Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C.

The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.

Acetylcholinesterase genes in the nematode Caenorhabditis elegans.

Acetylcholinesterase (AChE, EC 3.1.1.7) is responsible for the termination of cholinergic nerve transmission. It is the target of organophosphates and carbamates, two types of chemical pesticides being used extensively in agriculture and veterinary medicine against insects and nematodes. Whereas there is usually one single gene encoding AChE in insects, nematodes are one of the rare phyla where multiple ace genes have been unambiguously identified. We have taken advantage of the nematode Caenorhabditis elegans model to identify the four genes encoding AChE in this species. Two genes, ace-1 and ace-2, encode two major AChEs with different pharmacological properties and tissue repartition: ace-1 is expressed in muscle cells and a few neurons, whereas ace-2 is mainly expressed in motoneurons. ace-3 represents a minor proportion of the total AChE activity and is expressed only in a few cells, but it is able to sustain double null mutants ace-1; ace-2. It is resistant to usual cholinesterase inhibitors. ace-4 was transcribed but the corresponding enzyme was not detected in vivo.

Four genes encode acetylcholinesterases in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae. cDNA sequences, genomic structures, mutations and in vivo expression.

We report the full coding sequences and the genomic organization of the four genes encoding acetylcholinesterase (AChE) in Caenorhabditis elegans and Caenorhabditis briggsae, in relation to the properties of the encoded enzymes. ace-1 and ace-2, located on chromosome X and I, respectively, encode two AChEs (ACE-1 and ACE-2) that present 35% identity. The C-terminal end of ACE-1 is homologous to the C terminus of T subunits of vertebrate AChEs. ACE-1 oligomerizes into amphiphilic tetramers. ACE-2 has a hydrophobic C terminus of H type. It associates into glycolipid-anchored dimers. In C. elegans and C. briggsae, ace-3 and ace-4 are organized in tandem on chromosome II, with only 356 nt and 369 nt, respectively, between the stop codon of ace-4 (upstream gene) and the ATG of ace-3. ace-3 produces only 5 % of the total AChE activity. It encodes an H subunit that associates into dimers of glycolipid-anchored catalytic subunits, which are highly resistant to the usual AChE inhibitors, and which hydrolyze butyrylthiocholine faster than acetylthiocholine. ACE-4 is closer to ACE-3 (54 % identity) than to ACE-1 or ACE-2. The usual sequence FGESAG surrounding the active serine residue in cholinesterases is changed to FGQSAG in ace-4. ACE-4 was not detected by our current biochemical methods, although the gene is transcribed in vivo. However the level of ace-4 mRNAs is far lower than those of ace-1, ace-2 and ace-3. The ace-2, ace-3 and ace-4 transcripts were found to be trans-spliced by both SL1 and SL2, although these genes are not included in typical operons. The molecular bases of null mutations g72 (ace-2), p1304 and dc2 (ace-3) have been identified.

Structure and promoter activity of the 5' flanking region of ace-1, the gene encoding acetylcholinesterase of class A in Caenorhabditis elegans.

We report the structure and the functional activity of the promoter region of ace-1, the gene encoding acetylcholinesterase of class A in the nematode Caenorhabditis elegans. We found that ace-1 was trans -spliced to the SL1 spliced leader and that transcription was initiated at a cluster of multiple starts. There was neither a TATA nor a CAAT box at consensus distances from these starts. Interspecies sequence comparison of the 5' regions of ace-1 in C. elegans and in the related nematode Caenorhabditis briggsae identified four blocks of conserved sequences located within a sequence of 2.4 kilobases upstream from the initiator ATG. In vitro expression of CAT reporter genes in mammalian cells allowed the determination of a minimal promoter in the first 288 nucleotides. In phenotype rescue experiments in vivo, the ace-1 gene containing 2.4 kilobases of 5' flanking region of either C. elegans or C. briggsae was found to restore a coordinated mobility to the uncoordinated double mutants ace-1(-);ace-2(-)of C. elegans. This showed that the ace-1 promoter was contained in 2.4 kilobases of the 5' region, and indicated that cis -regulatory elements as well as coding sequences of ace-1 were functionally conserved between the two nematode species. The pattern of ace-1 expression was established through microinjection of Green Fluorescent Protein reporter gene constructs and showed a major mesodermal expression. Deletion analysis showed that two of the four blocks of conserved sequences act as tissue-specific activators. The distal block is a mesodermal enhancer responsible for the expression in body wall muscle cells, anal sphincter and vulval muscle cells. Another block of conserved sequence directs expression in pharyngeal muscle cells pm5 and three pairs of cephalic sensory neurons.

