Human Brain Acetylcholinesterase Research Articles

The Aptamer Ob2, a novel AChE inhibitor, restores cognitive deficits and alleviates amyloidogenesis in 5×FAD transgenic mice

Loss of cerebral cholinergic neurons and decreased levels of acetylcholine (ACh) are considered to be major factors causing cognitive dysfunction in Alzheimer’s disease (AD). Abnormally elevated levels of acetylcholinesterase (AChE) resulting in decreased levels of ACh are common in AD patients; thus, AChE inhibitors (AChEIs) are widely used for the treatment of AD. In our previous work, we acquired DNA aptamers Ob1, Ob2, and Ob3 against human brain AChE from systematic evolution of ligands by exponential enrichment (SELEX). In this study, we investigated the effect of these aptamers on learning and memory abilities, as well as the underlying mechanism in a 5×FAD transgenic AD mouse model. Here, we showed that only aptamer Ob2 exhibits a good inhibitory effect on both mouse and human AChE activity. In addition, chronic treatment with aptamer Ob2 significantly improved cognitive ability of 5×FAD mice in the Morris water maze. Moreover, the mechanism of aptamer Ob2 in 5×FAD mice may be associated with its inhibition of AChE activity, alleviation of the levels of Aβ by lowering the expression of β-secretase (BACE1), and activation of astrocytes in the brains of 5×FAD mice. These results indicate that aptamer Ob2 exhibits potential as an effective AChEI for the treatment of AD.

Emetine and Indirubin- 3- monoxime interaction with human brain acetylcholinesterase: A computational and statistical analysis.

Alzheimer's disease is a chronic neurodegenerative ailment and the most familiar type of dementia in the older population with no effective cure to date. It is characterized by a decrease in memory, associated with the mutilation of cholinergic neurotransmission. Presently, acetylcholinesterase inhibitors have emerged as the most endorsed pharmacological medications for the symptomatic treatment of mild to moderate Alzheimer's disease. This study aimed to research the molecular enzymatic inhibition of human brain acetylcholinesterase by a natural compound emetine and I3M. Molecular docking studies were used to identify superior interaction between enzyme acetylcholinesterase and ligands. Furthermore, the docked acetylcholinesterase-emetine complex was validated statistically using an analysis of variance in all tested conformers. In this interaction, H-bond, hydrophobic interaction, pi-pi, and Cation-pi interactions played a vital function in predicting the accurate conformation of the ligand that binds with the active site of acetylcholinesterase. The conformer with the lowest free energy of binding was further analyzed. The binding energy for acetylcholinesterase complex with emetine and I3M was -9.72kcal/mol and -7.09kcal/mol, respectively. In the current study, the prediction was studied to establish a relationship between binding energy and intermolecular energy (coefficient of determination [R2 linear = 0.999), and intermolecular energy and Van der wall forces (R2 linear = 0.994). These results would be useful in gaining structural insight for designing novel lead compounds against acetylcholinesterase for the effective management of Alzheimer's disease.

Molecular interaction of anti-cancer ligands with human brain acetylcholinesterase

There are a significant number of cases whereby cancer patients belonging to the old age group additionally suffer from cognition decline (a hallmark feature of Alzheimer’s disease). Hence, it is understandable that it would be a boon if certain drug molecules could provide health benefits to a patient suffering from cancer as well as Alzheimer’s disease. The objective of the work was to identify anticancer molecule(s) whose chemical-skeleton could be used as ‘seed’ for future design of dual-acting drugs against Alzheimer’s disease and cancer. The study employed criterion-based search, docking, SWISS-ADME-profiling, ▵ASA-calculations, molecular-overlay and ‘MoMA’-simulation to query possible binding of selected anticancer molecules with human brain acetylcholinesterase (AChE). Molecular interactions of all of the top ranking ligands were analyzed. ‘BOILED-egg’ model was employed to query brain-penetration of the ligands. A detailed molecular-simulation-analysis was performed. Snapshots of different stages of dynamic molecular interactions (selected from 254 pdb files) were captured by MoMA LigPath, a robotics inspired simulation algorithm. The study concluded that chemical skeletons of ‘Niraparib’ and ‘Ponatinib' might be used as ‘seed(s)’ for design of such drugs. If successfully materialized in future, this approach could decrease the total number of daily pills that an old patient needs to take. Furthermore, novel anticancer drugs could be synthesized that do not inhibit AChE (e.g. by removal/modification of moieties that are crucial to binding of anticancer drug to AChE) even if those happen to be ‘Blood Brain Barrier’-permeable. Alternatively, fresh AChE-inhibitors could be designed based on the scaffolds of the aforementioned anticancer drugs. Communicated by Ramaswamy H. Sarma

