Behavioral and immunological effects of exogenous butyrylcholinesterase in rhesus monkeys.

Although conventional therapies prevent organophosphate (OP) lethality, laboratory animals exposed to such treatments typically display behavioral incapacitation. Pretreatment with purified exogenous human or equine serum butyrylcholinesterase (Eq-BuChE), conversely, has effectively prevented OP lethality in rats and rhesus monkeys, without producing the adverse side effects associated with conventional treatments. In monkeys, however, using a commercial preparation of Eq-BuChE has been reported to incapacitate responding. In the present study, repeated administration of commercially prepared Eq-BuChE had no systematic effect on behavior in rhesus monkeys as measured by a six-item serial probe recognition task, despite 7- to 18-fold increases in baseline BuChE levels in blood. Antibody production induced by the enzyme was slight after the first injection and more pronounced following the second injection. The lack of behavioral effects, the relatively long in vivo half-life, and the previously demonstrated efficacy of BuChE as a biological scavenger for highly toxic OPs make BuChE potentially more effective than current treatment regimens for OP toxicity.

Stable complexes involving acetylcholinesterase and amyloid-beta peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer's fibrils.

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.

A monoclonal antibody against acetylcholinesterase inhibits the formation of amyloid fibrils induced by the enzyme.

A monoclonal antibody (mAb) 25B1 directed against fetal bovine-serum acetylcholinesterase (FBS AChE) was used to examine the ability of the cholinergic enzyme to promote the assembly of amyloid-beta peptides (A beta) into Alzheimers fibrils. This mAb binds to the peripheral anionic site of the enzyme and allosterically inhibits catalytic activity of FBS AChE. Several techniques, including thioflavine-T fluorescence, turbidity, and negative-staining at the electron microscopy level, were used to assess amyloid formation. Inhibition of amyloid formation was dependent on the molar ratio AChE:mAb 25B1, and at least 50% of the inhibition of the AChE promoting effect occurs at a molar ratio similar to that required for inhibition of the esterase activity. Our results suggest that mAb 25B1 inhibits the promotion of the amyloid fibril formation triggered by AChE by affecting the lag period of the A beta aggregation process.

Different effects of two peripheral anionic site-binding ligands on acetylcholinesterase active-site gorge topography revealed by electron paramagnetic resonance.

Both propidium and monoclonal antibody (mAb) 25B1 bind to the peripheral anionic site region of fetal bovine serum acetylcholinesterase (FBS AChE). Using electron paramagnetic resonance (EPR) with spin-labelled organophosphate specifically bound to the AChE active-site serine, we studied the effects of both ligands on the topography of the AChE active-site gorge. After incubation of FBS AChE with Fab fragments of mAb 25B1, freedom of motion of our spin label became more restricted, suggesting closing of the gorge. Stabilization against heat denaturation was also observed. No alterations in the freedom of motion or protection against heat denaturation could be detected after propidium binding. Our results demonstrate that two ligands binding to the peripheral anionic site region of AChE have different effects, suggesting a complex structure for this region of the molecule that allows various types of interactions with different ligands. We also demonstrate that EPR is a suitable tool for studying microtopographical alterations at the active sites of cholinesterases.

Characterization of monoclonal antibodies that inhibit the catalytic activity of acetylcholinesterases.

Monoclonal antibodies were generated against fetal bovine serum acetylcholinesterase and fetal bovine serum acetylcholinesterase inhibited by diisopropyl fluorophosphate or 7-(methylethoxyphosphinyloxy)-1-methylquinolinium iodide. Six monoclonal antibodies inhibited 70 to > 98% of the catalytic activity of fetal bovine serum acetylcholinesterase. Inhibition of serum acetylcholinesterase from several mammalia by four monoclonal antibodies showed broad cross-reactivity. In all cases, monoclonal antibodies bound to the native form of acetylcholinesterases. None reacted with serum butyrylcholinesterases from various species. Although all monoclonal antibodies inhibited catalytic activity of acetylcholinesterases, the site of interaction with acetylcholinesterase appeared to differ for several antibodies. Two types of acetylcholinesterase:monoclonal antibody complexes were formed: one between tetrameric forms and another between catalytic subunits within the tetramer. Monoclonal antibodies that inhibited acetylcholinesterase activity at > 98% also considerably slowed binding of diisopropyl fluorophosphate and other organophosphorus compounds to the acetylcholinesterase:monoclonal antibody complex. Binding of these monoclonal antibodies to acetylcholinesterase influenced function of the enzyme's peripheral anionic site. None of the antibodies bound to the esteratic site of acetylcholinesterase. Monoclonal antibodies caused changes in catalytic activity of acetylcholinesterase by interaction at a site remote from the catalytic site, presumably at the entrance to the active site gorge.

