Monoclonal Antibodies Specific for the Different Subunits of Asymmetric Acetylcholinesterase from Chick Muscle

The asymmetric (20S) acetylcholinesterase (AChE, EC 3.1.1.7) from 1-day-old chick muscle, purified on a column on which was immobilised a monoclonal antibody (mAb) to chick brain AChE, was used to immunise mice. Eight mAbs against the muscle enzyme were hence isolated and characterised. Five antibodies (4A8, 1C1, 10B7, 7G8, and 8H11) recognise a 110-kilodalton (kDa) subunit with AChE catalytic activity, one antibody (7D11) recognises a 72-kDa subunit with pseudocholinesterase or butyrylcholinesterase (BuChE, EC 3.1.1.8) catalytic activity, and two antibodies (6B6 and 7D7) react with the 58-kDa collagenous tail unit. Those three polypeptides can be recognised together in the 20S enzyme used, which is a hybrid AChE/BuChE oligomer. Antibodies 6B6 and 7D7 are specific for asymmetric AChE. Four of the mAbs recognising the 110-kDa subunit were reactive with it in immunoblots. Sucrose density gradient analysis of the antibody-enzyme complexes showed that the anti-110-kDa subunit mAbs cross-link multiple 20S AChE molecules to form large aggregates. In contrast, there is only a 2-3S increase in the sedimentation constant with the mAbs specific for the 72-kDa or for the 58-kDa subunit, suggesting that those subunits are more inaccessible in the structure to intermolecular cross-linking. The 4A8, 10B7, 7D11, and 7D7 mAbs showed cross-reactivity to the corresponding enzyme from quail muscle; however, none of the eight mAbs reacted with either enzyme type from mammalian muscle or from Torpedo electric organ. All eight antibodies showed immunocytochemical localisation of the AChE form at the neuromuscular junctions of chicken twitch muscles.     

A Globular, Not Asymmetric, Form of Acetylcholinesterase Is Expressed in Chick Motor Neurons: Down-Regulation Toward Maturity and After Denervation

Abstract: In vertebrate neuromuscular junctions, the postsynaptic specializations include the accumulation of acetylcholinesterase (AChE) at the synaptic basal lamina and the muscle fiber. Several lines of evidence indicate that the presynaptic motor neuron is able to synthesize and secrete AChE at the neuromuscular junctions. By using anti-AChE catalytic subunit, anti-butyrylcholinesterase (BuChE) catalytic subunit, and anti-AChE collagenous tail monoclonal antibodies, we demonstrated that the motor neurons of chick spinal cord expressed AChE in vivo and the predominant AChE was the globular form of the enzyme. Neither asymmetric AChE nor BuChE was detected in the motor neurons. The molecular mass of AChE catalytic subunit in the motor neuron was ∼105 kDa, which was similar to that of the globular enzyme from low-salt extracts of muscle; both of them were ∼5 kDa smaller than the asymmetric AChE from high-salt extracts of muscle. The level of AChE expression in the motor neurons decreased, as found by immunochemical and enzymatic analysis, during the different stages of the chick's development and after nerve lesion. Thus, the AChE activity at the neuromuscular junctions that is contributed by the presynaptic motor neurons is primarily the globular, not the asymmetric, form of the enzyme, and these contributions decreased toward maturity and after denervation.     

Generation of subunit-specific antibody probes for Torpedo acetylcholinesterase: cross-species reactivity and use in cell-free translations

The assembly of the collagen tailed A12 form of acetylcholinesterase (AChE) is regulated by muscle contraction. To begin to study this regulation, we derived antibody probes for the three subunits (100 kd, catalytic, and collagen tail) of AChE purified from Torpedo californica electric tissue. These included a polyclonal antiserum that recognizes all 3 subunits and 19 monoclonal antibodies; 16 of the monoclonals recognized the catalytic subunit, 2 recognized the tail subunit, and 1 recognized the 100 kd subunit on Western blots. We used immunohistochemical procedures to show that several of the anticatalytic and one of the antitail monoclonals cross-reacted with frog muscle AChE and Western blotting to show that several of the anticatalytic monoclonals cross-react with rat brain AChE. These antibodies were then used to immunoprecipitate AChE precursors from a cell-free translation system. There were generally three primary translation products, corresponding to the three enzyme subunits. Therefore, each subunit is probably derived from a separate mRNA. Occasionally there were two translation products corresponding to the catalytic subunit alone. The catalytic subunit was glycosylated following addition of canine microsomal membranes to the translation mix. The mRNA coding for this subunit appeared to be present in the poly(A)- RNA pool.     