AChE Inhibition Study for Alzheimer's Disease Treatment: An In-silico Study

Alzheimer's disease (AD) is the most common form of dementia associated with plaques and tangles in the brain. Several acetylcholinesterase inhibitors have been clinically used to delay or halt the progression of the disease. Solanadine (Snd) and gamma solamargine (Gsm) have been shown to inhibit acetylcholinesterase (AChE). The current study attempts to describe the molecular interactions between human brain AChE and inhibitors Snd and Gsm. The free energy of binding and estimated dissociation constant (Ki) for the 'Snd-AChE Catalytic Anionic site (CAS)-interaction' were determined to be –9.51 kcal/mol and 107.54 nM, respectively and the free energy of binding and estimated Ki for the 'Gsm -AChE CAS-interaction' were determined to be – 8.64 kcal/mol and 463.88 nM, respectively. Hydrophobic interactions, polar interactions, and hydrogen bonding play an important role in the correct positioning of Snd within the 'catalytic site' of AChE to permit docking, while hydrophobic interactions and polar interactions play a significant role in the correct positioning of Gsm within the 'catalytic site' of AChE to permit docking. It is hoped that the information provided in this study will help in the design of AChE-inhibitors as anti-Alzheimer agents.

Surface modification of pralidoxime chloride-loaded solid lipid nanoparticles for enhanced brain reactivation of organophosphorus-inhibited AChE: Pharmacokinetics in rat

The nanotechnological approach is an innovative strategy of high potential to achieve reactivation of organophosphorus-inhibited acetylcholinesterase in central nervous system. It was previously shown that pralidoxime chloride-loaded solid lipid nanoparticles (2-PAM-SLNs) are able to protect the brain against pesticide (paraoxon) central toxicity. In the present work, we increased brain AChE reactivation efficacy by PEGylation of 2-PAM-SLNs using PEG-lipid N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, sodium salt) (DSPE-PEG2000) as a surface-modifier of SLNs. To perform pharmacokinetic study, a simple, sensitive (LLOQ 1.0 ng/mL) high-performance liquid chromatography tandem mass spectrometry with atmospheric pressure chemical ionization by multiple reaction monitoring mode (HPLC-APCI-MS) was developed. The method was compared to mass spectrometry with electrospray ionization. The method was validated for linearity, accuracy, precision, extraction recovery, matrix effect and stability. Acetophenone oxime was used as the internal standard for the quantification of 2-PAM in rat plasma and brain tissue after intravenous administration. 2-PAM-DSPE-PEG2000-SLNs of mean size about 80 nm (PDI = 0.26), zeta-potential of −55 mV and of high in vitro stability, prolonged the elimination phase of 2-PAM from the bloodstream more than 3 times compared to free 2-PAM. An increase in reactivation of POX-inhibited human brain acetylcholinesterase up to 36.08 ± 4.3 % after intravenous administration of 2-PAM-DSPE-PEG2000-SLNs (dose of 2-PAM is 5 mg/kg) was achieved. The result is one of the first examples where this level of brain acetylcholinesterase reactivation was achieved. Thus, the implementation of different approaches for targeting and modifying nanoparticles’ surface gives hope for improving the antidotal treatment of organophosphorus poisoning by marketed reactivators.

Molecular interaction of investigational ligands with human brain acetylcholinesterase.

Alzheimer's disease, a neurodegenerative disorder continues to be an area of investigation by the international researchers' fraternity. Despite all the ongoing efforts, the effective set of promising cholinesterase inhibitors available in the market for patients' use is limited. Furthermore, the currently available drugs could provide only a palliative type of treatment instead of providing a complete cure or foolproof prevention. Hence, design/discovery of fresh drug molecules as acetylcholinesterase (AChE) inhibitors still remains an urgent requirement. The drug discovery platform, MCULE in the "structure-based virtual screening" (SBVS) mode was used for high throughput ligand screening of over five million structures targeted against the AChE catalytic site. A stepwise query was made for the SBVS input. The number of hits was narrowed down in consecutive succession via varied filtration criteria as AutoDock-Vina rankings, MCULE toxicity filtration, exclusion of ligands having less than four H-bond acceptors, filtration by ΔG cutoff, rule-of-five violation and SWISS ADME profiling. This was followed by holistic analysis of all the results, thereby leading to one promising ligand. The screened out drug molecule, MCULE-5872671137-0-1 exhibited a robust interaction with the AChE catalytic site involving 20 amino acid residues, an acceptable binding free energy of -10.2 kcal/mol in addition to a favorable SWISS ADME-profie showing no harmful effects on the human body. It can be carefully stated that the molecule, MCULE-5872671137-0-1, which is chemically (3S)-N-{4-[(4-chlorophenyl)sulfanyl]phenyl}-3-hydroxypyrrolidine-1-carboxamide could function as a significant "seed" ligand for future design of potent AChE inhibitors and/or novel neuro drugs built upon the seed-scaffold.

