilic reaction rate constants were affected, whereas for TMB-4(Trimedoxime), the affinities of the phosphorylated enzymes were significantly reduced. The fact that perturbations of the functional architecture of HuAChE active center can account for only some of the observed effects on the reactivation rates suggests that the binding modes of oxime to the phosphorylated and nonphosphorylated enzymes are considerably different and/or that interactions of the reactivators with the phosphoryl moieties play a dominant role in the reactivation process.
ESTHER : Grosfeld_1996_Mol.Pharmacol_50_639
PubMedSearch : Grosfeld_1996_Mol.Pharmacol_50_639
PubMedID: 8794905

Title : The architecture of human acetylcholinesterase active center probed by interactions with selected organophosphate inhibitors - Ordentlich_1996_J.Biol.Chem_271_11953
Author(s) : Ordentlich A , Barak D , Kronman C , Ariel N , Segall Y , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 271 :11953 , 1996
Abstract : The role of the functional architecture of human acetylcholinesterase (HuAChE) active center in facilitating reactions with organophosphorus inhibitors was examined by a combination of site-directed mutagenesis and kinetic studies of phosphorylation with organophosphates differing in size of their alkoxy substituents and in the nature of the leaving group. Replacements of residues Phe-295 and Phe-297, constituting the HuAChE acyl pocket, increase up to 80-fold the reactivity of the enzymes toward diisopropyl phosphorofluoridate, diethyl phosphorofluoridate, and p-nitrophenyl diethyl phosphate (paraoxon), indicating the role of this subsite in accommodating the phosphate alkoxy substituent. On the other hand, a decrease of up to 160-fold in reactivity was observed for enzymes carrying replacements of residues Tyr-133, Glu-202, and Glu-450, which are constituents of the hydrogen bond network in the HuAChE active center, which maintains its unique functional architecture. Replacement of residues Trp-86, Tyr-337, and Phe-338 in the alkoxy pocket affected reactivity toward diisopropyl phosphorofluoridate and paraoxon, but to a lesser extent that toward diethyl phosphorofluoridate, indicating that both the alkoxy substituent and the p-nitrophenoxy leaving group interact with this subsite. In all cases the effects on reactivity toward organophosphates, demonstrated in up to 10,000-fold differences in the values of bimolecular rate constants, were mainly a result of altered affinity of the HuAChE mutants, while the apparent first order rate constants of phosphorylation varied within a narrow range. This finding indicates that the main role of the functional architecture of HuAChE active center in phosphorylation is to facilitate the formation of enzyme-inhibitor Michaelis complexes and that this affinity, rather than the nucleophilic activity of the enzyme catalytic machinery, is a major determinant of HuAChE reactivity toward organophosphates.
ESTHER : Ordentlich_1996_J.Biol.Chem_271_11953
PubMedSearch : Ordentlich_1996_J.Biol.Chem_271_11953
PubMedID: 8662593

Title : Molecular Aspects of Catalysis and of Allosteric Regulation of Aceytlcholinesterases -
Author(s) : Shafferman A , Ordentlich A , Barak D , Kronman C , Ariel N , Leitner M , Segall Y , Bromberg A , Reuveny S , Marcus D , Bino T , Lazar A , Cohen S , Velan B
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. :189 , 1995
PubMedID:

Title : Contribution of aromatic moieties of tyrosine 133 and of the anionic subsite tryptophan 86 to catalytic efficiency and allosteric modulation of acetylcholinesterase - Ordentlich_1995_J.Biol.Chem_270_2082
Author(s) : Ordentlich A , Barak D , Kronman C , Ariel N , Segall Y , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 270 :2082 , 1995
Abstract : Substitution of Trp-86, in the active center of human acetylcholinesterase (HuAChE), by aliphatic but not by aromatic residues resulted in a several thousandfold decrease in reactivity toward charged substrate and inhibitors but only a severalfold decrease for noncharged substrate and inhibitors. The W86A and W86E HuAChE enzymes exhibit at least a 100-fold increase in the Michaelis-Menten constant or 100-10,000-fold increase in inhibition constants toward various charged inhibitors, as compared to W86F HuAChE or the wild type enzyme. On the other hand, replacement of Glu-202, the only acidic residue proximal to the catalytic site, by glutamine resulted in a nonselective decrease in reactivity toward charged and noncharged substrates or inhibitors. Thus, the quaternary nitrogen groups of substrates and other active center ligands, are stabilized by cation-aromatic interaction with Trp-86 rather than by ionic interactions, while noncharged ligands appear to bind to distinct site(s) in HuAChE. Analysis of the Y133F and Y133A HuAChE mutated enzymes suggests that the highly conserved Tyr-133 plays a dual role in the active center: (a) its hydroxyl appears to maintain the functional orientation of Glu-202 by hydrogen bonding and (b) its aromatic moiety maintains the functional orientation of the anionic subsite Trp-86. In the absence of aromatic interactions between Tyr-133 and Trp-86, the tryptophan acquires a conformation that obstructs the active site leading, in the Y133A enzyme, to several hundredfold decrease in rates of catalysis, phosphorylation, or in affinity to reversible active site inhibitors. It is proposed that allosteric modulation of acetylcholinesterase activity, induced by binding to the peripheral anionic sites, proceeds through such conformational change of Trp-86 from a functional anionic subsite state to one that restricts access of substrates to the active center.
ESTHER : Ordentlich_1995_J.Biol.Chem_270_2082
PubMedSearch : Ordentlich_1995_J.Biol.Chem_270_2082
PubMedID: 7836436

