Esomeprazole reduces sperm motility index by targeting the spermic cholinergic machinery: A mechanistic study for the association between use of proton pump inhibitors and reduced sperm motility index.

Recent studies have linked prolonged use of the most commonly prescribed proton pump inhibitors (PPIs) with declined human sperm function and infertility. Here, we report for the first time the most plausible underlying mechanism for this unwarranted secondary mode of action. We followed up on a recent serendipitous discovery in our laboratory regarding PPIs' off-target action and performed detailed pharmacodynamic analyses by combining in silico and in vitro studies to determine the off-target effect of one of the most commonly used PPI, esomeprazole, on the key human acetylcholine biosynthesizing enzyme, choline acetyltransferase (ChAT; EC 2.3.1.6). A pivotal enzyme in the spermic cholinergic system that governs the sperm motility, concentration and quality. Our results were conclusive and showed that both the racemic form, omeprazole and its pure S-enantiomer, esomeprazole, acted as potent mixed-competitive inhibitor of human ChAT with a global inhibition constant (Ki) of 88 nM (95%CI: 10-167 nM) for esomeprazole and 178 nM (95%CI: 140-230 nM) for the racemic drug omeprazole. Most importantly, esomeprazole substantially reduces both total number of motile sperm (by 36%, p < 0.001; and 21% p < 0.0001, at 10 and 100 nM, respectively) as well as the total number of sperm with progressive motility (by 42% p < 0.0016 and by 26% p < 0.0001, respectively) after 60 min relative to 20 min incubation in our ex vivo functional assay performed on ejaculated human sperm. In conclusion, this study presents a completely new perspective regarding PPIs secondary mode of action/unwarranted side effects and calls for further mechanistic and larger clinical studies to elucidate the role of PPIs in infertility.

Mutation of a serine near the catalytic site of the choline acetyltransferase a gene almost completely abolishes motility of the zebrafish embryo.

In zebrafish, the gene choline acetyltransferase a (chata) encodes one of the two ChAT orthologs responsible for the synthesis of acetylcholine. Acetylcholine (ACh) is essential for neuromuscular transmission and its impaired synthesis by ChAT can lead to neuromuscular junction disorders such as congenital myasthenic syndromes in humans. We have identified a novel mutation in the chata gene of zebrafish, chatatk64, in a collection of uncharacterised ENU-induced mutants. This mutant carries a missense mutation in the codon of a highly conserved serine changing it to an arginine (S102R). This serine is conserved among ChATs from zebrafish, rat, mice and chicken to humans. It resides within the catalytic domain and in the vicinity of the active site of the enzyme. However, it has not been reported so far to be required for enzymatic activity. Modelling of the S102R variant change in the ChAT protein crystal structure suggests that the change affects protein structure and has a direct impact on the catalytic domain of the protein which abolishes embryo motility almost completely.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost Cyprinus carpio.

Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio, the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates.  In this work, serial transverse sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases.

Stachys sieboldii Extract Supplementation Attenuates Memory Deficits by Modulating BDNF-CREB and Its Downstream Molecules, in Animal Models of Memory Impairment.

Cholinergic dysfunction, impaired brain-derived neurotrophic factor and cAMP response element binding protein (BDNF-CREB) signaling are one of the major pathological hallmarks of cognitive impairment. Therefore, improving cholinergic neurotransmission, and regulating the BDNF-CREB pathway by downregulating apoptosis genes is one strategy for inhibiting the etiology of dementia. This study evaluates the potential effects of Stachys sieboldii MIQ (SS) extract against cognitive dysfunction and its underlying mechanisms. SS supplementation for 33 days improved scopolamine-induced memory impairment symptoms in Morris water maze test and Y-maze test. SS reduced the acetylcholineesterase activity and significantly increase acetylcholine and cholineacetyltransferase activity in the brain. In the subsequent mechanism study, SS regulated the mRNA expression level of neuronal plasticity molecules such as (nerve growth factor) NGF, BDNF, CREB, and its downstream molecules such as Bcl-2 and Egr-1 by downregulating the neuronal apoptosis targets in both hippocampus and frontal cortex. Additionally, inward currents caused by SS in hippocampal CA1 neurons was partially blocked by the GABA receptor antagonist picrotoxin (50 µM), suggesting that SS acts on synaptic/extrasynaptic GABAA receptors. These findings indicate that SS may function in a way that is similar to nootropic drugs by inhibiting cholinergic abnormalities, and neuronal apoptosis targets and ultimately increasing the expression of BDNF-CREB.

