Tetrameric Acetylcholinesterase Research Articles

Tacrine Induces Endoplasmic Reticulum-Stressed Apoptosis via Disrupting the Proper Assembly of Oligomeric Acetylcholinesterase in Cultured Neuronal Cells.

Acetylcholinesterase inhibitors (AChEIs), the most developed treatment strategies for Alzheimer's disease (AD), will be used in clinic for, at least, the next decades. Their side effects are in highly variable from drug to drug with mechanisms remaining to be fully established. The withdrawal of tacrine (Cognex) in the market makes it as an interesting case study. Here, we found tacrine could disrupt the proper trafficking of proline-rich membrane anchor-linked tetrameric acetylcholinesterase (AChE) in the endoplasmic reticulum (ER). The exposure of tacrine in cells expressing AChE, e.g., neurons, caused an accumulation of the misfolded AChE in the ER. This misfolded enzyme was not able to transport to the Golgi/plasma membrane, which subsequently induced ER stress and its downstream signaling cascade of unfolded protein response. Once the stress was overwhelming, the cooperation of ER with mitochondria increased the loss of mitochondrial membrane potential. Eventually, the tacrine-exposed cells lost homeostasis and underwent apoptosis. The ER stress and apoptosis, induced by tacrine, were proportional to the amount of AChE. Other AChEIs (rivastigmine, bis(3)-cognitin, daurisoline, and dauricine) could cause the same problem as tacrine by inducing ER stress in neuronal cells. The results provide guidance for the drug design and discovery of AChEIs for AD treatment. SIGNIFICANCE STATEMENT: Acetylcholinesterase inhibitors (AChEIs) are the most developed treatment strategies for Alzheimer's disease (AD) and will be used in clinic for at least the next decades. This study reports that tacrine and other AChEIs disrupt the proper trafficking of acetylcholinesterase in the endoplasmic reticulum. Eventually, the apoptosis of neurons and other cells are induced. The results provide guidance for drug design and discovery of AChEIs for AD treatment.

Polyproline-rich peptides associated with Torpedo californica acetylcholinesterase tetramers

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

Development of a multivalent acetylcholinesterase inhibitor via dynamic combinatorial chemistry

In this study, we report the generation of a polymer based dynamic combinatorial library (DCL) using aldehyde-functionalized linear poly(glycidol) and hydrazide derivatives as initial building blocks. In combination with tetrameric acetylcholinesterase (AChE), a certain type of amplified acylhydrazone side chain is identified and further used for the synthesis of a multivalent AChE inhibitor. The cytotoxicity and inhibition properties of the multivalent inhibitor are evaluated, and the results indicate superior bioactivity compared to the commercial reference Edrophonium chloride.

Identification of Carboxylesterase, Butyrylcholinesterase, Acetylcholinesterase, Paraoxonase, and Albumin Pseudoesterase in Guinea Pig Plasma through Nondenaturing Gel Electrophoresis.

Drugs to protect against nerve agent toxicity are tested in animals. The current preferred small animal model is guinea pigs because their plasma bioscavenging capacity resembles that of NHP. We stained nondenaturing polyacrylamide slab gels with a variety of substrates, inhibitors, and antibodies to identify the esterases in heparinized guinea pig plasma. An intense band of carboxylesterase activity migrated behind albumin. Minor carboxylesterase bands were revealed after background activity from paraoxonase was inhibited by using EDTA. The major butyrylcholinesterase band was a disulfide-linked dimer. Incubation with the antihuman butyrylcholinesterase antibody B2 18-5 shifted the butyrylcholinesterase dimer band to slower migrating complexes. Carboxylesterases were distinguished from butyrylcholinesterase by their sensitivity to inhibition by bis-p-nitrophenyl phosphate. Acetylcholinesterase tetramers formed a complex with the antihuman acetylcholinesterase antibody HR2. Organophosphorus toxicants including cresyl saligenin phosphate, dichlorvos, and chlorpyrifos oxon irreversibly inhibited the serine esterases but not paraoxonase. Albumin pseudoesterase activity was seen in gels stained with α- or β-naphthyl acetate and fast blue RR. We conclude that guinea pig plasma has 2 types of carboxylesterase, butyrylcholinesterase dimers and 5 minor butyrylcholinesterase forms, a small amount of acetylcholinesterase tetramers, paraoxonase, and albumin pseudoesterase activity. A knockout mouse with no carboxylesterase activity in plasma is available and may prove to be a better model for studies of nerve agent toxicology than guinea pigs.