Title : Compilation of Evaluated Mutants of Cholinesterases -
Author(s) : Shafferman A , Kronman C , Ordentlich A
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. :481 , 1995
PubMedID:

Title : Denaturation of Recombinant Human Acetylcholinesterase -
Author(s) : Lebleu M , Clery C , Masson P , Reuveny S , Marcus D , Velan B , Shafferman A
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. :131 , 1995
PubMedID:

Title : Involvement of oligomerization, N-glycosylation and sialylation in the clearance of cholinesterases from the circulation - Kronman_1995_Biochem.J_311_959
Author(s) : Kronman C , Velan B , Marcus D , Ordentlich A , Reuveny S , Shafferman A
Ref : Biochemical Journal , 311 :959 , 1995
Abstract : The possible role of post-translational modifications such as subunit oligomerization, protein glycosylation and oligosaccharide processing on the circulatory life-time of proteins was studied using recombinant human acetylcholinesterase (rHuAChE). Different preparations of rHuAChE containing various amounts of tetramers, dimers and monomers are cleared at similar rates from the circulation, suggesting that oligomerization does not play an important role in determining the rate of clearance. An engineered rHuAChE mutant containing only one N-glycosylation site was cleared from the circulation more rapidly than the wild-type triglycosylated enzyme. On the other hand, hyperglycosylated mutants containing either four or five occupied N-glycosylation sites, analagous to those present on the slowly cleared fetal bovine serum acetylcholinesterase (FBS-AChE), were also cleared more rapidly from the bloodstream than the wild-type species. Furthermore, the two different tetraglycosylated mutants were cleared at different rates while the pentaglycosylated mutant exhibited the most rapid clearance profile. These results imply that though the number of N-glycosylation sites plays a role in the circulatory life-time of the enzyme, the number of N-glycan units in itself does not determine the rate of clearance. When saturating amounts of asialofetuin were administered together with rHuAChE, the circulatory half-life of the enzyme was dramatically increased (from 80 min to 19 h) and was found to be similar to that displayed by plasma-derived cholinesterases while desialylation of these enzymes caused a sharp decrease in the circulatory half-life to approximately 3-5 min. Determination of the average number of sialic acid residues per enzyme subunit of the five different N-glycosylation species generated, revealed that the rate of clearance is not a function of the absolute number of appended sialic acid moieties but rather of the number of unoccupied sialic acid attachment sites per enzyme molecule. Specifically, we demonstrate an inverse-linear relationship between the number of vacant sialic acid attachment sites and the values of the enzyme residence time within the bloodstream.
ESTHER : Kronman_1995_Biochem.J_311_959
PubMedSearch : Kronman_1995_Biochem.J_311_959
PubMedID: 7487957

Title : Electrostatic Attraction by Surface Charge does not Contribute to the Catalytic Efficiency of Acetylcholinesterase -
Author(s) : Barak D , Ordentlich A , Kronman C , Ber R , Bino T , Ariel N , Osman R , Velan B , Shafferman A
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. :223 , 1995
PubMedID:

Title : Signal-Mediated Cellular Retention and Subunit Assembly of Human Acetylcholinesterase -
Author(s) : Kronman C , Flashner Y , Shafferman A , Velan B
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. :293 , 1995
PubMedID:

Title : Amino Acids Determining Specificity to OP-Agents and Facilitating the Aging Process in Human Acetylcholinesterase -
Author(s) : Ordentlich A , Kronman C , Stein D , Ariel N , Reuveny S , Marcus D , Segall Y , Barak D , Velan B , Shafferman A
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. :221 , 1995
PubMedID:

Title : Allosteric modulation of acetylcholinesterase activity by peripheral ligands involves a conformational transition of the anionic subsite - Barak_1995_Biochemistry_34_15444
Author(s) : Barak D , Ordentlich A , Bromberg A , Kronman C , Marcus D , Lazar A , Ariel N , Velan B , Shafferman A
Ref : Biochemistry , 34 :15444 , 1995
Abstract : Replacement of residues Asp74, Trp286, and Tyr72, which are constituents of the peripheral anionic site (PAS) of human acetylcholinesterase (HuAChE), affected similarly both the binding and the inhibition constants of the PAS-specific ligand propidium, demonstrating that changes in the inhibitory activity are a direct consequence of altered binding to the PAS. In contrast, the active center HuAChE mutants W86A and Y133A show respective 350- and 25-fold increased resistance to inhibition by propidium but no change in binding affinities, demonstrating that the allosteric mechanism of PAS-mediated inhibition involves a conformational change of these Trp86 and Tyr133 residues rather than physical obstruction of substrate access by the inhibitor itself. These findings support the recent proposal that the allosteric mechanism operates via transition between active and nonactive conformations of the anionic subsite Trp86 and that replacement of Tyr133 by alanine may stabilize a nonactive Trp86 conformation that occludes the active center [Ordentlich et al. (1995) J. Biol. Chem. 270, 2082]. In further support of this mechanism and the role of Tyr133, we find that (a) the dissociation constants (Kd) for the noncovalent complexes of the irreversible inhibitors diisopropyl phosphorofluoridate or paraoxon with Y133A HuAChE are increased 20-500-fold, relative to either wild-type enzyme or its Y133F or W86A mutants; and (b) access of substrates such as 3,3-dimethylbutyl thioacetate is restored by removal of Trp86 from the Y133A enzyme (i.e., the W86A/Y133A mutant). We suggest that the conformational transition of Trp86 is coupled to the motions of the cysteine loop (Cys69-Cys96) of HuAChE and is inherent to the dynamics of the native enzyme.
ESTHER : Barak_1995_Biochemistry_34_15444
PubMedSearch : Barak_1995_Biochemistry_34_15444
PubMedID: 7492545