Novel ligands of Choline Acetyltransferase designed by in silico molecular docking, hologram QSAR and lead optimization.

Recent reports have brought back the acetylcholine synthesizing enzyme, choline acetyltransferase in the mainstream research in dementia and the cholinergic anti-inflammatory pathway. Here we report, a specific strategy for the design of novel ChAT ligands based on molecular docking, Hologram Quantitative Structure Activity Relationship (HQSAR) and lead optimization. Molecular docking was performed on a series of ChAT inhibitors to decipher the molecular fingerprint of their interaction with the active site of ChAT. Then robust statistical fragment HQSAR models were developed. A library of novel ligands was generated based on the pharmacophoric and shape similarity scoring function, and evaluated in silico for their molecular interactions with ChAT. Ten of the top scoring invented compounds are reported here. We confirmed the activity of α-NETA, the only commercially available ChAT inhibitor, and one of the seed compounds in our model, using a new simple colorimetric ChAT assay (IC50 ~ 88 nM). In contrast, α-NETA exhibited an IC50 of ~30 µM for the ACh-degrading cholinesterases. In conclusion, the overall results may provide useful insight for discovering novel ChAT ligands and potential positron emission tomography tracers as in vivo functional biomarkers of the health of central cholinergic system in neurodegenerative disorders, such as Alzheimer's disease.

Choline Acetyltransferase Mutations Causing Congenital Myasthenic Syndrome: Molecular Findings and Genotype-Phenotype Correlations.

Choline acetyltransferase catalyzes the synthesis of acetylcholine at cholinergic nerves. Mutations in human CHAT cause a congenital myasthenic syndrome due to impaired synthesis of ACh; this severe variant of the disease is frequently associated with unexpected episodes of potentially fatal apnea. The severity of this condition varies remarkably, and the molecular factors determining this variability are poorly understood. Furthermore, genotype-phenotype correlations have been difficult to establish in patients with biallelic mutations. We analyzed the protein expression of phosphorylated ChAT of seven CHAT mutations, p.Val136Met, p.Arg207His, p.Arg186Trp, p.Val194Leu, p.Pro211Ala, p.Arg566Cys, and p.Ser694Cys, in HEK-293 cells to phosphorylated ChAT, determined their enzyme kinetics and thermal stability, and examined their structural changes. Three mutations, p.Arg207His, p.Arg186Trp, and p.Arg566Cys, are novel, and p.Val136Met and p.Arg207His are homozygous in three families and associated with severe disease. The characterization of mutants showed a decrease in the overall catalytic efficiency of ChAT; in particular, those located near the active-site tunnel produced the most seriously disruptive phenotypic effects. On the other hand, p.Val136Met, which is located far from both active and substrate-binding sites, produced the most drastic reduction of ChAT expression. Overall, CHAT mutations producing low enzyme expression and severe kinetic effects are associated with the most severe phenotypes.

Polysaccharide nanofibers with variable compliance for directing cell fate.

