Acetylcholinesterase Subunits Research Articles

SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome.

The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5′ splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.

Reduced Expression of P2Y2 Receptor and Acetylcholinesterase at Neuromuscular Junction of P2Y1 Receptor Knock-out Mice.

ATP is co-stored and co-released with acetylcholine (ACh) at the pre-synaptic vesicles in vertebrate neuromuscular junction (nmj). Several lines of studies demonstrated that binding of ATP to its corresponding P2Y1 and P2Y2 receptors in the muscle regulated post-synaptic gene expressions. To further support the notion that P2Y receptors are playing indispensable role in formation of post-synaptic specifications at the nmj, the knock-out mice of P2Y1 receptor (P2Y1R (-/-)) were employed here for analyses. In P2Y1R (-/-) mice, the expression of P2Y2 receptor in muscle was reduced by over 50 %, as compared to P2Y1R (+/+) mice. In parallel, the expression of acetylcholinesterase (AChE) in muscle was markedly decreased. In the analysis of the expression of anchoring subunits of AChE in P2Y1R (-/-) mice, the proline-rich membrane anchor (PRiMA) subunit was reduced by 60 %; while the collagen tail (ColQ) subunit was reduced by 50 %. AChE molecular forms in the muscle were not changed, except the amount of enzyme was reduced. Immuno-staining of P2Y1R (-/-) mice nmj, both AChE and AChR were still co-localized at the nmj, and the staining was diminished. Taken together our data demonstrated that P2Y1 receptor regulated the nmj gene expression.

The Neuroligins and Their Ligands: from Structure to Function at the Synapse

The neuroligins are cell adhesion proteins whose extracellular domain belongs to the α/β-hydrolase fold family of proteins, mainly containing enzymes and exemplified by acetylcholinesterase. The ectodomain of postsynaptic neuroligins interacts through a calcium ion with the ectodomain of presynaptic neurexins to form flexible trans-synaptic associations characterized by selectivity for neuroligin or neurexin subtypes. This heterophilic interaction, essential for synaptic differentiation, maturation, and maintenance, is regulated by gene selection, alternative mRNA splicing, and posttranslational modifications. Mutations leading to deficiencies in the expression, folding, maturation, and binding properties of either partner are associated with autism spectrum disorders. The currently available structural and functional data illustrate how these two families of cell adhesion molecules bridge the synaptic cleft to participate in synapse plasticity and support its dynamic nature. Neuroligin partners distinct from the neurexins, and which may undergo either trans or cis interaction, have also been described, and tridimensional structures of some of them are available. Our study emphasizes the partnership versatility of the neuroligin ectodomain associated with molecular flexibility and alternative binding sites, proposes homology models of the structurally non-characterized neuroligin partners, and exemplifies the large structural variability at the surface of the α/β-hydrolase fold subunit. This study also provides new insights into possible surface binding sites associated with non-catalytic properties of the acetylcholinesterase subunit.

Readthrough Acetylcholinesterase Is Increased in Human Liver Cirrhosis

Background & AimsThere have been many studies on plasma butyrylcholinesterase in liver dysfunction. However, no data is available about acetylcholinesterase in human cirrhosis, although profound changes have been described in cirrhotic rat models.MethodsHuman serum and liver acetylcholinesterase and its molecular forms were determined enzymatically, after butyrylcholinesterase immunodepletion. The distinct species of acetylcholinesterase, with a distinct C-terminus, were determined by western blotting, and the level of liver transcripts by real-time PCR. Liver acetylcholinesterase was also evaluated by immunocytochemistry.ResultsIn patients with liver cirrhosis, the activity of plasma acetylcholinesterase (rich in light species), appeared to be apparently unaffected. However, the levels of the soluble readthrough (R) acetylcholinesterase form, an acetylcholinesterase species usually associated with stress and pathology, was increased compared to controls. Human liver acetylcholinesterase activity levels were also unchanged, but protein levels of the acetylcholinesterase-R and other acetylcholinesterase subunit species were increased in the cirrhotic liver. This increase in acetylcholinesterase protein expression in the cirrhotic liver was confirmed by PCR analysis. Immunohistological examination confirmed that acetylcholinesterase immunoreactivity is increased in parenchymal cells of the cirrhotic liver.ConclusionsWe demonstrate significant changes in acetylcholinesterase at the protein and mRNA levels in liver cirrhosis, with no difference in enzymatic activity. The altered expression of acetylcholinesterase protein may reflect changes in its pathophysiological role.

