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The α/β hydrolase fold family is perhaps the largest group of proteins presenting significant structural homology with divergent functions, ranging from catalytic hydrolysis to heterophilic cell adhesive interactions to chaperones in hormone production. All the proteins of the family share a common three-dimensional core structure containing the α/β hydrolase fold domain that is crucial for proper protein function. Several mutations associated with congenital diseases or disorders have been reported in conserved residues within the α/β-hydrolase fold domain of cholinesterase-like proteins, neuroligins, butyrylcholinesterase and thyroglobulin. These mutations are known to disrupt the architecture of the common structural domain either globally or locally. Characterization of the natural mutations affecting the α/β-hydrolase fold domain in these proteins has shown that they mainly impair processing and trafficking along the secretory pathway causing retention of the mutant protein in the endoplasmic reticulum. Studying the processing of α/β-hydrolase fold mutant proteins should uncover new functions for this domain, that in some cases require structural integrity for both export of the protein from the ER and for facilitating subunit dimerization. A comparative study of homologous mutations in proteins that are closely related family members, along with the definition of new three-dimensional crystal structures, will identify critical residues for the assembly of the α/β-hydrolase fold.

International audience; The complete knockout of the acetylcholinesterase gene (AChE) in the mouse yielded a surprising phenotype that could not have been predicted from deletion of the cholinesterase genes in Drosophila, that of a living, but functionally compromised animal. The phenotype of this animal showed a sufficient compromise in motor function that precluded precise characterization of central and peripheral nervous functional deficits. Since AChE in mammals is encoded by a single gene with alternative splicing, additional understanding of gene expression might be garnered from selected deletions of the alternatively spliced exons. To this end, transgenic strains were generated that deleted exon 5, exon 6, and the combination of exons 5 and 6. Deletion of exon 6 reduces brain AChE by 93% and muscle AChE by 72%. Deletion of exon 5 eliminates AChE from red cells and the platelet surface. These strains, as well as knockout strains that selectively eliminate the AChE anchoring protein subunits PRiMA or ColQ (which bind to sequences specified by exon 6) enabled us to examine the role of the alternatively spliced exons responsible for the tissue disposition and function of the enzyme. In addition, a knockout mouse was made with a deletion in an upstream intron that had been identified in differentiating cultures of muscle cells to control AChE expression. We found that deletion of the intronic regulatory region in the mouse essentially eliminated AChE in muscle and surprisingly from the surface of platelets. The studies generated by these knockout mouse strains have yielded valuable insights into the function and localization of AChE in mammalian systems that cannot be approached in cell culture or in vitro.

AChE is an alternatively spliced gene. Exons 2, 3 and 4 are invariantly spliced, and this sequence is responsible for catalytic function. The 3' alternatively spliced exons, 5 and 6, are responsible for AChE disposition in tissue [J. Massoulie, The origin of the molecular diversity and functional anchoring of cholinesterases. Neurosignals 11 (3) (2002) 130-143; Y. Li, S. Camp, P. Taylor, Tissue-specific expression and alternative mRNA processing of the mammalian acetylcholinesterase gene. J. Biol. Chem. 268 (8) (1993) 5790-5797]. The splice to exon 5 produces the GPI anchored form of AChE found in the hematopoietic system, whereas the splice to exon 6 produces a sequence that binds to the structural subunits PRiMA and ColQ, producing AChE expression in brain and muscle. A third alternative RNA species is present that is not spliced at the 3' end; the intron 3' of exon 4 is used as coding sequence and produces the read-through, unanchored form of AChE. In order to further understand the role of alternative splicing in the expression of the AChE gene, we have used homologous recombination in stem cells to produce gene specific deletions in mice. Alternatively and together exon 5 and exon 6 were deleted. A cassette containing the neomycin gene flanked by loxP sites was used to replace the exon(s) of interest. Tissue analysis of mice with exon 5 deleted and the neomycin cassette retained showed very low levels of AChE expression, far less than would have been anticipated. Only the read-through species of the enzyme was produced; clearly the inclusion of the selection cassette disrupted splicing of exon 4 to exon 6. The selection cassette was then deleted in exon 5, exon 6 and exons 5 + 6 deleted mice by breeding to Ella-cre transgenic mice. AChE expression in serum, brain and muscle has been analyzed. Another AChE gene targeted mouse strain involving a region in the first intron, found to be critical for AChE expression in muscle cells [S. Camp, L. Zhang, M. Marquez, B. delaTorre, P. Taylor, Knockout mice with deletions of alternatively spliced exons of Acetylcholinesterase, in: N.C. Inestrosa, E.O. Campus (Eds.), VII International Meeting on Cholinesterases, Pucon-Chile Cholinesterases in the Second Millennium: Biomolecular and Pathological Aspects. P. Universidad Catholica de Chile-FONDAP Biomedicina, 2004, pp. 43-48; R.Y.Y. Chan, C. Boudreau-Larivière, L.A. Angus, F. Mankal, B.J. Jasmin, An intronic enhancer containing an N-box motif is required for synapse- and tissue-specific expression of the acetylcholinesterase gene in skeletal muscle fibers. Proc. Natl. Acad. Sci. USA 96 (1999) 4627-4632], is also presented. The intronic region was floxed and then deleted by mating with Ella-cre transgenic mice. The deletion of this region produced a dramatic phenotype; a mouse with near normal AChE expression in brain and other CNS tissues, but no AChE expression in muscle. Phenotype and AChE tissue activities are compared with the total AChE knockout mouse [W. Xie, J.A. Chatonnet, P.J. Wilder, A. Rizzino, R.D. McComb, P. Taylor, S.H. Hinrichs, O. Lockridge, Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase. J. Pharmacol. Exp. Ther. 293 (3) (2000) 896-902].

