David Glick

Engineering of human cholinesterases explains and predicts diverse consequences of administration of various drugs and poisons

The acetylcholine hydrolyzing enzyme, acetylcholinesterase, primarily functions in nerve conduction, yet it appears in several guises, due to tissue-specific expression, alternative mRNA splicing and variable aggregation modes. The closely related enzyme, butyrylcholinesterase, most likely serves as a scavenger of toxins to protect acetylcholine binding proteins. One or both of the cholinesterases probably also plays a non-catalytic role(s) as a surface element on cells to direct intercellular interactions. The two enzymes are subject to inhibition by a wide variety of synthetic (e.g., organophosphorus and carbamate insecticides) and natural (e.g., glycoalkaloids) anticholinesterases that can compromise these functions. Butyrylcholinesterase may function, as well, to degrade several drugs of interest, notably aspirin, cocaine and cocaine-like local anesthetics. The widespread occurrence of butyrylcholinesterase mutants with modified activity further complicates this picture, in ways that are only now being dissected through the use of site-directed mutagenesis and heterologous expression of recombinant cholinesterases.

Defects in Pre-mRNA Processing as Causes of and Predisposition to Diseases

Humans possess a surprisingly low number of genes and intensively use pre-mRNA splicing to achieve the high molecular complexity needed to sustain normal body functions and facilitate responses to altered conditions. Because hundreds of thousands of proteins are generated by 25,000 to 40,000 genes, pre-mRNA processing events are highly important for the regulation of human gene expression. Both inherited and acquired defects in pre-mRNA processing are increasingly recognized as causes of human diseases, and almost all pre-mRNA processing events are controlled by a combination of protein factors. This makes defects in these processes likely candidates for causes of diseases with complicated inheritance patterns that affect seemingly unrelated functions. The elucidation of genetic mechanisms regulating pre-mRNA processing, combined with the development of drugs targeted at consensus RNA sequences and/or corresponding proteins, can lead to novel diagnostic and therapeutic approaches.

Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants.

To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3-variable AChE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural atypical BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the atypical BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.

In vitro phosphorylation of acetylcholinesterase at non-consensus protein kinase A sites enhances the rate of acetylcholine hydrolysis

Here, we report that the catalytic subunit of cAMP-dependent protein kinase (PKA) but not casein kinase II or protein kinase C phosphorylates recombinant human acetylcholinesterase (AChE) in vitro. This enhances acetylthiocholine hydrolysis up to 10-fold as compared to untreated AChE, while leaving unaffected the enzymes affinity for this substrate and for various active and peripheral site inhibitors. Alkaline phosphatase treatment enhanced the electrophoretic migration, under denaturing conditions, of part of the AChE proteins isolated from various mammalian sources and raised the isoelectric point of some of the treated AChE molecules, indicating that part of the AChE molecules are also phosphorylated in vivo. Enhancement of acetylthiocholine hydrolysis also occurred with Torpedo AChE, which has no consensus motif for PKA phosphorylation. Further, mutating the single PKA site in human AChE (threonine-249) did not prevent this enhancement, suggesting that in both cases it was due to phosphorylation at non-consensus sites. In vivo suppression of the acetylcholine hydrolyzing activity of AChE and consequent impairment in cholinergic neurotransmission occur under exposure to both natural and pharmacological compounds, including organophosphate and carbamate insecticides and chemical warfare agents. Phosphorylation of AChE may possibly offer a rapid feedback mechanism that can compensate for impairments in cholinergic neurotransmission, modulating the hydrolytic activity of this enzyme and enabling acetylcholine hydrolysis to proceed under such challenges.

Normal and Atypical Butyrylcholinesterases in Placental Development, Function, and Malfunction

Abstract1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous abortion and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development.2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta.3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with α-solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction.4. Atypical BuChE of serum or recombinant origin presented >10-fold weaker affinities than normal BuChE for cocaine and α-solanine. However, BuChE in the serum of a heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta.5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls or the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.

Acetylcholinesterase as a window onto stress responses

Abstract It has long been known that cholinergic neurotransmission is intimately associated with mammalian stress responses. Inhibition of acetylcholinesterase (AChE), like stress, elevates the levels of acetylcholine (ACh) in the short term, and both conditions induce some common long-lasting behavioral symptoms. Therefore, AChE manipulations provide an interesting window onto stress responses. Like many other stimuli, both stress and inhibition of AChE cause an increase in AChE gene expression that is also associated with a shift in its pre-mRNA splicing pattern. Of the several variants of AChE that arise due to alternative splicing, it is specifically the usually rare, soluble AChE-R variant that is up-regulated. Transgenic mice that over-express AChE also show many of the same symptoms as stress: erratic behavior following circadian light/dark shift, progressive failure of learning and memory, intensified long-term potentiation (LTP), development of neuropathologies, progressive muscle fatigue and degeneration of neuromuscular junctions. Altered expression of other cholinergic proteins in these mice, e.g. protein kinase CβII choline acetyltransferase and the high affinity choline transporter, suggests chronic feedback responses to the cholinergic imbalance. Stress-associated characteristics can be ameliorated in mice and humans by treatment with antisense agents that induce selective destruction of AChE-R, which provides further support for changes in alternative splicing, and in particular the accumulation of this variant, having a role in the etiology of stress responses.