Cells perceive their microenvironment through physical and mechanical cues, such as extracellular matrix topography or stiffness. In this study, we developed a polysaccharide scaffold that can provide combined substrate topography and matrix compliance signals to direct cell fate. Pullulan/dextran (P/D) nanofibers were fabricated with variable stiffness by in situ crosslinking during electrospinning. By varying the chemical crosslinking content between 10, 12, 14, and 16%, (denoted as STMP10, STMP12, STMP14, and STMP16 respectively), scaffold mechanical stiffness was altered. We characterized substrate stiffness by various methods. Under hydrated conditions, atomic force microscopy and tensile tests of bulk scaffolds were conducted. Under dry conditions, tensile tests of scaffolds and single nanofibers were examined. In addition, we evaluated the efficacy of the scaffolds in directing stem cell differentiation. Using human first trimester mesenchymal stem cells (fMSCs) cultured on STMP14 P/D scaffolds (Young's modulus: 7.84 kPa) in serum-free neuronal differentiation medium exhibited greatest extent of differentiation. Cells showed morphological changes and significantly higher expression of motor neuron markers. Further analyses by western blotting also revealed the enhanced expression of choline acetyltransferase on STMP14 (7.84 kPa) and STMP16 (11.08 kPa) samples as compared to STMP12 (7.19 kPa). Taken together, this study demonstrates that the stiffness of P/D nanofibers can be altered by differential in situ crosslinking during electrospinning and suggests the feasibility of using such polysaccharide nanofibers in supporting fMSC neuronal commitment.

Distribution of adrenergic and cholinergic nerve fibres within intrinsic nerves at the level of the human heart hilum.

OBJECTIVES: The disbalance between adrenergic (sympathetic) and cholinergic (parasympathetic) cardiac inputs facilitates cardiac arrhythmias, including the lethal ones. In spite of the fact that the morphological pattern of the epicardiac ganglionated subplexuses (ENsubP) has been previously described in detail, the distribution of functionally distinct axons in human intrinsic nerves was not investigated thus far. Therefore, the aim of the present study was to quantitatively evaluate the distribution of tyrosine hydroxylase (TH)- and choline acetyltransferase (ChAT)-positive axons within intrinsic nerves at the level of the human heart hilum (HH), since they are of pivotal importance for determining proper treatment options for different arrhythmias. METHODS: Tissue samples containing the intrinsic nerves from seven epicardiac subplexuses were obtained from nine human hearts without cardiac pathology and processed for immunofluorescent detection of TH and ChAT. The nerve area was measured and the numbers of axons were counted using microphotographs of nerve profiles. The densities of fibres were extrapolated and compared between subplexuses. RESULTS: ChAT-immunoreactive (IR) fibres were evidently predominant (>56%) in nerves of dorsal (DRA) and ventral right atrial (VRA) ENsubP. Within both left (LC) and right coronary ENsubP, the most abundant (70.9 and 83.0%, respectively) were TH-IR axons. Despite subplexal dependence, ChAT-IR fibres prevailed in comparatively thinner nerves, whereas TH-IR fibres in thicker ones. Morphometry showed that at the level of HH: (i) LC subplexal nerves were found to be the thickest (25 737 ± 4131 µm(2)) ones, whereas the thinnest (2604 ± 213 µm(2)) nerves concentrated in DRA ENsubP; (ii) the density of ChAT-IR axons was highest (6.8 ± 0.6/100 µm(2)) in the ventral left atrial nerves and lowest (3.2 ± 0.1/100 µm(2)) in left dorsal ENsubP and (iii) the density of TH-IR fibres was highest (15.9 ± 2.1/100 µm(2)) in LC subplexal nerves and lowest (4.4 ± 0.3/100 µm(2)) in VRA nerves. CONCLUSIONS: (i) The principal intrinsic adrenergic neural pathways in the human heart proceed via both coronary ENsubP that supply cardiac ventricles and (ii) the majority of cholinergic nerve fibres access the human heart through DRA and VRA ENsubP and extend towards the right atrium, including the region of the sinuatrial node.

Development of NMR spectroscopic methods for dynamic detection of acetylcholine synthesis by choline acetyltransferase in hippocampal tissue.

