Molecular aspects of carboxylesterase isoforms in comparison with other esterases

doi:10.1016/0378-4274(95)03493-5

Toxicology Letters

Volumes 82–83, December 1995, Pages 439-445

https://doi.org/10.1016/0378-4274(95)03493-5 Get rights and content

Abstract

The involvement of carboxylesterase, acetylcholinesterase, butyrylcholinesterase and cholesterol esterase in pharmacology and toxicology are well recognized. However, there are few papers concerning the comparative studies of these serine hydrolases in terms of molecular level. Recently, we have studied various aspects of carboxylesterases using cDNAs of carboxylesterase isozymes purified from 9 animal species and human liver microsomes, and found that there is high homology of the N-terminal amino acid sequences of the isozymes tested. On the other hand, we compared the amino acid sequences at the active site of the individual esterases and found that the sequences of all esterases tested are strictly conserved. These results strongly suggest that the esterases involved are classified into the serine hydrolase super family.

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In this study, responses of carboxylesterases, acetylcholinesterase, and stress protein Hsp70 were examined in the midgut and midgut tissue, and brain of fifth instar larvae of Lymantria dispar L. and Euproctis chrysorrhoea L. following chronic exposure to dietary fluoranthene. Specific carboxylesterase activity increased significantly in the midgut tissue of E. chrysorrhoea larvae treated with a lower fluoranthene concentration. The specific patterns of isoforms expression, recorded in larvae of both species, enable efficient carboxylesterase activity as a significant part of defense mechanisms. Increased Hsp70 concentration in the brain of L. dispar larvae points to a response to the proteotoxic effects of a lower fluoranthene concentration. Decreased Hsp70 in the brain of E. chrysorrhoea larvae in both treated groups can suggest induction of other mechanisms of defense. The results indicate the importance of the examined parameters in larvae of both species exposed to the pollutant, as well as their potential as biomarkers.
Hydrolysis of dibutyl phthalate and di(2-ethylhexyl) phthalate in human liver, small intestine, kidney, and lung: An in vitro analysis using organ subcellular fractions and recombinant carboxylesterases
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Phthalates are widely used plasticizers that are primarily and rapidly metabolized to monoester phthalates in mammals. In the present study, the hydrolysis of dibutyl phthalate (DBP) and di(2-ethylhexyl) phthalate (DEHP) in the human liver, small intestine, kidney, and lung was examined by the catalytic, kinetic, and inhibition analyses using organ microsomal and cytosolic fractions and recombinant carboxylesterases (CESs). The V_max (y-intercept) values based on the Eadie-Hofstee plots of DBP hydrolysis were liver > small intestine > kidney > lung in microsomes, and liver > small intestine > lung > kidney in cytosol, respectively. The CL_int values (x-intercept) were small intestine > liver > kidney > lung in both microsomes and cytosol. The V_max and CL_int or CL_max values of DEHP hydrolysis were small intestine > liver > kidney > lung in both microsomes and cytosol. Bis(4-nitrophenyl) phosphate (BNPP) effectively inhibited the activities of DBP and DEHP hydrolysis in the microsomes and cytosol of liver, small intestine, kidney, and lung. Although physostigmine also potently inhibited DBP and DEHP hydrolysis activities in both the microsomes and cytosol of the small intestine and kidney, the inhibitory effects in the liver and lung were weak. In recombinant CESs, the V_max values of DBP hydrolysis were CES1 (CES1b, CES1c) > CES2, whereas the CL_max values were CES2 > CES1 (CES1b, CES1c). On the other hand, the V_max and CL_max values of DEHP hydrolysis were CES2 > CES1 (CES1b, CES1c). These results suggest an extensive organ-dependence of DBP and DEHP hydrolysis due to CES expression, and that CESs are responsible for the metabolic activation of phthalates.
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Citation Excerpt :
In spite of interspecies differences that may occur related to affinity and reactivity towards inhibitors [16], and the use of different isoforms, AChE from electric eel (Electrophorus electricus) EeAChE, is largely reported in studies on novel reactivators against inhibitors or active compounds to address neurodegenerative diseases, being considered a trustable and affordable model [17,18]. Nerve agents are strictly regulated by the Chemical Weapons Convention (CWC) and its international watchdog, the Organization for the Prohibition of Chemical Weapons (OPCW), which oversees the CWC implementation and compliance [13]. Fig. 1 depicts examples of such toxic chemicals, the nerve agents sarin (5), soman (6), tabun (7), VX (8), the recently confirmed Novichok agents, e.g., A-234 (9), and the pesticide paraoxon (10).
Casualties caused by nerve agents, potent acetylcholinesterase inhibitors, have attracted attention from media recently. Poisoning with these chemicals may be fatal if not correctly addressed. Therefore, research on novel antidotes is clearly warranted. Pyridinium oximes are the only clinically available compounds, but poor penetration into the blood-brain barrier hampers efficient enzyme reactivation at the central nervous system. In searching for structural factors that may be explored in SAR studies, we synthesized and evaluated neutral aryloximes as reactivators for acetylcholinesterase inhibited by NEMP, a VX surrogate. Although few tested compounds reached comparable reactivation results with clinical standards, they may be considered as leads for further optimization.