Analysis of functional epitopes on the dengue 2 envelope (E) protein using monoclonal IgM antibodies.

Forty-two hybridomas secreting IgM antibody against dengue virus were derived from spleen cells of dengue 2 infected mice. Antibody from 27 of these recognised the E protein of this virus. Of the 22 antibodies which neutralised dengue 2, only two cross-reacted with other flaviviruses. These 22 antibodies recognised three discrete domains on dengue virions. Competitive binding studies with IgG monoclonal antibodies suggested that two of the three domains were recognised by both IgG and IgM antibodies and that there were two additional domains which may be recognised exclusively by either IgG or IgM antibodies. IgM antibodies reacting with domains recognised by IgG antibodies that enhanced infection of susceptible cells by dengue 2, had no enhancing properties. None of the IgM antibodies activated the serum complement system after reacting with dengue 2 virions.

Epitope mapping of form-specific and nonspecific antibodies to acetylcholinesterase.

We have mapped the epitopes to which two monoclonal antibodies against acetylcholinesterase (AChE) from Torpedo californica are directed. One antibody, 2C9, has equivalent affinity for both the 5.6S (amphiphilic) and 11S (hydrophilic) enzyme forms; the other, 4E7, recognizes only the amphiphilic form and has been shown previously to require an N-linked oligosaccharide residue on the protein. Isolation of cyanogen bromide peptides from the amphiphilic form and assay by a competition ELISA for 2C9 and by a direct binding ELISA for 4E7 identified the same peptide, residues 44-82, as containing epitopes against both antibodies. The epitope for 4E7 includes the oligosaccharide conjugated to Asp59, an N-linked glycosylation site not present in mouse AChE. A 20-amino-acid synthetic peptide, RFRRPEPKKPWSGVWNASTY, representing residues 44-63, was synthesized and found to inhibit completely 2C9 binding to 5.6S enzyme at molar concentrations comparable to those of the cyanogen bromide peptide. It was unreactive with 4E7. Fractionation of the synthetic peptide further localized the 2C9 epitope. Peptides RFRRPEPKKPW and KPWSGVWNASTY both reacted but less so than the entire synthetic peptide at equivalent molar concentrations, whereas the peptide RPEPKKPWSGVWNASTY was as effective as the larger synthetic peptide. The crystal structure of AChE shows the peptide to be on the surface of the molecule as part of a convex hairpin loop starting before the first alpha-helix.

Cholinesterases as scavengers for organophosphorus compounds: protection of primate performance against soman toxicity.

The present treatment for poisoning by organophosphates consists of multiple drugs such as carbamates, antimuscarinics, and reactivators in pre- and post-exposure modalities. Recently an anticonvulsant, diazapam, has been included as a post-exposure drug to reduce convulsions and increase survival. Most regimens are effective in preventing lethality from organophosphate exposure but do not prevent toxic effects and incapacitation observed in animals and likely to occur in humans. Use of enzymes such as cholinesterases as pretreatment drugs for sequestration of highly toxic organophosphate anticholinesterases and alleviation of side effects and performance decrements was successful in animals, including non-human primates. Pretreatment of rhesus monkeys with fetal bovine serum acetylcholinesterase protected them against lethal effects of soman (up to 5 LD50) and prevented signs of OP toxicity. Monkeys pretreated with fetal bovine serum acetylcholinesterase were devoid of behavioral incapacitation after soman exposure, as measured by serial probe recognition or primate equilibrium platform performance tasks. Use of acetylcholinesterase as a single pretreatment drug provided greater protection against both lethal and behavioral effects of potent organophosphates than current multicomponent drug treatments that prevent neither signs of toxicity nor behavioral deficits. Although use of cholinesterases as single pretreatment drugs provided complete protection, its use for humans may be limited, since large quantities will be required, due to the approximately 1:1 stoichiometry between organophosphate and enzyme. Bisquaternary oximes, particularly HI-6, have been shown to reactivate organophosphate-inhibited acetylcholinesterase at a rapid rate. We explored the possibility that enzyme could be continually reactivated in animals pretreated with fetal bovine serum acetylcholinesterase, followed by an appropriate dose of reactivator, and challenged with repeated doses of sarin. In in vitro experiments, stoichiometry greater than 1:400 for enzyme:sarin was achieved; in vivo stoichiometry in mice was 1:65. Pretreatment of mice with fetal bovine serum acetylcholinesterase and HI-6 amplified the effectiveness of exogenous enzyme as a scavenger for organophosphate.