Neuromuscular Junction Acetylcholinesterase Deficiency Responsive to Albuterol

Congenital myasthenic syndrome caused by endplate acetylcholinesterase deficiency constitutes a rare autosomal recessive disease. We describe a child with early-onset ptosis, complete ophthalmoplegia, facial and proximal muscle weakness, easy fatigability, a decremental electromyographic response, and a repetitive compound muscle action potential not improved by anti-acetylcholinesterase medication. Mutation analysis of the collagenic tail of endplate acetylcholinesterase (COLQ) that encodes the collagenic structural subunit of acetylcholinesterase revealed two canonic splice-site mutations: a previously identified IVS15+ 1G>A mutation and a novel IVS2- 1G>A mutation. Treatment with albuterol resulted in progressive improvement of muscle strength, exercise tolerance, and ophthalmoplegia. Further studies are needed of the efficacy of albuterol in different types of congenital myasthenic syndrome and the physiologic basis of its beneficial effects.

An induction effect of heat shock on the transcript of globular acetylcholinesterase in NG108-15 cells

Heat shock response, an induced transcription of a set of genes in response to high temperature, occurs in all organisms. In neurons, the catalytic subunit of acetylcholinesterase (AChE T) interacts with proline-rich membrane anchor (PRiMA) to form a globular tetrameric form (G 4 form). In this study, we examined the effects of heat shock on the transcription and protein assembly of AChE T in cultured NG108-15 cells. The transcription of AChE T was rapidly induced by heat shock at 40 °C, reaching a 15-fold increase in 3 h and decreasing thereafter. On the other hand, the level of PRiMA mRNA was not affected after the heat shock. In parallel with AChE T mRNA, the enzymatic activity of cellular AChE, in terms of G 1 and G 2 forms, was increased after heat shock; however, the PRiMA-linked G 4 remained unchanged. These results suggest that heat shock can induce the expression level of AChE T by the regulation of AChE T transcripts in NG108-15 cells.

Transcriptional control of different subunits of AChE in muscles: Signals triggered by the motor nerve-derived factors

Acetylcholinesterase (AChE) is a highly polymorphic enzyme. Alternative splicing in the 3′ region of the primary transcript generates different subunits that contain the same catalytic domain but with distinct carboxyl termini. In mammals, the AChE R variant produces a soluble monomer that is up-regulated in the brain during stress. The AChE H variant produces a GPI-anchored dimer that is mainly expressed in blood cells, while AChE T variant is largely predominant in the brain and muscle. AChE T subunits associate with a collagen tail subunit (ColQ) forming asymmetric AChE species (A 4, A 8, and A 12 AChE) in muscle, and also form amphiphilic tetramers associated with a proline-rich membrane anchor (PRiMA) as globular AChE (G 4 AChE) in brain and muscle. The formation of these AChE forms depends on the physiological status of the muscles, and on the innervating nerves. The motor nerves achieve this regulation by two distinct mechanisms: release of the trophic factor calcitonin gene-related peptide (CGRP) and nerve-evoked electrical activity, which differentially regulate the expression levels of AChE T, PRiMA and ColQ via different downstream signaling cascades. The regulatory mechanisms provided by the nerve are important to account for the different expression patterns of AChE and associated proteins in fast- and slow-twitch muscles.

