Electroanalysis of amino acid substitutions in bioengineered acetylcholinesterase.

This study reports the electrochemical profiling of Nippostrongylus brasiliensis acetylcholinesterase (AChE) wild-type and mutant proteins. An irreversible oxidation signal of electro-active tyrosine (Y), tryptophan (W) and cysteine (C) residues in five mutant proteins along with the wild-type AChE were detected using square-wave voltammetry (SWV) on screen-printed carbon electrodes. Significant differences were observed in the W303L, T65Y and M301W substituted proteins showing a 25-35% higher peak current intensity compared to the Y349Y and F345Y mutants. It was predicted that AChE substituted with electrochemically active residues would produce the greatest signals and this trend was observed in the T65Y, M301W and Y349L mutants. However, conformational changes in the proteins structure as a result of the substitutions appeared to be most influential on peak current intensities. This was demonstrated by the W303L and F345Y mutant enzymes. The current intensity of W303L was greatest despite the removal of its electro-active W residue whereas the F345Y mutant had the lowest peak value despite the addition of an electro-active Y residue. The preliminary results of this study demonstrate that SWV provides a promising tool to probe the presence of electro-active amino acid residues on the surface of a protein produced through bioengineering.

[Separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase from culture medium of genetic engineering Pichia pastoris].

To develop a simple, fast and highly efficient method for the separation and purification of recombinant Nippostrongylus brasiliensis acetylcholinesterase (NbAChE) from culture medium of genetic engineering Pichia pastoris, Q-Sepharose Fast Flow chromatographic medium was used. The chromatographic column was 20 cm x 3.5 cm i. d. The elution buffer A was 20 mmol/L NaH2PO4-Na2HPO4 (pH 8) and buffer B was 1 mol/L NaCl + 20 mmol/L NaH2PO4- Na2HPO4(pH 8). The elution gradient was nonlinear. It was firstly eluted with 10% B for 300 min, then with 30% B for 300 min, finally with 100% B for 300 min. The flow rate of mobile phase was 6 mL/min. The obtained recombinant NbAChE was proved to be homogeneous on sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and its relative molecular mass was estimated to be approximately 66 000, which was consistent with that reported in literature. The total activity recovery of this purification method was 52.6% and the purification factor was 3.87. The final specific activity of recombinant NbAChE was 2 837 U/mg. This chromatographic process is simple and highly efficient. It can be used to separate and purify recombinant NbAChE from culture medium of Pichia pastoris harboring NbAChE gene.

Screen-printed bienzymatic sensor based on sol-gel immobilized Nippostrongylusbrasiliensis acetylcholinesterase and a cytochrome P450 BM-3 (CYP102-A1) mutant.

Here, we describe the development of a bi-enzymatic biosensor that simplifies the sample pretreatment steps for insecticide detection, and opens the way for a highly sensitive detection of phosphorothionates in food. These compounds evolve their inhibitory activity towards acetylcholinesterases (AChEs) only after oxidation, which is performed in vivo by P450 monooxygenases. Consequently, phosphorothionates require a suitable sample pretreatment by selective oxidation to be detectable in AChE based systems. In this study, enzymatic phosphorothionate activation and AChE inhibition were integrated in a single biosensor unit. A triple mutant of cytochrome P450 BM-3 (CYP 102-A1) and Nippostrongylus brasiliensis AChE (NbAChE) was immobilized using a fluoride catalyzed sol-gel process. Different sol-gel types were fabricated and characterized regarding enzyme loading capacity and enzyme activity containment. The enzyme sol-gel itself already proved to be suitable for the highly sensitive detection of paraoxon and parathion in a spectrometric assay. A method for screen-printing of this enzyme sol-gel on thick film electrodes was developed. Finally, amperometric biosensors containing coimmobilized NbAChE and the cytochrome P450 BM-3 mutant were produced and characterized with respect to signal stability, organophosphate detection, and storage stability. The detection limits achieved were 1 microg/L for paraoxon and 10 microg/L for parathion, which is according to EC regulations the highest tolerable pesticide concentration in infant food.