Exercise modulates synaptic acetylcholinesterase at neuromuscular junctions

Acetylcholinesterase plays a major role in neuromuscular transmission and is regulated by neuromuscular activity. Since fast-twitch motor units are recruited with increased motor demand, we examined acetylcholinesterase regulation in rat leg muscles following treadmill training. Total acetylcholinesterase and specifically the membrane-bound tetramer increased in exercised fast-, but not slow-twitch muscles, while other isoforms remained unchanged. Synaptic acetylcholinesterase increased markedly in neuromuscular junctions of trained fibers, without concomitant changes in synaptic acetylcholine receptor, thus elevating synaptic acetylcholinesterase/receptor ratios. Electron microscopy showed that acetylcholinesterase increased in postjunctional folds and primary cleft, where it was added adjacent to the postsynaptic muscle membrane. Thus, although the primary acetylcholinesterase at the neuromuscular junction is the collagen-tailed asymmetric isoform associated with synaptic basal lamina, physiological demands such as strenuous exercise, or potentially pathological conditions, can selectively recruit the membrane-bound acetylcholinesterase tetramer to the synapse for optimal synaptic transmission.

Vectorially Imprinted Hybrid Nanofilm for Acetylcholinesterase Recognition

Effective recognition of enzymatically active tetrameric acetylcholinesterase (AChE) is accomplished by a hybrid nanofilm composed of a propidium‐terminated self‐assembled monolayer (Prop‐SAM) which binds AChE via its peripheral anionic site (PAS) and an ultrathin electrosynthesized molecularly imprinted polymer (MIP) cover layer of a novel carboxylate‐modified derivative of 3,4‐propylenedioxythiophene. The rebinding of the AChE to the MIP/Prop‐SAM nanofilm covered electrode is detected by measuring in situ the enzymatic activity. The oxidative current of the released thiocholine is dependent on the AChE concentration from ≈0.04 × 10−6 to 0.4 × 10−6m. An imprinting factor of 9.9 is obtained for the hybrid MIP, which is among the best values reported for protein imprinting. The dissociation constant characterizing the strength of the MIP‐AChE binding is 4.2 × 10−7m indicating the dominant role of the PAS‐Prop‐SAM interaction, while the benefit of the MIP nanofilm covering the Prop‐SAM layer is the effective suppression of the cross‐reactivity toward competing proteins as compared with the Prop‐SAM. The threefold selectivity gain provided by i) the “shape‐specific” MIP filter, ii) the propidium‐SAM, iii) signal generation only by the AChE bound to the nanofilm shows promise for assessing AChE activity levels in cerebrospinal fluid.

Research on the regulation of the spatial structure of acetylcholinesterase tetramer with high efficiency by AFM