Regulation of PRiMA-linked G 4 AChE by a cAMP-dependent signaling pathway in cultured rat pheochromocyoma PC12 cells

The catalytic subunit of acetylcholinesterase (AChE T) interacts with proline-rich membrane anchor (PRiMA) to form PRiMA-linked G 4 AChE on membrane surface for its cholinergic function. Cultured PC12 cells expressed the transcripts encoding AChE T and PRiMA I, but the expression of PRiMA II transcript was below detection. Upon the treatment of dibutyryl-cAMP (Bt 2-cAMP) and forskolin in cultured cells to stimulate the cAMP-dependent signaling pathway, the mRNA expressions of both AChE T and PRiMA I, as well as the enzymatic activity were up-regulated. More importantly, sucrose density gradient analysis revealed that both G 1 and G 4 AChE isoforms were increased in the Bt 2-cAMP-treated cultures. These results suggest that the regulation of PRiMA-linked G 4 AChE in terms of gene transcription and molecular assembly in the cultured PC12 cells could be mediated by a cAMP-dependent signaling mechanism.

Viral vector-medicated expression of human collagen Q in cultured cells

Congenital myasthenic syndromes are caused by mutations in molecules expressed at the neuromuscular junction. Collagen Q (ColQ) makes a triple helical structure and anchors the catalytic subunit of acetylcholinesterase (AChE) to the synaptic basal lamina in the form of asymmetric AChE. Mutations in the collagen Q gene ( COLQ) cause endplate AChE deficiency. As an initial step to develop a novel therapeutic strategy for endplate acetylcholinesterase deficiency, we expressed AChE species in cultured cells using retrovirus and adeno-associated virus (AAV). The retroviral vectors carried human ACHE and COLQ either in a single construct (EF1α- ACHE-IRES- COLQ) or in two separate constructs (EF1α- ACHE and EF1α- COLQ). We produced high-titer retroviruses using the PLAT-E retrovirus packaging cells. We also confirmed expression of asymmetric AChE in the PLAT-E cells. We infected NIH3T3 and confirmed expression of the transgenes by RT-PCR. The AAV vector carried human COLQ-IRES- EGFP downstream of the CMV promoter (pAAV-CMV- COLQ-IRES- EGFP). We produced recombinant AAV using HEK293 cells carrying pDF6 encoding the AAV6 capsid gene. We infected AAVHT1080 cells and confirmed expression of COLQ by RT-PCR and EGFP by flow cytometry. We are currently trying to achieve further higher expression levels of transgenes in cultured cells to make the current strategy applicable to an animal model.

Social defeat, a paradigm of depression in rats that elicits 22-kHz vocalizations, preferentially activates the cholinergic signaling pathway in the periaqueductal gray

Gene expression profiles in the periaqueductal gray (PAG) of adult Long-Evans rats as a function of a stressful social defeat in inter-male fighting encounters were examined. This social subordination model mimics prototypical behavioral changes that parallel aspects of clinical depression, has been postulated to simulate early changes in the onset of depression in the losers, and has been successfully utilized for the evaluation of antidepressant activity. The 22-kHz ultrasonic vocalizations (USVs) have been shown to reflect negative emotional states akin to anxiety and depression. Social defeat is the most robust and reliable method of eliciting these calls. The PAG has been shown to be a key brain region for the generation of 22-kHz ultrasonic vocalizations, and 22-kHz USVs have been shown to be controlled by the mesolimbic cholinergic system. In this present study, we examined gene expression changes in the PAG of social subordinate rats compared to dominant rats that do not Exhibit 22-kHz USVs. We found that social defeat significantly altered the genes associated with cholinergic synaptic transmission in the PAG. The most robust of these were the increased expression of the β2 subunit of the nicotinic acetylcholine receptor (CHRNB2) and the T subunit of acetylcholinesterase (ACHE) in the subordinate animals. These changes were corroborated by quantitative real-time polymerase chain reaction (qRT-PCR) and found to be exclusive to the PAG compared to seven other brain regions examined. These data suggest that cholinergic transmission in the PAG is involved in the generation of 22-kHz USVs and provide potential therapeutic targets for the treatment of affective disorders.