Existence of four acetylcholinesterase genes in the nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

Three genes, ace-1, ace-2 and ace-3, respectively located on chromosomes X, I and II, were reported to encode acetylcholinesterases (AChEs) of classes A, B and C in the nematode Caenorhabditis elegans. We have previously cloned and sequenced ace-1 in the two related species C. elegans and C. briggsae. We report here partial sequences of ace-2 (encoding class B) and of two other ace sequences located in close proximity on chromosome II in C. elegans and C. briggsae. These two sequences are provisionally named ace-x and ace-y, because it is not possible at the moment to establish which of these two genes corresponds to ace-3. Ace-x and ace-y are transcribed in vivo as shown by RT-PCR and they are likely to be included in a single operon.

Characterization of a null mutation in ace-1, the gene encoding class A acetylcholinesterase in the nematode Caenorhabditis elegans.

Two genes (ace-1 and ace-2) encode two major classes (A and B) of acetylcholinesterase (AChE) in the nematode Caenorhabditis elegans. A null mutation in ace-1 (allele p1000) suppresses all acetylcholinesterase activity of class A. We have identified an opal mutation TGG (W99)-->TGA (Stop) as the only alteration in the mutated gene. This leads to a truncated protein (98 instead of 620 amino acids) with no enzymatic activity. The mutation also reduces the level of ace-1 transcripts to only 10% of that in wild-type animals. This most likely results from a destabilization of mRNA containing the nonsense message. In contrast, compensation of class B by class A AChE in the null mutant strain ace-2 takes place with unchanged ace-1 mRNA level and enzymatic activity similar to class A AChE.

Potential phasic and tonic muscles express a common set of fast and slow myosin light chains and fast tropomyosin during early development of chick embryo.

We investigated the expression of myosin light chains and tropomyosin subunits during chick embryonic development of the anterior (ALD) and posterior (PLD) parts of the latissimus dorsi muscles. As early as day 8 in ovo, both muscles accumulate a common set of myosin light chains (LC) in similar ratios (LC1F: 55 per cent; LC2S: 25 per cent; LC2F: 12 per cent; LC1S: 8 per cent) and a common set of tropomyosin (TM) subunits (beta 2, beta 1, alpha 2F). Later during development, the slow components of the LC regularly disappear in the PLD and the fast components of the LC and the alpha 2FTM disappear in the ALD, so that the adult pattern is almost established at the time of hatching. Thus, early in development, the two muscles accumulate a common set of fast and slow myosin light chains and fast tropomyosin and some isoforms are repressed at a later stage during development. These data might suggest that during development, the regulatory mechanisms of muscle specific isoform expression differ from one contractile protein to another.

An evaluation of the hydrophobic interactions of chick muscle acetylcholinesterase by charge shift electrophoresis and gradient centrifugation.