Molecular Interaction of Anti-Diabetic Drugs With Acetylcholinesterase and Sodium Glucose Co-Transporter 2.

Type 2 Diabetes Mellitus (T2DM) and Alzheimer's disease (AD) are the two disorders which are known to share pertinent pathological and therapeutic links. Sodium glucose co-transporter-2 (SGLT2) and Acetylcholinesterase (AChE) are established inhibition targets for T2DM and AD treatments, respectively. Reports suggest that anti-diabetic drugs could be used for AD treatment also. The present study used molecular docking by Autodock4.2 using our "Click-By-Click"-protocol, Ligplot1.4.3 and "change in accessible surface area (ΔASA)-calculations" to investigate the binding of two investigational anti-diabetic drugs, Ertugliflozin and Sotagliflozin to an established target (SGLT2) and a research target (human brain AChE). Sotagliflozin appeared more promising for SGLT2 as well as AChE-inhibition with reference to ΔG and Ki values in comparison to Ertugliflozin. The ΔG and Ki values for "Sotagliflozin:AChE-binding" were -7.16 kcal/mol and 5.6 μM, respectively while the same were found to be -8.47 kcal/mol and 0.62 μM, respectively for its interaction with SGLT2. Furthermore, "Sotagliflozin:SGLT2-interaction" was subjected to (un)binding simulation analyses by "Molecular-Motion-Algorithms." This information is significant as the exact binding mode, interacting amino acid residues and simulation results for the said interaction have not been described yet. Also no X-ray crystal is available for the same. Finally, the results described herein indicate that Sotagliflozin could have an edge over Ertugliflozin for treatment of Type 2 diabetes. Future design of drugs based on Sotagliflozin scaffolds for treatment of Type 2 and/or Type 3 diabetes are highly recommended. As these drugs are still in late phases of clinical trials, the results described herein appear timely. J. Cell. Biochem. 118: 3855-3865, 2017. © 2017 Wiley Periodicals, Inc.

Cloning, expression, and characterization of human brain acetylcholinesterase in Escherichia coli using a SUMO fusion tag

The molecular structure of acetylcholinesterase (AChE) attracts interest because of its versatility and significant role in the cholinergic system. The main purpose of the present study was to clone a full-length cDNA sequence of human brain acetylcholinesterase (hAChE) into pET SUMO vector and express it successfully. The integrity of the constructed plasmid was confirmed by cross PCR. This recombinant construct was expressed in Escherichia coli BL21 (DE-3). In this work, we produced hexahistidine (6xHis) tagged fusion protein by isopropyl β-D-1-thiogalactopyranoside (IPTG) induction and purified using nickel (Ni2+) affinity chromatography. Using anti-His antibody, we detected ~90 kDa fusion protein. The expression of the hAChE gene in a microbial host resulted in good biological activity. Using the Ellman method, the recombinant AChE exhibited activity with optima at pH 9.0 glycine-NaOH buffer and room temperature. Kinetic parameters, KM and Vmax, were determined as 0.63 and 0.69, respectively.

A neuroinformatics study to compare inhibition efficiency of three natural ligands (Fawcettimine, Cernuine and Lycodine) against Human Brain Acetylcholinesterase

Enzyme-inhibition is considered as a potent therapeutic approach to the treatment of diseases associated with acetylcholinesterase (AChE). The present study elucidates molecular interactions of human brain AChE, with three natural ligands Lycodine, Cernuine and Fawcettimine for comparison. Docking between these ligands and enzyme was performed using ‘Autodock 4.2’. It was determined that polar and hydrophobic interactions play an important role in the correct positioning of Lycodine, Cernuine and Fawcettimine within the ‘catalytic site’ of AChE to permit docking. This approach would be helpful to understand the selectivity of the given drug molecule in the treatment of neurological disorder. Moreover, the present study confirms that Lycodine is a more efficient inhibitor of human brain AChE compared to Cernuine and Fawcettimine with reference to ΔG and Ki values.