Title : Acetylcholinesterase peripheral anionic site degeneracy conferred by amino acid arrays sharing a common core - Barak_1994_J.Biol.Chem_269_6296
Author(s) : Barak D , Kronman C , Ordentlich A , Ariel N , Bromberg A , Marcus D , Lazar A , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 269 :6296 , 1994
Abstract : Several of the residues constituting the peripheral anionic site (PAS) in human acetylcholinesterase (HuAChE) were identified by a combination of kinetic studies with 19 single and multiple HuAChE mutants, fluorescence binding studies with the Trp-286 mutant, and by molecular modeling. Mutants were analyzed with three structurally distinct positively charged PAS ligands, propidium, decamethonium, and di(p-allyl-N-dimethylaminophenyl)pentane-3-one (BW284C51), as well as with selective active center inhibitors, hexamethonium and edrophonium. Single mutations of residues Tyr-72, Tyr-124, Glu-285, Trp-286, and Tyr-341 resulted in up to 10-fold increase in inhibition constants for PAS ligands, whereas for multiple mutants up to 400-fold increase was observed. The 6th PAS element residue Asp-74 is unique in its ability to affect conformation of both the active site and the PAS (Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) EMBO J. 11, 3561-3568) as demonstrated by the several hundred-fold increase in Ki for D74N inhibition by the bisquaternary ligands decamethonium and BW284C51. Based on these studies, singular molecular models for the various HuAChE inhibitor complexes were defined. Yet, for the decamethonium complex two distinct conformations were generated, accommodating the quaternary ammonium group by interactions with either Trp-286 or with Tyr-341. We propose that the PAS consists of a number of binding sites, close to the entrance of the active site gorge, sharing residues Asp-74 and Trp-286 as a common core. Binding of ligands to these residues may be the key to the allosteric modulation of HuAChE catalytic activity. This functional degeneracy is a result of the ability of the Trp-286 indole moiety to interact either via stacking, aromatic-aromatic, or via pi-cation attractions and the involvement of the carboxylate of Asp-74 in charge-charge or H-bond interactions.
ESTHER : Barak_1994_J.Biol.Chem_269_6296
PubMedSearch : Barak_1994_J.Biol.Chem_269_6296
PubMedID: 8119978

Title : The back door hypothesis for product clearance in acetylcholinesterase challenged by site-directed mutagenesis - Kronman_1994_J.Biol.Chem_269_27819
Author(s) : Kronman C , Ordentlich A , Barak D , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 269 :27819 , 1994
Abstract : The active site of acetylcholinesterase is near the bottom of a long and narrow gorge. The dimensions of the gorge and the strong electrostatic field generated by the enzyme appear inconsistent with the enzyme's high turnover rate. Consequently, a "back door" mechanism involving movement of the reaction products through a transient opening near the active center was recently suggested. We investigated this hypothesis in human acetylcholinesterase by testing mutants at key residues (Glu-84, Trp-86, Asp-131, and Val-132) located near or along the putative back door channel. The turnover rates of all mutants tested, and in particular of V132K, where the channel is expected to be sealed by salt bridge Lys-132-Glu-452, are similar to that of the wild type enzyme. This indicates that the proposed back door is not a route for product clearance from the active site gorge of acetylcholinesterase and is probably of no functional relevance to its catalytic activity.
ESTHER : Kronman_1994_J.Biol.Chem_269_27819
PubMedSearch : Kronman_1994_J.Biol.Chem_269_27819
PubMedID: 7961709

Title : Electrostatic attraction by surface charge does not contribute to the catalytic efficiency of acetylcholinesterase - Shafferman_1994_EMBO.J_13_3448
Author(s) : Shafferman A , Ordentlich A , Barak D , Kronman C , Ber R , Bino T , Ariel N , Osman R , Velan B
Ref : EMBO Journal , 13 :3448 , 1994
Abstract : Acetylcholinesterases (AChEs) are characterized by a high net negative charge and by an uneven surface charge distribution, giving rise to a negative electrostatic potential extending over most of the molecular surface. To evaluate the contribution of these electrostatic properties to the catalytic efficiency, 20 single- and multiple-site mutants of human AChE were generated by replacing up to seven acidic residues, vicinal to the rim of the active-center gorge (Glu84, Glu285, Glu292, Asp349, Glu358, Glu389 and Asp390), by neutral amino acids. Progressive simulated replacement of these charged residues results in a gradual decrease of the negative electrostatic potential which is essentially eliminated by neutralizing six or seven charges. In marked contrast to the shrinking of the electrostatic potential, the corresponding mutations had no significant effect on the apparent bimolecular rate constants of hydrolysis for charged and non-charged substrates, or on the Ki value for a charged active center inhibitor. Moreover, the kcat values for all 20 mutants are essentially identical to that of the wild type enzyme, and the apparent bimolecular rate constants show a moderate dependence on the ionic strength, which is invariant for all the enzymes examined. These findings suggest that the surface electrostatic properties of AChE do not contribute to the catalytic rate, that this rate is probably not diffusion-controlled and that long-range electrostatic interactions play no role in stabilization of the transition states of the catalytic process.
ESTHER : Shafferman_1994_EMBO.J_13_3448
PubMedSearch : Shafferman_1994_EMBO.J_13_3448
PubMedID: 8062821