Choline acetyltransferase (ChAT) is the key enzyme for acetylcholine (ACh) synthesis and constitutes a reliable marker for the integrity of cholinergic neurons. Cortical ChAT activity is decreased in the brain of patients suffering from Alzheimer's and Parkinson's diseases. The standard method used to measure the activity of ChAT enzyme relies on a very sensitive radiometric assay, but can only be performed on post-mortem tissue samples. Here, we demonstrate the possibility to monitor ACh synthesis in rat brain homogenates in real time using NMR spectroscopy. First, the experimental conditions of the radiometric assay were carefully adjusted to produce maximum ACh levels. This was important for translating the assay to NMR, which has a low intrinsic sensitivity. We then used (15) N-choline and a pulse sequence designed to filter proton polarization by nitrogen coupling before (1) H-NMR detection. ACh signal was resolved from choline signal and therefore it was possible to monitor ChAT-mediated ACh synthesis selectively over time. We propose that the present approach using a labeled precursor to monitor the enzymatic synthesis of ACh in rat brain homogenates through real-time NMR represents a useful tool to detect neurotransmitter synthesis. This method may be adapted to assess the state of the cholinergic system in the brain in vivo in a non-invasive manner using NMR spectroscopic techniques.

New horizons for congenital myasthenic syndromes.

During the past five years an increasing number of patients have been diagnosed with congenital myasthenic syndromes (CMS) and a number of novel syndromes have been recognized and investigated. This presentation focuses on the CMS caused by defects in choline acetyltransferase, novel fast-channel syndromes that hinder isomerization of the acetylcholine receptor from the closed to the open state, the consequences of deleterious mutations in the intermediate filament linker plectin, altered neuromuscular transmission in a centronuclear myopathy, and two recently identified CMS caused by congenital defects in glycosylation.

Functional consequences and structural interpretation of mutations of human choline acetyltransferase.

Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes synthesis of acetylcholine from acetyl-CoA (AcCoA) and choline in cholinergic neurons. Mutations in CHAT cause potentially lethal congenital myasthenic syndromes associated with episodic apnea (ChAT-CMS). Here, we analyze the functional consequences of 12 missense and one nonsense mutations of CHAT in 11 patients. Nine of the mutations are novel. We examine expression of the recombinant missense mutants in Bosc 23 cells, determine their kinetic properties and thermal stability, and interpret the functional effects of 11 mutations in the context of the atomic structural model of human ChAT. Five mutations (p.Trp421Ser, p.Ser498Pro, p.Thr553Asn, p.Ala557Thr, and p.Ser572Trp) reduce enzyme expression to less than 50% of wild-type. Mutations with severe kinetic effects are located in the active-site tunnel (p.Met202Arg, p.Thr553Asn, and p.Ala557Thr) or adjacent to the substrate binding site (p.Ser572Trp), or exert their effect allosterically (p.Trp421Ser and p.Ile689Ser). Two mutations with milder kinetic effects (p.Val136Met and p.Ala235Thr) are also predicted to act allosterically. One mutation (p.Thr608Asn) below the nucleotide binding site of CoA enhances dissociation of AcCoA from the enzyme-substrate complex. Two mutations introducing a proline residue into an α-helix (p.Ser498Pro and p.Ser704Pro) impair the thermal stability of ChAT.

Peripheral type of choline acetyltransferase: biological and evolutionary implications for novel mechanisms in cholinergic system.

The peripheral type of choline acetyltransferase (pChAT) is an isoform of the well-studied common type of choline acetyltransferase (cChAT), the synthesizing enzyme of acetylcholine. Since pChAT arises by exons skipping, its amino acid sequence is similar to that of cChAT, except the lack of a continuous peptide sequence encoded by all the four exons from 6 to 9. While cChAT expression has been observed in both the central and peripheral nervous systems, pChAT is preferentially expressed in the peripheral nervous system. pChAT appears to be a reliable marker for the visualization of peripheral cholinergic neurons and their processes, whereas other conventional markers including cChAT have not been used successfully for it. In mammals like rodents, pChAT immunoreactivity has been observed in most, if not all, physiologically identified peripheral cholinergic structures such as all parasympathetic postganglionic neurons and most neurons of the enteric nervous system. In addition, pChAT has been found in many peripheral neurons that are derived from the neural crest. These include sensory neurons of the trigeminal ganglion and the dorsal root ganglion, and sympathetic postganglionic neurons. Recent studies moreover indicate that pChAT, as well as cChAT, appears ubiquitously expressed among various species not only of vertebrate mammals but also of invertebrate mollusks. This finding implies that the alternative splicing mechanism to generate pChAT and cChAT has been preserved during evolution, probably for some functional benefits.

