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.

Electrophorus acetylcholinesterase. Biochemical and electron microscope characterization of low ionic strength aggregates.

The "tailed" molecules of Electrophorus (electric eel) acetylcholinesterase aggregate under conditions of low ionic strength. These aggregates have been studied by sedimentation analysis and high-resolution electron microscopy. They consist of bundles of at least half a dozen molecules, the tails of which are packed side by side, to form the core of the structure. Although aggregation is normally fully reversible, aggregates were irreversibly stabilized by methylene blue-sensitized photo-oxidation. This process was shown to consist of a singlet oxygen oxidation reaction and probably involves methionine or histidine residues. It did not modify the structural or hydrodynamic characteristics of the aggregates.

Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function.

Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.

Patients with congenital myasthenia associated with end-plate acetylcholinesterase deficiency show normal sequence, mRNA splicing, and assembly of catalytic subunits.

A congenital myasthenic condition has been described in several patients characterized by a deficiency in end-plate acetylcholinesterase (AChE). The characteristic form of AChE in the end-plate basal lamina has the catalytic subunits disulfide linked to a collagen-like tail unit. Southern analysis of the gene encoding the catalytic subunits revealed no differences between patient and control DNA. Genomic DNA clones covering exon 4 and the alternatively spliced exons 5 and 6 were analyzed by nuclease protection and sequencing. Although allelic differences were detected between controls, we found no differences in exonic and intronic areas that might yield distinctive splicing patterns in patients and controls. The ACHE gene was cloned from genomic libraries from a patient and a control. Transfection of the cloned genes revealed identical species of mRNA and expressed AChE. Cotransfection of the genes expressing the catalytic subunits with a cDNA from Torpedo encoding the tail unit yielded asymmetric species that require assembly of catalytic subunits and tail unit. thus the catalytic subunits of AChE expressed in the congenital myasthenic syndrome appear identical in sequence, arise from similar splicing patterns, and assemble normally with a tail unit to form a heteromeric species.

Stability and secretion of acetylcholinesterase forms in skeletal muscle cells.

Muscle cells express a distinct splice variant of acetylcholinesterase (AChE(T)), but the specific mechanisms governing this restricted expression remain unclear. In these cells, a fraction of AChE subunits is associated with a triple helical collagen, ColQ, each strand of which can recruit a tetramer of AChE(T). In the present study, we examined the expression of the various splice variants of AChE by transfection in the mouse C2C12 myogenic cells in vitro, as well as in vivo by injecting plasmid DNA directly into tibialis anterior muscles of mice and rats. Surprisingly, we found that transfection with an ACHE(H) cDNA, generating a glycophosphatidylinositol-anchored enzyme species, produced much more activity than transfection with AChE(T) cDNA in both C2C12 cells and in vivo. This indicates that the exclusive expression of AChE(T) in mature muscle is governed by specific splicing. Interaction of AChE(T) subunits with the complete collagen tail ColQ increased enzyme activity in cultured cells, as well as in muscle fibers in vivo. Truncated ColQ subunits, presenting more or less extensive C-terminal deletions, also increased AChE activity and secretion in C2C12 cells, although the triple helix could not form in the case of the larger deletion. This suggests that heteromeric associations are stabilized compared with isolated AChE(T) subunits. Coinjections of AChE(T) and ColQ resulted in the production and secretion of asymmetric forms, indicating that assembly, processing, and externalization of these molecules can occur outside the junctional region of muscle fibers and hence does not require the specialized junctional Golgi apparatus.

Differences in expression of acetylcholinesterase and collagen Q control the distribution and oligomerization of the collagen-tailed forms in fast and slow muscles.

The collagen-tailed forms of acetylcholinesterase (AChE) are accumulated at mammalian neuromuscular junctions. The A(4), A(8), and A(12) forms are expressed differently in the rat fast and slow muscles; the sternomastoid muscle contains essentially the A(12) form at end plates, whereas the soleus muscle also contains extrajunctional A(4) and A(8) forms. We show that collagen Q (ColQ) transcripts become exclusively junctional in the adult sternomastoid but remain uniformly expressed in the soleus. By coinjecting Xenopus oocytes with AChE(T) and ColQ mRNAs, we reproduced the muscle patterns of collagen-tailed forms. The soleus contains transcripts ColQ1 and ColQ1a, whereas the sternomastoid only contains ColQ1a. Collagen-tailed AChE represents the first evidence that synaptic components involved in cholinergic transmission may be differently regulated in fast and slow muscles.