Polymorphism of acetylcholinesterase and identification of new molecular forms after sedimentation analysis

Acetylcholinesterase (AChE) is composed of several distinct molecular forms, which are identified and partly resolved by velocity sedimentation analysis on sucrose gradients. We made the assumption that each AChE form sediments as a peak of activity with a gaussian shape in the continuous sucrose gradient. We experimentally demonstrate that the complex AChE profiles can be decomposed in gaussian distributions of separate molecular entities. We performed a high salt-detergent extraction of AChE from mouse skeletal muscle and isolated fractions enriched in each particular from. These fractions were then submitted to a second sedimentation, to assess the stability and to further characterize each AChE form. Then, we calculated the statistical significance level of each AChE form and identified up to 9 separate molecular specifies in mouse adult muscle. These forms are the major "4 S", "6.5 S", "10 S", "12 S" and "16 S" and minor molecular active components of AChE. These results suggest complex structural interactions between catalytic and non catalytic subunits of AChE and do not simply fit the tailed asymmetric globular model of AChE with six molecular species.     

The electric organ ofDiscopyge tschudii: Its innervated face and the biology of acetylcholinesterase

An ultrastructural, histochemical, and biochemical study of the electric organ of the South American Torpedinid ray, Discopyge tschudii, was carried out. Fine structural cytochemical localization of acetylcholinesterase (AChE) indicated that most of the esterase was associated with the basal lamina. Electron microscopy indicated no marked differences in the electrocyte ultrastructure between Discopyge and Torpedo californica. Discopyge electric organ possessed three molecular forms, two asymmetric forms (16 S and 13 S) and one globular hydrophobic form (6.5 S). The asymmetric 16 S AChE form was solubilized by heparin, a sulfated glycosaminoglycan, suggesting that heparin-like macromolecules are involved in the binding of the enzyme to the basal lamina. Our results show that cell-free translated AChE peptides, synthesized using Discopyge electric organ poly(A+) RNA, correspond to a main band of 62,000 daltons which probably represents the catalytic subunit of the asymmetric AChE.     

Assembly and regulation of acetylcholinesterase at the vertebrate neuromuscular junction

The collagen-tailed form of acetylcholinesterase (ColQ-AChE) is the major if not unique form of the enzyme associated with the neuromuscular junction (NMJ). This enzyme form consists of catalytic and non-catalytic subunits encoded by separate genes, assembled as three enzymatic tetramers attached to the three-stranded collagen-like tail (ColQ). This synaptic form of the enzyme is tightly attached to the basal lamina associated with the glycosaminoglycan perlecan. Fasciculin-2 is a snake toxin that binds tightly to AChE. Localization of junctional AChE on frozen sections of muscle with fluorescent Fasciculin-2 shows that the labeled toxin dissociates with a half-life of about 36h. The fluorescent toxin can subsequently be taken up by the muscle fibers by endocytosis giving the appearance of enzyme recycling. Newly synthesized AChE molecules undergo a lengthy series of processing events before final transport to the cell surface and association with the synaptic basal lamina. Following co-translational glycosylation the catalytic subunit polypeptide chain interacts with several molecular chaperones, glycosidases and glycosyltransferases to produce a catalytically active enzyme that can subsequently bind to one of two non-catalytic subunits. These molecular chaperones can be rate limiting steps in the assembly process. Treatment of muscle cells with a synthetic peptide containing the PRAD attachment sequence and a KDEL retention signal results in a large increase in assembled and exportable AChE, providing an additional level of post-translational control. Finally, we have found that Pumilio2, a member of the PUF family of RNA-binding proteins, is highly concentrated at the vertebrate neuromuscular junction where it plays an important role in regulating AChE translation through binding to a highly conserved NANOS response element in the 3'-UTR. Together, these studies define several new levels of AChE regulation in electrically excitable cells.     

