Wild-type Acetylcholinesterase Research Articles

N‐linked glycosylation of dimeric acetylcholinesterase in erythrocytes is essential for enzyme maturation and membrane targeting

Acetylcholinesterase (AChE) is well-known for its cholinergic functions in the nervous system; however, this enzyme is also found in other tissues where its function is still not understood. AChE is synthesized through alternative splicing as splicing variants, with isoforms including read-through (AChE(R)), tailed (AChE(T)) and hydrophobic (AChE(H)). In human erythrocytes, AChE(H) is a glycophosphatidylinositol-linked dimer on the plasma membrane. Three N-linked glycosylation sites have been identified in the catalytic domain of human AChE. Here, we investigate the roles of glycosylation in assembly and trafficking of human AChE(H). In transfected fibroblasts, expression of AChE(H) was able to mimic the function of the dimeric form of AChE on the erythrocyte membrane. A glycan-depleted form was constructed by site-directed mutagenesis. By comparison with the wild-type AChE(H), the mutant had a much lower enzymatic activity and a much higher K(m) value. In addition, the mutant was dimerized in the endoplasmic reticulum, but was not trafficked to the Golgi apparatus. The results suggest that the glycosylation may affect AChE(H) enzymatic activity and trafficking, but not dimer formation. The present findings indicate the significance of N-glycosylation in controlling the biosynthesis of the AChE(H) dimer form.

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.

Translational Regulation of Acetylcholinesterase by the RNA-binding Protein Pumilio-2 at the Neuromuscular Synapse

Acetylcholinesterase (AChE) is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Our understanding of the molecular mechanisms underlying its regulation is incomplete, but they appear to involve both translational and post-translational events as well. Here, we show that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates. Immunoprecipitation of muscle cell extracts with a PUM2 specific antibody precipitated AChE mRNA, suggesting that PUM2 binds to the AChE transcripts in a complex. Gel shift assays using a bacterially expressed PUM2 RNA binding domain showed specific binding using wild type AChE 3'-UTR RNA segment that was abrogated by mutation of the consensus recognition site. Transfecting skeletal muscle cells with shRNAs specific for PUM2 up-regulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. Finally, we found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific proteins involved in modulating synaptic transmission, analogous to CNS synapses.

Neuropathological and Immunochemical Studies of Brain Parenchyma in Acetylcholinesterase Knockout Mice: Implications in Alzheimer's Disease

The 'cholinergic hypothesis', based on the correlation of the reduction of cholinergic activity in Alzheimer's disease (AD) with cognition and memory, is currently the most widely-held view for AD. Drug treatments for AD focus mainly on inhibition of acetylcholinesterase (AChE), and to some extent butyrylcholinesterase (BChE). In addition to changes in AChE in AD, there is a rise in the level of the sister enzyme BChE. However, the role of the two cholinesterases is poorly understood in vivo. We characterized several proteins immunohistochemically in brain sections from AChE nullizygote (AChE-/-) and wild type AChE+/+ mice. Previous studies had shown that AChE-/- mouse tissues are devoid of AChE activity and that the overall cholinesterase activity is significantly decreased in the knockout group [16]. Despite the differences of cholinesterase activity, we found no significant structural alterations between the experimental groups. Immunohistochemical examination revealed no neuronal, dendritic, astrocytic, synaptic, microglial, and endothelial differences between AChE-/- and AChE+/+ mice. Similarly, the histochemical examination showed no morphologic alterations between AChE-/- and AChE+/+ mice. Our studies show that neither the absence of AChE nor the presence exclusively of BChE is associated with neuroglial and vascular pathology.

Correlation between rate of enzyme-substrate diffusional encounter and average Boltzmann factor around active site.

The utility of the average Boltzmann factor around the active site of an enzyme as the predictor of the electrostatic enhancement of the substrate binding rate is tested on a set of data on wild-type acetylcholinesterase and 18 charge mutants recently obtained by Brownian dynamics simulations. A good correlation between the average Boltzmann factors and the substrate binding rate constants is found. The effects of single charge mutations on both the Boltzmann factor and the substrate binding rate constant are modest, i.e., < 5 fold increase or decrease. This is consistent with the experimental results of Shafferman et al. but does not support their suggestion that the overall rate of the catalytic reaction is not limited by the diffusional encounter of acetylcholinesterase and its substrate.

