The membrane-anchoring systems of vertebrate acetylcholinesterase and variant surface glycoproteins of African trypanosomes share a common antigenic determinant

Amphiphilic detergent-soluble acetylcholinesterase (AChE) from Torpedo is converted to a hydrophilic form by digestion with phospholipase C from Trypanosoma brucei or from Bacillus cereus. This lipase digestion uncovers an immunological determinant which crossreacts with a complex carbohydrate structure present in the hydrophilic form of all variant surface glycoproteins (VSG) of T. brucei. This crossreacting determinant is also detected in human erythrocyte AChE after digestion with T. brucei lipase. From these results we conclude that the glycophospholipid anchors of protozoan VSG and of AChE of the two vertebrates share common structural features, suggesting that this novel type of membrane anchor has been conserved during evolution.     

Conversion of human erythrocyte acetylcholinesterase from an amphiphilic to a hydrophilic form by phosphatidylinositol-specific phospholipase C and serum phospholipase D

Each catalytic subunit in the amphiphilic dimer of human erythrocyte acetylcholinesterase (AChE) is anchored in the plasma membrane exclusively by a glycoinositol phospholipid. In contrast to erythrocyte AChEs in other mammalian species, the human enzyme is resistant to direct cleavage by phosphatidylinositol-specific phospholipase C (PtdIns-specific PLC). The resistance is due to the existence of an additional fatty acyl chain on the inositol ring which blocks the action of PtdIns-specific PLC [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. In this report, nondenaturing polyacrylamide gel electrophoresis was applied to permit rapid and unambiguous distinction between amphiphilic AChE, in which each catalytic subunit binds one nonionic detergent micelle, and hydrophilic AChE, which does not interact with detergent. Deacylation of human erythrocyte AChE by an alkaline treatment with hydroxylamine rendered the amphiphilic AChE susceptible to PtdIns-specific PLC with the consequent release of hydrophilic AChE. Although serum anchor-specific phospholipase D (PLD) cleaves the intact human erythrocyte AChE anchor, this treatment, as judged by nondenaturing electrophoresis, did not release hydrophilic AChE. Hydroxylamine treatment before or after PLD digestion was necessary to achieve the conversion. These observations indicate that binding of a single detergent micelle was maintained when any of the three fatty acyl or alkyl groups in the human erythrocyte AChE anchor phospholipid were retained. For proteins that can be identified following nondenaturing gel electrophoresis, these procedures provide methods both for detecting glycoinositol phospholipid anchors resistant to PtdIns-specific PLC and for indicating fatty acyl and/or alkyl chains in these anchors.     

Acetylcholinesterases of the nematode Steinernema carpocapsae. Characterization of two types of amphiphilic forms differing in their mode of membrane association.

We analyzed the molecular forms of acetylcholinesterase (AChE) in the nematode Steinernema carpocapsae. Two major AChEs are involved in acetylcholine hydrolysis. The first class of AChE is highly sensitive to eserine (IC50 = 0.05 microM). The corresponding molecular forms are: an amphiphilic 14S form converted into a hydrophilic 14.5S form by mild proteolysis and two hydrophilic 12S and 7S forms. Reduction of the amphiphilic 14S form with 10 mM dithiothreitol produces hydrophilic 7S and 4S forms, indicating that it is an oligomer of hydrophilic catalytic subunits linked by disulfide bond(s) to a hydrophobic structural element that confers the amphiphilicity to the complex. Sedimentation coefficients suggest that 4S, 7S, 12S forms correspond to hydrophilic monomer, dimer, tetramer and that the 14S form is also a tetramer linked to one structural element. The second class of AChE is less sensitive to eserine (IC50 = 0.1 mM). Corresponding molecular forms are hydrophilic and amphiphilic 4S forms (monomers) and a major amphiphilic 7S form converted into a hydrophilic dimer by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C. This amphiphilic 7S form thus possesses a glycolipid anchor. It appears that Steinernema (a very primitive invertebrate) presents AChEs with two types of membrane association that closely resemble those described for amphiphilic G2 and G4 forms of AChE in more evolved animals.     

Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.     

