Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity

Ellen G. Duysen, Cynthia F. Bartels, Oksana Lockridge

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

Human butyrylcholinesterase (BChE) hydrolyzes cocaine to inactive metabolites. A mutant of human BChE, A328W, hydrolyzed cocaine 15-fold faster compared with wild-type BChE. Although the catalytic properties of human BChE secreted by Chinese hamster ovary (CHO) cells are identical to those of native BChE, a major difference became evident when the recombinant BChE was injected into rats and mice. Recombinant BChE disappeared from the circulation within minutes, whereas native BChE stayed in the blood for a week. Nondenaturing gel electrophoresis showed that the recombinant BChE consisted mainly of monomers and dimers. In contrast, native BChE is a tetramer. The problem of the short residence time was solved by finding a method to assemble the recombinant BChE into tetramers. Coexpression in CHO cells of BChE and 45 residues from the N terminus of the COLQ protein yielded 70% tetrameric BChE. The resulting purified recombinant BChE tetramers had a half-life of 16 h in the circulation of rats and mice. The 16-h half-life was achieved without modifying the carbohydrate content of recombinant BChE. The protective effect of recombinant wild-type and A328W mutant BChE against cocaine toxicity was tested by measuring locomotor activity in mice. Pretreatment with wild-type BChE or A328W tetramers at a dose of 2.8 units/g i.p. reduced cocaineinduced locomotor activity by 50 and 80%. These results indicate that recombinant human BChE could be useful for treating cocaine toxicity in humans.

Original languageEnglish (US)
Pages (from-to)751-758
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume302
Issue number2
DOIs
StatePublished - Jul 30 2002

Fingerprint

Butyrylcholinesterase
Cricetulus
Cocaine
Half-Life
Ovary
Locomotion

ASJC Scopus subject areas

  • Molecular Medicine
  • Pharmacology

Cite this

@article{ccf5c37e30224827bce18e392471c63e,
title = "Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity",
abstract = "Human butyrylcholinesterase (BChE) hydrolyzes cocaine to inactive metabolites. A mutant of human BChE, A328W, hydrolyzed cocaine 15-fold faster compared with wild-type BChE. Although the catalytic properties of human BChE secreted by Chinese hamster ovary (CHO) cells are identical to those of native BChE, a major difference became evident when the recombinant BChE was injected into rats and mice. Recombinant BChE disappeared from the circulation within minutes, whereas native BChE stayed in the blood for a week. Nondenaturing gel electrophoresis showed that the recombinant BChE consisted mainly of monomers and dimers. In contrast, native BChE is a tetramer. The problem of the short residence time was solved by finding a method to assemble the recombinant BChE into tetramers. Coexpression in CHO cells of BChE and 45 residues from the N terminus of the COLQ protein yielded 70{\%} tetrameric BChE. The resulting purified recombinant BChE tetramers had a half-life of 16 h in the circulation of rats and mice. The 16-h half-life was achieved without modifying the carbohydrate content of recombinant BChE. The protective effect of recombinant wild-type and A328W mutant BChE against cocaine toxicity was tested by measuring locomotor activity in mice. Pretreatment with wild-type BChE or A328W tetramers at a dose of 2.8 units/g i.p. reduced cocaineinduced locomotor activity by 50 and 80{\%}. These results indicate that recombinant human BChE could be useful for treating cocaine toxicity in humans.",
author = "Duysen, {Ellen G.} and Bartels, {Cynthia F.} and Oksana Lockridge",
year = "2002",
month = "7",
day = "30",
doi = "10.1124/jpet.102.033746",
language = "English (US)",
volume = "302",
pages = "751--758",
journal = "Journal of Pharmacology and Experimental Therapeutics",
issn = "0022-3565",
publisher = "American Society for Pharmacology and Experimental Therapeutics",
number = "2",

}

TY - JOUR

T1 - Wild-type and A328W mutant human butyrylcholinesterase tetramers expressed in Chinese hamster ovary cells have a 16-hour half-life in the circulation and protect mice from cocaine toxicity

AU - Duysen, Ellen G.

AU - Bartels, Cynthia F.

AU - Lockridge, Oksana

PY - 2002/7/30

Y1 - 2002/7/30

N2 - Human butyrylcholinesterase (BChE) hydrolyzes cocaine to inactive metabolites. A mutant of human BChE, A328W, hydrolyzed cocaine 15-fold faster compared with wild-type BChE. Although the catalytic properties of human BChE secreted by Chinese hamster ovary (CHO) cells are identical to those of native BChE, a major difference became evident when the recombinant BChE was injected into rats and mice. Recombinant BChE disappeared from the circulation within minutes, whereas native BChE stayed in the blood for a week. Nondenaturing gel electrophoresis showed that the recombinant BChE consisted mainly of monomers and dimers. In contrast, native BChE is a tetramer. The problem of the short residence time was solved by finding a method to assemble the recombinant BChE into tetramers. Coexpression in CHO cells of BChE and 45 residues from the N terminus of the COLQ protein yielded 70% tetrameric BChE. The resulting purified recombinant BChE tetramers had a half-life of 16 h in the circulation of rats and mice. The 16-h half-life was achieved without modifying the carbohydrate content of recombinant BChE. The protective effect of recombinant wild-type and A328W mutant BChE against cocaine toxicity was tested by measuring locomotor activity in mice. Pretreatment with wild-type BChE or A328W tetramers at a dose of 2.8 units/g i.p. reduced cocaineinduced locomotor activity by 50 and 80%. These results indicate that recombinant human BChE could be useful for treating cocaine toxicity in humans.

AB - Human butyrylcholinesterase (BChE) hydrolyzes cocaine to inactive metabolites. A mutant of human BChE, A328W, hydrolyzed cocaine 15-fold faster compared with wild-type BChE. Although the catalytic properties of human BChE secreted by Chinese hamster ovary (CHO) cells are identical to those of native BChE, a major difference became evident when the recombinant BChE was injected into rats and mice. Recombinant BChE disappeared from the circulation within minutes, whereas native BChE stayed in the blood for a week. Nondenaturing gel electrophoresis showed that the recombinant BChE consisted mainly of monomers and dimers. In contrast, native BChE is a tetramer. The problem of the short residence time was solved by finding a method to assemble the recombinant BChE into tetramers. Coexpression in CHO cells of BChE and 45 residues from the N terminus of the COLQ protein yielded 70% tetrameric BChE. The resulting purified recombinant BChE tetramers had a half-life of 16 h in the circulation of rats and mice. The 16-h half-life was achieved without modifying the carbohydrate content of recombinant BChE. The protective effect of recombinant wild-type and A328W mutant BChE against cocaine toxicity was tested by measuring locomotor activity in mice. Pretreatment with wild-type BChE or A328W tetramers at a dose of 2.8 units/g i.p. reduced cocaineinduced locomotor activity by 50 and 80%. These results indicate that recombinant human BChE could be useful for treating cocaine toxicity in humans.

UR - http://www.scopus.com/inward/record.url?scp=0036070598&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0036070598&partnerID=8YFLogxK

U2 - 10.1124/jpet.102.033746

DO - 10.1124/jpet.102.033746

M3 - Article

C2 - 12130740

AN - SCOPUS:0036070598

VL - 302

SP - 751

EP - 758

JO - Journal of Pharmacology and Experimental Therapeutics

JF - Journal of Pharmacology and Experimental Therapeutics

SN - 0022-3565

IS - 2

ER -