Title : Reversal of signal-mediated cellular retention by subunit assembly of human acetylcholinesterase - Velan_1994_J.Biol.Chem_269_22719
Author(s) : Velan B , Kronman C , Flashner Y , Shafferman A
Ref : Journal of Biological Chemistry , 269 :22719 , 1994
Abstract : The interrelationship between signal-mediated endoplasmic reticulum retention and control of subunit assembly in secreted complex proteins was examined in recombinant 293 cells expressing human acetylcholinesterase (HuAChE). This was achieved by analyzing the mutual effects of co-residing retention and dimerization signals on enzyme secretion by transfected cells. The function of putative signals within the COOH-terminal tetrapeptide CSDL of HuAChE was examined by site-directed mutagenesis. The CSDL tetrapeptide carries the free cysteine (Cys-580) involved in subunit assembly, yet it fails to function as a KDEL-type retention signal. This was demonstrated by mutations that increase similarity to the canonical retention signal (substitution of CSDL by KSDL) or those that deviate from it (substitution to CSAL). Cells expressing both types of mutants exhibited cell-associated HuAChE levels identical to that of wild type enzyme. Appendage of an engineered KDEL retention signal to a dimerization-impaired HuA-ChE subunit (the C580A mutant) resulted in intracellular retention of large amounts of fully active enzyme not prone to proteolytic degradation. On the other hand, attachment of KDEL to a native, dimerization-competent HuAChE polypeptide did not lead to intracellular retention and allowed efficient secretion of enzyme to the cell growth medium. Yet, appendage of KDEL to the native HuAChE led to some retardation in the transport of enzyme molecules through the Golgi apparatus, as manifested by increase in cellular population of endo H-resistant dimers, when compared with wild type enzyme. Taken together, these results indicate (alpha) that sub-unit dimerization mediated by the COOH-terminal cysteine of HuAChE can reverse the signal-mediated retention by masking recognition of KDEL by its cognate receptor and (b) that the native sequences of the acetylcholinesterase subunit polypeptide do not appear to function as a coupled retention/dimerization signal in the control of secretion of assembled enzyme molecules.
ESTHER : Velan_1994_J.Biol.Chem_269_22719
PubMedSearch : Velan_1994_J.Biol.Chem_269_22719
PubMedID: 8077224

Title : Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos - Seidman_1994_J.Neurochem_62_1670
Author(s) : Seidman S , Aziz-Aloya RB , Timberg R , Loewenstein Y , Velan B , Shafferman A , Liao J , Norgaard-Pedersen B , Brodbeck U , Soreq H
Ref : Journal of Neurochemistry , 62 :1670 , 1994
Abstract : Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DNA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2- and 3-day-old embryos within the same subcellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.
ESTHER : Seidman_1994_J.Neurochem_62_1670
PubMedSearch : Seidman_1994_J.Neurochem_62_1670
PubMedID: 8158119

Title : Role of tyrosine 337 in the binding of huperzine A to the active site of human acetylcholinesterase - Ashani_1994_Mol.Pharmacol_45_555
Author(s) : Ashani Y , Grunwald J , Kronman C , Velan B , Shafferman A
Ref : Molecular Pharmacology , 45 :555 , 1994
Abstract : Huperzine A (HUP), a natural, potent, 'slow,' reversible inhibitor of antiacetylcholinesterase (AChE), has been suggested to be superior to antiacetylcholinesterase drugs now being used for management of Alzheimer's disease. To delineate the binding site of human AChE (HuAChE) for HUP, the biochemical constants kon, koff, and Ki were determined for complexes formed between HUP and single-site (Y337F, Y337A, F295A, W286A, and E202Q) or double-site (F295L/F297V) mutants of recombinant HuAChE (rHuAChE). The kinetic and dissociation constants were compared with those obtained for wild-type rHuAChE and AChE from Torpedo californica. Results demonstrate that the inhibition of AChE by HUP occurs through association with residues located inside the active site 'gorge,' rather than at the rim of the gorge. Tyrosine at position 337 (Y337) is essential for inhibition of rHuAChE by HUP (Ki = 26 nM). An aromatic array constituted from residues Y337, F295, and probably W86 is likely to offer a multicontact subsite that interacts with the ammonium group and with both the exo-and endocyclic double bond moieties of HUP. Lack of the aromatic side chain in the position homologous to Y337 explains the poor inhibitory potency of HUP toward human butyrylcholinesterase (Ki > 20,000 nM). Replacement of the carboxylate-containing E202 by glutamine had only marginal effect on the stability of the complex formed between HUP and rHuAChE. The pH-rate profiles suggest that destabilization of the complex after proton gain cannot be attributed solely to protonation of E202. These findings are expected to establish HUP as a lead compound for the design of new anti-AChE drugs.
ESTHER : Ashani_1994_Mol.Pharmacol_45_555
PubMedSearch : Ashani_1994_Mol.Pharmacol_45_555
PubMedID: 8145739
Gene_locus related to this paper: human-ACHE