Prediction of Comparative Inhibition Efficiency for a Novel Natural Ligand, Galangin Against Human Brain Acetylcholinesterase, Butyrylcholinesterase and 5-Lipoxygenase: A Neuroinformatics Study

The present study elucidates molecular interactions of human acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and 5-lipoxygenase (5-LPO) with a novel natural ligand Galangin (GAL); and also with the well-known ligands Bisnorcymserine (BNC) and Cymserine for comparison. Docking between these ligands and enzymes were performed using 'Autodock4.2'. It was found that hydrophobic interactions play an important role in the correct positioning of BNC within the 'catalytic site' of AChE, BuChE and 5-LPO to permit docking while hydrogen bonds are significant in case of cymserine for the same. However, only polar interactions are significant in the correct positioning of GAL within the 'catalytic site' of AChE, BuChE and 5-LPO to permit docking. Such information may aid in the design of versatile AChE, BuChE and 5 LPO-inhibitors, and is expected to aid in safe clinical use of above ligands. Scope still remains in the determination of the three-dimensional structure of AChE-GAL, BuChE-GAL and 5-LPO-GAL complex by X-ray crystallography to certify the described data. Moreover, the present study confirms that GAL is a more efficient inhibitor of human brain AChE compared to BNC and cymserine, while in case of 5-LPO and human brain BuChE, BNC is a more efficient inhibitor compared to GAL and cymserine with reference to ΔG and Ki values.

Molecular Interaction of Human Brain Acetylcholinesterase with a Natural Inhibitor Huperzine-B: An Enzoinformatics Approach

The present study emphasizes the molecular interactions between human brain acetylcholinesterase (AChE) and the natural ligand Huperzine-B and its comparison to 'AChE-Tolserine interactions'. Docking between Huperzine-B and AChE was performed using 'Autodock4.2'. Hydrophobic interactions and hydrogen bonds both play an equally important role in the correct positioning of Huperzine-B within the 'catalytic site' of AChE to permit docking. However, docking of Tolserine to AChE is largely dominated by hydrophobic interactions. Such information may aid in the design of versatile AChE-inhibitors, and is expected to aid in safe clinical use of Huperzine-B. Scope still remains in the determination of the three-dimensional structure of AChE-Huperzine-B complex by X-ray crystallography to validate the described data. Furthermore, this study confirms that Huperzine-B is a more efficient inhibitor of human brain AChE compared to tolserine with reference to Ki and ΔG values.

A Neuroinformatics Study Describing Molecular Interaction of Cisplatin with Acetylcholinesterase: A Plausible Cause for Anticancer Drug Induced Neurotoxicity

Several chemotherapeutic drugs are known to cause significant clinical neurotoxicity, which can result in the early cessation of treatment. To identify and develop more effective means of neuroprotection it is important to understand the toxicity of these drugs at the molecular and cellular levels. This study describes molecular interactions between human brain acetylcholinesterase (AChE) and the well-known anti-neoplastic drug, Cisplatin. Docking between Cisplatin and AChE was performed using 'GOLD 5.0' and accessible surface area of protein before and after ligand binding was calculated by NACCESS version 2.1.1. Hydrophobic interactions and hydrogen bonds both play an equally important role in the correct positioning of Cisplatin within the 'acyl pocket' as well as 'catalytic site' of AChE to permit docking. Gold fitness score of 'Cisplatin- acyl domain of AChE' interaction and 'Cisplatin-CAS domain of AChE' interaction were 38.78 and 39.91, respectively and free binding energy was found to be -5.82 Kcal/mol and -5.79 Kcal/mol, respectively. During 'Cisplatin-CAS site of AChE enzyme' interaction, it was found that out of the three amino acids constituting the catalytic triad (S203, H447 and E334), two amino acid residues namely S203 and H447 interact with Cisplatin by hydrogen bonding and hydrophobic interaction, respectively. The values for 'accessible surface area' for the amino acid residues H447 and S203 were found to be reduced by 14.398 Å(2) and 3.894 Å(2), respectively after interaction with Cisplatin. Hence, Cisplatin might act as a potent inhibitor of AChE. Scope still remains in the determination of the three-dimensional structure of AChE-Cisplatin complex by X-ray crystallography to validate the described data. Moreover, such information may aid in the design of versatile AChE-inhibitors, and is expected to aid in safe clinical use of Cisplatin.