Mutations and impaired expression in the ACHE and BCHE genes: neurological implications

The acetylcholine hydrolysing cholinesterases control the termination of cholinergic signalling in multiple tissues and are targets for a variety of drugs, natural and man-made poisons and common insecticides. Molecular cloning and gene mapping studies revealed the primary structure of human acetyl- and butyrylcholinesterase and localized the corresponding ACHE and BCHE genes to the chromosomal positions 3q26-ter and 7q22, respectively. Several different point mutations in the coding region of BCHE were found to be particularly abundant in the Israeli population. Analytical expression studies in microinjected Xenopus oocytes have demonstrated that the biochemical properties of cholinesterases may be modified by rationalized site-directed mutagenesis and in chimeric ACHE/BCHE constructs. These properties are differently altered in the various allelic BCHE variants, conferring resistance to several anti-cholinesterases, which may explain the evolutionary emergence of these multiple alleles. At the clinical level, abnormal expression of both ACHE and BCHE and the in vivo amplification of the ACHE and BCHE genes has been variously associated with abnormal megakaryocytopoiesis, leukemias and brain and ovarian tumors. Moreover, antisense oligonucleotides blocking the expression of these genes were shown to interfere with hemocytopoiesis in culture, implicating these genes in cholinergic influence on cell growth and proliferation.

Autism, Stress, and Chromosome 7 Genes

Autism, a disorder affecting 4 out of 10,000 individuals in the general population, is classified as a pervasive developmental disorder. It becomes apparent by the third year of life and its characteristic impairments persist into adulthood. Whereas the inheritance of this disorder does not follow a simple Mendelian pattern, there is compelling evidence for strong genetic basis. This fact places autism in the category of complex genetic traits and predicts difficulties in any attempt to unravel the various genes that contribute to the disorder, genes that may even have different contributions in differen populations. Recently, the rising interest in autism has spawned quite a few multinational efforts aimed at identification of these genes. The projects, taking advantage of experimental approaches designed for the study of complex genetic traits, succeeded in identifying several candidate loci. However, none of these loci was identified with certainty. Among this group of candidate loci, several appear to be more prominent than the rest. The long arm of chromosome 7 (7q) is one of those. The main characteristics of autism are social and communicative impairments, as well as repetitive and stereotyped behaviors and interests. However, there are also reports of stress response-like behavior in autistic children. Such reports strongly suggest that autism is associated with inherited impairments in stress responses. However, this association has yet to be confirmed. An interesting link that relates impairments in stress responses to autism is found in the chromosome 7 gene ACHE, which encodes the enzyme acetylcholinesterase (AChE). The expression of this gene is robustly and persistently up-regulated under stress and was recently found to be affected by a novel mutation in the gene’s extended promoter. This chapter describes the recent advances in the identification of genes or gene loci that contribute to the autistic phenotype, concentrating on what appears to be one of the best candidate loci, 7q. It will further describe the reports that suggest an association between

Population Diversity of Point Mutations in the Human AChE and BCHE Genes Predicts Variable Responses to Anticholinesterase Drugs

On an experimental basis, patients with neurodegenerative diseases receive anti-cholinesterases (Iversen, 1993; Enz et al., 1993; Giacobini, 1993) to improve cognitive function (Alzheimer’s disease) or to reduce muscle spasms (Parkinson’s disease). These drugs are directed toward the nervous system acetyl- and butyrylcholinesterase (AChE, BuChE). However, the response of specific individuals to such drugs was found to be highly variable. Molecular genetics findings strongly suggest that this variability may be due to genomic diversity in the corresponding genes, primarily in the BuChE gene, BCHE.

Genetic Predisposition for Variable Response to Anticholinesterase Therapy Anticipated in Carriers of the Butyrylcholinesterase “Atypical” Mutation

Anticholinesterases were recently approved for treating patients suffering from Alzheimer’s disease (AD) in an attempt to balance their cholinergic system. These drugs are targeted at acetylcholinesterase (AChE) but also inhibit butyrylcholinesterase (BuChE), known for its numerous genetic variants. The most common of these is the “atypical” phenotype created through a replacement of Asp70 by Gly (D70G) due to a point mutation. The “atypical” enzyme causes prolonged postanesthesia apnea following succinylcholine administration for muscle relaxation and displays a considerably reduced sensitivity to various other inhibitors. The allelic frequency of “atypical” BuChE was studied in different populations and revealed distinct patterns particular to various ethnic groups. Recently, a relatively high allelic frequency of 0.06 was found in a population of Georgian Jews, differing by up to 4-fold from the incidence in other populations (Ehrlich et al., Genomics, in press). This implies that in groups of AD patients from diverse ethnic origins, a significant fraction of carriers of at least one allele of this mutation should be expected. To predict their responsiveness to anticholinesterase treatment, we examined the susceptibility of AChE, as compared to that of BuChE and the “atypical” BuChE variant, towards several anticholinesterases in use for AD treatment. IC50 values and rate constants reflecting inhibitor susceptibilities were calculated for various recombinant human cholinesterases produced in Xenopus oocytes and immobilized on microtiter plates through selective monoclonal antibodies. The reversible amino acridinium compound Tacrine, currently in use for AD therapy, displayed a 300-fold higher IC50 value for the “atypical” enzyme than for BuChE (1mM BtCh as substrate). Pseudo first order rate constants for inhibition of BuChEs by the carbamates heptyl-physostigmine (0.139 min−1, lOnM inhibitor), physostigmine (0.3 min−1, 1 μM inhib.) and SNZ-ENA713 (0.139 min−1, 10 μM inhib.) were found to be higher than or equal to those of AChE, suggesting that BuChE serves as a second primary target for these drugs. Moreover, the “atypical” variant of BuChE displayed considerably slower inactivation rates to these drugs (0.01 min−1, 0.025 min−1, and 0.01 min−1, respectively) as compared with the wild type BuChE. These findings predict that carriers of the D70G BuChE mutation would vary from other patients in their susceptibility to the above drugs, which potentially contributes to the wide variability of responses observed in clinical trials. Screening patients for D70G carriers should therefore precede anticholinesterase treatment.