Fasciculin, a selective peptidic inhibitor of acetylcholinesterase, is a member of the three-fingered peptide toxin superfamily isolated from snake venoms. The availability of a crystal structure of a fasciculin 2 (Fas2)-acetylcholinesterase complex affords an opportunity to examine in detail the interaction of this toxin with its target site. To this end, we constructed a synthetic fasciculin gene with an appropriate leader peptide for expression and secretion from mammalian cells. Recombinant wild-type Fas2, expressed and amplified in Chinese hamster ovary cells, was purified to homogeneity and found to be identical in composition and biological activities to the venom-derived toxin. Sixteen mutations at positions where the crystal structure of the complex indicates a significant interfacial contact point or determinant of conformation were generated. Two mutants of loop I, T8A/T9A and R11Q, ten mutants of the longest loop II, R24T, K25L, R27W, R28D, H29D, DeltaPro30, P31R, K32G, M33A, and V34A/L35A, and two mutants of loop III, D45K and K51S, were expressed transiently in human embryonic kidney cells. Inhibitory potencies of the Fas2 mutants toward mouse AChE were established, based on titration of the mutants with a polyclonal anti-Fas2 serum. The Arg27, Pro30, and Pro31 mutants each lost two or more orders of magnitude in Fas2 activity, suggesting that this subset of three residues, at the tip of loop II, dominates the loop conformation and interaction of Fas2 with the enzyme. The Arg24, Lys32, and Met33 mutants lost about one order of magnitude, suggesting that these residues make moderate contributions to the strength of the complex, whereas the Lys25, Arg28, Val34-Leu35, Asp45, and Lys51 mutants appeared as active as Fas2. The Thr8-Thr9, Arg11, and His29 mutants showed greater ratios of inhibitory activity to immunochemical titer than Fas2. This may reflect immunodominant determinants in these regions or intramolecular rearrangements in conformation that enhance the interaction. Of the many Fas2 residues that lie at the interface with acetylcholinesterase, only a few appear to provide substantial energetic contributions to the high affinity of the complex.

A soluble, monomeric form of acetylcholinesterase from mouse (mAChE), truncated at its carboxyl-terminal end, was generated from a cDNA encoding the glycophospholipid-linked form of the mouse enzyme by insertion of an early stop codon at position 549. Insertion of the cDNA behind a cytomegalovirus promoter and selection by aminoglycoside resistance in transfected HEK cells yielded clones secreting large quantities of mAChE into the medium. The enzyme sediments as a soluble monomer at 4.8 S. High levels of expression coupled with a one-step purification by affinity chromatography have allowed us to undertake a crystallographic study of the fasciculin-mAChE complex. Complexes of two distinct fasciculins, Fas1-mAChE and Fas2-mAChE, were formed prior to the crystallization and were characterized thoroughly. Single hexagonal crystals, up to 0.6 mm x 0.5 mm x 0.5 mm, grew spontaneously from ammonium sulfate solutions buffered in the pH 7.0 range. They were found by electrophoretic migration to consist entirely of the complex and diffracted to 2.8 A resolution. Analysis of initial X-ray data collected on Fas2-mAChE crystals identified the space group as P6(1)22 or P6(5)22 with unit cell dimensions a = b = 75.5 A, c = 556 A, giving a Vm value of 3.1 A3/Da (or 60% of solvent), consistent with a single molecule of Fas2-AChE complex (72 kDa) per asymmetric unit. The complex Fas1-mAChE crystallizes in the same space group with identical cell dimensions.