Molecular Interaction of the Antineoplastic Drug, Methotrexate with Human Brain Acetylcholinesterase: A Docking Study

This study describes molecular interactions between human brain acetylcholinesterase (AChE) and the well known anti-neoplastic drug, methotrexate (MTX) and its comparison to 'AChE-cyclophosphamide (CP) interactions' that we reported previously. Docking between MTX and AChE was performed using 'Autodock4.2'. Hydrophobic interactions and hydrogen bonds both play an equally important role in the correct positioning of MTX within the 'acyl pocket' as well as 'catalytic site' of AChE to permit docking. However, docking of CP to AChE is largely dominated by hydrophobic interactions. Such information may aid in the design of versatile AChE-inhibitors, and is expected to aid in safe clinical use of MTX. Scope still remains in the determination of the three-dimensional structure of AChE-MTX complex by X-ray crystallography to validate the described data. The current computational study supports our previous experimental study which concluded a mixed inhibition model for AChE-inhibition by MTX. Furthermore, the present report confirms that MTX is a more efficient inhibitor of human brain AChE compared to CP with reference to K(i) and ΔG values.

Interaction of Human Brain Acetylcholinesterase with Cyclophosphamide: A Molecular Modeling and Docking Study

This study describes the interaction between human acetylcholinesterase (AChE), a key regulator of central and peripheral cholinergic function, and the widely used nitrogen mustard alkylating agent, cyclophosphamide (CP). Modeling of the AChE sequence (NCBI Accession No: AAI05061.1) was performed using 'Swiss Model Workspace'. The protein-model was submitted to the Protein Model Database and was assigned accession number PM0077393. A plot showing normalized QMEAN scores versus protein size was made to compare the model with a non-redundant set of Protein Data Bank structures, which gave a Z-score QMEAN as -0.58. The predicted local error for the modeled structure was found to be well within tolerable limits. Z-score values for Cβ interaction, all atom interaction, solvation and torsion were found to be -1.10, -0.90, -0.06 and -0.40, respectively. Docking between CP and AChE was performed using 'Autodock4.2'. Apart from other interaction-types, six carbon atoms of CP (C1, C2, C3, C4, C6 and C7) were determined to be involved in hydrophobic interactions with amino acid residues Y121, W233, L323, F331, F335 and Y338 of the 'acyl pocket' within AChE. Five carbon atoms of CP (C2, C4, C5, C6 and C7) were involved in hydrophobic interactions with 3 amino acid residues within the enzyme's 'catalytic site'. In conclusion, hydrophobic interactions play a major role in the appropriate positioning of CP within the 'acyl pocket' as well as 'catalytic site' of AChE to permit suitable orientation and allow docking. This information may aid the design of more potent and versatile AChE-inhibitors as pharmacologic tools and drugs to characterize and treat neurological disorders, and additionally provides a model whose value can be quantitatively assessed by X-ray crystallographic analysis of the AChECP three-dimensional structure.

Antisense inhibition of acetylcholinesterase gene expression for treating cognition deficit in Alzheimer's disease model mice

To examine whether the selected antisense oligodeoxynucleotides (AS-ODN) targeting against human brain acetylcholinesterase (AChE) mRNA could improve the cognitive deficit in the Alzheimer's disease (AD) model mice induced by amyloid-β peptide (Aβ), we determined the time–effect relationship of AChE activity and the learning and memory after AS-ODN delivery. The results showed that the AChE activity decreased gradually along with time, initiating at 8 h and lasting 42 h. The time–effect curves of acetylcholine (ACh) behaved consistency with that of AChE activity. The animal cognition studies showed that in step-through test, the error number of the AS-ODN-treated AD model mice was significantly decreased, and the memory retention was increased. In the water maze performance, the swimming time obviously shortened. Our results indicated that antisense therapy is of potential use in the treatment of cognitive deficit in the Aβ model mice.

In vitro reactivation of sarin-inhibited brain acetylcholinesterase from different species by various oximes

In vitro as well as in vivo evaluation of the reactivating efficacy of various oximes against nerve agent-inhibited acetylcholinesterase has been usually done with the help of animal experiments. Nevertheless, previously published data indicate that the reactivation potency of oximes may be different in human and animal species, which may hamper the extrapolation of animal data to human data. Therefore, to better evaluate the efficacy of various oximes (pralidoxime, obidoxime, HI-6, K033) to reactivate brain acetylcholinesterase inhibited by sarin by in vitro methods, human, rat and pig brain acetylcholinesterase were used to calculate kinetic parameters for the reactivation. Our results show differences among the species, depending on the type of oxime, and indicate that data from animal experiments needs to be carefully evaluated before extrapolation to humans.

