Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase: evidence for CPT-11 drug activation.
Butyrylcholinesterases (BuChEs; acylcholine acylhydrolase; EC 3.1.1.8) have been demonstrated to convert the anticancer agent CPT-11 (irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) into its active metabolite SN-38 (7-ethyl-10-hydroxycamptothecin). In addition, significant differences in the extent of drug metabolism have been observed with BuChEs derived from different species. In an attempt to understand these differences, we have isolated the cDNA encoding a horse BuChE. Based upon the NH2-terminal amino acid sequence of a purified horse BuChE, we designed degenerate primers to amplify the coding sequence from horse liver cDNA. Following polymerase chain reaction and rapid amplification of the cDNA ends, we generated an 1850-bp DNA fragment, containing an 1806-bp open reading frame. The cDNA encodes a protein of 602 amino acid residues, including a 28-amino-acid NH2-terminal signal peptide. Furthermore, the DNA sequence and the deduced amino acid sequence revealed extensive homology to butyrylcholinesterase genes from several other species. In vitro transcription-translation of the cDNA produced a 66-kDa protein, identical to the size of native horse serum BuChE following removal of carbohydrate residues with endoglycosidase F. Additionally, transient expression of the cDNA in Cos-7 cells yielded extracts that exhibited cholinesterase activity and demonstrated a Km value for butyrylthiocholine of 106+/-9 nM. This extract converted the anticancer drug CPT-11 into SN-38, demonstrating that this drug can be activated by enzymes other than carboxylesterases.
Wierdl
M
M
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Morton
C L
CL
Danks
M K
MK
Potter
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D002091:Butyrylcholinesterase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D002166:Camptothecin / Q000031:analogs & derivatives / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D006736:Horses; D006801:Humans; D000077146:Irinotecan; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D011355:Prodrugs / Q000378:metabolism; D014176:Protein Biosynthesis; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D000972:Antineoplastic Agents, Phytogenic; D018076:DNA, Complementary; D011355:Prodrugs; D000077146:Irinotecan; D002091:Butyrylcholinesterase; D002166:Camptothecin