Title : Engineering resistance to 'aging' of phosphylated human acetylcholinesterase. Role of hydrogen bond network in the active center - Ordentlich_1993_FEBS.Lett_334_215
Author(s) : Ordentlich A , Kronman C , Barak D , Stein D , Ariel N , Marcus D , Velan B , Shafferman A
Ref : FEBS Letters , 334 :215 , 1993
Abstract : Recombinant human acetylcholinesterase (HuAChE) and selected mutants (E202Q, Y337A, E450A) were studied with respect to catalytic activity towards charged and noncharged substrates, phosphylation with organophosphorus (OP) inhibitors and subsequent aging of the OP-conjugates. Amino acid E450, unlike residues E202 and Y337, is not within interaction distance from the active center. Yet, the bimolecular rates of catalysis and phosphylation are 30-100 fold lower for both E450A and E202Q compared to Y337A or the wild type and in both mutants the resulting OP-conjugates show striking resistance to aging. It is proposed that a hydrogen bond network, that maintains the functional architecture of the active center, involving water molecules and residues E202 and E450, is responsible for the observed behaviour.
ESTHER : Ordentlich_1993_FEBS.Lett_334_215
PubMedSearch : Ordentlich_1993_FEBS.Lett_334_215
PubMedID: 8224249

Title : Evaluation of anchorage-dependent cell propagation systems for production of human acetylcholinesterase by recombinant 293 cells - Lazar_1993_Cytotech_13_115
Author(s) : Lazar A , Reuveny S , Kronman C , Velan B , Shafferman A
Ref : Cytotechnology , 13 :115 , 1993
Abstract : Production of recombinant human acetylcholinesterase (AChE) by a high producer human embryonic kidney cell line (293) was evaluated by three main cell propagation systems; surface propagator, fixed-bed reactor and stirred microcarrier cultures. The recombinant cell line expresses AChE levels as high as 10-20 mg/l/day. System productivities in either the surface propagator (multitray system), or in the fixed-bed reactor (polyurethane macroporous sponges) were 4-8 mg AChE/l/day during a production period of 8 days. Similar productive rates, yet longer production periods (up to 22 days), were obtained in microcarrier (MC) cultures using either polystyrene beads (Biosilon); collagen-coated dextran beads (Cytodex-3); or gelatin macroporous beads (Cultispher-G). Best results were obtained in an aggregate culture using cellulose beads charged with diethylaminoethyl (DEAE) groups, (Servacel), as carriers. In this culture, a system productivity of 6-10 mg/l/day was maintained for 28 days.
ESTHER : Lazar_1993_Cytotech_13_115
PubMedSearch : Lazar_1993_Cytotech_13_115
PubMedID: 7764576

Title : Dissection of the human acetylcholinesterase active center determinants of substrate specificity. Identification of residues constituting the anionic site, the hydrophobic site, and the acyl pocket - Ordentlich_1993_J.Biol.Chem_268_17083
Author(s) : Ordentlich A , Barak D , Kronman C , Flashner Y , Leitner M , Segall Y , Ariel N , Cohen S , Velan B , Shafferman A
Ref : Journal of Biological Chemistry , 268 :17083 , 1993
Abstract : Substrate specificity determinants of human acetylcholinesterase (HuAChE) were identified by combination of molecular modeling and kinetic studies with enzymes mutated in residues Trp-86, Trp-286, Phe-295, Phe-297, Tyr-337, and Phe-338. The substitution of Trp-86 by alanine resulted in a 660-fold decrease in affinity for acetythiocholine but had no effect on affinity for the isosteric uncharged substrate (3,3-dimethylbutylthioacetate). The results demonstrate that residue Trp-86 is the anionic site which binds, through cation-pi interactions, the quaternary ammonium of choline, and that of active center inhibitors such as edrophonium. The results also suggest that in the non-covalent complex, charged and uncharged substrates with a common acyl moiety (acetyl) bind to different molecular environments. The hydrophobic site for the alcoholic portion of the covalent adduct (tetrahedral intermediate) includes residues Trp-86, Tyr-337, and Phe-338, which operate through nonpolar and/or stacking interactions, depending on the substrate. Substrates containing choline but differing in the acyl moiety (acetyl, propyl, and butyryl) revealed that residues Phe-295 and Phe-297 determine substrate specificity of the acyl pocket for the covalent adducts. Phe-295 also determines substrate specificity in the non-covalent enzyme substrate complex and thus, the HuAChE F295A mutant exhibits over 130-fold increase in the apparent bimolecular rate constant for butyrylthiocholine compared with wild type enzyme. Reactivity toward specific butyrylcholinesterase inhibitors is similarly dependent on the nature of residues at positions 295 and 297. Amino acid Trp-286 at the rim of the active site "gorge" and Trp-86, in the active center, are essential elements in the mechanism of inhibition by propidium, a peripheral anionic site ligand. Molecular modeling and kinetic data suggest that a cross-talk between Trp-286 and Trp-86 can result in reorientation of Trp-86 which may then interfere with stabilization of substrate enzyme complexes. It is proposed that the conformational flexibility of aromatic residues generates a plasticity in the active center that contributes to the high efficiency of AChE and its ability to respond to external stimuli.
ESTHER : Ordentlich_1993_J.Biol.Chem_268_17083
PubMedSearch : Ordentlich_1993_J.Biol.Chem_268_17083
PubMedID: 8349597
Gene_locus related to this paper: human-ACHE , human-BCHE

