Mutant of Bungarus fasciatus acetylcholinesterase with low affinity and low hydrolase activity toward organophosphorus esters.

Enzymes hydrolysing highly toxic organophosphate esters (OPs) are promising alternatives to pharmacological countermeasures against OPs poisoning. Bungarus fasciatus acetylcholinesterase (BfAChE) was engineered to acquire organophosphate hydrolase (OPase) activity by reproducing the features of the human butyrylcholinesterase G117H mutant, the first mutant designed to hydrolyse OPs. The modification consisted of a triple mutation on the (122)GFYS(125) peptide segment, resulting in (122)HFQT(125). This substitution introduced a nucleophilic histidine above the oxyanion hole, and made space in that region. The mutant did not show inhibition by excess acetylthiocholine up to 80 mM. The k(cat)/K(m) ratio with acetylthiocholine was 4 orders of magnitude lower than that of wild-type AChE. Interestingly, due to low affinity, the G122H/Y124Q/S125T mutant was resistant to sub-millimolar concentrations of OPs. Moreover, it had hydrolysing activity with paraoxon, echothiophate, and diisopropyl phosphofluoridate (DFP). DFP was characterised as a slow-binding substrate. This mutant is the first mutant of AChE capable of hydrolysing organophosphates. However, the overall OPase efficiency was greatly decreased compared to G117H butyrylcholinesterase.

Markers of organophosphate exposure in human serum.

The goal of this work is to identify novel serum proteins that are labeled with organophosphates (OPs) and to create a protocol for identification using mass spectroscopy. The use of OP-labeled proteins for identification of exposure is useful because such proteins will remain in circulation for weeks (Van Der Schans et al., 2004). Currently, both butyrylcholinesterase (BChE) and albumin have been shown to bind OPs in blood. Peeples et al. (2005) showed that albumin is labeled by OPs, specifically 6-Nbiotinylaminohexyl isopropyl phosphorofluoridate hemihydrate, in living mice. Albumin is the major protein in human serum, and its reaction with OPs tends to overwhelm the identification of other proteins. In vitro studies of human serum require removal of the serum albumin without depleting the less abundant proteins. Following this step, identifying the remaining proteins is simply a matter of labeling the proteins with an OP, separating the labeled from nonlabeled proteins, and using Q-trap mass spectrometry for identification.

Increased hepatotoxicity and cardiac fibrosis in cocaine-treated butyrylcholinesterase knockout mice.

In mice, cocaine is detoxified to inactive products by butyrylcholinesterase (BChE) and carboxylesterase. In human beings, cocaine detoxification is primarily by BChE. The focus of this investigation was to elucidate the importance of BChE in reducing pathophysiological effects following cocaine exposure. Previous studies examining the effects of cocaine on BChE deficient animals relied on chemical inhibition of BChE with tetraisopropyl pyrophosphoramide (iso-OMPA). The creation of the BChE knockout mouse has provided a model for studying pathological effects of cocaine in mice free of chemical confounders. We hypothesized that mice with low or no BChE activity would have reduced cocaine metabolism, leading to hepatotoxicity and cardiomyopathy. A high-resolution in vivo imaging system recorded cardiac and respiratory function following treatment with a carboxylesterase inhibitor and a high dose of cocaine (100 mg/kg, intraperitoneally). The BChE-/- mice demonstrated depressed respiration through 12 hr after dosing and abnormal respiratory patterns consisting of a pause at full inspiration (apneusis), whereas BChE+/+ mice had recovered normal respiration rates by 30 min. after dosing and exhibited no apneusis. Liver and cardiac histology sections were analysed following a 20 mg/kg intraperitoneally dose of cocaine administered daily for 7 days. BChE-/- mice treated for 7 days with the chronic low dose showed significant hepatotoxicity and cardiac perivascular fibrosis compared to BChE+/+ mice. The observed functional changes following acute high-dose and chronic low-dose cocaine in BChE-/- and +/- mice warrants further investigation into the possibility of increased cocaine toxicity in human beings with BChE deficiency.