Stable expression of acetylcholinesterase and associated collagenic subunits in transfected RBL cell lines: production of GPI-anchored dimers and collagen-tailed forms.

We obtained a stable expression of acetylcholinesterase (AChE, E.C. 3.1.1.7) in the rat basoleukemia cell line, RBL-2H3, which possesses a well developed secretory pathway, but expresses only very little endogenous AChE. Metabolic labeling showed that AChEH and AChET, differing by C-terminal peptides encoded by alternatively spliced exons, were synthesized at a similar rate. When transfected with AChEH, RBL cells efficiently produced GPI-anchored dimers, which were mostly exposed at the cell surface, as shown both by activity and immunofluorescence labeling. In contrast, when transfected with AChET, RBL cells produced about tenfold less activity, which was essentially retained in the cell, and the enzyme could not be detected at the cell surface by immunolabeling. The fate of the enzyme is therefore determined by its C-terminal alternative peptides. We were also able to coexpress the AChET subunit with the collagenic Q subunit. The cells produced small but significant amounts of collagen-tailed forms, essentially A4. The expression of these different catalytic and structural subunits in stably transfected RBL cells will be useful to explore the regulated posttranslational processes involved in protein maturation and transport.

Expression of a cDNA encoding the glycolipid-anchored form of rat acetylcholinesterase.

We amplified by PCR and characterized a fragment of cDNA from rat spleen, encoding the distinctive C-terminal region of the acetylcholinesterase (AChE) H subunit. A recombinant vector encoding this subunit was constructed and expressed in COS cells: the H subunits produced glycophosphatidylinositol (GPI)-anchored dimers, showing that the spleen cDNA fragment contained a functional GPI cleavage/attachment site. Using PCR, we did not detect mRNAs encoding AChE H in rat muscle or hypothalamus. In the liver of 16-day rat embryos, we found both H and T transcripts, in agreement with the presence of both GPI-anchored dimers and amphiphilic monomers of type II. In addition, we detected 'read-through' (R) transcripts, in which regular introns are spliced, but the intervening sequence between the common exon 4 and the alternative exon 5 (H) is maintained.

Isolation of a cDNA clone for a catalytic subunit of Torpedo marmorata acetylcholinesterase.

We have constructed a cDNA library from Torpedo marmorata electric organ poly(A+) RNA in the lambda phage expression vector lambda gt11. This library has been screened with polyclonal anti-acetylcholinesterase antibodies. One clone, lambda AChE1, produced a fusion protein which was recognized by the antibodies and which prevented the binding of native acetylcholinesterase in an enzymatic immune assay. These results indicate that lambda AChE1 contains a cDNA insert coding for a part of a catalytic subunit of Torpedo acetylcholinesterase. The 200-base-pair cDNA insert hybridized to three mRNAs (14.5, 10.5 and 5.5 kb) from Torpedo electric organs. These mRNAs were also detected in Torpedo electric lobes.

Expression of a bovine vesicular monoamine transporter in COS cells.

Catecholamines are accumulated in vesicles by a proton gradient-dependent transport, which has mostly been studied in bovine chromaffin granules. The full sequence of a cDNA encoding a vesicular transporter from bovine chromaffin cells, bVMAT2, was recently reported. We now present an analysis of bVMAT2, expressed in transfected COS cells. Comparing the binding of a labelled ligand, [3H]TBZOH, and the rate of uptake, we find a much lower molecular turnover number than in chromaffin granules, probably indicating that a majority of expressed transporters are correctly folded and possess the ligand binding site but cannot actively transport monoamines because they are located in compartments which do not possess a proton gradient. The substrate specificity of uptake and its pharmacological sensitivity to various inhibitors closely resemble those previously observed in chromaffin granules. These results suggest that VMAT2 is the major transporter in bovine adrenal glands, and raise the question of the significance of the second related transporter, VMAT1, which is also expressed in this tissue.

A conformation-dependent monoclonal antibody against active chicken acetylcholinesterase.

We show that the C-131 monoclonal antibody, directed against chicken AChE, recognizes active chicken AChE, but not the SDS-denatured or heat-inactivated protein. Previous results indicated that C-131 only binds to the active enzyme, and not to inactive molecules which also occur in the embryonic chicken brain. In contrast with C-131, other monoclonal antibodies obtained in the same series, such as C-6 and C-54, also recognize denatured or inactive AChE. It is noteworthy that these antibodies all seem to react with a trypsin-sensitive peptide which is present in chicken but not in mammalian or Torpedo AChE, whereas the C-131 antibody binds trypsin-modified as well as intact molecules. These results show that C-131 is highly conformation-dependent, specific for active AChE. They confirm our previous conclusion that active and inactive molecules arise from different folding processes.