Immunochemical characterization of anti-acetylcholinesterase inhibitory monoclonal antibodies.

Monoclonal antibodies (mAbs) were prepared against native or DFP-inhibited Torpedo californica acetylcholinesterase and native or DFP-, MEPQ-, and soman-inhibited fetal bovine serum acetylcholinesterase. The cross reactivity of these antibodies with acetylcholinesterases from various species and their ability to inhibit catalytic activity were determined. Eight antibodies were found to inhibit catalytic activity of either Torpedo or fetal bovine serum enzyme. In all cases the antibodies bound to the native form of the enzymes and in some cases even to the denatured form. None of the antibodies recognized human or horse serum butyrylcholinesterase. Sucrose density gradient centrifugation of enzyme-antibody complexes provided two types of profiles, one with multiple peaks, indicating numerous complexes between tetrameric forms of the enzyme, and the other with single peaks, demonstrating complex formation within the tetrameric form. Different antibodies appeared to interact with slightly different regions, but in all cases the binding encompassed the peripheral anionic site. Decrease in catalytic activity of the enzyme was most likely caused by conformational changes in the enzyme molecule resulting from interaction with these mAbs.

Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins.

Based on the recently determined X-ray structures of Torpedo californica acetylcholinesterase and Geotrichum candidum lipase and on their three-dimensional superposition, an improved alignment of a collection of 32 related amino acid sequences of other esterases, lipases, and related proteins was obtained. On the basis of this alignment, 24 residues are found to be invariant in 29 sequences of hydrolytic enzymes, and an additional 49 are well conserved. The conservation in the three remaining sequences is somewhat lower. The conserved residues include the active site, disulfide bridges, salt bridges, and residues in the core of the proteins. Most invariant residues are located at the edges of secondary structural elements. A clear structural basis for the preservation of many of these residues can be determined from comparison of the two X-ray structures.

Use of cholinesterases as pretreatment drugs for the protection of rhesus monkeys against soman toxicity.

Purified fetal bovine serum acetylcholinesterase (FBS AChE) and horse serum butyrylcholinesterase (BChE) were successfully used as single pretreatment drugs for the prevention of pinacolyl methylphosphonofluoridate (soman) toxicity in nonhuman primates. Eight rhesus monkeys, trained to perform Primate Equilibrium Platform (PEP) tasks, were pretreated with FBS AChE or BChE and challenged with a cumulative level of five median lethal doses (LD50) of soman. All ChE-pretreated monkeys survived the soman challenge and showed no symptoms of soman toxicity. A quantitative linear relation was observed between the soman dose and the neutralization of blood ChE. None of the four AChE-pretreated animals showed PEP task decrements, even though administration of soman irreversibly inhibited nearly all of the exogenously administered AChE. In two of four BChE-pretreated animals, a small transient PEP performance decrement occurred when the cumulative soman dose exceeded 4 LD50. Performance decrements observed under BChE protection were modest by the usual standards of organophosphorus compound toxicity. No residual or delayed performance decrements or other untoward effects were observed during 6 weeks of post-exposure testing with either ChE.

Membrane-bound acetylcholinesterase: an early differentiation marker for skeletal myoblasts.

Cell-bound acetylcholinesterase (AChE) was found to be an early differentiation marker on embryonic chick skeletal myoblasts in mixed primary cell cultures. AChE biosynthesis was detected and characterized by (a) a sensitive microtiter assay, (b) use of selective inhibitors, and (c) with mono- and polyclonal antibodies. Both secreted and cell-bound AChE appeared on the first day in culture, at a time when no muscle cell fusion was observed. Characterization of this enzyme revealed that true AChE was bound and secreted by myoblasts. BW284c51, which permeates cell membranes poorly, inhibited all the cell-associated AChE activity on myoblasts, suggesting that the activity measured was on the outer cell surface. On the other hand, fibroblasts appeared to have no or very little bound enzyme and the low level of secreted enzyme activity had the characteristics of pseudo-, or butyrylcholinesterase. Polyclonal anti-Torpedo californica electroplax AChE antibody and several monoclonal antibodies were found to bind specifically to chick myoblasts. Since the cells had not been made permeable before antibody binding, a membrane-bound form of the enzyme was most likely being detected. The cell-bound true AChE was present in identifiable quantities from the first day of culture. Membrane-bound AChE can thus serve as an early differentiation marker for embryonic chick myoblasts in mixed primary cultures.