Five new naturally occurring mutations of the BCHE gene and frequencies of 12 butyrylcholinesterase alleles in a Brazilian population.

Human butyrylcholinesterase (BChE; EC 3.1.1.8) is codified by the BCHE gene (3q26.1-q26.2) in which 65 variants have been identified. BChE is a scavenger of organophosphorus and carbamate compounds and hydrolyzes succinylcholine, mivacurium and cocaine. The present study describes 12 naturally occurring BCHE mutations including five new mutations (K12R, G15G, V294M, G333C and R470W) identified in 366 blood donors from Southern Brazil. Exons 2 and 4 of the BCHE gene were examined by PCR-SSCA and samples with unexpected electrophoretic patterns were sequenced. The respective nucleotide substitution that characterizes each of the four new nonsynonymous mutations was introduced into BCHE cDNA by site directed mutagenesis and transfected into human embryonic kidney 293T cells and/or Chinese hamster ovary cells. The catalyzed hydrolysis of butyrylthiocholine (BTC) by BChE was measured by the Ellman method. Enzyme kinetic parameters obtained after the expression of the respective recombinant BChE evaluated the effects of the four nonsynonymous mutations. Thirty-four out of 366 individuals carried a BChE mutation in exon 2. The K variant mutation, A539T in exon 4, was present in one out of three persons. Gene expression showed that only one of the newly identified mutations (G333C) altered BChE activity, leading to a decrease of about 80% in relation to the wild-type enzyme.

Expression of three naturally occurring genetic variants (G75R, E90D, I99M) of the BCHE gene of human butyrylcholinesterase.

The present paper examined the effects of three non synonymous BCHE mutations (G75R, E90D and /99M) on enzyme kinetic parameters obtained after the expression of the respective recombinant BChEs. The respective nucleotide substitution that characterizes each of the three variants was introduced into BCHE cDNA by site directed mutagenesis and transfected into human embryonic kidney 293 T cells and Chinese hamster ovary cells (for E90D). BChE catalysed hydrolysis of butyrylthiocoline (BTC) was measured by Ellman method. The expression results showed that: (1) the activity of the G75R enzyme represents approximately 45% of the wild-type activity, whereas that of the I99M enzyme does not differ from the wild-type; (2) the E90D enzyme presents a silent phenotype; disruption of the salt bridge between E90 and R42 may cause the enzyme to be rapidly degraded inside the cells. In homozygous form the E90D enzyme may confer increased susceptibility to succinylcholine, but may delay cognitive impairment in aged individuals. BChE genotyping may become important for estimating prognosis, and the knowledge of the genetic variants of BChE in a particular population may be useful for carrying out the genotyping assays.

Naturally occurring mutation Leu307Pro of human butyrylcholinesterase in the Vysya community of India.

BACKGROUND: People with genetic variants of butyrylcholinesterase (EC 3.1.1.8, BChE) can have hours of prolonged apnea after a normal dose of succinylcholine or mivacurium. METHODS: Plasma samples from 226 people in the Vysya community in Coimbatore, India were tested for BChE activity. RESULTS: Nine unrelated individuals had no detectable activity. DNA sequencing revealed a novel mutation in exon 2 of the BCHE gene, responsible for the silent phenotype of human serum BChE. All silent BChE samples were homozygous for a point mutation at codon 307 (CTT-->CCT), resulting in substitution of leucine 307 by proline. Western blot analysis with a monoclonal antibody showed no BChE protein in plasma. Silent BChE plasma samples had no organophosphate-reactive BChE, as measured with FP-biotin. Expression of recombinant Leu307Pro BChE in cell culture confirmed that this mutant is expressed at very low levels. The proline substitution most likely destabilizes the BChE structure and causes the protein to be misfolded and rapidly degraded. CONCLUSIONS: This is the first report of a molecularly defined BChE mutation in the Indian population. The frequency of homozygous silent BChE in the Vysya community is 1 in 24, a value 4000-fold higher than the frequency of homozygous silent BChE in European and American populations.