The major protein of pancreatic zymogen granule membranes (GP-2) is anchored via covalent bonds to phosphatidylinositol

GP-2, the major integral protein characteristic of the pancreatic zymogen granule membrane can be released from the membrane by the action of a phosphatidylinositol specific phospholipase C (PI-PLC). In a hydrophobic/hydrophilic phase separation system using the non-ionic detergent Triton X-114, the membrane-bound form of the protein went from the detergent phase into the hydrophilic phase upon action of the phospholipase. PI-PLC solubilization of GP-2 unmasked an antigenic determinant similar to the cross-reacting determinant of the trypanosome variant surface glycoproteins. This determinant being a distinctive feature of the glycan moiety of phosphatidyl-inositol anchored membrane proteins, it established the glycosyl-phosphatidyl-inositol nature of the GP-2 membrane anchor. Since soluble GP-2 is also found in the contents of the granule and is secreted intact into the pancreatic juice, it is likely that one of the mechanisms responsible for its release could be a specific phospholipase. GP-2 is the first glycosyl-phosphatidyl-inositol-anchored protein that is integral to the membrane of an organelle and not located at the surface of the cell.     

The membrane-anchor of Paramecium temperature-specific surface antigens is a glycosylinositol phospholipid

The temperature-specific G surface antigen of Paramecium primaurelia strain 156 was biosynthetically labeled by [3H]myristic acid in its membrane-bound form, but not in its soluble form. It could be cleaved by a phosphatidylinositol-specific phospholipase C from Trypanosoma brucei or from Bacillus cereus which released its soluble form with the unmasking of a particular glycosidic immunodeterminant called the crossreacting determinant. The Paramecium enzyme, capable of converting its membrane-bound form into the soluble one, was inhibited by a sulphydril reagent in the same way as the trypanosomal lipase. From this evidence we propose that the Paramecium temperature-specific surface antigens are anchored in the plasma membrane via a glycophospholipid, and that an endogenous phospholipase C may be involved in the antigenic variation process.     

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.     

Determinants of substrate specificity of a second non-neuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We recently reported on a non-neuronal secreted acetylcholinesterase (AChE B) from the nematode parasite Nippostrongylus brasiliensis. Here we describe the primary structure and enzymatic properties of a second secreted variant, termed AChE C after the designation of native AChE isoforms from this parasite. As for the former enzyme, AChE C is truncated at the carboxyl terminus in comparison with the Torpedo AChE, and three of the 14 aromatic residues that line the active site gorge are substituted by nonaromatic residues, corresponding to Tyr70 (Ser), Trp279 (Asn) and Phe288 (Met). A recombinant form of AChE C was highly expressed by Pichia pastoris. The enzyme was monomeric and hydrophilic, and displayed a marked preference for acetylthiocholine as substrate. A double mutation (W302F/W345F, corresponding to positions 290 and 331 in Torpedo) rendered the enzyme 10-fold less sensitive to excess substrate inhibition and two times less susceptible to the bis quaternary inhibitor BW284C51, but did not radically affect substrate specificity or sensitivity to the 'peripheral site' inhibitor propidium iodide. In contrast, a triple mutant (M300G/W302F/W345F) efficiently hydrolysed propionylthiocholine and butyrylthiocholine in addition to acetylthiocholine, while remaining insensitive to the butyrylcholinesterase-specific inhibitor iso-OMPA and displaying a similar profile of excess substrate inhibition as the double mutant. These data highlight a conserved pattern of active site architecture for nematode secreted AChEs characterized to date, and provide an explanation for the substrate specificity that might otherwise appear inconsistent with the primary structure in comparison to other invertebrate AChEs.     

Altered GPI modification of insect AChE improves tolerance to organophosphate insecticides

The olive fruit fly Bactrocera oleae is the most destructive and intractable pest of olives. The management of B. oleae has been based on the use of organophosphate (OP) insecticides, a practice that induced resistance. OP-resistance in the olive fly was previously shown to be associated with two mutations in the acetylcholinesterase (AChE) enzyme that, apparently, hinder the entrance of the OP into the active site. The search for additional mutations in the ace gene that encodes AChE revealed a short deletion of three glutamines (??3Q) from a stretch of five glutamines, in the C-terminal peptide that is normally cleaved and substituted by a GPI anchor. We verified that AChEs from B. oleae and other Dipterans are actually GPI-anchored, although this is not predicted by the “big-PI” algorithm. The ??3Q mutation shortens the unusually long hydrophilic spacer that follows the predicted GPI attachment site and may thus improve the efficiency of GPI anchor addition. We expressed the wild type B. oleae AChE, the natural mutant ??3Q and a constructed mutant lacking all 5 consecutive glutamines (??5Q) in COS cells and compared their kinetic properties. All constructs presented identical K_m and k_cat values, in agreement with the fact that the mutations did not affect the catalytic domain of the enzyme. In contrast, the mutants produced higher AChE activity, suggesting that a higher proportion of the precursor protein becomes GPI-anchored. An increase in the number of GPI-anchored molecules in the synaptic cleft may reduce the sensitivity to insecticides.     