Transcriptional Control of Different Acetylcholinesterase Subunits in Formation and Maintenance of Vertebrate Neuromuscular Junctions

Acetylcholinesterase (AChE; EC 3.1.1.7) is a highly polymorphic enzyme (Massoulié, 2002). Asingle ACHE gene produces several types of catalytic subunits by alternative splicing, but a single splice variant, called type T (AChET), is expressed in adult mammalian muscle and brain. Catalytic subunits of AChET produce amphiphilic monomers and dimers, nonamphiphilic homotetramers, as well as heteromeric associations with anchoring proteins, ColQ (collagenous subunit) and PRiMA (proline-rich membrane anchor), which allow their functional localization in cholinergic synapses (Massoulié, 2002). ColQ characterizes the collagen-tailed forms (Aforms) of AChE and butyrylcholinesterase (BChE), which are localized in the basal lamina at neuromuscular junctions (NMJs) of vertebrates (Krejci et al., 1999); in these molecules (A4, A8, A12), one, two, or three tetramers of catalytic subunits are disulfide-linked to the strands of a triple helix of ColQ collagen. The cDNAs encoding ColQ, which have two transcripts, have been cloned: ColQ-1a predominantly in fast-twitch muscle, and ColQ-1 predominantly in slow-twitch muscle. The tetrameric globular (G4) form of AChE is characterized by linkage to PRiMA. PRiMAcDNA encodes a single-pass approximately 20-kDa type-I transmembrane protein and, similar to that of ColQ, contains a short PRAD (proline-rich attachment domain) that is able to organize AChE catalytic subunits into tetramers and anchor the enzyme at the surface of neuron and muscle (Massoulié, 2002).

The Association of Tetrameric Acetylcholinesterase with ColQ Tail: A Block Normal Mode Analysis

Acetylcholinesterase (AChE) rapidly hydrolyzes acetylcholine in the neuromuscular junctions and other cholinergic synapses to terminate the neuronal signal. In physiological conditions, AChE exists as tetramers associated with the proline-rich attachment domain (PRAD) of either collagen-like Q subunit (ColQ) or proline-rich membrane-anchoring protein. Crystallographic studies have revealed that different tetramer forms may be present, and it is not clear whether one or both are relevant under physiological conditions. Recently, the crystal structure of the tryptophan amphiphilic tetramerization (WAT) domain of AChE associated with PRAD ([WAT]4PRAD), which mimics the interface between ColQ and AChE tetramer, became available. In this study we built a complete tetrameric mouse [AChET]4–ColQ atomic structure model, based on the crystal structure of the [WAT]4PRAD complex. The structure was optimized using energy minimization. Block normal mode analysis was done to investigate the low-frequency motions of the complex and to correlate the structure model with the two known crystal structures of AChE tetramer. Significant low-frequency motions among the catalytic domains of the four AChE subunits were observed, while the [WAT]4PRAD part held the complex together. Normal mode involvement analysis revealed that the two lowest frequency modes were primarily involved in the conformational changes leading to the two crystal structures. The first 30 normal modes can account for more than 75% of the conformational changes in both cases. The evidence further supports the idea of a flexible tetramer model for AChE. This model can be used to study the implications of the association of AChE with ColQ.

The C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports homomeric and heteromeric interactions.

Acetylcholinesterase subunits of type T (AChET) possess an alternatively spliced C-terminal peptide (t peptide) which endows them with amphiphilic properties, the capacity to form various homo-oligomers and to associate, as a tetramer, with anchoring proteins containing a proline rich attachment domain (PRAD). The t peptide contains seven conserved aromatic residues. By spectroscopic analyses of the synthetic peptides covering part or all of the t peptide of Torpedo AChET, we show that the region containing the aromatic residues adopts an alpha helical structure, which is favored in the presence of lipids and detergent micelles: these residues therefore form a hydrophobic cluster in a sector of the helix. We also analyzed the formation of disulfide bonds between two different AChET subunits, and between AChET subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (QN)] possessing two cysteines upstream or downstream of the PRAD. This shows that, in the complex formed by four T subunits with QN (T4-QN), the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD. The formation of disulfide bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility.