The α/β-hydrolase fold superfamily of proteins is composed of structurally related members that, despite great diversity in their catalytic, recognition, adhesion and chaperone functions, share a common fold governed by homologous residues and conserved disulfide bridges. Non-synonymous single nucleotide polymorphisms within the α/β-hydrolase fold domain in various family members have been found for congenital endocrine, metabolic and nervous system disorders. By examining the amino acid sequence from the various proteins, mutations were found to be prevalent in conserved residues within the α/β-hydrolase fold of the homologous proteins. This is the case for the thyroglobulin mutations linked to congenital hypothyroidism. To address whether correct folding of the common domain is required for protein export, we inserted the thyroglobulin mutations at homologous positions in two correlated but simpler α/β-hydrolase fold proteins known to be exported to the cell surface: neuroligin3 and acetylcholinesterase. Here we show that these mutations in the cholinesterase homologous region alter the folding properties of the α/β-hydrolase fold domain, which are reflected in defects in protein trafficking, folding and function, and ultimately result in retention of the partially processed proteins in the endoplasmic reticulum. Accordingly, mutations at conserved residues may be transferred amongst homologous proteins to produce common processing defects despite disparate functions, protein complexity and tissue-specific expression of the homologous proteins. More importantly, a similar assembly of the α/β-hydrolase fold domain tertiary structure among homologous members of the superfamily is required for correct trafficking of the proteins to their final destination.

By examining inhibitor interactions with single and multiple site-specific mutants of mouse acetylcholinesterase, we have identified three distinct domains in the cholinesterase structure that are responsible for conferring selectivity for acetyl- and butyrylcholinesterase inhibitors. The first domain is the most obvious; it defines the constraints on the acyl pocket dimensions where the side chains of F295 and F297 primarily outline this region in acetylcholinesterase. Replacement of these phenylalanine side chains with the aliphatic residues found in butyrylcholinesterase allows for the catalysis of larger substrates and accommodates butyrylcholinesterase-selective alkyl phosphates such as isoOMPA. Also, elements of substrate activation characteristic of butyrylcholinesterase are evident in the F297I mutant. Substitution of tyrosines for F295 and F297 further alters the catalytic constants. The second domain is found near the lip of the active center gorge defined by two tyrosines, Y72 and Y124, and by W286; this region appears to be critical for the selectivity of bisquaternary inhibitors, such as BW284C51. The third domain defines the site of choline binding. Herein, in addition to conserved E202 and W86, a critical tyrosine, Y337, found only in the acetylcholinesterases is responsible for sterically occluding the binding site for substituted tricyclic inhibitors such as ethopropazine. Analysis of a series of substituted acridines and phenothiazines defines the groups on the ligand and amino acid side chains in this site governing binding selectivity. Each of the three domains is defined by a cluster of aromatic residues. The two domains stabilizing the quaternary ammonium moieties each contain a negative charge, which contributes to the stabilization energy of the respective complexes.