High yield production of a mutant Nippostrongylus brasiliensis acetylcholinesterase in Pichia pastoris and its purification.

The mutant M301A of the acetylcholinesterase B from Nippostrongylus brasiliensis (NbAChE) was produced in a high-cell-density fermentation of a recombinant methylotrophic yeast Pichia pastoris. Dissolved oxygen (DO) spikes were used as an indicator for feeding the carbon source. Wet cell weight (WCW) reached after 8 days a maximum value of 316 g/L and the OD600 at this time was 280. The acetylcholinesterase activity increased up to 6,600 U/mL corresponding to an expression rate of 2 g of NbAChE per liter supernatant. The specific activity of the mutant NbAChE was determined after purification as 3,300 U/mg. Active site titration with chlorpyrifos, a strong AChE inhibitor, yielded in a specific activity of 3,400 U/mg. The enzyme was secreted by Pichia pastoris. Therefore, it could be concentrated from culture broth by cross-flow-filtration (50 kDa cut-off membrane). It was further purified in one-step anion-exchange chromatography, using a XK 50/20 column filled with 125 mL Q Sepharose HP. Mutant NbAChE was purified 1.9-fold up to a purity of 97% and a yield of 87%. The isolated enzyme was nearly homogenous, as seen on the silver stained SDS-PAGE as well as by a single peak after gel filtration. This extraordinary high expression rate and the ease of purification is an important prerequisite for their practical application, for example in biosensors for the detection of neurotoxic insecticides.

Insecticide detection through protein engineering of Nippostrongylus brasiliensis acetylcholinesterase B.

The sensitivity of acetylcholinesterase (AChE) biosensors for insecticide detection could be increased substantially by engineering AChE B of Nippostrongylus brasiliensis. The introduction of 10 single and 4 double mutations into the AChE peptide chain led to an increase in sensitivity to 10 of the 11 insecticides tested. The combination of three mutants with the wild-type enzyme in a multienzyme biosensor array enabled the detection of 11 out of the 14 most important organophosphates and carbamates at concentrations below 10 microg/kg, the maximum residue limit of infant food. The detection limit for pirimiphos methyl could be reduced from 10 microg/L to a value as low as 1 ng/L (3.5 x 10(-)(12) mol/L). The newly created biosensors exhibited an extraordinary high storage stability. There was no loss of sensitivity of N. brasiliensis AChE B, immobilized on screen-printed, disposable electrodes, even after 17-month storage at room temperature.

Villus epithelial injury induced by infection with the nematode Nippostrongylus brasiliensis is associated with upregulation of granzyme B.

Intestinal parasite infections induce thymus-dependent villus atrophy, but the effector mechanisms directly responsible for the development of villus atrophy are not thoroughly understood. In this study, we analyzed the expression of cytotoxic factors or ligands in athymic nude rnu/rnu rats and their littermate euthymic rnu/+ rats infected with the nematode Nippostrongylus brasiliensis. Morphometric analyses showed that partial villus atrophy developed 10 days after infection in euthymic but not in athymic rats, whereas crypt hyperplasia occurred in both types of animal. Reverse transcription-polymerase chain reaction analyses of the isolated jejunal epithelial fraction showed that the development of villus atrophy in euthymic rats was positively correlated with an increase of granzyme B transcript levels but not with Fas ligand or tumor necrosis factor-alpha expression. In addition, the number of granzyme B-immunoreactive cells was increased significantly in euthymic rat villus epithelium and the propria mucosa after infection. The CD8+ cell number did not change significantly. Collectively, these findings showed that significant increases in the number of cells that express the cytotoxic factor granzyme B occur in the nematode-infected small intestine of immunocompetent hosts. The type of cells that express granzyme B and their role in the progression of enteropathy remain to be elucidated.

Mucin-type O-glycosylation in helminth parasites from major taxonomic groups: evidence for widespread distribution of the Tn antigen (GalNAc-Ser/Thr) and identification of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity.