Redesign of cosubstrate specificity and identification of important residues for substrate binding to hChAT.

In eukaryotes, choline acetyltransferase (ChAT) catalyzes the reversible formation of the neurotransmitter acetylcholine from choline and acetyl-CoA. ChAT belongs to a family of CoA-dependent enzymes that also includes the carnitine acyltransferases CrAT, CrOT, and CPTs. In contrast to CrOT and CPTs that are very active toward medium- and long-chain acyl-CoAs, respectively, CrAT and ChAT display activity toward only short-chain acyl-CoAs. We recently demonstrated the substrate and cosubstrate promiscuity of the wild-type human ChAT (hChAT). To extend the flexibility of this enzyme, we have generated a series of single, double, and triple hChAT mutants. Here we report the conversion of hChAT into choline octanoyltransferase (ChOT) and choline palmitoyltransferase (ChPT). The E337 and C550 residues (numbering from hChAT) were previously shown to dictate the acyl-CoA cosubstrate specificity in the carnitine series. Here we identify and demonstrate the importance of C551, in addition to E337 and C550, in contributing to the acyl-CoA specificity of hChAT. We also show that either C550 or C551 needs to be present for the transfer of medium- and long-chain acyl-CoAs by hChAT. By exploring the potential expansion of the tunnel on the substrate side, we demonstrate that residues M84, Y436, and Y552 play a critical role in binding and holding the choline substrate in the ChAT active site.

Axotomy alters alternative splicing of choline acetyltransferase in the rat dorsal motor nucleus of the vagus nerve.

Choline acetyltransferase of the peripheral type (pChAT) is a splice variant that lacks exons 6-9 of the common-type ChAT (cChAT); the role of pChAT remains unknown. We investigated the expression of pChAT and cChAT after axotomy to try to elucidate its function. In the dorsal motor nucleus of the vagus nerve (DMNV), nucleus ambiguus (NA), and hypoglossal nucleus (HN) of control rats, we observed neural expression of cChAT but no pChAT-positive neurons. Following nerve transection, we clearly detected pChAT-labeled neurons in the DMNV and weakly labeled neurons in the NA, but pChAT was not seen in the HN. In the DMNV, the mean number of cChAT-positive neurons decreased rapidly to 40.5% of control at 3 days post transection, and to 5.0% of control after 7 days. The number of cChAT-positive neurons then gradually increased and reached a plateau of about 25% of control value at 28 days post transection. pChAT-positive neurons did not appear until 7 days after transection. On the same day, pChAT mRNA was detected in the DMNV neurons by reverse transcription-polymerase chain reaction (RT-PCR) by using laser capture microdissection. The number of pChAT-positive neurons gradually decreased, and only 10% of the cholinergic neurons retained pChAT expression 56 days post transection. Double-immunofluorescence analysis showed that some of the DMNV neurons expressed both cChAT and pChAT upon recovery from axotomy. These results suggest that the expression of pChAT is associated with the regenerative or degenerative processes of motoneurons especially for general visceral efferents.

KAP, the accessory subunit of kinesin-2, binds the predicted coiled-coil stalk of the motor subunits.

Kinesin-2 is an anterograde motor involved in intraflagellar transport and certain other intracellular transport processes. It consists of two different motor subunits and an accessory protein KAP (kinesin accessory protein). The motor subunits were shown to bind each other through the coiled-coil stalk domains, while KAP was proposed to bind the tail domains of the motor subunits. Although several genetic studies established that KAP plays an important role in kinesin-2 functions, its exact role remains unclear. Here, we report the results of a systematic analysis of the KAP binding sites by using recombinant Drosophila kinesin-2 subunits as well as the endogenous proteins. These show that at least one of the coiled-coil stalks is sufficient to bind the N-terminal region of DmKAP. The soluble complex involving the recombinant kinesin-2 fragments is reconstituted in vitro at high salt concentrations, suggesting that the interaction is primarily nonionic. Furthermore, independent distant homology modeling indicated that DmKAP may bind along the coiled-coil stalks through a combination of predominantly hydrophobic interactions and hydrogen bonds. These observations led us to propose that KAP would stabilize the motor subunit heterodimer and help assemble a greater kinesin-2 complex in vivo.