Endplate acetylcholinesterase (AChE) consists of globular catalytic subunits attached to the basal lamina by a collagen-like tail. Different genes encode the catalytic subunit and the tail portion of the enzyme. Endplate AChE deficiency was reported previously in a single case (Engel et al., 1977, patient 1). We describe here our observations in four additional patients (patients 2-5). Three cases were sporadic; patients 2 and 3 were sisters. All had generalized weakness increased by exertion but ophthalmoparesis was not a constant feature. All had mild slowing of the pupillary light reflex; other dysautonomic features were absent. None benefited from anticholinesterase therapy. All patients had a decremental electromyogram response; in four of the five patients, single nerve stimuli evoked a repetitive response. Miniature endplate potential amplitude was reduced in patient 5 only. Endplate amplitudes and currents were prolonged but the open-time of the acetylcholine receptor ion channel was normal. In patients 1-4 the quantal content of the endplate potential was reduced due to a reduced number of readily releasable quanta. Quantitative electron microscopy revealed abnormally small nerve terminals, abnormal encasement of the presynaptic membrane by Schwann cells and degeneration of junctional folds and of organelles in the junctional sarcoplasm. Acetylcholinesterase was absent from all endplates of all patients by cytochemical and immunocytochemical criteria. Density gradient ultracentrifugation of muscle extracts from patients 1, 3, 4 and 5 revealed an absence of the collagen-tailed form of the enzyme in patients 1, 3 and 4 but not in patient 5. The kinetic properties of the residual AChE in muscle were normal. Erythrocyte AChE activity and Km values, determined in three patients, were also normal. Studies of the catalytic subunit gene of AChE in patients 2 and 3 revealed no abnormality in those exons that encode the domain to which the tail subunit binds. In patients 1-4 the molecular defect is likely to reside in the gene encoding the tail subunit of AChE, or in a protein necessary to assemble the catalytic and tail subunits. In patient 5, the absence of AChE from the endplate may be due to a faulty tail subunit, a defect in the basal lamina site that binds the tail subunit or failure of transport of the assembled asymmetric enzyme from the cell interior to the basal lamina. The cause of the weakness in these patients is not fully understood but possible mechanisms are discussed.

International audience; Acetylcholinesterase (AChE) terminates the action of acetylcholine at cholinergic synapses thereby preventing rebinding of acetylcholine to nicotinic postsynaptic receptors at the neuromuscular junction. Here we show that AChE is not localized close to these receptors on the postsynaptic surface, but is instead clustered along the presynaptic membrane and deep in the postsynaptic folds. Because AChE is anchored by ColQ in the basal lamina and is linked to the plasma membrane by a transmembrane subunit (PRiMA), we used a genetic approach to evaluate the respective contribution of each anchoring oligomer. By visualization and quantification of AChE in mouse strains devoid of ColQ, PRiMA or AChE, specifically in the muscle, we found that along the nerve terminus the vast majority of AChE is anchored by ColQ that is only produced by the muscle, whereas very minor amounts of AChE are anchored by PRiMA that is produced by motoneurons. In its synaptic location, AChE is therefore positioned to scavenge ACh that effluxes from the nerve by non-quantal release. AChE-PRiMA, produced by the muscle, is diffusely distributed along the muscle in extrajunctional regions.

Acetylcholinesterase (AChE) accumulates on axonal varicosities and is primarily found as tetramers associated with a proline-rich membrane anchor (PRiMA). PRiMA is a small transmembrane protein that efficiently transforms secreted AChE to an enzyme anchored on the outer cell surface. Surprisingly, in the striatum of the PRiMA knock-out mouse, despite a normal level of AChE mRNA, we find only 2–3% of wild type AChE activity, with the residual AChE localized in the endoplasmic reticulum, demonstrating that PRiMAin vivois necessary for intracellular processing of AChE in neurons. Moreover, deletion of the retention signal of the AChE catalytic subunit in mice, which is the domain of interaction with PRiMA, does not restore AChE activity in the striatum, establishing that PRiMA is necessary to target and/or to stabilize nascent AChE in neurons. These unexpected findings open new avenues to modulating AChE activity and its distribution in CNS disorders.

International audience; A mouse strain with a deleted acetylcholinesterase (AChE) gene (AChE knockout) shows a decreased inspiration time and increased tidal volume and ventilation .To investigate the respective roles of AChE in brain and muscle, we recorded respiration by means of whole-body plethysmography in knockout mice with tissue selective deletions in AChE expression. A mouse strain with the anchoring domains of AChE deleted (del E5+6 knockout mice) has very low activity in the brain and neuromuscular junction, but increased monomeric AChE in serum. A mouse strain with deletion of the muscle specific region of AChE (del i1RR knockout mice) exhibits no expression in muscle, but unaltered expression in the central nervous system. Neither strain exhibits the pronounced phenotypic traits observed in the complete AChE knockout strain. A third strain lacking the anchor molecule PRiMA, has no functional AChE and butyrylcholinesterase (BChE) in brain and an unaltered respiratory function. BChE inhibition by bambuterol decreases tidal volume and body temperature in del E5+6 and i1RR knockout strains, but not in PRiMA deletion or wild-type controls. We find that: (1) deletion of the full AChE gene is required for a pronounced alteration in respiratory phenotype, (2) BChE is involved in respiratory muscles contraction and temperature control in del E5+6 and i1RR knockout mice, and (3) AChE expression requiring a gene product splice to either exons 5 and 6 or regulated by intron1 influences temperature control.

