Solubilization of collagen-tailed molecular forms of acetylcholinesterase from several brain areas in different vertebrate species

The relative efficiency of a buffered medium containing a high salt concentration and EDTA as a means to solubilize collagen-tailed molecular forms of acetylcholinesterase has been examined in four brain areas of several species belonging to different vertebrate classes. This extraction procedure has proved successful in most cases, with the yield of tailed enzyme varying between less than 1 and 26% of the total tissue activity. The solubilization values are consistently higher in more primitive vertebrates than in mammals and, for a given species, are usually lower in the telencephalon than in other brain structures. Our results confirm that the vertebrate central nervous system contains collagen-tailed quaternary structural forms of acetylcholinesterase.     

Molecular structure of elongated forms of electric eel acetylcholinesterase.

Molecular forms of acetylcholinesterase extracted from fresh electric organ tissue of the electric eel are elongated structures in which a multi-subunit head is connected to a fibrous tail. The principal form, 18 S acetylcholinesterase, is of molecular weight approximately 1,050,000, contains about 12 catalytic subunits in its head, has a tail approximately 500 Å long, and aggregates reversibly at low ionic strength. Trypsin converts it to an 11 S globular tetramer devoid of the tail and lacking the capacity to aggregate in low-salt solutions.Amino acid analysis shows that elongated forms of acetylcholinesterase contain significant amounts of hydroxyproline and hydroxylysine, characteristic components of collagen, which are absent from 11 S acetylcholinesterase.Collagenase converts 18 S acetylcholinesterase to a 20 S form which no longer aggregates in low salt. Purified 20 S acetylcholinesterase has about half the hydroxyproline and hydroxylysine contents of the 18 S enzyme, and physicochemical measurements indicate the formation of a more symmetrical molecular structure without marked reduction in molecular weight.Sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reducing agent shows that in 18 S acetylcholinesterase half the catalytic subunits are present as dimers linked by disulfide bonds. The remaining subunits migrate as larger molecular species which contain significant amounts of hydroxylysine, are specifically modified by collagenase and are converted to dimers and monomers by trypsin.Sodium dodecyl sulfate/acrylamide gel electrophoresis with reducing agent reveals, in 18 S acetylcholinesterase, two polypeptides of molecular weights 45,000 and 47,000 which are absent in the 11 S tetramer. They are readily digested by collagenase under conditions which do not affect the catalytic subunits, with concomitant formation of a new 30,000 polypeptide.The above data can be rationalized by a model in which 18 S acetylcholinestorase contains three subunit tetramers, each linked by disulfides to one strand of a collagen triple helix. Sodium dodecyl sulfate detaches those subunit dimers which are not covalently linked to the tail; trypsin attacks the distal portion of the collagen triple helix releasing discrete tetramers, and collagenase specifically attacks the triple helix near its midpoint, producing a shortened structure in which the residual tail still holds the tetramers together, but destroying the capacity for self-association at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring acetylcholinesterase to the fibrillar matrix of the basement membrane.     

Two classes of collagen-tailed,asymmetric molecular forms of acetylcholinesterase in skeletal muscle: differential effects of denervation

Skeletal muscles of different vertebrate species contain, as it is the case in other cholinergic tissues, two classes of collagen-tailed, asymmetric forms (A-forms) of acetylcholinesterase (AChE). Class I A-forms are readily brought into solution in the presence of high salt, while class II A-forms do additionally require a chelating agent, such as EDTA, for solubilization. All A-forms aggregate at low ionic strength but only class II A-forms are reaggregated by excess Ca⁺⁺, even in the presence of 1M NaCl. This Ca⁺⁺-mediated aggregability of class II A-forms is slowly lost upon exposure to detergents such as Triton X-100.Although these two classes of AChE tailed forms seem to be present in endplate and non-endplate areas, and in both the extra- and intracellular compartments, class II A-forms are predominantly extracellular and endplate-specific, at least in the rat diaphragm. On the other hand, well-characterized fast- and slow-twitch muscles show no preference for either class of asymmetric AChE species. Upon denervation, class I A-forms are degraded faster and disappear earlier than their class II counterparts, which are still easily detectable 17 days after nerve section.Class I and class II AChE molecular species exist in similar relative proportions in many vertebrate muscles. Thus, collagen-tailed forms may be altogether more abundant, in skeletal muscle, than it was hitherto realized.It is expected that this further example of AChE polymorphism will contribute to a better understanding of cholinergic transmission in skeletal muscle and, more specially, of nerve-muscle interactions.     