The rate of thermal inactivation of Torpedo acetylcholinesterase is not reduced in the C231S mutant.

The rate of thermal inactivation of Torpedo AChE at pH 8.5 was increased by the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). At 30 degrees C or 37 degrees C, inactivation rates with 0.3 mM DTNB increased about 5-fold for the wild-type enzyme and for two site-specific mutants, D72S and V129R. The reversible active site inhibitor, ambenonium, completely stabilized the wild type enzyme and partially stabilized the D72S mutant. However, ambenonium did not protect against the destabilization introduced by DTNB, which still accelerated inactivation of D72S 5-fold. When the only free sulfhydryl group in AChE was removed by replacing cysteine 231 with serine, increased rates of thermal inactivation were observed. The inactivation rate increased by a factor of 2 to 3 for the single mutant (C231S) and by a factor of 5 for the double mutant V129R/C231S. Even in the C231S mutants, DTNB still had an additional effect. It increased the inactivation rate for C231S and V129R/C231 by a factor of about 1.5 to 3 beyond the rates seen in the absence of DTNB. Therefore, at least part of the destabilization seen with DTNB in enzymes that retain C231 does not involve reaction of DTNB with C231.

Site-directed mutagenesis of active-site-related residues in Torpedo acetylcholinesterase. Presence of a glutamic acid in the catalytic triad.

Site-directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterase. The recently determined atomic structure of this enzyme shows the conserved Glu-327, together with His-440 and Ser-200 as forming a catalytic triad, while the adjacent conserved Asp-326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp-326----Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu-327 to Gln or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.

Expression and regulation of the bovine vesicular monoamine transporter gene.

In monoaminergic cells, the neurotransmitter is accumulated into secretory or synaptic vesicles by a tetrabenazine- and reserpine-sensitive transporter, catalyzing an H+/monoamine antiport. The major vesicular monoamine transporter from bovine chromaffin cells was cloned, using sequences common to adrenal medulla and brain rat vesicular monoamine transporters. Its identity was confirmed by peptide sequences, determined from the purified protein. Surprisingly, the bovine adrenal medulla sequence, bVMAT2, is more related to the transporter from human and rat brain than to that from rat adrenal medulla. PCR amplification showed that bVMAT2 is expressed in both adrenal medulla and brain, in contrast with the situation reported in rats, where distinct genes appear to be expressed in brain (SVAT or MAT, now renamed rVMAT2) and in the adrenal medulla (CGAT, now renamed rVMAT1). In bovine chromaffin cells, long-term depolarization by KCl resulted in an increase in the level of bVMAT2 mRNA, in agreement with the previously observed increase in the transporter binding sites, suggesting that a coupling between stimulation, secretion and synthesis changes the composition of the secretory granule membrane.

Acetylcholinesterase from Bungarus venom: a monomeric species.

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non-amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N-glycans of the 'complex' and 'hybrid' types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non-denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.

Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function.

The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter-like back door near Trp84, a key- residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the omega loop, bearing Trp84, with the lid which sequesters the substrate in neutral lipases displaying structural homology with acetylcholinesterase, suggests a flap-like back door. Both possibilities were examined by site-directed mutagenesis. The shutter-like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap-like back door was tested by de novo insertion of a disulfide bridge which tethered the omega loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however, substantial mobility of the omega loop in the immediate vicinity of Trp84, even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible.

[Multiplicity of the molecular forms of acetylcholinesterase and thermal acclimatization in Carassius auratus]. / Multiplicité des formes moléculaires de l'acétylcholinestérase et acclimatation thermique chez le Carassius auratus

Multiple forms of acetylcholinesterase have been studied in Goldfish adapted to 15 degrees C and 25 degrees C. Three forms of enzyme (sedimenting at 18S, 12S and 5S) could be distinguished by sucrose gradient centrifugation. Their molecular and enzymatic properties did not vary with environmental temperature. It was noted that the relative proportion of the three forms differed between brain and muscle extracts, the 18S forms predominating in muscle (about 75 per cent) the 12S forms being more abundant in the brain, suggesting a physiological differentiation between these forms. The latter form varied from 48 per cent to 70 per cent in Goldfish adapted to 15 degrees and 25 degrees C respectively. The relative efficiency of saline buffer solubilization for each form also depended on the tissue used and in all cases decreased with rising environmental temperature. These two facts are taken to reflect interactions between acetylcholinesterase and membrane that depend on the nature of the membrane and probably involve the aliphatic chains of its phospholipids. The greater case of solubilization found in the low temperature adapted fish may be a function of the increased fluidity of the cellular membranes formed at lower temperatures.