The hydrophobic interactions of globular forms of acetylcholinesterase from adult and embryonic chick muscles have been analyzed by sucrose gradient centrifugation and non denaturing polyacrylamide gel electrophoresis. The presence of positively- or negatively-charged detergents influences the electrophoretic migrations of hydrophobic globular forms, whereas the mobility of hydrophilic components is unchanged. We defined an hydrophobicity index (HI) which quantitatively reflects this interaction. Globular forms of acetylcholinesterase were isolated in preparative sucrose gradients of muscle extracts. The G(1) form (5 S) appeared as a single band in electrophoresis, the G(2) form (7 S) under two and the G(4) form (11 S) under three electromorphs. The G(1) and the G(2) forms interacted with detergents: this resulted in a shift in their sedimentation in sucrose gradients upon removal of detergents, and in a modification of their electrophoretic migrations in the presence of charged detergents (HI = 1.0 for G(1), HI = 1.7 for G(2)). The G(4) form was heterogenous: one band (G(4f)) did not interact with detergent (HI = 0.1). The other variants (G(4i) and G(4s)) were clearly hydrophobic (HI = 0.5 and HI = I respectively). The hydrophilic and hydrophobic variants of the G(4) form however, were not resolved by sedimentation analysis performed in the presence of Triton X100, but their separation was improved in the presence of 10-oleyl-ether. Therefore, the combination of electrophoretic and sedimentation methods, as described in this paper, can be used successfully for subdividing a single molecular form (size isomer defined by hydrodynamic parameters) into several constituents differing by their hydrophobic interactions.

Polymorphism of acetylcholinesterase in adult Pieris brassicae heads. Evidence for detergent-insensitive and triton X-100-interacting forms.

Acetylcholinesterase (AChE, EC 3117) was extracted from Pieris brassicae heads, by successive homogenizations yielding low-salt soluble (LSS), detergent soluble (DS) and high-salt soluble (HSS) fractions. In all three extracts, two distinct AChE forms were observed, sedimenting at 7.3 and 6.5 S in the presence of Triton X-100. They accounted respectively for 20-40 and 60-80% of the total AChE activity. The 7.3 S component does not interact with Triton X-100 nor with sodium deoxycholate as indicated by sedimentation and electrophoretic migrations. Thus 7.3 S form is therefore considered as a hydrophilic component. The 6.5 S form binds detergent micelles and it aggregates in the absence of Triton X-100. Its electrophoretic mobility is increased in the presence of deoxycholate. This 6.5 S form is therefore a hydrophobic species. These two components have similar substrate and inhibitor specificities and probably correspond to different cellular locations of a single enzyme species. In contrast with vertebrate hydrophobic forms of AChE, the hydrophobic variant of Pieris AChE is not converted into the hydrophilic component by mild pronase treatment. We did not observe any additional form of AChE solubilized at high ionic strength, even in the presence of EDTA. Thus Pieris heads do not apparently contain collagen-tailed AChE molecules, similar to the asymmetric forms described for vertebrate AChE.

Analysis of molecular forms and pharmacological properties of acetylcholinesterase in several mosquito species.

Two acetylcholinesterases (AChE1 and AChE2) have recently been characterized in the common mosquito Culex pipiens. This situation appeared to be an exception among insects, where only one acetylcholinesterase gene had previously been repeatedly reported. In the present study, acetylcholinesterase was studied in five mosquito species: Aedes aegypti, Anopheles gambiae, Anopheles stephensi, Culiseta longeareolata and Culex hortensis, in order to test whether or not two different acetylcholinesterase enzymes could be detected as occurs in C. pipiens. Molecular forms and catalytic properties of the enzyme show that only one enzyme species was detected in the five species. This suggests that a duplication of a single locus Ace probably occurred recently in the phylogeny tree leading to C. pipiens, and produced two distinct acetylcholinesterases: AchE1 and AChE2.

Acetylcholinesterase in tentacles of Octopus vulgaris (Cephalopoda). Histochemical localization and characterization of a specific high salt-soluble and heparin-soluble fraction of globular forms.