Specific oligobodies against human brain acetylcholinesterase

In order to develop the specific oligobodies against human brain acetylcholinesterase (AChE) and distinguishes between human erythrocyte and brain AChEs, we applied the strategy of ‘target switching’ to obtain the specific polyclonal and monoclonal oligobodies. The specificity between human brain AChE and other ChEs was identified by Western blotting, dot blotting and enzyme protein binding assay (EPBA). The results showed that the oligobodies against the human brain AChE specifically immunoreacted with the human brain AChE and Torpedo AChE, not showing significant binding to AChE from human erythrocyte and butyrylcholinesterase (BChE) from human serum.

Synthetic antigenic decapeptides of human brain acetylcholinesterase cross-immunoreact with peptide-specific antibodies against Torpediniformes narcine timlei acetylcholinesterase

Antigenic decapeptides of human brain acetylcholinesterase (AChE) were investigated for immunoreactivity with the rabbit anti- Torpediniformes narcine timlei AChE polyclonal antibody (anti-narcine AChE polyclonal antibody). The decapeptides were synthesized using the multipin combinatorial chemical synthesis technique and biotinylated at N-terminals. Rabbit anti-narcine AChE polyclonal antibodies were purified by Protein A-Sepharose CL 4B column chromatography. Enzyme-linked immunosorbent assay (ELISA) was used for the assay of the reaction between the antigen and the antibody. Seven of 11 antigenic synthetic decapeptides of human brain AChE showed obvious immunoreactivity with the rabbit anti-narcine AChE polyclonal antibodies. The similarity of the AChE sequences of humans and Torpedo species were compared thereby with the epitopes indicated. The results indicate that the epitopes of human brain AChE and Torpedo AChEs have been highly conserved during evolution. In view of this, no N-glycosylation site was found in the antigenic decapeptides tested, they all belong to oligopeptide epitopes.

Epitopes of human brain acetylcholinesterase

The main purpose of the present work was to identify B-cell epitopes on human brain acetylcholinesterase (AChE) by the synthetic peptide approach. Five hundred and seventy-four decapeptides comprising amino acids No. n to n+9 (where n denotes the residue number of the 583 amino acids in the primary structure of human brain AChE and is an integer in the range 1–574) were synthesized, using the multipin combinatorial chemical synthesis technique, and biotinylated. Epitopes of human brain AChE were detected by enzyme-linked immunosorbent assay (ELISA) and compared with the predicted epitopes of human AChE by ‘Goldkey’ software. Among 574 synthetic decapeptides, 47 decapeptides at 11 antigenic regions showed immunoreactivity with mouse anti-human brain AChE polyclonal antibodies. The minimum sequence of epitope was defined at every antigenic region explored. The locations and sequences of the former ten continuous epitopes at the 11 antigenic regions of the human brain AChE had been identified as follows: TPVLVWIY (112–119), RTVLVSMNY (143–151), LLDQRLALQW (173–182), RRATQLAH (246–253), VFRFSFVPV (294∼302), KDEGSYFLVY (332–341), RVYA (424–427), LMRY (476–479), KAPQWPPY (496–503), GLRAQACAFW (523–532). The rate of hits of the predicted epitopes from the software came out at 33%. In our work, the epitopes of human AChE have been mapped by purified polyclonal antibody at eleven distinct sites in the primary structure.

Hydrophobic protein that copurifies with human brain acetylcholinesterase: amino acid sequence, genomic organization, and chromosomal localization.

The mechanism of attachment of acetylcholinesterase (AChE) to neuronal membranes in interneuronal synapses is poorly understood. We have isolated, sequenced, and cloned a hydrophobic protein that copurifies with AChE from human caudate nucleus and that we propose forms a part of a complex of membrane proteins attached to this enzyme. It is a short protein of 136 amino acids and has a molecular mass of 18 kDa. The sequence contains stretches of both hydrophobic and hydrophilic amino acids and two cysteine residues. Analysis of the genomic sequence reveals that the coding region is divided among five short exons. Fluorescence in situ hybridization localizes the gene to chromosome 6p21.32-p21.2. Northern blot analysis shows that this gene is widely expressed in the brain with an expression pattern that parallels that of AChE.