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 cocaine-induced locomotor activity by 50 and 80%. These results indicate that recombinant human BChE could be useful for treating cocaine toxicity in humans.
Footnotes
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This work was supported by U.S. Army Medical Research and Materiel Command Grant DAMD17-01-2-0036 and National Institutes of Health/National Institute on Drug Abuse Grant R01 DA011707 (to O.L.), and by a Center Grant to University of Nebraska Medical Center from the National Cancer Institute, Grant CA36727. The opinions or assertions contained herein belong to the authors and should not be construed as the official views of the U.S. Army or the Department of Defense.
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DOI: 10.1124/jpet.102.033746
- Abbreviations:
- BChE
- butyrylcholinesterase
- CHO
- Chinese hamster ovary
- AChE
- acetylcholinesterase
- RSV
- Rous sarcoma virus
- G418
- geneticin
- 293 cell
- human embryonic kidney 293cell
- Received January 29, 2002.
- Accepted April 15, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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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