Transverse sections of Octopus tentacles were stained for acetylcholinesterase (AChE) activity. An intense staining, that was suppressed by preincubation in 10(-5) M eserine, was detected in a number of neuronal cells, nerve fibres and neuromuscular junctions of intrinsic muscles of the arm. Octopus acetylcholinesterase was found as two molecular forms: an amphiphilic dimeric form (G2) sensitive to phosphatidylinositol phospholipase C and a hydrophilic tetrameric (G4) form. Sequential solubilization revealed that a significant portion of both G2 and G4 forms was recovered only in a high salt-soluble fraction (1 M NaCl, no detergent), Heparin (2 mg/ml) was able to solubilize G2 and G4 forms with the same efficiency than 1 M NaCl. The solubilizing effect of heparin was concentration-dependent and was reduced by protamine (2 mg/ml). This suggests that heparin operates through the dissociation of ionic interactions existing in situ between globular forms of AChE and cellular or extracellular polyanionic components. Interaction of AChE molecular forms with heparin has been reported so far in only a few instances and its physiological meaning is uncertain. G2 and G4 forms, interacting or not with heparin, all belong to a single pharmacological class of AChE. This suggests the existence of a single AChE gene. Amphiphilic and hydrophilic subunits thus likely result either from the processing of a single AChE transcript by alternative splicing (as in vertebrate AChE) or from a post-translation modification of a single catalytic peptide.

Expression of the A(12) form of acetylcholinesterase by developing avian leg muscle cells in vivo and during differentiation in primary cell cultures.

The accumulation of the molecular forms of acetylcholinesterase (AChE) has been studied in leg muscles during embryonic chick development and in cell cultures initiated with myoblasts obtained from embryos at different stages of development. The collagen-tailed, A(12) form appears in leg muscles as soon as day 5 in ovo. An early excision of the lumbar zone of the neural tube at day 2 1 2 in ovo severely delayed the morphological development. In leg muscles dissected at day 12 in ovo from operated embryos, we found that the total amount of AChE activity and particularly the proportion of A(12) form were dramatically reduced. Muscle cells were grown in vitro in a medium supplemented with fetal calf serum. In these conditions, chick muscle cells unequivocally synthesize the A(12) form when they originated from muscles which accumulated this form in vivo. In contrast, myoblasts obtained from 5-day old embryo leg muscles did not produce the A(12) form either in aneural cultures or in the presence of nerve cells. In relation with previous observations concerning chick myogenesis, we discuss the possibility that this difference reflects the existence of two types of myoblasts. This hypothesis would also explain the results of cocultures performed with nerve cells and normal or demedullated leg muscle myoblasts.

Four acetylcholinesterase genes in the nematode Caenorhabditis elegans.

Whereas a single gene encodes acetylcholinesterase (AChE) in vertebrates and most insect species, four distinct genes have been cloned and characterized in the nematode Caenorhabditis elegans. We found that ace-1 (mapped to chromosome X) is prominently expressed in muscle cells whereas ace-2 (located on chromosome I) is mainly expressed in neurons. Ace-x and ace-y genes are located in close proximity on chromosome II where they are separated by only a few hundred base pairs. The role of these two genes is still unknown.

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA.

Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetylcholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

Morphological and histochemical differentiation of intrafusal fibres in the posterior latissimus dorsi muscle of the developing chick.

Morphological and histochemical differentiation of neuromuscular spindles was studied in the posterior latissimus dorsi (PLD) of the chick during embryonic and post-hatching development. A rapid increase in the number of spindles takes place between the 13th and 15th of embryonic life. By the 15th day in ovo, the spindle capsule appears filled with numerous contiguous cells. Large sensory endings and small primitive motor endings are observed on intrafusal fibres. Ultrastructural observations of the nerve supply of the spindles confirm that each developing spindle receives one thick Ia axon with one to three thin gamma axons. The intracapsular space differentiates by the 17th day of embryonic development. All intrafusal fibres are morphologically of the nuclear-chain type, while two fibre types are distinguished as early as the 14th day of embryonic life, when myofibrillar ATPase activity is demonstrated after acid preincubation. These two histochemical types of intrafusal fibres are also described in the adult. The relation between these two histochemical types and different functional activity of intrafusal fibres is suggested.