Interactions with lectins indicate differences in the carbohydrate composition of the membrane-bound enzymes acetylcholinesterase and 5'-nucleotidase in different cell types.

We have examined the interactions of the membrane-bound enzymes, 5'-nucleotidase and acetylcholinesterase from bovine tissues with lectins and shown that glycosylation contributes significantly to the polymorphism of these enzymes, in a tissue-specific manner. Lectins which bind 5'-nucleotidase also inhibit its catalytic activity to various degrees. We found different specificities with 5'-nucleotidases from various cell types: for example lymphocyte 5'-nucleotidase did not interact with wheat germ agglutinin, in contrast with 5'-nucleotidases from hepatocyte and caudate nucleus membranes. Treatment with glycohydrolases, alpha-D-mannosidase and neuraminidase, suggested that the latter enzymes possess sialic residues which are absent in the lymphocyte enzyme. Interactions of acetylcholinesterase with lectins were demonstrated by sedimentation analysis and binding to immobilized lectins, but its activity was generally not affected. A notable exception was lymphocyte acetylcholinesterase which was inhibited by the fucose-binding Ulex europeus agglutinin. This inhibition was relieved by alpha-L-fucose but not by alpha-D-fucose and reduced after treatment with alpha-L-fucosidase. In addition this enzyme differs from acetylcholinesterases from other tissues by its higher Km value, although it appears immunologically equivalent. The different forms of acetylcholinesterase from the same tissue may differ in their interactions with lectins. In muscle for example G4 carries carbohydrate chains of the complex type whereas G1 appears to possess only the high mannose type. We discuss the possible relationships between these forms.

Monoclonal antibodies against acetylcholinesterase from electric organs of Electrophorus and Torpedo.

We studied the reactivity of monoclonal antibodies (mAbs) raised against acetylcholinesterase (AChE) purified from Electrophorus and Torpedo electric organs. We obtained IgG antibodies (Elec-21, Elec-106, Tor-3E5, Tor-ME8, Tor-1A5), all of them directed against the catalytic subunit of the corresponding species, with no significant cross-reactivity. These antibodies do not inhibit the enzyme and recognize all molecular forms, globular (G) and asymmetric (A). Tor-ME8 reacts specifically with the denatured A and G subunits of Torpedo AChE, in immunoblots. Several hybridomas raised against Electrophorus AChE produced IgM antibodies (Elec-39, Elec-118, Elec-121). These antibodies react with the A forms of Electrophorus electric organs and also with a subset of dimers (G2) from Torpedo electric organ. In addition, they react with a number of non-AChE components, in immunoblots. In contrast, they do not recognize AChE from other Electrophorus tissues or A forms from Torpedo electric organs.

A comparative Raman spectroscopic study of cholinesterases.

We report Raman spectra of various cholinesterases: lytic tetrameric forms (G4) obtained by tryptic digestion of asymmetric acetylcholinesterase (AChE) from Torpedo californica and Electrophorus electricus, a PI-PLC-treated dimeric form (G2) of AChE from T marmorata, and the soluble tetrameric form (G4) of butyrylcholinesterase (BuChE) from human plasma. The contribution of different types of secondary structure was estimated by analyzing the amide I band, using the method of Williams. The spectra of cholinesterases in 10 mM Tris-HCl (pH 7.0) indicate the presence of both alpha-helices (about 50%) and beta-sheets (about 25%), together with 15% turns and 10% undefined structures. In 20 mM phosphate buffer (pH 7.0), the spectra indicated a smaller contribution of alpha-helical structure (about 35%) and an increased beta-sheet content (from 25 to 35%). This shows that the ionic milieu profoundly affects either the conformation of the protein (AChE activity is known to be sensitive to ionic strength), or the evaluation of secondary structure, or both. In addition, we analyzed vibrations corresponding to the side chains of aromatic and aliphatic amino acids. In particular, the analyses of the tyrosine doublet (830-850 cm-1) and of the tryptophan vibration at 880 cm-1 indicated that these residues are predominantly 'exposed' on the surface of the molecules.