Dynamics of the acetylcholinesterase tetramer

Acetylcholinesterase rapidly hydrolyzes the neurotransmitter acetylcholine in cholinergic synapses, including the neuromuscular junction. The tetramer is the most important functional form of the enzyme. Two low-resolution crystal structures have been solved. One is compact with two of its four peripheral anionic sites (PAS) sterically blocked by complementary subunits. The other is a loose tetramer with all four subunits accessible to solvent. These structures lacked the C-terminal amphipathic t-peptide (WAT domain) that interacts with the proline-rich attachment domain (PRAD). A complete tetramer model (AChEt) was built based on the structure of the PRAD/WAT complex and the compact tetramer. Normal mode analysis suggested that AChEt could exist in several conformations with subunits fluctuating relative to one another. Here, a multiscale simulation involving all-atom molecular dynamics and Cα-based coarse-grained Brownian dynamics simulations was carried out to investigate the large-scale intersubunit dynamics in AChEt. We sampled the ns-μs timescale motions and found that the tetramer indeed constitutes a dynamic assembly of monomers. The intersubunit fluctuation is correlated with the occlusion of the PAS. Such motions of the subunits “gate” ligand-protein association. The gates are open more than 80% of the time on average, which suggests a small reduction in ligand-protein binding. Despite the limitations in the starting model and approximations inherent in coarse graining, these results are consistent with experiments which suggest that binding of a substrate to the PAS is only somewhat hindered by the association of the subunits.     

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.     

Molecular forms of acetylcholinesterase in mammalian smooth muscles

A comparative study of the molecular forms of acetylcholinesterase (AChE) was made in various smooth muscles (intestine, vas deferens, ciliary body, iris, nictitating membrane retractor, ureter, arteries, anococcygeus muscles) of some mammals (cat, guinea-pig, rat, rabbit, mouse), seeking for a correlation between the presence of 16 S (asymmetric, tailed) form of AChE in smooth muscles and their type of innervation defined by morphological criteria, as well as by the nature of the main neurotransmitters involved in their neuroeffector junctions. Contrary to previous assertions, many smooth muscles contain 16 S AChE, although all those examined here exhibited a proportion clearly less than that of striated muscles. There are large species-specific and individual variations in the percentage of 16 S AChE. The highest percentages of 16 S AChE were found in ciliary and iris muscles, which are provided with an individual (= multiunit) cholinergic innervation. The vas deferens muscles, which are also individually, but noradrenergically innervated contain practically no 16 S AChE. In the muscles having a fascicular (= unitary) innervation, the differences are striking: 16 S AChE is in rather high amount in intestine muscle layers, whereas it is very low or virtually absent in ureter or arterial muscles. Thus, the type of innervation is not clearly involved in the amount of 16 S AChE present in smooth muscles. As for the nature of neurotransmitter a clear correlation exists only in the case of individual innervation, in which only one neurotransmitter is involved or largely predominant.     

Molecular forms of acetylcholinesterase in Xenopus muscle

Xenopus adult muscle, whole Xenopus embryos, and cultured embryonic myocytes together contain five acetylcholinesterase forms which can be resolved by sucrose density gradient centrifugation. These are identified as the collagenase-sensitive asymmetric forms A12 and A8, and the globular forms G4, G2, and G1. Asymmetric forms rise in whole embryos during the period of neuromuscular synapse formation, but their rise is not prevented by tricaine methanesulfonate, which abolishes motor activity. Aneural myocyte cultures synthesize primarily asymmetric acetylcholinesterase, much of which is extracellular. Prior nerve contact is not required for its expression. The proportion of asymmetric forms is neither decreased by tetrodotoxin, nor enhanced by veratridine and aconitine. We conclude that muscle activity does not modulate the expression of asymmetric acetylcholinesterase in Xenopus.     