At the vertebrate skeletal neuromuscular junction (NMJ), two closely related enzymes can hydrolyze acetylcholine (ACh): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Advances in mouse genomics offer new approaches to assess the role of specific cholinesterases involved in neuromuscular transmission (Minic et al., 2003). AChE knockout mice provide a valuable tool for examining the effects of long-term complete and selective abolition of AChE activity (Xie et al., 2000). AChE and BChE genes encode two functional domains--the catalytic domain (exons 2, 3, and 4 of AChE, or exon 2 of BChE) and a C-terminal domain (exon 5 or 6 of AChE, or exon 3 of BChE)--that dictate the targeting of the enzymes (Massoulie, 2002). In mammals, the AChE gene produces three types of coding regions by deleting 5'- splice acceptor sites, which generate proteins; these proteins possess the same catalytic domain associated with distinct C-terminal peptides. AChE subunits of type R (readthrough) produce soluble monomers; they are expressed during development and are thought to be induced in the mouse brain by stress (Kaufer et al., 1998). AChE subunits of type H (hydrophobic) produce GPI-anchored dimers, mainly in blood cells. Subunits of type T (tailed) exist for both AChE and BChE. They represent the predominant AChE variant expressed in cholinergically innervated tissues (muscle and nerve). These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins ColQ (Krejci et al., 1997) and PRiMA (Perrier et al., 2002). At the NMJ, AChE is clustered by the interaction of the coding sequence of exon 6 with ColQ (Feng et al., 1999). The deletion of exons 5 and 6 in the AChE gene transforms anchored AChE into a soluble enzyme (Camp et al., 2004). The present study was designed to evaluate neuromuscular transmission and nicotinic ACh receptor (nAChR) distribution in muscles from mutant mice with deletions of these two spliced exons (AChE-del-exons-5+6-/-).

Fasciculin (FAS), a 61-amino acid peptide from snake toxin with three large loops formed by disulfide bonds, is an extremely potent (2 pM) inhibitor of acetylcholinesterase (AChE). Previously we have described the synthesis of recombinant DNA encoding various mutants of the snake toxin fasciculin attached to the leader peptide from erabutoxin, allowing secretion of the translated protein into the media of cultured cells (1). These mutants have now been stably expressed in Chinese Hamster Ovary cells (CHO-K1). The cells can be grown in the absence of serum, and we have acheived production levels of up to 500 nmole (3.5 mg) per liter of culture.

Muscle development involves a complex series of events. Mesodermal progenitor cells commit to become myoblasts, myoblasts fuse and, accompanied by cell cycle arrest, differentiate to myotubes. As a cell culture paradigm to a portion of the differentiation process we have chosen to study murine C2C12 cells. C2C12 myoblasts remain undifferentiated and proliferative when grown in the presence of serum due to cell cycle regulated gene products (1). Myoblasts produce virtually no acetylcholinesterase (AChE). Withdrawal of serum stops cell division and induces fusion of myoblasts into multinucleate myotubes which produce both cell associated and exported AChE.

International audience; The availability of a crystal structure and comparative sequences of the cholinesterases has provided templates suitable for analyzing the molecular bases of specificity of reversible inhibitors, carbamoylating agents and organophosphates. Site-specific mutagenesis enables one to modify the structures of both the binding site and peptide ligand as well as create chimeras reflecting one type of esterase substituted in the template of another. Herein we define the bases for substrate specificity of carboxylesters, the stereospecificity of enantiomeric alkylphosphonates and the selectivity of tricyclic aromatic compounds in the active center of cholinesterase. We also describe the binding loci of the peripheral site and changes in catalytic parameters induced by peripheral site ligands, using the peptide fasciculin.