Atomic force microscopy (AFM) was applied for obtaining structural information about acetylcholinesterase (AChE) tetramer (AChE G4) before and after reaction with S-acetylcholine iodide (S-ACh), in the presence or absence of propidium iodide (PI), an inhibitor for peripheral anionic sites (PAS). An iced-bath ultrasound was used to prepare the phospholipid membrane. Ves-fusion technique was applied for incorporating AChE G4 in a lipid layer on mica. Before reaction with substrates, the single AChE G4 particle was ellipsoid in shape with a clear border. It had a smooth surface with a central projection. The four subunits of a single enzyme particle were arranged tightly (no separated subunits being found, with an average size of 89 ± 7 nm in length, 68 ± 9 nm in width, and 6 ± 3 nm in height). After reaction with S-ACh in the absence of PI, the loose arrangement of subunits of AChE G4 was seen, with an average size of 104 ± 7 nm in length, 91 ± 5 nm in width, and 8 ± 2 nm in height. Also there was free-flowing space amongst the four subunits of the AChE G4. This was consistent with the results of the ×-ray diffraction crystallography and molecular dynamics studies. The apparent free space was the central path of AChE G4, changing from small to big, to small, to lateral door appearance, with an average size of 60 ± 5 nm in length and 51 ± 9 nm in width. The size of lateral door was 52 ± 5 nm in width and 32 ± 3 nm in depth on average. In the presence of PI, S-ACh could not cause topological structure changes of AChE G4. AFM verified that the central path might govern the turnover of the enzyme morphologically, and the interactions between PI and S-ACh might gate the creation of a central path and the opening of ACG in monomer; and the combination of S-ACh with peripheral anionic sites is conducive to the opening of ACG while PI can inhibit this action. Resolution at the inframolecular level is favorable in providing substantial information on how the spatial structure is adapted to the high efficiency of AChE molecules.

N-linked glycosylation of proline-rich membrane anchor (PRiMA) is not required for assembly and trafficking of globular tetrameric acetylcholinesterase

Acetylcholinesterase (AChE) is organized into globular tetramers (G4) by a structural protein called proline-rich membrane anchor (PRiMA), anchoring it into the cell membrane of neurons in the brain. The assembly of AChE tetramers with PRiMA requires the presence of a C-terminal “t-peptide” in the AChE catalytic subunit (AChET). The glycosylation of AChET is known to be required for its proper assembly and trafficking; however, the role of PRiMA glycosylation in the oligomer assembly has not been revealed. PRiMA is a glycoprotein containing two putative N-linked glycosylation sites. By using site-directed mutagenesis, the asparagine-43 was identified to be the N-linked glycosylation site of PRiMA. Abolishing glycosylation on mouse PRiMA appeared not to affect its assembly with AChET, the enzymatic properties of AChE, and the membrane trafficking of PRiMA-linked AChE tetramers. This result is contrary to the reports that glycosylation is essential for conformation and trafficking of membrane glycoproteins.

A tetrameric acetylcholinesterase from the parasitic nematode Dictyocaulus viviparus associates with the vertebrate tail proteins PRiMA and ColQ

Dictyocaulus viviparus causes a serious lung disease of cattle. Similar to other parasitic nematodes, D. viviparus possesses several acetylcholinesterase (AChE) genes, one of which encodes a putative neuromuscular AChE, which contains a tryptophan (W) amphiphilic tetramerization (WAT) domain at its C-terminus. In the current study, we describe the biochemical characterization of a recombinant version of this WAT domain-containing AChE. To assess if the WAT domain is biologically functional, we investigated the association of the recombinant enzyme with the vertebrate tail proteins, proline-rich membrane anchor (PRiMA) and collagen Q (ColQ), as well as the synthetic polypeptide poly-l-proline. The results indicate that the recombinant enzyme hydrolyzes acetylthiocholine preferentially and exhibits inhibition by excess substrate, a characteristic of AChEs but not butyrylcholinesterases (BChEs). The enzyme is inhibited by the AChE inhibitor, BW284c51, but not by the BChE inhibitors, ethopropazine or iso-OMPA. The enzyme is able to assemble into monomeric (G1), dimeric (G2), and tetrameric (G4) globular forms and can also associate with PRiMA and ColQ, which contain proline-rich attachment domains (PRADs). This interaction is likely to be mediated via WAT-PRAD interactions, as the enzyme also assembles into tetramers with the synthetic polypeptide poly-l-proline. These interactions are typical of AChET subunits. This is the first demonstration of an AChET from a parasitic nematode that can assemble into heterologous forms with vertebrate proteins that anchor the enzyme in cholinergic synapses. We discuss the implications of our results for this particular host/parasite system and for the evolution of AChE.