Title : Interrelations between assembly and secretion of recombinant human acetylcholinesterase - Kerem_1993_J.Biol.Chem_268_180
Author(s) : Kerem A , Kronman C , Bar-Nun S , Shafferman A , Velan B
Ref : Journal of Biological Chemistry , 268 :180 , 1993
Abstract : Transport and secretion of recombinant human acetylcholinesterase (rHuAChE) were studied in transfected human 293 cells expressing either the oligomerized soluble enzyme or a monomeric mutant derivative in which Cys-580 was substituted by alanine (C580A). In cells expressing the wild-type enzyme, the gradual assembly of newly synthesized intracellular rHuAChE monomers into oligomers occurs within the endoplasmic reticulum. Secretion of mature wild-type enzyme into the medium is efficient and appears to be exclusive to multimeric forms. Consistently, intracellular oligomers, but not monomers, are endoglycosidase H-resistant, indicating that only oligomers undergo terminal glycosylation in the wild-type enzyme. In contrast, in cells expressing the dimerization-defective C580A mutant, newly synthesized rHuAChE monomers undergo terminal glycosylation and are secreted into the medium as efficiently as wild-type multimers. No significant difference between the intracellular transport rates of wild-type rHuAChE oligomers and mutant C580A monomers was revealed by probing with specific lectins. In both systems, transport and processing prior to the trans-Golgi galactosylation compartment appear to be rate-limiting, whereas the following passage to the cell surface is rapid. In conclusion, we suggest that in the presence of a free cysteine at the COOH terminus of the rHuAChE polypeptide, secretion of monomers is not effectuated, whereas in its absence, monomers are exported from the endoplasmic reticulum and are capable of traversing the entire secretory pathway.
ESTHER : Kerem_1993_J.Biol.Chem_268_180
PubMedSearch : Kerem_1993_J.Biol.Chem_268_180
PubMedID: 8416926

Title : N-glycosylation of human acetylcholinesterase: effects on activity, stability and biosynthesis - Velan_1993_Biochem.J_296_649
Author(s) : Velan B , Kronman C , Ordentlich A , Flashner Y , Leitner M , Cohen S , Shafferman A
Ref : Biochemical Journal , 296 :649 , 1993
Abstract : The role of N-glycosylation in the function of human acetylcholinesterase (HuAChE) was examined by site-directed mutagenesis (Asn to Gln substitution) of the three potential N-glycosylation sites Asn-265, Asn-350 and Asn-464. Analysis of HuAChE mutants, defective in a single or multiple N-glycosylation sites, by expression in transiently or stably transfected human embryonal 293 kidney cells suggests the following. (a) All three AChE glycosylation signals are utilized, but not all the secreted molecules are fully glycosylated. (b) Glycosylation at all sites is important for effective biosynthesis and secretion; extracellular AChE levels in mutants defective in one, two or all three sites amounted to 20-30%, 2-4% and about 0.5% of wild-type level respectively. (c) Some glycosylation mutants display impaired stability, as reflected by increased susceptibility to heat inactivation; substitution of Asn-464 has the most pronounced effect on thermostability. (d) Abrogation of N-glycosylation has no detectable effect on the enzyme activity of HuAChE; all glycosylation mutants, including the triple mutant, hydrolyse acetylthiocholine efficiently, displaying Km, kcat. and kcat./Km values similar to those of the wild-type enzyme. (e) In most mutants, inhibition profiles with edrophonium and bisquaternary ammonium ligands are identical with those of wild-type enzyme; the Asn-350 mutants, however, exhibit a slight decrease in their affinity towards these ligands. (f) Elimination of oligosaccharide side chains has no detectable effect on the surface-related 'peripheral-site' functions; like the wild-type enzyme, all mutants were inhibited by propidium and by increased concentrations of acetylthiocholine.
ESTHER : Velan_1993_Biochem.J_296_649
PubMedSearch : Velan_1993_Biochem.J_296_649
PubMedID: 8280063

Title : Recombinant human acetylcholinesterase - Enzyme engineering -
Author(s) : Shafferman A , Velan B , Barak D , Kronman C , Ordentlich A , Flashner Y , Leitner M , Segal Y , Grosfeld H , Stein D , Ariel N
Ref : Medical Defense Bioscience Review , 3 :1097 , 1993
PubMedID:

Title : Acetylcholinesterase Catalysis - Protein Engineering Studies -
Author(s) : Shafferman A , Velan B
Ref : In Multidisciplinary approaches to cholinesterase functions - Proceedings of Fourth International Meeting on Cholinesterases , (Shafferman, A. and Velan, B., Eds) Plenum Press, New York :165 , 1992
PubMedID:

Title : Mutagenesis of human acetylcholinesterase. Identification of residues involved in catalytic activity and in polypeptide folding - Shafferman_1992_J.Biol.Chem_267_17640
Author(s) : Shafferman A , Kronman C , Flashner Y , Leitner M , Grosfeld H , Ordentlich A , Gozes Y , Cohen S , Ariel N , Barak D , Harel M , Silman I , Sussman JL , Velan B
Ref : Journal of Biological Chemistry , 267 :17640 , 1992
Abstract : Evidence for the involvement of Ser-203, His-447, and Glu-334 in the catalytic triad of human acetylcholinesterase was provided by substitution of these amino acids by alanine residues. Of 20 amino acid positions mutated so far in human acetylcholinesterase (AChE), these three were unique in abolishing detectable enzymatic activity (less than 0.0003 of wild type), yet allowing proper production, folding, and secretion. This is the first biochemical evidence for the involvement of a glutamate in a hydrolase triad (Schrag, J.D., Li, Y., Wu, M., and Cygler, M. (1991) Nature 351, 761-764), supporting the x-ray crystal structure data of the Torpedo californica acetylcholinesterase (Sussman, J.L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L. and Silman, I. (1991) Science 253, 872-879). Attempts to convert the AChE triad into a Cys-His-Glu or Ser-His-Asp configuration by site-directed mutagenesis did not yield effective AChE activity. Another type of substitution, that of Asp-74 by Gly or Asn, generated an active enzyme with increased resistance to succinylcholine and dibucaine; thus mimicking in an AChE molecule the phenotype of the atypical butyrylcholinesterase natural variant (D70G mutation). Mutations of other carboxylic residues Glu-84, Asp-95, Asp-333, and Asp-349, all conserved among cholinesterases, did not result in detectable alteration in the recombinant AChE, although polypeptide productivity of the D95N mutant was considerably lower. In contrast, complete absence of secreted human AChE polypeptide was observed when Asp-175 or Asp-404 were substituted by Asn. These two aspartates are conserved in the entire cholinesterase/thyroglobulin family and appear to play a role in generating and/or maintaining the folded state of the polypeptide. The x-ray structure of the Torpedo acetylcholinesterase supports this assumption by revealing the participation of these residues in salt bridges between neighboring secondary structure elements.
ESTHER : Shafferman_1992_J.Biol.Chem_267_17640
PubMedSearch : Shafferman_1992_J.Biol.Chem_267_17640
PubMedID: 1517212

Title : Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center - Shafferman_1992_EMBO.J_11_3561
Author(s) : Shafferman A , Velan B , Ordentlich A , Kronman C , Grosfeld H , Leitner M , Flashner Y , Cohen S , Barak D , Ariel N
Ref : EMBO Journal , 11 :3561 , 1992
Abstract : Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.
ESTHER : Shafferman_1992_EMBO.J_11_3561
PubMedSearch : Shafferman_1992_EMBO.J_11_3561
PubMedID: 1396557

Title : Production and secretion of high levels of recombinant human acetylcholinesterase in cultured cell lines: microheterogeneity of the catalytic subunit - Kronman_1992_Gene_121_295
Author(s) : Kronman C , Velan B , Gozes Y , Leitner M , Flashner Y , Lazar A , Marcus D , Sery T , Papier Y , Grosfeld H , Cohen S , Shafferman A
Ref : Gene , 121 :295 , 1992
Abstract : To allow for structural analysis of the human acetylcholinesterase (hAChE) subunit, a series of eukaryotic vectors was designed for efficient expression. Several eukaryotic multicistronic expression vectors were tested in various mammalian cell lines. All expression vectors contained the selectable neo gene under control of a weak promoter, while the hAChE cDNA was under control of the cytomegalovirus (CMV) immediate-early or Rous sarcoma virus long terminal repeat (RSV LTR) or simian virus 40 (SV40) early promoters. Optimal production and secretion of recombinant hAChE (rehAChE) was achieved in the embryonal kidney 293 cell line transfected either with the RSV-hAChE or with CMV-hAChE expression vectors. Clones expressing and secreting as much as 5-25 pg of enzyme per cell per 24 h were obtained without resorting to coamplification techniques or continuous maintenance of cells under selective pressure. The purified (specific activity of 6000 units per mg protein) homodimer and tetramer enzyme molecules displayed typical AChE biochemical properties: a Km value of 120 microM for acetylthiocholine; a kcat value of 3.9 x 10(5)/min, and selective by AChE-specific inhibitors. Catalytic subunit dimers (130 kDa) exhibit differential N-glycosylation patterns, and upon reduction resolve into 67- and 70-kDa monomeric subunits. These two forms appear as a single discrete 62-kDa band following deglycosylation by N-glycanase. The N-terminal amino acid sequence analysis of the purified mature enzyme suggests the existence of two alternative cleavage sites for the removal of the signal peptide, in which the 'mature' position 1 is either Ala31 or Gly33. Both of these positions conform with the consensus signal peptide recognition sequences and demonstrate bidirected processing of signal peptides on a native molecule.
ESTHER : Kronman_1992_Gene_121_295
PubMedSearch : Kronman_1992_Gene_121_295
PubMedID: 1446827

Title : The effect of elimination of intersubunit disulfide bonds on the activity, assembly, and secretion of recombinant human acetylcholinesterase. Expression of acetylcholinesterase Cys-580----Ala mutant - Velan_1991_J.Biol.Chem_266_23977
Author(s) : Velan B , Grosfeld H , Kronman C , Leitner M , Gozes Y , Lazar A , Flashner Y , Marcus D , Cohen S , Shafferman A
Ref : Journal of Biological Chemistry , 266 :23977 , 1991
Abstract : Site-directed mutagenesis was used to study the cysteine residue involved in the assembly of human acetylcholinesterase (HuAChE) catalytic subunits. Substitution of the cysteine at position 580 by alanine resulted in impairment of interchain disulfide bridge formation; the mutagenized enzyme (C580A) was secreted from recombinant cells in the monomeric form and failed to assemble into dimers. The mutant monomeric HuAChE did not differ from the native oligomeric enzyme neither in rate of catalysis nor in affinity to acetylthiocholine. Mutant monomers were also shown to retain the acetylcholinesterase characteristic sensitivity to high substrate concentrations. The mutation did not seem to affect the efficiencies of either synthesis or secretion of recombinant HuAChE polypeptides, as was demonstrated in cell lines derived from human embryonic kidney (293 cells) as well as from a human neuroblastoma (SK-N-SH). Furthermore, the mutation did not lead to an increase in accumulation of intracellular HuAChE polypeptides, suggesting that export of acetylcholinesterase from cells may not be coupled to subunit assembly.
ESTHER : Velan_1991_J.Biol.Chem_266_23977
PubMedSearch : Velan_1991_J.Biol.Chem_266_23977
PubMedID: 1748670