This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.

The C-terminal T peptide of acetylcholinesterase enhances degradation of unassembled active subunits through the ERAD pathway.

The catalytic domain of acetylcholinesterase AChE(T) subunits is followed by a C-terminal T peptide which mediates their association with the proline-rich attachment domain (PRAD) of anchoring proteins. Addition of the T peptide induced intracellular degradation and concomitantly reduced to variable degrees the secretion of AChE species differing in their oligomerization capacity and of human alkaline phosphatase. The T peptide forms an amphiphilic alpha-helix, containing a series of conserved aromatic residues. Replacement of two, four or five aromatic residues gradually suppressed degradation and increased secretion. Co-expression with a PRAD- containing protein induced the assembly of PRAD-linked tetramers in the endoplasmic reticulum (ER) and allowed partial secretion of a dimerization- defective mutant; by masking the aromatic side chains, hetero-oligomerization rescued this enzyme from degradation. Degradation was due to ERAD, since it was not blocked by brefeldin A but was sensitive to proteasome inhibitors. Kifunensine reduced degradation, suggesting a cooperativity between the glycosylated catalytic domain and the non-glycosylated T peptide. This system appears particularly well suited to analyze the mechanisms which determine the degradation of correctly folded multidomain proteins in the ER.

A distinct family of acetylcholinesterases is secreted by Nippostrongylus brasiliensis.

A third variant of acetylcholinesterase (AChE A) secreted by the parasitic nematode Nippostrongylus brasiliensis has been isolated which shows 63-64% identity to AChE B and AChE C, with a truncated carboxyl terminus and a short internal insertion relative to AChEs from other species. Three of the fourteen aromatic residues which line the active site gorge in Torpedo AChE are substituted by non-aromatic residues (Y70T, W279D and F288M). All three enzymes have 8 cysteine residues in conserved positions, including 6 which have been implicated in disulphide bonds in other AChEs. Phylogenetic analysis suggests that these enzymes form a distinct group which evolved after speciation and are most closely related to ACE-2 of Caenorhabditis elegans. Recombinant AChE A secreted by Pichia pastoris was monomeric and hydrophilic, with a substrate preference for acetylthiocholine and negligible activity against butyrylthiocholine. A model structure of AChE A built from the coordinates of the Torpedo californica AChE suggests that W345 (F331 in Torpedo) limits the docking of butyrylcholine. This model is consistent with mutational analysis of the nematode enzymes. Expression of AChE A is regulated at the transcriptional level independently of the other 2 secreted variants, with maximal expression by fourth stage larvae and young adult worms. These enzymes thus appear to represent an unusual family of AChEs with conserved structural features which operate outside the normal boundaries of known functions in regulation of endogenous neurotransmitter activity.

Blood group A glycosyltransferase occurring as alleles with high sequence difference is transiently induced during a Nippostrongylus brasiliensis parasite infection.

Neutral mucin oligosaccharides from the small intestine of control rats and rats infected with the parasite Nippostrongylus brasiliensis were released and analyzed by gas chromatography-mass spectrometry. Infected animals expressed seven blood group A-like structures that were all absent in the control animals. The blood group A nature of these epitopes was confirmed by blood group A reactivity of the prepared mucins, of which Muc2 was one. Transferase assays and Northern blotting on small intestines from infected animals showed that an alpha-N-acetylgalactosaminyltransferase similar to the human blood group A glycosyltransferase had been induced. The expression was a transient event, with a maximum at day 6 of the 13-day-long infection. The rat blood group A glycosyltransferase was cloned, revealing two forms with an amino acid similarity of 95%. Both types had blood group A transferase activity and were probably allelic because none of 12 analyzed inbred strains carried both types. The second type was found in outbred rats and in one inbred strain. First generation offspring of inbred rats of each type were heterozygous, further supporting the allelic hypothesis. The transient induction and the large allelic variation could suggest that glycosyltransferases are part of a dynamic system altering mucins and other glycoconjugates as a protecting mechanism against microbial challenges.