The synaptic acetylcholinesterase tetramer assembles around a polyproline II helix

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

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

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

Molecular forms of acetylcholinesterase in developing Torpedo embryos

We have studied the evolution of acetylcholinesterase molecular forms during the embryonic development of Torpedo marmorata, in the electric organs and in the electric lobes of the central nervous system. In the early stages of development (35 mm embryos, ‘myogenic phase’ of electric organ development), globular forms of acetylcholinesterase (G₄ and G₂) are abundant in both tissues and the collagen-tailed form A₁₂ is already present. In the electric organs, this form accumulates rapidly after the 55–60 mm stage (‘electrogenic phase’), when synapse formation first commences. Although the molecular characteristics of the collagen-tailed forms, and particularly their aggregation properties, do not appear to change during development, their solubilization requires higher concentrations of MgCl₂, as the electrocytes mature, suggesting that they become more tightly integrated in a better organized basal lamina. The smaller collagen-tailed form A₈ shows a transient increase which coincides approximately with the maximal accumulation of A₁₂, suggesting that it is an intermediate in its synthesis. The accumulation of the hydrophobic G₂, which eventually becomes predominent in the adult electric organs, lags behind that of A₁₂. The functional significance of this important fraction of acetylcholinesterase is therefore not that of a pool of precursor for the synthesis of A₁₂. In the electric lobes, the tetrameric form (G₄) is abundant during development, as well as G₂ and G₁ at certain stages, but the A₁₂ form is predominant in the adult.     

Cardiac troponin T forms a tetramer in vitro

Cardiac troponin T (cTnT) is a myofibrillar protein essential for calcium-dependent contraction. This property has led to functional studies of developmentally expressed cTnT isoforms and mutants identified in patients with hypertrophic cardiomyopathy. The release of cTnT into the serum following myocardial infarction has led to the development of antibody-based assays for measuring cTnT serum concentration. We examined the behavior of cTnT in solution. Recombinant human cTnT3, the dominant isoform in the adult human heart, was used. The protein was pure and functional, as demonstrated by SDS-PAGE and surface plasmon resonance. cTnT3 was found to bind specifically and in a concentration-dependent manner to cTnC. Routine size exclusion chromatography suggested a higher-than-expected MW for cTnT. Using analytical ultracentrifugation, we found cTnT3 in solution to be mainly in the form of a tightly bound tetramer at concentrations as low as 4 micromol/L. Our sedimentation velocity and transmission electron microscopy results indicate that the tetramer's shape is elongated rather than globular. CTnT's self-association in solution is an important consideration in the design and interpretation of experiments with the aim of understanding the biochemical and biophysical properties of cTnT, its isoforms, and its mutants.     

Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure

The effects of the mutation beta9(A6)Ser --> Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the beta9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher-molecular-weight oligomeric fraction (30%). Reversed-phase high-performance liquid chromatography and electrospray ionization mass spectrometry (ESI-MS) under denaturing conditions showed that the tetrameric fraction contained only normal alpha- and beta-chains, whereas the oligomeric fraction contained only normal alpha-chain and disulfide-linked beta(PA) dimer. Under native conditions, ESI-MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 A diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA-CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer-dimer allosteric interface. None of the other variants investigated formed such a large oligomer.     

Molecular forms of purified human erythrocyte membrane acetylcholinesterase investigated by crosslinking with diimidates.

Several molecular forms of human erythrocyte membrane acetylcholinesterase have been studied after crosslinking with bifunctional diimidates. The crosslinked products were analysed by centrifugation on linear sucrose density gradients containing Triton X-100. Molecular weights of covalently linked oligomers were estimated by sodium dodecylsulfate gel electrophoresis. It was shown that acetylcholinesterase crosslinked in absence of Triton X-100 consists of molecular forms built up by dimeric protomers. These dimers were identical with the enzymatically active species sedimenting with 6.5S in linear sucrose density gradients.     