The interaction of fasciculin 2 was examined with wild-type and several mutant forms of acetylcholinesterase (AChE) where Trp86, which lies at the base of the active center gorge, is replaced by Tyr, Phe, and Ala. The fasciculin family of peptides from snake venom bind to a peripheral site near the rim of the gorge, but at a position which still allows substrates and other inhibitors to enter the gorge. The interaction of a series of charged and uncharged carboxyl esters, alkyl phosphoryl esters, and substituted trifluoroacetophenones were analyzed with the wild-type and mutant AChEs in the presence and absence of fasciculin. We show that Trp86 is important for the alignment of carboxyl ester substrates in the AChE active center. The most marked influence of Trp86 substitution in inhibiting catalysis is seen for carboxyl esters that show rapid turnover. The extent of inhibition achieved with bound fasciculin is also greatest for efficiently catalyzed, charged substrates. When Ala is substituted for Trp86, fasciculin becomes an allosteric activator instead of an inhibitor for certain substrates. Analysis of the kinetics of acylation by organophosphates and conjugation by trifluoroacetophenones, along with deconstruction of the kinetic constants for carboxyl esters, suggests that AChE inhibition by fasciculin arises from reductions of both the commitment to catalysis and diffusional entry of substrate into the gorge. The former is reflected in the ratio of the rate constant for substrate acylation to that for dissociation of the initial complex. The action of fasciculin appears to be mediated allosterically from its binding site at the rim of the gorge to affect the orientation of the side chain of Trp86 which lies at the gorge base.

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.

Upon first inspection, acetylcholinesterase (AChE) and the nicotinic acetylcholine receptor (nAchR) share several functional and structural characteristics. Both recognize the neurotransmitter, acetylcholine, and elicit the respective actions of catalysis of the neurotransmitter and neurotransmitter-induced channel opening in a millisecond time frame. Their relative stoichiometries at various synapses are carefully regulated within precise limits. Homologous toxins, the fasciculins and a-neurotoxins (i. e., α-cobratoxin and α-bungaro-toxin), which have nearly identical tertiary structures, inhibit the two respective proteins at concentrations in the picomolar to subpicomolar range. Finally, the two proteins show coordinated expression during development.

Fasciculin (FAS), a 61 amino acid peptide found in venom of green mambas, is a potent inhibitor of acetylcholinesterases from most species (EC 3.1.1.7)(AChEs) (Karlsson et al., 1984). The crystal structure of FAS (Le Du et al., 1992) reveals close structural features with a larger family of “three finger” snake toxins such as erabutoxin, cardiotoxins, bungarotoxin and cobratoxin. These toxins, however, do not inhibit AChE while FAS at low concentrations does not bind to acetylcholine receptors (Endo and Tamiya, 1987; Karlsson et al., 1984).

Fasciculins are the only known peptide inhibitors of acetylcholinesterase (AChE) with a high degree of selectivity. They are found in mamba snake venoms and have been shown to display powerful inhibitory activity toward mammalian and fish AChE. To date, four iso-fasciculins have been characterized: fasciculin 1 (FASI) and fasciculin 2 (FAS2) from the venom of Dendroaspis angusticeps (Rodriguez-Ithurralde et al., 1983), toxin C from the venom of D. polylepis (Joubert and Taljaard, 1978), and fasciculin 3 (FAS3) from a venom of D. viridis (Marchot et al., 1993). The early pharmacological and biochemical studies of FAS2, carried out both in vivo and in vitro on various AChE-containing tissues (Karlsson et al., 1984; Lin et al., 1987), showed that i) FAS2 inhibits several (but not all) AChEs from different sources, ii) inhibition is of pseudo-irreversible type with Ki values of about 10−11 M., iii) FAS2 is able to displace propidium, known as a specific probe for a peripheral anionic site of AChE (See Harvey et al., 1984 for a review).

