Martine Arpagaus

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.

Proposed nomenclature for human butyrylcholinesterase genetic variants identified by DNA sequencing

Summary1.New information identifying nucleotide alterations of human butyrylcholinesterase allows the use of more specific nomenclature for the variants commonly known as atypical, fluoride, silent, and K variant.2.In addition to suggesting a system of trivial names and abbreviations, we provide a list of formal names that follow the guidelines of the Committee for Human Gene Nomenclature.3.It is suggested that formal names be included in publications whenever possible.

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.

Duplication of theAce.1 locus inCulex pipiens mosquitoes from the Caribbean

InCulex pipiens mosquitoes, AChE1 encoded by the locusAce.1 is the target of organophosphorus and carbamate insecticides. In several resistant strains homozygous forAce.1RR, insensitive AChE1 is exclusively found. An unusual situation occurs in two Caribbean resistant strains where each mosquito, at each generation, displays a mixture of sensitive and insensitive AChE1. These mosquitoes are not heterozygotes,Ace.1RS, as preimaginal mortalities cannot account for the lethality of both homozygous classes. This situation is best explained by the existence of twoAce.1 loci, coding, respectively, a sensitive and an insensitive AChE1. Thus, we suggest that in the Caribbean a duplication of theAce.1 locus occurred before the appearance of insecticide resistance at one of the two copies.

Mutations in the dystrophin-like dys-1 gene of Caenorhabditis elegans result in reduced acetylcholinesterase activity

Mutations of the Caenorhabditis elegans dystrophin/utrophin‐like dys‐1 gene lead to hyperactivity and hypercontraction of the animals. In addition dys‐1 mutants are hypersensitive to acetylcholine and acetylcholinesterase inhibitors. We investigated this phenotype further by assaying acetylcholinesterase activity. Total extracts from three different dys‐1 alleles showed significantly less acetylcholinesterase‐specific activity than wild‐type controls. In addition, double mutants carrying a mutation in the dys‐1 gene plus a mutation in either of the two major acetylcholinesterase genes (ace‐1 and ace‐2) display locomotor defects consistent with a strong reduction of acetylcholinesterases, whereas none of the single mutants does. Therefore, in C. elegans, disruption of the dystrophin/utrophin‐like dys‐1 gene affects acetylcholinesterase activity.

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.

Molecular cloning and expression of a full-length cDNA encoding acetylcholinesterase in optic lobes of the squid Loligo opalescens: a new member of the cholinesterase family resistant to diisopropyl fluorophosphate.

Abstract : Acetylcholinesterase cDNA was cloned by screening a library from Loligo opalescens optic lobes ; cDNA sequence analysis revealed an open reading frame coding for a protein of 610 amino acids that showed 20‐41% amino acid identity with the acetylcholinesterases studied so far. The characteristic structure of cholinesterase (the choline binding site, the catalytic triad, and six cysteines that form three intrachain disulfide bonds) was conserved in the protein. The heterologous expression of acetylcholinesterase in COS cells gave a recovery of acetylcholinesterase activity 20‐fold higher than in controls. The enzyme, partially purified by affinity chromatography, showed molecular and kinetic features indistinguishable from those of acetylcholinesterase expressed in vivo, which displays a high catalytic efficiency. Both enzymes are true acetylcholinesterase corresponding to phosphatidylinositol‐anchored G2a dimers of class I, with a marked substrate specificity for acetylthiocholine. The deduced amino acid sequence may explain some particular kinetic characteristics of Loligo acetylcholinesterase, because the presence of a polar amino acid residue (S313) instead of a nonpolar one [F(288) in Torpedo] in the acyl pocket of the active site could justify the high substrate specificity of the enzyme, the absence of hydrolysis with butyrylthiocholine, and the poor inhibition by the organophosphate diisopropyl fluorophosphate.

Release of periplasmic proteins of Brucella suis upon acidic shock involves the outer membrane protein Omp25

ABSTRACT The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.