Mutant acetylcholinesterases as potential detoxification agents for organophosphate poisoning

It has been demonstrated that cholinesterases (ChEs) are an effective mode of pretreatment to prevent organophosphate (OP) toxicity in mice and rhesus monkeys. The efficacy of ChE as a bioscavenger of OP can be enhanced by combining enzyme pretreatment with oxime reactivation, since the scavenging capacity extends beyond a stoichiometric ratio of ChE to OP. Aging has proven to be a major barrier to achieving oxime reactivation of acetylcholinesterase (AChE) inhibited by the more potent OPs. To further increase the stoichiometry of OP to ChE required, we have sought AChE mutants that are more easily reactivated than wild-type enzyme. Substitution of glutamine for glutamate (E 199) located at the amino-terminal to the active-site serine (S 200) in Torpedo AChE generated an enzyme largely resistant to aging. Here we report the effect of the corresponding mutation on the rate of inhibition, reactivation by 1-(2-hydroxyiminomethyl-1-pyridinium)-1-(4-carboxyaminopyridinium)-dimethyl ether hydrochloride (HI-6), and aging of mouse AChE inhibited by C( + )P(−)- and C(−)P(−)-epimers of soman. The E 202 to Q mutation decreased the affinity of soman for AChE, slowed the reactivation of soman-inhibited AChE by HI-6, and decreased the aging of mutant AChE. These effects were more pronounced with C(−)P(−)-soman than with C( + )P(−)-soman. In vitro detoxification of soman and sarin by wild-type and E 202Q AChE in the presence of 2 mM HI-6 showed that, E 202Q AChE was 2–3 times more effective in detoxifying soman and sarin than wild-type AChE. These studies show that these recombinant DNA-derived AChEs are a great improvement over wild-type AChE as bioscavengers. They can be used to develop effective methods for the safe disposal of stored OP nerve agents and potential candidates for pre- or post-exposure treatment for OP toxicity.

Mutation His322Asn in Human Acetylcholinesterase Does Not Alter Electrophoretic and Catalytic Properties of the Erythrocyte Enzyme

The YT blood group antigen is located on human red blood cell (RBC) acetylcholinesterase. Wild-type acetylcholinesterase, YT1, has histidine at codon 322, whereas the genetic variant of acetylcholinesterase, YT2, has asparagine. This mutation is located within exon 2 of the ACHE gene, an exon that is present in all alternatively spliced forms of acetylcholinesterase. Therefore, acetylcholinesterase in brain and muscle has the same mutation as RBC acetylcholinesterase. We compared the electrophoretic and kinetic properties of RBC acetylcholinesterases having His 322 or Asn 322. We found no differences in the isoelectric point, mobility on non-denaturing gel electrophoresis, affinity for acetylthiocholine, activity per milligram of RBC ghost protein, substrate inhibition constants, binding to the peripheral site ligand (propidium), and binding to active site ligands (tetrahydroaminoacridine and edrophonium). Thus, although the point mutation elicits antibody production in nonmatching blood transfusion recipients, it has no effect on the enzymatic properties of acetylcholinesterase.

Expression of recombinant acetylcholinesterase in a baculovirus system: kinetic properties of glutamate 199 mutants.

The glycophospholipid-linked, amphiphilic form of acetylcholinesterase (AChE) from Torpedo californica and the hydrophilic form from mouse were overexpressed in Sf9 insect cells using the baculovirus expression system. Recombinant baculovirus, constructed by inserting AChE cDNA's into the genome of Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedron promoter, yielded recombinant enzyme varying between 0.5 and 3.8 mg/L. The recombinant enzyme was glycosylated although it migrated slightly more rapidly in SDS gel electrophoresis than enzyme purified from the electric organ of Torpedo. Kinetic properties of the recombinant DNA- and tissue-derived enzymes are identical. The detailed catalytic properties and susceptibility to inhibitors were examined for two enzyme mutations of the glutamate residue N-terminal to the active site serine. The Glu199 to Gln mutation shifted both the Km and Kss to higher substrate concentrations and resulted in a kcat of 28% of the wild type. Mutation of Glu199 to Asp also yielded a reduction in kcat but with no change in Km. Substrate inhibition normally apparent in wild-type AChE was eliminated with the Asp mutation, suggesting that substrate catalysis and substrate inhibition are not directly linked. Both mutations decreased the affinity of reversible inhibitors and reduced the rates of phosphorylation and carbamoylation; these changes were more striking with the Gln199 mutation. Decarbamoylation rates were unaffected by these mutations. Glu199 is the charged residue found deep within the active center gorge close to the site of acetylcholine binding, and our findings indicate it influences, but is not essential for, efficient catalysis.