Engineering of a monomeric and low‐glycosylated form of human butyrylcholinesterase

Nachon, Florian; Nicolet, Yvain; Viguié, Nathalie; Masson, Patrick; Fontecilla‐Camps, Juan C.; Lockridge, Oksana

doi:10.1046/j.0014-2956.2001.02692.x

Cited by 128 publications

(120 citation statements)

References 46 publications

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“…Complexes-Recombinant human BChE suitable for crystallization was obtained, purified, and crystallized as described previously (17). No butyrate was present in the culture medium and none was added during any step of the purification procedure.…”

Section: Crystallization Of Recombinant Bche and Itsmentioning

confidence: 99%

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Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products

Nicolet

Lockridge

Masson

et al. 2003

Journal of Biological Chemistry

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572

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Cholinesterases are among the most efficient enzymes known. They are divided into two groups: acetylcholinesterase, involved in the hydrolysis of the neurotransmitter acetylcholine, and butyrylcholinesterase of unknown function. Several crystal structures of the former have shown that the active site is located at the bottom of a deep and narrow gorge, raising the question of how substrate and products enter and leave. Human butyrylcholinesterase (BChE) has attracted attention because it can hydrolyze toxic esters such as cocaine or scavenge organophosphorus pesticides and nerve agents. Here we report the crystal structures of several recombinant truncated human BChE complexes and conjugates and provide a description for mechanistically relevant non-productive substrate and product binding. As expected, the structure of BChE is similar to a previously published theoretical model of this enzyme and to the structure of Torpedo acetylcholinesterase. The main difference between the experimentally determined BChE structure and its model is found at the acyl binding pocket that is significantly bigger than expected. An electron density peak close to the catalytic Ser 198 has been modeled as bound butyrate.

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Section: Crystallization Of Recombinant Bche and Itsmentioning

confidence: 99%

“…We have recently published the engineering and crystallization of a monomeric and partially glycosylated recombinant human BchE (17). Here we report several crystal structures of BChE complexed with a substrate, products, and conjugated to soman after aging.…”

mentioning

confidence: 99%

Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products

Nicolet

Lockridge

Masson

et al. 2003

Journal of Biological Chemistry

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720

572

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“…2B). Natural tetramerization peptide was introduced to obtain the tetrameric form of rhBChE, which has shown better stability in the bloodstream than the monomeric enzyme, suggesting that tetramerization plays an even more important role than accurate glycosylation (23) (Fig. 2A).…”

Section: Resultsmentioning

confidence: 99%

“…However, none of these has yet been economically effective or received approval for administration to humans. Although the CHO cell expression system is very widely used for different US Food and Drug Administration-approved protein drugs (18)(19)(20)(21)(22), the recently reported CHO-based expression of BChE has delivered only modest production and thus cannot be adopted for medical application (23).…”

mentioning

confidence: 99%

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

Ilyushin

Смирнов

Belogurov

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD 50 of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.organophosphate | N-acetylneuraminic acid | Russian VX | pesticide | mass spectrometry

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“…BChE was expressed in Chinese hamster ovary (CHO) cells and secreted into serum-free culture medium, and purified by affinity and ion-exchange chromatography as described earlier (16). The BChE enzyme was a truncated monomer containing residues 1-529 whose tetramerization domain was deleted.…”

Section: Recombinant Human Butyrylcholinesterasementioning

confidence: 99%

Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-agents

Wandhammer

Carletti²,

Schans

et al. 2011

Journal of Biological Chemistry

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Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous timedependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P S adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX R -(؉) and VX S -(؊) solutions lead to the formation of the P S and P R adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX R -(؉) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX S -(؊). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P R enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P S enantiomer and ages.

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Engineering of a monomeric and low‐glycosylated form of human butyrylcholinesterase

Cited by 128 publications

References 46 publications

Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products

Crystal Structure of Human Butyrylcholinesterase and of Its Complexes with Substrate and Products

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo

Structural Study of the Complex Stereoselectivity of Human Butyrylcholinesterase for the Neurotoxic V-agents

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