Molecular forms of acetylcholinesterase from Torpedo californica: their relationship to synaptic membranes.

The 16S and 8S forms of acetylcholinesterase (AchE), which are composed of an elongated tail structure in addition to the more globular catalytic subunits, were extracted and purified from membranes from Torpedo californica electric organs. Their subunit compositions and quaternary structures were compared with 11S lytic enzyme which is derived from collagenase or trypsin treatment of the membranes and devoid of the tail unit. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence of reducing agent, appreciable populations of monomeric through tetrameric species are observed for the 11S form. Under the same conditions, the 16S form yields only monomer and dimer in addition to a higher molecular weight species. If complete reduction is effected, only the 80,000 molecular weight monomer is dominant for both the 11S and 16S forms. Cross-linking of the 11S form by dimethyl suberimidate followed by reduction yields monomer through tetramer in descending frequency, while the 16S form again shows a high molecular weight species. A comparison of the composition of the 11S and 16S forms reveals that the latter has an increased glycine content, and 1.1 and 0.3 mol % hydroxyproline and hydroxylysine, respectively. Collagenases that have been purified to homogencity and are devoid of amidase and caseinolytic activity, but active against native collagen, will convert 16S acetylcholinesterase to the 11S form. Thus, composition and substrate behavior of the 16S enzyme are indicative of the tail unit containing a collagen-like sequence. A membrane fraction enriched in acetylcholinesterase and components of basement membrane can be separated from the major portion of the membrane protein. The 16S but not the 11S form reassociates selectively with this membrane fraction. These findings reveal distinct similarities between the tail unit of acetylcholinesterase and basement membrane components and suggest a primary association of AchE with the basement membrane.     

Torpedo marmorata acetylcholinesterase; a comparison with the Electrophorus electricus enzyme. Molecular forms, subunits, electron microscopy, immunological relationship.

Electron microscopy, sequential degradation by hydrolytic enzymes and the physical-chemical properties of the molecular forms of Torpedo acetylcholinesterase indicate that these molecules are structurally related to each other in the same way as the molecular forms of Electrophorus acetylcholinesterase: all are derived from a complex structure in which three tetrameric groups of subunits are associated with a rod-like 'tail'. In aged preparations the catalytic subunits are split into fragments in a manner similar to those of Electrophorus acetylcholinesterase. Immunological cross-reaction between both enzymes demonstrates the occurrence of common antigenic sites. The enzymes from the two sources, however, are different in their molecular weights and susceptibility to hydrolytic enzymes. Also, Torpedo acetylcholinesterase does not precipitate with either isologous or heterologous antibodies.     

Anchorage of collagen-tailed acetylcholinesterase to the extracellular matrix is mediated by heparan sulfate proteoglycans

Heparan sulfate and heparin, two sulfated glycosaminoglycans (GAGs), extracted collagen-tailed acetylcholinesterase (AChE) from the extracellular matrix (ECM) of the electric organ of Discopyge tschudii. The effect of heparan sulfate and heparin was abolished by protamine; other GAGs could not extract the esterase. The solubilization of the asymmetric AChE apparently occurs through the formation of a soluble AChE-GAG complex of 30S. Heparitinase treatment but not chondroitinase ABC treatment of the ECM released asymmetric AChE forms. This provides direct evidence for the vivo interaction between asymmetric AChE and heparan sulfate residues of the ECM. Biochemical analysis of the electric organ ECM showed that sulfated GAGs bound to proteoglycans account for 5% of the total basal lamina. Approximately 20% of the total GAGs were susceptible to heparitinase or nitrous acid oxidation which degrades specifically heparan sulfates, and approximately 80% were susceptible to digestion with chondroitinase ABC, which degrades chondroitin-4 and -6 sulfates and dermatan sulfate. Our experiments provide evidence that asymmetric AChE and carbohydrate components of proteoglycans are associated in the ECM; they also indicate that a heparan sulfate proteoglycan is involved in the anchorage of the collagen-tailed AChE to the synaptic basal lamina.     

