Isolation and characterization of a cDNA encoding a horse liver butyrylcholinesterase: evidence for CPT-11 drug activation.

This research article is about the isolation of a specific gene encoding a horse liver enzyme, Butyrylcholinesterase (BuChE), and exploring its ability to metabolize the anticancer drug CPT-11 into its active form, SN-38, showing that enzymes other than carboxylesterases can activate this drug.

Isolation and Characterization of a cDNA Encoding Horse Liver Butyrylcholinesterase

• The study was centered on an enzyme called Butyrylcholinesterase (BuChE).
• Different species’ BuChEs have varying efficiencies in metabolizing and activating the anticancer drug CPT-11.
• To understand these differences, researchers isolated the cDNA (complementary DNA), which carries the gene coding for a horse liver BuChE.
• The isolation was based on the NH2-terminal amino acid sequence of a purified horse BuChE, which guided the design of degenerate primers for the amplification of the cDNA in horse liver.

Resulting DNA Fragment and cDNA

• By using polymerase chain reactions and rapid cDNA ends amplification, an 1850-bp DNA fragment carrying an 1806-bp open reading frame was generated.
• The cDNA inside this fragment encodes a protein of 602 amino acids, including a 28-amino-acid NH2-terminal signal peptide that is responsible for directing the protein to its appropriate location within the cell.

Homology to Other Butyrylcholinesterase Genes

• The DNA sequence and the translated amino acid sequence were found to show extensive homology, or similar structure and function, to butyrylcholinesterase genes from various other species.

Protein Production and Activity

• The in vitro transcription-translation of the cDNA produced a 66-kDa protein, a size identical to the native horse serum BuChE after carbohydrate residues removal.
• In a cell line (Cos-7), transient expression of the cDNA resulted in extracts that exhibited cholinesterase activity and showed a Km value (a measure of enzyme-substrate affinity) for butyrylthiocholine of 106+/-9 nM.
• This extract was able to convert CPT-11 into its active form SN-38, providing evidence that enzymes other than carboxylesterases can contribute to activating the anticancer drug.