A congenital myasthenic condition was described several years ago that is characterized by a deficiency in end-plate acetylcholinesterase (AChE); the absence of the inflammatory immune responses of myasthenia categorizes the condition as congenital (Engle, et al., 1977). Since that time several additional patients with a similar disorder have been identified. Patient blood samples were drawn for DNA extraction and isolation of lymphocytes which have been immortalized by Epstein-Barr virus transformation. To ascertain the genetic basis of the disease, we have examined the structure of the gene encoding the catalytic subunits of AChE by comparing patients and control individuals. Southern analysis revealed no differences between patient and control DNA, suggesting no major chromosomal rearrangements had occurred. PCR amplification of genomic DNA yielded clones covering exon 4 and the alternatively spliced exons 5 and 6; this region was analyzed by nuclease protection and sequencing. While allelic differences were detected, we found no differences in exonic or intronic areas which might give rise to distinctive splicing pattern differences in patients and control individuals. The ACHE gene was cloned from genomic libraries from one of the patients and a control. Identical patterns of mRNA production and species of expressed AChE were revealed when the cloned genes were transfected into COS and HEK cells. Cotransfection of the genes expressing the catalytic subunits from either patient or control with a gene expressing the tail unit from Torpedo AChE (Krejci et. al., 1991) yielded asymmetric species (18 + 14 S) which require assembly of catalytic subunits and tail unit. Thus, the catalytic subunits of AChE expressed in the congenital myasthenia syndrome appear identical in sequence, arise from similar splicing patterns and will assemble with a tail unit to form a heterologous species. These findings point to the genetic abnormality likely existing in the tail unit or in a protein responsible for assembly of the catalytic subunit with the tail subunit.

Acetyl- and butyrylcholinesterase have 51-54% sequence identity in mammalian species; they exhibit distinct substrate and inhibitor specificities. The crystal structure of acetylcholinesterase enables one to predict folding of related esterases as well as assign residues responsible for differences in substrate specificity. These predictions were tested by expression of esterase chimeras and site-specific mutants using mouse acetylcholinesterase as a template. Chimeras of acetylcholinesterase in which the amino-terminal 174 and the carboxyl-terminal 88 amino acids have been converted to the butyrylcholinesterase sequences still exhibit acetyl-like substrate specificity. Four nonconserved amino acids which are within the central sequence and appear to surround the acyl pocket, F295, R296, F297, and V300, have been mutated alone and in combination to the corresponding residues found in butyrylcholinesterase, L286, S287, I288, and G291. The V300 and R296 mutants slightly enhance butyrylthiocholine hydrolysis while the F295 and F297 mutants, alone and in combination, confer butyrylcholinesterase character by enhancing activity to butyrylthiocholine, and diminishing activity to acetylthiocholine. The F297 mutation eliminates substrate inhibition. F295 and F297 may form a clamp around the acetoxy methyl group. They have distinctive roles in affecting catalysis of the two acylcholines and precisely control acyl ester specificity. Comparison of the susceptibilities of the chimeras and site-specific mutants to cholinesterase-specific inhibitors isoOMPA, ethopropazine, and BW284c51 suggests that inhibitor selectivity for isoOMPA is attributable to residues limiting the size of the acyl pocket, while residues in the amino-terminal domain presumably near the lip of the gorge affect BW284c51 selectivity.(ABSTRACT TRUNCATED AT 250 WORDS)

The predominant degeneration of cholinergic neurons extending from the nucleus basalis of Meynert suggests that retarding the loss of cholinergic function may prove palliative in the treatment of Alzheimer’s disease. Use of selective acetylcholinesterase (AChE) inhibitors which are retained within the central nervous system is one widely considered approach. An alternative approach would be regulation of expression of proteins critical to cholinergic nervous system function. However, this approach, to be most effective, would require adjustment of subtle differences in gene expression. For example, enhancement of the transport and enzymatic processes involved in acetylcholine biosynthesis concomitant with a decrease in AChE expression might be therapeutically beneficial in facilitating the availability of neurotransmitter. However, AChE may play a role in the development of the nervous system (Layer and Sporns, 1987) and in the maintenance of synaptic and heterologous cell contacts (DeLa Escalera et al., 1990). Thus, a rational approach to therapeutic intervention in Alzheimer’s disease requires a detailed knowledge of the potential target. Recombinant DNA technology should prove useful in this regard. Already expression of cloned genes provides sources of human AChE (Soreq et al., 1990; Li et al., 1991) and site-directed mutants (Gibney et al., 1990; Soreq et al., 1991) add a new dimension to structure-activity relationships. Moreover, recombinant techniques should define selective trophic factors and trans-activating factors which influence expression of gene products essential to neurotransmission. In this chapter we describe a target of therapeutic attack, AChE, and how expression of its gene is regulated.