Protection of rhesus monkeys against soman and prevention of performance decrement by pretreatment with acetylcholinesterase.

The ability of acetylcholinesterase from fetal bovine serum (FBS AChE) to protect against soman, a highly toxic organophosphorus (OP) compound, was tested in rhesus monkeys. Intravenous administration of FBS AChE produced a minimal behavioral effect on the serial probe recognition task, a sensitive test of cognitive function and short-term memory. Pharmacokinetic studies of injected FBS AChE indicated a plasma half-life of 40 hr for FBS AChE in monkeys. Both in vitro and in vivo titration of FBS AChE with soman produced a 1:1 stoichiometry between organophosphate-inhibited FBS AChE and the cumulative dose of the toxic stereoisomers of soman. Administration of FBS AChE protected monkeys against the lethal effects of up to 2.7 LD50 of soman and prevented any signs of organophosphate intoxication, e.g., excessive secretions, respiratory depression, muscle fasciculations, or convulsions. In addition, monkeys pretreated with FBS AChE were devoid of any behavioral incapacitation after soman challenge, as measured by the serial probe recognition task. Compared to the current multicomponent drug treatment against soman, which does not prevent the signs or the behavioral deficits resulting from OP intoxication, use of FBS AChE as a single pretreatment drug provides significantly effective protection against both the lethal and the behavioral effects of soman.

Use of recombinant fusion proteins and monoclonal antibodies to define linear and discontinuous antigenic sites on the dengue virus envelope glycoprotein.

Sixteen overlapping fragments of the dengue-2 virus envelope (E) protein, expressed as trpE-E fusion products in Escherichia coli, were used to map the epitopes defined by a panel of 20 monoclonal antibodies (MAbs) by immunoblotting. Using this technique, the amino acid sequence of six antigenic domains on the E protein was characterized. Nonneutralizing MAbs were found to define either linear-specific, subcomplex-specific (amino acids 22-58), and complex-specific (amino acids 304-332) epitopes or a subcomplex conformational-dependent epitope requiring the presence of two closely linked amino acid sequences from the E protein, 60-97 and 298-397. Neutralizing MAbs, however, defined either group-reactive epitopes present on two overlapping domains (amino acids 60-135; amino acids 60-205) or type-, subcomplex-, complex-, subgroup-, and group-specific determinants (amino acids 298-397). These neutralizing epitopes were all found to be dependent upon disulfide bridges. Our results suggest that the maintenance of a topographical arrangement of discontinuous antigenic domains in the flavivirus E-protein is necessary to induce neutralizing and protective antibodies.

A microtiter assay for determining protein, acetylcholinesterase activity, and G418 (Neomycin) resistance in cultured cells.

The Coomassie brilliant blue assay for the determination of protein has been extended to rapidly and conveniently measure the protein concentration of cells growing in culture in a 96-well microtiter format. Modifications of the standard assay include sodium hydroxide to solubilize the cells and ovalbumin, instead of bovine serum albumin, as a protein standard. The procedure allows a large number of small samples to be assayed simultaneously. Two examples of its use, enzyme-specific activity and drug resistance, are shown. An assay for acetylcholinesterase activity in the same culture plate is demonstrated. G418, an inhibitor of cell protein synthesis, is frequently used to select for cells transfected with the neomycin resistance gene. The required concentration of G418 can be easily determined with this protein assay.

Processing, secretion, and immunoreactivity of carboxy terminally truncated dengue-2 virus envelope proteins expressed in insect cells by recombinant baculoviruses.

Two recombinant baculoviruses were constructed by inserting via the transfer vector pAcYM1 the genes coding for the structural proteins of dengue (DEN)-2 virus downstream from the polyhedrin promoter of Autographa californica nuclear polyhedrosis virus. The two recombinants differed in truncation of 26 and 71 amino acids, respectively, in the carboxy-terminal sequence of DEN-specific envelope (E) glycoprotein. Recombinant DEN-2 E glycoproteins were processed and transported to the surface of Spodoptera frugiperda Sf9 cells infected with both viruses. We show that about one-third of the E glycoprotein minus its whole C-terminal hydrophobic anchor domain was secreted into an endoglycosidase H-resistant form. The type-specific neutralizing epitopes were conserved in the recombinant proteins as shown with a panel of monoclonal antibodies.

Studies on the topography of the catalytic site of acetylcholinesterase using polyclonal and monoclonal antibodies.