Trimerization domain of the collagen tail of acetylcholinesterase.

In the collagen-tailed forms of cholinesterases, each subunit of a specific triple helical collagen, ColQ, may be attached through a proline-rich domain (PRAD) situated in its N-terminal noncollagenous region, to tetramers of acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). This heteromeric assembly ensures the functional anchoring of AChE in extracellulare matrices, for example, at the neuromuscular junction. In this study, we analyzed the influence of deletions in the noncollagenous C-terminal region of ColQ on its capacity to form a triple helix. We show that an 80-residue segment located downstream of the collagenous regions contains the trimerization domain, that it can form trimers without the collagenous regions, and that a pair of cysteines located at the N-boundary of this domain facilitates oligomerization, although it is not absolutely required. We further show that AChE subunits can associate with nonhelical collagen ColQ monomers, forming ColQ-associated tetramers (G4-Q), which are secreted or are anchored at the cell surface when the C-terminal domain of ColQ is replaced by a GPI-addition signal.

Effect of human acetylcholinesterase subunit assembly on its circulatory residence

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 740min respectively. As the level of sialylation decreased, this differential behaviour became less pronounced; eventually, after desialylation all oligoforms had the same MRT (5min). 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.

Characterization of acetylcholinesterase from Korean electric ray and comparison withTorpedo californica

This study has been undertaken to examine the acetylcholinesterase (AChE) of electric organ from korean electric ray (Narke japonica). Korean electric ray was caughted at Chungmu sea and transported to the laboratory, where electric organs were removed and stored at −70°C until used. Acetylcholinesterase(AChE) of electric organ was purified by affinity column that was prepared with dicaproyl-methylpyridinium linked to Sepharose 4B. Upon purification, the specific activities in Ellman unit were increased by 52 and 39 times for high salt soluble AChE (HSSE, 870.86 ΔOD/min/gram of tissue) and detergent soluble AChE(DSE, 105.42 ΔOD/min/gram of tissue), respectively. Each subunit of AChE separated by SDS polyacrylamide gel electrophoresis(SDS-PAGE) was transferred to immobilon P by western blotting and detected by mAbs raised against each subunit of AChE from electric organ ofTorpedo californica. Collagenic tail of AChE fromNarke japonica were identified by monoclonal antibody specific to collagenic tail of AChE fromTorpedo californica, likewise 103Kd protein of AChE fromNarke japonica was detected by monoclonal antibody specific to 103Kd of AChE fromTorpedo californica. However, molar ratio of three subunits of AChE fromNarke japonica is different from that ofTorpedo californica. Furthermore, catalytic subniit of AChE fromNarke japonica was not identified by monoclnal antibody specific to catalytic subunit of AChE fromTorpedo californica. These results showed differences in molecular structure of AChE fromNarke japonica and AChE fromTorpedo californica eventhough they showed same enzymatic activities.

Reversal of signal-mediated cellular retention by subunit assembly of human acetylcholinesterase.

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.

Cloning and analysis of chicken acetylcholinesterase transcripts from muscle and brain

We have isolated cDNA clones from an embryonic chicken muscle cDNA library which encodes the complete catalytic T subunit of acetylcholinesterase. The deduced polypeptide comprises 767 amino acids, shows ∼60% homology to acetylcholinesterases from other vertebrates and contains a 155 amino acid sequence inserted into the middle of the peptide which is unique to the chicken enzyme. Northern blots of embryonic chicken muscle and adult brain show three transcripts approximately 4.5, 5.5, and 6.0 kb hybridizing to a cDNA fragment of AChE. The 6.0 kb transcript is expressed transiently in embryonic muscle and is a major transcript in adult brain.