The solubilization of tetrameric alkaline phosphatase from human liver and its conversion into various forms by phosphatidylinositol phospholipase C or proteolysis

When membrane-bound human liver alkaline phosphatase was treated with a phosphatidylinositol (PI) phospholipase C obtained from Bacillus cereus, or with the proteases ficin and bromelain, the enzyme released was dimeric. Butanol extraction of the plasma membranes at pH 7.6 yielded a water-soluble, aggregated form that PI phospholipase C could also convert to dimers. When the membrane-bound enzyme was solubilized with a non-ionic detergent (Nonidet P-40), it had the Mr of a tetramer; this, too, was convertible to dimers with PI phospholipase C or a protease. Butanol extraction of whole liver tissue at pH 6.6 and subsequent purification yielded a dimeric enzyme on electrophoresis under nondenaturing conditions, whereas butanol extraction at pH values of 7.6 or above and subsequent purification by immunoaffinity chromatography yielded an enzyme with a native Mr twice that of the dimeric form. This high molecular weight form showed a single Coomassie-stained band (Mr = 83,000) on electrophoresis under denaturing conditions in sodium dodecyl sulfate, as did its PI phospholipase C cleaved product; this Mr was the same as that obtained with the enzyme purified from whole liver using butanol extraction at pH 6.6. These results are highly suggestive of the presence of a butanol-activated endogenous enzyme activity (possibly a phospholipase) that is optimally active at an acidic pH. Inhibition of this activity by maintaining an alkaline pH during extraction and purification results in a tetrameric enzyme. Alkaline phosphatase, whether released by phosphatidylinositol (PI) phospholipase C or protease treatment of intact plasma membranes, or purified in a dimeric form, would not adsorb to a hydrophobic medium. PI phospholipase C treatment of alkaline phosphatase solubilized from plasma membranes by either detergent or butanol at pH 7.6 yielded a dimeric enzyme that did not absorb to the hydrophobic medium, whereas the untreated preparations did. This adsorbed activity was readily released by detergent. Likewise, alkaline phosphatase solubilized from plasma membranes by butanol extraction at pH 7.6 would incorporate into phosphatidylcholine liposomes, whereas the enzyme released from the membranes by PI phospholipase C would not incorporate. The dimeric enzyme purified from a butanol extract of whole liver tissue carried out at pH 6.6 did not incorporate. We conclude that PI phospholipase C converts a hydrophobic tetramer of alkaline phosphatase into hydrophilic dimers through removal of the 1,2-diacylglycerol moiety of phosphatidylinositol. Based on these and others' findings, we devised a model of alkaline phosphatase's conversion into its various forms.     

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.     

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

Substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus examined using the resolved enantiomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate).

The substrate stereospecificity of phosphatidylinositol-specific phospholipase C from Bacillus cereus is examined using the resolved optical isomers of synthetic myo-inositol 1-(4-nitrophenyl phosphate), a chromogenic substrate for the phospholipase. The synthetic route employs mild acid-labile protecting groups and separation of the substituted myo-inositol enantiomers as the (-)-camphanyl ester diastereomers. Measurements of the initial rates of cleavage of the D and L enantiomers of the nitrophenyl substrate by phosphatidylinositol-specific phospholipase C from B. cereus show that this enzyme is essentially stereospecific for the D enantiomer. Under identical conditions, the rate of cleavage of the L isomer is less than 0.2% of that observed for the D isomer. The same is observed for the highly homologous enzyme from Bacillus thuringiensis. There is no measurable inhibition by the L enantiomer of the B. cereus enzyme acting on the D enantiomer, even when the molar ratio of L:D is 5, indicating that binding of the L enantiomer to the phospholipase is negligible. Thus, the enzyme active site is exquisitely sensitive to the stereochemistry of the myo-inositol group of the substrate.     

Characterization of the glycosyl-phosphatidylinositol-anchored human renal dipeptidase reveals that it is more extensively glycosylated than the pig enzyme.

