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.

Zebrafish acetylcholinesterase is encoded by a single gene localized on linkage group 7. Gene structure and polymorphism; molecular forms and expression pattern during development.

We cloned and sequenced the acetylcholinesterase gene and cDNA of zebrafish, Danio rerio. We found a single gene (ache) located on linkage group LG7. The relative organization of ache, eng2, and shh genes is conserved between zebrafish and mammals and defines a synteny. Restriction fragment length polymorphism analysis was allowed to identify several allelic variations. We also identified two transposable elements in non-coding regions of the gene. Compared with other vertebrate acetylcholinesterase genes, ache gene contains no alternative splicing at 5' or 3' ends where only a T exon is present. The translated sequence is 60-80% identical to acetylcholinesterases of the vertebrates and exhibits an extra loop specific to teleosts. Analysis of molecular forms showed a transition, at the time of hatching, from the globular G4 form to asymmetric A12 form that becomes prominent in adults. In situ hybridization and enzymatic activity detection on whole embryos confirmed early expression of the acetylcholinesterase gene in nervous and muscular tissues. We found no butyrylcholinesterase gene or activity in Danio. These findings make zebrafish a promising model to study function of acetylcholinesterase during development and regulation of molecular forms assembly in vivo.

Effects of carbofuran, diuron, and nicosulfuron on acetylcholinesterase activity in goldfish (Carassius auratus).

Juvenile goldfish (Carassius auratus) were exposed to three widely used pesticides; carbofuran, diuron, and nicosulfuron. Acetylcholinesterase (AChE) activity and molecular forms of AChE were first characterized in brain and skeletal muscle of unexposed fish. Skeletal muscle had higher AChE activity than brain (306 and 215 nmol/min/mg protein, respectively). In brain, four molecular forms of AChE were found: A12, G4, G2, and G1. In the muscle, three molecular forms were found A12, A8, and G2. AChE activity was then evaluated in both tissues of fish exposed to different concentration of pesticides (5, 50, and 500 microg/L) for 6, 12, 24, and 48 h. In brain, AChE activity was significantly inhibited during all the periods of exposure in response to 50 microg/L (19-28%) and 500 microg/L (85-87%) carbofuran. Such effect was observed in the muscle only at 500 microg/L (86-92%). Carbofuran had no effect on the distribution of molecular forms. Significant inhibitions (9-12%) of brain AChE activity were also observed in response to diuron and nicosulfuron at 500 microg/L during all periods of exposure and for 50 microg/L nicosulfuron after 24 and 48 h. This study pointed out short-term effects of exposure to sublethal concentrations of the three pesticides, ranging among different chemical families, on brain and muscle AChE in goldfish.

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.

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.

Decay-accelerating factor (CD55) and membrane inhibitor of reactive lysis (CD59) are released within exosomes during In vitro maturation of reticulocytes.

Exosomes are membrane vesicles released by reticulocytes during their maturation into erythrocytes. They have a clearing function because of their enrichment with some proteins known to decrease or disappear from the cell surface during maturation, eg, acetylcholinesterase (AChE) and transferrin receptor (TfR), respectively. To better understand the molecular events leading to protein sorting in exosomes, we analyzed the expression of glycosylphosphatidylinositol (GPI)-anchored proteins on the exosome surface through a technique involving bead coupling and flow cytometry immunodetection. The presence of AChE, decay-accelerating factor (DAF), membrane inhibitor of reactive lysis (MIRL), and lymphocyte function-associated antigen 3 (LFA-3) on the surface of exosomes obtained from normal and paroxysmal nocturnal hemoglobinuria (PNH) reticulocytes, suggests that (1) the GPI anchor is efficiently sorted during exosome formation, (2) exosome release could account for the observed discrepancy in GPI-protein expression between reticulocytes and erythrocytes from PNH patients, and (3) exosomes could have another physiologic function related to controlling membrane attack complex formation.

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.

aCHEdb: the database system for ESTHER, the alpha/beta fold family of proteins and the Cholinesterase gene server.