Acetylcholinesterase, an essential enzyme of the nervous system, rapidly terminates the action of acetylcholine released into the synapse. Acetylcholinesterase is also found (in lower abundance) in extrajunctional areas of muscle and nerve and on erythrocyte membranes. Hydrodynamic analyses of the native enzyme and characterization of its dissociated subunits have revealed multiple enzyme forms which can be divided into two classes: dimensionally asymmetric forms which are usually found within the synapse and contain a collagen-like structural subunit disulphide-linked to the catalytic subunits; and globular forms which appear to be widely distributed on the outer surface of cell membranes. Both forms have been characterized in the ray Torpedo californica and, although their catalytic behaviours seem to be identical, they differ slightly in amino-acid composition, peptide maps and reactivity with certain monoclonal antibodies. Here, we report the complete amino-acid sequence of an acetylcholinesterase inferred from the sequence of a complementary DNA clone. The 575-residue protein shows significant homology with the C-terminal portion of thyroglobulin.

A mixture of the 5.6S hydrophobic dimer and the asymmetric, tail-containing (17 + 13)S forms of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from Torpedo californica was used to immunize mice, and spleen cells from these mice were used to produce nine hybridoma lines secreting antibodies against acetylcholinesterase. Antibodies from one of the lines showed a 100-fold greater affinity for the 5.6S species when compared with the catalytic subunits of the (17 + 13)S species. This difference in specificity was retained after denaturation of the two acetylcholinesterase species. Another line produced antibody directed only to structural subunits of the (17 + 13)S species, whereas the remaining seven antibodies exhibited nearly equivalent crossreactivity for all of the forms of acetylcholinesterase. Tryptic peptides were generated from the catalytic subunits of the 5.6S and tail-containing acetylcholinesterase species, and high-pressure liquid chromatographic profiles show at least two distinct peptides in the catalytic subunits for each enzyme species. Some of these peptides exhibit retention times different from those of the identified glycopeptides. Thus, it is likely that the catalytic subunits of two molecular forms of acetylcholinesterase differ in primary structure and sites of antigenicity.

Treatment of C2-C12 mouse myoblasts with the immunosuppressant drug cyclosporin A (CsA) enhances the increase in acetylcholinesterase (AChE) expression observed during skeletal muscle differentiation. The enhanced AChE expression is due primarily to increased mRNA stability because CsA treatment increases the half-life of AChE mRNA, but not the apparent transcriptional rate of the gene. Neither tacrolimus (FK506), an immunosuppressive agent with a distinct structure, nor cyclosporine H, an inactive congener of CsA, alters AChE expression. The enhanced AChE expression is associated with the muscle differentiation process, but cannot be triggered by CsA exposure before differentiation. Myoblasts and myotubes of C2-C12 cells express similar amounts of cyclophilin A and FKBP12, immunophilins known to be intracellular-binding targets for CsA and tacrolimus, respectively. However, cellular levels of calcineurin, a calcium/calmodulin-dependent phosphatase known to be the cellular target of ligand-immunophilin complexes, increase 3-fold during myogenesis. Overexpression of constitutively active calcineurin in differentiating cells reduces AChE mRNA levels and CsA antagonizes such an inhibition. Conversely, overexpression of a dominant negative calcineurin construct increases AChE mRNA levels, which are further enhanced by CsA. Thus, a CsA sensitive, calcineurin mediated pathway appears linked to differentiation-induced stabilization of AChE mRNA during myogenesis.

The cholinesterases are serine hydrolases that show no global similarities in sequence with either the trypsin or the subtilisin family of serine proteases. The cholinesterase superfamily includes several esterases with distinct functions and other proteins devoid of the catalytic serine and known esterase activity. To identify the residues involved in catalysis and conferring specificity on the enzyme, we have expressed wild-type Torpedo acetylcholinesterase (EC 3.1.1.7) and several site-directed mutants in a heterologous system. Mutation of serine-200 to cysteine results in diminished activity, while its mutation to valine abolishes detectable activity. Two conserved histidines can be identified at positions 425 and 440 in the cholinesterase family; glutamine replacement at position 440 eliminates activity whereas the mutation at 425 reduces activity only slightly. The assignment of the catalytic histidine to position 440 defines a rank ordering of catalytic residues in cholinesterases distinct from trypsin and subtilisin and suggests a convergence of a catalytic triad to form a third, distinct family of serine hydrolases. Mutation of glutamate-199 to glutamine yields an enzyme with a higher Km and without the substrate-inhibition behavior characteristic of acetylcholinesterase. Hence, modification of the acidic amino acid adjacent to the serine influences substrate association and the capacity of a second substrate molecule to affect catalysis.