Acetylcholinesterase assay for rapid expression screening in liquid and solid media.

The synaptic enzyme acetylcholinesterase (AChE), which is the target of many insecticides and potential warfare agents, is implied in Alzheimer's disease and is a good potential candidate to be used in biosensors. This promotes a strong demand for production of recombinant AChE to be used in various studies. A promising expression system is the yeast Pichia pastoris, but the expression efficiency needs to be improved. Optimization studies require a rapid and efficient screening test to detect positive yeast colonies after transformation. Using indoxylacetate as a substrate, we designed a chromogenic test that is not interfered with by the culture media background color and, thus, is suitable for microplate screening. Moreover, it was possible to adapt the test for direct on-plate detection of AChE-expressing colonies.

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.

Nippostrongylus brasiliensis: infection induces upregulation of acetylcholinesterase activity on rat intestinal epithelial cells.

Expression of cholines terases and muscarinic acetylcholine receptors in the jejunal mucosa has been investigated during infection of rats with the nematode parasite Nippostrongylus brasiliensis. Selective expression of m3 receptors was observed on epithelial cells from uninfected rats and animals 7 days postinfection, and saturation binding with [(3)H]quinuclidinyl benzilate indicated that receptor expression on cell membranes was unaltered by infection. Butyrylcholinesterase was highly expressed in mucosal epithelia, but acetylcholinesterase was present at low levels in uninfected animals. In contrast, discrete foci of intense acetylcholinesterase activity were observed on the basement membrane of intestinal epithelial cells in animals infected with N. brasiliensis. This was demonstrated to be due to upregulation of expression of endogenous enzyme, which peaked at Day 10 postinfection and subsequently declined to preinfection levels. It is suggested that this occurs in response to hyper-activation of the enteric nervous system as a result of infection, and may benefit the host by limiting excessive fluid secretion due to cholinergic stimulation.

Cloning, expression, and properties of a nonneuronal secreted acetylcholinesterase from the parasitic nematode Nippostrongylus brasiliensis.

We have isolated a full-length cDNA encoding an acetylcholinesterase secreted by the nematode parasite Nippostrongylus brasiliensis. The predicted protein is truncated in comparison with acetylcholinesterases from other organisms such that the carboxyl terminus aligns closely to the end of the catalytic domain of the vertebrate enzymes. The residues in the catalytic triad are conserved, as are the six cysteines which form the three intramolecular disulfide bonds. Three of the fourteen aromatic residues which line the active site gorge in the Torpedo enzyme are substituted by nonaromatic residues, corresponding to Tyr-70 (Thr), Trp-279 (Asn), and Phe-288 (Met). High level expression was obtained via secretion from Pichia pastoris. The purified enzyme behaved as a monomeric hydrophilic species. Although of invertebrate origin and possessing the above substitutions in the active site gorge residues, the enzyme efficiently hydrolyzed acetylthiocholine and showed minimal activity against butyrylthiocholine. It displayed excess substrate inhibition with acetylthiocholine at concentrations over 2. 5 mM and was highly sensitive to both active site and "peripheral" site inhibitors. Northern blot analysis indicated a progressive increase in mRNA for AChE B in parasites isolated from 6 days postinfection.

Nippostrongylus brasiliensis: characterisation of a somatic amphiphilic acetylcholinesterase with properties distinct from the secreted enzymes.

We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.

Purification and properties of monomeric (G1) forms of acetylcholinesterase secreted by Nippostrongylus brasiliensis.