Acetylcholinesterase belongs to a family of proteins, the alpha/beta hydrolase fold family, whose constituents evolutionarily diverged from a common ancestor and share a similar structure of a central beta sheet surrounded by alpha helices. These proteins fulfil a wide range of physiological functions (hydrolases, adhesion molecules, hormone precursors) [Krejci,E., Duval,N., Chatonnet,A., Vincens,P. and Massoulié,J. (1991) Proc. Natl. Acad. Sci. USA , 88, 6647-6651]. ESTHER (for esterases, alpha/beta hydrolase enzymes and relatives) is a database aimed at collecting in one information system, sequence data together with biological annotations and experimental biochemical results related to the structure-function analysis of the enzymes of the family. The major upgrade of the database comes from the use of a new database management system: aCHEdb which uses the ACeDB program designed by Richard Durbin and Jean Thierry-Mieg. It can be found at http://www.ensam.inra.fr/cholinesterase

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.

The alpha/beta fold family of proteins database and the cholinesterase gene server ESTHER.

ESTHER (for esterases, alpha/betahydrolase enzyme and relatives) is a database of sequences phylogenetically related to cholinesterases. These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) sharing a similar structure of a central beta-sheet surrounded by alpha-helices. Among these proteins a wide range of functions can be found (hydrolases, adhesion molecules, hormone precursors). The purpose of ESTHER is to help comparison of structures and functions of members of the family. Since the last release, new features have been added to the server. A BLAST comparison tool allows sequence homology searches within the database sequences. New sections are available: kinetics and inhibitors of cholinesterases, fasciculin-acetylcholinesterase interaction and a gene structure review. The mutation analysis compilation has been improved with three-dimensional images. A mailing list has been created.

Existence of two acetylcholinesterases in the mosquito Culex pipiens (Diptera:Culicidae).

Two acetylcholinesterases (AChEs), AChE1 and AChE2, differing in substrate specificity and in some aspects of inhibitor sensitivity, have been characterized in the mosquito Culex pipiens. The results of ultracentrifugation in sucrose gradients and nondenaturing gel electrophoresis of AChE activity peak fractions show that each AChE is present as two molecular forms: one amphiphilic dimer possessing a glycolipid anchor and one hydrophilic dimer that does not interact with nondenaturing detergents. Treatment by phosphatidylinositol-specific phospholipase C converts each type of amphiphilic dimer into the corresponding hydrophilic dimer. Molecular forms of AChE1 have a lower electrophoretic mobility than those of AChE2. However, amphiphilic dimers and hydrophilic dimers have similar sedimentation coefficients (5.5S and 6.5S, respectively). AChE1 and AChE2 dimers, amphiphilic or hydrophilic, resist dithiothreitol reduction under conditions that allow reduction of Drosophila AChE dimers. In the insecticide-susceptible strain S-LAB, AChE1 is inhibited by 5 x 10(-4) M propoxur (a carbamate insecticide), whereas AChE2 is resistant. All animals are killed by this concentration of propoxur, indicating that only AChE1 fulfills the physiological function of neurotransmitter hydrolysis at synapses. In the insecticide-resistant strain, MSE, there is no mortality after exposure to 5 x 10(-4) M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 x 10(-4) M propoxur. The possibility that AChE1 and AChE2 are products of tissue-specific posttranslational modifications of a single gene is discussed, but we suggest, based on recent results obtained at the molecular level in mosquitoes, that they are encoded by two different genes.

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.

A cholinesterase genes server (ESTHER): a database of cholinesterase-related sequences for multiple alignments, phylogenetic relationships, mutations and structural data retrieval.

We have built a database of sequences phylogenetically related to cholinesterases (ESTHER) for esterases, alpha/beta hydrolase enzymes and relatives). These sequences define a homogeneous group of enzymes (carboxylesterases, lipases and hormone-sensitive lipases) with some related proteins devoid of enzymatic activity. The purpose of ESTHER is to help comparison and alignment of any new sequence appearing in the field, to favour mutation analysis of structure-function relationships and to allow structural data recovery. ESTHER is a World Wide Web server with the URL http://www.montpellier.inra.fr:70/cholinesterase.

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.

Solubilization, molecular forms, purification and substrate specificity of two acetylcholinesterases in the medicinal leech (Hirudo medicinalis).