Tetrameric (G4) Acetylcholinesterase: Structure, Localization, and Physiological Regulation

Abstract: Acetylcholinesterase (AChE), a highly conserved enzyme in the animal kingdom, is distributed throughout a wide range of vertebrate tissues where it is expressed as multiple molecular forms comprising different arrangements of catalytic and structural subunits. The major AChE form in the CNS is an amphiphilic globular tetramer (G₄ AChE) consisting of four identical catalytic subunits attached to cellular membranes by a hydrophobic noncatalytic subunit (P-subunit). This study focuses primarily on current data involving the structure of the G₄ AChE P-subunit, the expression and regulation of G₄ AChE during development and adulthood, and its role(s) in certain neurological disorders including Alzheimer's disease.     

Regulation of acetylcholinesterase synthesis and assembly by muscle activity. Effects of tetrodotoxin

The abundance and distribution of acetylcholinesterase (AChE) oligomeric forms expressed in skeletal muscle is strongly dependent upon the activity state of the cells. In this study, we examined several stages of AChE biogenesis to determine which ones were regulated by muscle activity. Inhibiting spontaneous contraction of tissue-cultured quail myotubes with tetrodotoxin (TTX) reduces AChE activity by approximately 30% of the levels found in actively contracting cells. This decrease is due primarily to the loss of 20 S asymmetric (collagen-tailed) AChE from TTX-treated cultures and is reflected in reduced pool sizes for both cell surface and intracellular AChE molecules. Using monoclonal anti-AChE antibodies to immunoprecipitate and quantify isotopically labeled enzyme molecules, we show that AChE down-regulation by TTX is not mediated through changes in the rates of synthesis or degradation of AChE polypeptide chains. Newly synthesized AChE polypeptides acquire enzymatic activity at the same rate in TTX-treated cultures as in actively contracting cells, however, a larger percentage of catalytically active dimers and tetramers are secreted from TTX-treated cultures compared with controls. These results suggest that TTX-induced down-regulation of asymmetric AChE occurs at the level of assembly of globular AChE molecules with collagen-like tail subunits in the Golgi apparatus, rather than through changes in the availability of catalytic subunits. Thus, post-translational mechanisms appear to play an important role in regulating the abundance and distribution of this important synaptic component in skeletal muscle.     

Gender-related differences in circadian rhythm of rat plasma acetyl- and butyrylcholinesterase: Effects of sex hormone withdrawal

The role of acetylcholinesterase (AChE) in the termination of the cholinergic response through acetylcholine (ACh) hydrolysis and the involvement of plasma butyrylcholinesterase (BuChE), mainly of hepatic origin, in the metabolism of xenobiotics with ester bonds is well known. Besides, BuChE has a crucial role in ACh hydrolysis, especially when selective anticholinesterases inhibit AChE. Herein, we analyzed the gender-related differences and the circadian changes of rat plasma cholinesterases. Plasma and liver cholinesterase activities were evaluated in control or 2–30-day castrated adult male and female rats. Plasma and liver AChE activities did not differ between genders and were not influenced by sex hormone deprivation. BuChE plasma activity was 7 times greater in female, reflecting gender differences in liver enzyme expression. Castration increased liver and plasma BuChE activity in male, while reduced it in female, abolishing gender differences in enzyme activity. Interestingly, female AChE and BuChE plasma activities varied throughout the day, reaching values 27% and 42% lower, respectively, between 2 p.m. and 6 p.m. when compared to the morning peaks at 8 a.m. Castration attenuated daily female BuChE oscillation. On the other hand, male plasma enzymes remained constant throughout the day. In summary, our results show that liver and plasma BuChE, but not AChE, expression is influenced by sex hormones, leading to high levels of blood BuChE in females. The fluctuation of female plasma BuChE during the day should be taken into account to adjust the bioavailability and the therapeutic effects of cholinesterase inhibitors used in cholinergic-based conditions such Alzheimer's disease.     

Monoclonal antibodies against chicken brain acetylcholinesterase. Their use in immunopurification and immunochemistry to demonstrate allelic variants of the enzyme