Limiting Role of Protein Disulfide Isomerase in the Expression of Collagen-tailed Acetylcholinesterase Forms in Muscle

The expression of acetylcholinesterase (AChE) in skeletal muscle is regulated by muscle activity; however, the underlying molecular mechanisms are incompletely understood. We show here that the expression of the synaptic collagen-tailed AChE form (ColQ-AChE) in quail muscle cultures can be regulated by muscle activity post-translationally. Inhibition of thiol oxidoreductase activity decreases expression of all active AChE forms. Likewise, primary quail myotubes transfected with protein disulfide isomerase (PDI) short hairpin RNAs showed a significant decrease of both the intracellular pool of all collagen-tailed AChE forms and cell surface AChE clusters. Conversely, overexpression of PDI, endoplasmic reticulum protein 72, or calnexin in muscle cells enhanced expression of all collagen-tailed AChE forms. Overexpression of PDI had the most dramatic effect with a 100% increase in the intracellular ColQ-AChE pool and cell surface enzyme activity. Moreover, the levels of PDI are regulated by muscle activity and correlate with the levels of ColQ-AChE and AChE tetramers. Finally, we demonstrate that PDI interacts directly with AChE intracellularly. These results show that collagen-tailed AChE form levels induced by muscle activity can be regulated by molecular chaperones and suggest that newly synthesized exportable proteins may compete for chaperone assistance during the folding process.

Enzymatic Activity versus Structural Dynamics: The Case of Acetylcholinesterase Tetramer

The function of many proteins, such as enzymes, is modulated by structural fluctuations. This is especially the case in gated diffusion-controlled reactions (where the rates of the initial diffusional encounter and of structural fluctuations determine the overall rate of the reaction) and in oligomeric proteins (where function often requires a coordinated movement of individual subunits). A classic example of a diffusion-controlled biological reaction catalyzed by an oligomeric enzyme is the hydrolysis of synaptic acetylcholine (ACh) by tetrameric acetylcholinesterase (AChEt). Despite decades of efforts, the extent to which enzymatic efficiency of AChEt (or any other enzyme) is modulated by flexibility is not fully determined. This article attempts to determine the correlation between the dynamics of AChEt and the rate of reaction between AChEt and ACh. We employed equilibrium and nonequilibrium electro-diffusion models to compute rate coefficients for an ensemble of structures generated by molecular dynamics simulation. We found that, for the static initial model, the average reaction rate per active site is ∼22–30% slower in the tetramer than in the monomer. However, this effect of tetramerization is modulated by the intersubunit motions in the tetramer such that a complex interplay of steric and electrostatic effects either guides or blocks the substrate into or from each of the four active sites. As a result, the rate per active site calculated for some of the tetramer structures is only ∼15% smaller than the rate in the monomer. We conclude that structural dynamics minimizes the adverse effect of tetramerization, allowing the enzyme to maintain similar enzymatic efficiency in different oligomerization states.

Cholinesterase activity in brain of senescence‐accelerated‐resistant mouse SAMR1 and its variation in brain of senescence‐accelerated‐prone mouse SAMP8

The early-onset, irreversible, severe deficits of learning and memory in the senescence-accelerated mouse (SAM)-prone/8 (SAMP8) support its use as an animal model for human dementias of early onset. Possible implication of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in cognitive dysfunction of SAMP8 mice was studied by comparing cholinesterase (ChE) expression in brains of SAMP8 mice and of their normal control, SAM-resistant/1 (SAMR1) mice. The level of ChE mRNAs was the same in SAMP8 and SAMR1 brains, which agreed with their equal AChE activity (3.09 +/- 1.45 vs. 3.07 +/- 1.44 mumol.hr(-1).mg protein(-1), U/mg), but not with a doubled BuChE activity in SAMP8 brain (0.14 +/- 0.05 vs. 0.07 +/- 0.02 U/mg; P < 0.01). This great increase in neural BuChE activity may contribute to cognitive deficit of SAMP8 mice. Hydrophilic (G(4) (H), 8%) and amphiphilic (G(4) (A), 74%) AChE tetramers, besides dimers and monomers (G(2) (A) + G(1) (A), 18%), were identified in SAMR1 brains. They also contained G(4) (H) BuChE forms (18%) as well as G(4) (A) (53%) and G(2) (A) + G(1) (A) (29%) species. Although SAMP8 brain displayed proportions of AChE and BuChE forms that were similar to those of SAMR1 brain, phenyl-agarose chromatography with detergent-free extracts showed a rise in the proportion of secretory G(4) (H) BuChE from 35% in SAMR1 to 44% in SAMP8 brain. The strong immunolabelling of glial fibrillary acidic protein (GFAP), a marker of reactive gliosis, in SAMP8 brain and the consideration of BuChE as a marker of glial cells suggest a relationship between phenotypic changes in neuroglial cells and the excess of BuChE activity in SAMP8 brain.

