Jean-Pierre Toutant

Existence of Two Acetylcholinesterases in the Mosquito Culex pipiens (Diptera: Culicidae)

Abstract: 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 × 10−4M 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 × 10−4M propoxur: AChE2 sensitivity to propoxur is unchanged, whereas AChE1 is now resistant to 5 × 10−4M 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.

Native Molecular Forms of Head Acetylcholinesterase from Adult Drosophila melanogaster: Quaternary Structure and Hydrophobic Character

Abstract: The native molecular forms of acetylcholinesterase (AChE) present in adult Drosophila heads were characterized by sedimentation analysis in sucrose gradients and by nondenaturing electrophoresis. The hydrophobic properties of AChE forms were studied by comparing their migration in the presence of Triton X100. 10‐oleyl ether, or sodium deoxycholate, or in the absence of detergent. We examined the polymeric structure of AChE forms by disulfide bridge reduction. We found that the major native molecular form is an amphiphilic dimer which is converted into hydrophilic dimer and monomer on autolysis of the extracts, or into a catalytically active amphiphilic monomer by partial reduction. The latter component exists only as trace amounts in the native enzyme. Two additional minor native forms were identified as hydrophilic dimer and monomer. Although a significant proportion of AChE was only solubilized in high salt, following extractions in low salt, this high salt‐soluble fraction contained the same molecular forms as the low salt‐soluble fractions: thus, we did not detect any molecular form resembling the asymmetric forms of vertebrate cholinesterases.

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.

Multiple ace genes encoding acetylcholinesterases of Caenorhabditis elegans have distinct tissue expression

ace‐1 and ace‐2 genes encoding acetylcholinesterase in the nematode Caenorhabditis elegans present 35% identity in coding sequences but no homology in noncoding regions (introns, 5′‐ and 3′‐untranslated regions). A 5′‐region of ace‐2 was defined by rescue of ace‐1;ace‐2 mutants. When green fluorescent protein (GFP) expression was driven by this regulatory region, the resulting pattern was distinct from that of ace‐1. This latter gene is expressed in all body‐wall and vulval muscle cells ( Culetto et al., 1999 ), whereas ace‐2 is expressed almost exclusively in neurons. ace‐3 and ace‐4 genes are located in close proximity on chromosome II ( Combes et al., 2000 ). These two genes were first transcribed in vivo as a bicistronic messenger and thus constitute an ace‐3;ace‐4 operon. However, there was a very low level of monocistronic mRNA of ace‐4 (the upstream gene) in vivo, and no ACE‐4 enzymatic activity was ever detected. GFP expression driven by a 5′ upstream region of the ace‐3;ace‐4 operon was detected in several muscle cells of the pharynx (pm3, pm4, pm5 and pm7) and in the two canal associated neurons (CAN cells). A dorsal row of body‐wall muscle cells was intensively labelled in larval stages but no longer detected in adults. The distinct tissue‐specific expression of ace‐1, ace‐2 and ace‐3 (coexpressed only in pm5 cells) indicates that ace genes are not redundant.

The α/β 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.

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.

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.

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.

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.

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.