Substrate binding and catalytic mechanism of human choline acetyltransferase.

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.

[The neurochemistry and neuropharmacology of diffuse Lewy body disease]. / Neuroquímica y neurofarmacología de la enfermedad difusa de cuerpos de Lewy.

INTRODUCTION AND DEVELOPMENT: The main neurochemical alteration in diffuse Lewy body disease (DLBD) is the cholinergic deficit in the cerebral cortex, which involves mainly cholin-acetyl-transferase. There have been also described dopamine deficiency and alterations affecting other neurotransmitters and neuromodulators, such as serotonin, noradrenaline, neuropeptides, etc. Cerebral perfusion and glucose metabolism studies usually show diffuse hypoperfusion or hypometabolism, with higher alteration of associative cortex, including occipital involvement. Several studies have shown increased markers of oxidative stress in brain and other tissues, suggesting its possible role in the pathogenesis of DLBD. CONCLUSIONS: Acetylcholinesterase inhibitors seem to improve cognitive and conductual symptoms, although their usefulness according evidence-based medicine criteria is weak. Some patients need atypical neuroleptics at low doses to get the symptomatic control of conductual alterations.

Computational approaches to model ligand selectivity in drug design.

To be effective, a designed drug must discriminate successfully the macromolecular target from alternative structures present in the organism. The last few years have witnessed the emergence of different computational tools aimed to the understanding and modeling of this process at molecular level. Although still rudimentary, these methods are shaping a coherent approach to help in the design of molecules with high affinity and specificity, both in lead discovery and in lead optimization. It is the purpose of this review to illustrate the array of computational tools available to consider selectivity in the design process, to summarize the most relevant applications, and to sketch the challenges ahead.

Surface-entropy reduction used in the crystallization of human choline acetyltransferase.

Human choline acetyltransferase (ChAT) synthesizes the neurotransmitter acetylcholine (ACh) from choline and acetyl-CoA. A crystal structure of human ChAT has been a long-standing goal in the neuronal signalling field. Milligram quantities of pure ChAT can be purified [Kim et al. (2005), Protein Expr. Purif. 40, 107-117], but exhaustive crystallization efforts failed to produce any crystals suitable for high-resolution structural studies. To obtain high-quality crystals of human ChAT, a truncation was made in a large poorly conserved loop region and high-entropy side chains were removed from the surface of the protein. The resulting 'entropy-reduced' ChAT (MR = 68.1 kDa) crystallizes readily and reproducibly and the crystals diffract X-rays to approximately 2.2 A. The availability of these crystals will allow us to study the structure of human ChAT on its own as well as in complex with its substrates and inhibitor molecules, leading to a greater understanding of its catalytic mechanism and regulation.

Two methods for large-scale purification of recombinant human choline acetyltransferase.

Choline acetyltransferase (ChAT) catalyzes the transfer of an acetyl group from acetyl-CoA to choline to produce the neurotransmitter acetylcholine (ACh). We have produced large quantities of pure human ChAT using two different bacterial expression systems. In the first, ChAT is fused to a chitin-binding domain via a self-cleavable linker allowing the release of ChAT without the use of proteases. In the second, ChAT is fused to a hexahistidine (His6) tag at the N-terminus with a linker incorporating a TEV protease cleavage site. In both cases, pure ChAT was produced that has a final specific activity of approximately 50 micromol ACh/min/mg and is suitable for structural characterization. Analysis of purified ChAT by Western blots and mass spectrometry revealed that the C-terminal 15 amino acids were slowly removed by endogenous proteolytic activity, to produce a stable 615 residue protein. Furthermore, we show that purified recombinant human ChAT is highly prone to oxidation, leading to the formation of covalent dimers and/or a loss of catalytic activity. Kinetic parameters of our purified proteins were obtained and, when compared to previously published constants for human placental ChAT, we found that recombinant human ChAT displays lower values for Michaelis and inhibition constants for ACh, which may be due to the complete absence of post-translational modifications.