Renal dipeptidase (EC 3.4.13.11) has been purified from human kidney cortex by affinity chromatography on cilastatin-Sepharose following solubilization with either n-octyl-beta-D-glucopyranoside or bacterial phosphatidylinositol-specific phospholipase C (PI-PLC). Phase separation in Triton X-114 revealed that the detergent-solubilized form was amphipathic and retained the glycosyl-phosphatidylinositol membrane anchor whereas the phospholipase solubilized form was hydrophilic. Both forms of the enzyme existed as a disulphide-linked dimer of two identical subunits of Mr 59,000 each. The glycosyl-phosphatidylinositol anchor of purified human renal dipeptidase was hydrolysed by a range of bacterial PI-PLCs and by a plasma phospholipase D. Mild acid treatment and nitrous acid deamination of the hydrophilic form revealed that the cross-reacting determinant, characteristic of the glycosyl-phosphatidylinositol anchor, was due exclusively to the inositol 1,2-cyclic phosphate ring epitope. The N-terminal amino acid sequences of the amphipathic and hydrophilic forms were identical, locating the membrane anchor at the C-terminus. The N-terminal sequence of human renal dipeptidase showed a high degree of similarity with that of the pig enzyme, and enzymic deglycosylation revealed that the difference in size of renal dipeptidase between these two species is due almost entirely to differences in the extent of N-linked glycosylation.     

Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells.

Incubation at 37 degrees C or treatment of granule membranes of chromaffin cells with Staphylococcus aureus phosphatidylinositol-specific phospholipase C converted from an amphiphilic to a hydrophilic form two proteins with molecular masses of 82 and 68 kDa respectively. Their release is time- and enzyme-concentration-dependent. We showed that they were immunoreactive with an anti-(cross-reacting determinant) antibody known to be revealed only after removal of a diacylglycerol anchor. Furthermore, the action of HNO2 suggests the presence of a non-acetylated glucosamine residue in the determinant. This is one of the first reports suggesting that a glycosylphosphatidylinositol anchor might exist in membranes other than the plasma membrane. We showed that the 68 kDa protein is probably not the subunit of dopamine (3,4-dihydroxyphenethylamine) beta-hydroxylase, an enzyme present in granules in both soluble and membrane-associated forms.     

Purification and properties of the membrane-bound form of acetylcholinesterase from chicken brain. Evidence for two distinct polypeptide chains

The major molecular form of acetylcholinesterase (AChE) from chicken brain is a membrane-bound glycoprotein with an apparent sedimentation coefficient of 11.4 S. Analysis of the purified protein by gel filtration, velocity sedimentation, and sodium dodecyl sulfate-gel electrophoresis shows that the solubilized enzyme is a globular tetramer with an apparent Mr = 420,000. This membrane-bound form of AChE is hydrophobic and readily aggregates in the absence of detergent. These aggregates are concentration-dependent, relatively stable in the presence of high salt concentrations, yet readily dissociate upon addition of detergent to the 11.4 S form, indicating that the interactions are hydrophobic. Polyclonal and monoclonal antibodies raised against chicken brain AChE purified by ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis precipitate AChE enzyme activity. However, these antibodies do not cross-react with the enzyme from chicken muscle which preferentially hydrolyses butyrylcholine. Immunoprecipitation of isotopically labeled enzyme molecules from tissue cultured brain cells and analysis by sodium dodecyl sulfate-gel electrophoresis shows that AChE consists of two polypeptide chains with apparent Mr = 105,000 (alpha) and 100,000 (beta) in a 1:1 ratio. Immunoblotting of brain AChE with either the polyclonal or monoclonal antibodies indicates that the alpha and beta chains share antigenic determinants. Furthermore, both polypeptide chains can be labeled with [3H]diisopropyl fluorophosphate, indicating that they each contain a catalytic site. This is the first indication that globular forms of AChE may consist of multiple polypeptide chains.     