Acetylcholinesterase (AChE) from 1-day chicken brain was enriched over 2000-fold by affinity chromatography using N-methylacridinium-Sepharose. This preparation was used to prepare monoclonal antibodies (mAb) directed against AChE, of which two were extensively characterised for further application. Both mAbs bound to the enzyme from the chicken with high affinity (Kd approximately 8 X 10(-10) M) and one mAb, in addition, recognised AChE from quail brain and muscle. Neither mAb cross-reacted with mammalian or fish AChE. Both mAbs recognised AChE in the endplate region of adult chicken skeletal muscle and bound with equal affinity to the three major oligomeric forms found in early ambryonic muscle. One mAb was used to immunopurify chicken brain AChE to homogeneity (over 12000-fold enrichment), with nearly complete recovery of the enzyme and without detectable proteolytic breakdown. The other mAb recognised AChE after immunoblotting and was used to screen crude brain extracts from individual chickens for allelic variations. Evidence is presented to show that two allelic forms occur, represented in SDS-PAGE by a doublet polypeptide of Mr approximately 110,000, this pattern is maintained after deglycosylation of the N-linked oligosaccharides. This variation was found throughout development and in both the brain and the muscle of individuals. We conclude that the gene encoding the catalytic subunit of chicken AChE is polymorphic with either one or two equally active alleles being expressed.     

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.     

Two types of asymmetric acetylcholinesterase in chick hindlimb muscle: Developmental profiles,in Vivo and in cell culture,and recovery after inactivation

1. We have analyzed the behavior of two types of asymmetric molecular forms (A forms) of acetylcholinesterase (AChE) during development of chick hindlimb muscle, in vivo and in cell culture, and upon irreversible inactivation of peroneal muscle AChE with diisopropylfluorophosphate (DFP) in vivo. 2. In agreement with previous developmental studies on chick muscle, globular forms of AChE (G forms) are predominant in chick hindlimb at early embryonic ages, being gradually replaced by A forms as hatching (and, therefore, onset of locomotion) approaches. Of the two A-form types, AI appears and accumulates significantly earlier than AII, so that A/G and II/I ratios higher than 1 are attained only at about hatching time. 3. Cultures prepared from 11-day chick embryo hindlimb myoblasts express both types of A forms, with a combined activity of 27% of total AChE after 12 days in culture. AI forms appear again earlier and are much more abundant than type II asymmetric species through the life span of cultures. 4. All AChE activity in the peroneal muscle is irreversibly inactivated by injection of DFP in vivo. The recovery of A forms follows the same sequence described for normal development, with a delayed and slower recovery of AII forms as compared with AI. 5. Several hypotheses involving tail polypeptides or tissue target molecules, or posttranslational interconversion, are proposed to help explain the earlier appearance and accumulation of AI forms in chick muscle.     

Subunit Association and Glycosylation of Acetylcholinesterase from Monkey Brain

Abstract: Cercopithecus monkey brain acetylcholinesterase (AChE; EC 3.1.1.7) consists of about 15% hydrophilic, salt-soluble enzyme and 83% amphiphilic, detergent-soluble enzyme. Sucrose density gradient centrifugation showed that hydrophilic, salt-soluble AChE was composed of about 85% tetramer (10.3S) and 15% monomer (3.3S). In amphiphilic, detergent-soluble AChE, 85% tetramer (9.7S), 10% dimer (5.7S), and 5% monomer (3.2S) were seen. The enzyme is N -glycosylated, and no O-linked carbohydrate could be detected. Use of two monoclonal antibodies, one directed against the catalytic subunit and the other against the hydrophobic anchor, gave new insights into the subunit assembly of brain AChE. It is shown that in tetrameric AChE, not all of the subunits are disulfide-bonded and that two populations of tetramers exist, one carrying one and the other carrying two hydrophobic anchors.     

Acetylcholinesterase from the invertebrate Ciona intestinalis is capable of assembling into asymmetric forms when co-expressed with vertebrate collagenic tail peptide

To learn more about the evolution of the cholinesterases (ChEs), acetylcholinesterase (AChE) and butyrylcholinesterase in the vertebrates, we investigated the AChE activity of a deuterostome invertebrate, the urochordate Ciona intestinalis, by expressing in vitro a synthetic recombinant cDNA for the enzyme in COS-7 cells. Evidence from kinetics, pharmacology, molecular biology, and molecular modeling confirms that the enzyme is AChE. Sequence analysis and molecular modeling also indicate that the cDNA codes for the AChE(T) subunit, which should be able to produce all three globular forms of AChE: monomers (G(1)), dimers (G(2)), and tetramers (G(4)), and assemble into asymmetric forms in association with the collagenic subunit collagen Q. Using velocity sedimentation on sucrose gradients, we found that all three of the globular forms are either expressed in cells or secreted into the medium. In cell extracts, amphiphilic monomers (G(1)(a)) and non-amphiphilic tetramers (G(4)(na)) are found. Amphiphilic dimers (G(2)(a)) and non-amphiphilic tetramers (G(4)(na)) are secreted into the medium. Co-expression of the catalytic subunit with Rattus norvegicus collagen Q produces the asymmetric A(12) form of the enzyme. Collagenase digestion of the A(12) AChE produces a lytic G(4) form. Notably, only globular forms are present in vivo. This is the first demonstration that an invertebrate AChE is capable of assembling into asymmetric forms. We also performed a phylogenetic analysis of the sequence. We discuss the relevance of our results with respect to the evolution of the ChEs in general, in deuterostome invertebrates, and in chordates including vertebrates.     