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Carboxylesterases (CarEs) play key roles in metabolism of specific hormones and detoxification of dietary and environmental xenobiotics in insects. We sequenced and characterized CarE cDNAs putatively derived from two different genes named LmCesA1 and LmCesA2 from the migratory locust, Locusta migratoria, one of the most important agricultural pests in the world. The full-length cDNAs of LmCesA1 (1892 bp) and LmCesA2 (1643 bp) encode 543 and 501 amino acid residues, respectively. The two deduced CarEs share a characteristic α/β-hydrolase structure, including a catalytic triad composed of Ser-Glu (Asp)-His and a consensus sequence GQSAG, which suggests that both CarEs are biologically active. Phylogenetic analysis grouped both LmCesA1 and LmCesA2 into clade A which has been suggested to be involved in dietary detoxification. Both transcripts were highly expressed in all the nymphal and adult stages, but only slightly expressed in eggs. Analyses of tissue-dependent expression and in situ hybridization revealed that both transcripts were primarily expressed in gastric caeca. RNA interference (RNAi) of LmCesA1 and LmCesA2 followed by a topical application of carbaryl or deltamethrin did not lead to a significantly increased mortality with either insecticide. However, RNAi of LmCesA1 and LmCesA2 increased insect mortalities by 20.9% and 14.5%, respectively, when chlorpyrifos was applied. These results suggest that these genes might not play a significant role in detoxification of carbaryl and deltamethrin but are most likely to be involved in detoxification of chlorpyrifos in L. migratoria.
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2011, Bioorganic and Medicinal Chemistry
Citation Excerpt :
This family of proteins share a common α/β hydrolase fold that consists of a mostly parallel 5 to 14-stranded β sheet surrounded by several α helices,16 and a high degree of overall structural homology. For example, the reported structures of carboxyesterases (EC 3.1.1.1), acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8)17–20 reveal a high degree of similarity in the N-terminal domain, especially around the active-site serine.21 It is interesting to note that an X-ray structure of pancreatic CEase has been solved at 1.6 Å by molecular replacement based on a truncated model of Torpedo californica acetylcholinesterase.16
We present a new class of inhibitors of pancreatic cholesterol esterase (CEase) based on ‘priviledged’ 5-benzylidenerhodanine and 5-benzylidene-2,4-thiazolidinedione structural scaffolds. The lead structures (5-benzylidenerhodanine 4a and 5-benzylidene-2,4-thiazolidinedione 4b) were identified in an in-house screening and these inhibited CEase with some selectivity over another serine hydrolase, acetylcholinesterase (AChE) (4a, CEase IC₅₀ = 1.76 μM vs AChE IC₅₀= 5.14 μM and 4b, CEase IC₅₀ = 5.89 μM vs AChE IC₅₀ >100 μM). A small library of analogs (5a–10a) containing a core amino acid in place of the glycerol group of the lead structures, was prepared to explore other potential binding interaction with CEase. These analogs inhibited CEase with IC₅₀ values ranging from 1.44 to 85 μM, with the majority exhibiting some selectivity for CEase versus AChE. The most potent compound of the library (10a) had 17-fold selectivity over AChE. We also report molecular docking (with CEase) and detailed kinetic analysis on the amino acid analogs to further understand the associated structure–activity relationships.
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Ghrelin circulates as acylated (AG) and unacylated (or desacyl) ghrelin (UAG). We aimed at clarifying the effect of age and sex on plasma deacylation and degradation of AG in vivo and in vitro in the rat. In vivo, we compared AG and UAG concentrations following administration of 1 μg AG intraperitoneally in rat neonates during the first 3 h of life. AG administration caused a 2–3 times increase in plasma AG concentrations contrasting with a ≈1000 times increase in UAG concentrations suggesting rapid deacylation of AG into UAG. In vitro, we demonstrated that AG degradation was greater in the fetus (97% over 30 min) and decreased progressively to 57% in adult animals (P < 0.001). Carboxylesterase and butyrylcholinesterase activities were determined during the fetal (day 21 of pregnancy) and postnatal period (days 1, 6, 13, 21 and 28) and in the adult rat and were found to increase with age (P < 0.001). While inhibition of carboxylesterase and butyrylcholinesterase did not affect AG deacylation, serine protease inhibitors decreased AG degradation in the adult rat (from 59% to 23%) and, to a lesser extent, in the rat neonate (from 92% to 57%) by reducing both deacylation and degradation into non-UAG metabolites. Our data suggest that degradation of AG into UAG and non-UAG metabolites is much faster in the fetus and in the rat neonate compared to the adult. We speculate that this process allows for fine tuning of the physiological effects of both AG and UAG.