Biochemical characterization of two distinct acetylcholinesterases possessing almost identical catalytic activity in the damselfly Vestalis gracilis

Most insects possess two different acetylcholinesterases (AChEs) (i.e., AChE1 and AChE2). It has been recently reported that only one AChE (either AChE1 or AChE2) has been selected as the main synaptic enzyme and it varies with different insect lineages (Kim et al., 2012, Kim and Lee, 2013). Interestingly, however, both AChE1 and AChE2 are almost equally active in a damselfly species, providing a unique example of the incomplete specialization of one AChE function after duplication, where, consequently, both AChE1 and AChE2 likely play a similar role in synaptic transmission. In this study, therefore, we investigated the tissue distribution patterns and the molecular and inhibitory properties of two AChEs (i.e., VgAChE1 and VgAChE2) from the Vestalis gracilis damselfly as a model species possessing two AChEs that are equally active. VgAChEs exhibited almost identical catalytic activity and were expressed in the central nervous system (CNS). The most predominant molecular form of both VgAChEs was a disulfide-bridged dimer, which is associated with the cell membrane via a glycosylphosphatidylinositol anchor. In an inhibition assay, however, VgAChE1 and VgAChE2 exhibited different sensitivities to organophosphate and carbamate insecticides depending on the structure of the inhibitors. These findings suggest that both VgAChEs have neuronal functions. In addition, soluble monomeric and cleaved molecular forms were detected in both the CNS and peripheral nervous system tissues by an AChE2-specific antibody, implying that VgAChE2 probably shares both neuronal and non-neuronal physiological functions in V. gracilis. Our results support the notion that both VgAChEs, paralogous of each other, are involved in synaptic transmission, with VgAChE2 being in the early stage of acquiring non-neuronal functions.     

Crystal structure of the phosphatidylinositol-specific phospholipase C from Bacillus cereus in complex with myo-inositol.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI-specific phospholipase C (PI-PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI-PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8-barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI-PLC in complex with its inhibitor, myo-inositol, at 2.6 A resolution (R factor = 19.5%). This substrate-like inhibitor interacts with a number of residues highly conserved among prokaryotic PI-PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI-PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.     

Characterization of Salt-Soluble Forms of Acetylcholinesterase from Bovine Brain

Abstract: The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimenting at 10.3S (∼40%), and a monomer sedimenting at 3.4S (∼60%). The enzyme is N -glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrates could be detected. Amino acid sequencing showed that the N terminus of SS-AChE is identical to that of DS-AChE. In tetrameric SS-AChE, two pairs of disulfide-linked dimers are associated by hydrophobic forces located in the C terminus. Antibodies were raised against a peptide identical to the last 10 amino acid residues of bovine brain DS-AChE. The peptide included the sequence of residues 574–583 (H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH) of the enzyme. The antibodies cross-reacted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of tetrameric SS-AChE at the C terminus led to the formation of an enzymatically active monomer, which did not react with anti-C-terminal antibody. Although the DS form of AChE contains a structural subunit that serves as membrane anchor, no anchor was detected in SS-AChE. Enzyme antigen immunoassays showed that SS-AChE reacted with all monoclonal antibodies directed against the catalytic subunit of DS-AChE, but not with monoclonal antibodies targeting the membrane-anchored subunits. From our results, we conclude that SS-AChE utilizes the same alternative splicing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor. The active monomer of SS-AChE is most likely derived from tetrameric forms by limited postsynthetic proteolysis.     

Acetylcholinesterase dynamics at the neuromuscular junction of live animals

At cholinergic synapses, acetylcholinesterase (AChE) is critical for ensuring normal synaptic transmission. However, little is known about how this enzyme is maintained and regulated in vivo. In this work, we demonstrate that the dissociation of fluorescently-tagged fasciculin 2 (a specific and selective peptide inhibitor of AChE) from AChE is extremely slow. This fluorescent probe was used to study the removal and insertion of AChE at individual synapses of living adult mice. After a one-time blockade of AChEs with fluorescent fasciculin 2, AChEs are removed from synapses initially at a faster rate (t(1/2) of approximately 3 days) and later at a slower rate (t(1/2) of approximately 12 days). Most of the removed AChEs are replaced by newly inserted AChEs over time. However, when AChEs are continuously blocked with fasciculin 2, the removal rate increases substantially (t(1/2) of approximately 12 h), and most of the lost AChEs are not replaced by newly inserted AChE. Furthermore, complete one-time inactivation of AChE activity significantly increases the removal of postsynaptic nicotinic acetylcholine receptors (AChRs). Finally, time lapse imaging reveals that synaptic AChEs and AChRs that are removed from synapses are co-localized in the same pool after being internalized. These results demonstrate a remarkable AChE dynamism and argue for a potential link between AChE function and postsynaptic receptor lifetime.