Cloning and expression of a rat acetylcholinesterase subunit: generation of multiple molecular forms and complementarity with a Torpedo collagenic subunit.

We obtained a cDNA clone encoding one type of catalytic subunit of acetylcholinesterase (AChE) from rat brain (T subunit). The coding sequence shows a high frequency of (G+C) at the third position of the codons (66%), as already noted for several AChEs, in contrast with mammalian butyrylcholinesterase. The predicted primary sequence of rat AChE presents only 11 amino acid differences, including one in the signal peptide, from that of the mouse T subunit. In particular, four alanines in the mouse sequence are replaced by serine or threonine. In northern blots, a rat AChE probe indicates the presence of major 3.2- and 2.4-kb mRNAs, expressed in the CNS as well as in some peripheral tissues, including muscle and spleen. In vivo, we found that the proportions of G1, G2, and G4 forms are highly variable in different brain areas. We did not observe any glycolipid-anchored G2 form, which would be derived from an H subunit. We expressed the cloned rat AChE in COS cells: The transfected cells produce principally an amphiphilic G1a form, together with amphiphilic G2a and G4a forms, and a nonamphiphilic G4na form. The amphiphilic G1a and G2a forms correspond to type II forms, which are predominant in muscle and brain of higher vertebrates. The cells also release G4na, G2a, and G1a in the culture medium. These experiments show that all the forms observed in the CNS in vivo may be obtained from the T subunit. By co-transfecting COS cells with the rat T subunit and the Torpedo collagenic subunit, we obtained chimeric collagen-tailed forms. This cross-species complementarity demonstrates that the interaction domains of the catalytic and structural subunits are highly conserved during evolution.

H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells, generate all types of globular forms.

We analyzed the production of Torpedo marmorata acetylcholinesterase (AChE) in transfected COS cells. We report that the presence of an aspartic acid at position 397, homologous to that observed in other cholinesterases and related enzymes (Krejci, E., N. Duval, A. Chatonnet, P. Vincens, and J. Massoulié. 1991. Proc. Natl. Acad. Sci. USA. 88:6647-6651), is necessary for catalytic activity. The presence of an asparagine in the previously reported cDNA sequence (Sikorav, J.L., E. Krejci, and J. Massoulié. 1987. EMBO (Eur. Mol. Biol. Organ.) J. 6:1865-1873) was most likely due to a cloning error (codon AAC instead of GAC). We expressed the T and H subunits of Torpedo AChE, which differ in their COOH-terminal region and correspond respectively to the collagen-tailed asymmetric forms and to glycophosphatidylinositol-anchored dimers of Torpedo electric organs, as well as a truncated T subunit (T delta), lacking most of the COOH-terminal peptide. The transfected cells synthesized similar amounts of AChE immunoreactive protein at 37 degrees and 27 degrees C. However AChE activity was only produced at 27 degrees C and, even at this temperature, only a small proportion of the protein was active. We analyzed the molecular forms of active AChE produced at 27 degrees C. The H polypeptides generated glycophosphatidylinositol-anchored dimers, resembling the corresponding natural AChE form. The cells also released non-amphiphilic dimers G2na. The T polypeptides generated a series of active forms which are not produced in Torpedo electric organs: G1a, G2a, G4a, and G4na cellular forms and G2a and G4na secreted forms. The amphiphilic forms appeared to correspond to type II forms (Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:776-785; Bon, S., J. P. Toutant, K. Méflah, and J. Massoulié. 1988. J. Neurochem. 51:786-794), which are abundant in the nervous tissue and muscles of higher vertebrates (Bon, S., T. L. Rosenberry, and J. Massoulié. 1991. Cell. Mol. Neurobiol. 11:157-172). The H and T catalytic subunits are thus sufficient to account for all types of known AChE forms. The truncated T delta subunit yielded only non-amphiphilic monomers, demonstrating the importance of the T COOH-terminal peptide in the formation of oligomers, and in the hydrophobic character of type II forms.