Title : Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme - Velan_1991_Cell.Mol.Neurobiol_11_143
Author(s) : Velan B , Kronman C , Grosfeld H , Leitner M , Gozes Y , Flashner Y , Sery T , Cohen S , Ben-Aziz R , Seidman S , Shafferman A , Soreq H
Ref : Cellular Molecular Neurobiology , 11 :143 , 1991
Abstract : 1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.
ESTHER : Velan_1991_Cell.Mol.Neurobiol_11_143
PubMedSearch : Velan_1991_Cell.Mol.Neurobiol_11_143
PubMedID: 1849451

Title : Cloning, expression and biological activity of a new variant of human interferon alpha identified in virus induced lymphoblastoid cells - Cohen_1985_Dev.Biol.Stand_60_111
Author(s) : Cohen S , Velan B , Grosfeld H , Shalita Z , Leitner M , Shafferman A
Ref : Developmental Biology Stand , 60 :111 , 1985
Abstract : A synthetic oligonucleotide complementary to a highly conserved sequence in the IFN-alpha gene family, was used to screen a Namalva cDNA library. Among the cDNA clones having typical IFN-alpha traits, one was distinct from previously characterized IFN-alpha cDNAs. E. coli cells carrying this recombinant cDNA plasmid express an alpha-interferon activity. The sequence of this IFN-cDNA is extremely homologous (99.5%) to that of the IFN-alpha J gene and is designated IFN-alpha J1. Several E. coli trp expression plasmids were constructed for efficient transcription and translation of the mature IFN-alpha J1. The maximal level of expression (5 X 10(3) molecules/cells) was obtained from plasmid pJ1-4. A synthetic consensus translation initiation sequence coupled to the trp p/o region (in pJ1-5) proved to be 10 times less effective in promoting metIFN production in bacteria, than the in-vitro mutated trpL initiation sequence carried on pJ1-4. The bacterial IFN-alpha J1 was purified (to over 90% purity) to a specific activity of 1.3 X 10(8) units/mg. The antiviral activity of the purified IFN-alpha J1 was compared with other highly purified IFN-alpha species (bacterial IFN alpha A and alpha C, leukocyte IFN-alpha 1, leukocyte IFN mixture and Namalva IFN preparation) on a large panel of mammalian cell cultures. IFN-alpha J1 exhibits a distinct antiviral activity.
ESTHER : Cohen_1985_Dev.Biol.Stand_60_111
PubMedSearch : Cohen_1985_Dev.Biol.Stand_60_111
PubMedID: 2995168

Title : Cloning of a bovine interferon-alpha gene subfamily and comparisons between genetically engineered and leukocyte bovine interferons - Velan_1985_Dev.Biol.Stand_60_355
Author(s) : Velan B , Cohen S , Grosfeld H , Shalita Z , Shafferman A
Ref : Developmental Biology Stand , 60 :355 , 1985
Abstract : Bovine peripheral leukocytes were virally induced for interferon production, and an acid stable, SDS stable, antiviral activity was detected in the preparation. This bovine interferon (BoIFN) was tested for its ability to induce an antiviral state in various mammalian cells and was found to be specific to cells from bovine origin. The BoIFN cross reacts with antibodies against human IFN-alpha but these antibodies do not neutralize the bovine IFN activity. Leukocyte BoIFN exhibits polymorphism upon Affi-Gel Blue chromatography and SDS-PAGE (16k and 24K). The virally induced leukocytes produce a 13S mRNA which upon translation in oocytes yields an active IFN molecule. Bovine genomic library was constructed and screened for BoIFN-alpha sequences, using human IFN-alpha probes. From the clones isolated, five were found to represent distinct genes. Sequence analysis indicate that these genes are closely related (94% homology). One of these genes was expressed in E. coli under the control of trp promoter operator. The physicochemical and biological properties of the bacterial BoIFN-alpha product resemble those of a subpopulation of natural BoIFN.
ESTHER : Velan_1985_Dev.Biol.Stand_60_355
PubMedSearch : Velan_1985_Dev.Biol.Stand_60_355
PubMedID: 3899795

Title : Mutations not altering the symmetrical sequences in the trp operator yield a constitutive phenotype - Grosfeld_1984_Mol.Gen.Genet_195_358
Author(s) : Grosfeld H , Cohen S , Velan B , Shalita Z , Shafferman A
Ref : Molecular & General Genetics , 195 :358 , 1984
Abstract : An E. coli trp promoter operator mutant was constructed, having two base pair alterations at position -4 and -1 relative to the transcription initiation site (+1). Expression of chloramphenicol acetyltransferase gene under this trp promoter operator suggests that it is almost fully constitutive. This trp Oc in vitro derived mutant differs from previously isolated Oc mutants in that its twofold symmetry sequence is identical to that of the wild type trp operator. The base substitution in the operator does not affect the functionality of the trp promoter. The trp Oc promoter DNA fragment is engineered so that it can be manipulated conveniently for efficient expression of various genes in E. coli.
ESTHER : Grosfeld_1984_Mol.Gen.Genet_195_358
PubMedSearch : Grosfeld_1984_Mol.Gen.Genet_195_358
PubMedID: 6092859