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Toxicology Letters

Abstract

References (17)

Complete amino acid sequence of fetal bovine serum acetylcholinesterase and its comparison in various regions with other cholinesterases

FEBS Lett.

Differential responses of rat hepatic microsomal carbox-ylesterase isozymes to glucocorticoid hormones and pregnenolone 16 α-carbonitrile

Biochem. Pharmacol.

A C-terminal signal prevents secretion of luminal ER proteins

Cell

The cholinesterases: from genes to proteins

Annu. Rev. Pharmacol. Toxicol.

Relationship between sequence conservation and three-dimensional structure in a large family of esterases, lipases, and related proteins

Protein Sci.

Cholinesterase-like domains in enzymes and structural proteins: functional and evolutionary relationships and identification of a catalytically essential aspartic acid

Isolation and characterization of acetylcholinesterase from Drosophila

J. Biol. Chem.

Role of carboxylesterases in xenobiotic metabolism

Cited by (71)

Effects of dietary fluoranthene on tissue-specific responses of carboxylesterases, acetylcholinesterase and heat shock protein 70 in two forest lepidopteran species

Hydrolysis of dibutyl phthalate and di(2-ethylhexyl) phthalate in human liver, small intestine, kidney, and lung: An in vitro analysis using organ subcellular fractions and recombinant carboxylesterases

Synthesis and in vitro evaluation of neutral aryloximes as reactivators of Electrophorus eel acetylcholinesterase inhibited by NEMP, a VX surrogate

RNA interference revealed the roles of two carboxylesterase genes in insecticide detoxification in Locusta migratoria

New cholesterol esterase inhibitors based on rhodanine and thiazolidinedione scaffolds

Ontogeny of acylated ghrelin degradation in the rat