Tetrameric Acetylcholinesterase: The Effects of Substrates Acetylcholine and Butyrocholine on Its Submolecular Structure Observed by Atomic Force Microscope

Tetrameric Acetylcholinesterase: The Effects of Substrates Acetylcholine and Butyrocholine on Its Submolecular Structure Observed by Atomic Force Microscope

Acetylcholinesterase Associates Differently with Its Anchoring Proteins ColQ and PRiMA

Acetylcholinesterase tetramers are inserted in the basal lamina of neuromuscular junctions or anchored in cell membranes through the interaction of four C-terminal t peptides with proline-rich attachment domains (PRADs) of cholinesterase-associated collagen Q (ColQ) or of the transmembrane protein PRiMA (proline-rich membrane anchor). ColQ and PRiMA differ in the length of their proline-rich motifs (10 and 15 residues, respectively). ColQ has two cysteines upstream of the PRAD, which are disulfide-linked to two AChE(T) subunits ("heavy" dimer), and the other two subunits are disulfide-linked together ("light" dimer). In contrast, PRiMA has four cysteines upstream of the PRAD. We examined whether these cysteines could be linked to AChE(T) subunits in complexes formed with PRiMA in transfected COS cells and in the mammalian brain. For comparison, we studied complexes formed with N-terminal fragments of ColQ, N-terminal fragments of PRiMA, and chimeras in which the upstream regions containing the cysteines were exchanged. We also compared the effect of mutations in the t peptides on their association with the two PRADs. We report that the two PRADs differ in their interaction with AChE(T) subunits; in complexes formed with the PRAD of PRiMA, we observed light dimers, but very few heavy dimers, even though such dimers were formed with the PQ chimera in which the N-terminal region of PRiMA was associated with the PRAD of ColQ. Complexes with PQ or with PRiMA contained heavy components, which migrated abnormally in SDS-PAGE but probably resulted from disulfide bonding of four AChE(T) subunits with the four upstream cysteines of the associated protein.

Tetrameric Mouse Acetylcholinesterase: Continuum Diffusion Rate Calculations by Solving the Steady-State Smoluchowski Equation Using Finite Element Methods

The tetramer is the most important form for acetylcholinesterase in physiological conditions, i.e., in the neuromuscular junction and the nervous system. It is important to study the diffusion of acetylcholine to the active sites of the tetrameric enzyme to understand the overall signal transduction process in these cellular components. Crystallographic studies revealed two different forms of tetramers, suggesting a flexible tetramer model for acetylcholinesterase. Using a recently developed finite element solver for the steady-state Smoluchowski equation, we have calculated the reaction rate for three mouse acetylcholinesterase tetramers using these two crystal structures and an intermediate structure as templates. Our results show that the reaction rates differ for different individual active sites in the compact tetramer crystal structure, and the rates are similar for different individual active sites in the other crystal structure and the intermediate structure. In the limit of zero salt, the reaction rates per active site for the tetramers are the same as that for the monomer, whereas at higher ionic strength, the rates per active site for the tetramers are ∼67%–75% of the rate for the monomer. By analyzing the effect of electrostatic forces on ACh diffusion, we find that electrostatic forces play an even more important role for the tetramers than for the monomer. This study also shows that the finite element solver is well suited for solving the diffusion problem within complicated geometries.