Polyclonal and monoclonal antibodies were generated against a synthetic peptide (25 amino acid residues) corresponding to the amino acid sequence surrounding the active site serine of Torpedo californica acetylcholinesterase (AChE). Prior to immunization, the peptide was either coupled to bovine serum albumin or encapsulated into liposomes containing lipid A as an adjuvant. To determine whether this region of AChE is located on the surface of the enzyme and thus accessible for binding to antibodies, or located in a pocket and thus not accessible to antibodies, the immunoreactivity of the antibodies was determined using enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, Western blots, and competition ELISA. The polyclonal antibody and several of the monoclonal antibodies failed to react with either Torpedo or fetal bovine serum AChE in their native conformations, but showed significant cross-reactivity with the denatured enzymes. Human serum butyrylcholinesterase, which has a high degree of amino acid sequence homology with these AChEs, failed to react with the same antibodies in either native form or denatured form. Chymotrypsin also failed to react with the monoclonal antibodies in either form. Eighteen octapeptides spanning the entire sequence of this region were synthesized on polyethylene pins, and epitopes of representative monoclonal antibodies were determined by ELISA. The reactivity of peptides suggest that a portion of the 25 mer peptide in AChE containing the active site serine is the primary epitope. It is not exposed on the surface of the enzyme and is most likely sequestered in a pocket-like conformation in the native enzyme.

Immunocytochemical localization of phosphatidylinositol-anchored acetylcholinesterase in excitable membranes of Torpedo ocellata.

In Torpedo electric organ much of the acetylcholinesterase is a 'globular' dimer (G2), anchored to the plasma membrane via covalently attached phosphatidylinositol and solubilized by a bacterial phosphatidylinositol-specific phospholipase C. This suggested that selective solubilization with phosphatidylinositol-specific phospholipase C, coupled with immunocytochemistry, might be used to localize G2 acetylcholinesterase in excitable tissues of Torpedo. Cryostat sections of electric organ, electromotor nerve, electric lobe and back muscle from Torpedo ocellata were labelled, using three different antibody preparations to Torpedo acetylcholinesterase, followed by a fluorescent second antibody, before and after exposure to the phospholipase. Sites of innervation on electrocytes and myofibers were labelled selectively, as were motor and electromotor nerves. In all these cases labelling was substantially diminished by prior exposure to the phospholipase. The results support our previous assignment, based on biochemical evidence, for a neuronal and synaptic localization of the G2 acetylcholinesterase in Torpedo. Electric lobe acetylcholinesterase appears insensitive to the phospholipase treatment and lacks certain epitopes present in both electric organ and electromotor nerve enzyme. This suggests that substantial processing of the G2 form occurs concomitantly with its movement from the electric lobe into the electromotor nerve.

Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases.

The complete amino acid sequence of a mammalian acetylcholinesterase from fetal bovine serum (FBS AChE) is presented. This enzyme has a high degree of sequence identity with other cholinesterases, liver carboxyesterases, esterase-6, lysophospholipase, and thyroglobulin. The locations of 191 amino acids in 10 regions of the FBS enzyme were compared with corresponding sequences of Torpedo, human, and Drosophila AChEs and human serum butyrylcholinesterase (BChE). In one region there is a marked difference in both the number of amino acids and their sequence between mammalian AChE and other AChEs and the human serum BChE. The amino acid sequence of FBS AChE showed overall homologies of 90% with human AChE, 60% with T. california AChE, 50% with human serum BChE, and 39% with Drosophila AChE in these regions.

Differences in conformational stability between native and phosphorylated acetylcholinesterase as evidenced by a monoclonal antibody.

Monoclonal antibody 25B1 generated against diisopropyl phosphorofluoridate inhibited fetal bovine serum acetylcholinesterase has been extensively characterized with respect to its anticholinesterase properties. This antibody demonstrated considerably different properties from previously reported inhibitory antibodies raised against acetylcholinesterase in terms of the degree of inhibition (greater than 98%), the high degree of specificity, and the stability of the antigen-antibody complex. Monoclonal antibody 25B1 appears to be directed against a conformational epitope located in close proximity to the catalytic center of the enzyme and was found to be most suitable for studying the stabilization of the active site of acetylcholinesterase against denaturation by heat or guanidine following phosphorylation by organophosphorus anticholinesterase compounds. This approach allowed the determination of stability rank order of various phosphorylated acetylcholinesterases. Among all the organophosphates tested, the combination of a methyl group and a negatively charged oxygen attached to the P atom, CH3P(O)(O-)-AChE, conferred the greatest protection to the active site of aged or nonaged organophosphoryl conjugates of acetylcholinesterase.