Butyrylcholinesterase-Mediated enhancement of the enzymatic activity of trypsin

1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline.2. Both AChE and BuChE have been shown to copurify with peptidases.3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with -amyloid protein in Alzheimer brain tissues.4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis.5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE.6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.     

Association of tailed acetylcholinesterase to lipidic membranes in mammalian skeletal muscle

Tailed acetylcholinesterase (AChE) was studied in three subcellular membrane fractions of mouse skeletal muscle: a fraction enriched in isolated motor endplates (C), an extrasynaptic membrane fraction (A) and a microsomal fraction (S). In the (C) fraction, tailed asymmetric 16S AChE required high salt conditions to be extracted, while in (A) and (S) microsomal membranes, a collagenase sensitive 16S form, was extracted by detergent alone. This apparent “hydrophobic” property suggests that there is a pool of 16S AChE which is probably bound to lipidic membranes. The detergent extractable (DE) 16S AChE was not concentrated in motor endplate-rich regions and differential inhibition of external and internal AChE demonstrated that it could have both intra- and extracellular locations in the adult differentiated muscle fibres.     

Reciprocal neural regulation of extrajunctional acetylcholinesterase and collagen Q in rat muscles--the role of calcineurin signaling

There are two main differences regarding acetylcholinesterase (AChE) expression in the extrajunctional regions of fast and slow rat muscles: (1) the activity of AChE catalytic subunits (G1 form) is much higher in fast than in slow muscles, and (2) the activity of the asymmetric forms of AChE (A(8) and A(12)) is quite high extrajunctionally in slow muscles but virtually absent in fast muscles. The latter is due to the absence of the expression of AChE-associated collagen Q (ColQ) in the extrajunctional regions of fast muscle fibers, in contrast to its ample expression in slow muscles. We showed that both differences are caused by different neural activation patterns of fast vs. slow muscle fibers, which determine the respective levels of mRNA of both proteins. Whereas the changes in AChE mRNA levels in fast and slow muscles, as well as the levels of ColQ mRNA levels in slow muscles, observed in response to exposing either slow or fast muscles to different muscle activation patterns, are completely reversible, the extrajunctional suppression of ColQ expression in fast muscle fibers seems to be irreversible. Calcineurin signaling pathway in muscles is activated by high-average sarcoplasmic calcium concentration resulting from tonic low-frequency muscle fiber activation pattern, typical for slow muscle fibers, but is inactive in fast muscle fibers, which are activated by infrequent high-frequency bursts of neural impulses. Application to rats of two inhibitors of calcineurin (tacrolimus-FK506 and cyclosporin A) demonstrated that the mRNA levels of both the AChE catalytic subunit and ColQ in the extrajunctional regions of the soleus muscle are regulated by the calcineurin signaling pathway, but in a reciprocal way. Under the conditions of low calcineurin activity, AChE expression is enhanced and that of ColQ is suppressed, and vice versa. Our results also indicated that different, calcineurin-independent regulatory pathways are responsible for the reduction of AChE expression during muscle denervation, and for maintaining high ColQ expression in the neuromuscular junctions of fast muscle fibers.     

Identification of a novel protein phosphatase catalytic subunit by cDNA cloning

A cDNA encoding a novel protein phosphatase catalytic subunit (protein phosphatase X) has been isolated from a rabbit liver library. It codes for a protein having 45% and 65% amino acid sequence identity, respectively, to the catalytic subunits of protein phosphatase 1 and protein phosphatase 2A from skeletal muscle. The enzyme is neither the hepatic form of protein phosphatase 1 or 2A, nor is it protein phosphatase 2B or 2C. The possible identity of protein phosphatase X is discussed.