Acetylcholinesterase (AChE) activity secreted by Nippostrongylus brasiliensis was resolved by sucrose density centrifugation and gel permeation chromatography in single peaks estimated at 4.3 S and 60-85 kDa, respectively. Sedimentation was unaffected by the inclusion of detergent. AChE was purified by affinity chromatography on 9-[Nbeta-(epsilon-aminocaproyl)-beta-aminopropylamino]-acridinium bromide hydrobromide-coupled sepharose 4B. Three forms of the enzyme (A, B and C) were distinguished by non-denaturating polyacrylamide gel electrophoresis, and displayed apparent masses of 74, 69 and 71 kDa respectively when resolved by SDS-PAGE. All three isoforms showed a preference for acetylthiocholine (ASCh) as substrate. They were highly sensitive to inhibition by the AChE-specific inhibitor bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with inhibitor concentration reducing initial activity by 50% (IC50) between 0.1 and 0.8 microM, but activity was unaffected by tetramonoisopropylpyrophosphortetramide (iso-OMPA) at concentrations up to 10 mM. We conclude that the secreted enzymes are authentic AChEs of hydrophilic monomeric (G1) form and broadly similar properties, but which can be distinguished by molecular mass, inhibitor sensitivities and the degree of excess substrate inhibition.

Characterization of a platelet-activating factor acetylhydrolase secreted by the nematode parasite Nippostrongylus brasiliensis.

Nippostrongylus brasiliensis, a small nematode parasite of the gastrointestinal tract of rodents, secretes an enzyme that cleaves the proinflammatory molecule platelet-activating factor to its inactive lyso-form. The enzyme activity of Ca(2+)-dependent and does not exhibit interfacial activation. It does not require the addition of reducing agents for maximal activity, and is not inhibited by thiol-active reagents. Sensitivity of inhibitors suggests the involvement of serine and histidine residues in the enzyme activity. As described for other platelet-activating factor acetylhydrolases, it cannot cleave, nor is it inhibited by, long-chain diacyl phospholipids that are typical substrates for phospholipases A2. The purified enzyme was resolved by SDS/PAGE as a heterodimer composed of two protein subunits with apparent molecular masses of 38 and 25 kDa. The properties of the nematode enzyme thus differ from those described for the mammalian enzymes, but are more closely related to those of an acetylhydrolase than a phospholipase.

Cysteine protease of the nematode Nippostrongylus brasiliensis preferentially evokes an IgE/IgG1 antibody response in rats.

Some cysteine proteases such as papain and those of mites and schistosomes have potent allergenic properties. To clarify the allergenicity of nematode cysteine proteases, the enzyme was purified from the intestinal nematode Nippostrongylus brasiliensis using cation exchange chromatography and gel filtration chromatography. The purified protease, of 16 kD and pI 8.5, showed maximum enzyme activity at pH 5.5 and substrate preference for Z-Phe-Arg-MCA. The specific inhibitors of cysteine protease leupeptin, iodoacetic acid, and E-64, completely suppressed the activity, indicating that the purified enzyme belongs to the cysteine protease family. Cysteine protease activity was found not only in somatic extract, but also in the excretory-secretory (ES) product of the nematode. When anti-cysteine protease immunoglobulin isotypes were examined in sera from rats infected with N. brasiliensis, a high level of IgG1 and a lower level of IgE antibody were detected. Depletion of IgG antibodies from the sera using protein G affinity columns resulted in a marked increase in reactivity of anti-cysteine protease IgE with the antigen, possibly due to the removal of competing IgG antibodies. In contrast to IgE and IgG1, production of anti-cysteine protease IgG2a was negligible. These results indicate that the nematode cysteine protease preferentially evokes an IgE/IgG1 antibody response.

The identification of a variant form of cystathionine beta-synthase in nematodes.

Characterization of the physical and catalytic properties of the enzyme responsible for nematode "activated L-serine sulfhydrase" activity (L-cysteine + R-SH-->cysteine thioether + H2S) has led to its identification as a novel, variant form (allelozyme) of cystathionine beta-synthase that is distinct from a mammalian-type synthase also present in nematodes. Additional work has demonstrated the ability of live Panagrellus redivivus to produce H2[35S] from exogenous L-[35S]cysteine and 2-mercaptoethanol, thus providing preliminary evidence for the in vivo operation of the activated L-serine sulfhydrase reaction in nematodes.