Molecular forms of chicken embryo acetylcholinesterase in vitro and in vivo. Isolation and characterization

The four molecular forms of chick embryo leg muscle acetylcholinesterase have been isolated by velocity sedimentation; their apparent sedimentation coefficients are 19.5 S, 11.5 S, 7.1 S, and 5.4 S. All four forms are glycoproteins, exhibit the same Km for acetylcholine, and are inhibited to the same extent by specific inhibitors of acetyl- and buryrylcholinesterase. Treatment of the 19.5 S form of acetylcholinesterase with trypsin generates an array of molecular forms, several of which have sedimentation coefficients identical with the naturally occurring forms. Collagenase treatment of the 19.5 S acetylcholinesterase results in a somewhat different pattern of acetylcholinesterase forms including a novel 20.6 S form. Only the 19.5 S acetylcholinesterase is sensitive to collagenase treatment. Our results indicate that the several acetylcholinesterase forms share a common catalytic subunit, and suggest that the molecular forms of acetylcholinesterase in the chick represent different ensembles of a common monomer. In culture, the muscle cells contain only the 11.5 and 7.1 S acetylcholinesterase forms; however, they also secrete substantial amounts of enzyme into the medium. These secreted acetylcholinesterases have sedimentation coefficients of 9 S and 15 S. The relative abundance of the different acetylcholinesterase molecular forms changes during muscle development, both in vivo and in vitro, suggesting that the assembly and distribution of this family of membrane glycoproteins is developmentally regulated.     

Molecular forms of hippocampal acetylcholinesterase and their changes following septal lesions in the rat.

Changes of acetylcholinesterase activity and its molecular forms, extracted by Triton X-100 and separated by polyacrylamide gel electrophoresis, were studied in the rat hippocampus following septal lesions. Detection of acetylcholinesterase was made densitometrically. While the total activity of acetylcholinesterase was decreased, its molecular forms exhibited a different pattern of changes: the heavy forms were decreased, while the light ones were increased. The results support the view that different acetylcholinesterase molecular forms serve different regulatory mechanisms.     

Molecular forms of acetylcholinesterase in frog skeletal muscle: effects of denervation

In the muscles of the frog, four main molecular forms of acetylcholinesterase are present, with sedimentation coefficients of 5.7, 10.4, 13 and 17.6 S. The heaviest forms, 13 S and 17.6 S are found in both nerve-free segments and endplates zones of sartorius muscle. They decrease in long-term denervation experiments. Consequently, these two forms are not specifically localized in endplates containing regions. However, they depend either on muscle activity or on neural influence or both.     

Molecular forms of acetylcholinesterase: their de novo synthesis in mouse neuroblastoma cells

Rat mouse AChE molecular forms are indistinguishable with respect to their sedimentation coefficients and their evolutive proportions during brain maturation. Among rat or mouse erythrocytes, rat C6 glial cells, and mouse 2A and NS 20 neuroblastoma cells, only neuroblastoma cells showed both the ES and HS molecular forms with a 1:1 proportion for NS 20 cells. All these cells lack a third molecular form (16S), which is present in rat and mouse superior cervical ganglia. After irreversible inhibition of pre-existing NS 20 neuroblastoma AchE, the ES form is first synthesized (de novo synthesis). The HS form begins to appear after a lag time of several hours and represents, 24 h after inhibition, only 15% of the total recovered activity, which is near the initial level. The initial relative proportions return by 2 to 3 days after inhibition. The recovery of the HS form is, for the most part, blocked by actinomycin D, which does not block the recovery of activity itself, which remains as an ES form. It seems that integration of the ES form into the HS form more probably depends on the synthesis of a new messenger RNA, which is required for the synthesis of either new AChE polypeptide chain, polymerization initiating protein or activating enzyme.