Two acetylcholinesterases (AChE) differing in substrate and inhibitor specificities have been characterized in the medical leech (Hirudo medicinalis). A 'spontaneously-soluble' portion of AChE activity (SS-AChE) was recovered from haemolymph and from tissues dilacerated in low-salt buffer. A second portion of AChE activity was obtained after extraction of tissues in low-salt buffer alone or containing 1% Triton X-100 [detergent-soluble (DS-) AChE). Both enzymes were purified to homogeneity by affinity chromatography on edrophonium- and concanavalin A-Sepharose columns. Denaturing SDS/PAGE under reducing conditions gave one band at 30 kDa for purified SS-AChE and 66 kDa for DS-AChE. Sephadex G-200 chromatography indicated a molecular mass of 66 kDa for native SS-AChE and of 130 kDa for DS-AChE. SS-AChE showed a single peak sedimenting at 5.0 S in sucrose gradients with or without Triton X-100, suggesting that it was a hydrophylic monomer (G1). DS-AChE sedimented as a single 6.1-6.5 S peak in the presence of Triton X-100 and aggregated in the absence of detergent. A treatment with phosphatidylinositol-specific phospholipase C suppressed aggregation and gave a 7 S peak. DS-AChE was thus an amphiphilic glycolipid-anchored dimer. Substrate specificities were studied using p-nitrophenyl esters (acetate, propionate and butyrate) and corresponding thiocholine esters as substrates. SS-AChE displayed only limited variations in Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. By contrast, DS-AChE displayed higher Km values with uncharged than with charged substrates. SS-AChE was more sensitive to eserine and di-isopropyl fluorophosphate (IC50 5 x 10(-8) and 10(-8) M respectively) than DS-AChE (5 x 10(-7) and 5 x 10(-5) M.

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.

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.

Acetylcholinesterase and butyrylcholinesterase expression in adult rabbit tissues and during development.

A large cDNA fragment covering the complete sequence of the mature catalytic subunit of rabbit acetylcholinesterase (AChE) has been cloned and sequenced. This sequence was compared to that of rabbit butyrylcholinesterase [BChE; Jbilo, O. & Chatonnet, A. (1990) Nucleic Acids Res. 18, 3990]. Amino acid sequences of AChE and BChE have 51% identity. They both possessed a choline-binding site W84, a catalytic triad S200-H440-E327 and six cysteine residues (positions 67-94, 254-265, 402-521) in conserved sequence positions to those that form three intrachain disulfide bonds in all cholinesterases (by convention, numbering of amino acids is that used for Torpedo AChE). Rabbit AChE had a larger number of aromatic residues lining the active-site gorge than rabbit BChE (14 compared to 8, respectively) and a smaller number of potential N-glycosylation sites (3 compared to 8, respectively). Both catalytic subunits have a hydrophilic C-terminus (catalytic subunits of type T). Expression of acetylcholinesterase and butyrylcholinesterase genes (ACHE and BCHE) was studied in rabbit tissues and during development by a correlation of Northern-blot analysis and enzymic activities. This correlation was rendered difficult by the presence of an eserine-resistant esterase active on butyrylthiocholine in serum, liver and lung. When the contribution of this carboxylesterase was taken into account, brain was found as the richest source of BChE followed by lung and heart. Rabbit liver had a very low content of BChE that correlated with the low BChE activity in plasma. During development, BCHE transcripts were detected as early as day 10 post coitum, whereas ACHE transcripts appeared only on day 12.

Promoter and transcription start site of human and rabbit butyrylcholinesterase genes.

Two kilobase segments of the 5'-untranslated regions of the human and rabbit butyrylcholinesterase (BCHE) genes were characterized. The sequences shared extensive identity except for a 333-base pair (bp) Alu repeat present only in human BCHE. One single transcription start site was found in both genes with the techniques of primer extension, amplification of the 5'-end of mRNA, and RNase protection. Cap sites in human and rabbit BCHE genes were found in strictly homologous positions. In human BCHE, the transcription start site was found 157 bp upstream of Met-28, the translation start site. Potential regulatory elements in both promoters included one AP1 site and multiple sites for topoisomerase, Oct-1 and PEA-3. Transient expression of BCHE-reporter gene constructs showed that a 194-bp fragment of the 5'-flanking region of human BCHE and a 570-bp fragment of rabbit BCHE were sufficient for promoting chloramphenicol acetyltransferase activity in HeLa cells. No consensus TATA and CAAT boxes were found. However, the sequence around the transcription start site exhibited homology with initiator elements found in other TATA-less promoters in developmentally regulated genes.