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

Functional localization of acetylcholinesterase (AChE) in vertebrate muscle and brain depends on interaction of the tryptophan amphiphilic tetramerization (WAT) sequence, at the C-terminus of its major splice variant (T), with a proline-rich attachment domain (PRAD), of the anchoring proteins, collagenous (ColQ) and proline-rich membrane anchor. The crystal structure of the WAT/PRAD complex reveals a novel supercoil structure in which four parallel WAT chains form a left-handed superhelix around an antiparallel left-handed PRAD helix resembling polyproline II. The WAT coiled coils possess a WWW motif making repetitive hydrophobic stacking and hydrogen-bond interactions with the PRAD. The WAT chains are related by an approximately 4-fold screw axis around the PRAD. Each WAT makes similar but unique interactions, consistent with an asymmetric pattern of disulfide linkages between the AChE tetramer subunits and ColQ. The P59Q mutation in ColQ, which causes congenital endplate AChE deficiency, and is located within the PRAD, disrupts crucial WAT-WAT and WAT-PRAD interactions. A model is proposed for the synaptic AChE(T) tetramer.

Effects of huperzine A on acetylcholinesterase isoforms in vitro: comparison with tacrine, donepezil, rivastigmine and physostigmine

Five inhibitors of acetylcholinesterase, huperzine A, donepezil, tacrine, rivastigmine and physostigmine, were compared with regard to their effects on different molecular forms of acetylcholinesterase in cerebral cortex, hippocampus, and striatum from the rat brain. In general, huperzine A preferentially inhibited tetrameric acetylcholinesterase (G4 form), while tacrine and rivastigmine preferentially inhibited monomeric acetylcholinesterase (G1 form). Donepezil showed pronounced selectivity for G1 acetylcholinesterase in striatum and hippocampus, but not in cortex. Physostigmine showed no form-selectivity in any brain region. In cortex, the most potent inhibitors of G4 acetylcholinesterase were huperzine A ( K i 7×10 −9 M) and donepezil ( K i 4×10 −9 M). The potent inhibitors of cortical G1 acetylcholinesterase were donepezil ( K i 3.5×10 −9 M) and tacrine ( K i 2.3×10 −8 M). In hippocampus, huperzine A and physostigmine were the most potent inhibitors of G4 acetylcholinesterase, while donepezil and tacrine were most potent against G1 acetylcholinesterase. In striatum, huperzine A and donepezil were the most potent against G4 acetylcholinesterase, while again donepezil was the most potent against G1. Although the inhibition constants ( K i) of these acetylcholinesterase inhibitors differed significantly from region to region, the nature of the inhibition did not vary. These results suggest that the use of acetylcholinesterase inhibitors in treatment of Alzheimer's disease must consider both form-specific and region-specific characteristics of acetylcholinesterase inhibition.

Muscle histopathology in schizophrenia

Acetylcholinesterase plays a major role in neuromuscular transmission and is regulated by neuromuscular activity. Since fast-twitch motor units are recruited with increased motor demand, we examined acetylcholinesterase regulation in rat leg muscles following treadmill training. Total acetylcholinesterase and specifically the membrane-bound tetramer increased in exercised fast-, but not slow-twitch muscles, while other isoforms remained unchanged. Synaptic acetylcholinesterase increased markedly in neuromuscular junctions of trained fibers, without concomitant changes in synaptic acetylcholine receptor, thus elevating synaptic acetylcholinesterase/receptor ratios. Electron microscopy showed that acetylcholinesterase increased in postjunctional folds and primary cleft, where it was added adjacent to the postsynaptic muscle membrane. Thus, although the primary acetylcholinesterase at the neuromuscular junction is the collagen-tailed asymmetric isoform associated with synaptic basal lamina, physiological demands such as strenuous exercise, or potentially pathological conditions, can selectively recruit the membrane-bound acetylcholinesterase tetramer to the synapse for optimal synaptic transmission.

Two Distinct Proteins Are Associated with Tetrameric Acetylcholinesterase on the Cell Surface

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

Conformational Flexibility of the Acetylcholinesterase Tetramer Suggested by X-ray Crystallography

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.