Sequence comparison of ACE-1, the gene encoding acetylcholinesterase of class A, in the two nematodes Caenorhabditis elegans and Caenorhabditis briggsae.

The ace-1 gene, which encodes acetylcholinesterase of class A, has been cloned and sequenced in C. briggsae and compared to its homologue in C. elegans. Both genes present an open reading frame of 1860 nucleotides. The percentages of identity are 80% and 95% at the nucleotide and aminoacid levels respectively. All residues characteristic of an acetylcholinesterase are found in conserved positions in C. briggsae ACE-1. The deduced C-terminus is hydrophilic, thus resembling the catalytic peptide T of vertebrate cholinesterases. Codon usage in both ace-1 genes appears to be lowly biased. This may indicate that these genes are lowly expressed. The splicing sites of the eight introns of ace-1 in C. elegans are conserved in C. briggsae, but introns are shorter in C. briggsae. No homology was found between intronic sequences in both species, except for the consensus border sequences.

cDNA sequence, gene structure, and cholinesterase-like domains of an esterase from Caenorhabditis elegans mapped to chromosome V.

The structure of an esterase gene from Caenorhabditis elegans has been determined by comparison of the sequences in genomic and cDNA clones. The gene was mapped close to the center of chromosome V (1.7 centimorgans to the left of dpy-11) and is therefore distinct from the gut esterase gene ges-1. It possessed 7 short introns. The 5' splice site of intron 3 presented the sequence GC instead of the usual GT that was found in the other six introns. The cDNA was trans-spliced with the short leader SL1. The open reading frame indicated that a protein of 557 aminoacids was encoded. The deduced aminoacid sequence did not present a signal peptide at the N-terminal but a potential N-myristoylation site (GXXXS) provided that the initiator methionine was removed. This protein should therefore remain intracellular. Comparison of this C. elegans sequence to other protein sequences in databases, as well as the analysis of the secondary structure in the protein showed that it belongs to the subgroup of esterases in the alpha/beta hydrolase fold family.

Expression of asymmetric forms of acetylcholinesterase during myogenesis in vitro.

Chick muscle cells differentiating in vitro in the absence of nerve cells produce asymmetric forms of acetylcholinesterase (AChE) only if they originate from muscles which accumulate these forms in ovo (i.e. after embryonic day 5). The presence of nerve cells does not induce the synthesis of A forms in cultures of 5 day-old myoblasts and does not increase their proportion in cultures of 7 day-old myoblasts. Thus, the capacity to synthesize (or assemble) the complex polymeric forms of AChE does not reflect a direct neural influence but might rather be considered as an intrinsic property of the "late" categories of myoblasts that sequentially occur during the differentiation of leg muscles. We studied the synthesis of ChE molecular forms in the mouse muscle C2 cell line. From these experiments we suggest that the synthesis of A forms (or their assembly) can take place as soon as the cells are withdrawn from the cell cycle, but does not require cell fusion by itself. These observations are related to other recent studies that challenge the validity of A forms as topographical/physiological markers of neuromuscular interactions.

[Effect of the type of muscle fiber innervation on the acquisition of myofibrillar ATPase properties]. / Influence du type d'innervation des fibres musculaires sur l'acquisition des propriétés de l'ATPase myofibrillaire.

We present some observations differing from the idea that neurons precisely control the differentiation of muscle fibre histochemical properties and show that, in a few cases, innervation does not influence the development of myofibrillar ATPase properties. Moreover, we report that a regional variation of the acid stability of myofibrillar ATPase may occur in the polar zone of intrafusal fibres in chick PLD muscle spindles, a zone devoid of innervation. This observation is related to a similar variation in the polar zone of rat intrafusal fibres (Kucera et al., 1978). New examples are presented supporting the conclusion that the differentiation of the properties of ATPase activity, and thus of fibre types, takes place, at least in some cases, independently of the type of innervation received.