Marta Grauso

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.

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.

Evidence for a molecular polymorphis of cholinesterase in Sepia officinalis (cephalopoda: decapoda)

1. 1. Three forms of cholinesterase were sequentially extracted from head and tentacles of Sepia officinalis and noted as low-salt (LSS), detergent (DS) and high-salt (HSS) soluble. They represent about 24, 30 and 46% of total activity. 2. 2. All enzyme forms seem to be amphiphilic proteins with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation (LSS form). 3. 3. The DS form is membrane-anchored by a phosphatidylinositol, while the HSS form is likely linked to some proteoglycan molecule of the extracellular matrix by ionic interactions. 4. 4. According to Vmax/Km values, all the enzymes are acetylcholinesterases, even if hydrolyze propionylthiocoline at the highest rate. 5. 5. Some kinetic and molecular properties of the studied enzymes are compared with those of other cholinesterases from vertebrates and invertebrates. Possible phylogenic and adaptive features are discussed.

Cholinesterase in Helix pomatia (Gastropoda: Stylommatophora): presence of a soluble (hemolymph) and a membrane-bound form

Abstract Two forms of cholinesterase (ChE) were detected in the gastropod mollusc Helix pomatia : a fully soluble (FS) ChE in the hemolymph, representing about 90% of total activity, and a detergent-soluble (DS) membrane-bound enzyme. The FS enzyme seems to be a stable complex forming a large particle with a sedimentation coefficient of 32 S. The DS ChE (estimated M r : 129,000) is likely to be an amphiphilic protein with hydrophobic domains interacting with non-ionic detergent (Triton X-100) and giving self-aggregation. Based on V max / K m values, the enzymes are an acetylcholinesterase (FS) and a butyrylcholinesterase (DS), even if they hydrolyze propionylthiocholine at the highest rate. FS ChE seems to discriminate among the substrates with an involvement of steric hindrance and hydrophobic forces; DS ChE shows a lower substrate specificity level. The study with inhibitors shows a far higher sensitivity of DS ChE to inhibition by edrophonium. Both FS and DS ChE are totally inhibited by 10 −5 M and 10 −4 M eserine, respectively. Some kinetic and molecular features of FS and DS ChE from H. pomatia are compared with those of other invertebrate enzymes.

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.

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.

Molecular Cloning and Characterization of a cDNA Encoding AChE from Optic Lobe of Loligo Opalescences

The optic lobe of Cephalopods shows the highest acetylcholine content at any neural tissue. Therefore, we intended to characterize acetylcholinesterase in such at organ from Loligo opalescens and then to perform cloning and sequencing of the relative cDNA from an expression library of the same species. The purified enzyme shows a high catalytic efficiency like that of vertebrate AChEs. It is present with two distinct forms, a G2 amphiphilic one (90% of total activity), likely anchored to the cell membrane through a phosphatidylinositol tail, and a G2 hydrophilic enzyme. The cDNA regarding the prevailing amphiphilic form has been cloned, sequenced and expressed in COS cells. It shows that the aminoacid residues of catalytic triad (Ser, His, Glu) are in conserved position, as well as the aromatic residues at the border of catalytic gorge. The aminoacid sequence deduced from cDNA shows a hydrophobic C-terminus. The enzyme obtained from expression in COS cells displays pharmacological and kinetic features quite similar to those of the enzyme extracted and purified from the fresh tissue (sensitivity to AChE-specific inhibitors, excess- substrate inhibition, membrane anchoring via phosphatidylinositol).

MOLECULAR POLYMORPHISM OF ACETYLCHOLINESTERASE IN HIRUDO MEDICINALIS

Two different cholinesterases were extracted from Hirudo medicinalis that differ in molecular forms and in substrate and inhibitor specificity. Spontaneous-soluble (SS) activity was recovered from tissue dilaceration in low salt buffer, and detergent-soluble (DS) was solubilized by LS buffer+1% Triton. Both enzymes were purified to homogeneity on edrophonium-sepharose followed by chromatography on Concanavalin A-sepharose. Purified enzymes were studied by SDS-PAGE in reducing conditions. SS enzyme gave two bands at 30 and 66 kDa, while only one band was seen at 66 kDa for DS enzyme. Mr of the purified ChEs evaluated by sephadex G 200 chromatography were about 66000 for SS enzyme and 130000 for DS. Sedimentation analysis of crude and purified enzyme preparations was performed with or without Triton in the gradient. SS ChE showed a single peak sedimenting at 5.0 S in both conditions. This result suggests that SS enzyme is a hydrophylic monomer (G1). DS enzyme showed a single peak of activity at 6.5 S and aggregated in the absence of detergent in the gradient. PI-PLC suppressed the aggregation. DS ChE is thus an amphiphilic dimer anchored to the membrane via a glycosylphosphatidylinositol. Kinetic study was carried out using p-nitrophenyl and thiocholine esters. Both enzymes appear to hydrolyze propionyl esters best but are also active on butyryl and acetyl derivatives. Both ChEs were inhibited by excess substrate and should thus be referred to as acetylcholinesterases. SS AChE displayed marked similarity between the sets of Km values with charged and uncharged substrates, suggesting a reduced influence of electrostatic interactions in the enzyme substrate affinity. Unlike SS enzyme, ionic interactions strongly affect the formation of DS AChE substrate complex (far higher Km values with uncharged substrates). SS AChE was more sensitive to inhibition by eserine and DFP (IC50 = 10−7 and 10−8 M respectively) than DS enzyme (IC50 = 10−6 and 10−5 M).

Acetylcholinesterase from Octopus vulgaris (Cephalopoda)

Octopus acetylcholinesterase (AChE) is found as two molecular forms: an amphiphilic dimeric form (G2) sensitive to phosphatidylinositol phospholipase C and a hydrophilic tetrameric (G4) form. G2 and G4 forms belong to a single pharmacological class of AChE. Thus they likely result from a post-transcriptional or post-translational processing of a single AChE gene. Sequential solubilizations reveal that a significant portion of both G2 and G4 forms can be recovered only in a High Salt-Soluble fraction (IM NaCl, no detergent). Heparin (2mg/ml) was able to solubilize G2 and G4 forms with the same efficiency than 1M NaCl. The solubilizing effect of heparin was concentration-dependent and was reduced by protamine (2mg/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.